University of Veterinary Medicine Hannover

University of Veterinary Medicine Hannover

Generation and characterization of antibodies against Rift Valley fever phlebovirus and evaluation of their

therapeutic efficacy in a mouse model

Inaugural-Dissertation to obtain the academic degree Doctor medicinae veterinariae

(Dr. med. vet.)

submitted by Benjamin Gutjahr

Suhl

Hannover 2019

Academic supervision: Prof. Dr. Martin H. Groschup Institute of Novel and Emerging Infectious Diseases Friedrich-Loeffler-Institut Greifswald-Insel Riems

1. Referee: Prof. Dr. Martin H. Groschup Institute of Novel and Emerging Infectious Diseases Friedrich-Loeffler-Institut Greifswald-Insel Riems

2. Referee: Prof. Dr. Stefanie Becker Research Center for Emerging Infections and Zoonoses / Institute for Parasitology University of Veterinary Medicine Hannover

Day of the oral examination: 30th of October, 2019

Sponsorship:

This work was supported by the Zoonoses Anticipation and Preparedness Initiative(ZAPI) in the frame of the Innovative Medicines Initative (IMI) of the European Commission.

To my family

TABLE OF CONTENTS

1 INTRODUCTION .......................................................................... 01

2 LITERATURE REVIEW ............................................................... 03

2.1 History and naming of Rift Valley fever phlebovirus ................................. 03

2.2 Epidemiology ................................................................................................ 03

2.3 Classification ................................................................................................. 06

2.4 Morphology and genome characterization ................................................. 06

2.5 Replication ..................................................................................................... 07

2.6 Transmission ................................................................................................. 09

2.6.1 Enzootic / inter-epidemic cycle ......................................................................... 09

2.6.2 Epizootic / epidemic cycle ................................................................................ 09

2.6.3 Human exposure routes .................................................................................. 10

2.7 Clinical signs ................................................................................................. 10

2.8 Pathology and histopathology ..................................................................... 12

2.9 Diagnostic ...................................................................................................... 12

2.10 Vaccination and treatment ........................................................................... 13

2.11 Monoclonal antibodies ................................................................................. 14

2.12 Variable heavy chain domain of a heavy chain antibody .......................... 15

3 MATERIALS AND METHODS ..................................................... 18

3.1 Animal trials .................................................................................................. 18

3.2 Serological assays ........................................................................................ 18

3.3 PCR ................................................................................................................ 18

3.4 Extraction and purification kits.................................................................... 19

4 MANUSCRIPT I:

A single domain antibody with high affinity to

Rift Valley fever phlebovirus strains ......................................... 20

4.1 Abstract ......................................................................................................... 20

4.2 Introduction ................................................................................................... 20

4.3 Materials and methods ................................................................................. 21

4.4 Results ........................................................................................................... 26

4.5 Discussion ..................................................................................................... 27

4.6 Figures ........................................................................................................... 28

4.7 Acknowledgments ........................................................................................ 33

5 MANUSCRIPT II:

Two monoclonal antibodies against glycoprotein Gn

fully protect mice from Rift Valley fever

challenge by cooperative effects .............................................. 34

5.1 Abstract ......................................................................................................... 34

5.2 Author summary ........................................................................................... 34

5.3 Introduction ................................................................................................... 35

5.4 Materials and methods ................................................................................. 36

5.5 Results ........................................................................................................... 39

5.6 Discussion ..................................................................................................... 42

5.7 Figures ........................................................................................................... 44


6 MANUSCRIPT III:

Live vaccine virus yields a neutralizing monoclonal

antibody against Rift Valley fever phlebovirus ....................... 53

6.1 Abstract ......................................................................................................... 53

6.2 Short communication ................................................................................... 53

6.3 Tables ............................................................................................................. 56

6.4 Figures ........................................................................................................... 57


7 GENERAL DISCUSSION ............................................................ 60

8 SUMMARY .................................................................................. 65

9 ZUSAMMENFASSUNG ............................................................... 67

10 BIBLIOGRAPHY .......................................................................... 69

11 SUPPLEMENTS .......................................................................... 90

11.1 Manuscript I ................................................................................................... 90

11.2 Manuscript II .................................................................................................. 93

12 AUTHORS´ CONTRIBUTION .................................................... 112

13 ACKNOWLEDGMENTS ............................................................ 114

Manuscripts extracted from the doctorate project:

1. B. Gutjahr, M. Rissmann, M. Keller, M. H. Groschup, M. Eiden; A single domain antibody with high affinity to Rift Valley fever phlebovirus strains (to be submitted)

2. B. Gutjahr, M. Keller, M. Rissmann, F. von Arnim, S. Jäckel, S. Reiche, R. Ulrich, M. H. Groschup and M. Eiden; Two monoclonal antibodies against glycoprotein Gn fully protect mice from Rift Valley Fever challenge by cooperative effects; PLOS Neglected Tropical Diseases (submitted)

3. B. Gutjahr, M.Eiden, M. Rissmann, C. Mroz, S. Reiche, M. H. Groschup; Live vaccine virus yields a neutralizing monoclonal antibody against Rift Valley fever phlebovirus (to be submitted)

Parts of the results have been presented in conference:

Junior Scientist Zoonoses Meeting, Langen, Germany, 2017: “Generation and application of monoclonal antibodies and Nanobodies against Rift Valley fever virus MP-12 strain” (Poster presentation)

National Symposium on Zoonoses Research in Germany, Berlin, Germany, 2017: “Novel monoclonal antibodies and alpaca derived single domain antibody fragments (VHH) against Rift Valley fever virus” (Poster presentation)

National Symposium on Zoonoses Research in Germany, Berlin, Germany, 2018: “Neutralizing monoclonal antibodies and alpaca derived single domain antibody fragments against Rift Valley fever virus” (Oral presentation)

IMI 10th Anniversary Scientific Symposium, Brussels, Belgium, 2018: “Novel peptide vaccines against Rift valley fever virus” (Poster presentation; 2nd place for the best poster)

7th European Seminar in Virology, Padova, Italy, 2019: “Monoclonal antibodies protect mice in Rift Valley fever challenge model” (Oral and poster presentation)

LIST OF ABBREVATIONS

µg Microgram

µl Microliter

µm Micrometer

µM Micromolar

aa amino acid

ABC Avidin-biotin-peroxidase complex

AEC 3-amino-9-ethylcarbazole

ABTS 2. 2′-azino di-ethylbenzothiazoline sulphonic acid

ANOVA Analysis of variance

Arbovirus Arthropod-borne virus

Arg Arginine

BATV Batai virus

BHK-21 Baby hamster kidney-21

bp Base pair

BSL-3 Biosafety level 3

BUNV Bunyamwera virus

cDNA Complementary deoxyribonucleic acid

CDR Complementarity determining region

CH Constant domain of the heavy chain

CI Combination index

Cys Cysteine

Cy3 Cyanine 3

DAPI 4′, 6-Diamidin-2-phenylindol

DC-Sign Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin

DENV Dengue virus

DIVA Differentiating Infected from Vaccinated Animals

DMEM Dulbecco´s Modified Eagle Medium

dpi Days post inoculation

DRI Dose-reduction index

E. coli Escherichia coli

EC50 Effective concentration of 50 %

EGFR Epidermal Growth Factor Receptor

ELISA Enzyme linked immunosorbent assay

EMA European medicines agency

ER Endoplasmatic reticulum

EURL European directive

F(a) Fraction affected

Fab Fragment antigen-binding

Fc Fragment crystallisable

FDA Food and drug administration

FLI Friedrich-Loeffler-Institut

FR Framework region

Gc Glycoprotein c (-terminal)

Glu Glutamine

Gn Glycoprotein n (-terminal)

h Hour

HCAb Heavy chain antibody

HIV Human immunodeficiency virus

HRP Horseradish peroxidase

IC50 Inhibitory concentration of 50 %

ICTV International Committee on Taxonomy of Viruses

IFN-β Beta-Interferon

IgG Immunoglobulin G

IgM Immunoglobulin M

IgNAR Immunoglobulin new antigen receptor

IgW Immunoglobulin W

IHC Immunohistochemistry

(I)IFA (Indirect) immunofluorescence assay

IMGT International Immunogenetics Information System®

i. p. Intraperitoneal

i. v. Intravenous

kDa Kilo Dalton

L Light

LALLF Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern

LB Luria-Bertani

L segment Large segment

M Molar

mAb Monoclonal antibody

mg Milligram

min Minute

ml Milliliter

mM Millimolar

MOI Multiplicity of infection

mRNA Messenger ribonucleic acid

M segment Medium segment

NaN Not a number

ND50 Neutralizing dose of 50 %

Ni-NTA Nickel-nitrilotriacetic acid

nm Nanometer

nM Nanomolar

Np Nucleoprotein

nplr N-Parameter logistic regression

NSm Nonstructural protein, encoded on the medium segment

NSs Nonstructural protein, encoded on the small segment

n. s. not significant

n. t. not tested

PBMC Peripheral blood mononuclear cell

PBS Phosphate buffered saline

PBST PBS containing 0.1 % Tween

PCR Polymerase chain reaction

PCV2 Porcine circovirus 2

PEG Polyethylene glycol

PEP Post-exposure treatment

PFA Paraformaldehyde

PFU Plaque forming unit

Phe Phenylalanine

PKR Double-stranded RNA-dependent protein kinase

pmol Picomole

PrEP Prophylactic exposure treatment

RANKL Receptor Activator of NF-κB Ligand

RNA Ribonucleic acid

RNP Ribonucleoprotein

rpm Rounds per minute

RPMI Roswell Park Memorial Institute

RSV Respiratory syncytial virus

RT-PCR Reverse transcriptase polymerase chain reaction

RT-qPCR Quantitative real-time reverse transcriptase polymerase chain reaction

RT Room temperature

RVF Rift Valley fever

RVFV Rift Valley fever phlebovirus

s second

S / P % Sample-to-positive-ratio

scFv Single chain variable fragment

sdAb Single domain antibody

SDS Sodiumdodecyl-sulfate

SNT Serum neutralization test

SPF Specific-pathogen-free

spp. Species

S segment Small segment

TCID50 50 % tissue culture infective dose

TFIIH Transcription factor II Human

TMB 3. 3´, 5. 5´-Tetramethylbenzidin

Trp Tryptophan

TY Tryptone yeast extract medium

u Unit

USA United States of America

VL Variable domain of the light chain

VHH Variable heavy chain domain of a heavy chain antibody

VH Variable domain of the heavy chain

VNT Virus neutralization test

VT Virus titration

LIST OF TABLES

Table 6.1: Overview of BALB / c mice, immunized with different antigens, the final neutralizing titer and number of generated mAbs 56

Table 6.2: Overview of all RVFV specific generated mAbs and their results in different tests 56

LIST OF FIGURES

Figure 2.1: Taxonomy of Rift Valley fever phlebovirus 06

Figure 2.2: Schematic of the replication cycle of RVFV 08

Figure 2.3: Schematic overview of naturally occurring antibodies in (I) Camelidae, (II) Chondrichthyes and (III) a sdAb with possible multimeres 16

Figure 4.1: Protein sequence of VHH 9F2 28

Figure 4.2: Biochemical analysis of recombinant VHH 9F2 29

Figure 4.3: In-house IIFA and commercial IIFA (E, J without DAPI) with VHH 9F2 using different RVFV strains 29

Figure 4.4: Data are shown as semi-quantitative signal strength (0-3) in IIFA using VHH 9F2 in serial dilutions against different RVFV strains 30

Figure 5.1: ELISA analysis of mAbs Gn3 and Gn32 using recombinant glycoprotein Gn as antigen 44

Figure 5.2: mAb Gn3 and Gn32 neutralize RVFV in two serum neutralization tests 45

Figure 5.3: Survival curves and cumulative mean clinical scores of tested groups 46

Figure 5.4: PCR results of brain, liver and cruor and ELISA results of serum from heart blood of individual mice 47

Figure 5.5: Histopathological and immunohistochemical findings in different tissues of three individual mice 48

Figure 6.1: Exemplary IIFA images with (A-C) strong positive mAbs, (D) weak positive mAb, (E) negative control and (F) positive control (mAb against RVFV Gn) 57

Figure 6.2: mAbs were used in ELISA for detecting RVFV Gn, Gc, Np, NSs and NSm 58

Figure 6.3: Western blot analysis 58

Supplemental figure 4.1: A and B show different PCR products that were amplified during generation of VHHs 31

Supplemental figure 4.2: Three consecutive rounds of panning were done and colonies were counted after each step 32

https://en.wikipedia.org/wiki/Chondrichthyes

Supplemental figure 4.3: In-house IIFA with BUNV (A, C) and BATV (B, D) 33

Supplemental figure 5.1: Clinical score sheet for daily examination of infected mice 49

Supplemental figure 5.2: Virus replication from PCR positive tissues, determined as virus titer (log[TCID50 / ml]) 50

Supplemental figure 5.3: Liver, spleen, brain and lung were examined for virus in IHC (A) and histopathology (B) 51

Supplemental figure 5.4: Alignment of amino acid sequences of RVFV strain 35 / 74 MP-12 52

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1 INTRODUCTION

The Rift Valley fever phlebovirus (RVFV) is a zoonotic arbovirus belonging to the genus of Phlebovirus that is widespread in large parts of Africa and on the Arabian Peninsula. Main vectors for transmission are mosquitos of the genera Aedes, Culex and Anopheles. The virus is maintained by transovarial transmission in Aedes species and is therefore able to survive in the dry season. Heavy rainfall events, e. g. El Niño, cause mass hatching of RVFV infected mosquitos and subsequent transmission to ruminants like sheep, goats and cattle. So-called abortion storms with an abortion rate of up to 100 % in pregnant ewes are a typical sign for Rift Valley fever (RVF) infections. The mortality rate in newborns is significantly higher compared to older animals that suffer from less severe symptoms. Humans are mainly infected by direct contact with infected animals and tissues such as blood or abortion material, or by inhalation of infectious aerosols. Employees like veterinarians, slaughterhouse worker or farmers are therefore exposed to a higher risk of infection. Infected humans show mostly unspecific flu-like symptoms. In severe cases, mainly ocular complications can be observed, but jaundice and hemorrhagic manifestations or neurological disorders are observed in 1-2 % of reported cases.

As RVFV has spread rapidly throughout Africa and Arabia in recent decades and due to anthropogenic factors like climate change and global trade, there is a potential danger that the virus can also reach Europe among other continents through competent vectors or infected animals. Therefore, the improvement and development of new diagnostic assays to monitor RVFV endemic areas and immunological naïve regions is of major importance to limit further spread of the virus as well as to prevent large outbreaks in endemic areas at an early stage.

In a first study, the generation of anti-RVFV single domain antibodies (sdAb) or variable heavy chain domain of a heavy chain antibody (VHH) was established. In addition to conventional IgG antibodies, animals of the Camelidae family produce so-called heavy chain antibodies (HCAbs). Their antigen-binding side is composed of only one variable heavy chain and is therefore, with a size of 15 kDa, only a tenth of the size of conventional antibodies. Consequently, they can reach hidden epitopes and are also exceptionally stable. Similar to monoclonal antibodies (mAbs), single domain antibodies have been evaluated as a possible diagnostic and therapeutic tool against various viruses (e. g. Marburg virus, DENV, PCV2, RSV, Influenza), but up to now there are no published data regarding RVFV specific VHHs. Therefore, in the here described set of experiments established protocols have been modified to generate RVFV specific sdAbs from the blood of an alpaca (Vicugna pacos) immunized with RVFV vaccine strain MP-12. The specific sdAb sequence was isolated over consecutive PCR steps enabling us to ultimately generate a cDNA library of different VHHs. The specificity was gained by multiple panning rounds on RVFV infected cells. As final result, a specific sdAb was obtained for the first time, detecting different RVFV strains even in a low nM range in an in-house immunofluorescence assay (IIFA).

In order to prevent the spread of the Rift Valley fever phlebovirus in endemic countries, several commercially available exclusively veterinary RVF vaccines (e. g. strain Smithburn either inactivated or even alive) are used. However, there are substantial

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safety concerns, as severe adverse reactions have been described, including fetal malformations and teratogenic effects. There are currently several attempts to produce new safe and effective vaccines for the active immunization of animals and humans. This is accompanied by a lack of approved antivirals against RVF. Common antivirals such as Ribavirin and Favipiravir show only a limited effect in animal models. A promising therapeutic approach is therefore the generation and use of highly neutralizing (monoclonal) antibodies like already described for other viral infections such as rabies, influenza or Ebola.

To date, there is little and mostly outdated information on neutralizing antibodies against RVFV and no published detailed in vivo studies with mAbs used as antivirals against RVF infection. So in the second part of this study a neutralizing mAb alone and in combination with a non-neutralizing mAb was evaluated for protective efficacy in a lethal RVFV mouse challenge model using highly virulent RVFV strain 35 / 74. Both antibodies originated from immunization of mice with recombinant glycoprotein Gn. Intravenous application of both mAbs were performed before and after infection (prophylactic-exposure and post-exposure) of BALB / c mice. In addition to survival, daily blood samples and organic tissues were tested for viral load and virus replication as well as examined by immunohistochemistry and histology. The immune response was assessed by ELISA and SNT. Especially the simultaneous application of both monoclonal antibodies showed complete protection accompanied by a strong decrease of viral loads and elevated antibody response of mice´ immune system.

The last aim was to develop new neutralizing antibodies against RVFV by a conventional approach. Most of the literature known about monoclonal antibodies against RVFV dates back to the 1980s and 1990s where few neutralizing antibodies were generated by immunizing mice with RVF glycoproteins Gn or Gc. As a new approach, we generated RVFV mAbs by immunizing four BALB / c mice with RVFV strain MP-12 followed by immunization of four BALB / c mice with virulent RVFV strain 35 / 74. After the development of a strong neutralizing antibody response, hybridoma cells were generated and mAbs were screened for RVFV specificity in an IIFA and subsequently tested in a serum neutralization test (SNT). In total, 14 individual antibodies were determined by specific staining in IIFA and one single mAb exhibited neutralizing activity. This showed that immunizing mice with full virus is an option to generate neutralizing antibodies.

When combined, our studies point towards new approaches for the generation of different RVFV specific antibody variants. Due to its high affinity, sdAb could be used as a promising novel diagnostic tool. Furthermore, the use of neutralizing antibodies as antiviral agents is a promising therapeutic option to combat RVF.

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2 LITERATURE REVIEW

2.1 History and naming of Rift Valley fever phlebovirus

The first Rift Valley fever phlebovirus (RVFV) reports originated from Daubney et al. (1931) in Kenya, when they investigated an outbreak with a high mortality rate in lambs on a farm near Lake Naivasha, in the Rift Valley. They noticed a previously unknown disease in sheep and cattle. In addition to a high mortality in lambs, they observed a high abortion rate of pregnant ewes and thus coined the expression abortion storm. The disease was particularly devastating in young lambs and death occurred within 24 h. The liver is considered the most severely affected organ; hence, the disease was initially designated as “enzootic hepatitis”. During this outbreak, 5,000 animals died and the first human cases were described in which affected scientists and veterinarians showed signs of infection. Eventually about 200 humans were affected without fatalities. Due to the typical fever in humans and the location of the first outbreak, enzootic hepatitis was soon renamed to Rift Valley fever (RVF). The outbreak was associated to unusually high precipitation followed by a vastly increased occurrence of mosquitos. In order to stop the described outbreak, sheep were moved to a farm at a higher altitude. The condition of sheep soon improved and subsequently mosquitos were identified as vectors. Additionally, an important role in the described events was assigned to unusually heavy rainfalls (Daubney et al., 1931). Retrospectively, Montgomery most likely described the first occurrence of RVF in 1913 when an unnamed mortality among lambs with RVF typical symptoms was described in a report of the Department of Agriculture for British East Africa (nowadays known as the Republic of Kenya) in 1912 (McMillen and Hartman, 2018). In 2015, RVFV was renamed by the ICTV (International Committee on Taxonomy of Viruses) to Rift Valley fever phlebovirus (ICTV (2019)).

2.2 Epidemiology

Probably the first cases of affected lambs were reported in 1912 in Kenya by Montgomery (1913) and later by Daubney and Hudson at a farm near Lake Naivasha in the Rift Valley in Kenya (Daubney et al., 1931). Further cases were observed over the years in Kenya until 1937 (Weiss, 1957). In the late 1940s, RVFV spread over the Kenyan boarder and reached Tanzania (Nanyingi et al., 2015). Since the 1950s, about 11 epizootics occurred on average every 3.6 years in Kenya and neighboring countries (Murithi et al., 2011). Outbreaks at the Horn of Africa occurred mainly at the end of the year during warm El Niño Southern Oscillation events, which caused heavy rainfalls and subsequent massive mosquito hatching (Linthicum et al., 2016). Most recent outbreak in East Africa appeared 2017 and 2018 in parts of Eastern Lake States of South Sudan (WHO, 2018a). In South Africa, the first large epizootic outbreak appeared 1950-1951 with 100,000 affected sheep and half a million aborted lambs (Gerdes, 2004). Since then, several recurrent epizootics have been reported over the years in South Africa (Pienaar and Thompson, 2013). First human fatality cases caused by RVF have been reported in

4

South Africa in 1974 (Mcintosh et al., 1980). All outbreaks were correlated with exceptional high precipitation due to the cool La Niña phase (Linthicum et al., 2016). So far, the last epidemic in South Africa occurred between 2010 and 2011 with a large number of affected humans and animals (Archer et al., 2013). In 1977, RVFV emerged for the first time outside Sub-Saharan Africa in Egypt (Laughlin et al., 1979). The epidemic affected 200,000 humans and led to 600 fatal cases, the highest numbers of human cases to date (McMillen and Hartman, 2018). Furthermore, this RVF outbreak was devastating for the Egyptian economy, as half of all animals in the country were affected. The virus might have emerged from the import of infected livestock or via infected mosquitos from Sudan (McMillen and Hartman, 2018), where an epizootic occurred in 1976 (Eisa et al., 1980). Another driving factor for this huge outbreak was the completion of the Aswan dam built to regulate the irrigation of the Nile delta (Kortekaas, 2014). By creating lake Aswan, large breeding sites for mosquitos were formed. Moreover, mosquito species of the Culex pipiens-complex were associated with a RVF outbreak for the first time (Hoogstraal et al., 1979). In West Africa RVFV was isolated for the first time in Nigeria in 1967, probably due the importation of infected cattle (Meegan and Bailey, 1989). The first major West African epidemic was eventually observed in southern Mauritania and northern Senegal about 20 years later. Similar to the events in Egypt, this major outbreak was associated with the construction of the Diama dam at the Senegal river. This epidemic led to a loss of 220 human lifes (Digoutte and Peters, 1989). Several other epidemics appeared in Mauritania and Senegal afterwards, again associated with rainfall events (Caminade et al., 2014), in particular for the first time in 2010 in non-endemic northern Mauritania (El Mamy et al., 2011). The most recent outbreak in West Africa was reported 2018 in Gambia with at least one fatal human case (WHO, 2018b). In contrast to East Africa, where outbreaks are associated with heavy precipitation over a long period of time, outbreaks in West Africa follow a typical pattern of precipitation event during the late monsoon season when ponds are filled. During this period (August-October), an absence of rain can occur for at least a week, followed by further heavy rainfall. Aedes vexans is a widespread and well-known example of a mosquito species adapted to West African climate. The eggs can handle an extensive drought period after oviposition if it is followed by a rainy season long or heavy enough to allow full larval development. Consequently, mosquito larvae tend to hatch in the upcoming year during monsoon season. Under certain circumstances, a second generation of mosquitos might hatch from September to October for a second time. Since Culex species might find conditions to hatch concurrently at this time, this mosquito burden combined with a large amount of livestock around filled ponds, present perfect conditions for a RVF outbreak (Caminade et al., 2014). In 1979, RVFV spread from the African continent to Madagascar for the first time (Linthicum et al., 2016) caused an epizootic in 1990-1991 (Morvan et al., 1992) followed by another one in 2008-2009 (Lancelot et al., 2017). In 2000, RVFV eventually emerged on the peninsula of Arabia where simultaneous outbreaks in the southern coastal provinces Asir and Jizan of Saudi Arabia and in the El Zuhrah district of the Hodeidah governorate in Yemen occured (Kortekaas, 2014). These outbreaks caused 2,000 human cases with about 245 fatal cases (Al-Hazmi et al., 2003; Madani et al., 2003) and thousands of infected animals (Balkhy and Memish,

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2003). There is strong evidence that RVFV was introduced by infected animals from the Horn of Africa. The fact that the virus isolated in this outbreak showed a strong phylogenetic similarity to a strain from Kenya in 1996-1997 and furthermore, that many small ruminants are imported from the Horn of Africa for religious events like the Hajj support this suspicion (Bird et al., 2009; Bird et al., 2007; McMillen and Hartman, 2018). Once again, the epidemics / epizootics were triggered by heavy rainfalls. Many reported cases of human infections correlate with visits in flooded areas, pointing towards mosquitos as the major source for infection (Jupp et al., 2002; Madani et al., 2003). The most recent and ongoing outbreaks are reported 2018-2019 on the French island Mayotte (WHO, 2019a) and in the Central African Republic (WHO, 2019b). Finally, an exceptional RVF case from China in 2016 was reported, when a Chinese forklift worker, who lived and worked for two years in Angola, was diagnosed in Beijing hospital for a RVF infection. He was probably infected by mosquito bites, as there was no known animal contact in the patient´s history (Liu et al., 2017).

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2.3 Classification

The species Rift Valley fever phlebovirus belongs to genus Phlebovirus, one genus among the family of Phenuiviridae of the Bunyavirales order. In total, the family Phenuiviridae comprises of 11 different genera and altogether 57 individual species (ICTV, 2019). A brief taxonomic overview is shown in Figure 2.1, displaying only a selection of corresponding families and genera. All species of Phlebovirus are part of the heterogenetic arbovirus group including sandfly fever species and the Uukuniemi and Bhanja group (Matsuno et al., 2015). Most members are transmitted by sandflies, but also mosquitos or ceratopogonid midges of the genus Culicoides have been shown to act the role of vectors. A remarkable peculiarity of the RVFV is that it is the only virus within the sandfly fever group that can also be transmitted via aerosols (Xu et al., 2007).

Figure 2.1:

Taxonomy of Rift Valley fever phlebovirus.

2.4 Morphology and genome characterization

The spherical virion of the RVFV reaches a diameter of about 80-120 nm, is enveloped and consists of two glycoproteins, Gn and Gc, that are arranged as surface subunits with 5-8 nm length and attached to the membrane bilayer (Ikegami, 2012). The virion has an icosahedral symmetry with 120-122 glycoprotein capsomers arranged in a lattice with T = 12 (Pepin et al., 2010). The RVFV genome is divided into a small (S), medium (M) and large (L) single stranded RNA segment, which are each assembled as a ribonucleoprotein (RNP). The terminal sequence of the 3' and 5' ends of the RNPs are complementary to each other, forming a panhandle structure that is visible in

7

electron microscopy (Pepin et al., 2010). The M and L segments have a negative polarity, with the L segment encoding the RNA-dependent RNA polymerase and the M segment coding for glycoproteins Gn and Gc and the non-structural protein NSm (14 kDa protein) which are expressed as a 78 kDa precursor protein. The RNA-dependent RNA polymerase is needed for RNA replication and RNA transcription (Boshra et al., 2011). Glycoproteins Gn and Gc mediate the virus entry into cells and are the main targets for neutralizing antibodies (Spiegel et al., 2016). Furthermore, they are required for an efficient virus assembly and maturation (Boshra et al., 2011). In addition, there seems to be an interaction between the cytoplasmic tail of the glycoproteins and the internal RNP layer forming a multilayered structure (Bouloy and Weber, 2010; Pepin et al., 2010). NSm suppresses virus-induced apoptosis (Won et al., 2007) and is a prerequisite for replication in mosquitos (Kading et al., 2014). The ambisense S segment encodes for the nucleoprotein Np and in opposite polarity for the non-structural protein NSs (Pepin et al., 2010). The nucleoprotein is important for virus assembly and encapsidates viral RNA to form the ribonucleoprotein, which enables transcription as well as replication (Ikegami, 2012). It also induces a humoral and T-cell immune response (Boshra et al., 2011). NSs is the main virulence factor for RVFV, since the virus uses it to evade the host's antiviral immune response (Pepin et al., 2010). It forms a filamentous structure in the nucleus of infected cells (Sall et al., 1997) and targets the IFN-β promoter region (Le May et al., 2008), which suppresses the interferon response at an early (3-4 h) phase post-infection (Pepin et al., 2010). Furthermore, at later stages, it interacts with the p44 subunit protein of TFIIH and TFIIH p62, ultimately leading to a general transcriptional shutdown (Le May et al., 2004; Habjan et al., 2009; Kalveram et al., 2011). Additionally, NSs downregulates PKR for preventing viral translation suppression (Pepin et al., 2010). Interestingly, Moutailler et al. (2011) showed that the NSs gene remains stable, only when the virus is alternately passaged on between mammalian and mosquito hosts. Although both non-structural proteins have important functions in pathogenicity, the virus replication is not hampered by a lack of these proteins (Gerrard et al., 2007; Muller et al., 1995).

2.5 Replication

The replication cycle of RVFV runs in the cytoplasm (Figure 2.2) and starts after virus entry via receptor-mediated endocytosis. Glycoproteins like the lectin DC-SIGN play the most important role as receptors for attachment and cell entry (Spiegel et al., 2016). Virus attachment induced clustering of DC-SIGN followed by internalization. During this process, the cytoplasmic tail of the lectin triggers an endocytosis signal and cellular uptake by early endosomes followed by dissociation of DC-SIGN (Lozach et al., 2011). In addition to DC-SIGN, heparan sulfate also plays an important role for virus attachment and entry (Boer et al., 2012). Furthermore, clathrin-dependent (Wichgers Schreur and Kortekaas, 2016) as well as clathrin-independent mechanisms such as caveolin-1-mediated endocytosis have been proposed (Harmon et al., 2012). In late endosomes, a decrease in pH leads to membrane fusion and subsequent release of RNPs into the cytoplasm (Spiegel et al., 2016). The decrease in pH is presumably induced by the Gc protein, which harbors a structure similar to class II membrane fusion proteins (Dessau and Modis, 2013). Due to protonation of histidine residues in

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Gc, the membrane fusion in late endosomes is initiated and finally viral RNA and proteins are released into the cytoplasm (de Boer et al., 2012).

Figure 2.2:

Schematic of the replication cycle of RVFV. Reprinted from Wichgers Schreur and Kortekaas (2016).

The primary transcription of genomic RNA into mRNA and subsequent replication of genomic RNA is catalyzed by RNA dependent RNA polymerase in the cytoplasm. Since the genomic RNA lacks a 5’ cap structure, the host cell derived capped mRNA fragments are used for synthesis of viral mRNA by a „cap snatching” mechanism

(Ikegami, 2012). The mRNA coding for the S and L segments are translated in free ribosomes, whereas the M segment mRNA is translated in membrane bound ribosomes at the endoplasmic reticulum (ER) (Wichgers Schreur and Kortekaas,

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2016). Compared to other RNA viruses, mRNA synthesis is closely coupled to the translation of viral proteins (Barr, 2007). Segment encapsidation and assembly of new viral particles is performed by nucleoprotein and the cytoplasmic tail of Gn. The so-formed RNPs first replicate randomly in the cytoplasm and are then recruited to the Golgi apparatus by the glycoproteins (Wichgers Schreur and Kortekaas, 2016). Only the Gn protein targets the localization in the Golgi apparatus, which is necessary for correct maturation of the particles (Pepin et al., 2010). Wichgers Schreur and Kortekaas (2016) showed that RVFV does not have a specific and selective genome packaging strategy and only about 10 % of all virion particles incorporate all three segments. Finally, assembled virions are released from the cell via exocytosis by budding from specific membrane sites (Wichgers Schreur and Kortekaas, 2016).

2.6 Transmission

2.6.1 Enzootic / inter-epidemic cycle

RVFV transmission is divided into the enzootic / inter-epidemic and the epizootic / epidemic cycle. The transition to an epidemic cycle is mediated mainly by long-lasting rainfall and flooding that cause rapid multiplication of mosquito vectors within outbreak regions (Linthicum et al., 2016). The most important primary vector are floodwater mosquito species of the genus Aedes that use seasonally flooded depressions (so-called dambos or pans), for oviposition (Vloet et al., 2017). A unique characteristic of Aedes spp. is the vertical transmission of RVFV into laid eggs, which allows the virus to persist in the environment for a long time (Bird et al., 2009). It is still unknown whether this is important for the survival of RVFV or whether there is a still unidentified reservoir host (Bird and McElroy, 2016). In the inter-epidemic cycle, infected mosquitos hatch at a moderate rate during a normal rainy season. The zoophilic Aedes (Vloet et al., 2017) then infect ungulates (wildlife and livestock) by blood feeding. Monitoring studies demonstrate a low seroprevalence in corresponding species during inter-epidemic cycle (Britch et al., 2013; Rissmann et al., 2017a). Enzootic cycles can continue for several years until the occurrence of a new epizootic. Human cases are observed only sporadically during this cycle (Vloet et al., 2017).

2.6.2 Epizootic / epidemic cycle

Aedes are also important as primary vectors for the shift to an epizootic / epidemic, which is primarily triggered by a permanent increase of the mosquito population. Factors promoting mosquito expansions are artificially dammed waters as in Egypt or West Africa (Meegan et al., 1979; Digoutte and Peters, 1989) or usually heavy seasonal rainfalls (Bird et al., 2009). During these events, up to one million mosquitos can hatch from a 1,800 m2 habitat within a comparatively short period of time (Linthicum et al., 1985). The epidemic starts with the mass hatching of infected Aedes mosquitos and a subsequent infection of amplifying hosts. The number of RVFV competent secondary vectors like Culex and Mansonia also multiplies rapidly in these

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flooded areas. These mosquitos are considered to be the most important species for horizontal transmission (Linthicum et al., 1983, 1984; Mcintosh et al., 1972). Anthrophilic mosquitos like Anopheles also represent secondary vectors, allowing the virus to spread further into the human population (Vloet et al., 2017).

2.6.3 Human exposure routes

In most outbreaks, it is difficult to tell whether humans are infected by mosquitos or infected animals, as they are normally exposed to both (Kamal, 2011). Although sporadic infections of humans by mosquitos were observed (Liu et al., 2017), the direct contact with infected animals involves a significantly higher risk of infection. The handling of tissue and blood from infected animals poses a high risk; especially the contact to abortion material seems to lead to a particularly severe disease progression (Mcintosh et al., 1980; Vanvelden et al., 1977; LaBeaud et al., 2015; Anyangu et al., 2010). In addition to direct contact, the inhalation of infectious aerosols can also lead to a RVFV infection, as the virus is very stable under these conditions (Miller et al., 1963). Therefore, shepherds and farmers, but also veterinarians and slaughterhouse workers are considerably exposed (LaBeaud et al., 2015; Anyangu et al., 2010). In a recent study, Grossi-Soyster et al. (2019) also showed that people who drink milk from infected cows, sheep or goats seem to have a higher risk of infection. However, humans are dead-end hosts, with no transmissions from person to person, as described for other pathogens (McMillen and Hartman, 2018).

2.7 Clinical signs

Among ruminants, sheep are the most susceptible species to RVF infections, which is characterized by a high rate of abortions in pregnant sheep and a high mortality rate of newborn lambs (Daubney et al., 1931). Susceptibility is mainly dependent of the age of the animals. Therefore, the mortality rate of lambs is 90 % (Daubney et al., 1931) and that of adults only 20 % (Easterday et al., 1962). Lambs show high fever, abdominal pain, increased respiration rate and hemorrhages in the gastrointestinal tract (Coetzer, 1977, 1982)). Older animals usually suffer from fever (39-40 °C), diarrhea, nasal discharge and reduced activity (Easterday et al., 1962). So-called abortion storms (including malformations and still birth) occur in pregnant ewes (Gerdes, 2004). Breed-specific differences in susceptibility apply, e. g. Yansaka sheep died acutely after few days post subcutaneous infection (Ikegami and Makino, 2011), while European breed sheep survived (Busquets et al., 2010). Goats show similar symptoms but are usually less severely affected (Hartman, 2017). Among ruminants, cattle are the least susceptible species. The mortality rate is only 5- 10 % for adult animals and typical symptoms are weakness, anorexia, bloody diarrhea and nasal discharge (Hartman, 2017). Abortions can also occur. Mortality rates in calves range from 10-70 % with symptoms similar to those in lambs and kids (Bird et al., 2009). Studies on wild ruminants are rare, but an infection study with African buffalos

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(Syncercus caffer) showed an abortion of one out of two pregnant animals and a high viremia (Bird et al., 2009). Camels are generally less susceptible to RVFV infections and rarely show signs of a disease. Clinical symptoms, if they occur, are rather mild. Pregnant animals are the exception since high abortion rates are reported. Moreover, severe clinical diseases up to fatalities were also seen during a RVF outbreak in northern Mauritania: Either a hyper acute form leading to sudden death in less than 24 hours or a severe illness with fever, ataxias, dyspnea, bloody nasal discharge, eye diseases, hemorrhagic symptoms and abortions were observed. These animals died within a few days if hemorrhagic signs occurred. (El Mamy et al., 2011) Mice are also highly susceptible to RVFV and the clinical course is similar to that of newborn lambs. Mice die either after a few days from a severe hepatitis, yet without displaying obvious clinical signs until then or survive this acute phase, but develop neurological symptoms (e. g. hind leg paralysis) and eventually die after 8-9 dpi (days post inoculation) as a result of an encephalitis (Smith et al., 2010; Ikegami and Makino, 2011). Most human patients infected with RVFV suffer from a self-limiting febrile disease. The incubation periods last usually 4-6 days (Ikegami and Makino, 2011) followed by suddenly appearing symptoms like headache, aching limbs and fever for 3-5 days (Laughlin et al., 1979). In some cases, biphasic fever (Gear, 1951) or other symptoms such as abdominal pain, joint and muscle pain or nausea are also observed (Gear, 1951). These forms are not fatal, but can last for several weeks. Only 1-2 % of patients suffer from a severe disease (Bird et al., 2009; McMillen and Hartman, 2018). As the liver is the most heavily affected organ liver enzymes in blood rise sharply and blood clotting disorders follow (Madani et al., 2003). In addition to the typical flu-like symptoms, the patient may develop jaundice, renal failure and typical hemorrhagic symptoms, such as bloody urine / stool, blood vomiting, bleeding under the skin or into / from gingival bleeding (Laughlin et al., 1979). These severe diseases are often fatal within one week (Meegan, 1981). Another serious form of the disease is associated with neurological disorders. Severe and persistent symptoms such as hallucination, disorientation, vertigo or paralysis can develop after days up to weeks of the typical febrile disease, which finally leads to encephalitis (Vanvelden et al., 1977). Decerebrate posturing is often associated with fatal outcome and patients often suffer from permanent hemiparesis (Laughlin et al., 1979). Visual disturbances occur in 10 % of all infected patients regardless of the severity of the disease (Al-Hazmi et al., 2005). Symptoms such as photophobia, impaired vision due to uveitis, retinitis or bleeding into the latter develop within 1-3 weeks after the onset of the original disease. The initial handicap may disappear within weeks or months or continue permanently (Al-Hazmi et al., 2005; Siam et al., 1980; Daubney et al., 1931). Severe progressions with terminal illness correlate significantly with high virus titers in the blood (Bird et al., 2007).

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2.8 Pathology and histopathology

The liver as the main target organ shows an enlarged, soft, friable texture with disseminated pale foci in both animals and humans in gross pathology (Bird et al., 2009). Despite the severe hepatitis in the mouse, only multifocal distributed pale foci are often seen (Smith et al., 2010). Histology shows hepatocellular necrosis with varying severity and infiltration of neutrophil granulocytes and macrophages depending on age. Lymph nodes are often enlarged and especially in young animals also the spleen. In histology, lymphoid necrosis can be observed in the lymph nodes and in the white pulp of the spleen (Bird et al., 2009). In lambs and kids, histology also shows a congestion of the lung, hemorrhages in the abomasum and karyorrhexis in the kidney (Coetzer, 1977, 1982). In addition, infected mice show meningoencephalitis with neuronal necrosis and microhemorrhagia around 8 dpi in histology in multiple regions of the brain (Smith et al., 2010). By immunohistochemistry, viral antigen can be detected in many different tissues, including the wall of small blood vessels, kidney and spleen, but especially in the hepatocytes of the liver (Van der Lugt et al., 1996).

2.9 Diagnostics

Information on kinetics and distribution of the virus is necessary to determine the status of the disease and to ascertain acute or already passed infections in animals. Infected sheep usually show a viremia of 1-6 dpi with titers of around 1x106-1x108 PFU / ml. From 4 dpi until 60 dpi, IgM antibodies, while IgG antibodies typically appear after 9 dpi and persist for at least 120 days (Bird et al., 2009). Detection methods for RVFV include virus isolation on mammalian cells or inoculation of suckling mice and the RVFV genome detection by quantitative real-time reverse transcriptase PCR (RT-qPCR). Numerous protocols are available targeting the S (Garcia et al., 2001), M (Drosten et al., 2002) or L segment (Bird et al., 2007). Moreover, antigen detection can be performed by ELISA (Fukushi et al., 2012) and by immunohistochemical methods (Rissmann et al., 2017b). Serological standard procedures for antibody detection are ELISA and the virus neutralization test (VNT), which is also considered being the gold standard due to its sensitivity and specificity (Wichgers Schreur et al., 2017). The disadvantage of most VNTs is that they need elaborate laboratory conditions and are based on virulent RVFV strains that must be handled under BSL-3 conditions (OIE, 2019). However, there are now numerous efforts to develop tests with attenuated strains like vaccine strain MP-12 (Wichgers Schreur et al., 2017). Most ELISAs, including commercial IgG / IgM kits, are based on the highly immunogenic Np protein (Fafetine et al., 2007; van Vuren and Paweska, 2009; Paweska et al., 2005; Paweska et al., 2008; Paweska et al., 2007), but work also with Gn antigen (Jackel et al., 2013a). Finally, several immunofluorescence assays have been established that deal with attenuated as well as virulent RVFV isolates (OIE, 2019) like the commercial available kit from EUROIMMUN Medizinische Labordiagnostika AG.

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2.10 Vaccination and treatment

In 1940, Smithburn created the first live vaccine by intracerebrally passaging a virus isolate from Uganda in mice (Smithburn, 1949; Smithburn et al., 1948). Smithburn vaccine is administered once and is most widely used in veterinary medicine in endemic countries. However, this vaccine still exhibits residual teratogenic virulence, which can affect fetuses in pregnant animals (Kortekaas, 2014). The first human vaccine NDBR-103 was obtained by inactivating the RVFV Entebbe strain with formaladehyde (Randall et al., 1962). This vaccine was further developed into the TSI-GSD 200 vaccine (Rusnak et al., 2011). Both vaccines were applied to laboratory personnel whereby the TSI-GSD 200 has been reported for 19 years to show good immunogenicity and safety profile. However, a three-step immunization and a recommended boost after 6 months is still required (Rusnak et al., 2011). Two further important live vaccine strains are MP-12 and Clone 13. MP-12 originated from the virulent strain ZH548 through 12 passages in the presence of the mutagenic 5-fluoruracil (Morrill et al., 1987) leading to several mutations in all segments (Saluzzo and Smith, 1990). Clone 13 was obtained from a purified plaque clone of a virus strain isolated from a human case (Muller et al., 1995) and exhibits a 69 % deletion in the NSs region (Vialat et al., 2000). MP-12 elicits no clinical symptoms in mice, sheep, cattle or rhesus macaques demonstrating low residual virulence and strong immunogenicity (Morrill et al., 1987; Morrill et al., 1997; Morrill and Peters, 2003). Two out of 16 Clone 13 infected mice died during neurological phase (Kortekaas, 2014). Nevertheless, a good protection has been reported in sheep and cattle (Dungu et al., 2010; von Teichman et al., 2011). However, several studies provided evidence for fetal death and miscarriages regarding both vaccines (Hunter et al., 2002; Morrill et al., 2013a; Makoschey et al., 2016). Nevertheless, MP-12 strain is the only approved vaccine with a conditional license in the USA (Bird and McElroy, 2016). Clone 13 was commercialized by Onderstepoort Biological Products in South Africa 2010 and used in the field vaccinations (Kortekaas, 2014). Two further live attenuated viruses were produced by either deleting both NSs and NSm coding regions from the virulent strain ZH501 or by deleting exclusively the NSm coding region in strain MP-12. Both viruses show strong protection in different animal models without further complications in pregnant animals (Bird et al., 2008; Bird et al., 2011; Morrill et al., 2013b; Weingartl et al., 2014). An additional advantage of these strains is their use as a DIVA (Differentiating Infected from Vaccinated Animals) vaccine (Hartman, 2017). Finally, a four-segmented RVFV was developed where the M segment has been divided into two segments encoding either Gn or Gc. In addition, the NSs coding sequence was removed. In first animal trials, this vaccine shows a comparable strong protection without teratogenic effects (Schreur et al., 2017). Various experimental vaccines based on vectors such as Modified-Vaccinia-Ankara virus (Lopez-Gil et al., 2013), alphavirus replicons (Gorchakov et al., 2007), non-replicating chimpanzee adenoviruses (Warimwe et al., 2016) or Newcastle disease virus backbone (Kortekaas et al., 2012) provide good protection in various animal species. However, the utilization is often restricted by the great scope of registration costs (Kortekaas, 2014). Ribavirin and Favipiravir were tested for possible antiviral treatments. Ribavirin demonstrated high efficacy in vitro, but only weak effects in the mouse model (Atkins

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and Freiberg, 2017). Since it was already used against other hemorrhagic pathogens, it was deployed during the RVF outbreak on the Arabian Peninsula, but the treatment was stopped due to adverse effects (Hartman, 2017). Favipiravir exhibited better protection in the mouse model but only when the treatment started already six hours post infection. Several small molecule candidates are also currently under development (Atkins and Freiberg, 2017).

2.11 Monoclonal antibodies

Monoclonal antibodies are standard reagents for diagnostics and therapeutics and are normally obtained from murine hybridoma cells produced by fusion of single B-lymphocytes with murine myeloma cells. In 1975, Kohler and Milstein describe the method of producing monoclonal antibodies in unlimited amounts for the first time (Kohler and Milstein, 1975). Usually mice (especially BALB / c mice, but also rabbits) are immunized against a particular epitope of an antigen followed by extraction of B-lymphocytes from the spleen. Subsequently, the spleen cells are removed and fused with myeloma cells, which generates so-called hybridoma cells. These hybridoma cells are then cultured in vitro in selective medium where only these cells survive as they have inherited immortality from the myeloma cells and additionally selective-resistance from the primary B-lymphocytes. The initial culture of hybridomas contains a mixture of different primary B-lymphocyte clones. Each individual clone can be separated by dilution into different culture wells and screened for specific antibody reactivity (Kohler and Milstein, 1975; Liu, 2014). In addition to classical in vivo protocols, there are also numerous new in vivo, but also in vitro techniques. For example, the phage display technology could be used to produce and analyze neutralizing HIV mAbs by epitope mapping as well as for the identification of ligand targets (Delhalle et al., 2012). Another technique is the isolation and characterization of antigen-specific plasmablasts by a fluorescence based method, subsequent antibody gene cloning and the generation of individual monoclonal antibodies (Clargo et al., 2014). Further methods are proteomics-directed cloning of mAbs from serum and deep sequencing of paired antibodies encoding genes from B cells (Salazar et al., 2017). Since its discovery, the most important application of mAbs has been therapeutic treatment. In addition to the complete antibody, separate antigen-binding fragments (Fab), Fc (Fragment crystallisable) and scFv (single chain variable fragment) were also used (dos Santos et al., 2018). In 2017, a total of 61 mAbs and 11 Fc fusion proteins were approved by the FDA and / or EMA (ACTIP, 2017; Lagasse et al., 2017; Strohl, 2018). Seven out of 15 products of the best selling drugs in 2018 including those in first place all contained monoclonal antibodies (GEN, 2019). Although mAbs are most commonly used in oncology (dos Santos et al., 2018), they also play an important role as potential new antivirals. New therapeutic mAbs are currently generated against many different viral pathogens. Neutralizing antibodies against HIV are an important approach. Thus, various institutes were able to isolate broad and potent neutralizing mAbs, mostly from infected donors. These mAbs are already in various clinical trials such as 4E10, 2F5, 2G12 in phase 1 / 2, VRC01 and

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3BNC117 in phase 2 and Ibalizumab even in phase 3 (Caskey et al., 2015; Henrich and Kuritzkes, 2013; Wu et al., 2011; University, 2005; Salazar et al., 2017). Another important example is rabies. Although there is already a post-exposure prophylaxis, which consists of polyclonal immunoglobulins from immunized humans or horses, produced batches are limited and may have different efficiencies due to their origin. Therefore, new mAbs are generated to possibly replace these immunoglobulins. The mAb CL 184 isolated from an immunized donor or the mAb RAB-1 generated from a humanized mouse have shown safety and efficacy in several clinical trials (Bakker et al., 2008; Sloan et al., 2007; Salazar et al., 2017). In recent years, one urgent application was the treatment of Ebola infections. During an outbreak in West Africa in 2014-2015, the mAb cocktail ZMapp, which worked well in animal experiments, was used in form of a clinical trial (Dodd et al., 2016). Unfortunately, the statistical values were not convincing. However, in a recent outbreak in the Democratic Republic of the Congo, the cocktail REGN-EB3 and the mAb 114 isolated from a surviving Ebola patient showed a more promising effect than the ZMapp (Maxmen, 2019; Qiu et al., 2014; Salazar et al., 2017). As already shown by these examples, the origin of the monoclonal antibodies plays a decisive role. The disadvantage of murine mAb, i. e. adverse effects when administered in humans, was recognized already early on (Hwang and Foote, 2005). Therefore, many methods were developed to humanize murine mAbs (Morrison et al., 1984; Jones et al., 1986). On the other hand, new technologies are used to produce human mAbs directly from humanized mice or humans (dos Santos et al., 2018). In addition to therapy, mAbs are also used in other areas. In a broad variety of different diagnostic tests (e. g. ELISA, IFA, Western Blot) they are often used as secondary antibodies, either labelled or unlabeled since they are commercially available separately from a wide range of suppliers.

2.12 Variable heavy chain domain of a heavy chain antibody

The first description of so-called heavy chain antibodies dates back to 1993 where antibodies devoid of the light chain were detected in the serum of camels. In contrast to conventional IgG antibodies, these antibodies lack any of the light chains as well as the constant domain of the heavy chain 1 (CH-1) and were designated as heavy chain antibodies (HCAb). These IgG subclasses exist for all members of the Camelidae (50-80 % of total antibodies in Old World camels and 10-25 % in New World camels (Blanc et al., 2009)) (Hamers-Casterman et al., 1993). A similar antibody type was found in nurse sharks (Ginglymostoma cirratum) and in general throughout the class of Chondrichthyes (Greenberg et al., 1995). Obviously, the emergence of these antibodies seems to have provided an evolutionary advantage to these animals (Flajnik et al., 2011). The variable domain of the heavy chain alone was designated variable heavy chain domain of a heavy chain antibody (VHH) and is now widely used for the development of therapeutics and diagnostics. VHH genes originate from the same H locus as the classic variable domain of the heavy chain (VH) but exhibit specific differences (Muyldermans, 2013). Several VH derived hydrophobic amino acids (aa), which are important for binding to the light (L) chain are replaced by hydrophilic amino acid variants (Padlan, 1994). In conventional antibodies,


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CH-1 and hydrophobic amino acids are important for binding to a chaperone protein system, which allows the connection to the L chain (Padlan, 1994). Thus, the VHH circumvents this system, which leads to the removal of CH-1 by mRNA splicing during translation in the endoplasmic reticulum (Nguyen et al., 2001). VHH has only three instead of six complementarity determining regions (CDR) that are however significantly longer compared to conventional ones. Especially the CDR 3 represents the major part of the antigen-binding site and exhibits specific folding to compensate the lacking variable domain of the light chain (VL) (Transue et al., 1998; Muyldermans et al., 1994; Desmyter et al., 1996). The CDR structure is stabilized by disulfide bridges between the CDR1 and 3 in camels and the CDR 2 and 3 in llamas (Govaert et al., 2012). As a consequence, the paratope of the VHHs displays a convex shape and can bind particularly to (hidden) cavities on the antigen surface that are not accessible for conventional antibodies due to their size (De Genst et al., 2006). VHHs can be well generated from HCAb sequences and remain stable as single domain antibodies (Siontorou, 2013). A single VHH is of 2.5 nm in diameter, 4 nm high and only 15 kDa small (Muyldermans, 2013; Siontorou, 2013). Thus, it is the smallest intact antigen-binding fragment with full antigen-binding capacity and was therefore also named “Nanobody” (Cortez-Retamozo et al., 2004; Siontorou, 2013).

Figure 2.3:

Schematic overview of naturally occuring antibodies in (I) Camelidae, (II) Chondrichthyes and (III) a sdAb with possible multimeres.


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There are now various protocols that can be used to produce antibodies against certain antigens (Ghahroudi et al., 1997; Vincke et al., 2012; Pardon et al., 2014). For Nanobody production animals from the family of Camelidae (generally camels or llamas) are immunized with corresponding antigens, normally small proteins. Subsequently peripheral blood mononuclear cells (PBMCs) can be isolated and RNA can be extracted. Using consecutive PCR steps, specific VHH DNA is amplified, cloned into phage display vectors (e. g. pMECS or pHEN4) and finally are transformed into E. coli TG1 to generate a primary library of about 106 clones. Also naïve, semi-synthetic or synthetic VHH libraries can be used if the target antigen is toxic or causes other adverse effects in the animal. By phage display (or alternatively bacterial display, yeast display, intracellular 2 hybrid selection, ribosome display) specific antibodies are obtained via serial panning steps by exposing the phage library to an immobilized antigen. In general, immobilized antigens adsorbed on a microtiter plates are used. During this process, antigen-binding phages are rescued, while non-adherent ones are washed away. Bound phage can be recovered from the surface, reinfected into bacteria and re-grown for the next round of enrichment. Usually three panning rounds are performed to obtain a specific sub-library. From this, individual clones are subsequently expressed and tested again for specific binding. Finally, individual antigen-specific sdAbs are obtained (Vincke et al., 2012; Pardon et al., 2014; Muyldermans, 2013). VHHs are very stable at various temperatures and can partially withstand temperatures up to 90 °C (van der Linden et al., 1999). They also show high stability in the presence of detergents or denaturants (Dolk et al., 2005) which makes them especially suitable for oral administration (Hussack et al., 2011). Due to their small size, rapid clearance and high sequence similarity to human VH, sdAb show a low immunogenicity in humans (Cortez-Retamozo et al., 2004) that is further enhanced by humanization of VHH derived sequences (Vincke et al., 2009). Various VHH applications were reported including nanotraps that recognize fluorescent proteins for cellular analyses (Muyldermans, 2013; Rothbauer et al., 2008; De Genst et al., 2010) as well as Nanobodies against carcinoma markers for noninvasive in vivo imaging of tumors (Vaneycken et al., 2010; Vosjan et al., 2011). Another application is intracellular targeting in the form of intrabodies (Steeland et al., 2016). As a consequence of the afore mentioned findings, the possible therapeutic use of VHH has been investigated in many fields: Oncology, neurodegenerative diseases, infectious diseases, inflammation processes, envenoming or toxicity (Salvador et al., 2019). This implies drug delivery systems like so-called lactobodies that are expressed by lactobacilli and protect against virus-induced diarrhea (Pant et al., 2006). Furthermore, there are many in vivo approaches in cancer treatment such as tumor-penetrating 4NC2 sdAb that targets EGFR for the therapy against gastric cancer (Sha et al., 2015a, 2015b). Regarding antiviral therapies, two antibodies have already passed Phase I: The trivalent antibody ALX-0171 against respiratory syncytial virus, which is currently in phase IIa and ARP1, a rice based sdAb for oral administration against rotavirus infection, currently in phase III (Steeland et al., 2016). Four antibodies against inflammatory diseases are currently evaluated in different clinical stages, three for the treatment of rheumatoid arthritis and one for psoriasis (Steeland et al., 2016). Finally, a trivalent sdAb targeting RANK ligand is currently in Phase II against breast cancer derived bone metastasis (Samadfam et al., 2012).

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3 MATERIALS AND METHODS

The following section provides an overview of materials and methods used in this work. Detailed descriptions are found in the corresponding sections of manuscripts I-III.

3.1 Animal trials

In the animal trials, BALB / c mice were used in manuscripts II and III and an alpaca (Vicugna pacos) in manuscript I. Mice were immunized and infected via intraperitoneal route. Antibodies were applied intravenously via the tail vein. For alpaca immunization, live virus was administered subcutaneously. Mice immunized with RVFV MP-12 received a total of four boosters, while virulent virus (III) and monoclonal antibodies (mAbs; II) were administered only once. Blood was taken from the mice either via submandibular venipuncture or after euthanasia from the heart, in alpaca from the jugular vein. Serum was used for serological tests, while PBMCs (Peripheral blood mononuclear cell) were obtained from heparinized whole blood. Infected mice were observed for either 10 (III) or 14 (II) days and their health status was assessed on the basis of a score sheet. During necropsy, different tissues were removed for PCR, virus titration, immunohistochemistry (IHC) and histopathology (II) or the spleen for the generation of hybridoma cells (III).

3.2 Serological assays

An in-house immunofluorescence assay (IIFA; Mroz et al., 2018) was used for the screening and characterization of anti-RVFV antibodies in manuscripts I and III. In addition, a commercial IIFA kit (EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany) was used for single domain antibody (sdAb) characterization in manuscript I. An in-house ELISA (Jackel et al., 2014) with the antigens Gn, Gc, Np, NSm and NSs was used for further characterization of produced antibodies (III). In manuscript II serum was used in a commercial competition ELISA from IDvet (IDvet, Grabels, France) based on the Np. Serum from alpaca (I), mice (III), and generated antibodies (II and III) were tested in a serum neutralization test (SNT) based on RVFV strain MP-12 (Rissmann et al., 2017b). MAbs and serum of mice (II) were further tested in a SNT based on RVFV strain 35 / 74.

3.3 PCR

For the protocol to obtain sdAb (I), the Promega Reverse Transcription System (Promega, Madison, USA) with oligo (dT) 15 primer was used in Reverse-Transcriptase-PCR (RT-PCR). Phusion High-Fidelity PCR Kit (Thermo Fisher Scientific, Waltham, USA) with the primers CALL001 and CALL002 or with the primers GIII and MP57 was used in conventional PCR. Primers VHH-BACK and PMCF were used in nested PCR (Vincke et al., 2012; Pardon et al., 2014). For the detection of RVFV RNA in II a RT-qPCR was performed with the Quanti Tect® Probe RT-PCR Kit (Qiagen, Hilden, Germany) (Bird et al., 2007).

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3.4 Extraction and purification kits

PBMCs (I) were obtained using Lymphocyte Separation Medium LSM 1077 (PAA Laboratories GmbH, Pasching, Austria). RNA was obtained from PBMCs by phenol-chlorophorm extraction using the RNeasy® Mini Kit (Qiagen). In manuscript II, RNA was obtained for subsequent PCR from different tissues and cruor using the NucleoMag® VET Kit (MACHEREY-NAGEL, Düren, Germany) and King Fisher™ Flex Purification System (Thermo Fisher Scientific). In manuscript I, PCR amplification products were obtained and purified with the QIAquick® Gel Extraction Kit (Qiagen) from an agarose gel. In addition, PCR products were purified for further use as templates, after restriction digestion or for preparation for sequencing with the QIAquick® PCR Purfication Kit (Qiagen). Expressed sdAbs were purified via Ni-NTA columns (Jackel et al., 2014) due to their his-tag.

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4 MANUSCRIPT I:

A single domain antibody with high affinity to different Rift Valley fever phlebovirus strains

Benjamin Gutjahr1, Melanie Rissmann1, Markus Keller1, Martin H. Groschup1, Martin Eiden1*

1 Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany

*Corresponding author: [email protected], Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany

4.1 Abstract

Rift Valley fever phlebovirus (RVFV) is a mosquito-borne hemorrhagic fever virus, which causes large outbreaks affecting humans and livestock throughout Africa and the Arabian Peninsula. Diagnostic assays for the detection of antibodies, RVFV genomes and antigens are available, but continuous methodical improvements are needed to deepen knowledge on epidemiology, molecular virology and diagnosis, but also for assaying efficacy of innocuous vaccines and therapeutics. Therefore, we have developed a single domain antibody to RVFV as an alternative to conventional antibodies, which consists of a single variable heavy chain domain of a heavy chain antibody (VHH). These unique antibodies are part of the immune system of Camelid species and of few other taxa. Because of their small size, high solubility and affinity as well as excellent stability and cost-effective production, their use may be advantageous for the above purposes. We therefore generated a RVFV immune phage library from peripheral blood mononuclear cells of an alpaca (Vicugna pacos), which was immunized with the RVF vaccine strain MP-12. By biopanning the library directly with virus infected fixed cells a VHH antibody was identified, which bound with high affinity to one of the major virus immunogens, the nucleoprotein. Biochemical and cell analysis confirmed a high specificity and sensitivity of this novel antibody.

4.2 Introduction

Rift Valley fever phlebovirus (RVFV) belongs to the family Phenuiviridae, genus Phlebovirus and contains a single stranded tripartite genome of negative polarity. It consists of three segments L, M and S. The L segment encodes for the RNA-dependent RNA polymerase, the M segment for two glycoproteins Gn and Gc and for the non-structural NSm protein. The S segment expresses the nucleoprotein Np and in ambisense direction the non-structural protein NSs (Ikegami, 2012). A large variety of mosquitos can transmit the virus and facilitates the emergence of RVF epidemics

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(Linthicum et al., 2016). This coincides with the transovarial transmission especially in Aedes spp.. Excessive rainfalls usually precede large outbreaks as they lead to mass hatching of infected mosquitos. Culex spp. and other mosquitos subsequently transmit virus as secondary vectors (McMillen and Hartman, 2018; Pepin et al., 2010). RVFV is distributed over vast regions of Africa and since 2000 also on the Arabian Peninsula. Re-occurring outbreaks were monitored mainly in Sub-Saharan countries like South Africa, Mauritania, Tanzania and Kenya, but also far north as Egypt (Nanyingi et al., 2015). Abortion rates of up to 100 % can be observed in pregnant ungulates (in particular sheep, goat and camels) and a similar mortality was seen in infected newborn lambs or kids. Adult animals mainly suffer from moderate symptoms, i. e. develop fever, viremia, diarrhea, nasal discharge and decreased activity (Pepin et al., 2010; Hartman, 2017; El Mamy et al., 2011). Most human infections are self-limiting febrile illnesses. However, in more severe cases patients develop ocular and neurological disorders or hemorrhagic fever (McMillen and Hartman, 2018). Even though many diagnostic methods were established for the detection of RVFV (Bird et al., 2007; Fafetine et al., 2007; Jackel et al., 2013a), novel methods are needed to improve diagnostic assays and clinical applications. Limits to current methods are based on certain disadvantages of conventional antibodies like multi-subunit structure, high molecular mass, thermal instability and high production costs. A promising alternative are heavy chain antibodies (HCAb) found in the family Camelidae that are devoid of light chains. They comprise two constant domains (CH-2 and CH-3), a hinge region, and an antigen-binding or variable heavy chain domain (VHH) (Hamers-Casterman et al., 1993). VHH or single domain antibodies (sdAb) are the smallest, naturally occurring, antigen-binding units with a molecular mass of 15 kDa (Cortez-Retamozo et al., 2004). Their small size allows a rapid penetration of tissues and enables construction of engineered multivalent formats with higher avidity than monoclonal antibodies (mAbs) (Wilken and McPherson, 2018). A broad range of different VHHs are already generated as diagnostic and therapeutic attempts in different fields of research (Siontorou, 2013; De Meyer et al., 2014). Nevertheless, no sdAbs have been generated against RVFV so far. In our study, we modified known protocols (Pardon et al., 2014; Vincke et al., 2012) for the generation of RVFV specific VHHs. Panning of the generated phage library was performed against whole virus in infected and fixed cells. A single antibody with a high affinity to the nucleoprotein of Rift Valley fever phlebovirus was generated and analyzed.

4.3 Materials and methods

4.3.1 Immunization of alpaca

Samples were derived from alpacas (Vicugna pacos) that were immunized in a previous study with RVFV MP-12 vaccine strain (Rissmann et al., 2017b). Heparinized whole blood fractions were selected from alpaca #A3.

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4.3.2 Isolation of peripheral blood mononuclear cells (PBMCs)

To isolate PBMCs, 10 ml heparinized whole blood was taken and mixed with the same volume of phosphate buffered saline (PBS). 20 ml of this blood mixture were laid carefully over 14 ml of Lymphocyte Separation Medium LSM 1077 (PAA Laboratories GmbH, Pasching, Austria) in 50 ml Falcon tubes. Tubes were centrifuged at 1200 g, at room temperature (RT), for 20 min without acceleration and brake. PBMCs forming a white ring at the interphase were meticulously collected and mixed with the same volume of PBS. PBMCs were then centrifuged again at 300 g for 10 min. The supernatant was decanted and the pellet was resolved with 5 ml PBS before being centrifuged again. After the last step, the supernatant was decanted and the pellet was used for further RNA extraction.

4.3.3 Amplification of VHHs

For RNA extraction, PBMCs were processed with Trizol® LS reagent (Life technologies, Carlsbad, USA) and total RNA was collected using RNeasy® Mini Kit (Qiagen, Hilden, Germany) according to manufacturer´s instructions. cDNA was generated by reverse transcriptase PCR (RT-PCR) using Promega Reverse Transcription System (Promega, Madison, USA) with oligo (dT) 15 primer. cDNA was taken as template for the next conventional PCR step using Phusion High-Fidelity PCR Kit (Thermo Fisher Scientific, Waltham, USA) and primer CALL001 and CALL002 (Vincke et al., 2012). The PCR product was run on a 1 % agarose gel and the requested fragment at 700 bp was cut out and purified with QIAquick® Gel Extraction Kit (Qiagen). Purified DNA was used as template for nested PCR with Phusion High-Fidelity PCR Kit (Thermo Fisher Scientific) and primer VHH-BACK and PMCF (Vincke et al., 2012). PCR product was run on a 2 % agarose gel and the desired fragment at 400 bp was cut out and purified with QIAquick® Gel Extraction Kit (Qiagen). (PCR protocols are listed in Data file S4.1).

4.3.4 Cloning and library construction

Purified VHH DNA and pMECS vector were digested with restriction enzymes NotI and PstI (NEB, Frankfurt am Main, Germany) as described by Vincke et al. (2012). Insert and vector were ligated in a 3:1 ratio with T4-Ligase (NEB) following manufacturer´s instructions. The purified ligated product was eventually transformed via electroporation into freshly prepared competent E. coli TG1 cells (Zymo Research, Irvine, USA) as described before (Vincke et al., 2012). Electroporated cells were plated on Luria-Bertani (LB) agar plates with ampicillin and glucose. Colonies were checked via PCR for positive clones in correct orientation with primers GIII and MP57 (Vincke et al., 2012) and Phusion High-Fidelity PCR Kit (Thermo Fisher Scientific). (PCR protocols are listed in Data file S4.1).

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4.3.5 Cell culture infection

Vero 76 cells (received from the Collection of Cell Lines in Veterinary Medicine, Friedrich-Loeffler-Institut, Germany) were seeded in 96 well cell culture plates and were infected with RVFV MP-12 after reaching a confluency of about 90 %. For this procedure, supernatants were removed and 100 µl of a MP-12 solution was added to the wells at a multiplicity of infection (MOI) of 1 for panning and of 0.5 for indirect immunofluorescence assay (IIFA) respectively. After 1 h incubation at 37 °C, 5 % CO2, wells were overlaid with 100 µl DMEM with 2 % fetal calf serum, penicillin and streptomycin. After 24 h incubation at 37 °C and 5 % CO2, supernatants were removed and cells were fixed with 4 % PFA (Paraformaldehyde, Sigma Aldrich, St. Louis, USA).

4.3.6 Panning

After infecting the library with M13KO7 helper phages (NEB), panning was done following the protocol of Pardon et al. (2014). MP-12 infected cells were used as panning matrix. Briefly, for each round of panning, PBS of fixed cells was discarded and washed five times with 200 µl PBS containing 0.1 % Tween (PBST). After that, cells were blocked with 200 µl Pierce protein free blocking buffer (Thermo Fisher Scientific) for 2 h at RT and washed again five times with PBST. Afterwards, cells were incubated with 2x1011 phages for 2 h at RT, followed by 10 times washing with PBST. To elute phages carrying antigen-specific antibodies, wells were incubated with 100 µl trypsin (0.25 mg / ml) for 30 min. To inactivate trypsin, 8.33 µl Pefabloc (4 mg / ml; Sigma- Aldrich) was added to the 100 µl supernatant. Freshly grown E. coli TG1 cells were infected with phages and a serial dilution was plated on LB agar with ampicillin and glucose, to count numbers of phages. This was repeated consecutive for three rounds of panning. After that, 48 colonies of the three panning rounds were randomly picked and incubated in a 96 well plate with 200 µl 2xTY (tryptone yeast extract) medium (ampicillin, glucose and glycerol) overnight at 37 °C. Grown bacteria of each well were used for generation of periplasmic extract containing individual single-domain antibodies as described by Pardon et al. (2014). Periplasmic extracts were screened in an in-house IIFA.

4.3.7 In-house indirect immunofluorescence assay

Antibodies were screened by an in-house IIFA as published before (Mroz et al., 2018), but with some modifications. In short, fixed cells were permeabilized with 0.1 % Triton X (Sigma- Aldrich) for 10 min at RT. After washing, 0.1 M glycine (Carl Roth, Karlsruhe, Germany) was added to cells for 10 min at RT. After a further washing step, cells were incubated in blocking reagent (PBS containing 20 % bovine serum albumin [Merck Millipore, Berlington, USA], 0.2 % Tween-20 [Sigma-Aldrich], 30 % glycerine [Carl Roth] and 0.02 % sodium azide [Carl Roth]) for 30 min. Subsequently single domain antibodies were diluted 1:5 in blocking reagent and incubated on cells for 1 h at RT. Furthermore, three washing steps for 5 min were done respectively. After that, cells were incubated with a 1:500 diluted MonoRabᵀᴹ rabbit anti camelid VHH antibody [iFluor 488] (Genscript Biotech, Piscataway Township, USA) and 1:20,000 diluted

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DAPI (Carl Roth) for 1 h at RT following a washing step. After a final washing step, 50 µl PBS was added to each well and fluorescence staining was evaluated by fluorescence microscopy (Nikon, Minato, Japan). Fluorescence intensity was described as strong (3), medium (2), weak (1) or not detectable (0). Furthermore, for defining EC50 (Effective concentration of 50%), RVFV specific sdAb was used in a serial dilution following the same protocol. Beside with RVFV MP-12, this protocol was also performed with Clone 13 and 35 / 74 on BHK-21 cells (Collection of Cell Lines in Veterinary Medicine, Friedrich-Loeffler-Institut, Germany) and with ZH501 on Vero E6 cells (Collection of Cell Lines in Veterinary Medicine, Friedrich-Loeffler-Institut, Germany) respectively. For investigations on cross reactivities, an IIFA was performed with the same protocol, but using Bunyamwera virus (BUNV) or Batai virus (BATV) on infected Vero E6 cells.

4.3.8 Commercial IIFA

A commercial RVFV IIFA slide test kit (EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany) was used with modifications. In short, a serial dilution series of RVFV specific sdAb in sampling buffer was applied to a slide with RVFV ZH548-infected and uninfected VeroE6 cells. After a 30 min incubation at RT, slides were washed for 10 min with PBS and 0.2 % Tween, followed by a further incubation with a 1:500 diluted MonoRabᵀᴹ rabbit anti camelid VHH antibody [iFluor 488] (Genscript Biotech) for 30 min, RT in the dark. The slides were washed again for 10 minutes and covered with a coverslip for a subsequent examination using a fluorescence microscope (Nikon). Fluorescence intensity was described as strong (3), medium (2), weak (1) or not detectable (0).

4.3.9 Sequencing of VHH

A PCR was done with positive clones using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) and Primers GIII and MP57 (Vincke et al., 2012). PCR product was purified with QIAquick® PCR Purification Kit and sent with primers GIII and MP57 to Eurofins Genomics (Eurofins Genomics, Ebersberg, Germany). Amino acids (aa) are numbered according to IMGT, the international ImMunoGeneTics information system®, (http://www.imgt.org, Lefranc et al., 2003).

4.3.10 Expression and purification

A culture of with specific clones was grown in 500 ml LB medium containing 100 µg / ml ampicillin and 0.1 % glucose at 37 °C and expressed at optical density of 0.6 with 1 mM isopropylthiogalactoside (Thermo Fisher Scientific). After 4h, cells were harvested by a centrifugation step. Due to the terminal his-tag, sdAb was purified under native conditions via affinity chromatography using Ni-NTA columns as described earlier (Jackel et al., 2014). Finally, the complete eluate was concentrated to a volume of about 1 ml with Amicon Ultra-15 centrifugal filter units (Merck KGaA) following manufacturer´s instructions.

http://www.imgt.org/

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4.3.11 SDS Page and Western blotting

Single domain antibody was analyzed by SDS polyacrylamide gel electrophoresis (SDS Page) in a 16 % acrylamide gel. 20 μl of each protein fraction was mixed with 20 μl loading buffer (1 % SDS, 25 mM Tris / HCl pH 7.4, 0.5 % mercaptoethanol, 0.001 % bromophenol blue) and incubated at 95 °C for 5 min before loading the acrylamide gel. Staining was performed with Instant Blue™ (Expedeon AG, Heidelberg, Germany). The antibody was also analyzed by Western blot analysis (Mroz et al., 2018). Proteins were transferred to PVDF membranes by semi-dry electroblotting and subsequently blocked for 30 min at RT with 5 % skim milk in PBST and followed by an incubation with a mouse anti his-tag antibody in a 1:2,000 dilution (Merck KGaA). After a washing step (three times for 10 min in PBST), the membrane was incubated for 1 h with an alkaline phosphatase-conjugated goat-anti-mouse antibody (Dianova, Hamburg, Germany) in a 1:2,000 dilution and simultaneously with Precision Protein™ Strep Tactin-AP Conjugate (Bio Rad Laboratories, Hercules, USA) in a 1:5,000 dilution. After a second washing step, the proteins were visualized by chemiluminescence with CDP-Star substrate (Tropix, Sigma Aldrich) and Versadoc Imaging system and Image Lab Software (both Bio-Rad Laboratories), respectively. For antigen detection, E. coli expressed RVFV Np (Jackel et al., 2014) was subjected to SDS Page and transferred to a PVDF membrane. VHH was used as primary antibody and incubated over night at 4 °C. Following a secondary antibody rabbit anti camelid VHH antibody, conjugated to horseradish peroxidase (MonoRabᵀᴹ, Genscript Biotech), in a 1:1,000 dilution was applied. Protein was visualized by SuperSignal® West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and Versadoc Imaging system and Image Lab Software (Bio-Rad)

4.3.12 Serum neutralization test

100 µl TCID50 (50% tissue culture infective dose) of MP-12 was added to a duplicate of serial twofold diluted RVFV specific sdAb, followed by a 30 min incubation at 37 °C, 5 % CO2. Finally, 3x105 Vero 76 cells were added to each well and incubated for 6 d at 37 °C, 5 % CO2. Neutralizing dose of 50 % (ND50) were expressed as the reciprocal of the serum dilution that still inhibited > 50 % of cytopathogenic effect (Rissmann et al., 2017b).

4.3.13 Data analysis

Data were graphed and EC50 values were obtained by fitting the data to a 4-Parameter Logistic Regression model using GraphPad prism 8 (GraphPad Software, San Diego, USA).

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4.4 Results

An alpaca was immunized with 106 TCID50 / ml RVFV MP-12. 24 days post inoculation, when the alpaca showed a SNT titer of 320, heparinized blood was taken and PBMCs were isolated immediately followed by RNA extraction. Using primers CALL001 (targeting the signal sequences of camelid VHH genes) and CALL002 (targeting CH-2 sequence of camelid IgG) a 700 bp fragment was generated in two consecutive PCR steps (Supplemental figure 4.1 A). This fragment contained the VHH, hinge and CH-2 region and was used as template for subsequent amplification in a nested PCR. The resulting 400 bp fragment (Supplemental figure 4.1 B), generated by primers PMCF and VHHBACK, comprised the VHH region. After the fragment was ligated into the pMECS vector and electroporated into E. coli TG1 cells, a primary library with 4x106 clones was generated. A specific sub-library was created using phage display system in three consecutive panning steps with RVFV MP-12 infected Vero76 cells. In the first two steps, no apparent difference was detected in comparison to the control. Panning step three however, revealed a significant enrichment (Supplemental figure 4.2). After three panning steps, 48 recovered clones were expressed and screened for RVFV specificity in an in-house IIFA where one individual clone (9F2) exhibited a strong and specific signal. Subsequently, the sequence of the clone was determined and numbered according to IMGT. The antibody consists of four framework regions (FR) and three complementarity determining regions (CDR) and total sequence length of 112 amino acids. The CDR1 consists of eight, CDR2 of eight and CDR3 of five aa, accordingly. Cys are located at position 23 (FR1) and at position 104 (FR3). The hydrophilic amino acids Phe, Glu, Arg and Arg could be identified in FR2 at positions 42, 49, 50 and 52. In addition was a Trp at position 118 of FR4. (Figure 4.1) The purified 9F2 was subjected to SDS Page, Coomassie staining and Western blotting (Figure 4.2) and revealed a molecular mass of about 16 kDa. Detection by Western blot was carried out with anti-his antibodies that target the C-terminal his-tag of the recombinant VHH. Furthermore, recombinant RVFV nucleoprotein was visualized by the VHH 9F2 at a molecular mass of about 26 kDa. For further characterization, VHH 9F2 was analyzed in serial dilutions in both in-house and commercial IIFA using different attenuated (Clone 13, MP-12) and virulent RVFV (ZH501, ZH548, 35 / 74) strains. VHH 9F2 exhibited high affinity to different strains in a concentration dependent manner (Figure 4.3 and 4.4). The highest affinity was achieved to RVFV strain ZH501 with an EC50 of 0.23 µg / ml. The lowest affinity was seen for RVFV strain ZH548 and an EC50 value of 1.85 µg / ml. Virus antigen detection was possible at nanomolar concentrations of 23.19-115.63 nM and 5.8-14.7 nM respectively. (Detailed concentrations and EC50 calculation can be seen in Data file S4.2) SdAb was further tested for cross reactivity in an in-house IIFA using Bunyamwera and Batai virus infected cells. No signals against BUNV nor against BATV were observed. Finally a serum neutralization test was performed with VHH 9F2 but no inhibitory effect could be determined.

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4.5 Discussion

SdAbs provide valuable tools in many fields of biomedicine research including in vivo or in vitro diagnosis of infection, selective tumor targeting, toxin neutralization or oral immunotherapy (Siontorou, 2013). For this reason, in this study VHHs against RVFV were generated to exploit the biophysical properties like small size, increased hydrophilicity and high stability for diagnostic and further therapeutic applications to treat this devastating disease. We were able to successfully select a high affinity sdAb from a RVFV vaccine strain MP-12 immunized phage display library. In our study, alpaca immunized with RVFV vaccine strain MP-12 was used as antibody source. MP-12 induces high immunity in humans and animal hosts and elicits high neutralizing titers of up to 1:1,280 in alpacas (Rissmann et al., 2017b). Although the HCAb to total IgG ratio in alpacas is considerably lower as in camels (Blanc et al., 2009), they are much easier to handle and have been used for the generation of VHH before (Vance et al., 2017). After PCR amplification, the corresponding VHH fragments were ligated into a phagemid vector and transferred to E. coli TG1 cells via electroporation, resulting in a primary library of 4x106 clones. Similar sizes were described in other studies (Vance et al., 2013). No single antigen was selected as panning target, but cells infected with MP-12 to obtain a full range of antibodies binding to all possible virus proteins. After the third round of panning, the highest specificity was obtained. A RVFV specific sdAb was identified during initial screening in the in-house IIFA. Several rounds of panning are leading to highly affine antibodies, but the diversity of different CDR3 families gets lost (Pardon et al., 2014), why only one RVFV specific CDR3 family was obtained in this study. The sequence of the sdAb corresponds to a typical VHH sequence and consists of four FR and three CDR. Usually the CDR3 is the longest of the three CDRs (Muyldermans, 2013) and on average consists of 17.8 amino acids (Maass et al., 2007). However, in our case a very short sequence of about five amino acids was found. In addition, 9F2 was able to detect recombinant Np by Western blot, which indicates that this short sequence binds to a linear epitope. Furthermore, the sequence for VHH shows typical hydrophilic amino acids in FR2 and also two Cys, which form a disulfide bond. Both the hydrophilic aa and the disulfide bond play an important role in solubility and stability of the protein (Muyldermans, 2013; Lefranc et al., 2003). It is not surprising that 9F2 is directed against the antigen Np, since Np is a major immunogen of RVFV (van Vuren et al., 2007). Furthermore, no neutralization could be identified in a SNT, which is primarily expected from antibodies directed against RVFV Gn / Gc protein (Spiegel et al., 2016). In serial dilution, 9F2 was tested for sensitivity against different virulent and attenuated strains in IIFA. The results show, that 9F2 has a strong affinity to representatives of phylogenetic lineage A (ZH501, ZH548, MP-12), lineage E (Clone 13) and lineage L (35 / 74) (Grobbelaar et al., 2011). The affinity to the virus is very strong and detection / visualization is possible in the range of about 5-115 nM. EC50 values are in a comparable range with commercially available Np mAbs and mAbs from an ELISA kit (Immune Technology Corp., New York, USA; Creative Diagnostics®, New York, USA). Even though 9F2 showed broad affinities to RVFV, no cross reactivity was observed against BUNV or BATV, members of related Orthobunyavirus family, similar

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as Fafetine et al. (2013) wherefore it is well suited as diagnostic tool. In summary, a protocol to generate specific sdAbs against different RVFV antigens was established. 9F2 shows a high affinity to the RVFV Np, but no reactivity against BUNV or BATV. Therefore, it is very suitable for diagnostic use regarding competition ELISA, antigen capture ELISA (Fafetine et al., 2007; Martín-Folgar, 2010) or a lateral flow immunoassay (Wanja et al., 2011). Furthermore, the antibody is well suited for immunohistochemistry for the mice infection model due to the good tissue penetration (Steeland et al., 2016) and without unspecific background like seen by murine monoclonal antibodies. Compared to mAbs VHHs can be produced cheaply and easily, and used for field studies due to their high stability (De Meyer et al., 2014; Muyldermans, 2013). Since neutralizing sdAbs are already considered as therapeutic antibodies (Salvador et al., 2019; Steeland et al.,2016), the phage library could be used as a starting point for generating neutralizing sdAbs by panning and screening with RVFV Gn / Gc.

4.6 Figures

Figure 4.1:

Protein sequence of VHH 9F2. FR = Framework region, CDR = Complementarity determining region. “.” missing amino acid according to IMGT numbering.

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Figure 4.2:

Biochemical analysis of recombinant VHH 9F2. Lane 1: Western blot analysis of recombinant RVFV Np protein. Detection was carried out with VHH 9F2. Lane 2: Western blot analysis of VHH 9F2. Detection was carried out with anti-his monoclonal antibody. Lane 3: SDS-PAGE of recombinant VHH 9F2. M = marker

Figure 4.3:

In-house IIFA and commercial IIFA (E, J without DAPI) with VHH 9F2 using different RVFV strains. Expression of nucleoprotein Np was monitored with VHH 9F2 (green) and cell nuclei with DAPI (blue). A-E infected cells and F-J not infected cells. A, F RVFV strain Clone 13, B, G strain MP-12, C, H strain 35 / 74, D, I strain ZH501 and E, J strain ZH548.

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Figure 4.4:

Data are shown as semi-quantitative signal strength (0-3) in IIFA using VHH 9F2 in serial dilutions against different RVFV strains. A regression model is fitted using 4-Parameter Logistic.

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Supplemental figure 4.1:

A and B show different PCR products that were amplified during generation of VHHs.

A shows first PCR product with sequences of conventional antibody (900 bp) and of

HCAb (700 bp). B shows final sequence of VHH.

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Three consecutive rounds of panning were done and colonies were counted after each

step.

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In-house IIFA with BUNV (A, C) and BATV (B, D). A, B shows sera as positive control

for the two viruses, respectively and for C, D was 9F2 used for detection. Red

fluorescence shows detected sera and blue DAPI staining of cell nucleus.

4.7 Acknowledgments:

Vector pMECS was kindly provided by Serge Muyldermans (Vrije Universiteit Brussel, Brussels, Belgium). Rift Valley fever strains 35 / 74 and ZH501 were kindly provided by Jeroen Kortekaas (Wageningen Bioveterinary Research, Lelystad, the Netherlands). We kindly acknowledge the technical assistance of Birke Böttcher (FLI).

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5 MANUSCRIPT II:

Two monoclonal antibodies against glycoprotein Gn fully protect mice from Rift Valley fever challenge by cooperative effects

Benjamin Gutjahr1, Markus Keller1, Melanie Rissmann1, Felicitas von Arnim1, Susanne Jäckel1,2, Sven Reiche3, Reiner Ulrich3,4, Martin H. Groschup1 and Martin Eiden1*


2 Saxon State Laboratory of Health and Veterinary Affairs, Jägerstraße 8 / 10, 01099 Dresden, Germany

3 Department of Experimental Animal Facilities and Biorisk Management, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany

4 Institute of Veterinary Pathology, Leipzig University, An den Tierkliniken 33, 04103 Leipzig, Germany

*Corresponding author: [email protected] Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany

5.1 Abstract

Rift Valley fever phlebovirus (RVFV) is a zoonotic arbovirus that causes severe disease in humans and ruminants. The infection is characterized by abortions in pregnant animals, high mortality in neonates as well as febrile illness in humans that develop in 1 % of cases encephalitis or hemorrhagic fever. There is presently no specific antiviral treatment for RVFV infection available. In this study, two monoclonal antibodies (mAbs), raised against glycoprotein Gn, were applied in a therapeutic study. Prophylactic and post-exposure treatment of neutralizing mAb Gn3 in RVFV infected mice showed only moderate efficacy (58.3 % survival in both applications). However, a combination therapy together with non-neutralizing mAb Gn32 demonstrated nearly complete protection (83.3 % survival) and a subsequent post-exposure trial protected 100 % of the animals from a lethal challenge dose. The increase of mAb efficacy is probably based on cooperative neutralization effects. These data suggest that a combination therapy with mAbs Gn3 and Gn32 could be an effective post-exposure treatment for patients infected with RVFV.

5.2 Author summary

Rift Valley fever phlebovirus represents an acute viral disease affecting animals especially livestock and humans and is responsible for widespread outbreaks throughout Africa and on the Arabian Peninsula. The virus causes abortions and high mortality especially in young animals, whereas the symptoms in humans range from

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mild flu-like illness to severe hemorrhagic manifestations that can be lethal. So far, no antiviral therapeutics for animals nor humans were available. Therefore, we evaluated two monoclonal antibodies - one neutralizing and one non-neutralizing - in a mouse model for therapeutic treatment against Rift Valley fever. We selected these antibodies since they exhibited cooperative effects in vitro. During Rift Valley fever phlebovirus infection in mice, the applied neutralizing antibody alone showed only partial protection. In contrast, a combined application with both antibodies, lead to a complete protection in one treatment group (100 % survival). A detailed pathological and molecular analysis clearly indicated a strong reduction of virus replication in target tissues of treated mice. Taken together, these results identified two monoclonal antibodies with strong antiviral effects against Rift Valley fever infection, which are promising candidates for human therapy.

5.3 Introduction

Rift Valley fever phlebovirus (RVFV) belongs to the family Phenuiviridae, genus Phlebovirus and contains a single stranded tripartite genome of negative polarity. It consists of three segments that code for nucleoprotein (S segment), two glycoproteins Gn, Gc (M segment) and a RNA dependent RNA polymerase (L segment). In addition, two non-structural proteins are encoded by the S segment (NSs) and M segment (NSm), respectively (Pepin et al., 2010). More than 50 mosquito species including Anopheles, Aedes and Culex spp. have been associated with RVFV transmission, either by isolation of the virus from field-collected mosquitos or by successful experimental transmission in laboratory setting (Linthicum et al., 2016; Vloet et al., 2017). During inter-epizootic periods, the virus may persist by vertical transmission in Aedes mosquitos and the deposition of infected eggs. Subsequent heavy rainfalls and flooding lead to hatching of RVFV infected vectors causing epidemics. The virus emerged during the 1970ths and 1980ths throughout Africa and entered the Arabian Peninsula in 2000 (Nanyingi et al., 2015). Main focus with re-occurrent epidemics are Egypt and Sub-Saharan countries (e. g. Mauritania, South-Africa, Tanzania, Kenya) (Nanyingi et al., 2015). Based on this emergence and due to numerous susceptible mosquito species that are also found in Europe, there is a significant risk for introduction and subsequent dissemination of RVFV in Europe (Chevalier, 2010). Especially in pregnant ruminants (sheep, goats and cattle), the virus causes high abortion rates up to 100 % in sheep and displays a similar mortality in infected newborn lambs. Generally increasing age reduces susceptibility, but adult animals can also be affected and suffer from weakness, anorexia, diarrhea, bloody nasal discharge and jaundice. Apart from abortions, no clinical symptoms can be observed in camels; however, (hyper) acute clinical courses are described in an outbreak in northern Mauritania (El Mamy et al., 2011). Human infection mainly results from direct contact or inhalation during the handling of infected animals and tissues. Most infections display non-specific flu-like symptoms (Bird et al., 2009). Severe cases are associated mostly with ocular complications (e. g. retinitis, reduced vision) and in 1-2 % of the cases with jaundice and hemorrhagic manifestations. Severe complications can be also associated with encephalitis (Al-Hazmi et al., 2003; Alrajhi et al., 2004; Madani et al., 2003). Up to now, no approved antiviral therapeutics nor approved vaccine for Rift

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Valley fever (RVF) is available (Kortekaas, 2014). Known antivirals like Ribavirin or Favipiravir showed only limited efficacy in animal models (Atkins and Freiberg, 2017). Additional small molecules are currently still under development (Atkins and Freiberg, 2017). Most recently, rabbit and human derived monoclonal antibodies were shown to be protective in mice against Rift Valley fever infections (Allen et al., 2018; Wang et al., 2019). In our study, we analyzed the therapeutic application of two murine monoclonal antibodies (mAbs) raised against glycoprotein Gn in a mouse model for Rift Valley fever. The combined application of a neutralizing with a non-neutralizing antibody led to complete protection against an otherwise lethal dose of RVFV attributed to cooperative interference. These results open up new therapeutic treatment possibilities.

5.4 Materials and methods

5.4.1 Virus and cell culture

Rift Valley fever strain 35 / 74 (accession number: JF784386-88) was propagated in BHK-21 cells (Collection of Cell Lines in Veterinary Medicine, Friedrich-Loeffler-Institut, Germany). The virus titer was determined using a 50 % tissue culture infective dose (TCID50) assay, calculated as described by Spearman and Kärber (Mayr, 1977). Briefly, 100 µl of 10-fold serial diluted strain 35 / 74 were added to 90 % confluent monolayers of BHK-21 cells. After incubation at 37 °C, 5 % CO2 for 6 days, plates were fixed with neutral buffered formalin and stained with crystal violet. Virus titration: Samples tested positive in quantitative real-time RT-PCR (RT-qPCR) were used for virus isolation on BHK-21 cells. Corresponding homogenates were diluted from 10-1-10-8 in DMEM supplemented with penicillin-streptomycin and 2 % fetal calf serum. 100 µl of each dilution was added in quadruplicates on a 96 well cell plate seeded with a 90 % confluent monolayer of BHK-21 cells. After incubation for 1 h at 37 °C, 5 % CO2, each well was overlaid with 100 µl medium, incubated at 37 °C, 5 % CO2 for 6 days und subsequently fixed with neutral buffered formalin and stained with crystal violet.

5.4.2 Animals and experimental design

For the mouse challenge model, 3-6 month old BALB / c mice were transferred from in-house specific-pathogen-free (SPF) facility into BSL-3 containment and kept in isolated ventilated cages. The experiments were approved by the competent authority of the Federal State of Mecklenburg-Western Pomerania, Germany, on the basis of national and European legislation, namely EURL 63 / 2010 for the protection of animals used for experiments (LALLF 7221.3-1.1-048 / 17). Following an initial clinical examination, all animals have been accustomed to the new surroundings prior to the initiation of experiments. All animals were fed with commercial feed and had access to water ad libitum.

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In total 78 mice were included in this study and allocated in six groups with equal proportion of male and female individuals. 72 mice were inoculated intraperitoneally with 200 µl 102,7 TCID50 / ml of RVFV strain 35 / 74. Six control animals were treated only with mAbs (mAb control group). From 72 infected mice, 24 individuals were treated with phosphate buffered saline (PBS; PBS control group) and 48 individuals with different antibody regimen. Antibodies (200 µg in 100 µl PBS) were applied intravenously in the tail vein either 30 min before virus challenge (prophylactic exposure treatment, PrEP) or 30 min after virus challenge (post-exposure treatment, PEP). Accordingly mice were treated prophylactically with mAb Gn3 (n = 12, Gn3 PrEP group) or post-exposure (n = 12, Gn3 PEP group) or with a simultaneous administration of mAb Gn3 and Gn32 - again prophylactically (n = 12, Gn3 + Gn32 PrEP group) or post-exposure (n = 12, Gn3 + Gn32 PEP group), referred as “combi groups”. Blood samples were taken daily by submandibular venipuncture from corresponding mice starting from 1 dpi (days post inoculation) till 13 dpi. Mice behavior and clinical anomalies were scored twice daily by a defined score sheet (Supplemental figure 5.1). The animals were inspected for the presence of signs of disease including ruffled fur, lethargy, immobility and bleedings. Depending on the course of the disease, the listed symptoms can occur in individual degrees of severity. Corresponding values were added in an individual score sheet. The humane endpoint was defined as a cumulative score ≥ 8. Mice were euthanized with an overdose of Isofluran CP 1 ml / ml (CP-Pharma, Burgdorf, Germany) followed by heart puncture at 13 dpi or when termination criteria from the score sheet were met earlier.

5.4.3 Molecular detection of RVFV-specific RNA

RNA was extracted from liver, brain and blood cruor with NucleoMag® VET Kit (MACHEREY-NAGEL, Düren, Germany) and King Fisher™ Flex Purification System (Thermo Fisher Scientific, Waltham, USA), according to the manufacturer´s instructions. As internal extraction and PCR control, MS2 bacteriophage was added to each sample (Ninove et al., 2011). The presence of RVFV-derived RNA was determined by a RT-qPCR (Bird et al., 2007). A synthetic RNA comprising the target region of the RT-qPCR was utilized for quantification (Jackel et al., 2013b).

5.4.4 Serology

Monoclonal IgG1 antibodies Gn3 and Gn32 were raised against recombinant bacterially (E. coli) expressed ectodomain of RVFV glycoprotein Gn and were reactive in ELISA, indirect immunofluorescence assay (IIFA) and Western blot (Jackel et al., 2014). Neutralizing activity of mAb Gn3 and mAb Gn3 in combination with Gn32 was determined in a serum neutralization test (SNT) using RVF vaccine strain MP-12 (Rissmann et al., 2017b). Briefly, 100 TCID50 of MP-12 was added to duplicates of serial twofold diluted mAbs. Following an incubation of 30 min at 37 °C and 5 % CO2, 3x105 Vero 76 cells (Collection of Cell Lines in Veterinary Medicine, Friedrich-Loeffler-Institut, Germany) were added to each well. Plates were incubated at 37 °C, 5 % CO2 for 6 days. Neutralizing doses of 50 % (ND50) were expressed as the reciprocal of the

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serum dilution that still inhibited > 50 % of cytopathogenic effect. Furthermore, dilution series of mAbs and heat-inactivated sera of heart puncture was tested in the same SNT protocol but using strain 35 / 74 on BHK-21 cells. As a positive control neutralizing serum of a cattle immunized with recombinant, bacterially (E. coli) expressed ectodomain of RVFV glycoprotein Gn and as negative control serum of a naïve sheep were used. Mouse sera were analyzed with the ID Screen® RVFV competition multi-species ELISA (ID Vet, Montpellier, France) according to the manufacturer’s instructions. The ELISA is based on the detection of antibodies (IgM / IgG) against the RVFV nucleoprotein Np.

5.4.5 Epitope mapping

A peptide library of RVFV Gn was generated by synthesizing oligopeptides with 20 amino acid (aa) length and 12 amino acid overlap by JPT (Berlin, Germany). Peptide assay was similar performed as already described in (Lagatie et al., 2014). In short, NUNC Immobilizer Streptavidin plates (Thermo Fisher Scientific) were coated with 2.5 µM / well peptides at 4 °C overnight. After that, plates were blocked with 3 % skimmed milk for 1 h, room temperature. Furthermore, plates were incubated with monoclonal antibodies (1:100 diluted in blocking buffer) for 1 h, 37 °C. After that, secondary goat anti mouse HRP (Horseradish peroxidase) antibody (Dianova, Hamburg, Germany) was used as detection of specific binding mAbs and incubated for 1 h, 37 °C. Finally, TMB (3.3′, 5.5′-Tetramethylbenzidin) was added and incubated for 10 min. Reaction was stopped with 1 M sulphuric acid and read out in an ELISA reader at 450 nm. Threefold washing steps with tris-buffered saline and 0.1 % Tween was done between single steps.

5.4.6 Pathological examination and immunohistochemistry

Necropsy was performed according to standard procedures under a work bench at BSL-3 conditions. Specimen from brain, lung, liver and spleen were fixed during necropsy in 4 % neutral buffered formaldehyde for at least more than 21 days, processed, embedded in paraffin wax, sectioned at 2-4 µm thickness, and stained with hematoxylin and eosin. The grade of characteristic histopathological lesions (hepatitis, encephalitis, lymphoid depletion, follicular hyperplasia) was semiquantitatively assessed on a 0-3 scale as follows: 0 = no obvious findings, 1 = mild, 2 = moderate and 3 = severe findings. Immunohistochemistry (IHC) was performed with the avidin–biotin–peroxidase complex method (ABC, Elite PK6100; Vector Laboratories, Burlingame, USA) with 3-amino-9-ethylcarbazole (AEC, Dako, Glostrup, Denmark) as chromogen and hematoxylin counterstain. The primary antibody used for the detection of RVFV nucleoprotein was a heat-inactivated serum of a sheep immunized with recombinant RVFV MP12-strain nucleoprotein (internal code: S24NP) in a dilution of 1:4,000. As positive control, Vero 76 cells were infected with MP-12 strain, pelleted by centrifugation (1,500 rpm, 20 min, 12 °C) 24 h post infection, and processed and embedded in paraffin wax similar to the tissue samples. Negative controls consisted

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of uninfected Vero 76 cells and replacement of the primary antibody by sera of a non-immunized sheep on serial sections, respectively. The distribution of the RVFV antigen was semiquantitatively assessed for each organ on a 0-3 scale as follows: 0 = no viral antigen, 1 = focal or oligofocal, 2 = multifocal, and 3 = confluent to diffuse immunoreactive cells.

5.4.7 Data and statistical analysis

EC50 (Effective concentration of 50 %) and IC50 (Inhibitory concentration of 50 %) values were obtained by fitting the data to a 4-Parameter Logistic Regression model (Wickham, 2009) using the nplr package in R. (nplr: N-Parameter Logistic Regression; R package version 0.1-7 R Core Team (2018). R: A language and environment for statistical computing. R Foundation for Statistical computing, Vienna, Austria). CI (Combination index) and DRI (Dose-reduction index) values were obtained by CompuSyn software (ComboSyn, Inc., Paramus, USA) to quantify synergy. Kaplan-Meier survival curves were analyzed by the log rank test and virological data (viral RNA, virus titration and pathological scores) compared using an ANOVA (Kruskal-Wallis H) test and correlation was done with Pearson product moment correlation with SigmaPlot (Systat Software, San Jose, USA). Data were graphed using Microsoft Excel 2016 (Microsoft Corporation, Redmond, USA). See also Data file S5.1.

5.5 Results

5.5.1 In vitro characterization of mAbs Gn3 and Gn32

The binding of both monoclonal antibodies Gn3 and Gn32 to recombinant RVFV Gn ectodomain was assessed in ELISA yielding EC50 values of about 1.29 µg / ml and 0.38 µg / ml (Figure 5.1). Co-incubation of both mAbs showed a shift to an increased binding activity (EC50 = 0.49 µg / ml) probably based on cooperative interactions. An epitope mapping study on mAbs Gn3 and Gn32 was performed using biotinylated oligopeptides with 20 amino acid length and 12 aa overlap covering the whole sequence of glycoprotein Gn ectodomain. Gn32 targeted two adjacent peptides at the outermost n-terminus of Gn protein (encompassing sequence: PGKGHNYIDGMT, Data file S5.2) in contrast to Gn3 that did not react with any of the linear epitopes. Both mAbs were also assessed in serum neutralization test (Figure 5.2). Only mAb Gn3 exhibited neutralizing activity and reached a 50 % neutralizing concentration (IC50) against RVFV vaccine strain MP-12 at 33.0 µg / ml. As already seen in ELISA, co-incubation of mAbs Gn3 with Gn32 showed a synergistic effect and reduced IC50 to 24.6 µg / ml (Figure 5.2 A). IC50 values of mAb Gn3 against virulent RVFV strain 35/74 were 4-5 times higher showing a concentration of 147.2 µg / ml and in combination with Gn32 of 100.6 µg / ml (Figure 5.2 B, Data file S5.3). The synergistic effects of both mAbs were additionally quantified by so-called combination index and dose-reduction index as described earlier (Howell et al., 2017). The calculation was carried out by CompuSyn software, which basically quantifies synergistic effects of drug

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combinations (Chou, 2010; Chou and Talalay, 1984). The CI, which is divided into 3 categories (<1: synergistic effects, 1: additive effects, >1: antagonistic effects) revealed F(a)55 (Fraction affected) values of 0.39 and 0.31 for MP-12 and strain 35 / 74, respectively. The DRI indicates how many folds of dose-reduction for each compound at a given effect are allowed in synergistic combination. The corresponding values were 2.59 for MP-12 and 3.17 for strain 35 / 74 (detailed data used for calculation and CI and DRI at other F(a) are listed in Data file S5.4).

5.5.2 In vivo protective efficacy of mAbs Gn3 and Gn32 against lethal RVFV challenge

To determine whether mAbs Gn3 and Gn32 had therapeutic activity, BALB / c mice inoculated i. p. with RVFV strain 35 / 74, were subjected to a prophylactic exposure (PrEP) and a post-exposure (PEP) antibody treatment. PrEP and PEP studies were carried out with Gn3 alone or in combination with non-neutralizing Gn32 (combi group). Unfortunately, one mouse of PEP combi group died during antibody application. PrEP and PEP treatment of Gn3 in RVF infected mice (n = 12 in both groups) showed medium efficacy (about 58.3 % survival in both applications). However, a prophylactic combination therapy together with non-neutralizing mAb Gn32 demonstrated nearly complete protection (83.3 % survival) and a subsequent post-exposure trial protected 100 % of the animals from a lethal challenge dose (Figure 5.3 A). In this group, only one individual displayed a transient clinical sign (ruffled fur). In contrast, the PBS control group presented a survival rate of only 16.67 %. Diseased mice showed typical symptoms like ruffled fur, lethargy and acute death around 3-6 dpi and symptoms of encephalitis like hind limb paralysis on 8 dpi, when there was no effective antibody protection. Mean clinical score of corresponding groups is depicted in Figure 5.3 B. The compilation of individual scores is found in Data file S5.5. Non-infected mice (mAb control group) showed no signs of antibody incompatibility (Data file S5.5). Analysis of tissues (brain and liver) as well as cruor in necropsied mice showed a general strong reduction of viral RNA, dependent on treatment regimen (Figure 5.4 A). Main target tissue was liver with an average value of 2.3x105 copies / mg tissue, followed by cruor (7.6x104 copies / mg tissue) and brain (1.2x104 copies / mg tissue) in the untreated group. A significant lower level was observed in both Gn3 groups as well as the combi PrEP group regarding cruor samples and in Gn3 PrEP and combi PrEP regarding brain. However, high viral RNA loads remained in the liver tissues of these groups (mean values: 7x104-1.7x105 copies / ml). In contrast, no viral RNA was detected in liver of the combi PEP group and low amounts of RNA (mean value: 1 copy / mg) in the brain of three individuals as well as 1.8 copies / mg tissue in cruor of one animal indicating a nearly complete protection. In addition, all PCR positive samples were used for virus titration on BHK-21 cells (Supplemental figure 5.2). The results demonstrate a high correlation with the PCR results. In particular, there was no live virus in tissues from the combi PEP group. A compilation for all individuals is seen in Data file S5.6. The daily blood samples revealed viremia in the PBS control group during 2-6 dpi in PCR and virus titration. All therapy groups showed a shortened viremic phase, whereas both Gn3 groups still having peaks similar to those of the PBS control group.

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The combi groups showed delayed viremia and especially combi PEP group showed a distinct lower viral load during viremic phase. See detailed data in Data file S5.7. Sera from necropsied animals were further tested in a competition ELISA (Figure 5.4 B). The lowest number of seropositive samples (4 / 24) were observed in the PBS control group probably due to rapid virus spread and onset on disease. Interestingly in both pre-exposure groups (Gn3 and combi group) a distinct higher number of animals were seropositive (7 and 9 out of 12, respectively) compared to the post-exposure group (4 out of 12 and 6 out of 11, respectively). Finally, sera were tested for neutralizing activity, which correlated in most cases with ELISA positive results. The titer ranged from 1:10 up to 1:60 (see Data file S5.6 summarizing complete individual serological and molecular data). All necropsied animals were subjected to comprehensive histopathological and immunohistochemical analysis including semiquantitative assessment of virus antigen (nucleoprotein) distribution in brain, liver, spleen and lung (Supplemental figure 5.3). Furthermore, the severity of the typical lesions was assessed employing four different histopathological scores (hepatitis, encephalitis, lymphoid depletion and follicular hyperplasia within the spleen). Thereby, three basic combinations of pathological findings and antigen distribution were observed (Figure 5.5): Pattern I (acute, high-grade hepatitis) includes animals with acute RVF infection that show predominantly high-grade, acute, confluent to diffuse, necrotizing hepatitis with Councilman corpuscles and intranuclear, irregular, eosinophilic inclusion bodies (Cowdry type B). Furthermore, these mice showed low to moderate lymphoid necrosis, apoptosis and / or depletion of the white pulp of the spleen. Pattern I is mostly found in individuals of the untreated PBS control group, which are characterized by accumulation of RVF antigen mainly in liver and spleen. Main target cells for viral antigen were hepatocytes and macrophages in the red pulp and follicular dendritic cells in the spleen. In the lung, alveolar macrophages or interstitial and intravascular phagocytes were stained whereas in brain primarily neurons and rarely microglia were antigen positive. Pattern II was observed in only few mice, which survived the acute, diffuse, necrotizing hepatitis and succumbed around 8 dpi. They exhibited a more subacute hepatitis with more pronounced infiltrates of macrophages and heterophils, and an acute, focal to multifocal, necrotizing polioencephalitis. Notably there was no RVFV antigen in the livers of these mice at the time of necropsy but in the lesioned areas within the brain. These mice also display lymphatic apoptosis, necrosis and depletion in the spleen. Pattern III was observed in surviving animals and is characterized mainly by no obvious findings or low-grade, subacute, multifocal, periportal and randomized, lymphohistiocytic infiltrates with infrequent heterophils in the liver. Furthermore, some of these mice displayed low-to moderate follicular hyperplasia of the white pulp in the spleen. These findings were observed in the combi groups, especially the PrEP group. Mice of the PEP group showed a significant lower incidence of hepatitis or were asymptomatic. Both groups exhibited a considerably lower antigen signal in liver, a lower incidence of lymphoid depletion but a significant higher incidence of follicular hyperplasia compared to the PBS control group. Mice treated only with single mAb Gn3 showed in both groups (PrEP and PEP) mixed histopathological phenotypes or no findings at all. In general, a reduction of viral antigen was observed especially in the liver. The Gn3 PEP group showed also a

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significant lower incidence of lymphoid depletion compared to the PBS treated mice. Individual data regarding IHC and histopathology are deposited in Data file S5.6.

5.6 Discussion

In this study, RVF neutralizing mAb Gn3 in combination with Gn32 was applied in a mouse RVF infection model demonstrating complete protection against virus challenge. It is the first trial about in vivo protection of cooperative mouse monoclonal antibodies against RVF. Similar antibody prevention studies against RVF were carried out very recently by applying human (Wang et al., 2019) or rabbit (Allen et al., 2018) derived mAbs. Furthermore, we conducted a detailed in-depth molecular and histopathological analysis of antibody treatment in infected mice. MAbs Gn3 and Gn32 (as well as mixtures of both) showed high binding affinity to Gn in ELISA which are in the same EC50 range as RVFV-neutralizing rabbit mAbs (Allen et al., 2018). In contrast, neutralizing activity exhibited IC50 values of about 100 µg / ml that are significantly lower compared to rabbit antibodies (~3.0 µg / ml) and human antibodies which were effective in the nanogram range (Wang et al., 2019). However, the values are only comparable to a limited extent, since different neutralization or alternative assays were used (SNT vs plaque reduction neutralization test vs FACS based assays). In each case, mAb application led to protection against RVFV infection. Interestingly, for an efficient neutralization of virulent challenge strain 35 / 74 a distinctly higher antibody concentration was needed compared to vaccine strain MP-12, although both strains exhibited only four amino acid differences within glycoprotein Gn sequence (Supplemental figure 5.4). Via PepScan analysis the main epitope of non-neutralizing mAb Gn32 could be successfully elucidated. The corresponding sequence PGKGHNYIDGMT is located in domain I of Gn and directly neighbors antigenic site A described in Wang et al. (2019). Gn3 showed no distinct signal in PepScan, indicating a conformational epitope. Even though Gn32 has no neutralizing activity in vitro, it increases significantly the neutralizing potency of Gn3. This synergistic effect was supported by the CompuSyn software with values of a CI of 0.39 / 0.31 and a DRI of 2.59 / 3.17. Similar effects were already described from studies with mAbs targeting other viruses, e. g. HIV triple and quadriple mAb combinations with CI of 0.2-0.8 and DRI of 2- 39.9 (Li et al., 1998). Synergistic effects of RVFV neutralizing antibodies were previously reported from in vitro studies (Besselaar and Blackburn, 1992). We therefore hypothesize that the mechanistic basis for this “cooperative neutralization” (Howell et al., 2017) is based on the binding of Gn32 to glycoprotein Gn, which induces a conformational change and makes the epitope of mAb Gn3 more accessible. Thus, the induced conformational change allows a comprehensive and more effective binding of mAb Gn3. A similar cooperative effect of non-neutralizing mAbs that improve the activity of otherwise poorly neutralizing mAbs has been originally described for mAbs against HIV (Li et al., 1998). In any case, domain I of RVFV Gn plays an important role in antibody-mediated neutralization and protection, since additional neutralizing mAbs against RVFV cluster in this domain (Wang et al., 2019). Application of Gn3 alone protected more than half of the group against a severe RVFV infection. In combination with non-neutralizing Gn32, 83 % of the

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prophylactically treated group survived, whereas post-exposure treatment provided complete protection. Complete protection was correlated with a significant reduction of viral load in all tested tissues and the incapability of further replication. The combined PEP antibody regimen could even completely eradicate the virus from the liver, which represents the main target organ of RVFV. Especially in the unprotected PBS control group, strain 35 / 74 causes an infection with marked liver tropism, leading to a fulminant acute diffuse necrotizing hepatitis that has already been shown with other RVFV strains in mice (Smith et al., 2010; Ikegami et al., 2017). Importantly, all antibody treated groups showed a decrease and shortening in viremia. PBS control group showed a viremia from 2-6 dpi, as reported earlier (Smith et al., 2010). Especially in combi groups, viremia was clearly reduced, shortened and further delayed. Detailed histopathological and immunohistological analyses elucidated specific differences during the course of RVFV infection. In acute cases, coalescing to diffuse RVFV antigen was detectable mainly in hepatocytes, but also in multifocal macrophages and follicular dendritic cells in the spleen. The livers displayed characteristic severe, acute, coalescing to diffuse necrotizing hepatitis, and many hepatocytes displayed morphological changes reminiscent of apoptotic cell death as well as intranuclear Cowdry type B inclusion bodies (Ulrich, 2019). These typical findings in the liver and spleen have been already noted during studies with other RVFV strains (Ikegami et al., 2017). Furthermore, a moderate lymphoid depletion in the spleen was seen, which may lead to lymphopenia, and is probably part of a fatal systemic inflammatory response syndrome as a consequence of fulminant hepatitis. Lymphocyte apoptosis in spleens of infected mice that had died 3 dpi was reported previously (Smith et al., 2010). These findings, which mainly occurred in the PBS control group, were confirmed by a strong positive correlation between lymphoid depletion and the incidence of hepatitis (Pearson product moment correlation, r = 0.922; p = 0.0258* (*significant p<0.05)), RVFV-positive liver (r = 0.815; p = 0.0927) and cruor (r = 0.877; p = 0.0509). MAb application indeed revealed a clearly differentiated picture of disease progression: Mice that survived the RVFV infection showed mainly only low-grade, multifocal, lymphohistiocytic lesions in the liver, together with follicular hyperplasia in the spleen. MAbs could not completely neutralize the virus, but minimized viral replication, which could be successfully repelled by a successful and strong immune response in the mouse. This led to lymphocytosis as found in vaccine (Kamal, 2009) and mouse infection studies (Smith et al., 2010) in form of follicular hyperplasia. Therefore antibody treatment is characterized by a strong positive correlation between follicular hyperplasia and positive ELISA (r = 0.975; p = 0.00484*) and SNT (r = 0.921; p = 0.0261*) values and by a negative correlation with hepatitis (r = - 0.888; p = 0.0445*), virus replication in liver (r = - 0.925; p = 0.0242*) and viremia (r = - 0.93; p = 0.0219*). These findings were mainly seen in the combi groups, where a significantly low-grade hepatitis and frequent follicular hyperplasia was observed. In summary, this study demonstrated an efficient protection against RVFV by application of a combined mAb regimen in a RVF mouse challenge model. Further studies will now try to determine the specific epitope of Gn3 antibody to find a comprehensive molecular explanation for the cooperative and protective effects. In addition, both murine mAbs will be converted into humanized or fully human variants

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to finally evaluate them in clinical trials and to avoid well-known side effects and treatment failures (dos Santos et al., 2018).

5.7 Figures

Figure 5.1:

ELISA analysis of mAbs Gn3 and Gn32 using recombinant glycoprotein Gn as antigen. Experiments were repeated three times.

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Figure 5.2:

mAb Gn3 and Gn32 neutralize RVFV in two serum neutralization tests. Data are shown as percentage neutralization and a regression model is fitted using 4-Parameter Logistic. (A) SNT using RVFV-strain MP-12. (B) SNT using RVFV-strain 35 / 74 Experiments were performed in duplicate.

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Figure 5.3:

Survival curves and cumulative mean clinical scores of tested groups. (A) Efficacy of antibody protection was assessed by survival analysis. A Kaplan-Meier log rank test was performed; n. s. between mAb-Gn3-treated (PrEP/PEP) and PBS control, p<0.001 between mAB-Gn3-Gn32-treated (PrEP) and PBS control, p<0.001 between mAb-Gn3-Gn32-treated (PEP) and PBS control, n. s. between mAb-Gn3-Gn32-treated (PrEP/PEP) and Gn3 (PrEP/PEP). (B) Clinical scores were monitored and recorded daily after infection. n. s. not significant

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Figure 5.4:

PCR results of brain, liver and cruor and ELISA results of serum from heart blood of individual mice. (A) Virus loads were assessed by quantitative real-time RT-PCR with RVFV specific primers and quantified with a synthetic calibrator in samples from liver, brain and cruor. The results represent the virus loads [log10 (copies / mg tissue)] of each group in a box blot diagram. Significance was analyzed by ANOVA (Kruskal-Wallis H) test (*p<0.05) (B) Antibodies in mice serum samples at day of death were determined by a commercial competition ELISA kit targeting antibodies against nucleoprotein. Results are shown as sample-to-positive ratio for each individual mouse. Horizontal line marks ELISA cut off.

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Figure 5.5:

Histopathological and immunohistochemical findings in different tissues of three individual mice. Light microscopy revealed three principally different patterns of lesions and antigen distribution in the livers (first row, A-F), brains (second row, G-L), spleens (third row, M-R), and lungs (fourth row, S-X) of mice, which succumbed due to the infection within the first 6 days (left column: P18-865, mouse #25, 3 dpi, A, B, G, H, M, N, S, T) later in the time course of the disease (middle column: P18-870, mouse #30, 8 dpi, C, D, I, J, O, P, U, V) and those, which survived until the end of the observation period (right column, P18-878, mouse #78, 13 dpi, E, F, K, L, W, X and P18-851, mouse #68, 13 dpi Q, R). (A) The mice dying until 6 dpi display mainly a severe, acute, diffuse, necrotizing hepatitis with Councilman corpuscles (arrows), suggestive of hepatocellular apoptosis. (B) These mice show coalescing to diffuse antigen within hepatocytes. (M) There are few apoptotic or necrotic lymphocytes in the white matter of the spleen (arrow). (C) The few mice dying later than 6 dpi display multifocal infiltrations of macrophages and lymphocytes (arrow) and hepatocellular vacuolar

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degeneration within the livers. (D) There is no antigen present within the liver. (I) There are oligofocal necrotic neurons and glia (arrows) within the brain, interpreted as necrotizing polioencephalitis. (J) There is multifocal, neuronal antigen accumulation. (O) There are many apoptotic or necrotic lymphocytes (arrow) leading to lymphatic depletion within the spleen. (Q) There is follicular hyperplasia with enlarged follicles with a pale lymphoblast-rich center and a darker peripheral zone of more differentiated lymphocytes. (A, C, E, G, I, K, M, O, Q, S, U, W) hematoxylin-eosin. (B, D, F, H, J, L, N, P, R, T, V, X) Immunohistochemistry employing the avidin-biotin-peroxidase-complex method for RVFV nucleoprotein with AEC as chromogen (red-brown) and hematoxylin counterstain (blue). (A-X) Bars = 50 µm.


Clinical score sheet for daily examination of infected mice.

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Virus replication from PCR positive tissues, determined as virus titer (log[TCID50 / ml]).

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Liver, spleen, brain and lung were examined for virus in IHC (A) and histopathology (B).

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Alignment of amino acid sequences of RVFV strain 35 / 74 MP-12.


Rift Valley fever strain 35 / 74 was kindly provided by Jeroen Kortekaas (Wageningen Bioveterinary Research, Lelystad, the Netherlands). We kindly acknowledge the technical assistance of Rebecca König, Birke Böttcher, Katrin Schwabe and Silvia Schuparis (FLI) as well as the support in data analysis by Filip Cierniak and Jana Schulz (FLI).

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6 MANUSCRIPT III: Live vaccine virus yields a neutralizing monoclonal antibody

against Rift Valley fever phlebovirus

Benjamin Gutjahr1, Martin Eiden1, Melanie Rissmann1, Claudia Mroz1, Sven Reiche2, Martin H. Groschup1*


2 Department of Experimental Animal Facilities and Biorisk Management, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany

*Corresponding author: [email protected] Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems.

6.1 Abstract

Rift Valley fever phlebovirus (RVFV) causes repeatedly serious epidemics and epizootics in vast regions of Africa and on the Arabian Peninsula. RVFV is an arbovirus, which is transmitted by numerous mosquito species. The purpose of this study was to generate neutralizing monoclonal antibodies (mAbs) against RVFV, which has been rather difficult in the past. As there are no convincing therapeutic drugs, the passive immunization by administering potent mAbs may be a promising alternative intervention approach. BALB / c mice were initially immunized with vaccine strain MP-12, whereas three further mice were boosted with virulent strain 35 / 74 and one mouse was additionally immunized with 35 / 74 only. Immunofluorescence screening of hybridoma cell supernatants finally revealed 14 anti-RVFV specific mAbs and one of these (mAb 2A4) displayed a neutralizing effect in serum neutralization test.

6.2 Short communication

Rift Valley fever phlebovirus (RVFV) is a viral zoonosis that can be transmitted by arthropods. In most cases, human infections result from a contact with contaminated animal tissues. However, human infections have also been caused by mosquito bites (Liu et al., 2017). The virus has been isolated for the first time in Kenya in 1931 in the Rift Valley (Daubney et al., 1931) and was responsible for serious epizootic diseases including epidemics such as the one in 1977 / 78 in Egypt with 200,000 human cases reported, including 600 fatalities (Nanyingi et al., 2015). In 2000, the first outbreak occurred on the Arabian Peninsula. Repeated outbreaks have been reported from Egypt and Sub-Saharan African countries, such as Kenya,Tanzania and Uganda as well as Mauritania and South Africa (Nanyingi et al., 2015). Due to the high vector competence and the rapid spread, there is a great concern that the virus could also

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reach Europe (Chevalier, 2010). So far, there is no treatment for Rift Valley fever (RVF) (Kortekaas, 2014) as known antiviral compounds like Favipiravir and Ribavirin show only a limited effect and sometimes even a possible worsening of the symptoms (Hartman, 2017; Atkins and Freiberg, 2017). Currently there are no approved vaccines or specific therapies to prevent or treat RVFV in humans. Most literature known about neutralizing monoclonal antibodies against RVFV, reaches back in the 1980s and 1990s (e. g. Saluzzo et al., 1989a, 1989b); Besselaar et al., 1991), whereas in recent years, first efforts have been undertaken to utilize monoclonal antibodies as a means of therapeutic treatment (Allen et al., 2018; Wang et al., 2019). Most generated neutralizing RVFV mAbs are based on immunization with individual Gn / Gc protein (Besselaar and Blackburn, 1992; Allen et al., 2018; Wang et al., 2019). In this study however, an alternative approach was done by immunizing mice with live virus. For mice immunization, RVF strain MP-12 was propagated on Vero 76 cells as already described (Rissmann et al., 2017b). RVFV strain 35 / 74 was propagated in BHK-21 cells. Both cell lines were obtained from the Collection of Cell Lines in Veterinary Medicine, Friedrich-Loeffler-Institut, Germany. The virus titer was determined using a 50 % tissue culture infective dose (TCID50) assay, calculated as described by Mayr (1977). Briefly, 100 µl of 10-fold serial diluted strain 35 / 74 were added to 90 % confluent monolayers of BHK-21 cells. After incubation at 37 °C, 5 % CO2 for 6 days, plates were fixed with neutral buffered formalin and stained with crystal violet. Four BALB / c mice were immunized intraperitoneally with attenuated RVFV MP-12 on days 0, 30, 60 and 90 post immunization. A final boost was carried out three days before spleen cells were fused with SP2 / 0 myeloma cells. Hybridoma cell generation was done like published before (Mroz et al., 2018). As an additional approach, spleen cells were removed from a mouse immunized with 102.7 TCID50 / ml RVFV 35 / 74 after 10 days post inoculation (dpi) and hybridoma cells were produced as described above. Additional pooled spleen cells of three mice, immunized with 102.7 TCID50 / ml RVFV MP-12 and boostered at 21 dpi with 102.7 TCID50 / ml RVFV 35 / 74 were used at day 31 for hybridoma cell generation. The experiments were approved by the competent authority of the Federal State of Mecklenburg-Western Pomerania, Germany, on the basis of national and European legislation, namely EURL 63 / 2010 for the protection of animals used for experiments (LALLF 7221.3-1.1-048 / 17 and 7221.3-2.5-004 / 10). Hybridoma cell supernatants were screened primarily in an in-house indirect immunofluorescence assay (IIFA) as published before (Mroz et al., 2018). An indirect ELISA was done like described in Jackel et al. (2014), coated with E. coli expressed Gn, Gc, Np, NSm and NSs protein for determining the directed antigen of individual mAb. For the further characterization of ELISA positive mAb a Western blot was performed as described earlier (Mroz et al., 2018). Finally, all mAbs were screened in a serum neutralization test (SNT) as published in Rissmann et al. (2017b). The neutralizing dose of 50 % (ND50) was expressed as the reciprocal of the serum dilution that still inhibited > 50 % of cytopathogenic effect. Serum with a ND50 value over 10 (mAbs over 2) were determined positive, while values under 10 (mAbs under 2) were determined negative. Immunization with both an attenuated and a virulent RVFV strain induced neutralizing titers in BALB / c mice up to values of about 640 (ND50) with the exception of

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mouse #I. 108 (Table 6.1). In total 14 RVFV specific mAbs have been generated by screening with an IIFA. The titer thereby mostly correlates with the number of generated antibodies ranging from one mAb of a mouse with low neutralizing titer up to nine mAbs from mouse #442 with the highest titer (Table 6.2). Only three mAbs were obtained from the 35 / 74 infected mice. The low yield can be explained by the removal of the spleen after only 10 days, which prevents the maturation of IgG response and may be a further reason for the significantly lower signal in IIFA than seen in mAbs generated by MP-12 immunized mice. In general, during RVFV infections, unspecific IgM antibodies were detected earliest at 4 dpi and the first IgG antibodies in the blood at 9 dpi (Pepin et al., 2010). Even an alternative immunization scheme with booster of MP-12 infected mice with the virulent strain did not induce a significant increase of neutralizing titer and subsequent mAb recovery. Therefore, only one additional RVFV specific mAb was obtained. All 14 antibodies displayed a specific staining of viral antigens within the cytoplasm in an IIFA (Figure 6.1). To determine specific epitopes, mAbs were analyzed in ELISA by five recombinant E. coli expressed viral proteins (Np, NSs, NSm, Gn, Gc). Only one mAb (2F12) bound to recombinant Np protein, which is one of the most immunogenic RVFV antigens (van Vuren et al., 2007) (Figure 6.2). This binding to a linear epitope was confirmed by Western blot (Figure 6.3). These findings indicate that the majority of mAbs recognizes conformational epitopes that are yet to be determined. Only one of the generated 14 mAbs showed weak neutralization, which most likely refers to binding to Gn or Gc derived epitope. Both glycoproteins are the solely targets to effectively inhibit virus propagation (Spiegel et al., 2016). In most cases, neutralizing epitopes base on properly folded proteins and harbor conformational or discontinuous epitopes. Therefore, epitope mapping through linear peptides or unprocessed protein fragments will not or only insufficiently lead to revealing the epitope. Interestingly, a cooperative effect between the neutralizing mAbs 2A4 and Gn3 (ND50 = 2, Jackel et al., 2014) was observed during co-incubation, which more than doubled the neutralizing effect. However, as the specific epitopes could not be determined so far, underlying molecular mechanisms for this phenomenon remain unclear. A similar effect has already been described for other RVFV (Besselaar and Blackburn, 1992) and Ebolavirus derived mAbs (Howell et al., 2017).

In summary, neutralizing mAbs can be generated by immunizing mice with live virus, however screening with Gn / Gc may reveal a higher number.

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6.3 Tables

Table 6.1:

Overview of BALB / c mice, immunized with different antigens, the final neutralizing titer and number of generated mAbs.

mAb IIFA ELISA (Antigen) SNT (ND50)

#397

2G7 +++ - -

#442

1D5 +++ - -

2F1 +++ - -

2A4 ++ - 10

2B8 +++ - -

2F12 +++ Np -

3A3 +++ - -

6C3 +++ - -

6H6 +++ - -

6H11 +++ - -

#444

6E7 +++ - -

#I.108

1F5 + n. t. -

2H11 + n. t. -

#II.106,107,108

1G8 + n. t. -

Table 6.2:

Overview of all RVFV specific generated mAbs and their results in different tests. Strong (+++), medium (++) or (+) weak fluorescent signal, n. t. = not tested

BALB / c mouse Antigen SNT (ND50) № of generated mAbs

#397 MP-12 240 1

#442 MP-12 640 9

#443 MP-12 80 -

#444 MP-12 480 1

#I.108 35 / 74 <10 2

#II.106,107,108 MP-12+ 35 / 74 320,60,80 1

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6.4 Figures

Figure 6.1:

Exemplary IIFA images with (A-C) strong positive mAbs, (D) weak positive mAb, (E) negative control and (F) positive control (mAb against RVFV Gn). Red fluorescence shows mAbs detecting RVFV and blue DAPI staining of cell nucleus.

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Figure 6.2:

mAbs were used in ELISA for detecting RVFV Gn, Gc, Np, NSs and NSm.

Figure 6.3:

Western blot analysis: Lane 1: mAb 2F12 was used as detection for RVFV Np protein. M= marker

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Rift Valley fever strain 35 / 74 was kindly provided by Jeroen Kortekaas (Wageningen Bioveterinary Research, Lelystad, the Netherlands). We kindly acknowledge the technical assistance of Sven Sander and Birke Böttcher (FLI).

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7 GENERAL DISCUSSION

The Rift Valley fever phlebovirus (RVFV) is an arbovirus found within the genus Phlebovirus. It is considered to be one of the most important zoonoses in Africa. The virus is the only known member of the genus that can cause hemorrhagic fever and severe, sometimes fatal, diseases in both animals and humans (Strauss and Strauss, 2008; Pepin et al., 2010). Major outbreaks have occurred over the last century, including the epidemics in South Africa in 1950 / 51 with more than half a million of affected animals and over 100,000 deaths (Nanyingi et al., 2015). Furthermore, large human associated outbreaks were registered, such as the largest outbreak to date in Egypt in 1977, with up to 200,000 cases and 600 human fatalities (Nanyingi et al., 2015), posing a public health threat with huge socio-economic implications on trade and economic growth. In recent decades, the virus has emerged from the Rift Valley in Kenya to large parts of Africa, adjacent islands and the Arabian Peninsula (Nanyingi et al., 2015). Due to climate change and global trade, the risk of virus introduction into new areas and continents through competent vectors and infected animals is increasing (Chevalier, 2010). Sporadic cases of Rift Valley fever (RVF) infected people traveling from Africa to Europe (Tong et al., 2019) or China (Liu et al., 2017) have been reported. Since the RVF cases in Europe harbored high viral titers in whole blood while travelling through several countries with competent vectors (e. g. Aedes albopictus and Culex pipiens) there is a significant risk for the introduction of RVFV in RVF free areas (Tong et al., 2019). In general, due to the spread of the virus as well as the continuous circulation in endemic areas, an integrated surveillance, preparedness and control system should be implemented to perform risk assessments with a focus on public health threats (Hassan et al., 2017). Therefore, numerous diagnostic assays (Jackel et al., 2013a; Paweska et al., 2008; van Vuren et al., 2007; Bird et al., 2007; Drosten et al., 2002; Lindahl et al., 2019; Wichgers Schreur et al., 2017; Ellis et al., 2014) have been developed in recent years to perform serological and molecular screening studies during epidemic and inter-epidemic periods to rapidly identify acute infections and to intervene as quickly as possible in early outbreak events. Furthermore, immunological naïve areas that are potentially at risk can be monitored with said diagnostic tools. However, despite many efforts and activities in the field of public health, outbreaks were still reported regularly, e. g. in Uganda, Kenya, Rwanda, Gambia in 2018 (OIE, 2018a, 2018b; WHO, 2018b, 2018c) with some still ongoing in Mayotte (WHO, 2019a) and Central African Republic (WHO, 2019b), affecting both humans and livestock alike. In addition, the search for the specific mammalian reservoir host of RVFV is still open (Bird and McElroy, 2016). Additional information and continuous improvement of existing methods is therefore needed to obtain further input regarding the virus circulation and distribution in animals and to perform effective control measures. This especially includes veterinary vaccines to prevent the spread of the virus in endemic regions and to ensure long-term protection. Even though first veterinary vaccines are commercially accessible for endemic regions, there is no fully licensed RVFV vaccine for human nor veterinary use available so far (Kortekaas, 2014; Bird and McElroy, 2016). In addition, there are no approved antivirals authorized for animals or humans. To date, drugs like Ribavirin and Favipiravir provide only limited improvements and are known to show adverse effects (Atkins and Freiberg, 2017; Hartman, 2017).

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Therefore, the key challenge of this thesis was the generation, characterization and evaluation of monoclonal and for the first time of single domain antibodies and their application for virus imaging and antiviral therapy. In a first study (manuscript I) an approach was carried out to generate single domain antibodies against RVFV from immunized alpacas (Vicugna pacos). Alpacas like other members of the Camelidae family produce heavy chain antibodies whose antigen-binding site consists of a single variable heavy chain domain (VHH) that is subsequently selected for downstream applications. After immunization with vaccine strain MP-12 (Rissmann et al., 2017b), a high neutralizing titer against RVFV was determined. From peripheral blood mononuclear cells (PBMC) corresponding VHH-sequences were obtained via PCR. Subsequently, a VHH phage library from the B-cell transcriptome was synthesized, which contained 4×106 clones. Library selection against RVF derived antigens was carried out in three rounds of panning using cells infected with RVFV strain MP-12. As a result, VHH (9F2) could be recovered that demonstrated high reactivity against the nucleoprotein, which makes it suitable for diagnostic use. This was confirmed by the detection of isolates from three different phylogenetic lineages A, E and L (Grobbelaar et al., 2011) via indirect immunofluorescence assay (IIFA) with concentrations in the nM range. The antibody showed the typical VHH structure of four framework regions (FR) interspersed with three complementarity determining regions (CDR) (Muyldermans, 2013). The relatively short third CDR sequence consists of only five amino acids. This short CDR 3 and the detection of the nucleoprotein Np in Western blot suggests the recognition of a presumably linear epitope. No neutralizing activity was seen in serum neutralization test, a result that was expected from Gn / Gc derived VHHs only. Future application of this single domain antibody (sdAb) will include indirect and competitive Np based ELISAs, which is facilitated by their general thermal stability and an overall comparatively simple and cost-effective production procedure. In addition, by fusing fluorescent protein sequences (e. g. GFP) to 9F2 the newly generated fusion proteins may serve as versatile tools to observe cellular Np kinetics or processing by using fluorescence microscopy and live cell imaging (Muyldermans, 2013; Steeland et al., 2016). Finally, the generated phage library may act as starting point for further screenings of Gn / Gc derived VHHs to receive potential neutralizing sdAbs for subsequent therapeutic application. However, 9F2 should not be excluded from further therapeutic treatments since even non-neutralizing applications were promising: One study demonstrated that immunization with Np encoding cDNA provided at least partial protection in infected mice, although no neutralizing titer was detectable (Lagerqvist et al., 2009). A further search for neutralizing antibodies was performed in a conventional approach by immunization of mice with virulent (strain 35 / 74) or attenuated (vaccine strain MP-12) live virus (manuscript III). Both inoculated strains induced neutralizing titers in corresponding animals (ND50= 20 - 640). The resulting monoclonal antibodies (mAbs) were identified by screening supernatants on MP-12 infected cells via IIFA. Remarkably, most RVFV specific mAbs were recovered from the individual with the highest titer (9 out of 14). All mAbs were evaluated for reactivity against five different E.coli expressed RVFV proteins including glycoprotein Gn, Gc, nucleoprotein Np and non-structural proteins NSs, NSm by an in-house ELISA. Only one mAb (mAb 2F12) could detect Np protein in ELISA and in an additional Western blot, which indicates a specificity of the

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remaining clones to conformational epitopes. Only one single mAb (mAb 2A4) showed neutralizing activity with a low ND50 titer of about 10. Further studies should elucidate, whether they react against Gn, Gc as prerequisite for virus neutralization as previously shown for effective anti-RVF antibodies (Allen et al., 2018; Wang et al., 2019). A promising approach would be individual expression of Gn and Gc proteins in mammalian cells since correct folding and glycosylation are more likely to recognize conformational epitopes (Sahdev et al., 2008). A specific binding to these epitopes can then be determined by IIFA. Interestingly one mAb 2A4 induced cooperative effects in a serum neutralization test (SNT) when co-incubated with mAb Gn3 (see manuscript II) leading to a ND50 of about 30. Besides immunization with full virus, immunization of mice with single (recombinant) antigens is considered a standard method to generate mouse-derived monoclonal antibodies. By applying this method, a set of monoclonal antibodies against structural (Np, Gn, Gc) as well as non-structural antibodies (NSm, and NSs) was generated that exhibited reactivity against corresponding proteins in Western blot, IIFA and immunohistochemistry (Jackel et al., 2014; Rissmann et al., 2017b; Mroz et al., 2018). Further evaluation of this panel for neutralizing activity using serum neutralization test revealed one single neutralizing antibody (Gn3), which targets glycoprotein Gn. Neutralization is one main criterion when mAbs are assessed for therapeutic applications. Effective antibody treatment against Ebola, Western equine encephalitis virus or Hantavirus (Brannan et al., 2019; Burke et al., 2018; Garrido et al., 2019) are among many examples for this application. In the main part of this study (manuscript II), antibodies were selected after evaluation in vitro and were subsequently applied in vivo for their efficacy against RVF. Because neither effective antivirals nor approved application is available so far (Kortekaas, 2014; Atkins and Freiberg, 2017), there is an urgent need for a successful treatment against RVF. Therfore, antibody treatment is one method of choice and recent studies have already shown that neutralizing individual rabbit / human derived mAbs against RVF have a protective effect in mice (Allen et al., 2018; Wang et al., 2019). Although there is a long history of antibodies against RVF (e. g. Saluzzo et al., 1989a, 1989b; Besselaar et al., 1991), there are only few reports about neutralizing mAbs and their evaluation in vitro (Besselaar and Blackburn, 1992). In our work mAb Gn3 was used as starting point for co-incubation with a set of non-neutralizing antibodies derived from immunization against Gn protein (Jackel et al., 2014). Finally one non-neutralizing mAb (Gn32) was identified that induced in combination with Gn3 synergistic or cooperative effects by enhancing the neutralizing titer. Both mAbs showed a high affinity to Gn in ELISA with EC50 values of 1.29 µg / ml (Gn3) and 0.38 µg / ml (Gn32) in a similar range as other neutralizing rabbit mAbs (Allen et al., 2018) directed against Gn and also similar with 9F2 to Np. In SNT analyses with two different RVFV strains, a cooperative effect could be observed, where the non-neutralizing Gn32 significantly increased the neutralization of Gn3. MAb Gn32 recognizes the linear epitope PGKGHNYIDGMT, which is located in the immediate vicinity of antigenic site A (Wang et al., 2019) on domain I of Gn. This region is also detected by other neutralizing mAbs from a previous study (Wang et al., 2019). In contrast, Gn3 does not target a linear epitope which gives evidence for a yet to determined conformational epitope. Furthermore, the measured cooperative effect could further be confirmed in silico by the CompuSyn software, which shows a

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cooperative combination index (CI) and dose-reduction index (DRI) value similar to other mAbs (Li et al., 1998). The basis of this effect seems to be a conformational change of the antigen triggered by Gn32, which allows a more effective binding of the neutralizing antibody to his epitope. In a next step, mAb Gn3 was tested alone as well as in combination with Gn32 in vivo, in a mouse infection model. Mice were infected i. p. with strain 35 / 74 in a dose of 102.7 TCID50 / ml. MAb Gn3 was applied alone in a prophylactic exposure (Gn3 PrEP) and as post-exposure (Gn3 PEP) treatment, as well as in a combined approach with mAb Gn32. Furthermore, one control group was mock treated with PBS (Phosphate buffered saline). Over a period of 14 days, the health status of the mice was assessed daily on the basis of a score sheet. In the PBS control group, only two out of 24 animals survived, while in both Gn3 groups seven out of 12 animals survived to the end of the study. As already seen in vitro, combination groups showed a significantly higher survival curve, with only two of the 12 animals in the combi PrEP group dying and all animals in the combi PEP group surviving. The mean additive scores of the individual groups also accurately reflected the course of disease and survival. Despite significant higher IC50 values in vitro, treatment with Gn3 and especially with combination groups showed similar survival rates as reported from other studies (Allen et al., 2018; Wang et al., 2019). The high survival rate also correlates with a low virus dissemination in the mouse and corresponding tissues as revealed by IHC, PCR and virus titration of liver, brain and cruor. Especially in the combi groups, there is a significant reduction in viral loads and strong inhibition of replication in all tissues. The combi PEP group was even able to completely neutralize the virus in the main target organ, the liver. However, in the PBS control group, the RVFV strain 35 / 74 showed clear liver tropism, leading to a fulminant acute, diffuse necrotizing hepatitis, as already described with other strains in mice (Smith et al., 2010; Ikegami et al., 2017). In addition, a 5-day long viremia with a very high viral load could be detected in this group, which was also notified in other studies (Smith et al., 2010). In contrast, the therapeutic groups showed a significantly shortened and reduced viremia that was – in the case of the combined application – additionally delayed. Further histopathological and immunohistological examinations clearly highlighted differences in the various courses of the disease: RVFV antigen accumulated in liver and spleen was mainly detected in hepatocytes, but also in macrophages and follicular dendritic cells in the spleen in acute phases, which was mainly seen in the PBS control group. Livers displayed characteristic severe, acute, coalescing to diffuse necrotizing hepatitis, and many hepatocytes showed morphological changes reminiscent of apoptotic cell death as well as intranuclear Cowdry type B inclusion bodies (Ulrich, 2019). Such findings were also recognized in other studies in the corresponding organs (Ikegami et al., 2017). Moderate lymphoid depletion was also observed in the spleen, which can lead to lymphopenia. This is probably part of a fatal systemic inflammatory response syndrome resulting from fulminant hepatitis. Similar findings have been observed in infected mice at 3 dpi (days post inoculation) (Smith et al., 2010). These findings also showed a strong positive correlation between the incidence of lmyphoid depletion and hepatitis, PCR positive liver and cruor. In the mAb-treated groups, however, the picture was completely different. Surviving mice showed only low-grade, multifocal, lymphohistiocytic lesions in the liver, in

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combination with follicular hyperplasia in the spleen. The comprehensive approach of PCR, virus titration and serological assay results demonstrated that the antibodies did not completely neutralize the virus, but could reduce the viral loads in infected mice just sufficient enough to produce an anti-RVFV humoral immune response. In histology, this became manifest as lymphocytosis in the form of follicular hyperplasia, which was previously observed in vaccine and mouse infection studies (Smith et al., 2010; Kamal, 2009). This conclusion is supported by a strong positive correlation between the incidence of follicular hyperplasia and positive ELISA and SNT values as well as a negative correlation with hepatitis and virus negative liver and PCR values from the blood. These findings mainly occurred in the combi groups, which complies with the overall picture. Summarizing the second part of this work, it could be shown that neutralizing mAbs can be successfully applied for antiviral treatment against RVFV. Especially the combination of two mAbs revealed a complete protection in mice although residual virus could still be detected. Due to the incomplete neutralization, the residual risk of a teratogenic effect should also be clarified by further studies. Although other studies also demonstrated full protection by applied antibodies (Allen et al., 2018; Wang et al., 2019) our in-depth study illustrated detailed effects of the used antibodies for the first time. Further investigations should follow in order to determine the exact epitope of Gn3 and thus gain a better insight into the molecular mechanism of the observed cooperative effect. In addition, the mAbs will be humanized in order to enable a further treatment in humans without adverse effects. In summary, in this work a broad range of novel monoclonal antibodies and for the first time, a single domain antibody was generated that will be assayed for further diagnostic and therapeutic applications. In addition, we could demonstrate complete protection of RVF infected mice by simultaneous application of a neutralizing mAb, in combination with a non-neutralizing mAb. The effect is based on cooperative effects of both antibodies that were thoroughly investigated by molecular, serological and pathological investigations.

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8 SUMMARY

Generation and characterization of antibodies against Rift Valley fever phlebovirus and evaluation of their therapeutic efficacy in a mouse model

Benjamin Gutjahr

Rift Valley fever phlebovirus (RVFV) is one of the most important pathogens of the genus Phlebovirus as it causes severe diseases in humans and livestock. While the majority of infected humans only suffers from a self-limiting febrile disease, severe progression can occur in 1-2 % of cases, often with retinitis and neurological disorders or even as fatal hemorrhagic fever syndrome due to severe hepatitis. Ruminants are the most susceptible animals among livestock where RVFV can provoke so-called abortion storms and mortality rates in newborn animals of up to 100 %, while adult animals usually only suffer from subacute fever symptoms. Humans are primarily infected due to contact to infectious tissues and fluids from viremic animals and only rarely by mosquitos. RVFV has spread in the last decades to many African countries, its adjacent islands and to the Arabian Peninsula and has caused repeated outbreaks throughout the continent, thereby effecting major socio-economic losses. Moreover, given climate changes and the global trade of animals and goods, RVFV may even spread to Europe and is therefore a feasible emerging disease threat. To date there is no approved human vaccine and no therapy available. Antibodies are promising candidates for prophylactic and therapeutic interventions against RVF virus infection. Therefore, this study dealt with the generation and characterization of novel antibodies against RVF and their therapeutic assessment. In a first study (manuscript I) we were able to establish a modified protocol for the generation of single domain antibodies (sdAb) against RVFV by immunization of an alpaca (Vicugna pacos) to produce heavy chain antibodies (HCAb) lacking the corresponding light chain. The RVFV immunized alpaca displayed a high neutralizing antibody titer, which enabled the amplification of variable heavy chain domains (VHH) of HCAbs from the RNA of stimulated peripheral blood mononuclear cells. VHH sequences were ligated into the phagemid vector and a phage library was generated. After three consecutive rounds of panning, RVFV specific antibodies were selected by screening via an immunofluorescence assay (IIFA). The clone harbored a typical VHH sequence and exhibited a significant reactivity against the nucleoprotein (Np) in Western blot and a high affinity in the nM range to RVFV strains from three different phylogenetic lineages. Therefore, the sdAb can be used as a new diagnostic tool for the detection, processing and visualization of Np protein. Additionally, possible therapeutic treatments will be examined in further studies. Manuscript II therefore describes an efficacy study on two monoclonal antibodies (mAb Gn3 and Gn32) raised against recombinant Gn protein for their in vitro and in vivo efficacy against RVFV. Gn3 displayed neutralizing activity in a cell culture model, Gn 32 was inactive. However, co-incubation of Gn32 and Gn3 led to a significantly higher neutralization compared to Gn3 alone, demonstrating cooperative effects. Subsequently, RVFV infected mice were treated with Gn3 alone or in combination with

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Gn32 by either a post-exposure or a pre-exposure prophylaxis application. Application of Gn3 alone and the combination led to higher survival rate compared to the untreated control group. Subsequent studies in PCR, virus titration and immunohistochemistry showed a reduction of viral loads in all tissues that was significant in the combination groups. One application – post-exposure of combined Gn3 and Gn32 – fully protected mice what was shown by a 100 % survival rate. Thus, we successfully demonstrated for the first time that mouse-derived mAbs in a combined antibody regimen can be a promising approach for an antiviral therapy. In a third study (manuscript III), novel monoclonal antibodies were obtained by immunizing mice with either the vaccine strain MP-12 or the virulent RVFV strain 35 / 74. Both strains induced high neutralizing antibody titers in mice. Mice were therefore used for the generation of mAb secreting hybridoma cells and 14 new mAbs with high affinity to the virus in infected cells measured in an IIFA were obtained. Out of those 14 mAbs only one mAb showed ELISA reactivity against recombinant Np indicating that all others targeted conformational or discontinuous epitopes. Only one single clone displayed a low neutralizing activity in serum neutralization test, which makes this mAb an additional therapeutic candidate.

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9 ZUSAMMENFASSUNG

Generierung und Charakterisierung von Antikörpern gegen das Rifttal-Fieber-Virus und die Evaluation derer therapeutischer Wirksamkeit in einem Mausmodell

Benjamin Gutjahr

Das Rifttal-Fieber-Virus (Rift Valley fever phlebovirus, RVFV) ist ein von Stechmücken (Culicidae) übertragener Erreger innerhalb der Ordnung Bunyavirales, der schwere Erkrankungen bei Mensch und Tier auslöst. Auch wenn die Mehrheit der Menschen nur eine selbstlimitierende Fiebererkrankung durchläuft, können in 1-2 % der Fälle schwere Verläufe auftreten. Sie umfassen Retinitis, neurologische Störungen und schwere Hepatitiden mit einem oft tödlich verlaufenden hämorrhagischen Fieber. Besonders empfänglich sind kleine Wiederkäuer bei denen in infizierten Muttertieren typische massenhafte Verlammungen (sog. „abortion storms“) auftreten können. Der Mensch wird im Gegensatz zu Tieren hauptsächlich über infizierte Tiere, vor allem aber durch viruslastiges Tiergewebe und nur selten direkt von Stechmücken infiziert. Aufgrund einer hohen Vektor-Kompetenz hat sich das Virus in den letzten Jahrzehnten über weite Teile Afrikas, die angrenzenden Inseln und die Arabische Halbinsel ausgebreitet und stellt ein enormes globales Gesundheitsrisiko dar. Das Virus verursacht wiederholt Ausbrüche auf dem gesamten Kontinent mit einer hohen Zahl an betroffenen Menschen und Tieren mit schwerwiegenden sozioökonomischen Folgen. Gegenwärtig gibt es keine spezifische Therapie gegen das RVFV doch stellen Antikörper vielversprechende Kandidaten für prophylaktische und therapeutische Behandlungen dar. Ziel der Arbeit war daher neue und neuartige Antikörper gegen RVFV zu generieren, zu charakterisieren und als mögliche Therapeutika in Infektionsstudien einzusetzen. In einer ersten Studie (Manuskript I) wurden Einzeldomänenantikörper (sdAb) gegen RVFV durch die Immunisierung eines Alpakas (Vicugna pacos) mit RVFV gewonnen. Nach Isolierung und Amplifikation entsprechender Antikörpersequenzen in einer Phagenbibliothek konnte mit Hilfe eines neuen Selektionsverfahrens via Immunfluoreszenztest (IIFA) eine antigenbindende Domäne (VHH) identifiziert werden, die eine starke Bindung an das Nukleoprotein (Np) des Virus aufweist. Der 15 kDa große Antikörper zeigte zudem eine hohe Reaktivität in nanomolarer Konzentration für phylogenetisch verschiedene RVFV-Stämme in dem IIFA. Der neu gefundene sdAb eignet sich daher als neues Diagnosewerkzeug für die Detektion und Visualisierung des Np Proteins. Das angewendete Protokoll soll nun im Rahmen zukünftiger Screenings für die Gewinnung von sdAb vor allem gegen die viralen Glykoproteine eingesetzt werden, die als aussichtsreiche neutralisierende Antikörper-Kandidaten für mögliche therapeutische Behandlungen gelten. Der Hauptteil (Manuskript II) beschäftigte sich mit der therapeutischen Evaluierung von zwei murinen monoklonalen Antikörpern (mAbs) in einem Mausinfektionsmodell für das Rifttal-Fieber-Virus. Beide Antikörper (Gn3 und Gn32) wurden durch die Immunisierung mit rekombinanten Gn Protein hergestellt, wobei Gn3 neutralisierende

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Aktivität aufweist. Durch Ko-inkubation mit Gn32 konnte eine signifikant höhere Aktivität erzielt werden, die sich auf kooperative Effekte zurückführen lässt. Anschließend wurden RVFV-infizierte Mäuse durch Zugabe von Gn3 allein oder in Kombination mit Gn32 im Rahmen einer Präexpositions- und einer Postexpositionsprophylaxe behandelt. Die Anwendung von Gn3 allein und in Kombination führten zu einer höheren Überlebensrate im Vergleich zur unbehandelten Kontrollgruppe. Nachfolgende Analysen mittels PCR, Virustitration und Immunhistochemie zeigten eine Reduktion der Viruslast in allen Geweben, die in den Kombinationsgruppen signifikant war. Die kombinierte Anwendung von Gn3 und Gn32 als Postexpositionsprophylaxe zeigte dabei einen vollständigen Schutz (100 %ige Überlebensrate). Damit konnten wir erstmals nachweisen, dass eine Behandlung mit murinen monoklonalen Antikörpern vollständig vor einer RVFV-Infektion schützen kann. In einer letzten Studie (Manuskript III) wurden neue monoklonale Antikörper durch Immunisierung von Mäusen mit dem Impfstamm MP-12 oder dem virulenten RVFV-Stamm 35 / 74 gewonnen. Die unterschiedlichen Impfregime führten zu hohen neutralisierenden Titern bei Mäusen, die die Erzeugung von 14 neuen anti-RVFV mAbs ermöglichten. Mit allen Antikörpern kann das Virus in infizierten Zellen mittels indirekter Immunfluoreszenz nachgewiesen werden. Bei einem einzelnen mAb wurde Reaktivität gegenüber rekombinantem Np in einem ELISA festgestellt. Die anderen mAbs erkennen vermutlich konformationsabhängige oder diskontinuierliche Epitope. Ein einzelner Klon zeigte zudem im Serumneutralisationstest eine geringe neutralisierende Wirkung, die ihn zu einem zusätzlichen therapeutischen Kandidaten macht.

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90

11 SUPPLEMENTS

11.1 Manuscript I:

Data file S4.1: PCR protocols

RT-PCR components:

Components Concentration

Reverse transcription buffer (10x)

dNTP mixture 10 mM

RNAsin 40 u / µl

AMV 25 u / µl

Oligo (dT) 15 primer 500 µg / ml

H20

RNA

Amplification cycle:

Cycle Temperature (°C) Time (min)

1 42 15

2 95 5

Conventional PCR components:


Phusion HF Buffer (5x)

dNTP 10 mM

CALL001 10 pmol

CALL002 10 pmol

MgCl2 10 mM

Phusion DNA Polymerase 2 u / µl

H20

cDNA

91

Nested PCR components:



dNTP 10 mM

PMCF 10 pmol

VHH-BACK 10 pmol


H20

DNA

Amplification cycle (conventional and nested PCR):

Cycle Temperature (°C) Time

1 98 30 s

2-36 98 10 s

55 15 s

72 30 s

37 72 7 min

Clone PCR components:



dNTP 10 mM

GIII 10 pmol

MP57 10 pmol


H20

DNA

Amplification cycle:

Cycle Temperature (°C) Time

1 98 5 min

2-29 98 45 s

55 45 s

72 45 s

30 72 10 min

Data file S4.2: EC50 calculation

92

Concentration for calculation of EC50:

ZH548 MP12 Clone 13 35/74 ZH501

Dilution µg/ml signal strength Dilution µg/ml signal strength Dilution µg/ml signal strength Dilution µg/ml signal strength Dilution µg/ml signal strength

1 380 3 1 380 3 1 380 3 1 380 3 1 380 3

2 190 3 2 190 3 2 190 3 2 190 3 2 190 3

4 95 3 4 95 3 4 95 3 4 95 3 4 95 3

8 47,5 3 8 47,5 3 8 47,5 3 8 47,5 3 8 47,5 3

16 23,75 3 16 23,75 3 16 23,75 3 16 23,75 3 16 23,75 3

32 11,875 3 32 11,875 3 32 11,875 3 32 11,875 3 32 11,875 3

64 5,9375 2 64 5,9375 3 64 5,9375 3 64 5,9375 3 64 5,9375 3

128 2,96875 2 128 2,96875 2 128 2,96875 2 128 2,96875 2 128 2,96875 3

256 1,484375 1 256 1,484375 2 256 1,484375 2 256 1,484375 2 256 1,484375 2

512 0,7421875 1 512 0,7421875 1 512 0,7421875 2 512 0,7421875 2 512 0,7421875 2

1024 0,3710938 1 1024 0,3710938 1 1024 0,3710938 1 1024 0,3710938 1 1024 0,3710938 2

2048 0,1855469 0 2048 0,1855469 1 2048 0,1855469 1 2048 0,1855469 1 2048 0,1855469 1

4096 0,0927734 0 4096 0,0927734 0 4096 0,0927734 1 4096 0,0927734 1 4096 0,0927734 1

8192 0,0463867 0 8192 0,0463867 0 8192 0,0463867 0 8192 0,0463867 0 8192 0,0463867 0

16384 0,0231934 0 16384 0,0231934 0 16384 0,0231934 0 16384 0,0231934 0 16384 0,0231934 0

32768 0,0115967 0 32768 0,0115967 0 32768 0,0115967 0 32768 0,0115967 0 32768 0,0115967 0

µg/ml nM µg/ml nM µg/ml nM µg/ml nM µg/ml nM

EC50 1,848 115,63 0,8905 55,66 0,4156 25,98 0,4156 25,98 0,2352 14,7

Last detection 0,3711 23,19 0,1855 11,59 0,0928 5,8 0,0928 5,8 0,0928 5,8

Results from EC50 calculation with GraphPad Prism 8:

log(inhibitor) vs. response -- Variable slope (four parameters)ZH548 MP-12 Clone 13 35/74 ZH501

Best-fit values

Bottom -0,06657 -0,08143 -0,3275 -0,3275 -0,3652

Top 3,119 3,096 3,12 3,12 3,053

LogIC50 0,2666 -0,05038 -0,3813 -0,3813 -0,6285

HillSlope 0,9413 0,9452 0,697 0,697 0,8597

IC50 1,848 0,8905 0,4156 0,4156 0,2352

Span 3,186 3,177 3,448 3,448 3,419

95% CI (profile likelihood)

Bottom -0,5347 to 0,2570 -0,7237 to 0,3023 -2,999 to 0,2970 -2,999 to 0,2970 -1,866 to 0,1923

Top 2,830 to 3,512 2,838 to 3,425 2,822 to 3,671 2,822 to 3,671 2,824 to 3,344

LogIC50 0,005303 to 0,5046 -0,3600 to 0,1951 -1,456 to -0,007513 -1,456 to -0,007513 -1,188 to -0,3436

HillSlope 0,5809 to 1,501 0,5695 to 1,544 0,3229 to 1,188 0,3229 to 1,188 0,4929 to 1,377

IC50 1,012 to 3,196 0,4365 to 1,567 0,03496 to 0,9828 0,03496 to 0,9828 0,06484 to 0,4533

Goodness of Fit

Degrees of Freedom 12 12 12 12 12

R squared 0,971 0,9696 0,9621 0,9621 0,9682

Sum of Squares 0,7532 0,7602 0,8253 0,8253 0,6993

Sy.x 0,2505 0,2517 0,2623 0,2623 0,2414

Number of points

# of X values 16 16 16 16 16

# Y values analyzed 16 16 16 16 16

93

11.2 Manuscript II:

Data File S5.1: Statistics

The log rank statistic for the survival curves (P = <0,001). Data of S5 were used for calculation

Survival curve (Fig. 3A) N

Column 1 PBS group 24

Column 2 Gn3 PrEP 12

Column 3 Gn3 PEP 12

Column 4 Gn3+Gn32 combi PrEP group 12

column 5 Gn3+Gn32 combi PEP group 11

All Pairwise Multiple Comparison Procedures (Holm-Sidak method):

Overall significance level = 0,05

Comparisons Statistic P Value Significant?

Column 1 vs. Column 5 18,064 0,000214 Yes

Column 1 vs. Column 4 13,681 0,00195 Yes

Column 1 vs. Column 2 5,77 0,123 No








Kruskal-Wallis One Way Analysis of Variance on Ranks

All Pairwise Multiple Comparison Procedures (Dunn's Method) : Data of S6 were used for calculation

brain liver cruor

Viral RNA (Fig. 4A) N N N

Column 1 PBS group 24 24 21

Column 2 Gn3 PrEP 12 12 12

Column 3 Gn3 PEP 12 12 10

Column 4 Gn3+Gn32 combi PrEP group 12 12 12

Column 5 Gn3+Gn32 combi PEP group 11 11 11

brain

Comparison Diff of RanksQ P<0,05

Col 1 vs Col 5 33,784 4,495 Yes

Col 1 vs Col 4 21,708 2,975 Yes

Col 1 vs Col 2 20,625 2,826 Yes

Col 1 vs Col 3 8,792 1,205 No

Col 3 vs Col 5 24,992 2,901 Yes

Col 3 vs Col 4 12,917 1,533 No

Col 3 vs Col 2 11,833 1,404 Do Not Test

Col 2 vs Col 5 13,159 1,527 No



94

liver


Col 1 vs Col 5 40,083 5,233 Yes

Col 1 vs Col 4 28,792 4,002 Yes

Col 1 vs Col 3 18,375 2,554 No


Col 2 vs Col 5 26,833 3,079 Yes

Col 2 vs Col 4 15,542 1,871 No


Col 3 vs Col 5 21,708 2,491 No



cruor


Col 1 vs Col 5 31,225 4,37 Yes

Col 1 vs Col 4 26,202 3,772 Yes

Col 1 vs Col 2 23,202 3,34 Yes

Col 1 vs Col 3 21,552 2,922 Yes

Col 3 vs Col 5 9,673 1,153 No




Col 2 vs Col 4 3 0,383 Do Not Test


Kruskal-Wallis One Way Analysis of Variance on Ranks

All Pairwise Multiple Comparison Procedures (Dunn's Method) : Data of S6 were used for calculation

Pathological scoring (Fig. S2A and B) liver

lymphoid

depletion

follicular

hyperplasia hepatitis

N N N N

Col 7,10,11,12 Combi PrEP 12 12 12 12

Col 19,22,23,24 Combi PEP 11 11 11 11

Col 31.34,35,36 PBS 24 23 23 24

Col 43, 46,47,48 Gn3 PrEP 12 12 12 12

Col 55,58,59,60 Gn3 PEP 12 12 12 12

liver


Col 19 vs Col 31 24,862 3,308 Yes

Col 7 vs Col 31 20,688 2,835 Yes

Col 55 vs Col 31 14,604 2,001 No


lymphoid depletion


Col 22 vs Col 34 27,109 3,634 Yes

Col 10 vs Col 34 23,817 3,286 Yes

Col 46 vs Col 34 19,317 2,665 Yes

Col 58 vs Col 34 14,317 1,976 No

95

Data file S5.2: Epitope mapping

follicular hyperplasia


Col 11 vs Col 35 25,333 3,496 Yes

Col 23 vs Col 35 18,773 2,516 Yes

Col 59 vs Col 35 14,5 2,001 No


hepatitis


Col 24 vs Col 36 24,741 3,292 Yes

Col 12 vs Col 36 17,271 2,367 No



Pearson Product Moment Correlation

Col 65 Col 66 Col 68 Col 69 Col 70

Correlation coefficient Col 64 -0,886 0,922 0,815 0,877 -0,774

p value(<0,05) 0,0455 0,0258 9,27E-02 0,0509 0,124

N 5 5 5 5 5

Col 66 Col 68 Col 69 Col 70 Col 71

Correlation coefficient Col 65 -0,888 -0,925 -0,93 0,975 0,921

p value(<0,05) 0,0445 0,0242 0,0219 0,00484 0,0261

N 5 5 5 5 5

Col 64 lymphoid depletion

Col 65 follicular hyperplasia

Col 66 hepatitis

Col 68 PCR liver

Col 69 PCR cruor

Col 70 ELISA

Col 71 SNT

Data used for calculation:

Groups Lymphatic depletion Follicular

hyperplasia

Hepatitis Encepha-

litis

Liver Cruor

ELISA SNT

mean value of animals (score)

mean value

of animals

(score)

mean value

of animals

(score)

mean value

of animals

(score)

mean value

of animals

(copies/mg)

mean value

of animals

(copies/mg)

mean value

of positive

animals

(%)

mean value

of positive

animals

(%)

Combi PrEP 0,166666667 0,8333333 1,0833333 0,25 10004,884 676,87548 75 91,67

Combi PEP 0 0,6363636 0,6363636 0,0909091 0 1 54,55 72,73

PBS 1,260869565 0 2,375 0,0833333 237312,19 76271,053 18,18 20

Gn3 PrEP 0,666666667 0,5 1,25 0,1666667 70309,362 4254,9373 58,33 58,33

Gn3 PEP 0,333333333 0,4166667 1,5 0,25 169254,89 20407,135 44 77,78

96

Gn Test Sequence peptide AA Gn3 Gn32

>Peptide_01x BXEDPHLRNRPGKGHNYIDGMT1 - 20 0,06 3,7505

>Peptide_02 BXPGKGHNYIDGMTQEDATCKP9 - 28 0,0914 3,9171

>Peptide_03 BXDGMTQEDATCKPVTYAGACS17 - 36 0,0716 0,1183

>Peptide_04x BXTCKPVTYAGACSSFDVLLEK25 - 44 0,0711 0,0506

>Peptide_05 BXGACSSFDVLLEKGKFPLFQS33 - 52 0,0825 0,0713

>Peptide_06 BXLLEKGKFPLFQSYAHHRTLL41 - 60 0,0699 0,064

>Peptide_07x BXLFQSYAHHRTLLEAVHDTII 49 - 68 0,0714 0,054

>Peptide_08 BXRTLLEAVHDTIIAKADPPSC 57 - 76 0,0887 0,0996

>Peptide_09 BXDTIIAKADPPSCDLLSAHGN 65 - 84 0,0484 0,0964

>Peptide_10x BXPPSCDLLSAHGNPCMKEKLV73 - 92 0,0584 0,8369

>Peptide_11 BXAHGNPCMKEKLVMKTHCPND81 - 100 0,0621 0,0744

>Peptide_12 BXEKLVMKTHCPNDYQSAHHLN89 - 108 0,0539 0,1319

>Peptide_13x BXCPNDYQSAHHLNNDGKMASV97 - 116 0,0633 0,0807

>Peptide_14 BXHHLNNDGKMASVKCPPKYEL105 - 124 0,0687 0,0679

>Peptide_15 BXMASVKCPPKYELTEDCNFCR113 - 132 0,0662 0,0983

>Peptide_16x BXKYELTEDCNFCRQMTGASLK121 - 140 0,0509 0,0563

>Peptide_17 BXNFCRQMTGASLKKGSYPLQD129 - 148 0,0462 0,7031

>Peptide_18 BXASLKKGSYPLQDLFCQSSED137 - 156 0,0555 0,0529

>Peptide_19x BXPLQDLFCQSSEDDGSKLKTK145 - 164 0,053 0,3622

>Peptide_20 BXSSEDDGSKLKTKMKGVCEVG153 - 172 0,0529 0,0496

>Peptide_21 BXLKTKMKGVCEVGVQALKKCD161 - 180 0,047 0,059

>Peptide_22x BXCEVGVQALKKCDGQLSTAHE169 - 188 0,0504 0,0574

>Peptide_23 BXKKCDGQLSTAHEVVPFAVFK177 - 196 0,0581 0,0551

>Peptide_24 BXTAHEVVPFAVFKNSKKVYLD185 - 204 0,0488 0,0486

>Peptide_25x BXAVFKNSKKVYLDKLDLKTEE193 - 212 0,0449 0,1138

>Peptide_26 BXVYLDKLDLKTEENLLPDSFV201 - 220 0,0503 0,064

>Peptide_27 BXKTEENLLPDSFVCFEHKGQY209 - 228 0,0576 0,0815

>Peptide_28x BXDSFVCFEHKGQYKGTMDSGQ217 - 236 0,0503 0,0673

>Peptide_29 BXKGQYKGTMDSGQTKRELKSF225 - 244 0,0443 0,0603

>Peptide_30 BXDSGQTKRELKSFDISQCPKI233 - 252 0,0505 0,0734

>Peptide_31x BXLKSFDISQCPKIGGHGSKKC241 - 260 0,0666 0,0807

>Peptide_32 BXCPKIGGHGSKKCTGDAAFCS249 - 268 0,0535 0,1459

>Peptide_33 BXSKKCTGDAAFCSAYECTAQY257 - 276 0,0482 0,0937

>Peptide_34x BXAFCSAYECTAQYANAYCSHA265 - 284 0,0519 0,0697

>Peptide_35 BXTAQYANAYCSHANGSGIVQI273 - 292 0,0559 0,0606

>Peptide_36 BXCSHANGSGIVQIQVSGVWKK281 - 300 0,0535 0,0706

>Peptide_37x BXIVQIQVSGVWKKPLCVGYER289 - 308 0,0535 0,0755

>Peptide_38 BXVWKKPLCVGYERVVVKRELS297 - 316 0,055 0,0757

>Peptide_39 BXGYERVVVKRELSAKPIQRVE305 - 324 0,0534 0,0544

>Peptide_40x BXRELSAKPIQRVEPCTTCITK313 - 332 0,0542 0,0466

>Peptide_41 BXQRVEPCTTCITKCEPHGLVV321 - 340 0,0473 0,0684

>Peptide_42 BXCITKCEPHGLVVRSTGFKIS 329 - 348 0,051 0,7632

>Peptide_43x BXGLVVRSTGFKISSAVACASG337 - 356 0,0527 0,0624

>Peptide_44 BXFKISSAVACASGVCVTGSQS345 - 364 0,0497 0,0805

>Peptide_45 BXCASGVCVTGSQSPSTEITLK353 - 372 0,0464 0,0979

>Peptide_46x BXGSQSPSTEITLKYPGISQSS361 - 380 0,0492 0,0797

>Peptide_47 BXITLKYPGISQSSGGDIGVHM 369 - 388 0,0477 0,0646

>Peptide_48 BXSQSSGGDIGVHMAHDDQSVS377 - 396 0,0596 0,0523

>Peptide_49x BXGVHMAHDDQSVSSKIVAHCP385 - 404 0,0462 0,0775

>Peptide_50 BXQSVSSKIVAHCPPQDPCLVH393 - 412 0,0531 0,083

>Peptide_51 BXAHCPPQDPCLVHDCIVCAHG401 - 420 0,0521 0,0699

>Peptide_52x BXCLVHDCIVCAHGLINYQCHT409 - 428 0,048 0,0712

>Peptide_53 BXCAHGLINYQCHTALSAFVVV417 - 436 0,0478 0,1311

>Peptide_54 BXQCHTALSAFVVVFVFSSIAI 425 - 444 0,0452 0,0692

>Peptide_55x BXFVVVFVFSSIAIICLAILYR 433 - 452 0,0483 0,0812

>Peptide_56 BXSIAIICLAILYRVLKCLKIA 441 - 460 0,0461 0,052

>Peptide_57 BXILYRVLKCLKIAPRKVLNPL 449 - 468 0,0476 0,0987

>Peptide_58x BXLKIAPRKVLNPLMWITAFIR 457 - 476 0,0524 0,0631

>Peptide_59 BXLNPLMWITAFIRWIYKKMVA 465 - 484 0,0484 0,0898

>Peptide_60 BXAFIRWIYKKMVARVADNINQ 473 - 492 0,0471 0,0637

>Peptide_61x BXKMVARVADNINQVNREIGWM481 - 500 0,0454 0,0566

>Peptide_62 BXNINQVNREIGWMEGGQLVLG489 - 508 0,0545 0,5386

>Peptide_63 BXIGWMEGGQLVLGNPAPIPRH497 - 516 0,0515 0,0881

>Peptide_64x BXLVLGNPAPIPRHAPIPRYST 505 - 524 0,0548 0,049

>Peptide_65 BXIPRHAPIPRYSTYLMLLLIV 513 - 532 0,0763 0,0808

>Peptide_66 BXPIPRYSTYLMLLLIVSYASA 518 - 537 0,0991 0,1138

98

Data file S5.3: EC50 and IC50 calculation

Fig.1 ELISA fits:

Gn3 Gn32

value value

npar 4 npar 4

params.bottom 4.14837e-02 params.bottom 4.62708e-02

params.top 9.13609e-01 params.top 9.46424e-01

params.xmid 8.61936e-02 params.xmid -4.27657e-01

params.scal 1.96978e+00 params.scal 1.59518e+00

params.s 1.00000e+00 params.s 1.00000e+00

GOF 9.93557e-01 GOF 9.89457e-01

weightedGOF 9.99893e-01 weightedGOF 9.99814e-01

stdErr 0.0316608 stdErr 0.04005807

weighted stdErr 0.02761195 weighted stdErr 0.03609953

trapezoid 3.06068 trapezoid 3.64066

Simpson 3.02756 Simpson 3.60647

xInfl 0.0861936 xInfl -0.427657

yInfl 0.477546 yInfl 0.496348

Log10(EC50) 0.1089195 Log10(EC50) -0.423238

EC50 1.28505e+00 EC50 3.77365e-01

[95%] [1.12456e+00 | 1.47372e+00] [95%] [3.06354e-01 | 4.65456e-01]

date (Y-m-d) 2019-04-02 date (Y-m-d) 2019-04-02

nplr version 0.1.7 (2016-12-25) nplr version 0.1.7 (2016-12-25)

R version 3.5.2 (2018-12-20) R version 3.5.2 (2018-12-20)

Gn3+Gn32

value

npar 4

params.bottom 3.79946e-02

params.top 9.38683e-01

params.xmid -3.21513e-01

params.scal 1.75559e+00

params.s 1.00000e+00

GOF 9.92697e-01

weightedGOF 9.99881e-01

stdErr 0.0339645

weighted stdErr 0.02937417

trapezoid 3.5009

Simpson 3.46675

xInfl -0.321513

yInfl 0.488339

Log10(EC50) -0.3086988

EC50 4.91248e-01

[95%] [4.20256e-01 | 5.76991e-01]

date (Y-m-d) 2019-04-02

nplr version 0.1.7 (2016-12-25)

R version 3.5.2 (2018-12-20)

99

Fig. 2 SNT fits:

A MP12:

Gn3 Gn3+Gn32

value value

npar 4 npar 4

params.bottom -1.82790e-03 params.bottom -9.60328e-04

params.top 1.01021e+00 params.top 1.01775e+00

params.xmid 1.52787e+00 params.xmid 1.39401e+00



GOF 9.95093e-01 GOF 9.407e-01


stdErr 0.03365182 stdErr 0.1169808




xInfl 1.52787 xInfl 1.39401

yInfl 0.50419 yInfl 0.508393

Log10(IC50) 1.527067 Log10(IC50) 1.391181

IC50 3.36563e+01 IC50 2.4614e+01

[95%] [3.28416e+01 | 3.45e+01] [95%] [2.09436e+01 | 2.87556e+01]

date (Y-m-d) 2019-04-04 date (Y-m-d) 2019-04-04



B

35/74

Gn3 Gn3+Gn32

value value

npar 4 npar 4

params.bottom -1.53276e-03 params.bottom -3.43922e-04

params.top 9.12087e-01 params.top 9.99470e-01

params.xmid 2.15389e+00 params.xmid 2.00262e+00



GOF 6.77302e-01 GOF 5.64376e-01


stdErr 0.2728898 stdErr 0.3170623




xInfl 2.15389 xInfl 2.00262

yInfl 0.455277 yInfl 0.499563

Log10(IC50) 2.167875 Log10(IC50) 2.002748

IC50 1.47189e+02 IC50 1.00635e+02

[95%] [1.05181e+02 | 2.09276e+02] [95%] [6.92891e+01 | 1.45344e+02]

date (Y-m-d) 2019-04-02 date (Y-m-d) 2019-04-02



100

Data used for ELISA: Data used for SNT:

MP12 35/74

Gn3 Gn32 Combi Gn3 Combi Gn3 Combi

µg/ml OD OD ODNeutraliza-

tion (%)µg/ml µg/ml

Neutraliza-

tion (%)µg/ml µg/ml

2000 3,3013999 3,2369001 3,2198999 100 100 100 100 400 400

500 3,2165999 3,2182 3,1703999 100 50 50 100 200 200

100 3,1954 3,233 3,1098001 50 33.33 25 50 133.33 133.33

25 2,9769001 3,1566 3,3176999 0 25 16.67 0 100 100

15 3,2116001 3,1422 3,1801 0 16.67 12.5 0 50 50

5 2,9816 3,1119001 3,0926001 100 100 100 100 400 400

2,5 2,6688001 3,1217 3,0941 100 50 50 100 200 266.67

1 1,5465 2,9709001 2,9073999 50 33.33 25 50 133.33 200

1 1,3078001 2,78 2,2699001 0 25 16.67 0 100 133.33

0,5 0,6269 1,6686 1,7183 0 16.67 12.5 0 50 50

0,25 0,3654 1,2361 0,7812 100 100 100 100 600 200

0,1 0,2065 0,6186 0,3805 100 50 50 100 400 133.33

0,05 0,1409 0,3087 0,2005 50 33.33 16.67 50 266.67 100

0,025 0,1061 0,2076 0,1382 0 25 12.5 0 200 50

0,01 0,1667 0,1294 0,1132 0 16.67 6.25 0 50 25

0,005 0,1714 0,1062 0,1204

2000 3,2727001 3,4187 3,2509

500 3,1407001 3,2237 3,1372001

100 3,0799 3,2038 3,1768

25 2,9381001 3,1747 3,2923

15 3,0179 3,2086 3,2725

5 3,1166 3,1807001 3,0373001

2,5 2,6626999 3,1349001 3,0195999

1 1,3071001 3,062 2,7842

1 1,2315 2,8103001 2,2987001

0,5 0,6618 1,8459001 1,7627

0,25 0,3459 1,2697999 0,841

0,1 0,2064 0,619 0,3863

0,05 0,1251 0,3119 0,2057

0,025 0,0989 0,1957 0,1468

0,01 0,0991 0,1241 0,1109

0,005 0,092 0,1272 0,1058

2000 3,2047999 3,2493999 3,3162999

500 3,0253999 3,1644001 3,1696

100 3,0271001 3,1421001 3,1918001

25 3,0037 3,2117 3,1475999

15 2,9339001 3,1829 3,2690001

5 2,7976 3,0767 3,1136999

2,5 2,6152999 3,0739999 3,0904

1 1,3269 3,0288 2,8611

1 0,9738 2,7855999 2,2744999

0,5 0,6303 1,5979 1,6822

0,25 0,291 1,2410001 0,8876

0,1 0,192 0,6082 0,3742

0,05 0,1262 0,2955 0,199

0,025 0,0992 0,1874 0,1503

0,01 0,092 0,1245 0,1103

0,005 0,09 0,1151 0,104

101

Data File S5.4: Combination index and dose-reduction index

Concentrations used for calculation Concentrations used for calculation Concentrations used for calculation

SNT MP-12 SNT 35/74 ELISA

Dosis(µg/ml) F(a)* Dosis(µg/ml) F(a)* Dosis(µg/ml) F(a)*

Gn3: 25 0.01 Gn3: 133,33 0.01 Gn3: 0,5 0.2

33 0.5 147,2 0.5 1 0.39

50 0.99 177,78 0.99 15 0.99

Gn32: 25 0.01 Gn32: 133,33 0.01 Gn32: 0,5 0.52

33 0.01 147,2 0.01 1 0.88

50 0.01 177,78 0.01 15 0.99

Gn3+Gn32: 15 0.01 Gn3+Gn32: 57,6 0.01 Gn3+Gn32: 0,5 0.53

24,6 0.5 100,6 0.5 1 0.79

50 0.99 144,4 0.99 15 0.99

102

CI Data for Drug Combo: combi (Gn3+Gn32 [1:1]) CI Data for Drug Combo: combi (Gn3+Gn32 [1:1]) CI Data for Drug Combo: combi (Gn3+Gn32 [1:1])

F(a) CI Value Total Dose(µg/ml) F(a) CI Value Total Dose(µg/ml) F(a) CI Value Total Dose(µg/ml)

0.05 NaN* 17,89 0.05 NaN 69,88 0.05 0,81 0,04

0.1 NaN 19,75 0.1 NaN 75,39 0.1 0,81 0,07

0.15 NaN 21,00 0.15 NaN 79,02 0.15 0,81 0,11

0.2 NaN 21,99 0.2 NaN 81,86 0.2 0,81 0,14

0.25 NaN 22,85 0.25 NaN 84,29 0.25 0,81 0,17

0.3 NaN 23,62 0.3 NaN 86,4645 0.3 0,81 0,21

0.35 NaN 24,34 0.35 NaN 88,4915 0.35 0,81 0,25

0.4 NaN 25,04 0.4 NaN 90 0.4 0,81 0,30

0.45 NaN 25,73 0.45 NaN 92,3311 0.45 0,81 0,35

0.5 NaN 26,42 0.5 NaN 94,23 0.5 0,82 0,41

0.55 0,39 27,1354 0.55 0,31 96,17 0.55 0,82 0,48

0.6 0,39 27,8812 0.6 0,32 98,19 0.6 0,82 0,56

0.65 0,40 28,6808 0.65 0,32 100,344 0.65 0,83 0,66

0.7 0,40 29,5608 0.7 0,33 102,696 0.7 0,83 0,79

0.75 0,41 30,5609 0.75 0,34 105,35 0.75 0,84 0,96

0.8 0,41 31,7473 0.8 0,34 108,472 0.8 0,84 1,20

0.85 0,42 33,2456 0.85 0,35 112,377 0.85 0,85 1,58

0.9 0,43 35,3455 0.9 0,36 117,781 0.9 0,87 2,26

0.95 0,45 39,0208 0.95 0,38 127,063 0.95 0,90 4,04

0.97 0,46 41,8664 0.97 0,40 134,11 0.97 0,93 6,11

103

Data file S5.5: Clinical score

DRI Data for Drug Combo: combi (Gn3+Gn32 [1:1]) DRI Data for Drug Combo: combi (Gn3+Gn32 [1:1]) DRI Data for Drug Combo: combi (Gn3+Gn32 [1:1])

F(a) Dose Gn3 Dose Gn32 DRI Gn3 DRI Gn32 F(a) Dose Gn3 Dose Gn32 DRI Gn3 DRI Gn32 F(a) Dose Gn3 Dose Gn32 DRI Gn3 DRI Gn32

0.05 27,59 NaN 3,08379 NaN 0.05 137,90 NaN 3,95 NaN 0.05 0,23 0,03 10,90 1,39

0.1 29,21 NaN 2,95747 NaN 0.1 141 NaN 3,74778 NaN 0.1 0,23 0,03 9,26 1,43

0.15 30,26 NaN 2,88 NaN 0.15 143,402 NaN 3,6297 NaN 0.15 0,23 0,03 8,37 1,46

0.2 31,079 NaN 2,82622 NaN 0.2 145,026 NaN 3,54326 NaN 0.2 0,23 0,03 7,75 1,48

0.25 31,7698 NaN 2,78108 NaN 0.25 146,381 NaN 3,47342 NaN 0.25 0,23 0,03 7,28 1,49

0.3 32,3858 NaN 2,74223 NaN 0.3 147,575 NaN 3,41353 NaN 0.3 0,23 0,03 6,89 1,51

0.35 32,9557 NaN 2,70741 NaN 0.35 148,668 NaN 3,36004 NaN 0.35 0,23 0,03 6,55 1,52

0.4 33,498 NaN 2,67524 NaN 0.4 149,697 NaN 3,31075 NaN 0.4 0,23 0,03 6,26 1,54

0.45 34,0263 NaN 2,64475 NaN 0.45 150,692 NaN 3,26416 NaN 0.45 1,05 0,03 5,98 1,55

0.5 34,5521 NaN 2,61521 NaN 0.5 151,672 NaN 3,21914 NaN 0.5 1,05 0,03 5,72 1,56

0.55 35,086 Infinit 2,58599 Infinit 0.55 152,659 Infinit 3,17475 Infinit 0.55 1,05 0,03 5,48 1,57










*NaN = Not a Number (Value could not be calculated)

104

Treatment Animal number 1 2 3 4 5 6 7 8 9 10 11 12 13

25 0 0 8

26 0 0 0 0 0 8

27 0 0 8

28 0 0 0 0 8

29 0 0 0 0 0 0 0 0 0 0 0 0 0

30 0 0 0 0 0 0 0 8

31 0 0 0 0 0 8

32 0 0 0 0 0 8*

33 0 0 0 0 0 0 0 0 0 0 0 0 0

34 0 0 0 0 0 8*

35 0 0 8

36 0 0 8

73 0 0 0 8

74 0 0 8*

75 0 0 0 0 0 0 0 0 0 0 0 0 0

76 0 0 0 8

77 0 0 8*

78 0 0 0 0 0 0 0 0 0 0 0 0 0

79 0 0 0 8*

80 0 0 0 0 0 0 8

81 0 0 8*

82 0 0 0 0 0 0 0 8

83 0 0 8*

84 0 0 0 0 0 0 8*

number of

surviving animals24 24 24 17 13 12 8 6 4 4 4 4 4

total score 0 0 32 16 8 16 16 16 0 0 0 0 0

mean score 0 0 1,3 0,9 0,6 1,3 2 2,7 0 0 0 0 0

cumulative score 0 0 2,3 4,1 4,7 7,4 9,4 12,1 12,1 12,1 12,1 12,1 12,1

* found dead days post inoculation

PBS

105


97 0 0 0 0 0 0 0 0 0 0 0 0 0

98 0 0 0 0 0 0 0 0 0 0 0 0 0

99 0 0 0 0 0 0 0 0 0 0 0 0 0

100 0 0 0 0 0 0 0 8

101 0 0 0 0 0 0 0 0 0 0 0 0 0

102 0 0 0 0 0 0 0 0 0 0 0 0 0

103 0 0 0 8*

104 0 0 0 0 8*

105 0 0 0 0 0 8

106 0 0 0 0 0 0 0 0 0 0 0 0 0

107 0 0 0 0 0 0 0 0 0 0 0 0 0

108 0 0 8

number of


total score 0 0 8 8 8 8 0 8 0 0 0 0 0

mean score 0 0 0,7 0,7 0,8 0,9 0 1 0 0 0 0 0

cumulative score 0 0 0,7 1,4 2,2 3,1 3,1 4,1 4,1 4,1 4,1 4,1 4,1

85 0 0 0 0 0 0 0 0 0 0 0 0 0

86 0 0 0 0 0 0 0 0 0 0 0 0 0

87 0 0 0 0 0 0 0 0 0 0 0 0 0

88 0 0 0 0 0 0 0 0 0 0 0 0 0

89 0 0 0 0 0 0 0 0 0 0 0 0 0

90 0 0 0 0 0 0 0 0 0 0 0 0 0

91 0 0 0 0 0 0 0 0 0 0 0 0 0

92 0 0 0 8*

93 0 0 0 8

94 0 0 0 8*

95 0 0 0 0 0 8

96 0 0 0 8*

number of


total score 0 0 0 32 0 8 0 0 0 0 0 0 0

mean score 0 0 0 2,7 0 1 0 0 0 0 0 0 0

cumulative score 0 0 0 2,7 2,7 3,7 3,7 3,7 3,7 3,7 3,7 3,7 3,7


Gn3 PrEP

Gn3 PEP

106


1 0 0 0 0 2 8

2 0 0 0 0 0 2 0 0 0 0 0 0 0

3 0 0 0 0 0 0 0 0 0 0 0 0 0

4 0 0 0 0 0 0 0 0 0 0 0 0 0

5 0 0 0 0 0 0 0 0 0 0 0 0 0

6 0 0 0 0 0 8

7 0 0 0 0 0 0 0 0 0 0 0 0 0

8 0 0 0 0 0 0 0 0 0 0 0 0 0

9 0 0 0 0 0 0 0 0 0 0 0 0 0

10 0 0 0 0 0 0 0 0 0 0 0 0 0

11 0 0 0 0 0 0 0 0 0 0 0 0 0

12 0 0 0 0 0 0 0 0 0 0 0 0 0

number of


total score 0 0 0 0 2 18 0 0 0 0 0 0 0

mean score 0 0 0 0 0,2 1,5 0 0 0 0 0 0 0

cumulative score 0 0 0 0 0,2 1,7 1,7 1,7 1,7 1,7 1,7 1,7 1,7

13 0 0 0 0 0 0 0 0 0 0 0 0 0

14 0 0 0 0 0 0 0 0 0 0 0 0 0

16 0 0 0 0 0 0 0 0 0 0 0 0 0

17 0 0 0 0 0 0 0 0 0 0 0 0 0

18 0 0 0 0 0 3 0 0 0 0 0 0 0

19 0 0 0 0 0 0 0 0 0 0 0 0 0

20 0 0 0 0 0 0 0 0 0 0 0 0 0

21 0 0 0 0 0 0 0 0 0 0 0 0 0

22 0 0 0 0 0 0 0 0 0 0 0 0 0

23 0 0 0 0 0 0 0 0 0 0 0 0 0

24 0 0 0 0 0 0 0 0 0 0 0 0 0

number of


total score 0 0 0 0 0 3 0 0 0 0 0 0 0

mean score 0 0 0 0 0,0 0,25 0 0 0 0 0 0 0

cumulative score 0 0 0 0 0 0,25 0,25 0,25 0,25 0,25 0,25 0,25 0,25


Gn3+Gn32

combi

PrEP

Gn3+Gn32

combi PEP

107

Data file S5.6: PCR, serology, histology


67 0 0 0 0 0 0 0 0 0 0 0 0 0

68 0 0 0 0 0 0 0 0 0 0 0 0 0

69 0 0 0 0 0 0 0 0 0 0 0 0 0

70 0 0 0 0 0 0 0 0 0 0 0 0 0

71 0 0 0 0 0 0 0 0 0 0 0 0 0

72 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 00 0 0 0 0 0

0 0 0 0 0

cumulative score 0 0 0 0

0

mean score 0 0 0 0 0 0 0 0

0 0 0 0 0 00 0 0 0 0 0


mAb

controltotal score

108

Treatment Animal number necropsy (dpi) cruor liver brain cruor liver brain loung spleen liver brainlymphoid

depletion

follicular

hyperplasiahepatitis encephalitis

25 3 225000 164507,04 25 56234,133 3.162.278 3.162 100,18518 <10 1 2 3 0 2 0 3 0

26 6 8930 179693,25 10,906897 5623,4133 1000000 316 80,096296 <10 2 2 3 0 2 0 3 0

27 3 181000 273770,95 2609,6654 31622,777 562.341 1.778 n.t. n.t. 0 1 3 0 1 0 3 0

28 5 84100 277087,38 567,90123 5623,4133 562.341 316 105,31852 <10 0 n.t. 3 0 n.t. n.t. 3 0

29 13 296 53,240741 33,098592 0 0 0 93,429626 <10 0 0 0 0 0 0 0 0

30 8 53,8 532,12435 285000 0 0 3.162.278 23,570369 10 0 1 0 2 2 0 2 1

31 6 219 39563,91 182,30769 10000 5.623 3.162 23,62963 n.t. 0 0 3 0 2 0 3 0

32 6 n.t. 127480,62 7,5418502 n.t. 100.000 3.162 n.t. n.t. 0 0 3 0 1 0 3 0

33 13 0 9,5248619 0,9293785 0 0 0 101,34814 <10 0 0 0 0 0 0 1 0

34 6 2120 188626,37 244,66667 178 0 6.761 0 0 3 0 2 0 3 0

35 3 54800 931658,29 977,77778 562341,33 1.778.279 1.778 100,42963 <10 0 1 3 0 1 0 3 0

36 3 37700 21286,486 228,56089 6.761 316227,766 5.623 n.t. n.t. 0 2 3 0 1 0 3 0

73 4 115000 398192,77 1721,0811 100000 1000000 316 103,40741 <10 2 2 3 0 2 0 3 0

74 3 17600 697538,46 199,55128 147910,84 1000000 676 102,79259 <10 0 0 3 0 1 0 3 0

75 13 69,6 125,26066 0 0 0 0 95,911113 <10 0 0 0 0 0 0 0 0

76 4 481000 171932,2 1847,619 56234,133 1.778.279 676 104,05185 <10 0 1 3 0 2 0 3 0

77 3 246000 240959,6 1189,1892 316227,77 10.000 562 103,37777 n.t. 0 1 3 0 1 0 3 0

78 13 98,5 602 0 0 1.000 0 103,57778 <10 0 0 0 0 0 0 0 0

79 4 n.t. 794491,53 242,69896 n.t. 1.778 100 n.t. n.t. 1 2 3 0 2 0 3 0

80 7 854 2336,6812 12,724638 178 0 1.778 20,31111 10 0 0 2 0 1 0 3 1

81 3 139000 907602,34 51,727941 562 31622,7766 316 111,54074 n.t. 0 0 3 0 2 0 3 0

82 8 60,5 14,454887 96,2 0 0 562 36,459258 30 0 0 0 0 0 0 0 0

83 4 8710 273369,57 45,451613 31622,777 63.096 1.000 87,703703 <10 0 2 3 0 2 0 3 0

84 7 n.t. 4058,5253 286,60969 n.t. 0 3.162 n.t. n.t. 0 0 3 0 2 0 3 0

97 13 0 11,671674 7,0146628 0 0 0 22,244444 <10 0 0 0 0 0 1 0 0

98 13 0 26,033994 2,1759082 0 0 0 30,37037 30 0 0 0 0 0 2 1 0

99 13 0 3,597973 0 0 0 0 12,296296 20 0 0 0 0 0 1 1 0

100 8 0 1,8369369 0 0 0 0 47,733333 15 0 0 0 0 0 1 1 0

101 13 0 0,843787 12,513514 0 0 0 24,222221 20 0 0 0 1 0 1 0 2

102 13 0 1,3431611 0 0 0 0 18,688888 30 0 0 0 0 0 0 0 0

103 4 17395,129 46265,06 8,993576 10000 630.957 3.162 101,73333 <10 1 3 3 0 3 0 3 0

104 5 34349,65 407333,33 18,493298 3162,2777 630.957 1.778 100,84444 <10 1 2 3 0 2 0 3 0

105 6 760,93613 87296,296 28,38125 0 31622,7766 562 30,962963 10 0 0 3 0 2 0 3 0

106 13 0 4,2072072 21,482412 0 0,0 56,2 23,777778 15 0 0 0 0 0 0 0 0

107 13 0 8,1245791 0 0 0,0 0 104,73333 <10 0 0 0 0 0 0 0 0

108 3 4,3817134 302760 360,92843 3162,2777 630.957 1.778 114,21482 <10 0 1 3 0 1 0 3 0

IHC (NP antigen) HistopathologyPCR values [copies/mg] Serum

neutralisation

test [titer]

ELISA

[S/N%]

PBS

Gn3 PrEP

Virustitration [TCID50/ml]

negative

positive

borderline

n.t. not tested

109

Data file S5.7: Daily blood samples

Treatment Animal number necropsy (dpi) cruor liver brain cruor liver brain loung spleen liver brainlymphoid

depletion

follicular

hyperplasiahepatitis encephalitis

85 13 0 1,0412451 48,680352 0 0 562 45,133334 30 0 0 0 0 0 0 0 0

86 13 0 0 291,68831 0 0 56 24,837036 15 0 0 0 0 0 0 0 1

87 13 0 0 11,159664 0 0 0 17,940741 15 0 0 0 1 0 2 0 2

88 13 0 0 0 0 0 0 48,399999 10 0 0 0 0 0 0 1 0

89 13 0 0 12,703226 0 0 177,8 30,096296 15 0 0 0 0 0 2 1 0

90 13 0 0 0 0 0 0 45,044443 30 0 0 0 0 0 1 1 0

91 13 0 2,7486577 0 0 0 0 33,148147 20 0 0 0 0 0 0 0 0

92 4 161841,09 37251,082 389,16479 31622,777 100.000 1.778 n.t. n.t. 1 2 3 0 0 0 3 0

93 4 39177,168 60487,805 151,12412 3162,2777 316227,766 562 108,34074 <10 0 2 3 0 1 0 3 0

94 4 n.t. 296341,46 64,387352 n.t. 316227,766 177,8 n.t. n.t. 1 2 3 0 1 0 3 0

95 6 3053,1008 1063698,6 96,041667 31622,777 1.995.262 316 87,837032 <10 1 3 3 0 2 0 3 0

96 4 n.t. 573275,86 702,11268 n.t. 63.096 1.000 n.t. n.t. 1 2 3 0 0 0 3 0

1 6 6709,8943 59025,07 44,893451 562.341 316227,77 10000 61,762963 <10 0 0 3 0 0 0 3 0

2 13 18,4 25,757692 2,1017467 0 0 0 23,251851 10 0 0 0 0 0 1 1 0

3 13 0 3,1459283 150,04587 0 0 100 40,614816 60 0 0 0 0 0 1 1 2

4 13 0 0 0 0 0 0 33,933332 20 0 0 0 0 0 1 1 0

5 13 0 0 8,3553114 0 0 0 98,266662 10 0 0 0 0 0 0 0 0

6 6 1390 61000 20,984375 316,22777 31.623 0 33,362962 15 0 2 3 1 2 0 3 0

7 13 0 4,6349206 6,9893048 0 0 0 36,459258 40 0 0 0 0 0 1 0 1

8 13 0 0 0 0 0 0 23,696296 20 0 0 0 0 0 1 1 0

9 13 0 0 0,5830769 0 0 0 33,703704 45 0 0 0 0 0 1 1 0

10 13 0 0 0 0 0 0 30,533333 30 0 0 0 0 0 2 1 0

11 13 0 0 80,535714 0 0 0 30,333333 20 0 0 0 0 0 1 1 0

12 13 0 0 0 0 0 0 38,074073 20 0 0 0 0 0 1 0 0

13 13 8,6308411 0 0 0 0 0 44,755555 20 0 0 0 0 0 1 0 0

14 13 0 0 0 0 0 0 99,407406 <10 0 0 0 0 0 0 0 0

16 13 0 0 1,3382353 0 0 0 21,925926 15 0 0 1 0 0 1 2 0

17 13 0 0 0 0 0 0 94,288886 <10 0 0 0 0 0 0 0 0

18 13 0 0 4,5 0 0 0 22,148147 40 0 0 0 1 0 0 1 1

19 13 0 0 0 0 0 0 89,533327 15 0 0 0 0 0 0 0 0

20 13 0 0 0 0 0 0 27,118519 40 0 0 0 0 0 1 1 0

21 13 0 0 13,930876 0 0 0 26,422222 30 0 0 0 0 0 2 1 0

22 13 0 0 0 0 0 0 104,63704 <10 0 0 0 0 0 0 1 0

23 13 0 0 0 0 0 0 24,585185 30 0 0 0 0 0 1 1 0

24 13 0 0 0 0 0 0 22,799999 15 0 0 0 0 0 1 0 0

IHC (NP antigen) Histopathology

Gn3+Gn32

combi PEP

group

PCR values [copies/mg] Serum

neutralisation

test [titer]

ELISA

[S/N%]

Gn3 PEP

Gn3+Gn32

combi PrEP

Virustitration [TCID50/ml]

negative

positive

borderline

n.t. not tested

110

VT= virus titration

111

negative positive not tested

Animal

number

Necropsied

dpi1dpi 2dpi 3dpi 4dpi 5dpi 6dpi 7dpi 8dpi 9dpi

25 3 0

26 6 0 2,3(5)

27 3 0

28 5 0

29 13 0 1,75(3,25)

30 8 3,24(5) 1,34(1,75)

31 6

32 6

33 13 1,54(0) 0

34 6

35 3

36 3

73 4 0

74 3 0

75 13 0 0

76 4 0,93(1,75)

77 3 1,14(4,25)

78 13 0 0

79 4 3,96(5,25)

80 7 5(4,25)

81 3 5,15(3,75)

82 8 2,66(0)

83 4 5,14(3)

84 7 3,92(3,25)

85 13 0 0 0

86 13 0 0 0

87 13 0 0

88 13 0 0

89 13 0 0

90 13 0

91 13 0 0

92 4 5,77(5,5)

93 4 3,84(4)

94 4

95 6 1,44(0)

96 4

97 13 0 0 0

98 13 0 0 0

99 13 0 0 0

100 8 0 0

101 13 0 0

102 13 0 0

103 4 3,49(4)

104 5 2,33(4)

105 6 3,51(3)

106 13 1,91(2)

107 13 0

108 3

1 6 3,95(5,25)

2 13 2,84(3,75) 0

3 13 0 0

4 13 0,82(0)

5 13 0 0

6 6 0

7 13 0 0

8 13 0 0

9 13 0 0

10 13 0

11 13 0,39(1,75)

12 13 0,4(0)

13 13 2,38(2,75) 0,14(0)

14 13 0 0

16 13 0

17 13 0 0

18 13 1,62(2,5)

19 13 0 0

20 13 0,25(0) 0

21 13 0 0

22 13 0

23 13 0,93(1,75)

24 13 0

Gn3+Gn32

combi PEP

Gn3+Gn32

combi PrEP

PBS

Gn3 PrEP

Gn3 PEP

112

12 AUTHORS´ CONTRIBUTION

Benjamin Gutjahr:

Manuscript I:

Establishment of the complete protocol for single domain antibody generation against

RVFV at the FLI for the first time using published protocols as template.

Characterization of the generated antibodies including sequence analysis, Western

blot, IIFA (under BSL-2 conditions) and SNT (BSL-3 conditions). Data analysis (e. g.

EC50 calculation) and first draft of the manuscript.

Manuscript II:

In vitro characterization (e. g. antibody purification, ELISA and SNT [under BSL-2 or

BSL3 conditions respectively] for identifying cooperative effects and EC50 / IC50

calculations of antibodies). Planning and realization of animal trials (e. g. virus

propagation, mouse infection i. p. and antibody administration i. v., checking health

status twice daily, daily blood sampling and dissection of mice; all under BSL-3

conditions). Analysis of animal samples (e. g. tissue preparation and RNA extraction

with following RT-qPCR, SNT and ELISA under BSL-3 conditions) and pathological

preparation (support in tissue preparation for histological and IHC analysis;

prescreening of histological and IHC slices, final readings by Reiner Ulrich).

Furthermore, data analysis (e. g. statistical analysis, calculations of CI and DRI) and

writing first draft of the manuscript.

Manuscript III:

Hybridoma cell selection after cell fusions of MP-12 immunized mice. Immunization of

mice with virulent 35 / 74, followed by generation of fusions and hybridoma cell

selection (under BSL-3 conditions). Screening of all generated hybridoma cell

supernatants with IIFA and following SNT analysis. ELISA and Western blot assays

and writing the first draft of the manuscript.

Martin H. Groschup and Martin Eiden supervised all studies scientificly.

Markus Keller supported me in molecular biological methodologies and in the

realization of the animal trials.

113

Melanie Rissmann introduced me in BSL-3 working, showed me practical methodology

of IIFA and SNT and gave support in the animal trials.

Sven Reiche introduced me into the practical methodology for hybridoma cell and

following mAb generation and purification methodologies. Claudia Mroz prepared

fusions of MP-12 immunized myeloma cells for mAb generation.

Felicitas von Arnim supported me in animal trials and tissue preparation.

114

13 ACKNOWLEDGMENTS

First of all, I would like to thank Prof. Dr. M. H. Groschup and Dr. Martin Eiden for their supervision and support during my time as doctoral student. I am grateful for the opportunity to work at the Institute of Novel and Emerging Infectious Diseases and for being a part of this unique project. I always appreciated your trust in me and my work. I would like to thank both of you for all constructive discussions, helpful suggestions and encouraging words during the last years and for revising my thesis and the contributing manuscripts, especially in the last weeks, when time was running out.

Furthermore, I would like to thank IMI-ZAPI and all involved colleagues, especially the team of Jeroen Kortekaas for the support, fruitful discussions and inspirations.

Moreover, I would like to thank Markus Keller for coordinating the project and it´s belonging animal trials.

Great thanks to Dr. Sven Reiche and his lab for their great support in generating monoclonal antibodies and sharing their experiences with me and to Dr. Reiner Ulrich and his lab for all the valuable advices and technical support in terms of pathology.

A special thank goes to Dr. Melanie Rissmann, who introduced me to all important methods regarding to RVFV in the laboratory, but also in the stable. For all your advices, fruitful discussions and helping hand, whenever it was needed during the last three years.

Moreover, I would like to thank the ZAPI animal trial team for an interesting and enjoyable time no matter how exhausting days could have been. Furthermore, I am very grateful for Bärbel Hammerschmidt and all animal keepers´ help that made all the animal experiments possible.

Special thanks are also warranted to the whole team of the lab of Dr. Martin Eiden and many other colleagues for great three years and all the fun we had; additionally to Birke Böttcher and Martina Abs for the excellent technical support.

My very special thanks go to Dr. Melanie Rissmann and my brother for proofreading my thesis in a very short time.

But most of all I would like to thank my mother, father, brother and grandparents, my girlfriend Laura and her family and finally my best friend Sascha for your unlimited optimism and encouragement through all the years. I would not have come so far without your outstanding support at all times - thanks for always being there!

115

Erklärung nach § 6 Abs. 2 Nr. 7

Versicherung an Eides statt

Hiermit versichere ich an Eides statt, dass ich die Dissertation:

“Generation and characterization of antibodies against Rift Valley fever phlebovirus

and evaluation of their therapeutic efficacy in a mouse model”

selbstständig verfasst habe. Ich habe keine entgeltliche Hilfe von Vermittlungs- bzw.

Beratungsdiensten (Promotionsberater oder anderer Personen) in Anspruch

genommen. Niemand hat von mir unmittelbar oder mittelbar entgeltliche Leistungen

für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten

Dissertation stehen.

Ich habe die Dissertation an folgenden Institutionen angefertigt:

Institut für neue und neuartige Tierseuchenerreger

Friedrich-Loeffler-Institut

Bundesforschungsinstitut für Tiergesundheit

Greifswald – Insel Riems

Die Dissertation wurde bisher nicht für eine Prüfung oder Promotion oder für einen

ähnlichen Zweck zur Beurteilung eingereicht.

Ich erkläre, über die Bedeutung der Versicherung an Eides statt informiert worden zu

sein. Mir wurde der Inhalt der folgenden Vorschriften des Strafgesetzbuches bekannt

gegeben: § 156 StGb – Falsche Versicherung an Eides statt.

Benjamin Gutjahr

University of Veterinary Medicine Hannover

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