NEWSLETTER ISSUE 1 - OCTOBER 2015 NEWSLETTER ISSUE 1 - OCTOBER 2015 This project is supported...

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ISSUE 1 - OCTOBER 2015

www.pathco.org

Dear colleagues, dear friends and supporters of PathCo,

We are pleased to present you the first issue of the Newsletter of Pathogen COinfection: HIV, Tuberculosis, Malaria and Hepatitis C virus - PathCo - Project.

PathCo Newsletter will be filled with interesting information mainly for all groups external to the PathCo consortium that may have an interest in our research and progress.

Please don't hesitate to forward this mail to anyone who could also be interested in reading it. If they want toreceive their own Newsletter in the future they can write at [email protected]. If you're not interested in receivingour Newsletter anymore, you can unsubscribe via mail.

In order to contribute to the contents of the Newsletter, please send news, photos and other material relatedto PathCo areas of research at [email protected]. We hope you will enjoy reading our latest news.

Best regards,The PatchCo management team

This project is supported through Coordination Theme 1 (Health) of the European Community's FP7. Grant agreement number HEALTH-F3-2012-305578

The PathCo Project

PathCo addresses the challenge of a Seventh Framework Programme 2012 Call project on Co-infection ofHIV/AIDS, malaria, tuberculosis and/or hepatitis. The project officially kicked off November 18th, 2012. PathCoproject will aid to improve our understanding of pathogen co-infection effect(s) on host innate and adaptive immune responses and to develop new approaches to dissect pathogen interactions.

The PathCo project brings together powerful multidisciplinary technologies that will improve our understanding of the complex interactions between infectious agents and the host immuneresponse that will significantly improve the management of co-infection associated disease.

Recent developments in each of the disease disciplines enable the design of model systems that support pathogen co-infection, highlighting the timeliness of PathCo’s mission to study the biological and immunological consequences of co-infection(s).

The PathCo consortium consists of ten beneficiaries from five EU countries (The Netherlands, UK, France,Germany and Italy) and one beneficiary from non-EU countries (South Africa).

PathCo team is a well-balanced team of immunologists, virologists, clinicians, statisticians, epidemiologists withexpertise in HIV/AIDS, TB, malaria and hepatitis C infection research and has the most appropriate scientific andtechnical background as well as the animal models and instrumentation required to fulfill the goals of this projectand to succeed in its mission. PathCo beneficiaries will have access to well-characterised established cohorts ofco-infected patients from different geographical locations. Given the widespread global nature of the pathogensunder study it is imperative that we are not biased in our selection of patients.

The consortium is co-ordinated by the University of Liverpool (Dr. Bill Paxton, Project Coordinator) and will receive over €5.9 milion funding over 5 years from 1st November 2012.

The list of PathCo beneficiaries and more information on the project can be found on the website (www.pathco.org)

NEWSLETTER ISSUE 1 - OCTOBER 2015

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PathCo Project Second Annual Meeting

On the 18th, 19th and 20th of May 2015, the Second Annual Meeting of PathCo Project was held inRome (Italy). PathCo Second Annual Meeting has been organized only for the partners of the project and members of all beneficiary institutions attended the meeting in Rome.

The meeting was opened by the welcome addressof the Project Coordinator (Bill Paxton, Universityof Liverpool) that also underlined the main aimsand expectations of the PathCo Project. ProjectCoordinator’s talk was followed by the presentations of the scientific work packages(WPs) by WP Leaders. WP presentations were focused on the main successes, issues and challenges obtained during the third year of theproject as well as on most activities and objectivesforeseen for the next period of reference.

The PathCo scientific and financial reportingaspects were also revised and discussed during the meeting. Finally, the dissemination and training activities as well as the strategic tools to be realizedto progressively improve the visibility of the projectwere discussed and agreed by the PathCo Consortium members.

The second PathCo annual meeting was closed by the report of the PathCo Scientific and Ethical Advisory Board members focused on main strengths and weaknesses of the project and giving crucial suggestions to PathCo Consortium members on how optimize resources and efforts during the fourth year

of the project.

During the second day a joint meeting was heldbetween PathCo and PEACHI projects bothfocused on HCV, HIV-1 and co-infection. The goal of the Prevention of Hepatitis CVirus (HCV) and HIV-1 Co-Infections -PEACHI – project (www.peachi.eu) is to develop simple, affordable and effective vaccinestrategies that can be given alone or in combination to prevent hepatitis C virus (HCV),human immunodeficiency virus type 1 (HIV-1) andco-infection.

PROJECT NEWS

PathCo Consortium in Rome

PathCo-PEACHI joint meeting

http://www.peachi.eu/



www.pathco.org



Innate and adaptive immune responses in HCV infections.Heim MH, Thimme R J Hepatol. 2014 Nov;61(1 Suppl): S14-25

Hepatitis C virus has been identified a quarter of a decade ago as a leading cause ofchronic viral hepatitis that can lead to cirrhosis and hepatocellular carcinoma. Onlya minority of patients can clear the virus spontaneously during acute infection. Elimination of HCV during acute infection correlates with a rapid induction ofinnate, especially interferon (IFN) induced genes, and a delayed induction of adaptive immune responses. However, the majority of patients is unable to clearthe virus and develops viral persistence in face of an ongoing innate and adaptive immune response.

The virus has developed several strategies to escape these immune responses.For example, to escape innate immunity, the HCV NS3/4A protease can efficientlycleave and inactivate two important signalling molecules in the sensory pathways that react toHCV pathogen-associated molecular patterns (PAMPs) to induce IFNs, i.e., the mitochondrial anti-viralsignalling protein (MAVS) and the Toll-IL-1 receptor-domain containing adaptor-inducing IFN-β (TRIF). Despitethese escape mechanisms, IFN-stimulated genes (ISGs) are induced in a large proportion of patients with chronicinfection. Of note, chronically HCV infected patients with constitutive IFN-stimulated gene (ISG) expression havea poor response to treatment with pegylated IFN-α (PegIFN-α) and ribavirin. The mechanisms that protect HCVfrom IFN-mediated innate immune reactions are not entirely understood, but might involve blockade of ISG protein translation at the ribosome, localization of viral replication to cell compartments that are not accessibleto anti-viral IFN-stimulated effector systems, or direct antagonism of effector systems by viral proteins. Escapefrom adaptive immune responses can be achieved by emergence of viral escape mutations that avoid recognitionby antibodies and T cells. In addition, chronic infection is characterized by the presence of functionally and phenotypically altered NK and T cell responses that are unable to clear the virus but most likely contribute tothe ongoing liver disease.

In this review, the authors will summarize current knowledge about the role of innate and adaptive immune responses in determining the outcome of HCV infection.

Open Access publications produced by PathCo Project

Innate and adaptive immune responses in HCV infections

Markus H. Heim1,2,⇑, Robert Thimme3,⇑

1Division of Gastroenterology and Hepatology, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland; 2Department of

Biomedicine, University of Basel, Hebelstrasse 20, 4031 Basel, Switzerland; 3Department of Medicine, Clinic for Gastroenterology,

Hepatology, Endocrinology, Infectious Diseases, University Hospital Freiburg, Freiburg, Germany

SummaryHepatitis C virus has been identified a quarter of a decade ago as a

leading cause of chronic viral hepatitis that can lead to cirrhosis

and hepatocellular carcinoma. Only a minority of patients can

clear the virus spontaneously during acute infection. Elimination

of HCV during acute infection correlates with a rapid induction of

innate, especially interferon (IFN) induced genes, and a delayed

induction of adaptive immune responses. However, the majority

of patients is unable to clear the virus and develops viral persis-

tence in face of an ongoing innate and adaptive immune

response. The virus has developed several strategies to escape

these immune responses. For example, to escape innate

immunity, the HCV NS3/4A protease can efficiently cleave and

inactivate two important signalling molecules in the sensory

pathways that react to HCV pathogen-associated molecular pat-

terns (PAMPs) to induce IFNs, i.e., the mitochondrial anti-viral

signalling protein (MAVS) and the Toll-IL-1 receptor-domain-

containing adaptor-inducing IFN-b (TRIF). Despite these escape

mechanisms, IFN-stimulated genes (ISGs) are induced in a large

proportion of patients with chronic infection. Of note, chronically

HCV infected patients with constitutive IFN-stimulated gene

(ISG) expression have a poor response to treatment with pegylat-

ed IFN-a (PegIFN-a) and ribavirin. The mechanisms that protect

HCV from IFN-mediated innate immune reactions are not entirely

understood, but might involve blockade of ISG protein translation

at the ribosome, localization of viral replication to cell compart-

ments that are not accessible to anti-viral IFN-stimulated effector

systems, or direct antagonism of effector systems by viral

proteins. Escape from adaptive immune responses can be

achieved by emergence of viral escape mutations that avoid rec-

ognition by antibodies and T cells. In addition, chronic infection is

characterized by the presence of functionally and phenotypically

altered NK and T cell responses that are unable to clear the virus

but most likely contribute to the ongoing liver disease. In this

review, we will summarize current knowledge about the role of

innate and adaptive immune responses in determining the out-

come of HCV infection.

� 2014 European Association for the Study of the Liver. Published

by Elsevier B.V. All rights reserved.IntroductionHepatitis C virusHepatitis C virus (HCV) infects 130 to 170 million persons world-

wide [1]. HCV is parenterally transmitted, mainly due to injection

drug use and unsafe transfusions and therapeutic injections [2].

Acute HCV infections (AHC) are often oligo- or asymptomatic

[3]. In 70–80% of those infected, the virus persists and the infec-

tion becomes chronic. Spontaneous clearance of HCV is rare in

the chronic phase of the infection. In most patients, chronic hep-

atitis C (CHC) leads to some degree of liver fibrosis, and in 15–25%

of patients cirrhosis develops after 10 to 40 years [4]. Patients

with CHC and cirrhosis are at increased risk for liver failure and

for developing hepatocellular carcinoma [5].

Innate immunity and interferons

Innate immune responses are the first line of defence against

viral infections and interferons (IFNs) are the central cytokines

responsible for the induction of an antiviral state in cells and

for the activation and regulation of the cellular components of

innate immunity, such as natural killer (NK) cells [6]. Type I IFNs

(comprising several IFN-a and one IFN-b) and type III IFNs (IFN-

k1, -k2, and -k3; also designated IL29, IL28A, and IL28B) are pro-

duced by cells infected with viruses and by key sentinel cells of

the innate immune system: macrophages and dendritic cells

(DCs). Importantly, macrophages and DCs do not have to be

infected by viruses in order to produce IFNs. Instead, they

constantly sample material from the outside, including virus

Journal of Hepatology 2014 vol. 61 j S14–S25

Basic

Keywords: Interferon; Hepatitis C virus; Innate immunity; Jak-STAT; CD8+ T cells;

T cell exhaustion; Viral escape.

Received 28 May 2014; received in revised form 29 June 2014; accepted 30 June 2014

⇑ Addresses: Department of Biomedicine, University of Basel, Zentrum für Lehre

und Forschung, Hebelstrasse 20, 4031 Basel, Switzerland. Tel.: +41 61 265 33 62;

fax: +41 61 265 38 47 (M.H. Heim). Department of Medicine II, University

Hospital Freiburg, Hugstetter Str. 55, Freiburg 79106, Germany (R. Thimme).

E-mail addresses: [email protected] (M.H. Heim), robert.thimme@

uniklinik-freiburg.de (R. Thimme).

Abbreviations: AHC, acute hepatitis C; CHC, chronic hepatitis C; HCV, hepatitis C

virus; IFN, interferon; IRF, interferon regulatory factor; ISG, interferon-stimulated

gene; MAVS, mitochondrial anti-viral signalling protein; Mda5, melanoma diffe-

rentiation antigen 5; NK cells, natural killer cells; PAMP, pathogen-associated

molecular patterns; PBMC, peripheral blood mononuclear cell; PHH, primary

human hepatocytes; PIAS, protein inhibitor of activated STAT; RIG-1, retinoic acid

inducible gene-1; TLR, toll like receptor; USP18, ubiquitin specific peptidase 18.

Journal of Hepatology Update: Hepatitis C

The vaccines are based on novel and powerful viral vectors for in vivo delivery of antigens. PEACHI Consortiumconsists of cutting edge clinical and scientific expertise, partnered with industry. In the last two years PEACHIConsortium members have employed viral vectored technology to develop the most immunogenic HIV-1 andHCV vaccines described to date.

The joint meeting included several lectures on subjects related to HCV/HIV-1 co-infection biology, animal models for HCV/HIV co-infections and new HCV vaccines in HIV-1 infected individuals. More important, lots ofdiscussions amongst PathCo and PEACHI partners have taken place during the whole joint meeting.

http://www.pathco.org/documents/publications/Heim_MH_and_Thimme_R_2014_J_Hepatology.pdf





www.pathco.org



Colorectal Mucus Binds DC-SIGN and Inhibits HIV-1 Trans-Infection of CD4+ T-Lymphocytes.Stax MJ, Mouser EE, van Montfort T, Sanders RW, de Vries HJ, Dekker HL, Herrera C,Speijer D, Pollakis G, Paxton WA. PLoS One. 2015 Mar 20;10(3):e0122020

Bodily secretions, including breast milk and semen, contain factors that modulateHIV-1 infection.Since anal intercourse caries one of the highest risks for HIV-1 transmission, ouraim was to determine whether colorectal mucus (CM) also contains factors interfering with HIV-1 infection and replication. CM from a number of individualswas collected and tested for the capacity to bind DC-SIGN and inhibit HIV-1cis- or trans-infection of CD4+ T-lymphocytes.

To this end, a DC-SIGN binding ELISA, a gp140 trimer competition ELISA andHIV-1 capture/transfer assays were utilized. Subsequently the authors of thismanuscript aimed to identify the DC-SIGN binding component through biochemicalcharacterization and mass spectrometry analysis. CM was shown to bind DC-SIGN and competeswith HIV-1 gp140 trimer for binding. Pre-incubation of Raji-DC-SIGN cells or immature dendritic cells(iDCs) with CM potently inhibits DC-SIGN mediated trans-infection of CD4+ T-lymphocytes with CCR5 and CXCR4using HIV-1 strains, while no effect on direct infection is observed. Preliminary biochemical characterization demonstrates that the component seems to be large (>100kDa), heat and proteinase K resistant, binds in a α1–3mannose independent manner and is highly variant between individuals. Immunoprecipitation using DC-SIGN-Fc coated agarose beads followed by mass spectrometry indicated lactoferrin (fragments) and its receptor (intelectin-1)as candidates. Using ELISA the authors showed that lactoferrin levels within CM correlate with DCSIGN binding capacity.

In conclusion, CM can bind the C-type lectin DC-SIGN and block HIV-1 trans-infection of both CCR5 and CXCR4using HIV-1 strains. Furthermore, reported data indicate that lactoferrin is a DC-SIGN binding component of CM.These results indicate that CM has the potential to interfere with pathogen transmission and modulate immune responses at the colorectal mucosa.

Obtaining the Plasmodium falciparum full life cycle and observations on the P. ovale liverstages in humanized mice.Soulard V, Bosson-Vanga H, Lorthiois A, Roucher C, Franetich JF, Zanghi G, Bordessoulles M, Tefit M, Thellier M,Morosan S, Le Naour G, Capron F, Suemizu H, Snounou G, Moreno-Sabater A, Mazier D. Nat Comm 2015 Jul 24;6:7690.

Experimental studies of Plasmodium parasites that infect humans are restricted by their host specificity. Humanizedmice offer a means to overcome this and further provide the opportunity to observe the parasites in vivo. Theauthors of this manuscript improve on previous protocols to achieve efficient double engraftment of TK-NOGmice by human primary hepatocytes and red blood cells. Thus, the authors obtain the complete hepatic

RESEARCH ARTICLEColorectal Mucus Binds DC-SIGN and

Inhibits HIV-1 Trans-Infection of CD4+

T-LymphocytesMartijn J. Stax 1☯

, Emily E. I. M. Mouser 1☯, Thijs van Montfort 1, Rogier W. Sanders1,2, Henry

J. C. de Vries 3,8, Henk L. Dekker 4, Carolina Herrera 5, Dave Speijer 6, Georgios Pollakis 1,7,

William A. Paxton1,7*1 Laboratory of Experimental Virology, Department of Medical Microbiology, Centre for Infection and

Immunity, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands, 2 Department

of Microbiology and Immunology, Weill Medical College of Cornell University, New York, United States of

America, 3 Department of Dermatology, Medical Centre of the University of Amsterdam, Amsterdam, The

Netherlands, 4 Mass Spectrometry of Biomacromolecules, Swammerdam Institute for Life Sciences,

University of Amsterdam, Amsterdam, the Netherlands, 5 Division of Infectious Diseases, Faculty of

Medicine, Imperial College, London, United Kingdom, 6 Department of Medical Biochemistry, Medical

Centre of the University of Amsterdam, Amsterdam, the Netherlands, 7 Department of Clinical Infection,

Microbiology and Immunology, Institute of Infection and Global Health, University of Liverpool, Liverpool,

United Kingdom, 8 STI outpatient clinic, Cluster Infectious Diseases, Public Health Service Amsterdam and

Centre for Infections and Immunity Amsterdam, Academic Medical Center, University of Amsterdam,

Amsterdam, the Netherlands☯ These authors contributed equally to this work.

* [email protected]

Bodily secretions, including breast milk and semen, contain factors that modulate HIV-1 in-

fection. Since anal intercourse caries one of the highest risks for HIV-1 transmission, our

aim was to determine whether colorectal mucus (CM) also contains factors interfering with

HIV-1 infection and replication. CM from a number of individuals was collected and tested

for the capacity to bind DC-SIGN and inhibit HIV-1 cis- or trans-infection of CD4+T-lympho-

cytes. To this end, a DC-SIGN binding ELISA, a gp140 trimer competition ELISA and HIV-1

capture/ transfer assays were utilized. Subsequently we aimed to identify the DC-SIGN

binding component through biochemical characterization and mass spectrometry analysis.

CM was shown to bind DC-SIGN and competes with HIV-1 gp140 trimer for binding. Pre-in-

cubation of Raji-DC-SIGN cells or immature dendritic cells (iDCs) with CM potently inhibits

DC-SIGN mediated trans-infection of CD4+T-lymphocytes with CCR5 and CXCR4 using

HIV-1 strains, while no effect on direct infection is observed. Preliminary biochemical char-

acterization demonstrates that the component seems to be large (>100kDa), heat and pro-

teinase K resistant, binds in a α1–3 mannose independent manner and is highly variant

between individuals. Immunoprecipitation using DC-SIGN-Fc coated agarose beads fol-

lowed by mass spectrometry indicated lactoferrin (fragments) and its receptor (intelectin-1)

as candidates. Using ELISA we showed that lactoferrin levels within CM correlate with DC-

SIGN binding capacity. In conclusion, CM can bind the C-type lectin DC-SIGN and block

HIV-1 trans-infection of both CCR5 and CXCR4 using HIV-1 strains. Furthermore, our data

PLOSONE | DOI:10.1371/journal.pone.0122020 March 20, 2015

1 / 13

OPEN ACCESSCitation: Stax MJ, Mouser EEIM, van Montfort T,

Sanders RW, de Vries HJC, Dekker HL, et al. (2015)

Colorectal Mucus Binds DC-SIGN and Inhibits HIV-1

Trans-Infection of CD4 +T-Lymphocytes. PLoS ONE

10(3): e0122020. doi:10.1371/journal.pone.0122020

Academic Editor: Stefan Pöhlmann, German

Primate Center, GERMANYReceived: November 25, 2014

Accepted: February 9, 2015Published: March 20, 2015

Copyright: © 2015 Stax et al. This is an open access

article distributed under the terms of the Creative

Commons Attribution License, which permits

unrestricted use, distribution, and reproduction in any

medium, provided the original author and source are

credited.Data Availability Statement: All relevant data are

within the paper.Funding: This work was partly funded through a

Dutch AIDS fonds grant (2005024), a SKE grant from

the University of Amsterdam (WAP), and two

European Community’s Seventh Framework

Programme Grants [41642 (IDEA) and 305578

(PathCo)]. The funders had no role in study design,

data collection and analysis, decision to publish, or

preparation of the manuscript.Competing Interests: The corresponding author

William Paxton states that he acts as an Academic

http://www.pathco.org/documents/publications/Stax_MJ_et_al_PLoS_One_2015.pdf

http://www.pathco.org/documents/publications/Soulard_V_Nat_Comm_2015.pdf






www.pathco.org



development of P. falciparum, the transition to the erythrocytic stages, their subsequentmultiplication, and the appearance of mature gametocytes over an extended period

of observation. Furthermore, using sporozoites derived from two P. ovale-infectedpatients, the authors show that human hepatocytes engrafted in TK-NOG mice

sustain maturation of the liver stages, and the presence of late-developing schizonts indicate the eventual activation of quiescent parasites. Thus, TK-NOG

mice are highly suited for in vivo observations on the Plasmodium species ofhumans.

Patterns of HIV, TB, and non-communicable disease multi-morbidity in peri-urban SouthAfrica - a cross sectional study.Oni T, Youngblood E, Boulle A, McGrath N, Wilkinson RJ, Levitt, NS. BMC Infect Dis. 2015 Jan 17;15:20.

Many low and middle-income countries are experiencing colliding epidemics of chronic infectious (ID) and non-communicable diseases (NCD). As a result, the prevalence of multiple morbidities (MM) is rising.

The authors of this manuscript conducted a study to describe the epidemiology of MM in a primary care clinic in Khayelitsha. Adults with at least one of HIV, tuberculosis (TB), diabetes (DM), and hypertension (HPT) were identifiedbetween Sept 2012-May 2013 on electronic databases. Using unique patient identifiers, drugs prescribed across all facilities in the province were linked to each patient and each drug class assigned a condition.

Results reported in the manuscript show that these 4 diseases accounted for 45% of all prescription visits. Among 14364 chronic disease patients, HPT was the most commonmorbidity (65%). 22.6% of patients had MM, with an increasing prevalence with age; and ahigh prevalence among younger antiretroviral therapy (ART) patients (26% and 30% in18-35 yr and 36-45 year age groups respectively). Among these younger ART patientswith MM, HPT and DM prevalence was higher than in those not on ART.

The authors highlight the co-existence of multiple ID and NCD. This presents bothchallenges (increasing complexity and the impact on health services, providers andpatients), and opportunities for chronic diseases screening in a population linked tocare. It also necessitates re-thinking of models of health care delivery and requirespolicy interventions to integrate and coordinate management of co-morbid chronicdiseases.

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RESEARCH ARTICLE

Open Access

Patterns of HIV, TB, and non-communicable

disease multi-morbidity in peri-urban South

Africa- a cross sectional study

Tolu Oni 1,2,3*, Elizabeth Youngblood 4, Andrew Boulle 2,3, Nuala McGrath 5,6, Robert J Wilkinson 2,4,7,8

and Naomi S Levitt 4,9

AbstractBackground: Many low and middle-income countries are experiencing colliding epidemics of chronic infectious

(ID) and non-communicable diseases (NCD). As a result, the prevalence of multiple morbidities (MM) is rising.

Methods: We conducted a study to describe the epidemiology of MM in a primary care clinic in Khayelitsha. Adults

with at least one of HIV, tuberculosis (TB), diabetes (DM), and hypertension (HPT) were identified between Sept

2012-May 2013 on electronic databases. Using unique patient identifiers, drugs prescribed across all facilities in the

province were linked to each patient and each drug class assigned a condition.

Results: These 4 diseases accounted for 45% of all prescription visits. Among 14364 chronic disease patients, HPT

was the most common morbidity (65%). 22.6% of patients had MM, with an increasing prevalence with age; and a

high prevalence among younger antiretroviral therapy (ART) patients (26% and 30% in 18-35 yr and 36–45 year age

groups respectively). Among these younger ART patients with MM, HPT and DM prevalence was higher than in

those not on ART.Conclusions: We highlight the co-existence of multiple ID and NCD. This presents both challenges (increasing

complexity and the impact on health services, providers and patients), and opportunities for chronic diseases

screening in a population linked to care. It also necessitates re-thinking of models of health care delivery and

requires policy interventions to integrate and coordinate management of co-morbid chronic diseases.

Keywords: HIV, Tuberculosis, Hypertension, Diabetes, Multimorbidity

BackgroundThe concept of multi-morbidity (MM), defined as the co-

existence of more than one chronic condition in one per-

son, is well recognized, usually within the context of older

age [1]. Patients with MM have increased utilization of

health care, a reduced quality of life and poorer health out-

comes [1-4]. A recent systematic review of MM patterns

described a non-random pattern of MM for which com-

mon pathophysiological mechanisms underlie each disease

constellation [1]. However all studies in this review were

conducted in high-income settings, predominantly in older

age populations, and included only non-communicable

diseases (NCD). In low- and middle-income countries

(LMIC), with burgeoning urbanisation, not only is the

prevalence of NCD increasing, it is occurring alongside

chronic infectious diseases. Thus patterns of MM will

differ.South Africa is the most urbanised country in sub-

Saharan Africa with 62% of the country’s population liv-

ing in cities [1, 5]. The rapid and unplanned nature of

this demographic shift affects life choices and opportun-

ities; contributing to epidemiological transition with an

increase in unhealthy dietary patterns, a decrease in

physical activity and a rising NCD burden [2-4, 6, 7].

South Africa has the highest burden of hypertension

(HPT) in the >50 years old population and among the

* Correspondence: [email protected]

1Division of Public Health Medicine, School of Public Health and Family

Medicine, University of Cape Town, Room 2.24, Entrance 5, Falmouth

building Anzio road, Observatory 7925, Cape Town, South Africa

2Health Impact Assessment Directorate, Western Cape Department of Health,

Cape Town, South AfricaFull list of author information is available at the end of the article© 2015 Oni et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative

Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and

reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain

Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,

unless otherwise stated.

Oni et al. BMC Infectious Diseases (2015) 15:20

DOI 10.1186/s12879-015-0750-1

http://www.pathco.org/documents/publications/Oni_t_et_al_BMC_Infectious_Diseases_2015.pdf








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Cytotoxic mediators in paradoxical HIV-tuberculosis immune reconstitution inflammatory syndrome.Wilkinson KA, Walker NF, Meintjes G, Deffur A, Nicol MP, Skolimowska KH, MatthewsK, Tadokera R, Seldon RR, Maartens G, Rangaka MX, Besra G, Wilkinson RJ. J Immunol. 2015 Feb 15;194(4):1748-54. OI

Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS)frequently complicates combined antiretroviral therapy and antituberculosis therapy in HIV-1-coinfected tuberculosis patients. The immunopathological mechanisms underlying TB-IRIS are incompletely defined, and improved understanding is required to derive new treatments and to reduce associatedmorbidity and mortality. The authors of this manuscript performed longitudinaland cross-sectional analyses of human PBMCs from paradoxical TB-IRIS patients and non-IRIS controls (HIV-TB-coinfected patients commencing antiretroviral therapy who did not develop TB-IRIS). Freshly isolated PBMCstimulated with heat-killed Mycobacterium tuberculosis H37Rv (hkH37Rv) wereused for IFN-γ ELISPOT and RNA extraction. Stored RNA was used for microarray andRT-PCR, whereas corresponding stored culture supernatants were used for ELISA. Stored PBMCwere used for perforin and granzyme B ELISPOT and flow cytometry. There were significantly increased IFN-γresponses to hkH37Rv in TB-IRIS, compared with non-IRIS PBMC (p = 0.035). Microarray analysis of hkH37Rv-stimulated PBMC indicated that perforin 1 was the most significantly upregulated gene, with granzyme B amongthe top five (log2 fold difference 3.587 and 2.828, respectively), in TB-IRIS.

Downstream experiments using RT-PCR, ELISA, and ELISPOT confirmed the increased expression and secretionof perforin and granzyme B. Moreover, granzyme B secretion reduced in PBMC from TB-IRIS patients during corticosteroid treatment. Invariant NKT cell (CD3+Va24+) proportions were higher in TB-IRIS patients (p = 0.004) and were a source of perforin. Data reported in this manuscript implicate the granule exocytosis pathway in TB-IRIS pathophysiology. Further understanding of the immunopathogenesis of this condition will facilitate development of specific diagnostic and improved therapeutic options.

The impact of HIV exposure and maternal Mycobacterium tuberculosis infection on infantimmune responses to Bacille Calmette-Guérin vaccination.Jones CE, Hesseling A, Tena-Coki NG, Scriba TJ, Chegou, NN, Kidd M. Wilkinson, R.J., Kampmann, B. AIDS. 2015 Jan 14;29(2):155-65.

The objective of this study was to assess the effect of maternal HIV and Mycobacterium tuberculosis (Mtb) infection oncellular responses to bacille Calmette-Guérin (BCG) immunization.

To this aim, samples were collected from mother-infant pairs at delivery. Infants were BCG-vaccinated at 6 weeks ofage and a repeat blood sample was collected from infants at 16 weeks of age. BCG-specific T-cell proliferation and intracellular cytokine expression were measured by flow cytometry. Secreted cytokines and chemokines in cell culturesupernatants were analysed using a Multiplex assay.

The Journal of Immunology

Cytotoxic Mediators in Paradoxical HIV–Tuberculosis

Immune Reconstitution Inflammatory Syndrome

Katalin A. Wilkinson,* ,†,‡,1Naomi F. Walker,* ,x,1

Graeme Meintjes,* ,†,xArmin Deffur,*

Mark P. Nicol,* ,{,‖Keira H. Skolimowska,* ,x

Kerryn Matthews,* Rebecca Tadokera,*

Ronnett Seldon,* Gary Maartens, †Molebogeng X. Rangaka,* Gurdyal S. Besra, # and

Robert J. Wilkinson* ,†,‡,xTuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) frequently complicates combined antiretroviral ther-

apy and antituberculosis therapy in HIV-1–coinfected tuberculosis patients. The immunopathological mechanisms underlying TB-IRIS

are incompletely defined, and improved understanding is required to derive new treatments and to reduce associated morbidity and

mortality. We performed longitudinal and cross-sectional analyses of human PBMCs from paradoxical TB-IRIS patients and non-IRIS

controls (HIV-TB–coinfected patients commencing antiretroviral therapy who did not develop TB-IRIS). Freshly isolated PBMC

stimulated with heat-killed Mycobacterium tuberculosis H37Rv (hkH37Rv) were used for IFN-g ELISPOT and RNA extraction. Stored

RNAwas used for microarray and RT-PCR, whereas corresponding stored culture supernatants were used for ELISA. Stored PBMC

were used for perforin and granzyme B ELISPOTand flow cytometry. There were significantly increased IFN-g responses to hkH37Rv

in TB-IRIS, compared with non-IRIS PBMC (p = 0.035). Microarray analysis of hkH37Rv-stimulated PBMC indicated that perforin 1

was the most significantly upregulated gene, with granzyme B among the top five (log2 fold difference 3.587 and 2.828, respectively), in

TB-IRIS. Downstream experiments using RT-PCR, ELISA, and ELISPOT confirmed the increased expression and secretion of perforin

and granzyme B. Moreover, granzyme B secretion reduced in PBMC from TB-IRIS patients during corticosteroid treatment. Invariant

NKT cell (CD3+Va24+) proportions were higher in TB-IRIS patients (p = 0.004) and were a source of perforin. Our data implicate the

granule exocytosis pathway in TB-IRIS pathophysiology. Further understanding of the immunopathogenesis of this condition will

facilitate development of specific diagnostic and improved therapeutic options. The Journal of Immunology, 2015, 194: 1748–1754.

H uman immunodeficiency virus-1 is recognized as the

strongest predisposing factor to tuberculosis (TB), and

TB is the commonest cause of death in HIV-1–infected

persons in Africa (1, 2). However, otherwise beneficial dual

therapy for HIV-1 and TB is frequently complicated by the oc-

currence of the TB-associated immune reconstitution inflamma-

tory syndrome (TB-IRIS), an early complication of combination

antiretroviral therapy (ART).

Two forms of TB-IRIS are recognized: paradoxical, which

occurs in patients established on antituberculosis therapy before

ART, but who develop recurrent or new TB symptoms and clinical

features after ART initiation; and unmasking TB-IRIS in patients

not receiving treatment for TB when ART is started, but who

present with active TB within 3 mo of starting ART (3). Para-

doxical TB-IRIS affects ∼15.9% of all HIV-1–infected patients

commencing ART while on TB treatment, and up to 54% in some

populations, causing considerable morbidity and mortality (4, 5).

Immunosuppressive corticosteroid therapy improves symptoms

and reduces hospital admissions, but is not without adverse events,

and is potentially detrimental in cases of drug-resistant TB (6–8).

Specific diagnostic tools and treatments for TB-IRIS are lacking,

and understanding the pathogenesis of this condition is important

to assist in the development of more specific therapies.

Risk factors for TB-IRIS, such as low CD4 count and dissem-

inated TB disease at presentation, suggest that a pathological

immune reaction to mycobacterial Ags during immune recovery is

responsible. We previously described highly dynamic Ag-specific

CD4 T cell IFN-g responses in the first weeks after ART initiation

in both TB-IRIS and control patients in response to early secretory

antigenic target-6, 38-kDa cell wall–associated Ag, and a-crys-

*Clinical Infectious Diseases Research Initiative, University of Cape Town, Cape Town,

7925 South Africa; †Department of Medicine, University of Cape Town, Cape Town,

7925 South Africa; ‡Medical Research Council National Institute for Medical Research,

London NW7 1AA, United Kingdom; xDivision of Medicine, Imperial College London,

London W2 1PG, United Kingdom; {Division of Medical Microbiology, University of

Cape Town, Cape Town, 7925 South Africa; ‖National Health Laboratory Service, Cape

Town, 7925 South Africa; and #School of Biosciences, University of Birmingham,

Birmingham B15 2TT, United Kingdom

1K.A.W. and N.F.W. contributed equally to this manuscript.

ORCID: 0000-0002-9796-2040 (K.A.W.).

Received for publication August 19, 2014. Accepted for publication December 6,

2014.This work was supported by Wellcome Trust Grants 081667, 084323, 088316,

094000, and 085251; Medical Research Council of the United Kingdom Grant

U.1175.02.002.00014.01; European and Developing Countries Clinical Trials

Partnership Grant IP.07.32080.002; and European Union Grants PIRSES-GA-

2011-295214 and FP7-Health-F3-2012-305578.

The sequences presented in this article have been submitted to National Center for

Biotechnology Information’s Gene Expression Omnibus repository (http://www.ncbi.

nlm.nih.gov/geo/query/acc.cgi?acc=GSE48237) under accession number GSE48237.

Preliminary data were reported at the Host Response in Tuberculosis Keystone Sym-

posia, March 13–18, 2013 (Poster X7 4041), Whistler, British Columbia, Canada.

Address correspondence and reprint requests to Dr. Katalin A. Wilkinson, Room

N2.09.B3, Wernher Beit North Building, Institute of Infectious Disease and Molec-

ular Medicine, Faculty of Health Sciences, Observatory 7925, South Africa. E-mail

address: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: ART, antiretroviral therapy; hk, heat-killed; iNKT,

invariant NKT; IQR, interquartile range; IRIS, immune reconstitution inflammatory

syndrome; MOI, multiplicity of infection; TB, tuberculosis.

This is an open-access article distributed under the terms of the CC-BY 3.0 Unported

license.

Copyright � 2015 The Authors 0022-1767/15

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402105

http://www.pathco.org/documents/publications/Wilkinson_K_A_et_al_J_Immunol_2015.pdf

http://www.pathco.org/documents/publications/Jones_CE_et_al_AIDS_2015.pdf






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Obtained results showed that one hundred and nine (47 HIV-exposed and 62 HIV-unexposed) mother-infants pairs were recruited after delivery and followed longitudinally. At birth, proportions of mycobacteria-specific proliferating T cellswere not associated with either in-utero HIV exposure or maternal Mtb sensitization. However, in-utero HIV exposure affected infant-specific T-cell sub-sets [tumour necrosis factor-alpha (TNF-α) single positive proliferating CD4+T cells and interferon-gamma (IFN-γ),TNF-α dual-positive CD4+ T cells]. Levels of TNF-α protein in cell culture supernatants were also significantlyhigher in HIV-exposed infants born to Mtb-sensitized mothers. In the presence of maternal Mtb sensitization, frequencies of maternal and newborn BCG-specific proliferating CD4+ T cells were positively

correlated. Following BCG vaccination, there was no demonstrable effect of HIVexposure or maternal Mtb infection on infant BCG-specific T-cell proliferative responses

or concentrations of secreted cytokines and chemokines.

The authors conclude that effects of maternal HIV and Mtb infection on infant immune profiles at birth are transient only, and HIV-exposed, noninfected infants have the same potential to respond to and be protected byBCG vaccination as HIV-unexposed infants.

Immune reconstitution inflammatory syndrome in HIV-infected patients.Walker NF, Scriven J, Meintjes G, Wilkinson RJ. HIV AIDS (Auckl). 2015 Feb 12;7:49-64..

Access to antiretroviral therapy (ART) is improving worldwide. Immune reconstitution inflammatory syndrome (IRIS)is a common complication of ART initiation.

In this review, the authors provide an overview of clinical and epidemiological features ofHIV-associated IRIS, current understanding of pathophysiological mechanisms, availabletherapy, and preventive strategies.

The spectrum of HIV-associated IRIS is described, with a particular focus on three important pathogen-associated forms: tuberculosis-associated IRIS, cryptococcal IRIS,and Kaposi’s sarcoma IRIS. While the clinical features and epidemiology are well described, there are major gaps in the understanding of pathophysiology and as aresult therapeutic and preventative strategies are suboptimal. Timing of ART initiationis critical to reduce IRIS-associated morbidity. Improved understanding of the pathophysiology of IRIS will hopefully enable improved diagnostic modalities andbetter targeted treatments to be developed.

Theimpac

t ofHIV

exposu

re andmater

nal

Mycobac

terium

tubercu

losis in

fection

on

infant i

mmuneresp

onses t

o bacille

Calmette

-Guerin

vaccina

tion

Christin

e E.Jon

esa,b

,c , Annek

e C.Hes

seling

d , Nonto

bekoG

. Tena-C

okia,b ,

Thomas J

. Scriba

e , Novel

N. Cheg

ouf , M

artin Kid

dg ,

Robert

J. Wilkin

sonh,i,

j andBea

te Kampmann

a,b,k

Objective

: The obje

ctive of t

hisstud

y is to assess the

effect o

f mater

nalHIV

and

Mycobac

terium tub

erculosi

s (Mtb)

infectio

n oncell

ularresp

onses t

o bacille

Calmette

-

Guerin

(BCG)

immunizatio

n.

Design:

A mother–in

fantcoh

ortstud

y.

Methods:

Samples

were coll

ected from

mother–in

fantpair

s atdeli

very. In

fants w

ere

BCG-vac

cinated

at 6wee

ks of ag

e and a

repeat b

loodsam

plewas

collecte

d from infa

nts

at 16wee

ksof

age. BCG

-specific

T-cell pro

liferatio

n andintr

acellul

arcyto

kine

express

ionwer

e measured

byflow

cytometry

. Secret

edcyto

kines and

chemokin

es

in cellcult

uresup

ernatant

s were

analyse

d using a Multi

plexassa

y.

Results

: One

hundred

andnine

(47HIV

-expose

d and62

HIV-un

expose

d) mothe

r–

infants

pairs wer

e recruite

d after deli

veryand

followe

d longitud

inally.

Atbirt

h,

propor

tions of m

ycobac

teria-sp

ecific pro

liferatin

g T cells wer

e notasso

ciated

with

either i

n-utero

HIVexp

osure or m

aternal

Mtb sensitiz

ation. H

owever,

in-utero

HIV

exposu

re affected

infant-s

pecific

T-cell su

bsets [t

umournec

rosis fa

ctor-alp

ha (TNF-a

)

single pos

itivepro

liferatin

g CD4þ T cell

s and inte

rferon-

gamma (IFN

-g),TNF

-a dual-

positive

CD4þ T cell

s]. Leve

ls of TN

F-apro

teinin cell

culture

superna

tants w

erealso

significa

ntlyhigh

er in HIV

-expose

d infants

born to Mtb-s

ensitize

d mothers.

In the

presenc

e of mater

nalMtb sen

sitizatio

n, frequ

encies

of mater

naland

newbor

n BCG-

specific

prolifer

ating C

D4þ T ce

lls were

positive

ly corre

lated. F

ollowin

g BCG vac

cina-

tion, th

erewas

nodem

onstrab

le effect o

f HIV exp

osure or m

aternal

Mtb infectio

n on

infant B

CG-spe

cificT-ce

ll proli

ferative

respons

es or co

ncentra

tions of

secreted

cyto-

kines an

d chemokin

es.

Conclus

ion:Effe

ctsof m

aternal

HIVand

Mtb infectio

n oninfa

nt immune

profile

s at

birth are

transien

t only, and

HIV-ex

posed,

noninfe

ctedinfa

ntshav

e thesam

e

potent

ialto resp

ondto and

bepro

tected

byBCG

vaccin

ation as H

IV-unex

posed

infants.

� 2015 Wolte

rs Kluw

er Healt

h, Inc.

Allrigh

ts rese

rved.

a Academ

ic Departm

entof P

ediatric

s, Imperi

al Colle

ge London

, Londo

n, UK,

b Institut

e of Infec

tious D

iseases

andMolec

ular

Medicine,

Univers

ityof C

apeTow

n, Cape

Town, S

outhAfri

ca,c Pae

diatric

Infectio

us Disea

sesRes

earch Gro

up,St G

eorge’s,

Univers

ityof L

ondon,

London

, UK,

dDesmond

TutuTB

Centre,

Departm

entof P

ediatric

s and Chi

ld Health,

Faculty

of Healt

h

Science

s, Stelle

nbosch

Univers

ity,Stel

lenbosc

h,e Sou

th African

Tubercu

losis Va

ccine Init

iative, In

stitute of I

nfectiou

s Disea

ses

andMolec

ularMedic

ine,Uni

versity

of Cape

Town, C

apeTow

n,f DST

/NRF Cen

treof E

xcellen

ce forBio

medical T

ubercul

osis

Researc

h andMRC

Centre

forTub

erculosi

s Resear

ch,Div

ision of M

olecular

Biology

andHum

an Genetic

s, Depa

rtment

of

Biomedic

al Scien

ces,Fac

ultyof M

edicine

andHea

lthScie

nces, S

tellenbo

schUni

versity,

gDivisio

n of Statis

ticsand

Actuari

al

Science

s, Facu

ltyof S

cience,

Univers

ityof S

tellenbo

sch, St

ellenbo

sch, So

uthAfri

ca,hMRC

Nationa

l Institu

te forMedic

al

Researc

h, Mill H

ill,Lon

don, UK

,i Clin

icalInfe

ctious D

iseases

Researc

h Initiativ

e, Institu

te of In

fectious

Disease

s and Molec

ular

Medicine,

Univers

ity of Ca

pe Town

, Cape T

own, So

uthAfri

ca,j Div

ision of

Medicine,

Imperial C

ollege L

ondon,

London

, UK, an

d

k Vaccino

logyThe

me, Medic

al Resea

rchCou

ncilUni

t, Banju

l, The Gam

bia.

Corresp

ondenc

e to Christin

e E. Jones

, Paedia

tricInfe

ctious D

iseases

Researc

h Group,

Mail point

J2C, Le

vel2, J

enner W

ing,St

George’

s, Unive

rsityof L

ondon,

London

SW17

0RE, U

K.

Tel:+44

208725

2788; f

ax:+44

20872

50716;

e-mail:

cjones@

sgul.ac

.uk

Receive

d: 15 Aug

ust201

4; revis

ed:30

October 2

014; ac

cepted:

31Octob

er 2014

.

DOI:10

.1097/Q

AD.000

000000

000053

6

ISSN026

9-9370

Copyrig

ht Q201

5 Wolters K

luwer H

ealth, I

nc.All

rights r

eserved

. This is

an open acc

essarti

cledist

ributed

under t

he

Creativ

e Commons

Attribut

ionLice

nse4.0,

which per

mits unre

stricted

use, di

stributio

n, and

reprod

uction

in anymediu

m, provid

ed

theorig

inalwor

k is prop

erlycite

d.

155

© 2015 Walker et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0)

License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further

permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on

how to request permission may be found at: http://www.dovepress.com/permissions.php

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49

R E V I E W

open access to scientific and medical research

Open Access Full Text Article

http://dx.doi.org/10.2147/HIV.S42328

in HIV-infected patients

Naomi F Walker 1–3James Scriven2–4Graeme Meintjes 1–3Robert J Wilkinson1,2,51Department of Medicine, Imperial

College London, London, UK;

2Clinical Infectious Diseases Research

Initiative, Institute of Infectious

Disease and Molecular Medicine, Town, South Africa; 3Department of

Cape Town, South Africa; 4Liverpool

School of Tropical Medicine, Liverpool,

UK; 5MRC National Institute of

Medical Research, London, UK

Correspondence: Robert J Wilkinson

Clinical Infectious Diseases Research Initiative, Room 3.03.05,

Wolfson Pavilion, Institute of Infectious

Diseases and Molecular Medicine (IDM), Cape Town, South Africa Tel 27 21 406 6084 Fax 27 21 406 6796 Email [email protected]

Abstract: Access to antiretroviral therapy (ART) is improving worldwide. Immune reconstitution

inflammatory syndrome (IRIS) is a common complication of ART initiation. In this review, we

provide an overview of clinical and epidemiological features of HIV-associated IRIS, current

understanding of pathophysiological mechanisms, available therapy, and preventive strategies.

The spectrum of HIV-associated IRIS is described, with a particular focus on three important

pathogen-associated forms: tuberculosis-associated IRIS, cryptococcal IRIS, and Kaposi’s

sarcoma IRIS. While the clinical features and epidemiology are well described, there are major

gaps in our understanding of pathophysiology and as a result therapeutic and preventative

strategies are suboptimal. Timing of ART initiation is critical to reduce IRIS-associated mor-

bidity. Improved understanding of the pathophysiology of IRIS will hopefully enable improved

diagnostic modalities and better targeted treatments to be developed.

Keywords: antiretroviral therapy, tuberculosis, IRIS, diagnosis, complications

IntroductionAntiretroviral therapy (ART) has dramatically reduced HIV-associated mortality,

which decreased from a peak of 2.3 million in 2005 to 1.6 million in 2012.1–3 This

reflects improvement in access to ART in the last decade, especially for HIV-infected

patients in low- and middle-income countries, where the number of patients receiving

ART has increased more than 30-fold (from 300,000 in 2002 to 9.7 million in 2012)

and life expectancy is increasing.3,4 It is now recognized that commencement of ART

earlier in HIV infection improves outcomes, and international guidelines have been

updated to reflect this.5 Tuberculosis (TB) is now considered to be an indication for

ART irrespective of CD4 count, as ART reduces mortality in TB patients.6

However, ART initiation is not without risk of complications, particularly in the

first 6 months.7,8 HIV-associated immune reconstitution inflammatory syndrome (IRIS)

has emerged as an important early complication of ART initiation, associated with

considerable morbidity and mortality, particularly in patients who commence ART with

advanced immunosuppression.9 In this condition, immune recovery following ART

initiation associates with a pathological inflammatory response, usually directed toward

microbial antigens. Although there is considerable clinical and pathophysiological

heterogeneity, key features include clinical deterioration in the first weeks to months

of ART, with evidence of localized tissue inflammation with or without a systemic

inflammatory response.In this review, we provide an overview of clinical and epidemiological features of

HIV-associated IRIS, current understanding of pathophysiological mechanisms, available

http://www.pathco.org/documents/publications/Walker_NF_et_al_HIV_AIDS_2015.pdf







www.pathco.org



The road to drug resistance in Mycobacterium tuberculosis.Koch A and Wilkinson RJ. Genome Biol. 2014 Nov 13;15(11):520.

Sequencing of serial isolates of extensively drug-resistant tuberculosis highlightshow drug resistance develops within a single patient and reveals unexpected levelsof pathogen diversity.

Tricks to Translating TB Transcriptomics.Deffur A, Wilkinson RJ, Coussens AK. Ann Transl Med. 2015 May;3(Suppl 1):S43.

Transcriptomics and other high-throughput methods are increasingly applied to questions relating to tuberculosis (TB)pathogenesis. Whole blood transcriptomics has repeatedly been applied to define correlates of TB risk and has producednew insight into the late stage of disease pathogenesis. In a novel approach, authors of a recently published study inScience Translational Medicine applied complex data analysis of existing TB transcriptomic datasets, and in vitro models, inan attempt to identify correlates of protection in TB, which are crucially required for the development of novel TB diagnostics and therapeutics to halt this global epidemic. Utilizing latent TB infection (LTBI) as a surrogate of protection,they identified IL-32 as a mediator of interferon gamma (IFNγ)-vitamin D dependent antimicrobial immunity and amarker of LTBI.

In this manuscript, the authors provide a review of all TB whole blood transcriptomic studies to date in the context of identifying correlates of protection, discuss potentialpitfalls of combining complex analyses originating from such studies, the importance ofdetailed metadata to interpret differential patient classification algorithms, the effect ofdiffering circulating cell populations between patient groups on the interpretation ofresulting biomarkers and we decipher weighted gene co-expression network analysis(WGCNA), a recently developed systems biology tool which holds promise of identifying novel pathway interactions in disease pathogenesis.

In conclusion, the authors of this manuscript propose the development of an integrated OMICS platform and open access to detailed metadata, in order forthe TB research community to leverage the vast array of OMICS data being generated with the aim of unraveling the holy grail of TB research: correlates of protection.

RESEAR

CHHIG

HLIGH

T

Theroad

to drugresis

tance in

Mycobac

terium

tuberculo

sis

Anastasia

Koch1 and

Robert Jo

hn Wilkinson

2,3,4*

Seerelat

ed research http

://genom

ebiology

.com/201

4/15/11/

490

Abstrac

t

Sequenci

ng of ser

ial isolate

s ofexte

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ug-resist

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tuberculo

sis highli

ghtshow

drugresis

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within a

single pa

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reveals u

nexpecte

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f

pathogen

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Tubercu

losis(TB

) remains

a crucia

l public

health prob

-

lem, wi

th increasi

ng drugresis

tance posi

ng a challeng

e

to curre

nt contr

ol effort

s. Treat

mentregi

mensfor d

rug-

suscepti

ble TB are

onerous

, requirin

g aminim

umof s

ix

months of

treatment

withfour

antitube

rcular d

rugs. Th

ere

arepatie

ntswho

develop

multi-drug

-resistan

t (MDR)

, ex-

tensively

drug-res

istant (X

DR)and

totally drug

-resistan

t

(TDR) form

s, which

aresucc

essively

morediffi

cultto

treat. In

these circu

mstances,

treatment

regimens

involve

theuse

of alarge

r number

of less-e

ffective drug

s, which

havea na

rrower th

erapeuti

c margin

.

In many

bacteria,

drug-resi

stance d

eterminan

ts are ca

rried

on mobilegene

tic elements

. Howev

er, in Mycob

acterium

tubercul

osis(Mtb),

drugresis

tance is

exclusiv

ely assoc

iated

withpoin

t mutat

ionsand

chromosom

al rearra

ngements

.

Poor or

intermitten

t therap

y haslong

beenthou

ghtto

be themajor

explanat

ionfor

drugresis

tance, a

nd it is

believed

thatdrug

-resistan

t strains

develop

through

the

sequenti

al fixati

on of asmall s

et of mutat

ions, suc

h that

thepath

ogensam

plesonly

a small propo

rtionof p

os-

sibleevol

utionary

paths [1]

.

Theappl

ication

of whole

-genome sequ

encing (WGS)

hasreve

aledprev

iously u

nderappr

eciated

levels of

genetic

diversity

within circ

ulating

Mtb populat

ions, an

d the

implicatio

ns of th

is diver

sityfor

transmissio

n anddis-

easeoutc

omes areincr

easingly

being ackn

owledge

d.

* Corresp

ondence

: r.j.wilkin

son@imperia

l.ac.uk

2 Clinical I

nfectiou

s Disease

s Resear

ch Initiative,

Institute

of Infecti

ousDise

ases

andMole

cular Me

dicine, U

niversity

of Cape

Town, O

bservato

ry, Cape

Town

7925, Sou

th Africa

3MRCNati

onalInsti

tutefor M

edical Re

search, L

ondon NW7 1A

A, UK

Fulllist o

f author

information

is availab

le atthe

endof th

e article

By contras

t, mycob

acterial

heteroge

neity with

in a sin-

gle host,

andany

concom

itant bio

logical o

r clinica

l sig-

nificance

, has be

en explored

butseld

omdoc

umented.

In a study pub

lished in this

issue of G

enome Biol

ogy,

Eldholm

andcolle

agues appl

y WGSto inve

stigate the

evolutio

n fromdrug

-sensitiv

e toXDR

-TBwith

in a single

patient

[2].This

addsto an emergi

ng body of e

vidence

thatsugg

eststhat

intra-hos

t micro

bialdive

rsityis sub-

stantial

andmight

havesign

ificant c

onsequen

ceswhe

n

inferrin

g transm

ission. T

hereare

fewinsta

nces, if a

ny, in

theliter

ature whe

re thishas

beeninve

stigated

in such

detail.

Mapping

drug-res

istant t

ubercul

osisin a sing

le

patient

Thestud

y teamtook

advantag

e of nine

serial ba

cterial

isolates c

ollected

froma sin

glepati

ent,from

thetime of

firstdiag

nosis of d

rug-susc

eptible

TBthro

ughto the

developm

entof XDR

-TB. The

infection

ultimately

re-

solved follo

wingthe

addition

of linezo

lid to thetrea

tment

regimen - lin

ezolid is kn

ownto be a

moderately

effective

treatment

for drug-

resistant

TBwhe

n other op

tions ha

ve

failed [2].

Illumina

sequenci

ng was a

pplied to the

isolates,

which were

collected

overa pe

riodof 4

2 months, ac

hiev-

inga hig

h depth of c

overage.

By relax

ingthe

stringenc

y of

filters a

pplied to dete

ct single

-nucleoti

de polymorph

isms

(SNPs),

theauth

orsobse

rvedunex

pected leve

ls ofhete

ro-

geneity w

ithinindi

vidual sa

mples. It

is worth

mentionin

g

thatDNA

wasnot

extracte

d from sing

le coloni

es -inste

ad

‘loopfuls’

of bacte

rialcells

wereharv

ested for

DNA. Th

is

approach

allowed the

group to dete

ct SNPs

present

at a

frequenc

y ofappr

oximately

25%at si

tes with a m

inimum

read-dep

th of 50. O

f the35 S

NPsiden

tified in

thisway

, 20

weretran

sient an

d 15 eventual

ly becam

e fixed. T

welve of

theobse

rvedmutat

ionswere

associate

d withdrug

resist-

ance, and

phenotyp

ic resista

ncewas

observed

at the sa

me

time that ge

notypic r

esistance

emerged.

© 2014Koch

andWilkins

on; licen

seeBioM

ed Central L

td. The li

censee h

as exclusi

ve rights

to distrib

ute this a

rticle, in a

ny

medium,

for 12 month

s followin

g itspubl

ication. Af

ter this tim

e, the art

icle is ava

ilableunde

r theterm

s ofthe C

reative C

ommons

Attributio

n License

(http://cre

ativecom

mons.org

/licenses/b

y/4.0), wh

ich permits un

restricted

use,distr

ibution, a

nd reprod

uction in

anymediu

m, provid

ed the ori

ginal wo

rk isprop

erlycred

ited.The

Creative

Common

s Public D

omain Dedic

ationwaiv

er (http://

creativec

ommons.o

rg/public

domain/z

ero/1.0/) a

pplies to

the data m

adeavail

ablein th

is article,

unless ot

herwise s

tated.

Kochand

Wilkinson

Genome

Biology 2

014,15:5

20

http://ge

nomebio

logy.com

/2014/15

/11/520

Page 1 of 7

© Annals of Translational Medicine. All rights reserved.

Ann Transl Med 2015;3(S1):S43

www.atmjournal.org

Editorial

Tricks to translating TB transcriptomics

Armin Deffur 1,2, Robert J. Wilkinson 1,2,3,4, Anna K. Coussens 1

1Clinical Infectious Diseases Research Initiative, Institute of Infectious Disease and Molecular Medicine, 2Department of Medicine, Faculty of Health

Sciences, University of Cape Town and Groote Schuur Hospital, Observatory, 7925, South Africa; 3The Francis Crick Institute, Mill Hill Laboratory,

London, NW7 1AA, UK; 4Department of Medicine, Imperial College London, W2 1PG, UK

Correspondence to: Anna K. Coussens, PhD. Clinical Infectious Diseases Research Initiative, Institute of Infectious Disease & Molecular Medicine,

University of Cape Town, Anzio Rd, Observatory, 7925, South Africa. Email: [email protected].

Abstract: Transcriptomics and other high-throughput methods are increasingly applied to questions relating to

tuberculosis (TB) pathogenesis. Whole blood transcriptomics has repeatedly been applied to define correlates of

TB risk and has produced new insight into the late stage of disease pathogenesis. In a novel approach, authors of a

recently published study in Science Translational Medicine applied complex data analysis of existing TB transcriptomic

datasets, and in vitro models, in an attempt to identify correlates of protection in TB, which are crucially required

for the development of novel TB diagnostics and therapeutics to halt this global epidemic. Utilizing latent TB

infection (LTBI) as a surrogate of protection, they identified IL-32 as a mediator of interferon gamma (IFN )-

vitamin D dependent antimicrobial immunity and a marker of LTBI. Here, we provide a review of all TB whole-

blood transcriptomic studies to date in the context of identifying correlates of protection, discuss potential pitfalls

of combining complex analyses originating from such studies, the importance of detailed metadata to interpret

differential patient classification algorithms, the effect of differing circulating cell populations between patient

groups on the interpretation of resulting biomarkers and we decipher weighted gene co-expression network

analysis (WGCNA), a recently developed systems biology tool which holds promise of identifying novel pathway

interactions in disease pathogenesis. In conclusion, we propose the development of an integrated OMICS platform

and open access to detailed metadata, in order for the TB research community to leverage the vast array of OMICS

data being generated with the aim of unraveling the holy grail of TB research: correlates of protection.

Keywords: Biomarker; tuberculosis (TB); protective immunity; correlates of risk; microarray; systems biology

Submitted Apr 09, 2015. Accepted for publication Apr 09, 2015.

doi: 10.3978/j.issn.2305-5839.2015.04.12

View this article at: http://dx.doi.org/10.3978/j.issn.2305-5839.2015.04.12

IntroductionIn 2013, an estimated 9.0 million people developed

tuberculosis (TB) and 1.5 million died from the disease (1).

Despite ongoing research efforts and ever-increasing

knowledge of how Mycobacterium tuberculosis (M.tb)

interferes with the human immune response, we are still far

from developing the diagnostic and therapeutic approaches

that would reduce the severity of this global epidemic.

Vaccines form the cornerstone of potential eradication

strategies of TB, yet the biological factors that provide

protection from disease progression (so-called biological

correlates of protection), which would assist design and

testing of new vaccine candidates are lacking. A better

understanding of natural protective immunity to TB would

facilitate these attempts. Following inhalation of M.tb

bacilli, latent TB infection (LTBI) is the most common

outcome. It is considered that in LTBI the growth of the

bacilli is contained by the coordinated host innate and

adaptive immune response, preventing disease progression,

but there is failure to completely eradicate all organisms,

such that an underlying asymptomatic infection persists.

This makes LTBI a particularly useful model system for the

discovery of protective correlates.

In the last 8 years, 15 transcriptomic studies have

been published that use whole-genome gene expression

microarrays in an effort to gain a broader understanding

of the human response to M.tb infection, during various

http://www.pathco.org/documents/publications/Deffur_A_et_al_Ann_Transl_Med_2015.pdf

http://www.pathco.org/documents/publications/Koch_A_and_Wilkinson_RJ_Genome_Biol_2015.pdf







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A novel mouse model for stable engraftment of a human immune system and human hepatocytes. Strick-Marchand H, Dusséaux M, Darche S, Huntington ND, Legrand N, Masse-RansonG, Corcuff E, Ahodantin J, Weijer K, Spits H, Kremsdorf D, Di Santo JP. PLoS One. 2015 Mar 17;10(3):e0119820.

Hepatic infections by hepatitis B virus (HBV), hepatitis C virus (HCV) and Plasmodiumparasites leading to acute or chronic diseases constitute a global health challenge.The species tropism of these hepatotropic pathogens is restricted to chimpanzeesand humans, thus model systems to study their pathological mechanisms are severely limited. Although these pathogens infect hepatocytes, disease pathologyis intimately related to the degree and quality of the immune response.

As a first step to decipher the immune response to infected hepatocytes, theauthors of this manuscript developed an animal model harboring both ahuman immune system (HIS) and human hepatocytes (HUHEP) in BALB/cRag2-/- IL-2Rγc-/- NOD.sirpa uPAtg/tg mice. The extent and kinetics of human hepatocyteengraftment were similar between HUHEP and HIS-HUHEP mice. Transplanted human hepatocyteswere polarized and mature in vivo, resulting in 20-50% liver chimerism in these models. Human myeloid and lymphoid cell lineages developed at similar frequencies in HIS and HIS-HUHEP mice, and splenic and hepaticcompartments were humanized with mature B cells, NK cells and naïve T cells, as well as monocytes and dendriticcells.

Taken together, the results reported in the manuscript demonstrate that HIS-HUHEP mice can be stably (> 5months) and robustly engrafted with a humanized immune system and chimeric human liver. This novel HIS-HUHEP model provides a platform to investigate human immune responses against hepatotropic pathogens andto test novel drug strategies or vaccine candidates.

RESEARCH ARTICLEA Novel Mouse Model for Stable Engraftment

of a Human Immune System and Human

HepatocytesHelene Strick-Marchand1,2, Mathilde Dusséaux1,2, Sylvie Darche1,2, Nicholas

D. Huntington1,2¤a, Nicolas Legrand3¤b, Guillemette Masse-Ranson1,2, Erwan Corcuff 1,2¤b,

James Ahodantin 4, KeesWeijer 3, Hergen Spits 3, Dina Kremsdorf 4, James P. Di Santo1,2*

1 Innate Immunity Unit, Department of Immunology, Institut Pasteur, Paris, France, 2 Institut National de la

Santé et de la Recherche Médicale (INSERM) U668, Paris, France, 3 Academic Medical Center at the

University of Amsterdam, Amsterdam, The Netherlands, 4 Institut National de la Santé et de la Recherche

Médicale (INSERM) U845, Faculté de Médecine Paris Descartes, Paris, France

¤a Current address: Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

¤b Current address: AXENIS, Institut Pasteur, Paris, France.

* [email protected]

Hepatic infections by hepatitis B virus (HBV), hepatitis C virus (HCV) and Plasmodium para-

sites leading to acute or chronic diseases constitute a global health challenge. The species

tropism of these hepatotropic pathogens is restricted to chimpanzees and humans, thus

model systems to study their pathological mechanisms are severely limited. Although these

pathogens infect hepatocytes, disease pathology is intimately related to the degree and

quality of the immune response. As a first step to decipher the immune response to infected

hepatocytes, we developed an animal model harboring both a human immune system (HIS)

and human hepatocytes (HUHEP) in BALB/c Rag2 -/-IL-2Rγc -/-NOD.sirpa uPA tg/tgmice.

The extent and kinetics of human hepatocyte engraftment were similar between HUHEP

and HIS-HUHEPmice. Transplanted human hepatocytes were polarized and mature in

vivo, resulting in 20–50% liver chimerism in these models. Human myeloid and lymphoid

cell lineages developed at similar frequencies in HIS and HIS-HUHEP mice, and splenic

and hepatic compartments were humanized with mature B cells, NK cells and naïve T cells,

as well as monocytes and dendritic cells. Taken together, these results demonstrate that

HIS-HUHEPmice can be stably (> 5 months) and robustly engrafted with a humanized im-

mune system and chimeric human liver. This novel HIS-HUHEPmodel provides a platform

to investigate human immune responses against hepatotropic pathogens and to test novel

drug strategies or vaccine candidates.

IntroductionInfectious diseases that target the liver, including Plasmodium parasites and hepatitis B and C

viruses (HBV, HCV), affect over 600 million people worldwide. The restricted species tropism

PLOSONE | DOI:10.1371/journal.pone.0119820 March 17, 2015

1 / 14

a11111

OPEN ACCESSCitation: Strick-Marchand H, Dusséaux M, Darche S,

Huntington ND, Legrand N, Masse-Ranson G, et al.

(2015) A Novel Mouse Model for Stable Engraftment

of a Human Immune System and Human

Hepatocytes. PLoS ONE 10(3): e0119820.

doi:10.1371/journal.pone.0119820

Academic Editor: Gordon Langsley, Institut national

de la santé et de la recherche médicale—Institut

Cochin, FRANCEReceived: November 20, 2014Accepted: January 16, 2015

Published: March 17, 2015Copyright: © 2015 Strick-Marchand et al. This is an

open access article distributed under the terms of the

Creative Commons Attribution License, which permits

unrestricted use, distribution, and reproduction in any

medium, provided the original author and source are

credited.Data Availability Statement: All relevant data are

within the paper and its Supporting information files.

Funding: This study was supported by Bill & Melinda

Gates foundation global health program grant #

37869. Agence Nationale de la recherche

programme Emergence grant # ANR-11-EMMA-026.

Agence Nationale de la recherche programme

LABEX grant # ANR-10-LBX-73. Agence Nationale

de Recherches sur le Sida et les hépatites virales

grant # 2013–105. European Commission Seventh

Framework Programme PathCO grant #

CD81 is required for rhoptry discharge during host cell invasion by Plasmodium yoelii sporozoites.Risco-Castillo V, Topçu S, Son O, Briquet S, Manzoni G, Silvie O. Cell Microbiol. 2014 Oct;16(10):1533-48.

Additional publications generated by PathCo Project

http://www.ncbi.nlm.nih.gov/pubmed/24798694

http://www.pathco.org/documents/publications/Strick_Marchand_H_et_al_PLoS_One_2015.pdf





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PROJECT BROCHURE

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PROJECT WEBSITE

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PathCo additional communication tools

PATHOGEN

CO'INFECTION:

HIV‐1, Tuberculosis,

Malaria and

hepatitis C virus

UNIVERSITY OF BIRMINGHAMJane McKeating ‐ Gurdyal BesraUnited Kingdomwww.birmingham.ac.ukINSTITUT NATIONAL DE LA SANTÉET DE LA RECHERCHE MÉDICALEOlivier Silvie ‐ Francewww.inserm.fr

UNIVERSITAETSKLINIKUM FREIBURGRobert Thimme ‐ Germanywww.uniklinik‐freiburg.de

UNIVERSITY OF OXFORDTao Dong ‐ United Kingdomwww.ox.ac.uk

IMPERIAL COLLEGE OF SCIENCE,TECHNOLOGY AND MEDICINECarolina Herrera ‐ Xiao‐Ning XuUnited Kingdom ‐ www3.imperial.ac.ukUNIVERSITY OF CAPE TOWNRobert Wilkinson ‐ South Africawww.uct.ac.za

INSTITUT PASTEURJames Di Santo ‐ Francewww.pasteur.fr

ALTA RICERCA E SVILUPPOIN BIOTECNOLOGIE S.R.L.U.Riccardo Bertini ‐ Italywww.altaweb.eu

PATHCO PARTNERS

PATHOGEN CO'INFECTION:HIV‐1, Tuberculosis, Malaria and hepatitis C virus

COORDINATOR:Prof William A Paxton PhD DICDepartment of Clinical Infection, Microbiology and ImmunologyInstitute of Infection and Global HealthUniversity of Liverpool216e, Ronald Ross Building8 West Derby StreetLiverpool L69 [email protected]

www.pathco.org

EC Contribution: € 5.909.690Duration: 60 monthsStarting date: 01/11/2012

UNIVERSITY OF LIVERPOOLBill Paxton ‐ United Kingdomwww.liv.ac.uk

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