Characterization and Optimization of RepliVax Dengue: U ...
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Characterization and Optimization of RepliVax ® Dengue: a single cycle tetravalent vaccine candidate Christina M. Gallo, Raul C. Gomila, John A. Catalan, Alexander Sousa, Ana P. Goncalvez and Konstantin Pugachev Viral Vectors and Delivery, Sanofi Pasteur, Cambridge, MA 02139 Abstract Dengue virus (DENV) is the most prevalent vector-borne virus that afflicts humans, with close to 2.5 billion people living in areas where the virus can be transmitted. Currently, there are no vaccines or antiviral medications against DENV. Sanofi Pasteur’s live-attenuated vaccine (LAV) candidate against DENV, ChimeriVax TM Dengue (CYD), has shown promising results in SE Asia and the Americas. Overall efficacy against symptomatic disease is between 55 and 65 percent, while reducing the hospitalization rate by 80 percent and protection against severe manifestation of the disease by 95 percent over the 25 month Active Surveillance period [1,2]. The vaccine regimen consists of three doses over a one year period to induce a balanced immune response. We are currently investigating the potential of using the RepliVax® platform to produce a tetravalent DENV vaccine consisting of single-cycle replication viruses. These flavivirus-based replication-defective viruses could eliminate potential replication interference among the four replication-competent viruses in the LAV, potentially allowing for a smaller window of immunization. The four RepliVax® Dengue (RVDEN) prototypes have been constructed. The prM/E genes of DENV serotypes 1-4 were cloned into the RepliVax® West Nile virus (RVWN) backbone, which consists of the WN virus nonstructural (NS) genes and a truncated capsid (C) protein [3]. Viral RNA was in- vitro transcribed and transfected into baby hamster kidney (BHK) helper cells expressing WN C protein to generate virus. Serial passaging of the transfected cells was necessary to increase titers for all four RVDEN viruses, with serotypes 3 and 4 requiring more extensive passaging than 1 and 2. Sequencing of viruses recovered from the supernatant after cell passage 10 revealed adaptive mutations acquired by the viruses in the structural and NS regions (Table 2). Interestingly, a conserved mutation in the stem region of the envelope (Env) of RVWNDEN 3 and -4 was found. Introduction of the Env stem mutation by reverse genetics into RVWNDEN increased viral titers after transfection at least 100 fold (Figure 5). Preliminary results show that Env stem mutation in combination with the NS mutations provide a growth advantage over the Env stem mutation only (Figure 6). We are currently testing the immunogenicity in mice of the newly generated RVDEN 1-3 (Table 3). Introduction Infection with DENV causes up to 100 million cases of Dengue fever each year, and as many as 450,000 cases of the more severe manifestations, such as Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS). DENV is a member of the Flavivirus genus of the Flaviviridae family, which includes enveloped, positive-strand RNA viruses. The virus genome is translated as a polyprotein that is processed into 10 proteins, including three structural (C, prM, and Env) and seven NS proteins that encode the enzymatic activities needed for replication. There are four different serotypes of DENV, and infection with a DENV serotype creates lasting immunity against that specific serotype. Infection by other DENV serotypes has been linked to the more severe DHF and DSS. Therefore, to create a successful and broadly applicable vaccine against DENV, the vaccine must induce balanced immunity against all four DENV serotypes. Currently, there are no approved vaccines against DENV on the market, although Sanofi Pasteur has recently developed a chimeric LAV candidate, ChimeriVax TM Dengue, based on the Yellow Fever (YF) 17D vaccine backbone with the prM/E genes from each of the four DENV serotypes. To provide balanced immunity against all four serotypes, ChimeriVax TM Dengue is administered in three doses six months apart. RepliVax® is a single cycle, replication-defective virus reduces the potential for replication interference amongst serotypes after immunization. Our current RVDEN candidate contains a WNV backbone (5’ and 3’ UTR regions, truncated C protein and NS proteins) with the DENV prM and E proteins. Here we describe the generation and characterization of RVDEN viruses for all four DENV serotypes as part of a tetravalent DENV vaccine formulation, currently being tested in mice. Potential approaches to increase viral titers of all four serotypes for manufacturing are discussed. Overview of RepliVax® Technology Conclusions/Next Steps 1. Initial characterization of RVWNDEN4 shows similar immunogenicity in mice as the replication-competent CYD4, suggesting that the RepliVax ® platform can be used to create a viable Dengue vaccine candidate. 2. RVWNDEN serum-free virus stocks corresponding to the four DENV serotypes were produced for use in mouse and non-human primate immunogenicity studies . 3. Adaptive mutations were found in all virus stocks, including a conserved Env stem mutation in RVWNDEN3 and 4. 4. Viruses with reverse engineered Env stem plus NS mutations show increased titers after transfection, and are highly promising candidates for manufacturing of RVWNDEN3 and 4. 5. Monovalent and tetravalent formulations of all four RVWNDEN constructions are being tested in mice to characterize immunogenicity. Next steps: 1. Characterize the effect of the Env stem mutation on Env secretion using a VLP expression system. 2. Determine the optimal NS mutations in the RVWN backbone that enhance titers for all four RVWNDEN constructs. 3. Determine if RVWNDEN1 and 2 constructs with the Env stem mutation produce higher viral titers than wild-type RVWNDEN1 and 2. 4. Determine the immunogenicity of the RVWNDEN1-4 formulations in mice. References/Acknowledgements 1. Capeding, M.R., et al., Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet, 2014. 2. Villar, L., et al., Efficacy of a Tetravalent Dengue Vaccine in Children in Latin America. N Engl J Med, 2014. 3. Rumyantsev, A.A., et al., Characterization of the RepliVax platform for replication- defective flavivirus vaccines. Vaccine, 2011. 29(32): p. 5184-94. 4. Schmidt, A.G., P.L. Yang, and S.C. Harrison, Peptide Inhibitors of Dengue-Virus Entry Target a Late-Stage Fusion Intermediate. PLoS Pathog, 2010. 6(4): p. e1000851. Acknowledgments: Thank you to the Viral Vector and Delivery research team for your support and guidance, especially Raul Gomila, as well as Ana Goncalvez. I would also like to thank Northeastern University and Sanofi Pasteur for this opportunity to present our group’s work. West Nile virus E prM Non-structural Dengue Figure 1: To construct RepliVax® Dengue (RVDEN), the prM/E genes of RepliVax® WNV were replaced with the prM/E genes of DENV-1,-2,-3 and - 4. West Nile NS genes provide the backbone for RV-DENV, with a deletion in the structural Capsid protein. This deletion prevents infectious particles from forming when the virus infects naïve cells. T T T C C Helper cell Immunization Naïve cell VLPs CMI No spread NT-Abs NS C protein in trans Replication defective virus C Figure 2: When packaged in a substrate cell line expressing West Nile virus Capsid protein in trans, RepliVax® Dengue forms a replication-defective virus which is capable of only a single round of infection. Infection with RepliVax® mimics a viral infection, activating a the T-cell response and producing virus-like particles (VLPs) that stimulate the production of neutralizing antibodies. The RepliVax® platform can offer an extremely attenuated vaccine candidate that produces substantial immunogenicity in vivo. Passage FFU/mL P0 P5 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 RVWNDEN3-WT RVWNDEN3 Env #3 RVWNDEN3 Env #3 + NS Mutations 6,7 Passage FFU/mL P0 P5 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 RVWNDEN4-WT RVWNDEN4 Env #4 RVWNDEN4 Env #4 + NS Mutations 8,9,10 Env Mutation 5 Mutation 11 Mutation 12 E ∆C prM Nonstructural Mutations Provide Boost in Titers for Envelope Mutants Figure 5: Envelope and NS mutations were reverse engineered by site-directed mutagenesis into RVWNDEN expressing plasmids, which where then transfected into BHK WN C helper cells. Supernatants were collected at each cell passage and used to infect naïve Vero cells to determine titers in focus forming units (FFU). (A)RVDEN3. The Env and Env + NS mutations show titers at P0, compared to undetectable titers for the wild-type, and continue to show at least 10 fold higher titers though P5, indicating better overall replication than the wild-type. Sequencing of the P10 wild-type virus showed it acquired the Env #3 mutation; neither the Env nor Env + NS mutations acquired additional mutations. (B)RVDEN4. The Env and Env + NS mutants show 100 fold higher titers at P0 than the wild-type. At passage 5, the wild-type shows similar replication to the virus with Env + NS mutations. Expression of Env from Plasmids Containing DEN prM/E Sequences Figure 7: Immunofluorescence staining of HEK-293FT cells 72 hours post-transfection with DNA plasmids expressing DENV-4 prM/E under the control of the CMV promoter. Staining shows that both the DENV-4 wild-type and DENV-4 Env #4 mutant express Env protein. Western Blot optimization is currently ongoing to quantify differences in secretion between the wild-type DENV-4 and Env #4 mutant. The prM/E genes from all four DENV serotypes will be tested using this assay to determine if higher titers can be correlated with increased Env secretion. Anti- DENV- Env (1A5) Visible pVAX1 DENV4 WT prM/E 5μg DNA pVAX1 DENV4 Env mutation #3 prM/E 5μg DNA Mock No DNA Hours post Infection FFU/mL 48 72 96 120 144 10 1 10 2 10 3 10 4 10 5 10 6 RVWNDEN4-WT RVWNDEN4 Env #3 RVWNDEN4 Env #3 + NS Mutations 8,9,10 RVWNDEN4 Induces Similar Immunogenicity to CYD4 in Mice Dose (5 Log 10 FFU) PRNT 50 titer (Log 10 Dilution) Mock RVD4 (Env mutation #3) RVD4 (WT Env) CYD4 0 1 2 3 4 Prime Boost Virus Titer (FFU/ml) Volume (ml) RVWNDEN 1 3.70E+07 12 RVWNDEN 2T 1.38E+06 12 RVWNDEN 3 8.00E+06 10 RVWNDEN 4 1.22E+07 30 RVWNDEN Serum-Free Stocks for Immunogenicity Studies and A.A. Adaptations Table 1: Serum-free virus stocks were generated for each of the four DENV serotypes. Large-scale virus cultures were grown on BHK WN C expressing cells. Viruses were concentrated using tangential flow filtration (TFF) and are currently being tested for immunogenicity in mice. RVWNDEN1 RVWNDEN2T RVWNDEN3 RVWNDEN4 Env Mutation 1* Env Mutation 2 Env Mutation 3 Env Mutation 3 Mutation 1 Mutation 3* Mutation 6 Mutation 8* Mutation 2 Mutation 4* Mutation 7 Mutation 9* Mutation 5* Mutation 10 Table 2: Virus RNA was isolated from viral particles, reverse transcribed and sequenced to screen for mutations after passaging. Adaptive mutations were found in all four serotypes, with a mutation at the same amino acid position (Env mutations 3 and 4) in the stem region of Env found in RVDEN-3 and -4. *Mixed population Group Virus Mice per group Prime dose Day 1 Bleed Day 30, PRNT Boost dose Day 31 Bleed Day 51, PRNT Bleed Days 90,180 PRNT 1 Mock 5 - x - x X,X 2 RVWNDEN1 5 5 logFFU x 5 logFFU x X,X 3 RVWNDEN2 5 5 logFFU x 5 logFFU x X,X 4 RVWNDEN3 5 5 logFFU x 5 logFFU x X,X 5 RVWNDEN4 5 5 logFFU x 5 logFFU x X,X 6 RVWNV-tetra 5 5 logFFU x 5 logFFU x X,X Conserved Envelope Stem Region Important in Viral Fusion Figure 4: The conserved mutation found in RVDENV-3,4 stocks is located in the stem region of the Env protein, the major protein involved viral membrane fusion. (A) A representation of the dimeric Env protein before being exposed to a pH lower than 6.2. (B) After being exposed to low pH, Env dissociates and folds into a trimeric confirmation. The hydrophobic fusion loop formed then binds into the outer layer of the target membrane bilayer, followed by further rearrangement that completes viral fusion. The stem region plays an important role in the formation of the hydrophobic fusion loop.(C) Color coded schematic of the domains of the Env protein, with the stem region magnified [4]. Envelope and Nonstructural Mutations Display Increased Viral Replication in RVDEN4 Ongoing Mouse Study to Evaluate Monovalent and Tetravalent Formulations of RVWNDEN Table 3: Monovalent and tetravalent formulations of RVWNDEN are currently being tested in mice to determine immunogenicity. Mice were inoculated with 5log FFU doses (IP) and will be given an identical boost 31 days later. Additionally, RVWNDEN4 is being tested using subcutaneous (SC) and intramuscular (IM) routes of immunization (groups not shown). Figure 3: Two formulations of RVDEN4 were tested for immunogenicity in mice and compared to ChimeriVax TM Dengue- 4 (CYD4). Both RVDEN4 formulations showed a similar effect after receiving a 31 day boost after initial inoculation, showing RVDENV exhibits comparable immunogenicity to CYD4. The Envelope mutant of RVDEN4 showed similar PRNT titers to a virus with a wild-type Env, suggesting that the stem mutation does not affect immunogenicity. Figure 6: Cell-passaged viruses at P5 (Figure 5) were used to infect BHK WN C cells at a multiplicity of infection (MOI) of 0.01. Supernatant was collected every 24 hours for seven days and then titrated to quantify viral growth. Results indicate that the wild- type virus (that has accumulated additional adaptations) grew to the highest titers, followed by the virus with Env stem and NS mutations. When reverse transcribed and sequenced, the wild-type virus showed three adaptive mutations, including an Env stem mutation at the same A.A. position as tested and two novel NS mutations as shown in the diagram below. The viruses with the Env stem mutation and Env stem plus NS mutations did not acquire additional mutations after passaging. A B Results E prM E prM NS ∆C ∆C ∆C Modified from [4] WN Nonstructural Proteins
Transcript of Characterization and Optimization of RepliVax Dengue: U ...
Characterization and Optimization of RepliVax® Dengue: a single cycle tetravalent vaccine candidate
Christina M. Gallo, Raul C. Gomila, John A. Catalan, Alexander Sousa, Ana P. Goncalvez and Konstantin Pugachev
Viral Vectors and Delivery, Sanofi Pasteur, Cambridge, MA 02139
AbstractDengue virus (DENV) is the most prevalent vector-borne virus that afflicts humans,
with close to 2.5 billion people living in areas where the virus can be transmitted.Currently, there are no vaccines or antiviral medications against DENV. Sanofi Pasteur’slive-attenuated vaccine (LAV) candidate against DENV, ChimeriVaxTM Dengue (CYD), hasshown promising results in SE Asia and the Americas. Overall efficacy againstsymptomatic disease is between 55 and 65 percent, while reducing the hospitalizationrate by 80 percent and protection against severe manifestation of the disease by 95percent over the 25 month Active Surveillance period [1,2]. The vaccine regimenconsists of three doses over a one year period to induce a balanced immune response.
We are currently investigating the potential of using the RepliVax® platform toproduce a tetravalent DENV vaccine consisting of single-cycle replication viruses. Theseflavivirus-based replication-defective viruses could eliminate potential replicationinterference among the four replication-competent viruses in the LAV, potentiallyallowing for a smaller window of immunization. The four RepliVax® Dengue (RVDEN)prototypes have been constructed. The prM/E genes of DENV serotypes 1-4 werecloned into the RepliVax® West Nile virus (RVWN) backbone, which consists of the WNvirus nonstructural (NS) genes and a truncated capsid (C) protein [3]. Viral RNA was in-vitro transcribed and transfected into baby hamster kidney (BHK) helper cellsexpressing WN C protein to generate virus. Serial passaging of the transfected cells wasnecessary to increase titers for all four RVDEN viruses, with serotypes 3 and 4 requiringmore extensive passaging than 1 and 2. Sequencing of viruses recovered from thesupernatant after cell passage 10 revealed adaptive mutations acquired by the virusesin the structural and NS regions (Table 2). Interestingly, a conserved mutation in thestem region of the envelope (Env) of RVWNDEN 3 and -4 was found. Introduction ofthe Env stem mutation by reverse genetics into RVWNDEN increased viral titers aftertransfection at least 100 fold (Figure 5). Preliminary results show that Env stemmutation in combination with the NS mutations provide a growth advantage over theEnv stem mutation only (Figure 6). We are currently testing the immunogenicity in miceof the newly generated RVDEN 1-3 (Table 3).
IntroductionInfection with DENV causes up to 100 million cases of Dengue fever each year, and as
many as 450,000 cases of the more severe manifestations, such as Dengue HemorrhagicFever (DHF) and Dengue Shock Syndrome (DSS). DENV is a member of the Flavivirus genusof the Flaviviridae family, which includes enveloped, positive-strand RNA viruses. The virusgenome is translated as a polyprotein that is processed into 10 proteins, including threestructural (C, prM, and Env) and seven NS proteins that encode the enzymatic activitiesneeded for replication. There are four different serotypes of DENV, and infection with aDENV serotype creates lasting immunity against that specific serotype. Infection by otherDENV serotypes has been linked to the more severe DHF and DSS. Therefore, to create asuccessful and broadly applicable vaccine against DENV, the vaccine must induce balancedimmunity against all four DENV serotypes. Currently, there are no approved vaccinesagainst DENV on the market, although Sanofi Pasteur has recently developed a chimericLAV candidate, ChimeriVaxTM Dengue, based on the Yellow Fever (YF) 17D vaccinebackbone with the prM/E genes from each of the four DENV serotypes. To providebalanced immunity against all four serotypes, ChimeriVaxTM Dengue is administered inthree doses six months apart.
RepliVax® is a single cycle, replication-defective virus reduces the potential forreplication interference amongst serotypes after immunization. Our current RVDENcandidate contains a WNV backbone (5’ and 3’ UTR regions, truncated C protein and NSproteins) with the DENV prM and E proteins. Here we describe the generation andcharacterization of RVDEN viruses for all four DENV serotypes as part of a tetravalentDENV vaccine formulation, currently being tested in mice. Potential approaches toincrease viral titers of all four serotypes for manufacturing are discussed.
Overview of RepliVax® Technology
Conclusions/Next Steps1. Initial characterization of RVWNDEN4 shows similar immunogenicity in mice as the
replication-competent CYD4, suggesting that the RepliVax® platform can be used tocreate a viable Dengue vaccine candidate.
2. RVWNDEN serum-free virus stocks corresponding to the four DENV serotypes wereproduced for use in mouse and non-human primate immunogenicity studies .
3. Adaptive mutations were found in all virus stocks, including a conserved Env stemmutation in RVWNDEN3 and 4.
4. Viruses with reverse engineered Env stem plus NS mutations show increased titersafter transfection, and are highly promising candidates for manufacturing ofRVWNDEN3 and 4.
5. Monovalent and tetravalent formulations of all four RVWNDEN constructions arebeing tested in mice to characterize immunogenicity.
Next steps: 1. Characterize the effect of the Env stem mutation on Env secretion using a VLP
expression system.2. Determine the optimal NS mutations in the RVWN backbone that enhance titers for all
four RVWNDEN constructs.3. Determine if RVWNDEN1 and 2 constructs with the Env stem mutation produce higher
viral titers than wild-type RVWNDEN1 and 2.4. Determine the immunogenicity of the RVWNDEN1-4 formulations in mice.
References/Acknowledgements
1. Capeding, M.R., et al., Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet, 2014.2. Villar, L., et al., Efficacy of a Tetravalent Dengue Vaccine in Children in Latin America. N Engl J Med, 2014.3. Rumyantsev, A.A., et al., Characterization of the RepliVax platform for replication-defective flavivirus vaccines. Vaccine, 2011. 29(32): p. 5184-94.4. Schmidt, A.G., P.L. Yang, and S.C. Harrison, Peptide Inhibitors of Dengue-Virus Entry Target a Late-Stage Fusion Intermediate. PLoS Pathog, 2010. 6(4): p. e1000851.
Acknowledgments: Thank you to the Viral Vector and Delivery research team for your support and guidance, especially Raul Gomila, as well as Ana Goncalvez. I would also like to thank Northeastern University and Sanofi Pasteur for this opportunity to present our group’s work.
West Nile virus
EprM Non-structural
DengueFigure 1: To construct RepliVax®Dengue (RVDEN), the prM/E genes ofRepliVax® WNV were replaced withthe prM/E genes of DENV-1,-2,-3 and -4. West Nile NS genes provide thebackbone for RV-DENV, with adeletion in the structural Capsidprotein. This deletion preventsinfectious particles from formingwhen the virus infects naïve cells.
T
T
T
C
C
Helper cell
Immunization
Naïve cell
VLPs
CMI
No spread
NT-Abs
NS
C protein in trans
Replication
defective virus
C
Figure 2: When packaged in asubstrate cell line expressing WestNile virus Capsid protein in trans,RepliVax® Dengue forms areplication-defective virus which iscapable of only a single round ofinfection. Infection with RepliVax®mimics a viral infection, activatinga the T-cell response andproducing virus-like particles(VLPs) that stimulate theproduction of neutralizingantibodies. The RepliVax® platformcan offer an extremely attenuatedvaccine candidate that producessubstantial immunogenicity invivo.
P a s s a g e
FF
U/m
L
P0
P5
1 0 0
1 0 1
1 0 2
1 0 3
1 0 4
1 0 5
1 0 6
1 0 7
R V W N D E N 3 -W T
R V W N D E N 3 E n v # 3
R V W N D E N 3 E n v # 3 + N S M u ta tio n s 6 ,7
P a s s a g e
FF
U/m
L
P0
P5
1 0 0
1 0 1
1 0 2
1 0 3
1 0 4
1 0 5
1 0 6
1 0 7
R V W N D E N 4 -W T
R V W N D E N 4 E n v # 4
R V W N D E N 4 E n v # 4 + N S M u ta tio n s 8 ,9 ,1 0
Env Mutation 5 Mutation 11
Mutation 12
E∆C
prM
Nonstructural Mutations Provide Boost in Titers for Envelope Mutants
Figure 5: Envelope and NS mutations were reverse engineered by site-directedmutagenesis into RVWNDEN expressing plasmids, which where then transfected into BHKWN C helper cells. Supernatants were collected at each cell passage and used to infectnaïve Vero cells to determine titers in focus forming units (FFU). (A)RVDEN3. The Env andEnv + NS mutations show titers at P0, compared to undetectable titers for the wild-type,and continue to show at least 10 fold higher titers though P5, indicating better overallreplication than the wild-type. Sequencing of the P10 wild-type virus showed it acquiredthe Env #3 mutation; neither the Env nor Env + NS mutations acquired additionalmutations. (B)RVDEN4. The Env and Env + NS mutants show 100 fold higher titers at P0than the wild-type. At passage 5, the wild-type shows similar replication to the virus withEnv + NS mutations.
Expression of Env from Plasmids Containing DEN prM/E Sequences
Figure 7: Immunofluorescence staining of HEK-293FT cells 72 hours post-transfection withDNA plasmids expressing DENV-4 prM/E under the control of the CMV promoter. Stainingshows that both the DENV-4 wild-type and DENV-4 Env #4 mutant express Env protein.Western Blot optimization is currently ongoing to quantify differences in secretion betweenthe wild-type DENV-4 and Env #4 mutant. The prM/E genes from all four DENV serotypeswill be tested using this assay to determine if higher titers can be correlated with increasedEnv secretion.
An
ti-
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V-
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(1A
5)
Vis
ible
pVAX1 DENV4 WT prM/E 5µg DNA
pVAX1 DENV4Env mutation #3 prM/E
5µg DNA
MockNo DNA
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R V W N D E N 4 E n v # 3
R V W N D E N 4 E n v # 3 + N S M u ta tio n s 8 ,9 ,1 0
RVWNDEN4 Induces Similar Immunogenicity to CYD4 in Mice
D o s e (5 L o g 1 0 F F U )
PR
NT
50
tit
er (
Lo
g1
0D
ilu
tio
n)
M o c k R V D 4
(E n v m u ta t io n # 3 )
R V D 4
(W T E n v )
C Y D 4 0
1
2
3
4P rim e
B o o s t
Virus Titer (FFU/ml) Volume (ml)
RVWNDEN 1 3.70E+07 12
RVWNDEN 2T 1.38E+06 12
RVWNDEN 3 8.00E+06 10
RVWNDEN 4 1.22E+07 30
RVWNDEN Serum-Free Stocks for Immunogenicity Studies and A.A. Adaptations
Table 1: Serum-free virusstocks were generated for eachof the four DENV serotypes.Large-scale virus cultures weregrown on BHK WN Cexpressing cells. Viruses wereconcentrated using tangentialflow filtration (TFF) and arecurrently being tested forimmunogenicity in mice.
RVWNDEN1 RVWNDEN2T RVWNDEN3 RVWNDEN4
Env Mutation 1*
Env Mutation 2
EnvMutation 3
Env Mutation 3
Mutation 1 Mutation 3* Mutation 6 Mutation 8*
Mutation 2 Mutation 4* Mutation 7 Mutation 9*
Mutation 5* Mutation 10
Table 2: Virus RNA wasisolated from viral particles,reverse transcribed andsequenced to screen formutations after passaging.Adaptive mutations werefound in all four serotypes,with a mutation at the sameamino acid position (Envmutations 3 and 4) in thestem region of Env found inRVDEN-3 and -4. *Mixed population
Group Virus Mice per group
Prime doseDay 1
Bleed Day 30, PRNT
Boost dose Day
31
Bleed Day 51,
PRNT
Bleed Days 90,180PRNT
1 Mock 5 - x - x X,X
2 RVWNDEN1 5 5 logFFU x 5 logFFU x X,X
3 RVWNDEN2 5 5 logFFU x 5 logFFU x X,X
4 RVWNDEN3 5 5 logFFU x 5 logFFU x X,X
5 RVWNDEN4 5 5 logFFU x 5 logFFU x X,X
6 RVWNV-tetra 5 5 logFFU x 5 logFFU x X,X
Conserved Envelope Stem Region Important in Viral Fusion
Figure 4: The conserved mutation found in RVDENV-3,4 stocksis located in the stem region of the Env protein, the majorprotein involved viral membrane fusion. (A) A representationof the dimeric Env protein before being exposed to a pH lowerthan 6.2. (B) After being exposed to low pH, Env dissociatesand folds into a trimeric confirmation. The hydrophobic fusionloop formed then binds into the outer layer of the targetmembrane bilayer, followed by further rearrangement thatcompletes viral fusion. The stem region plays an importantrole in the formation of the hydrophobic fusion loop.(C) Colorcoded schematic of the domains of the Env protein, with thestem region magnified [4].
Envelope and Nonstructural Mutations Display Increased Viral Replication in RVDEN4
Ongoing Mouse Study to Evaluate Monovalent and Tetravalent Formulations of RVWNDEN
Table 3: Monovalent and tetravalent formulations of RVWNDEN are currently being tested in mice to determine immunogenicity. Mice wereinoculated with 5log FFU doses (IP) and will be given an identical boost 31 days later. Additionally, RVWNDEN4 is being tested using subcutaneous(SC) and intramuscular (IM) routes of immunization (groups not shown).
Figure 3: Two formulations ofRVDEN4 were tested forimmunogenicity in mice andcompared to ChimeriVaxTM Dengue-4 (CYD4). Both RVDEN4 formulationsshowed a similar effect afterreceiving a 31 day boost after initialinoculation, showing RVDENVexhibits comparable immunogenicityto CYD4. The Envelope mutant ofRVDEN4 showed similar PRNT titersto a virus with a wild-type Env,suggesting that the stem mutationdoes not affect immunogenicity.
Figure 6: Cell-passaged viruses at P5 (Figure5) were used to infect BHK WN C cells at amultiplicity of infection (MOI) of 0.01.Supernatant was collected every 24 hoursfor seven days and then titrated to quantifyviral growth. Results indicate that the wild-type virus (that has accumulated additionaladaptations) grew to the highest titers,followed by the virus with Env stem and NSmutations. When reverse transcribed andsequenced, the wild-type virus showedthree adaptive mutations, including an Envstem mutation at the same A.A. position astested and two novel NS mutations asshown in the diagram below. The viruseswith the Env stem mutation and Env stemplus NS mutations did not acquire additionalmutations after passaging.A B
Results
EprMEprM NS
∆C
∆C∆C
Modified from [4]
WN Nonstructural Proteins
