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Replicating VSV-SIV Chimeric Viral Vector Immunogenicity in Indian Rhesus Macaques

Immunogenicity of Replicating VSV-SIV Chimeric Viral Vector in Indian Rhesus Macaques
Gavin Morrow, Christy Jurgens, Arban Domi, John Coleman, Palka Sharma, Rebecca Powell, Ian Ouellette, Joanne DeStefano, Jennifer Martinez, Heather Arendt, B. Rasmussen, C. Richter King, Christopher Parks (International AIDS Vaccine Initiative (IAVI) Design and Development Laboratory, Brooklyn New York, USA 11220)
Live attenuated vaccines have been developed to prevent viral diseases including smallpox and measles; however, for HIV this is not practical. Therefore, we developed replication-competent vesicular stomatitis virus (VSV) vectors in which VSV glycoprotein (G) was replaced with SIVenv and SIVgag producing chimeric viruses (VSV-SIV).
Chimeric vaccines were tested in two NHP studies to compare systemic and mucosal immunization routes. NHPs were primed with VSV-SIV and boosted with Adenovirus-5 containing SIVgag and SIVenv (Ad5-SIV). In the first study, VSV-SIV was administered intramuscularly (IM) and in the second, VSV-SIV was delivered by combined intranasal (IN) and oral (OR) inoculation. Additionally, to improve mucosal uptake, some animals in the second study were vaccinated with VSV G-pseudotyped chimeric virus.
In the 1st study, NHPs were primed 3 times, IM 6 weeks apart with 104,106 or 108 pfu of VSV-SIV followed by an Ad5-SIV boost. Humoral immune responses were dose dependent with the highest dose inducing SIVenv antibodies after each prime.  Following the Ad5 boost, antibody titers increased 3-5 logs and pseudovirus-neutralizing activity was detected in the 106 and 108 groups.  In the 2nd study, 108 pfu VSV-SIV was administered mucosally (IN/OR) and in one group, VSV-SIV was pseudotyped with VSV-G. Mucosal immunization induced humoral responses of similar magnitude to IM while G-pseudotyped virus significantly enhanced Env-specific antibody and pseudovirus neturalization. Gag antibody titers were low after VSV-SIV but priming was evident with a 5 log increase in titers following boost. T-cell responses were low but detectable and increased significantly following Ad5-SIV boost indicative of priming by the VSV. NHPs in the 2nd study are being SIVmac239 challenged.
VSV-SIV chimeras are safe and immunogenic at 108 pfu when administered to macaques systemically and mucosally. VSV-SIV immunization induced pseudovirus neutralizing antibodies and immunogenicity is improved by G-pseudotyping broadening the VSV cell tropism.
•Live attenuated vaccines induce protective immunity by exposing the immune system to a subclinical virus infection.  This vaccine approach has controlled many highly infectious viruses.
•Because the attenuated virus replicates, it engages innate immune system receptors and cellular pathways for antigen processing and presentation soon after infection.  Moreover, because progeny virus is produced, the immune system is exposed to authentic particulate multivalent antigens that induce responses against multiple viral proteins. 
•Using a live attenuated HIV vaccine to vaccinate people is not practical, thus a replicating vector must be used to deliver HIV immunogens in the context of a viral infection.
•The negative-strand RNA virus, vesicular stomatitis virus (VSV), is attractive for development of a replicating HIV vaccine vector because:
-It is immunogenic in primates
-It can be delivered mucosally potentially inducing stronger mucosal immunity 
-Expression of viral and foreign proteins can be modulated simply by gene location in the viral genome
-Replication occurs in the cytoplasm; there is no DNA intermediate and no integration into the host DNA.
-Recombination does not occur
-Natural human infection is infrequent limiting pre-existing immunity
-Human infection is not associated with serious illness
•Prototype chimeric VSV-SIV vaccines were constructed with genes from SIVmac239. 
•The transcription unit encoding the VSV attachment and entry glycoprotein (G) was replaced with genes encoding SIV Env (gp160) and SIV Gag.  Thus the virus is dependent on functional Env for replication. 
•Cells infected with VSV-SIV chimeras bud virus-like particles (VLPs) and VSV bullets contain Env.
•VSV-SIV chimeric vaccines can be transiently pseudotyped with G to increase uptake of the vaccine dose.
•Immunogenicity of chimeric VSVΔG-SIV GagEnv  and G pseudotyped chimeras were evaluated after intramuscular or mucosal immunization  of non-human primates.
•SIV-Gag and -Env serum antibody titers determined by multiplex microsphere immunoassay.
•Neutralizing antibodies were evaluated using pseudoviruses prepared with SIV Env
Figure 1. Schematic of VSV-SIV vector constructs.
Genes encoding SIV Gag and Env were inserted into the 4th and 5th positions respectively. Infection of permissive cells results in the budding of VSV bullets coated in Env trimers and VLPs containing both Gag and Env. Transient transfection of VSV chimera with VSV G results in VSV bullets expressing both surface Env trimers and VSV G.
Figure 2A & 2B. VSV-SIV vector immunization induces  humoral responses. Sera from vaccinated and control NHPS were analyzed for antibodies against SIV Env (A) and Gag (B) following VSV immunization and Ad5 boost. Env Abs were detected in the 108pfu group after each VSV immunization. After  Ad5 there was a significant increase in Env Abs titers in the 106pfu group and a further increase in titers in the 108pfu group. Gag Abs were induced only following the  Ad5 boost.
Figure 2C. rVSV∆G-SIVGagEnv induces neutralizing antibodies.  The neutralization activity of serum of vaccinated and control NHPs was tested in a TZMbl assay. During the VSV immunization phase, neutralizing Abs were detected against a Tier 1A pseudovirus in the 108pfu group.   Following Ad5 we observed a significant increase in neutralizing Abs in the 106pfu group. Neutralizing Ab titers were detected at similar levels in the 108pfu group following the Ad5 boost. No significant titers could be detected in either group against Tier 2 and 3 SIV pseudoviruses.
Figure 3A & 3B. Mucosal delivery of VSV vectors induces humoral responses. At various time points post 1st and 2nd VSV immunizations sera from immunized and control NHPs was tested for Abs against SIV Env and Gag. High titer Env Abs were detected in the VSV G pseudotyped group (red) which further increased following Ad5 boosting. Low level Env Abs could be detected in the VSVGagEnv group (green) following VSV and these were significantly boosted following Ad5. Gag Abs were detected only after boosting with Ad5.
Figure 4A, 4B & 4C. Mucosal immunization with VSV vectors induces neutralizing antibodies.  Following VSV vector immunization and Ad5 boost sera from all NHPs was analyzed for the presence of neutralizing Abs.  Neutralizing Abs against Tier 1A and Tier 2 pseudoviruses could be detected in both VSVGagEnv (green) and VSV G pseudotyped (red) groups following VSV. In the VSV G pseudotyped group, Abs titers increased significantly against both Tier 1A and Tier 2 compared to the control group (blue). Titers remained constant following the boost against Tier 1A for the VSVGagEnv group, however an increase in titers was seen against Tier 2 pseudoviruses. Neutralizing Ab titers could detected against the Tier 3 pseudovirus SIVmac251.30 in both groups following VSV vector immunization and after Ad5 boost. DOC=day of challenge
Figure 5. Humoral immunity induced by VSV vectors does not affect SIVmac239 replication.  At 31 weeks post 1st VSV immunization all NHPs entered into a repeated low dose intrarectal challenge. NHPs were challenged biweekly with 150TCID50  of pathogenic SIVmac239 until they were confirmed infected by PCR. NHPs were monitored weekly for changes in virus load. At the end of 8 intrarectal challenges, 100% of NHPs were confirmed infected in the VSVGagEnv group, 66% in the VSV G pseudotyped  and control groups. No significant difference in peak or set point viral load could be observed between groups.
•VSV vectors are safe in Rhesus macaques when delivered at high doses. No adverse events occurred following  the intramuscular (IM) or intranasal/sublingual (mucosal) immunization with either rVSV∆G-SIVGag4Env5∆CT or VSV-G pseudotyped-rVSV∆G-SIVGag4Env5∆CT vectors.
•VSV vectors elicit strong anti-Env antibody responses following IM or mucosal immunization which can be further enhanced after boosting with Ad5-SIVEnv and –SIVGag.
•VSV vector immunization via IM and mucosal route induces neutralizing antibodies to Tier 1A (IM and mucosal), Tier 2 (mucosal) and  Tier 3 (mucosal) pseudoviruses.
•The humoral  immunity elicited by VSV vectors, both binding and neutralizing antibodies are unable to control infection of the highly pathogenic virus strain SIVmac239.
This research was supported by The Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Discovery and donors that support IAVI.   The views and conclusions expressed in this poster are those of the authors and do not necessarily reflect the opinions of donors or funding agencies.  We also thank David Montefiori’s lab for providing reagents and protocols for conducting  SIV pseudovirus neutralization assays.
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