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CDV vectors as vaccine candidates

CDV-SIV Vectors as Vaccine Candidates
Gavin Morrow, Jason Zhang, Arban Domi, Olivia Wallace, John Coleman, Palka Sharma, Rebecca Powell, Ian Ouellette, Beth. Rasmussen, C. Richter King, Christopher Parks (International AIDS Vaccine Initiative (IAVI), AIDS Vaccine Design and Development Laboratory, Brooklyn New York, USA 11220)
The development of a live attenuated HIV vaccine is not considered to be an option at this time. Our approach is to use unrelated live viruses as vectors to deliver an AIDS vaccine by developing a replication-competent, recombinant canine distemper virus vectors expressing HIV genes. As a proof of concept we developed two rCDV vectors containing simian immunodeficiency virus (SIV) genes encoding Gag and Env proteins (CDV-SIV).
Non-human primates were immunized intranasally (IN) at weeks 0 and 6. Two animals received CDV-SIV vectors at 10^7pfu and a control animal received empty CDV vector.  All animals received an Ad5-SIVGag and Ad5-SIVEnv boost and were subsequently challenged with SIVmac239 by intrarectal route.
CDV/SIV genome copies could be detected in intestinal tissue, cells from bronchalveolar lavage and in the oral cavity (4 weeks post 2nd immunization). Low level Env specific T-cell responses were detected after each CDV immunization. Env antibodies were detected following the 1st CDV immunization and were further boosted following 2nd CDV immunization. After Ad5-SIV boost, a robust increase in Env and Gag specific T-cell responses was seen in both BAL and PBMCs. Env antibody titers increased and Gag antibodies were detected following Ad5-SIV. All animals were intrarectally challenged with increasing doses of pathogenic SIVmac239. One immunized monkey became infected after one challenge. The remaining animals, resistant to two subsequent IR challenges, were ultimately infected intravenously. One CDV-SIV immunized NHP showed a peak viral load 3 logs lower than the control and although being infected both the immunized NHPs have maintained low viral loads for several months compared to the control animal.
rCDV vectors were shown to be safe and induce immune responses in a prime/boost regimen resulting in sustained control of SIV replication to low levels. A repeat study is currently underway to investigate these results further.
•An alternative strategy to live-attenuated HIV vaccines is to express HIV immunogens using live viral vectors that can mimic an HIV infection.
•CDV is being investigated as a vector because it naturally infects via mucosal surfaces with subsequent spread to lymphoid cells (1).
•CDV is a member of the morbillivirus genus in paramyxoviridae family. Unlike measles virus (MV), a related morbillivirus, CDV causes diseases in a variety of carnivores, but is not associated with any human illness (2).
•The CDV genome is a single-stranded, non-segmented, negative-sense RNA that contains six transcription units arranged 3’-N-P-M-F-H-L-5’ (3). Foreign genes were introduced into the CDV genome as additional transcriptional units using established reverse genetics and recombinant virus rescue systems (4, 5).
•By delivering CDV vectors mucosally we hope to stimulate mucosal immunity, and as the vector replicates in vivo, deliver immunogens to sites of HIV replication.
•We have developed rCDV vectors that express SIV Env and Gag proteins.
•Our previous studies in ferrets have demonstrated that CDV delivered mucosally is both safe and immunogenic.

•The Onderstepoort canine vaccine strain of CDV was used as the genetic background for vector construction. The vaccine virus was adapted to Vero cells after which a clonal isolate was derived and a genomic cDNA was prepared. Modified genomic clones were constructed with SIV Env inserted into the 6th position (A) or Gag in the 1st position (B).
•Multiplex Microsphere Immunoassay to determine Env and Gag antibodies in NHP serum: Env gp130 and Gag p55 proteins were conjugated to Bio-plex COOH microbeads (Bio-Rad). Conjugated microbeads were incubated with NHP serum to capture Env and Gag antibodies.  Captured antibodies were detected using biotin-conjugated anti-monkey IgG followed by Streptavidin-R-Phycoerythrin. Samples were read using a Bio-Plex 200 System (6).
•Interferon-γ (IFNγ) ELISPOT Assay: PBMCs isolated from peripheral blood and bronchoalveolar lavage fluid were cultured in wells coated with anti-IFNγ and incubated overnight with SIV-Env or SIV-Gag peptide pools. Captured IFNγ was detected using a HRP labeled secondary Ab and TMB substrate and the resulting spots quantified.
•Quantification of viral loads in plasma from SIV-infected macaques: RNA was extracted from plasma and SIV RNA genome copy numbers were quantified by RT-qPCR. Gag specific primers and probes were used for viral genomic RNA amplification and detection ( 7).  
Figure 1. Schematic of rCDV-SIV vector genomes. Genes encoding a soluble Env (Env∆TMCT) was inserted in the 6th gene position in the CDV genome. Gag with a wt myristoylation signal was inserted in the 1st gene position.  Viral mRNA synthesis is highest from position 1 and decreases downstream.
Figure 2. Pilot NHP study for evaluation of CDV-SIV vaccination
Two NHPs were immunized with 107pfu of rCDV-SIVEnv∆TMCT and rCDV-SIVGagMyr+ delivered intranasally followed by intramuscular boost with 2x1010pfu of Ad5-Env and Ad5-Gag vectors.  A third NHP was vaccinated with CDV-empty followed by Ad5-Gag and Ad5-Env.  NHPs were challenged with pathogenic SIVmac239 at a starting dose of 150TCID50. NHPs that remained uninfected after 3 mucosal challenges were infected by intravenous inoculation with 600TCID50.
Figure 3. CDV-SIV immunization induces humoral immune responses to SIV inserts. Sera was taken from immunized NHPs at various time points post CDV-SIVGag/Env prime, Ad5-SIVGag/Env boost and SIV challenge and analyzed for antibodies to SIV-Env and -Gag inserts. After one immunization Env Abs could be detected, which were further boosted after a 2nd CDV immunization. Ad5 boosting further increased Env Ab titers. Gag Abs were detected after Ad5 boosting.  Upon SIV infection there is a sharp increase in both Env and Gag Ab titers.
Figure 4. CDV-SIV prime/Ad5-SIV boost induces SIV specific T-cell responses in peripheral blood.
At various time points following CDV-SIVGag/Env prime and Ad5-SIVGag/Env boost PBMCs were used in a IFNγ ELISPOT assay to determine the level of SIV-Gag and –Env specific T cell responses using a single Gag and 3 Env peptide pools. Low level IFNγ  T cell responses could be observed following CDV immunization, however, these responses were significantly increased following Ad5-SIV administration.
Figure 5. CDV-SIV prime/Ad5-SIV boost induces SIV specific T-cell responses in brochoalveolar lavage (BAL) fluid.
At various time points following CDV-SIVGag/Env prime and Ad5-SIVGag/Env boost BAL was taken and implemented in a IFNγ ELISPOT assay to determine the number of SIV-Gag and –Env specific T cell using a single Gag and 3 Env peptide pools. Low level IFNγ  T cell responses could be seen in the Env peptide pools after CDV immunization, however, these responses were significantly increased following Ad5 administration, particularly against the Gag and Env 2 peptide pools.
Figure 6. Reduced SIVmac239 virus load in NHPs vaccinated with CDV-SIV/Ad5-SIV prime/boost regimen.
All NHPs were intrarectally challenged with an escalating doses of SIVmac239 until they were infected or intravenously challenged to ensure infection. One CDV-SIV+Ad5-SIV NHP (blue) infected by the IR route showed a decrease in peak viral load and maintained a low setpoint viral load out to 40 weeks post-infection.  The other 2 NHPs were infected IV after which a high peak viral load was observed. The CDV-SIV+Ad5-SIV NHP (red)  controlled virus for greater than 10 weeks before an increase in viral load occurred.  The NHP vaccinated with CDV-empty/Ad5-SIV (green) had a significantly higher setpoint virus load compared to the 2 CDV-SIV+Ad5-SIV vaccinated NHPs .
•rCDV-SIVEnv∆TMCT and rCDV-SIVGagMyr+ can be administered IN to NHPs without observing adverse reactions.
•CDV-SIV vectors induce humoral immune responses that are boosted by Ad5-SIV.
•CDV-SIV vectors induce cellular immune responses that are boosted by Ad5-SIV vectors.
•CDV-SIV vector prime/ Ad5-SIV boost appears to induce immunity that can significantly decrease SIVmac239 replication.  Studies with larger numbers of animals will be necessary to confirm this preliminary result.
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