sites were amplified from plasma RNA of all SIVmac239 pos- itive samples. The amplified PCR products were sequenced with. 454 pyrosequencing technology.
1
National Microbiology Laboratory, National HIV and Retrovirology Laboratory, Winnipeg, MB, Canada, 2University of Manitoba, Medical Microbiology, Winnipeg, MB, Canada, 3 National Microbiology Laboratory, Winnipeg, MB, Canada, 4 University of Santiago de Compostela, Santiago de Compostela, Spain, 5University of Nebraska-Lincoln, Nebraska-Lincoln, NE, United States Background: HIV-1 protease mediates the cleavage of Gag, Gag-Pol and Nef precursor polyproteins in a highly specific and temporally regulated manner. Because a total of 12 cleavage reactions are required to generate a mature virion, generating focused immune response targeting the sequences surrounding the protease cleavage sites (PCS) could drive viral mutations to its disadvantage. We have conducted a proof of concept study with Cynomolgus macaques and pathogenic SIVmac239 as a model and used a modified recombinant vesicular stomatitis vector and nanocarriers to deliver 12 20-amino acid antigens. We showed that a vaccine targeting the sequences surrounding the 12 protease cleavage sites is promising at prevention of HIV-1 infection and disease progression. In this study we systematically analyzed breakthrough viruses of vaccinated and control macaques. Methods: The sequences surrounding the 12 protease cleavage sites were amplified from plasma RNA of all SIVmac239 positive samples. The amplified PCR products were sequenced with 454 pyrosequencing technology. The amino acid and frame shift mutations were analyzed and correlated with viral load and CD4 counts. Regression analysis was conducted to correlate the viral mutations with viral load and CD4 counts. WebLogo was used to plot the amino acid mutations. Results: Extensive mutations were detected around PCS and both conserved and non-conserved mutations are correlated with lower viral load (p < 0.0001). The breakthrough viruses from the vaccinated macaques carry significantly higher mutations than the controls. Longitudinal analysis revealed that the high rate of non-conserved and conserved amino acid mutations along the sequences surrounding the PCS lead to the reduction and diminishing of viral load. Conclusions: The pathogenic SIVmac239 is extremely vulnerable to any amino acid alternations around PCS and focusing immune response to sequences surrounding the PCS of HIV-1 can drive amino acid mutations and lead to complete viral control.
Background: HIV-1 protease mediates the cleavage of Gag, Gag-Pol and Nef precursor polyproteins in a highly specific and temporally regulated manner. Because a total of 12 cleavage reactions are required to generate a mature virion, generating focused immune response targeting the sequences surrounding the protease cleavage sites (PCS) could drive viral mutations to its disadvantage. We have conducted a pilot study to investigate the feasibility and effectiveness of a vaccine targeting the sequences around the 12 PCS using Cynomolgus macaques and pathogenic SIVmac239 as a model. Methods: Twelve recombinant VSVpcs were used to immunize 12 Cynomolgus macaques and nanopackaged PCS peptides were used as a boost. The immunized macaques and 6 controls were repeatedly challenged intrarectally with an increased dosage of SIVmac239. Antibody and T cell responses to the PCS peptides, CD4 + and CD8 + T cell counts and challenge dosage were monitored. 454 Pyrosequencing was conducted to analyze break-through viruses and the amino acid mutations surrounding the PCS sites were correlated with viral load. Results: Antibody and T cell responses to the 12 PCS protected macaques against higher dosage of SIVmac239 intrarectal challenge (p = 0.005, R = 0.42). The vaccine group maintained higher CD4 + counts (p = 0.0002) than the controls weeks after being infected. Analysis of viral mutations around 12 PCS of 276 samples (14 to 20 sampling points/monkey) detected extensive mutations. These mutations, both conserved and nonconserved amino substitutions around PCS, are correlated with lower viral load (p < 0.0001). Conclusions: Our study with nonhuman primates and pathogenic SIVmac239 as a model showed that a vaccine targeting the sequences surrounding the 12 protease cleavage sites is promising at prevention of HIV-1 infection and disease progression. It demonstrated that the pathogenic SIVmac239 is extremely vulnerable to any amino acid alternations around PCS. Targeting PCS of HIV-1 could be an effective vaccine approach.
P41.19 Antibodies to CD52g, a Secreted Sperm-coating Antigen, Agglutinate Seminal Leukocytes and Prevent their Infiltration into Vaginal Epithelium Jai G. Marathe1, Joseph Politch2, Ayesha Islam2, Kevin Whaley3, Thomas Moench4, Deborah J. Anderson5 1
David Tang1, David La1, Rupert Capina1, Xin-Yang Yuan1, Jorge Correia-Pinto2, Cecilia Prego2, Maria Alonso2, Christina Barry3, Richard Pilon1, Christina Daniuk1, Mikaela Nykoluk1, Stephane Pillet3, Tomasz Bielawny1, Jeffrey Tuff1, Chris Czarnecki1, Philip Lacap1, Gary Wong3, Shaun Tyler3, Ben Liang3, Zhe Yuan4, Qingsheng Li4, Terry Blake Ball1, Paul Sandstrom1, Gary Kobinger3, Francis Plummer3,5, Ma Luo1,5 1
Boston University, Medicine, Boston, MA, United States, Boston University, Obstetrics and Gynecology, Boston, MA, United States, 3Mapp Biopharmaceutical, Inc., San Diego, CA, United States, 4ReProtect Inc., Baltimore, MD, United States, 5 Boston University, Obstetrics and Gynecology, Medicine, Microbiology, Boston, MA, United States 2
P41.18 Sequences Surrounding the 12 Protease Cleavage Sites are Good Targets for Both Prophylactic and Therapeutic HIV Vaccines
National Microbiology Laboratory, National HIV and Retrovirology Laboratory, Winnipeg, MB, Canada, 2University of Santiago de Compostela, Santiago de Compostela, Spain, 3 National Microbiology Laboratory, Winnipeg, MB, Canada, 4 University of Nebraska-Lincoln, Nebraska-Lincoln, NE, United States, 5University of Manitoba, Medical Microbiology, Winnipeg, MB, Canada
Background: Our program is studying the topical use of monoclonal antibodies (mAbs) for contraception and HIV prevention. mAbs directed against CD52g, an antigen secreted into the male genital tract and inserted into sperm membranes, potently agglutinate sperm and are being developed for contraceptive use. We are also seeking strategies to prevent cellassociated HIV transmission mediated by HIV-infected seminal white blood cells (sWBC). In this study, we investigated whether CD52g also attaches to sWBC, and whether anti-CD52g mAbs agglutinate these cells and/or inhibit their interaction with the vaginal epithelium. Methods: Dylight 633-conjugated MSH8, a mouse anti-CD52g mAb (gift of J. Herr), was used in flow cytometry experiments to
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