Intensified Efforts toward HIV Vaccine
Report from the Keystone Joint Symposia on HIV Pathogenesis and HIV Vaccines
From March 25-30, 2007, nearly 800 HIV researchers gathered in skier’s paradise --Whistler, British Columbia, Canada-- for the Keystone Joint Symposia on HIV Pathogenesis and HIV vaccines. This report highlights some of the work relevant to HIV vaccine development discussed at this meeting.
Simian Studies Offer Hope
Vaccine testing in monkeys weeds out poorly immunogenic vaccines sooner and moves immunogenic vaccines forward quicker. Monkeys vaccinated with SIVmac239Δ3 (missing Nef, Vpr, LTR genes) contain the matched virus (SIVmac239) challenge by generating broad CD4+ and CD8+ T cell responses and neutralizing antibodies targeting multiple epitopes. Dr. David Watkins at the University of Wisconsin in collaboration with IAVI, tested if this vaccine can also be effective against a SIV strain that differs from the vaccine strain. Ten vaccinated monkeys were challenged six months later intravenously with SIVsmE660. Compared to control animals, immunized animals suppressed peak plasma viremia levels by 2 log values (~105 viral RNA copies/mL). Vaccinated animals expressing Mamu B*08 and B*17 alleles (n=4) exerted better control over SIVsmE660 replication, with one animal reducing viral load to <200 vRNA copies/ml at set-point. In this animal massive expansion of B*08 epitopes Vif RL9 and Env KL9 occurred after challenge. In ELISPOT assays, CD4+ T cells from vaccinated animals responded against 4 Gag epitopes. Thus, adaptive responses may control acute infection as the infection rages uncontrolled in the nonvaccinated Mamu B*08 and B*17 animals. This work in monkeys suggest that vaccine induced protective immune responses in humans may depend on the HLA genotype of the vaccinees.
However other vaccine studies belie this suggestion. Dr. Marjorie Robert-Guroff at the National Cancer Institute presented two studies that demonstrated the efficacy of replication-competent Ad5-based vaccine platform in controlling SIV infection in monkeys. Previously Guroff’s team had primed monkeys with replication competent Ad5-SIV expressing multiple SIV genes (Env, Rev, Gag, Nef) and boosted with envelope protein. Among 11 monkeys that survived the first challenge study, 8 animals controlled a second SIVmac251 challenge one year after the first encounter with the virus. Viral load soared when the vaccinees were depleted of CD8+ T cells but reversed when CD8+ T cells were restored (Malkevitch et al. Virol 353, 83, 2006). So the vaccine imparted enduring immune responses in the animals without influence from their Mamu alleles.
Marchio et al. (Blood 105, 2802, 2005) showed that Tat released into the extracellular milieu by HIV-infected cells promotes viral spread into neighboring cells through its interaction with gp120. In fact, Tat antibodies with antibody dependent cellular cytotoxicity (ADCC) activity target Tat-coated cells in vitro. Guroff reasoned that priming the immune system to recognize Tat may also fend off a full-blown HIV infection (Demberg et al. J. Virol. 81, 3414, 2007). A multigenic combination of Tat/Env or Tat/Env/Gag/Nef significantly reduced the chronic viral load by 3-4 log values, but SIV Tat alone failed to protect monkeys against SHIV89.6P infection. High titers (1:1000) of Tat and Env antibodies with ADCC activity detected postchallenge as well as effector and central memory T cell responses to SIV antigens may have contributed to viremic control in vaccinated animals.
Vulnerable Spots on HIV
To disable the virus prior to its entry into host cells, a vaccine will need to provoke viral envelope binding neutralizing antibody production by B-cells, along with T cell responses. This is easier said than done. Through creative strategies researchers are engineering viable candidates that elicit neutralizing antibodies in humans.
The envelope spikes on the viral membrane exhibits unusual flexibility prior to engagement with CD4, so the antibody binding sites are often masked. After binding CD4, the envelope becomes more rigid. By introducing three cavity filling amino acid residues and four disulfide bonds, Dr. Peter Kwong at the National Institute of Allergy and Infectious Diseases and his colleagues constrained the gp120 monomer with disulfide bonds, thus compelling the molecule to expose the initial CD4 contact site, an area susceptible to neutralizing antibodies. This stabilized form binds to CD4, CD4-induced antibodies and the broadly neutralizing antibody IgGb12 (Nature 445, 732, 2007). Identifying a vulnerable site on the envelope surface recognized by antibodies constitutes the first step in vaccine design. Designing immunogens is the second step.
Hunting the protein database, Kwong’s team found a few proteins that contained segments that bore structural likeness to the b12 epitope. The researchers modified these proteins to more closely resemble the b12 binding site on gp120. The ability of these hybrid proteins to generate b12-like neutralizing antibodies is under testing in guinea pigs. A similar exercise is also being performed for the binding site recognized by another broadly neutralizing antibody, 2F5 that recognizes a conserved site on gp41.
Dr. Ian Wilson at Scripps Research Institute focused on sites bound by two broadly neutralizing antibodies, 2G12 and 4E10. 2G12 binds to dense clusters of mannose structures (oligomannose) on the surface of gp120. Oligomannose complexes containing 8 or 9 mannoses residues bound tightly to 2G12 (Calarese et al. PNAS 102, 13372, 2005). Dendrimers –branched tree-like molecules– displaying an array of 27-29 Man9 clusters showed high affinity interactions with 2G12. Additionally BSA and Qb bacteriophage are also being used as rigid base for displaying oligomannoses. A glycan array based assay will be used to rapidly screen for carbohydrate immunogens. 4E10 binds to a helical segment on gp41 defined by the core residues NWFDIT in the membrane proximal region (MPER). The peptide was locked into a-helical shape by adding up to 9 amino acids to the core on the C-terminal side and introducing either a-aminobutyric acid or linking cysteine and ornithine side chains as a cyclic thioether (Brunel et al. J Virol. 80, 1680, 2006; Cardoso et al. J Mol Biol. 365, 1533, 2007). Extending the sequence and constraining the peptide enhanced its affinity for 4E10. The helical epitope was grafted into gp120 by replacing V1/V2 loop with the MPER. Rabbits injected with the resultant antigen as a DNA prime/protein boost regimen elicited neutralizing antibodies, none of which bound the graft peptide (Law et al. J Virol. 81, 4272, 2007). The poor immunogenicity of the 4E10 epitope could be attributed to its high hydrophobicity that might cause aggregation in vivo, to tolerance, or need for the peptide to be presented as a trimer or in liposomes. So despite our knowledge of epitope sequence and structure, structure-based vaccine designers face significant hurdles.
Pursuing New Viral Vectors
Vectors deliver immunogens to the body safely to build immune defenses against a disease causing organism. All promising SIV vaccines constructed to date fail to impart sterilizing immunity as the vaccine generated SIV-specific T cells are short-lived. Dr. Ronald Desrosiers at Harvard Medical School and Dr. Louis Picker at Oregon Health and Science University probed if antigen persistence would improve vaccine efficacy. Desrosiers’ group picked herpesvirus and Picker’s team opted for cytomegalovirus (CMV) as delivery vehicles for HIV vaccines due to their ability to accommodate large gene inserts.
Ron Desrosiers and coworkers used monkey herpesvirus, rhesus macaque rhabdinovirus (RRV) to express SIV genes Gag, Env, or a fusion of Rev, Tat, Nef (Retanef). In the 5 immunized monkeys that received a single shot of RRV-SIV constructs, 3-5% of CD8+ T cells recognized gag at 15 weeks post-vaccination. But anti-SIV Env antibodies were undetectable despite protein synthesis from SIV genes. Vaccinated animals challenged (18 weeks post-vaccination) with SIVmac239 lowered their viral load by 37 fold (2 weeks post-challenge) compared to non-immunized animals. Following challenge a rapid rise in anti-SIV antibodies occurred. Since Gag expression under CMV promoter control was robust, so CMV promoter now drives production of all SIV genes. But the safety of human herpesvirus, KSHV as vaccine vector and effect of prior immunity has yet to be tested.
As CMV quintessentially induces CD4+ and CD8+ effector memory T cells, Louis Picker’s crew expressed SIV Gag, Env or Retanef in CMV vector. Monkeys were vaccinated sequentially with the three genes. In the immunized monkeys, SIV-specific T cells account for 6.7% of the total CD8+ T cell population. A large proportion of these cells release IL-2, TNF-a, and IFN-g. Even 18 months later, CMV vector can be detected in animals. Picker suggested that combining the CMV vaccine with Ad5-based vaccines may induce robust protective immune responses.
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