Important note: Information in this article was accurate in September/ November 2000. The state of the art may have changed since the publication date. 
A newcomer to the AIDS vaccine meeting scene, the "First International Conference on Vaccine Development and Immunotherapy" was held in Palm Beach, Florida from 28 June - 1 July. Sponsored by the International Medical Press and IAVI, it attracted about 150 researchers. Much of the focus was on vaccine design and testing, including novel vectors and macaques studies, and on immune evasion and correlates of protection. There was also a presentation on the VaxGen trial (see coverage in Durban report, p.3) and several reports about therapeutic vaccines.
Following are selected highlights from the meeting.
Mark Newman from Epimmune (San Diego) presented a novel approach to designing DNA vaccines against epitopes that are conserved in different HIV subtypes and recognized by multiple, common class I-restricted HLA alleles (to maximize immunogenicity across diverse populations).
To identify such epitopes, they began by scanning the sequences of different HIV-1 strains to find regions containing the known epitope-binding motif for three common class I HLA alleles (HLA-A2, A3 and B7). Motifs common to many virus strains were then synthesized as peptides of 8-11 amino acids and screened to find those which bound well to the HLA proteins. The 43 (out of 1000) selected peptides were then tested for immunogenicity in mice and humans, the latter according to whether they were recognized by CTLs from HIV-infected people with HLA-A2 or A3 phenotypes. Newman estimated that the resulting 43 peptides would be immunogenic in 87% of the general population.
He also predicted that this type of vaccine would require at least 16 CTL epitopes (2 Gag, 8 Pol, 3 Env, 1 Nef, 2 Vpr) for a single HLA haplotype, or 48 epitopes for three haplotypes (needed to achieve coverage across many populations). Epimmune has prepared a 20-epitope vaccine and is working towards one with 40.
One concern about prospects for an HIV vaccine is the virus' ability to mutate rapidly and thereby escape host immune responses. But there have been few documented cases of this, especially when it comes to cellular immunity. Here Todd Allen of the Wisconsin Regional Primate Center (Madison, WI) reported a clear case of CTL escape, which revolved around a previously undefined epitope in the Tat protein. He also showed that it occurred several weeks earlier than other known cases. (The work is now published in Nature 2000 Sep 21;407(6802):386-90).
Allen's team found that when 10 Mamu-A*01-positive rhesus macaques were infected with the pathogenic SIVmac239, they generated CTL responses against four out of six CTL epitopes tested (all from Gag or Tat). But 8 weeks later, all 10 animals harbored SIV variants with mutations in the same Tat epitope; in 5 animals, this variant had completely replaced the original strain. Complete viral sequencing from two animals at 4 weeks showed that, with one exception, the changes in Tat were the only ones present. In Mamu-A*01-negative macaques infected with SIV, 4/8 also showed Tat variants.
Allen also reported that the presence of variants correlated with initial control of viremia. By week 4, when wild-type virus began to be replaced by variant, all Mamu-A*01-positive and negative animals harboring Tat variants had begun to clear their initial, acute viremia. But the Mamu-A*01-negative animals without Tat variants were much less successful: 2/4 of these animals progressed to simian AIDS within 6 months, the only group to do so. Presumably this is because without CTL directed to Tat, there was neither immune pressure to generate escape variants, nor could the animals control viremia as well. Allen concluded that rapidly escaping CTL epitopes such as this one represent promising vaccine targets. (For more on Tat-based vaccines, see articles on the "Gallo Meeting," and "Debate on Tat").
Ron Desrosiers' group at Harvard Medical School has developed several classes of mutant SIVmac239 viruses that are more sensitive to neutralization and show altered properties in monkeys compared with the wild-type strain (which is difficult to neutralize). Their findings could therefore have important implications for vaccine design.
One set of mutants was created by removing 2 (out of 7) N-linked carbohydrate attachment sites in the V1-V2 loop of gp120, exposing the underlying protein domains. Macaques infected with one of 3 different mutants of this type (all fully replication-competent) initially showed high viral loads similar to wild-type virus. But by setpoint their load was significantly lower, suggesting that the animals can clear the mutant, neutralization-sensitive virus more effectively.
Similarly, macaques infected with an SIVmac239 strain lacking 5 N-linked carbohydrate attachment sites brought high post-challenge viral loads down to undetectable levels following the appearance of neutralizing antibodies. These monkeys were protected when challenged with SIVmac251, a highly related but heterogeneous and pathogenic virus strain.
A third class of mutants contained a 101 amino acid deletion that removed the entire V1/V2 region, also resulting in a replication-competent virus that is cleared at viral setpoint, and which Desrosiers said is one of the most neutralization-sensitive SIV strains his lab has encountered. When questioned about the potential of this latter mutant as a live attenuated vaccine, he cautioned that it has not been well-characterized in terms of safety, and said that tests are planned in combination with nef deletions, which also attenuate SIV.
Leo Stomatatos from Aaron Diamond AIDS Research Center in New York presented results consistent with Desrosiers' studies. He removed 30 amino acids from the V2 loop of gp140 in a primary HIV isolate. Two rhesus macaques immunized with env-DNA from this virus (and boosted with Env protein) generated antibody higher titers against gp140 than did wild-type env-DNA. Sera from these monkeys neutralized heterologous primary HIV strains.
Thomas Dubensky of Chiron Corporation (Emeryville, California) described the company's program to develop HIV vaccines based on alphavirus replicon particles, in particular Sindbis virus. The advantages of these vectors are that they target human dendritic cells (which play a key role in presenting antigen to the immune system) and that they induce more potent T-cell responses compared to conventional vectors. Chiron has isolated Sindbis variants that grow to high titers in human dendritic cells and is using them for prototype vaccine development. The company has also developed efficient packaging cell lines, overcoming a potential stumbling block for future large-scale vaccine production with this vector.
In further describing the HIV-Sindbis particles, Dubensky reported that constructs expressing HIV p55 gag or gp140 env infect immature human and mouse dendritic cells very efficiently in vitro. Moving to small animals, mice immunized intradermally with Sindbis particles showed infected, immature dendritic cells at the site of injection, and these cells migrated to the draining lymph nodes and underwent maturation. Intranasal immunization was also effective, eliciting high-level expression of CTLs systemically and in draining lymph nodes. Chiron researchers are now testing a variety of vector constructs (expressing env and gag) in prime-boost regimens in 44 macaques.
David Knipe of Harvard Medical School presented rhesus macaque data on two herpes simplex viruses (HSV-1) engineered to express SIV Env and Nef proteins. Interest in this approach stems from the fact that humans maintain persistent immune responses against herpes viruses, so HSV vectors may offer a way to generate long-lasting immunity. Knipe also speculated on their potential for a dual herpes-HIV vaccine, since replication-defective HSV strains have protected mice against HSV challenge.
Here he showed data on 7 macaques immunized (and boosted twice) with either a replication-competent construct, called K81, or the replication-deficient d81, plus 3 control animals. All animals were challenged rectally with homologous SIVmac239 22 weeks after the last boost. At 40 weeks post-challenge, 2 of the 7 immunized animals were completely protected (based on absence of viremia, viral p27 antigen and recoverable virus). Overall, peak viral load levels in immunized animals were 1.2 logs lower than control animals, a statistically significant difference. (These data are now published in J Virol 2000 Sep;74(17):7745-54).
Knipe's group is now immunizing monkeys with a second-generation HSV-2 recombinant that expresses higher levels of SIV proteins. He also reported that Avant Immunotherapeutics, a Massachusetts-based biotechnology company, is developing his modified HSV-2 strain as a genital herpes vaccine.
Some researchers in the audience expressed concern that vaccination with HSV vectors might result in latent infection. Knipe hasn't fully tested this premise but noted that no viral DNA was detected in mice injected with double-mutant HSV-2 viruses.
Jeffrey Ulmer of the Chiron Corporation discussed potential ways to enhance the generally weak immune responses elicited by DNA vaccines. These include modifying vectors to target antigens to specific cellular compartments or using novel adjuvants. But even with improved vaccine designs, Ulmer said, inefficient uptake of antigen by host cells could result in sub-optimal vaccines.
In one approach to improving DNA delivery, Chiron is investigating PLG microparticles pre-coated with a positively-charged surfactant that can then absorb negatively charged molecules like DNA (or proteins, virus particles or vectors). Animal studies show some promise. In one set of experiments, modified PLG particles adsorbed with HIV-gag DNA were used to immunize mice, which were then challenged with a lethal dose of vaccinia expressing HIV-gag. The result was 100-fold more potent CD8 T-cell responses, and 100-1000-fold more antibodies, than animals immunized with naked DNA alone. In macaques, animals primed with p55gag-DNA on PLG and then boosted with p55 protein on PLG showed both humoral and cellular responses, while immunization with only p55 protein on PLG failed to induce CTL. Ulmer also noted that DNA-PLG particle delivery targets antigen-presenting cells.
Vicki Burkitt is a U.S.-based science writer whose work has appeared in GMHC Treatment Issues, HIV Plus and other publications.
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