Scientific Publications

With the human immunodeficiency virus type 1 (HIV-1) epidemic expanding at increasing speed, development of a safe and effective vaccine remains a high priority. One of the most central vaccine platforms considered is plasmid DNA. However, high doses of DNA and several immunizations are typically needed to achieve detectable T-cell responses. In this study, a Semliki Forest virus replicon DNA vaccine designed for human clinical trials, DREP.HIVA, encoding an antigen that is currently being used in human trials in the context of a conventional DNA plasmid, pTHr.HIVA, was generated. It was shown that a single immunization of DREP.HIVA stimulated HIV-1-specific T-cell responses in mice, suggesting that the poor immunogenicity of conventional DNA vaccines may be enhanced by using viral replicon-based plasmid systems. The results presented here support the evaluation of Semliki Forest virus replicon DNA vaccines in non-human primates and in clinical studies.

The elicitation of broadly neutralizing antibodies directed against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, gp120 and gp41, remains a major challenge. Attempts to utilize monomeric gp120 as an immunogen to elicit high titers of neutralizing antibodies have been disappointing. Envelope glycoprotein constructs that better reflect the trimeric structure of the functional envelope spike have exhibited improved immunogenicity compared with monomeric gp120. We have described soluble gp140 ectodomain constructs with a heterologous trimerization motif; these have previously been shown to elicit antibodies in mice that were able to neutralize a number of HIV-1 isolates, among them primary isolate viruses. Recently, solid-phase proteoliposomes retaining the envelope glycoproteins as trimeric spikes in a physiologic membrane setting have been described. Here, we compare the immunogenic properties of these two trimeric envelope glycoprotein formulations and monomeric gp120 in rabbits. Both trimeric envelope glycoprotein preparations generated neutralizing antibodies more effectively than gp120. In contrast to monomeric gp120, the trimeric envelope glycoproteins elicited neutralizing antibodies with some breadth of neutralization. Furthermore, repeated boosting with the soluble trimeric formulations resulted in an increase in potency that allowed neutralization of a subset of neutralization-resistant HIV-1 primary isolates. We demonstrate that the neutralization is concentration-dependent, is mediated by serum IgG and that the major portion of the neutralizing activity is not directed against the gp120 V3 loop. Thus, mimics of the trimeric envelope glycoprotein spike described here elicit HIV-1-neutralizing antibodies that could contribute to a protective immune response and provide platforms for further modifications to improve the efficiency of this process.

The conserved membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 is a target of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10, and is an important lead for vaccine design. However, immunogens that bear MPER epitopes so far have not elicited neutralizing antibodies in laboratory animals. One explanation is that the immunogens fail to recreate the proper molecular environment in which the epitopes of 2F5 and 4E10 are presented on the virus. To explore this molecular environment, we used alanine-scanning mutagenesis across residues 660 to 680 in the MPER of a pseudotyped variant of HIV-1(JR-FL), designated HIV-1(JR2), and examined the ability of 2F5 and 4E10 to neutralize the Ala mutant viruses. The results show that the only changes to produce neutralization resistance to 2F5 occurred in residue D, K, or W of the core epitope (LELDKWANL). Likewise, 4E10 resistance arose by replacing one of three residues; two (W and F) were in the core epitope, and one (W) was seven residues C-terminal to these two (NWFDISNWLW). Importantly, no single substitution resulted in resistance of virus to both 2F5 and 4E10. Surprisingly, 8 out of 21 MPER Ala mutants were more sensitive than the parental pseudovirus to 2F5 and/or 4E10. At most, only small differences in neutralization sensitivity to anti-gp120 monoclonal antibody b12 and peptide T20 were observed with the MPER Ala mutant pseudoviruses. These data suggest that MPER substitutions can act locally and enhance the neutralizing activity of antibodies to this region and imply a distinct role of the MPER of gp41 during HIV-1 envelope-mediated fusion. Neutralization experiments showing synergy between and T20 and 4E10 against HIV-1 are also presented. The data presented may aid in the design of antigens that better present the MPER of gp41 to the immune system.

Gene transfer vectors based on recombinant adeno-associated virus (rAAV) are simple, versatile, and safe. While the conventional applications for rAAV vectors have focused on delivery of therapeutic genes, we have developed the system for delivery of vaccine antigens. In particular, we are interested in generating rAAV vectors for use as a prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine. To that end, we constructed vaccine vectors that expressed genes from the simian immunodeficiency virus (SIV) for evaluation in the monkey SIV model. After a single intramuscular dose, rAAV/SIV vaccines elicited SIV-specific T cells and antibodies in macaques. Furthermore, immunized animals were able to significantly restrict replication of a live, virulent SIV challenge. These data suggest that rAAV vaccine vectors induced biologically relevant immune responses, and thus, warrant continued development as a viable HIV-1 vaccine candidate.

Efficient immune responses to HIV-1 gene products are essential elements to the development and design of an effective vaccine. Ideally, both humoral and cellular responses will be optimally elicited. It is therefore important to elucidate any factors that might limit the immunogenicity of HIV-1 proteins that are likely to be included in an effective vaccine. Since the HIV-1 exterior envelope glycoprotein gp120 is a major target for neutralizing antibodies, it is a virtual certainty that this gene product will be a component of any vaccine that seeks to elicit neutralizing antibody responses from the host humoral immune system. We report here the testing of several HIV-1 gp120 variants derived from a primary isolate that appears deficient in eliciting immune responses at both the level of CD4+ help and consequently in the generation of high-affinity IgG antibody responses in small animals. Factors limiting an effective immune response include (a) envelope glycoprotein strain variation decreasing functional T-cell help, (b) alteration of the glycosylation patterns of gp120 by expression in different cell types, and (c) the native structure of gp120 itself, which may limit the elicitation of effective T-cell help during natural infection or during parenteral immunization in adjuvant. Such limiting factors and others should be considered in the design and testing of gp120-based immunogens in small animals and possibly in primates as well.

The core of the gp120 glycoprotein from human immunodeficiency virus type 1 (HIV-1) is comprised of three major structural domains: the outer domain, the inner domain, and the bridging sheet. The outer domain is exposed on the HIV-1 envelope glycoprotein trimer and contains binding surfaces for neutralizing antibodies such as 2G12, immunoglobulin G1b12, and anti-V3 antibodies. We expressed the outer domain of HIV-1(YU2) gp120 as an independent protein, termed OD1. OD1 efficiently bound 2G12 and a large number of anti-V3 antibodies, indicating its structural integrity. Immunochemical studies with OD1 indicated that antibody responses against the outer domain of the HIV-1 gp120 envelope glycoprotein are rare in HIV-1-infected human sera that potently neutralize the virus. Surprisingly, such outer-domain-directed antibody responses are commonly elicited by immunization with recombinant monomeric gp120. Immunization with soluble, stabilized HIV-1 envelope glycoprotein trimers elicited antibody responses that more closely resembled those in the sera of HIV-1-infected individuals. These results underscore the qualitatively different humoral immune responses elicited during natural infection and after gp120 vaccination and help to explain the failure of gp120 as an effective vaccine.

CD8(+) T lymphocytes (CD8-TL) select viral escape variants in both human immunodeficiency virus and simian immunodeficiency virus (SIV) infections. The frequency of CD8-TL viral escape as well as the contribution of escape to overall virus diversification has not been assessed. We quantified CD8-TL selection in SIV infections by sequencing viral genomes from 35 SIVmac239-infected animals at the time of euthanasia. Here we show that positive selection for sequences encoding 46 known CD8-TL epitopes is comparable to the positive selection observed for the variable loops of env. We also found that >60% of viral variation outside of the viral envelope occurs within recognized CD8-TL epitopes. Therefore, we conclude that CD8-TL selection is the dominant cause of SIV diversification outside of the envelope.

Infectious diseases are responsible for approximately 25% of global mortality, especially in children aged younger than 5 years. Much of the burden of infectious diseases could be alleviated if appropriate mechanisms could be put in place to ensure access for all children to basic vaccines, regardless of geographical location or economic status. In addition, new safe and effective vaccines should be developed for a variety of infections against which no effective preventive intervention measure is either available or practical. The public, private, and philanthropic sectors need to join forces to ensure that these new or improved vaccines are fully developed and become accessible to the populations in need as quickly as possible.

We have devised a novel approach for producing simian immunodeficiency virus (SIV) strains and, potentially, human immunodeficiency virus type 1 (HIV-1) strains that are limited to a single cycle of infection. Unlike previous lentiviral vectors, our single-cycle SIV is capable of expressing eight of the nine viral gene products and infected cells release immature virus particles that are unable to complete subsequent rounds of infection. Single-cycle SIV (scSIV) was produced by using a two-plasmid system specifically designed to minimize the possibility of generating replication-competent virus by recombination or nucleotide reversion. One plasmid carried a full-length SIV genome with three nucleotide substitutions in the gag-pol frameshift site to inactivate Pol expression. To ensure inactivation of Pol and to prevent the recovery of wild-type virus by nucleotide reversion, deletions were also introduced into the viral pol gene. In order to provide Gag-Pol in trans, a Gag-Pol-complementing plasmid that included a single nucleotide insertion to permanently place gag and pol in the same reading frame was constructed. We also mutated the frameshift site of this Gag-Pol expression construct so that any recombinants between the two plasmids would remain defective for replication. Cotransfection of both plasmids into 293T cells resulted in the release of Gag-Pol-complemented virus that was capable of one round of infection and one round of viral gene expression but was unable to propagate a spreading infection. The infectivity of scSIV was limited by the amount of Gag-Pol provided in trans and was dependent on the incorporation of a functional integrase. Single-cycle SIV produced by this approach will be useful for addressing questions relating to viral dynamics and viral pathogenesis and for evaluation as an experimental AIDS vaccine in rhesus macaques.

HIV entry to host cells begins with binding of the viral envelope protein to CD4 molecules on the host cell surface. This binding initiates conformational changes in the envelope protein that result in binding to a coreceptor (CCR5 or CXCR4), exposure of a previously hidden domain in the viral protein, insertion of a viral fusion peptide into the host-cell membrane and fusing the viral and cell membranes. Each of these steps provides an opportunity for intervention to prevent viral entry, and a number of agents targeting these steps are in development. Studies of coreceptor inhibitors and fusion inhibitors have indicated the presence of host and viral factors that can result in variability of antiretroviral effect. Improved understanding of these factors will help to guide clinical use of these new agents. This article summarizes a presentation by Robert W. Doms, MD, PhD, at the International AIDS Society-USA course in Chicago in May 2004.

Candidate human immunodeficiency virus (HIV) vaccine strategies that induce strong cellular immune responses protect rhesus macaques that are infected with recombinant simian/human immunodeficiency virus SHIV89.6p from acute CD4+ T-cell loss and delay progression to AIDS. However, similar strategies have not proven as efficacious in the simian immunodeficiency virus (SIV)mac model of AIDS, an infection that causes a slow, steady loss of CD4+ T-cell function and numbers in rhesus macaques similar to that caused by HIV-1, the principal cause of AIDS in humans. Efforts to increase vaccine efficacy by repeated boosting with the same vector are quickly limited by rising anti-vector immune responses. Here, the sequential use of three different vectors (DNA, Semliki Forest virus and modified vaccinia virus Ankara) encoding the same SIVmac structural and regulatory antigens was investigated and demonstrated to prevent or slow the loss of CD4+ T-cells after mucosal challenge with the highly pathogenic SIVmac251 strain. Of particular interest was an inverse association between the extent of T-helper 2 cytokine responses and steady-state virus load. Although limited in the number of animals, this study provides important proof of the efficacy of the triple-vector vaccine strategy against chronic, progressive CD4+ T-cell loss in the rigorous SIVmac/rhesus macaque model of AIDS.

We compared the human immunodeficiency virus type 1 (HIV-1)-specific cellular immune responses elicited in nonhuman primates by HIV-1 gag-expressing replication-defective adenovirus serotype 5 (Ad5) or poxvirus vectors, used either alone or in combination with each other. The responses arising from a heterologous Ad5 priming-poxvirus boosting regimen were significantly greater than those elicited by homologous regimens with the individual vectors or by a heterologous poxvirus priming-Ad5 boosting regimen. The heterologous Ad5 priming-poxvirus boosting approach may have potential utility in humans as a means of inducing high levels of cellular immunity.

Cytotoxic T lymphocytes (CTL) are associated with control of immunodeficiency virus infection but also select for variants that escape immune recognition. Declining frequencies of epitope-specific CTL frequencies have been correlated with viral escape in individual hosts. However, escape mutations may give rise to new epitopes that could be recognized by CTL expressing appropriate T-cell receptors and thus still be immunogenic when escape variants are passed to individuals expressing the appropriate major histocompatibility complex class I molecules. To determine whether peptide ligands that have been altered through escape can be immunogenic in new hosts, we challenged naïve, immunocompetent macaques with a molecularly cloned simian immunodeficiency virus (SIV) bearing common escape mutations in three immunodominant CTL epitopes. Responses to the altered peptides were barely detectable in fresh samples at any time after infection. Surprisingly, CTL specific for two of three escaped epitopes could be expanded by in vitro stimulation with synthetic peptides. Our results suggest that some escape variant epitopes evolving in infected individuals do not efficiently stimulate new populations of CTL, either in that individual or upon passage to new hosts. Nevertheless, escape variation may not completely abolish an epitope's immunogenicity. Moreover, since the mutant epitope sequences did not revert to wild type during the study period, it is possible that low-frequency CTL exerted enough selective pressure to preserve epitope mutations in viruses replicating in vivo.