Scientific Publications

Potent, broadly HIV-1 neutralizing antibodies (nAbs) may be invaluable for the design of an AIDS vaccine. 4E10 is the broadest HIV-1 nAb known to date and recognizes a contiguous and highly conserved helical epitope in the membrane-proximal region of gp41. The 4E10 epitope is thus an excellent target for vaccine design as it is also highly amenable to peptide engineering to enhance its helical character. To investigate the structural effect of both increasing the peptide length and of introducing helix-promoting constraints in the 4E10 epitope, we have determined crystal structures of Fab 4E10 bound to an optimized peptide epitope (NWFDITNWLWYIKKKK-NH(2)), an Aib-constrained peptide epitope (NWFDITNAibLWRR-NH(2)), and a thioether-linked peptide (NWFCITOWLWKKKK-NH(2)) to resolutions of 1.7 A, 2.1 A, and 2.2 A, respectively. The thioether-linked peptide is the first reported structure of a cyclic tethered helical peptide bound to an antibody. The introduced helix constraints limit the conformational flexibility of the peptides without affecting interactions with 4E10. The substantial increase in affinity (10 nM versus 10(4) nM of the IC(50) of the original KGND peptide template) is largely realized by 4E10 interaction with an additional helical turn at the peptide C terminus that includes Leu679 and Trp680. Thus, the core 4E10 epitope was extended and modified to a WFX(I/L)(T/S)XX(L/I)W motif, where X does not play a major role in 4E10 binding and can be used to introduce helical-promoting constraints in the peptide epitope.

We conducted a prospective study to evaluate methods of detecting clients with sexually transmitted diseases (STDs) who were acutely coinfected with human immunodeficiency virus (HIV) in Lilongwe, Malawi.

Candidate human immunodeficiency virus type 1 (HIV-1) vaccines focusing on T-cell induction, constructed as pTHr.HIVA DNA and modified vaccinia virus Ankara (MVA).HIVA, were delivered in a heterologous prime-boost regimen. The vaccines were tested in several hundred healthy or HIV-1-infected volunteers in Europe and Africa. Whilst larger trials of hundreds of volunteers suggested induction of HIV-1-specific T-cell responses in <15 % of healthy vaccinees, a series of small, rapid trials in 12-24 volunteers at a time with a more in-depth analysis of vaccine-elicited T-cell responses proved to be highly informative and provided more encouraging results. These trials demonstrated that the pTHr.HIVA vaccine alone primed consistently weak and mainly CD4(+), but also CD8(+) T-cell responses, and the MVA.HIVA vaccine delivered a consistent boost to both CD4(+) and CD8(+) T cells, which was particularly strong in HIV-1-infected patients. Thus, whilst the search is on for ways to enhance T-cell priming, MVA is a useful boosting vector for human subunit genetic vaccines.

A successful HIV vaccine would have a substantial impact on acquisition of infection, progression of disease among the infected, or infectiousness of the infected. Current vaccine candidates are anticipated to have their major effect on viremia, however, with the expectation that this would induce or be concordant with a reduced rate of AIDS, death, or infectiousness. Although direct assessment of disease progression or infectiousness may be impractical, available potential surrogates for these endpoints may be misleading. This article summarizes the proceedings of a National Institute of Allergy and Infectious Disease-sponsored workshop to explore the use of surrogate endpoints for licensure of an HIV vaccine. Early, medium, and late endpoints were discussed, along with challenges such as surrogate validity, the confounding effect of antiretroviral therapy initiation, and potential selection bias in the vaccine and placebo recipients who become infected. Results from 5 hypothetic HIV vaccine clinical trials with ambiguously successful results were presented to an expert panel for interpretation and discussion of next steps. Key recommendations included assessing magnitude and durability of surrogate effects, generalization across populations, and directed improvement of vaccines. Use of acquisition and a postinfection surrogate as coprimary endpoints was supported, along with use of composite endpoints and exploration of heterogeneity in vaccine efficacy by characteristics of the host and virus.

Live-vector human immunodeficiency virus (HIV) vaccines are an integral part of a number of HIV vaccine regimens currently under evaluation that have yielded promising results in pre-clinical testing. In this report, a non-pathogenic protozoan parasitic vector, Leishmania tarentolae, which shares common target cells with HIV-1, was used to express full-length HIV-1 Gag protein. Immunization of BALB/c mice with recombinant L. tarentolae led to the expansion of HIV-1 Gag-specific T cells and stimulated CD8(+) T cells to produce gamma interferon in response to specific viral Gag epitopes. A booster immunization with recombinant L. tarentolae elicited effector memory HIV-1 Gag-specific CD4(+) T lymphocytes and increased antibody titres against HIV-1 Gag. Most importantly, immunization of human tonsillar tissue cultured ex vivo with Gag-expressing L. tarentolae vaccine vector elicited a 75% decrease in virus replication following exposure of the immunized tonsils to HIV-1 infection. These results demonstrated that recombinant L. tarentolae is capable of eliciting effective immune responses in mice and human systems, respectively, and suggest that this novel non-pathogenic recombinant vaccine vector shows excellent promise as a vaccination strategy against HIV-1.

Because increasing numbers of HIV vaccine candidates are being tested globally, it is essential to differentiate vaccine- from virus-induced antibodies. Most of the currently tested vaccines contain multiple viral components. As a result, many vaccine recipients give positive results in FDA-licensed HIV serodetection tests. We have identified conserved sequences in Env-gp41 and Gag-p6, which are recognized soon after infection but are not included in most HIV vaccine candidates. A new HIV serodetection assay, the HIV-SELECTEST, was established that distinguishes between vaccine-induced antibodies and seroconversion due to true HIV infections. It is important to make this assay globally relevant, because many clinical trials are conducted around the world where most HIV infections are due to non-B subtype HIV-1. Therefore, the current study examined the reactivity of plasma samples from >3,000 infections with diverse HIV subtypes worldwide. The HIV-SELECTEST performed at >99% specificity and sensitivity. Both recent and established infections with clades A, B, C, D, E, F, G, J, and CRFs were detected. Antibodies elicited by other vaccinations or infections endemic to the clinical trial sites did not react in this assay. Therefore, HIV-SELECTEST could be an important differential diagnostic tool for HIV vaccine trials, blood banks, and population screening worldwide.

Allocation of funds to program areas where they may have an impact is critical to the success of any HIV control program. We examined the cost-effectiveness of providing first-line treatment for male trichomoniasis in Malawi, a condition not commonly considered in syndromic management throughout sub-Saharan Africa.

During natural HIV infection, an array of host receptors are thought to influence virus attachment and the kinetics of infection. In this study, to probe the interactions of HIV envelope (Env) with various receptors, we assessed the inhibitory properties of various anti-Env monoclonal antibodies (mAbs) in binding assays. To assist in detecting Env in attachment assays, we generated Fc fusions of full-length wild-type gp120 and several variable loop-deleted gp120s. Through investigation of the inhibition of Env binding to cell lines expressing CD4, CCR5, DC-SIGN, syndecans or combinations thereof, we found that the broadly neutralizing mAb, 2G12, directed to a unique carbohydrate epitope of gp120, inhibited Env-CCR5 binding, partially inhibited Env-DC-SIGN binding, but had no effect on Env-syndecan association. Furthermore, 2G12 inhibited Env attachment to primary monocyte-derived dendritic cells, that expressed CD4 and CCR5 primary HIV receptors, as well as DC-SIGN, and suggested that the dual activities of 2G12 could be valuable in vivo for inhibiting initial virus dissemination and propagation.

The aim of this article is to describe progress in research and development of vaccines to prevent HIV/AIDS, emphasizing clinical trials and human studies published during the past year.

A major challenge in developing an HIV-1 vaccine is to identify immunogens and their delivery methods that can elicit broad neutralizing antibodies against primary isolates of different genetic subtypes. Recently, we demonstrated that priming with DNA vaccines expressing primary HIV-1 envelope glycoprotein (Env) followed by recombinant Env protein boosting was successful in generating positive neutralizing antibody responses against a clade B primary HIV-1 isolate, JR-FL, that was not easily neutralized. In the current study, we examined whether the DNA priming plus recombinant protein boosting approach delivering a polyvalent primary Env formulation was able to generate neutralizing antibodies against primary HIV-1 viral isolates from various genetic subtypes. New Zealand White rabbits were first immunized with DNA vaccines expressing one, three or eight primary HIV-1 gp120 antigens delivered by a gene gun followed by recombinant gp120 protein boosting. Neutralizing antibody responses were examined by two independently executed neutralization assays: the first one was a single round infection neutralization assay against a panel of 10 primary HIV-1 isolates of subtypes A, B, C and E and the second one used the PhenoSense assay against a panel of 12 pseudovirues expressing primary HIV-1 Env antigens from subtypes A, B, C, D and E as well as 2 pseudoviruses expressing the Env antigens from MN and NL4-3 viruses. Rabbit sera immunized with the DNA priming plus protein boosting approach, but not DNA vaccine alone or Env protein alone, were capable of neutralizing 7 of 10 viruses in the first assay and 12 of 14 viruses in the second assay. More importantly, sera immunized with the polyvalent Env antigens were able to neutralize a significantly higher percentage of viruses than the sera immunized with the monovalent antigens. Our results suggest that DNA priming followed by recombinant Env protein boosting can be used to deliver polyvalent Env-antigen-based HIV-1 vaccines to elicit neutralizing antibody responses against viruses with diverse genetic sequence variations.

A double-blind randomized phase I trial was conducted in human immunodeficiency virus type 1 (HIV-1)-negative subjects receiving vaccines vectored by plasmid DNA and modified vaccinia virus Ankara (MVA) expressing HIV-1 p24/p17 gag linked to a string of CD8(+) T-cell epitopes. The trial had two groups. One group received either two doses of MVA.HIVA (2x MVA.HIVA) (n=8) or two doses of placebo (2x placebo) (n=4). The second group received 2x pTHr.HIVA followed by one dose of MVA.HIVA (n=8) or 3x placebo (n=4). In the pTHr.HIVA-MVA.HIVA group, HIV-1-specific T-cell responses peaked 1 week after MVA.HIVA vaccination in both ex vivo gamma interferon (IFN-gamma) ELISPOT (group mean, 210 spot-forming cells/10(6) cells) and proliferation (group mean stimulation index, 37), with assays detecting positive responses in four out of eight and five out of eight subjects, respectively. No HIV-1-specific T-cell responses were detected in either assay in the 2x MVA.HIVA group or subjects receiving placebo. Using a highly sensitive and reproducible cultured IFN-gamma ELISPOT assay, positive responses mainly mediated by CD4(+) T cells were detected in eight out of eight vaccinees in the pTHr.HIVA-MVA.HIVA group and four out of eight vaccinees in the 2x MVA.HIVA group. Importantly, no false-positive responses were detected in the eight subjects receiving placebo. Of the 12 responders, 11 developed responses to previously identified immunodominant CD4(+) T-cell epitopes, with 6 volunteers having responses to more than one epitope. Five out of 12 responders also developed CD8(+) T-cell responses to the epitope string. Induced T cells produced a variety of anti-viral cytokines, including tumor necrosis factor alpha and macrophage inflammatory protein 1 beta. These data demonstrate that prime-boost vaccination with recombinant DNA and MVA vectors can induce multifunctional HIV-1-specific T cells in the majority of vaccinees.