Scientific Publications

The ability to induce broadly neutralizing antibodies should be a key component of any forthcoming vaccine against human immunodeficiency virus type 1. One potential vaccine candidate, monomeric gp120, has generally failed to elicit such antibodies. We postulated that gp120 might be a better immunogen if it could be engineered to preferentially bind known broadly neutralizing antibodies. In a first study, we found that four alanine substitutions on the perimeter of the so-called Phe-43 cavity of gp120 could reduce binding of weakly neutralizing CD4-binding site antibodies (R. Pantophlet, E. O. Saphire, P. Poignard, P. W. H. I. Parren, I. A. Wilson, and D. R. Burton, J. Virol. 77:642-658, 2003), while slightly enhancing binding of the potent, broadly neutralizing antibody b12. In the present study, we sought to reduce or abolish the binding of a wider range of nonneutralizing antibodies, by incorporating extra N-glycosylation motifs at select positions into the hypervariable loops and the gp120 core. A hyperglycosylated mutant containing seven extra glycosylation sequons (consensus sequences) and the four alanine substitutions described above did not bind an extensive panel of nonneutralizing and weakly neutralizing antibodies, including a polyclonal immunoglobulin preparation (HIVIG) of low neutralizing potency. Binding of b12, at lowered affinity, and of four antibodies to the C1 and C5 regions was maintained. Removal of N- and C-terminal residues in the C1 and C5 regions, respectively, reduced or abolished binding of the four antibodies, but this also adversely affected b12 binding. The hyperglycosylated mutant and its analogues described here are novel antigens that may provide a new approach to eliciting antibodies with b12-like neutralizing properties.

We previously described a human immunodeficiency virus type 1 (HIV-1) envelope mutant that introduces a disulfide bridge between the gp120 surface proteins and gp41 transmembrane proteins (J. M. Binley, R. W. Sanders, B. Clas, N. Schuelke, A. Master, Y. Guo, F. Kajumo, D. J. Anselma, P. J. Maddon, W. C. Olson, and J. P. Moore, J. Virol. 74:627-643, 2000). Here we produced pseudovirions bearing the mutant envelope and a reporter gene to examine the mutant's infectious properties. These pseudovirions attach to cells expressing CD4 and coreceptor but infect only when triggered with reducing agent, implying that gp120-gp41 dissociation is necessary for infection. Further studies suggested that virus entry was arrested after CD4 and coreceptor engagement. By measuring the activities of various entry inhibitors against the arrested intermediate, we found that gp120-targeting inhibitors typically act prior to virus attachment, whereas gp41 inhibitors are able to act postattachment. Unexpectedly, a significant fraction of antibodies in HIV-1-positive sera neutralized virus postattachment, suggesting that downstream fusion events and structures figure prominently in the host immune response. Overall, this disulfide-shackled virus is a unique tool with potential utility in vaccine design, drug discovery, and elucidation of the HIV-1 entry process.

The HIV-1 env oligomer is structured such that conserved, neutralizing epitopes are obscured by gp120 variable loops. We have studied the ability of an IgG2 human monoclonal antibody (hmAb), F425 B4e8 (B4e8), dependent upon the base of the V3 loop, to induce conformational changes in the env oligomer.

Passaged simian-human immunodeficiency virus (SHIV)-HXBc2P 3.2 exhibits resistance to neutralization by most antibodies and soluble CD4 compared with the parental SHIV-HXBc2; these SHIVs are neutralized equivalently by 2G12 antibody. 2G12 antibody bound proteolytically processed, cell surface envelope glycoproteins from these viruses equivalently; by contrast, other antibodies bound less efficiently to HXBc2P 3.2 envelope glycoproteins than to HXBc2 envelope glycoproteins. We have examined the influence of proteolytic processing of the envelope glycoprotein precursor on antigenicity, comparing antibody binding to cleaved and uncleaved cell surface envelope glycoproteins and to uncleaved soluble trimeric envelope glycoproteins. All envelope glycoproteins bound neutralizing antibodies better than nonneutralizing antibodies, suggesting that their general topology is similar. Differences between cleaved HXBc2 and HXBc2P 3.2 envelope glycoproteins in binding a given antibody, which correlated with susceptibility to neutralization, were not evident in uncleaved envelope glycoproteins. These results indicate that proteolytic processing allows subtle but biologically important adjustments in the conformation of HIV-1 envelope glycoproteins.

Recombinant adeno-associated virus (rAAV) vectors are promising human gene transfer vectors, because they mediate long-term gene expression in vivo. The vector DNA form responsible for sustained gene expression has not been clearly defined, but it has been presumed that the vector integrates to some degree and persists in this manner. Using two independent methods, we were unable to identify rAAV integrants in mouse muscle. In the first approach, we were unable to recover host cell-vector DNA junctions from a lambda phage library generated using transduced mouse muscle DNA that contained a high vector copy number. Following this result, we devised a PCR assay based on the principle that integrated rAAV vector sequences could be amplified using primers specific for mouse interspersed repetitive sequences (B1 elements). Using this assay, we analyzed transduced mouse muscle DNA isolated from 6 to 57 weeks after injection and did not detect amplification above background levels. Based on the demonstrated sensitivity of the assay, these results suggested that >99.5% of vector DNA was not integrated. Additional analyses using a novel DNA exonuclease showed that the majority of the rAAV vector DNA in muscle persisted over time as transcriptionally active monomeric and concatameric episomes.

A novel, experimental subunit human immunodeficiency virus (HIV) vaccine, SFV.HIVA, was constructed. This consists of Semliki Forest virus (SFV), which is a suitable vaccine vector for use in humans, and a passenger gene encoding HIVA, which is an immunogen derived from HIV-1 clade A that is being currently tested in clinical trials of combined DNA- and modified vaccinia virus Ankara (MVA)-vectored vaccines in Oxford (UK) and Nairobi (Kenya). In the mouse, the SFV.HIVA vaccine was highly immunogenic for T cell-mediated immune responses and induced T cell memory that lasted for at least 6 months. SFV.HIVA was also compared to the vaccines currently used in the clinical trials and was shown to be as effective in T cell induction as pTHr.HIVA DNA but less immunogenic than MVA.HIVA. When tested in a prime-boost regimen, SFV.HIVA-induced responses could be boosted by MVA.HIVA. This work is a part of a long-term effort to build a panel of subunit vaccines expressing a common immunogen, which will allow both a direct comparison of various vaccine vectors and combined vaccination regimens in humans and provide more flexibility and/or a potential optimization of vaccinations for individuals based on their pre-existing anti-vector immunity.

Despite an urgent need for a prophylactic vaccine against human immunodeficiency virus (HIV) type 1, progress in this area has been slow. The initial euphoria after identifying and sequencing the causative agent of the acquited immunodeficiency syndrome (AIDS) was followed by a realization that for HIV, traditional vaccine approaches would not be applicable. Frustrations with the induction of neutralizing antibodies led to the development of new vaccine focusing on the induction of cytotoxic T-lymphocytes (CTLs). While CTLs cannot confer sterilizing immunity, there are encouraging data from animal models suggesting that these vaccines may increase the threshold of infection and delay the onset of AIDS in humans. The CTL hypothesis and the possibility that some non-neutralizing antibodies may assist CTLs in the prophylaxis against HIV have yet to be tested in phase III efficacy trials.

Entry of HIV-1 virions into cells is a complex and dynamic process carried out by envelope (Env) glycoproteins on the surface of the virion that promote the thermodynamically unfavorable fusion of highly stable viral and target cell membranes. Insight gained from studies of the mechanism of viral entry allowed insight into the design of novel inhibitors of HIV-1 entry, several of which are now in clinical trials. This review highlights the mechanism by which viral and cellular proteins mediate entry of HIV-1 into permissive cells, with an emphasis on targeting this process in the design of novel therapies that target distinct steps of the entry process, including antagonizing receptor binding events and blocking conformational changes intimately involved in membrane fusion.

To describe study design, methods and baseline findings of a behavioural intervention alone and in combination with improved management of sexually transmitted diseases (STDs) aimed at reducing HIV incidence and other STDs.

Although several human immunodeficiency virus (HIV) vaccine approaches have elicited meaningful antigen-specific T-cell responses in animal models, no single vaccine candidate has engendered antibodies that broadly neutralize primary isolates of HIV type 1 (HIV-1). Thus, there remains a significant gap in the design of HIV vaccines. To address this issue, we exploited the existence of rare human monoclonal antibodies that have been isolated from HIV-infected individuals. Such antibodies neutralize a wide array of HIV-1 field isolates and have been shown to be effective in vivo. However, practical considerations preclude the use of antibody preparations as a prophylactic passive immunization strategy in large populations. Our concept calls for an antibody gene of choice to be transferred to muscle where the antibody molecule is synthesized and distributed to the circulatory system. In these experiments, we used a recombinant adeno-associated virus (rAAV) vector to deliver the gene for the human antibody IgG1b12 to mouse muscle. Significant levels of HIV-neutralizing activity were found in the sera of mice for over 6 months after a single intramuscular administration of the rAAV vector. This approach allows for predetermination of antibody affinity and specificity prior to 'immunization' and avoids the need for an active humoral immune response against the HIV envelope protein.

The enzyme-linked immunosorbent (ELISPOT) assay, which enumerates peripheral blood mononuclear cells (PBMCs) releasing interferon gamma (IFN-gamma) on specific antigen stimulation, is becoming the assay of choice for evaluation of vaccine-induced cell-mediated immune responses in many clinical trials. A properly conducted trial requires the assays to be validated, especially should the trial lead to vaccine licensure. Here, the design and validation of an ELISPOT assay are described for use in clinical trials of candidate human immunodeficiency virus (HIV) vaccines, using a particular immunogen termed HIVA. This assay employs eight pools of 20 to 23 peptides each: seven pools are derived from the immunogen and one pool is derived from cytotoxic T cell epitopes of common human viruses serving as an internal positive control. The validation determined that first, the overall variation of a positive response of approximately 500 spot-forming units (SFU)/10(6) cells was 21%, while second, the average of 5 SFU/10(6) cells was detected for the seven HIVA-derived pools in HIV-uninfected individuals; third, a positive response to a peptide added to the assay pools was not occluded by the other pool peptides; fourth, the frequencies detected in fresh PBMCs were 2- to 3-fold higher compared with the same samples that had been cryopreserved; and finally, all seven HIV-derived pools induced IFN-gamma responses in PBMCs isolated from HIV-infected individuals. The limits of the validation of assays involving biological responses of living cells are discussed.

Without going into the details of the devastation that human immunodeficiency virus (HIV) infection causes especially in the developing world, the best hope for changing the course of this epidemic is development of a safe, effective, accessible prophylactic HIV vaccine. While the inaccessibility of potentially neutralising epitopes on primary HIV isolates has hampered the development of envelope-based vaccines, there is a number of new potent technologies capable of inducing high levels of circulating virus-specific CD8(+) cytotoxic T lymphocytes (CTL). Our original finding that a successive immunisation with DNA and modified vaccinia virus Ankara (MVA) vaccines expressing a common immunogen is a potent way of inducing CD8(+) CTL, which has been since reinforced by us and others, prompted us to test this approach in humans. With the view of proceeding into a high-risk cohort in Kenya for the efficacy trial, we designed the immunogen, termed HIVA, to match the HIV strain responsible locally for over 70% infections. It consists of a consensus clade A gag p24/p17 and a string of clade A-derived CTL epitopes. Pre-clinical studies demonstrated high immunogenicities of both the pTHr.HIVA and MVA.HIVA vaccines. In mice, these induced strong T cells-mediated immune responses which lasted at least 155 days. In rhesus macaques, the prime-boost immunisation elicited T cell responses specific for multiple HIV-derived epitopes. Phase I trials in healthy low-risk volunteers have commenced in Oxford and Nairobi, and the preliminary immunogenicity analysis from the Oxford site indicated that both vaccine components alone induced T cell responses in a majority of volunteers. These results have boosted expectations for the prime-boost vaccinations.