Scientific Publications

Murine leukemia viruses (MLVs) have been classified as N-tropic (N-MLV) or B-tropic (B-MLV), depending on their ability to infect particular mouse strains. The early phase of N-MLV infection is blocked in the cells of several mammalian species, including humans. This block is mediated by a dominant host factor that targets the viral capsid soon after virus entry into the cell has been achieved. A similar block to HIV-1 in rhesus monkey cells is mediated by TRIM5alpha. Here we show that human TRIM5alpha is both necessary and sufficient for the restriction of N-MLV in human cells. Rhesus monkey TRIM5alpha, which potently blocks HIV-1 infection, exhibited only modest inhibition of N-MLV infection. B-MLV was resistant to the antiviral effects of both human and rhesus monkey TRIM5alpha; susceptibility to TRIM5alpha-mediated restriction was conferred by alteration of residue 110 of the B-MLV capsid protein to the amino acid found in the N-MLV capsid. Our results demonstrate that species-specific variation in TRIM5alpha governs its ability to block infection by diverse retroviruses.

The increasing incidence of resistance to current HIV-1 therapy underscores the need to develop antiretroviral agents with new mechanisms of action. Integrase, one of three viral enzymes essential for HIV-1 replication, presents an important yet unexploited opportunity for drug development. We describe here the identification and characterization of L-870,810, a small-molecule inhibitor of HIV-1 integrase with potent antiviral activity in cell culture and good pharmacokinetic properties. L-870,810 is an inhibitor with an 8-hydroxy-(1,6)-naphthyridine-7-carboxamide pharmacophore. The compound inhibits HIV-1 integrase-mediated strand transfer, and its antiviral activity in vitro is a direct consequence of this ascribed effect on integration. L-870,810 is mechanistically identical to previously described inhibitors from the diketo acid series; however, viruses selected for resistance to L-870,810 contain mutations (integrase residues 72, 121, and 125) that uniquely confer resistance to the naphthyridine. Conversely, mutations associated with resistance to the diketo acid do not engender naphthyridine resistance. Importantly, the mutations associated with resistance to each of these inhibitors map to distinct regions within the integrase active site. Therefore, we propose a model of the two inhibitors that is consistent with this observation and suggests specific interactions with discrete binding sites for each ligand. These studies provide a structural basis and rationale for developing integrase inhibitors with the potential for unique and nonoverlapping resistance profiles.

Cytomegalovirus (CMV) infection is the most common intrauterine infection in the United States, and it exacts a heavy toll when it infects children and immunocompromised individuals. A CMV vaccine was assigned the highest priority by the Institute of Medicine in its 1999 assessment of targets for vaccine development. The priority was based on the cost and human suffering that would be alleviated by reducing the disease burden of congenital CMV infection. The National Vaccine Advisory Committee and invited experts examined the prospects for a CMV vaccine and the actions needed to bring about successful vaccine development at a National Vaccine Program Office workshop in October 2000. This article summarizes information about the changing epidemiology of CMV and immune responses to infection and immunity, and it reviews the current status of several vaccine candidates. Support of government agencies for CMV vaccine research and development is critical to address this need.

For the development of human immunodeficiency virus type 1 (HIV-1) vaccines, traditional approaches inducing virus-neutralizing antibodies have so far failed. Thus the effort is now focused on elicitation of cellular immunity. We are currently testing in clinical trials in the United Kingdom and East Africa a T-cell vaccine consisting of HIV-1 clade A Gag-derived immunogen HIVA delivered in a prime-boost regimen by a DNA plasmid and modified vaccinia virus Ankara (MVA). Here, we describe engineering and preclinical development of a second immunogen RENTA, which will be used in combination with the present vaccine in a four-component DNA/HIVA-RENTA prime-MVA/HIVA-RENTA boost formulation. RENTA is a fusion protein derived from consensus HIV clade A sequences of Tat, reverse transcriptase, Nef and gp41. We inactivated the natural biological activities of the HIV components and confirmed immunogenicities of the pTHr.RENTA and MVA.RENTA vaccines in mice. Furthermore, we demonstrated in mice and rhesus monkeys broadening of HIVA-elicited T-cell responses by a parallel induction of HIVA- and RENTA-specific responses recognizing multiple HIV epitopes.

CXCR4 is a co-receptor along with CD4 for human immunodeficiency virus type 1 (HIV-1). We investigated the role of N-linked glycosylation in the N-terminus of CXCR4 in binding to HIV-1 gp120 envelope glycoproteins. Gp120s from CXCR4 (X4) and CCR5 (R5) using HIV-1 strains bound more efficiently to non-N-glycosylated than to N-glycosylated CXCR4 proteoliposomes in a CD4-dependent manner. Similar results were observed in binding studies using non-N-glycosylated or N-glycosylated CXCR4 expressed on cells. Mutation of the N-glycosylation site N11 in CXCR4 (N11Q-CXCR4) enhanced CD4-dependent binding of X4 and R5 gp120s and allowed more efficient entry of viruses pseudotyped with X4 or R5 HIV-1 envelope glycoproteins. However, the binding of R5 gp120 to N11Q-CXCR4 and entry of R5 HIV-1 viruses into cells expressing N11Q-CXCR4 were 20- and 100- to 1000-fold less efficient, respectively, than the levels achieved using X4 gp120 or X4 HIV-1 viruses. Binding of stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and SDF-1alpha-induced signaling were reduced by the N11Q mutation. These findings demonstrate that N-glycosylation at N11 inhibits the binding of CXCR4 to X4 and R5 HIV-1 gp120, and provide a better understanding of the structural elements of CXCR4 involved in HIV-1 Env-co-receptor interactions.

Passively transferred neutralizing antibodies can block lentivirus infection, but their role in postexposure prophylaxis is poorly understood. In this nonhuman-primate study, the effects of short-term antibody therapy on 5-year disease progression, virus load, and host immunity were explored. We reported previously that postinfection passive treatment with polyclonal immune globulin with high neutralizing titers against SIVsmE660 (SIVIG) significantly improved the 67-week health of SIVsmE660-infected Macaca mulatta macaques. Four of six treated macaques maintained low or undetectable levels of virus in plasma, compared with one of ten controls, while two rapid progressors controlled viremia only as long as the SIVIG was present. SIVIG treatment delayed the de novo production of envelope (Env)-specific antibodies by 8 weeks (13). We show here that differences in disease progression were also significant at 5 years postinfection, excluding rapid progressors (P = 0.05). Macaques that maintained

Development of a vaccine against human immunodeficiency virus type 1 (HIV-1) is the main hope for controlling the acquired immunodeficiency syndrome pandemic. An ideal HIV vaccine should induce neutralizing antibodies, CD4+ helper T cells, and CD8+ cytotoxic T cells. While the induction of broadly neutralizing antibodies remains a highly challenging goal, there are a number of technologies capable of inducing potent cell-mediated responses in animal models, which are now starting to be tested in humans. Naked DNA immunization is one of them. This review focuses on the stimulation of HIV-specific T cells and discusses in the context of the current 'state-of-art' of DNA vaccines, the areas where this technology might assist either alone or as a part of more complex vaccine formulations in the HIV vaccine development.

In cells of Old World and some New World monkeys, dominant factors restrict human immunodeficiency virus type 1 (HIV-1) infections after virus entry. The simian immunodeficiency virus SIV(mac) is less susceptible to these restrictions, a property that is determined largely by the viral capsid protein. For this study, we altered exposed amino acid residues on the surface of the HIV-1 capsid, changing them to the corresponding residues found on the SIV(mac) capsid. We identified two distinct pathways of escape from early, postentry restriction in monkey cells. One set of mutants that were altered near the base of the cyclophilin A-binding loop of the N-terminal capsid domain or in the interdomain linker exhibited a decreased ability to bind the restricting factor(s). Consistent with the location of this putative factor-binding site, cyclophilin A and the restricting factor(s) cooperated to achieve the postentry block. A second set of mutants that were altered in the ridge formed by helices 3 and 6 of the N-terminal capsid domain efficiently bound the restricting factor(s) but were resistant to the consequences of factor binding. These results imply that binding of the simian restricting factor(s) is not sufficient to mediate the postentry block to HIV-1 and that SIV(mac) capsids escape the block by decreases in both factor binding and susceptibility to the effects of the factor(s).

The synthetic peptide T-20, which corresponds to a sequence within the C-terminal heptad repeat region (HR2) of the human immunodeficiency virus type 1 (HIV-1) gp41 envelope glycoprotein, potently inhibits viral membrane fusion and entry. Although T-20 is thought to bind the N-terminal heptad repeat region (HR1) of gp41 and interfere with gp41 conformational changes required for membrane fusion, coreceptor specificity determined by the V3 loop of gp120 strongly influences the sensitivity of HIV-1 variants to T-20. Here, we show that T-20 binds to the gp120 glycoproteins of HIV-1 isolates that utilize CXCR4 as a coreceptor in a manner determined by the sequences of the gp120 V3 loop. T-20 binding to gp120 was enhanced in the presence of soluble CD4. Analysis of T-20 binding to gp120 mutants with variable loop deletions and the reciprocal competition of T-20 and particular anti-gp120 antibodies suggested that T-20 interacts with a gp120 region near the base of the V3 loop. Consistent with the involvement of this region in coreceptor binding, T-20 was able to block the interaction of gp120-CD4 complexes with the CXCR4 coreceptor. These results help to explain the increased sensitivity of CXCR4-specific HIV-1 isolates to the T-20 peptide. Interactions between the gp41 HR2 region and coreceptor-binding regions of gp120 may also play a role in the function of the HIV-1 envelope glycoproteins.

An increasingly large number of antiviral agents that prevent entry of human immunodeficiency virus (HIV) into cells are in preclinical and clinical development. The envelope (Env) protein of HIV is the major viral determinant that affects sensitivity to these compounds. To understand how changes in Env can impact entry inhibitor sensitivity, we introduced six mutations into the conserved coreceptor binding site of the R5 HIV-1 strain YU-2 and measured the effect of these changes on CD4 and coreceptor binding, membrane fusion levels and rates, virus infection, and sensitivity to the fusion inhibitors enfuvirtide (T-20) and T-1249, the CCR5 inhibitor TAK-779, and an antibody to CD4. The mutations had little effect on CD4 binding but reduced CCR5 binding to various extents. In general, reductions in coreceptor binding efficiency resulted in slower fusion kinetics and increased sensitivity to TAK-779 and enfuvirtide. In addition, low CCR5 binding usually reduced overall fusion and infection levels. However, one mutation adjacent to the bridging sheet beta21 strand, P438A, had little effect on fusion activity, fusion rate, infectivity, or sensitivity to enfuvirtide or T-1249 despite causing a marked reduction in CCR5 binding and a significant increase in TAK-779 sensitivity. Thus, our findings indicate that changes in the coreceptor binding site of Env can modulate its fusion activity, infectivity, and entry inhibitor sensitivity by multiple mechanisms and suggest that reductions in coreceptor binding do not always result in prolonged fusion kinetics and increased sensitivity to enfuvirtide.

We investigated the protective efficacy of a systemic triple vector (DNA/rSFV/rMVA)-based vaccine against mucosal challenge with pathogenic simian immunodeficiency virus (SIV) in cynomolgus monkeys. Animals were immunized at week 0 with DNA (intradermally), at weeks 8 and 16 with recombinant Semliki Forest virus (rSFV, subcutaneously) and finally, at week 24, with recombinant modified vaccinia virus Ankara strain (rMVA, intramuscularly). Both DNA and recombinant viral vectors expressed a wide range of SIV proteins (Gag, Pol, Tat, Rev, Env and Nef). This immunization strategy elicited cell-mediated rather than humoral responses that were especially increased following the last boost. Upon intrarectal challenge with pathogenic SIVmac251, three of the four vaccinated monkeys dramatically abrogated virus load to undetectable levels up to 41 weeks after challenge. A major contribution to this vaccine effect appeared to be the T-cell-mediated immune response to vaccine antigens (Gag, Rev, Tat, Nef) seen in the early phase of infection in three of the four vaccinated monkeys. Indeed, the frequency of T-cells producing antigen-induced IFN-gamma mirrored virus clearance in the vaccinated and protected monkeys. These results, reminiscent of the efficacy of live attenuated virus vaccines, suggest that vaccination with a combination of many viral antigens can induce a robust and stable vaccine-induced immunity able to abrogate virus replication.