Control of persistent viral infection relies particularly on cell-mediated immunity comprised of CD4+ and CD8+ T cells. There is accumulating evidence on the relevance of soluble factor(s) secreted by CD8+ and CD4+ T cells in controlling HIV replication in vivo. While the β chemokines RANTES, MIP1-α and MIP1-β collectively account for the suppression of R5 viruses, yet information is lacking on the identity of the molecules involved in the suppression of X4 viruses. Proteins that inhibit the replication of X4 HIV isolates were purified from the conditioned media (CM) of immortalized CD8+ and CD4+ T cell lines from HIV+ long-term non-progressors subjects (LTNPs) and identified as the β chemokines macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC) and I-309. These chemokines are secreted primarily by CD4+ T cells but also by CD8+T cells. CD4+ T cells of asymptomatic HIV+ individuals secreted significantly higher levels of MDC and TARC compared with subjects who progressed to AIDS. Recombinant human MDC, TARC and I309 induced a dose dependent inhibition of X4 viruses. A cocktail of neutralizing antibodies against MDC, TARC and I309 abrogated the inhibition of the replication of X4 viruses mediated by the endogenous chemokines in PBMC and CD8-depleted PBMC cells acutely infected in vitro. While the β chemokines RANTES, MIP1-α and MIP1-β suppress R5 viruses by blocking their entry into host cells the mechanism of inhibition of X4 viruses mediated by MDC, TARC and I-309 is a post entry mechanism of suppression. These molecules represent a major component of the soluble anti-X4 activity of T cells, suggesting that the mechanism whereby CD8+ and CD4+ T cells contribute to the control of HIV-1 replication may relate to the secretion of MDC, TARC and I-309.These results may be relevant to HIV pathogenesis.

Control of persistent viral infection relies particularly on cell-mediated immunity comprised of CD4+ and CD8+ T cells. There is accumulating evidence on the relevance of soluble factor(s) secreted by CD8+ and CD4+ T cells in controlling HIV replication in vivo. While the α chemokines RANTES, MIP1-α and MIP1-β collectively account for the suppression of R5 viruses, yet information is lacking on the identity of the molecules involved in the suppression of X4 viruses. Proteins that inhibit the replication of X4 HIV isolates were purified from the conditioned media (CM) of immortalized CD8+ and CD4+ T cell lines from HIV+ long-term non-progressors subjects (LTNPs) and identified as the α chemokines macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC) and I309. These chemokines are secreted primarily by CD4+ T cells but also by CD8+T cells. CD4+ T cells of asymptomatic HIV+ individuals secreted significantly higher levels of MDC and TARC compared with subjects who progressed to AIDS. Recombinant human MDC, TARC and I309 induced a dose dependent inhibition of X4 viruses. A cocktail of neutralizing antibodies against MDC, TARC and I309 abrogated the inhibition of the replication of X4 viruses mediated by the endogenous chemokines in a dose dependent manner in PBMC and CD8-depleted PBMC cells acutely infected in vitro. These studies demonstrate that MDC, TARC and I309 represent a major component of the soluble anti-X4 activity of T cells, suggesting that the mechanism whereby CD8+ and CD4+ T cells contribute to the control of HIV-1 replication may relate to the secretion of these molecules.

ABSTRACT A major challenge for the development of an effective HIV vaccine is to elicit neutralizing antibodies against a broad array of primary isolates. Monomeric gp120-based vaccine approaches have not been successful in inducing this type of response, prompting a number of approaches designed to recreate the native glycoprotein complex that exists on the viral membrane. Gag-Env pseudovirions are noninfectious viruslike particles that recreate the native envelope glycoprotein structure and have the potential to generate neutralizing antibody responses against primary isolates. In this study, an inducible cell line was created in order to generate Gag-Env pseudovirions for examination of neutralizing antibody responses in guinea pigs. Unadjuvanted pseudovirions generated relatively weak anti-gp120 responses, while the use of a block copolymer water-in-oil emulsion or aluminum hydroxide combined with CpG oligodeoxynucleotides resulted in high levels of antibodies that bind to gp120. Sera from immunized animals neutralized a panel of human immunodeficiency virus (HIV) type 1 primary isolate viruses at titers that were significantly higher than that of the corresponding monomeric gp120 protein. Interpretation of these results was complicated by the occurrence of neutralizing antibodies directed against cellular (non-envelope protein) components of the pseudovirion. However, a major component of the pseudovirion-elicited antibody response was directed specifically against the HIV envelope. These results provide support for the role of pseudovirion-based vaccines in generating neutralizing antibodies against primary isolates of HIV and highlight the potential confounding role of antibodies directed at non-envelope cell surface components.

Several monosaccharide-centered multivalent HIV-1 gp41 peptides containing the sequence of DP178 were synthesized. Conformational studies showed that multivalent assembly enhanced the α-helical content of the peptide. Therefore, 2-, 3-, or 4-α-helix bundles of peptide DP178 could be obtained by assembling the peptide on a suitable bi-, tri-, or tetravalent template. Immunization studies indicated that while peptide DP178 alone was poorly immunogenic, the tetravalent peptide MVP-1 raised high titers of antibodies in mice that recognize not only peptide DP178 but also the native HIV-1 glycoprotein gp41, even in the absence of a carrier protein or adjuvant. The study suggests that carbohydrate-centered multivalent peptides provide not only a model for mimicking protein α-helix-bundle structure, but also an effective immunogen for raising high-titer antibodies against HIV-1 envelope glycoprotein gp41.

Human neutrophil alpha-defensin 4 (HNP4) is more effective than HNP1-3 in protecting human peripheral blood mononuclear cells from infection by both X4 and R5 HIV-1 strains. HNP4 binds to both CD4 and gp120 approximately two orders of magnitude weaker than does HNP1, and is less effectively sequestered by glycosylated serum proteins than HNP1. These results suggest that the HIV-1 inhibition by HNP4 stems at least partially from a unique and lectin-independent property of HNP4 with CD4 and/or gp120. Our finding identifies an anti-HIV-1 property of HNP4 and may have implications in the development of new antiviral agents for AIDS therapy.

Seventeen women who were persistently uninfected by human immunodeficiency virus type 1 (HIV-1), despite repeated sexual exposure, and 12 of their HIV-positive male partners were studied for antiviral correlates of non-transmission. Thirteen women had > or = 1 immune response in the form of CD8 cell noncytotoxic HIV-1 suppressive activity, proliferative CD4 cell response to HIV antigens, CD8 cell production of macrophage inflammatory protein-1 beta, or ELISPOT assay for HIV-1-specific interferon-gamma secretion. The male HIV-positive partners without AIDS had extremely high CD8 cell counts. All 8 male partners evaluated showed CD8 cell-related cytotoxic HIV suppressive activity. Reduced CD4 cell susceptibility to infection, neutralizing antibody, single-cell cytokine production, and local antibody in the women played no apparent protective role. These observations suggest that the primary protective factor is CD8 cell activity in both the HIV-positive donor and the HIV-negative partner. These findings have substantial implications for vaccine development.

The antigenic diversity, rapid genetic integration into host cell DNA, and immune evasion tactics of human immunodeficiency virus type 1 (HIV-1) create formidable obstacles to the development of an effective vaccine against it. In spite of this, the advent of conformationally constrained HIV-1 Env and gp120 immunogens has made it feasible to formulate HIV-1 vaccines that induce broadly cross-reactive neutralizing antibodies and afford protection through humoral mechanisms. This paper reviews recent advances made by the authors toward the development of an HIV-1 vaccine that elicits such antibodies in both the mucosal and systemic immune compartments.

The identification of HIV envelope structures that generate broadly cross-reactive neutralizing antibodies is a major goal for HIV-vaccine development. In this study, we evaluated one such structure, expressed as either a gp120-CD4 or a gp140-CD4 complex, for its ability to elicit a neutralizing antibody response. In rhesus macaques, covalently crosslinked complexes of soluble human CD4 (ShCD4) and HIV-1(IIIB) envelope glycoproteins (gp120 or gp140) generated antibodies that neutralized a wide range of primary HIV-1 isolates regardless of the coreceptor usage or genetic subtype. Ig with cross-reactive neutralizing activity was recovered by affinity chromatography with a chimeric single-chain polypeptide containing sequences for HIVBaL gp120 and a mimetic peptide that induces a CD4-triggered envelope structure. These results suggest that covalently crosslinked complexes of the HIV-1 surface envelope glycoprotein and CD4 elicit broadly neutralizing humoral responses that, in part, may be directed against a novel epitope(s) found on the HIV-1 envelope.

Specific conformational changes in the envelope glycoprotein gp120 of the human immunodeficiency virus type-1 (HIV-1) may be critical for eliciting a broadly neutralizing immune response against primary virus isolates. Since the interaction of gp120 with its receptor, CD4, induces conformational perturbations in both molecules, gp120-CD4 complexes should present unique immunogenic features that may include novel epitopes for broadly neutralizing antibodies. To test this hypothesis, we raised polyclonal antiserum against covalently crosslinked gp120-CD4 complexes in a goat and examined the ability of the anti-complex antibodies to neutralize primary and laboratory-adapted HIV-1 isolates. In cell-free neutralization assays with HIV-1MN, the antiserum demonstrated the ability to neutralize primary virus more effectively than the laboratory-adapted isolate. The neutralizing capacity of the anti-complex serum extended to primary isolates from distant genetic clades A, D, and E, although the degree of neutralization was found to vary among the clades. The neutralizing activity of the serum was composed of two components. The first component included anti-CD4 antibodies that recognized epitopes outside the gp120 binding site; the second was independent of CD4 reactivity and was retained after removal of cell surface anti-CD4 reactivity by repeated absorption with CD4-positive cells. These results demonstrate that gp120-CD4 complexes can elicit a unique polyclonal antibody response that is relevant to the neutralization of primary isolates of HIV-1.

We have studied perturbation of the gp120/gp41 envelope complex of HIV-1 in the presence of the mannose-specific lectin succinyl Con A (SC) and compared the effect with that observed in the presence of soluble CD4 (sCD4). SC did not inhibit the binding of gp120 to CD4. Both sCD4 and SC inhibited syncytium formation induced by HIV-1-infected Molt3/HIV-1IIIB cells. The infectivity of HIV-1 was markedly reduced when the virions were preincubated with SC or when SC was mixed simultaneously with virus and cells. The conformation of gp120 was altered in the presence of SC as evidenced by an increased susceptibility of the principal neutralizing epitope (V3 loop) to thrombin digestion. SC treatment of [35S]-methionine-labeled virions derived from Molt3/HIV-1IIIB cells resulted in the dissociation of gp120 from the viral membrane. The effect was less pronounced than that observed with sCD4. These results suggest that although interacting with different regions of gp120, the mannose-specific lectin alters the conformation of the glycoprotein in a manner similar to that induced by sCD4, causing destabilization of the gp120/gp41 complex.

Previously we raised a rabbit monospecific antibody (C2003) against a synthetic peptide derived from a sequence within the C-terminal portion of the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1). This sequence is found to be conserved in the predicted amino acid sequence of a related lentivirus, the equine infectious anemia virus (EIAV). It was previously determined that the C2003 antibody could cross-react with native EIAV RT and directly inhibit the DNA polymerase activity of the enzyme. We have now fractionated EIAV RT by immunoaffinity chromatography with immobilized C2003 antibody. The procedure yielded an equimolar mixture of two proteins of 66 and 51 kDa associated with both DNA polymerase and RNase H activities. When the EIAV RT proteins were examined by in situ activity gel assays, polymerase activity was found to be principally associated with the 66-kDa component. The fidelity of DNA synthesis by EIAV RT was found to be equivalent to that of HIV-1 RT and lower than that of AMV RT. These observations indicate that the RTs of EIAV and HIV-1 share similar structural and functional properties.