Progress toward the development of a bacterial vaccine vector that induces high-titer long-lived broadly neutralizing antibodies against HIV-1.

Conformationally constrained HIV-1 Env and gp120 immunogens induce broadly cross-reactive neutralizing antibodies. Thus, it is now feasible to rationally design an HIV-1 vaccine that affords protection through humoral mechanisms. This paper reviews our progress toward the development of an oral bacterial vaccine vector that is capable of delivering an HIV-1 DNA vaccine to host lymphoid tissues and inducing broadly neutralizing antibodies to HIV-1 in the mucosal and systemic immune compartments.

Immunogenicity of DNA vaccines that direct the coincident expression of the 120 kDa glycoprotein of human immunodeficiency virus and the catalytic domain of cholera toxin.

Passive antibody studies unequivocally demonstrate that sterilizing immunity against lentiviruses is obtainable through humoral mechanisms. In this regard, DNA vaccines represent an inexpensive alternative to subunit vaccine for mass vaccination programs designed to induce such responses to human immunodeficiency virus type I (HIV-1). At present, however, this vaccine modality has proven relatively ineffective at inducing humoral responses. In this report, we describe the immunogenicity of DNA vaccines that direct the coincident expression of the cholera toxin catalytic domain (CTA1) with that of the human immunodeficiency virus type I gp120 through genes either encoded in individual plasmids or in a single dicistronic plasmid. In BALB/cJ mice, coincident expression of CTA1 in either a separate plasmid or in the dicistronic plasmid in the DNA vaccines induced serum IgG responses to gp120 that were at least 1000-fold greater, and remained elevated longer than, the analogous responses in mice vaccinated with a DNA vaccine that expressed gp120 alone. In addition, mice vaccinated with CTA1 and gp120 produced significantly more gp120-specific IFN-gamma ELISPOTs than mice vaccinated with the gp120 DNA vaccine. Combined, these data show that the adjuvant properties of cholera toxin can be harnessed in DNA vaccine modalities.

Antigenic properties of the human immunodeficiency virus envelope during cell-cell fusion.

Human immunodeficiency virus (HIV) fusion and entry involves sequential interactions between the viral envelope protein, gp120, cell surface CD4, and a G-protein-coupled coreceptor. Each interaction creates an intermediate gp120 structure predicted to display distinct antigenic features, including key functional domains for viral entry. In this study, we examined the disposition of these features during the fusion of HeLa cells expressing either HIV(HXB2) envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various times and then arrested. The cells were then examined for reactivity with antibodies directed against receptor-induced epitopes on gp120. Analyses of cells arrested by cooling to 4( degrees )C revealed that antibodies against the CD4-induced coreceptor-binding domain, i.e., 17b, 48d, and CG10, faintly react with Env cells even in the absence of target cell or soluble CD4 (sCD4) interactions. Such reactivity increased after exposure to sCD4 but remained unchanged during fusion with target cells and was not intensified at the Env-target cell interface. Notably, the antibodies did not react with Env cells when treated with a covalent cross-linker either alone or during fusion with target cells. Immunoreactivity could not be promoted or otherwise altered on either temperature arrested or cross-linked cells by preventing coreceptor interactions or by using a 17b Fab. In comparison, two other gp120-CD4 complex-dependent antibodies against epitopes outside the coreceptor domain, 8F101 and A32, exhibited a different pattern of reactivity. These antibodies reacted with the Env-target cell interface only after 30 min of cocultivation, concurrent with the first visible transfer of cytoplasmic dye from Env to target cells. At later times, the staining surrounded entire syncytia. Such binding was entirely dependent on the formation of gp120-CD4-CXCR4 tricomplexes since staining was absent with SDF-treated or coreceptor-negative target cells. Overall, these studies show that access to the CD4-induced coreceptor-binding domain on gp120 is largely blocked at the fusing cell interface and is unlikely to represent a target for neutralizing antibodies. However, new epitopes are presented on intermediate gp120 structures formed as a result of coreceptor interactions. Such findings have important implications for HIV vaccine approaches based on conformational alterations in envelope structures.

Mucosal and systemic HIV-1 Env-specific CD8(+) T-cells develop after intragastric vaccination with a Salmonella Env DNA vaccine vector.

CD8(+) T-cell responses provide beneficial antiviral immunity against human immunodeficiency virus 1 (HIV-1). In this study, we show that intragastric vaccination with a Salmonella HIV-1 Env DNA vaccine vector generates Env-specific CD8(+) T-cells, both in mucosal and systemic lymphoid tissue. By contrast, intramuscular vaccination with the Env DNA vaccine alone only induced systemic CD8(+) T-cells. To our knowledge, this is the first report showing both mucosal and systemic CD8(+) T-cell responses following vaccination with a Salmonella vaccine vector. These data suggest that this mode of HIV-1 DNA vaccine delivery will be advantageous over parenterally administered HIV-1 DNA vaccines.

Perforin-low memory CD8+ cells are the predominant T cells in normal humans that synthesize the beta -chemokine macrophage inflammatory protein-1beta.

The synthesis of antiviral beta-chemokines has joined cytolysis as a potential mechanism for the control of HIV-1 infection by CD8(+) T cells. Recent evidence suggests that these two effector functions can diverge in some individuals infected with HIV-1; however, little is known about the CD8(+) T cell subsets in normal individuals that synthesize antiviral beta-chemokines. In this report, we have used mutliparameter flow cytometry to characterize the T cell subsets that secrete the antiviral beta-chemokine macrophage inflammatory protein (MIP)-1beta. These studies have shown: (i) CD8(+) cells are the predominant T cell subset that synthesizes MIP-1beta; (ii) MIP-1beta and IFN-gamma are synthesized congruently in most CD8(+) T cells; however, significant numbers of these cells synthesize only one of these effector molecules; (iii) approximately 60% of the CD8(+) T cells that synthesize MIP-1beta lack perforin; (iv) MIP-1beta is synthesized with approximately equal frequency by CD28(+) and CD28(-) subpopulations of CD8(+) T cells; (v) MIP-1beta is synthesized by three distinct CD8(+) T cell subsets defined by the expression of CD45R0 and CD62L; and (vi) MIP-1beta is not synthesized in short-term cultures of naive CD8(+) T cells. These results demonstrate substantial subset heterogeneity of MIP-1beta synthesis among CD8(+) T cells and suggest that these subsets should be evaluated as correlates of protective immunity against HIV-1.

Expression and characterization of a single-chain polypeptide analogue of the human immunodeficiency virus type 1 gp120-CD4 receptor complex.

The infection of CD4(+) host cells by human immunodeficiency virus type 1 (HIV-1) is initiated by a temporal progression of interactions between specific cell surface receptors and the viral envelope protein, gp120. These interactions produce a number of intermediate structures with distinct conformational, functional, and antigenic features that may provide important targets for therapeutic and vaccination strategies against HIV infection. One such intermediate, the gp120-CD4 complex, arises from the interaction of gp120 with the CD4 receptor and enables interactions with specific coreceptors needed for viral entry. gp120-CD4 complexes are thus promising targets for anti-HIV vaccines and therapies. The development of such strategies would be greatly facilitated by a means to produce the gp120-CD4 complexes in a wide variety of contexts. Accordingly, we have developed single-chain polypeptide analogues that accurately replicate structural, functional, and antigenic features of the gp120-CD4 complex. One analogue (FLSC) consists of full-length HIV-1BaL gp120 and the D1D2 domains of CD4 joined by a 20-amino-acid linker. The second analogue (TcSC) contains a truncated form of the gp120 lacking portions of the C1, C5, V1, and V2 domains. Both molecules exhibited increased exposure of epitopes in the gp120 coreceptor-binding site but did not present epitopes of either gp120 or CD4 responsible for complex formation. Further, the FLSC and TcSC analogues bound specifically to CCR5 (R5) and blocked R5 virus infection. Thus, these single-chain chimeric molecules represent the first generation of soluble recombinant proteins that mimic the gp120-CD4 complex intermediate that arises during HIV replication.

Higher macrophage inflammatory protein (MIP)-1alpha and MIP-1beta levels from CD8+ T cells are associated with asymptomatic HIV-1 infection.

To test the hypothesis that beta-chemokine levels may be relevant to the control of HIV in vivo, we compared RANTES, MIP-1alpha, and MIP-1beta production from purified CD8(+) T cells from 81 HIV-infected subjects and from 28 uninfected donors. Asymptomatic HIV(+) subjects produced significantly higher levels of MIP-1alpha and MIP-1beta, but not RANTES, than uninfected donors or patients that progressed to AIDS. In contrast, beta chemokines in plasma were either nondetectable or showed no correlation with clinical status. The high beta-chemokine-mediated anti-HIV activity was against the macrophage tropic isolate HIV-1(BAL), with no demonstrable effect on the replication of the T-cell tropic HIV-1(IIIB). These findings suggest that constitutive beta-chemokine production may play an important role in the outcome of HIV-1 infection.

The role of chemokines in human immunodeficiency virus infection.

Chemokines that bind to human immunodeficiency virus (HIV) co-receptors are potent and selective inhibitors of HIV infection. Therefore, ever since our discovery of this activity, we have proposed their role in controlling HIV infection as a third arm of the immune response, i.e. in concert with humoral and cellular responses. Research carried out in our laboratory, and performed independently by other groups, has recently strengthened this concept. Here, we critically analyze the evidence indicating the positive contribution of chemokines to HIV infection, their involvement with cognate and innate immunity, and the potential for their use in combating HIV infection.

Soluble complexes of regulated upon activation, normal T cells expressed and secreted (RANTES) and glycosaminoglycans suppress HIV-1 infection but do not induce Ca(2+) signaling.

Chemokines comprise a family of low-molecular-weight proteins that elicit a variety of biological responses including chemotaxis, intracellular Ca(2+) mobilization, and activation of tyrosine kinase signaling cascades. A subset of chemokines, including regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha), and MIP-1beta, also suppress infection by HIV-1. All of these activities are contingent on interactions between chemokines and cognate seven-transmembrane spanning, G protein-coupled receptors. However, these activities are strongly inhibited by glycanase treatment of receptor-expressing cells, indicating an additional dependence on surface glycosaminoglycans (GAG). To further investigate this dependence, we examined whether soluble GAG could reconstitute the biological activities of RANTES on glycanase-treated cells. Complexes formed between RANTES and a number of soluble GAG failed to induce intracellular Ca(2+) mobilization on either glycanase-treated or untreated peripheral blood mononuclear cells and were unable to stimulate chemotaxis. In contrast, the same complexes demonstrated suppressive activity against macrophage tropic HIV-1. Complexes composed of (125)I-labeled RANTES demonstrated saturable binding to glycanase-treated peripheral blood mononuclear cells, and such binding could be reversed partially by an anti-CCR5 antibody. These results suggest that soluble chemokine-GAG complexes represent seven-transmembrane ligands that do not activate receptors yet suppress HIV infection. Such complexes may be considered as therapeutic formulations for the treatment of HIV-1 infection.

Spontaneous and antigen-induced production of HIV-inhibitory beta-chemokines are associated with AIDS-free status.

The beta-chemokines RANTES, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta suppress infection by macrophage-tropic strains of HIV and simian immunodeficiency virus (SIV) by binding and down-regulating the viral coreceptor, CCR5. Accordingly, we have examined whether higher levels of CCR5 ligands are associated with a more favorable clinical status in AIDS. A cross-sectional study of 100 subjects enrolled in the Multicenter AIDS Cohort Study at the Baltimore site was conducted to measure chemokine production and lymphocyte proliferation by peripheral blood mononuclear cells (PBMC). Statistical analyses of the data revealed that the production of HIV-suppressive beta-chemokines by HIV antigen-stimulated PBMC was significantly higher in HIV-positive subjects without AIDS compared with subjects with clinical AIDS. Increased chemokine production was also correlated with higher proliferative responses to HIV antigens. Both parameters were significantly lower in the AIDS versus non-AIDS group. Notably, significantly higher levels of MIP-1alpha were also observed with unstimulated PBMC from seronegative subjects at risk for HIV infection released as compared with seropositive and non-Multicenter AIDS Cohort Study seronegative subjects. The association of chemokine production with antigen-induced proliferative responses, more favorable clinical status in HIV infection, as well as with an uninfected status in subjects at risk for infection suggests a positive role for these molecules in controlling the natural course of HIV infection.

HIV infection and pathogenesis: what about chemokines?

Several chemotactic cytokines, or chemokines. inhibit HIV replication by blocking or down regulating chemokine receptors that serve as entry cofactors for the virus. Although the role of chemokine receptors in HIV pathogenesis has been the subject of intense interest, chemokines are comparatively less seriously considered as potential correlates of protection from HIV infection and disease progression. However, a critical analysis of newly available data reveals substantial evidence to support a beneficial role for chemokines in HIV infection and disease. In this review we summarize the results of such studies and their promising implications for HIV infection.

A new monoclonal antibody, mAb 4A12, identifies a role for the glycosaminoglycan (GAG) binding domain of RANTES in the antiviral effect against HIV-1 and intracellular Ca2+ signaling.

The beta-chemokine RANTES (regulated on activation, normal T cell expressed and secreted) suppresses the infection of susceptible host cells by macrophage tropic strains of HIV-1. This effect is attributed to interactions of this chemokine with a 7-transmembrane domain receptor, CCR5, that is required for virus-cell fusion and entry. Here we identify domains of RANTES that contribute to its biological activities through structure-function studies using a new monoclonal antibody, mAb 4A12, isolated from mice immunized with recombinant human RANTES. This monoclonal antibody (mAb) blocked the antiviral activity of RANTES in infectivity assays with HIV-1Bal, and inhibited the mobilization of intracellular Ca2+ elicited by RANTES, yet recognized this chemokine bound to cell surfaces. Epitope mapping using limited proteolysis, reversed phase high-performance liquid chromatography, and mass spectrometry suggest that residues 55-66 of RANTES, which include the COOH-terminal alpha-helical region implicated as the glycosaminoglycan (GAG) binding domain, overlap the determinant recognized by mAb 4A12. This is supported by affinity chromatography studies, which showed that RANTES could be eluted specifically by heparin from a mAb 4A12 immunoaffinity matrix. Removal of cell surface GAGs by enzymatic digestion greatly reduced the ability of mAb 4A12 to detect RANTES passively bound on cell surfaces and abrogated the ability of RANTES to elicit an intracellular Ca2+ signal. Taken together, these studies demonstrate that the COOH-terminal alpha-helical region of RANTES plays a key role in GAG-binding, antiviral activity, and intracellular Ca2+ signaling and support a model in which GAGs play a key role in the biological activities of this chemokine.

Chemokine receptors and chemokines in HIV infection.

Suppression of HIV by chemokines represents a special case in virology and immunology where soluble molecules other than antibodies inhibit infection by a specific virus. The basis for this inhibition is that HIV has evolved to use certain chemokine receptors as "coreceptors" for entry into host cells. Human genotypes that reduce or prevent coreceptor expression are strongly associated with protection against infection and slower disease progression. We suggest that local production of certain chemokines can produce a similar modulation of coreceptor expression, and mounting evidence indicates that chemokine release is a major determinant of protection from HIV infection. Here we review this evidence and explore future avenues for investigating the role of chemokines in controlling HIV infection.

Beta-chemokines and protection from HIV type 1 disease.

A small revolution has occurred in the field of AIDS research. A number of chemokines, most of which belong to the CC or beta family, were found by us and others to inhibit HIV infection potently and specifically. The mechanism of such inhibition was shown to be at the level of receptor binding, as these chemokines are binding to receptors that mediate HIV infection. Therefore, chemokines effectively block entry of HIV. Although chemokines have a natural function as chemoattractants, it is intriguing to think that in crossing their path with the virus, they constitute the first example of a naturally occurring soluble molecule, other than antibodies, that can specifically prevent infection. Thus, chemokines could play a role in protective immunity against HIV infection together with other classic correlates, such as neutralizing antibodies and cytotoxic T cells, and some clinical studies suggest that this is indeed the case. Here we review and analyze some of the basic and clinical science that led to the elucidation of the role of chemokines and their receptor in protection from HIV infection.

C-C chemokine RANTES and HIV long terminal repeat-driven gene expression.

The C-C chemokines RANTES, MIP-1alpha, and MIP-1beta have been characterized as constituents of an HIV- and SIV-suppressive factor released by CD8+ cells. Furthermore, it has been demonstrated that chemokine receptors cooperate in HIV entry. However, these proteins are also known to have an effect on multiple intracellular signaling cascades that may affect the process of transcription. In the present study we demonstrate that treatment of CD4+ T cells with these chemokines or with cell supernatants from HTLV-I-immortalized CD8+ T cells results in significant reduction in the abundance of HIV-1-specific RNA as analyzed by Northern blot hybridization. To examine the possibility that such suppressive factors may inhibit HIV RNA transcription, we studied the effect of RANTES, the most effective HIV-suppressive chemokine, on basal and Tat-induced HIV-directed LTR expression of a reporter gene. Neither recombinant RANTES nor conditioned medium from CD8+ cells significantly altered HIV-1 LTR-directed chloramphenicol acetyltransferase expression in either transiently or stably transfected CD4+ T cell lines, either in the presence or in the absence of Tat. These results suggest that C-C chemokines do not inhibit viral RNA transcription.

Inhibition of HIV-1 infection by the beta-chemokine MDC.

CD8(+) T lymphocytes from individuals infected with human immunodeficiency virus-type 1 (HIV-1) secrete a soluble activity that suppresses infection by HIV-1. A protein associated with this activity was purified from the culture supernatant of an immortalized CD8(+) T cell clone and identified as the beta-chemokine macrophage-derived chemokine (MDC). MDC suppressed infection of CD8(+) cell-depleted peripheral blood mononuclear cells by primary non-syncytium-inducing and syncytium-inducing isolates of HIV-1 and the T cell line-adapted isolate HIV-1IIIB. MDC was expressed in activated, but not resting, peripheral blood mononuclear cells and binds a receptor on activated primary T cells. These observations indicate that beta-chemokines are responsible for a major proportion of HIV-1-specific suppressor activity produced by primary T cells.

The V3 domain of the HIV-1 gp120 envelope glycoprotein is critical for chemokine-mediated blockade of infection.

The ability of CD8 T cells derived from human immunodeficiency virus (HIV)-infected patients to produce soluble HIV-suppressive factor(s) (HIV-SF) has been suggested as an important mechanism of control of HIV infection in vivo. The C-C chemokines RANTES, MIP-1 alpha and MIP-1 beta were recently identified as the major components of the HIV-SF produced by both immortalized and primary patient CD8 T cells. Whereas they potently inhibit infection by primary and macrophage-tropic HIV-1 isolates, T-cell line-adapted viral strains tend to be insensitive to their suppressive effects. Consistent with this discrepancy, two distinct chemokine receptors, namely, CXCR4 (ref. 7) and CCR5 (ref. 8), were recently identified as potential co-receptors for T-cell line-adapted and macrophage-tropic HIV-1 isolates, respectively. Here, we demonstrate that the third hypervariable domain of the gp 120 envelope glycoprotein is a critical determinant of the susceptibility of HIV-1 to chemokines. Moreover, we show that RANTES, MIP-1 alpha and MIP-1 beta block the entry of HIV-1 into cells and that their antiviral activity is independent of pertussis toxin-sensitive signal transduction pathways mediated by chemokine receptors. The ability of the chemokines to block the early steps of HIV infection could be exploited to develop novel therapeutic approaches for AIDS.

Inhibition of human immunodeficiency virus type 1 and vaccinia virus infection by a dominant negative factor of the interferon regulatory factor family expressed in monocytic cells.

ICSBP is a member of the interferon (IFN) regulatory factor (IRF) family that regulates expression of type I interferon (IFN) and IFN-regulated genes. To study the role of the IRF family in viral infection, a cDNA for the DNA-binding domain (DBD) of ICSBP was stably transfected into U937 human monocytic cells. Clones that expressed DBD exhibited a dominant negative phenotype and did not elicit antiviral activity against vesicular stomatitis virus (VSV) infection upon IFN treatment. Most notably, cells expressing DBD were refractory to infection by vaccinia virus (VV) and human immunodeficiency virus type 1 (HIV-1). The inhibition of VV infection was attributed to defective virion assembly, and that of HIV-1 to low CD4 expression and inhibition of viral transcription in DBD clones. HIV-1 and VV were found to have sequences in their regulatory regions similar to the IFN-stimulated response element (ISRE) to which IRF family proteins bind. Accordingly, these viral sequences and a cellular ISRE bound a shared factor(s) expressed in U937 cells. These observations suggest a novel host-virus relationship in which the productive infection of some viruses is regulated by the IRF-dependent transcription pathway through the ISRE.

Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells.

Evidence suggests that CD8+ T lymphocytes are involved in the control of human immunodeficiency virus (HIV) infection in vivo, either by cytolytic mechanisms or by the release of HIV-suppressive factors (HIV-SF). The chemokines RANTES, MIP-1 alpha, and MIP-1 beta were identified as the major HIV-SF produced by CD8+ T cells. Two active proteins purified from the culture supernatant of an immortalized CD8+ T cell clone revealed sequence identity with human RANTES and MIP-1 alpha. RANTES, MIP-1 alpha, and MIP-1 beta were released by both immortalized and primary CD8+ T cells. HIV-SF activity produced by these cells was completely blocked by a combination of neutralizing antibodies against RANTES, MIP-1 alpha, and MIP-1 beta. Recombinant human RANTES, MIP-1 alpha, and MIP-1 beta induced a dose-dependent inhibition of different strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV). These data may have relevance for the prevention and therapy of AIDS.