Maria Luisa Visciano

Human Immunodeficiency Virus Type 1 Envelope gp120 Induces a Stop Signal and Virological Synapse Formation in Noninfected CD4+ T Cells

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-infected T cells form a virological synapse with noninfected CD4+ T cells in order to efficiently transfer HIV-1 virions from cell to cell. The virological synapse is a specialized cellular junction that is similar in some respects to the immunological synapse involved in T-cell activation and effector functions mediated by the T-cell antigen receptor. The immunological synapse stops T-cell migration to allow a sustained interaction between T-cells and antigen-presenting cells. Here, we have asked whether HIV-1 envelope gp120 presented on a surface to mimic an HIV-1-infected cell also delivers a stop signal and if this is sufficient to induce a virological synapse. We demonstrate that HIV-1 gp120-presenting surfaces arrested the migration of primary activated CD4 T cells that occurs spontaneously in the presence of ICAM-1 and induced the formation of a virological synapse, which was characterized by segregated supramolecular structures with a central cluster of envelope surrounded by a ring of ICAM-1. The virological synapse was formed transiently, with the initiation of migration within 30 min. Thus, HIV-1 gp120-presenting surfaces induce a transient stop signal and supramolecular segregation in noninfected CD4+ T cells.

Identification of an N-Linked Glycosylation in the C4 Region of HIV-1 Envelope gp120 That Is Critical for Recognition of Neighboring CD4 T Cell Epitopes

The heavy glycosylation of HIV-1 envelope gp120 shields this important Ag from recognition by neutralizing Abs and cytolytic CD8 T cells. However, very little work has been done to understand the influence of glycosylation on the generation of gp120 epitopes and their recognition by MHC class II-restricted CD4 T cells. In this study, three conserved glycans (linked to N406, N448, and N463) flanking the C4 region of gp120 that contains many known CD4 T cell epitopes were disrupted individually or in combination by asparagine-to-glutamine substitutions. The mutant proteins lacking the N448 glycan did not effectively stimulate CD4 T cells specific for the nearby C4 epitopes, although the same mutants were recognized well by CD4 T cells specific for epitopes located in the distant C1 and C2 regions. The loss of recognition was not due to amino acid substitutions introduced to the mutant proteins. Data from trypsin digestion and mass spectrometry analyses demonstrated that the N448 glycan removal impeded the proteolytic cleavage of the nearby C4 region, without affecting more distant sites. Importantly, this inhibitory effect was observed only in the digestion of the native nondenatured protein and not in that of the denatured protein. These data indicate that the loss of the N448 glycan induces structural changes in the C4 region of gp120 that make this specific region more resistant to proteolytic processing, thereby restricting the generation of CD4 T cell epitopes from this region. Hence, N-linked glycans are critical determinants that can profoundly influence CD4 T cell recognition of HIV-1 gp120.

The use of immune complex vaccines to enhance antibody responses against neutralizing epitopes on HIV-1 envelope gp120

The capacity of immune complexes to augment antibody (Ab) responses is well established. The enhancing effects of immune complexes have been attributed mainly to Fc-mediated adjuvant activity, while the ability of Abs to induce antigenic alterations of specific epitopes as a result of immune complex formation has been less well studied. Previously we have shown that the interaction of anti-CD4-binding site (CD4bs) Abs with HIV-1 gp120 induces conformation changes that lead to enhanced antigenicity and immunogenicity of neutralizing epitopes in the V3 loop. The present study shows that significant increases in the antigenicity of the V3 and C1 regions of gp120 were attained for several subtype B gp120s and a subtype C gp120 upon immune complex formation with the anti-CD4bs monoclonal Ab (mAb) 654-D. Such enhancement was observed with immune complexes made with other anti-CD4bs mAbs and anti-V2 mAbs, but not with anti-C2 mAbs, indicating this activity is determined by antigen specificity of the mAb that formed the immune complex. When immune complexes of gp120(LAI)/654-D and gp120(JRFL)/654-D were tested as immunogens in mice, serum Abs to gp120 and V3 were generated at significantly higher titers than those induced by the respective uncomplexed gp120s. Notably, the anti-V3 Ab responses had distinct fine specificities; gp120(JRFL)/654-D stimulated more cross-reactive anti-V3 Abs than gp120(LAI)/654-D. Neutralizing activities against viruses with heterologous envelope were also detected in sera of mice immunized with gp120(JRFL)/654-D, although the neutralization breadth was still limited. Overall this study shows the potential use of gp120/Ab complexes to augment the immunogenicity of HIV-1 envelope gp120, but further improvements are needed to elicit virus-neutralizing Ab responses with higher potency and breadth.

Characterization of antibodies that inhibit HIV gp120 antigen processing and presentation.

Antibodies to the CD4‐binding site (CD4bs) of HIV‐1 envelope gp120 have been shown to inhibit MHC class II presentation of this antigen, but the mechanism is not fully understood. To define the key determinants contributing to the inhibitory activity of these antibodies, a panel of anti‐CD4bs monoclonal antibodies with different affinities was studied and compared to antibodies specific for the chemokine receptor‐binding site or other gp120 regions. Anti‐CD4bs antibodies that completely obstruct gp120 presentation exhibit three common properties: relatively high affinity for gp120, acid‐stable interaction with gp120, and the capacity to slow the kinetics of gp120 proteolytic processing. None of these antibodies prevents gp120 internalization into APC. Notably, the broadly virus‐neutralizing anti‐CD4bs IgG1b12 does not block gp120 presentation as strongly, because although IgG1b12 has a relatively high affinity, it dissociates from gp120 more readily at acidic pH and only moderately retards gp120 proteolysis. Other anti‐gp120 antibodies, regardless of their affinities, do not affect gp120 presentation. Hence, high‐affinity anti‐CD4bs antibodies that do not dissociate from gp120 at endolysosomal pH obstruct gp120 processing and prevent MHC class II presentation of this antigen. The presence of such antibodies could contribute to the dearth of anti‐gp120 T helper responses in chronically HIV‐1‐infected patients.

Antibodies to the CD4-binding site of HIV-1 gp120 suppress gp120-specific CD4 T cell response while enhancing antibody response

BackgroundThe binding of Abs to the CD4-binding site (CD4bs) of HIV-1 envelope gp120 has been shown to obstruct the processing and generation of helper epitopes from this antigen, resulting in poor presentation of various gp120 epitopes by MHC class II to CD4 T cells. However, the physiologic significance of these inhibitory anti-CD4bs Abs in vivo has remained unclear. In this study, we evaluated the immunologic effects of anti-CD4bs Abs in vivo using a murine model.ResultsAnimals were immunized with recombinant envelope proteins with or without CD4-binding activity (designated CD4bs+ Env and CD4bs– Env, respectively). As expected, anti-CD4bs Abs were generated only after immunization with CD4bs+ Env and not with CD4bs– Env. The presence of anti-CD4bs Abs was associated with lower levels of envelope-specific lymphoproliferation in animals immunized with CD4bs+ Env. To further determine the specific role of the anti-CD4bs Abs, we immunized mice with gp120 in the presence of an inhibitory anti-CD4bs mAb or a non-inhibitory anti-gp120 mAb. The data show that the presence of anti-CD4bs mAb reduced CD4 T cell responses to gp120. However, we also detected significantly higher titers of anti-gp120 Abs following immunization with gp120 and the anti-CD4bs mAb.ConclusionAnti-CD4bs Abs can exert discordant effects on the gp120-specific CD4 T cell and Ab responses in vivo, indicating the importance of these particular Abs in influencing both the cellular and the humoral immune responses against HIV-1.

Targeting a Neutralizing Epitope of HIV Envelope Gp120 by Immune Complex Vaccine

There are formidable challenges in developing HIV vaccines that elicit potent neutralizing antibodies against a broad array of HIV-1 isolates. The key targets for these neutralizing antibodies are the viral envelope antigens gp120 and gp41. Although broadly reactive neutralizing epitopes on gp120 and gp41 have been mapped and studied extensively, these epitopes are poorly immunogenic. Indeed, various vaccine candidates tested in preclinical and clinical trials do not generate antibodies against these epitopes. Hence, novel immunogen designs to augment the immunogenicity of these neutralizing epitopes are wanted. In this review, a unique immunogen design strategy that exploits immune complexes of gp120 and selected anti-gp120 monoclonal antibodies (mAb) to elicit neutralizing antibodies against cross-reactive V3 epitopes is discussed. The ability of these complexes to stimulate neutralizing antibodies is dictated by fine specificity and affinity of mAbs used to form the complexes, indicating the contribution of Fab-mediated activity, rather than conventional Fc-mediated enhancement. Further improvement of V3 immunogenicity is attainable by forming immune complexes with gp120 mutants lacking site-specific N-linked glycans. The increased V3 immunogenicity on the mutated gp120/mAb complexes correlates with enhancement of in vitro antibody recognition (antigenicity) and proteolytic resistance of V3 epitopes when presented on the complexes. These insights should provide guidelines for the development of more potent immunogens that target not only the prototypic V3 epitopes but also other broadly reactive epitopes on the HIV envelope.

Characterization of Antibodies That Inhibit HIV gp120 Antigen Processing and Presentation

Antibodies to the CD4-binding site (CD4bs) of HIV-1 envelope gp120 have been shown to inhibit MHC class II presentation of this antigen, but the mechanism is not fully understood. To define the key determinants contributing to the inhibitory activity of these antibodies, a panel of anti-CD4bs monoclonal antibodies with different affinities was studied and compared to antibodies specific for the chemokine receptor-binding site or other gp120 regions. Anti-CD4bs antibodies that completely obstruct gp120 presentation exhibit three common properties: relatively high affinity for gp120, acid-stable interaction with gp120, and the capacity to slow the kinetics of gp120 proteolytic processing. None of these antibodies prevents gp120 internalization into APC. Notably, the broadly virus-neutralizing anti-CD4bs IgG1b12 does not block gp120 presentation as strongly, because although IgG1b12 has a relatively high affinity, it dissociates from gp120 more readily at acidic pH and only moderately retards gp120 proteolysis. Other anti-gp120 antibodies, regardless of their affinities, do not affect gp120 presentation. Hence, high-affinity anti-CD4bs antibodies that do not dissociate from gp120 at endolysosomal pH obstruct gp120 processing and prevent MHC class II presentation of this antigen. The presence of such antibodies could contribute to the dearth of anti-gp120 T helper responses in chronically HIV-1-infected patients.

P05-03. The use of immune complexes to enhance antibody responses against neutralizing epitopes on HIV envelope

Results The data show that gp120/Ab complexes display higher reactivity with mAbs to the neutralizing epitopes on the V3 loop. Enhanced Ab responses to the V3 loop were detected in mice immunized with thegp120/Ab complexes. Importantly, potent neutralizing activity was observed in the sera immunized with the complexes, but not in the sera of mice immunized with uncomplexed gp120. However, the neutralization was highly restricted to the homologous HIV-1LAI strain. When the immune complexes were prepared using gp120 from a more representative subtype B HIV-1 strain (JRFL), high titers of Ab responses with distinct fine specificity were induced in the sera of immunized mice, but the breadth of serum neutralizing activity was still limited. Conclusion The binding of Abs to gp120 alters the antigenicity and immunogenicity of gp120, leading to enhanced potency of Ab responses against the neutralizing V3 epitopes. However, further studies are necessary to improve the breadth of the neutralizing Ab responses. from AIDS Vaccine 2009 Paris, France. 19–22 October 2009

Effect of site-specific de-glycosylation on HIV gp120-specific CD4 T cell responses

Background Virus specific CD4+ T helper response is critical for maintenance of effective immunity against chronic viral infections. Vigorous HIV-specific T cell responses were found associated with control of viremia in HIV infected individuals. However, in most of the HIV infected individuals, virus specific CD4+ T cell responses are very low or undetectable. While the virus envelope is a critical target for the immune responses, this antigen is poorly immunogenic, especially for CD4+ T cells. One of the possible reasons is the heavy glycosylation of the envelope glycoproteins. In this study, we examined the effects of site-specific de-glycosylation on the presentation of gp120 antigen to the CD4+ T cells.

Role of anti-CD4-binding site antibodies in modulating gp120-specific CD4 T cell and antibody responses

Background MHC II presentation of antigenic peptides to CD4 T cell is critical for the initiation of the primary immune response as well as for the maintenance of the secondary immune response. Previously we have shown that gp120-specific human CD4+ T cell responses are inhibited in the presence of antibodies to the CD4-binding site of gp120 (CD4bs) [1,2]. But the role of these antibodies in modulating the immune responses to gp120 in vivo is not yet determined.