A Single-Cycle Glycoprotein D Deletion Viral Vaccine Candidate, ΔgD-2, Elicits Polyfunctional Antibodies That Protect against Ocular Herpes Simplex Virus.

Herpes simplex virus 1 (HSV-1) is a leading cause of infectious blindness, highlighting the need for effective vaccines. A single-cycle HSV-2 strain with the deletion of glycoprotein D, ΔgD-2, completely protected mice from HSV-1 and HSV-2 skin or vaginal disease and prevented latency following active or passive immunization in preclinical studies. The antibodies functioned primarily by activating Fc receptors to mediate antibody-dependent cellular cytotoxicity (ADCC). The ability of ADCC to protect the immune-privileged eye, however, may differ from skin or vaginal infections. Thus, the current studies were designed to compare active and passive immunization with ΔgD-2 versus an adjuvanted gD subunit vaccine (rgD-2) in a primary lethal ocular murine model. ΔgD-2 provided significantly greater protection than rgD-2 following a two-dose vaccine regimen, although both vaccines were protective compared to an uninfected cell lysate. However, only immune serum from ΔgD-2-vaccinated, but not rgD-2-vaccinated, mice provided significant protection against lethality in passive transfer studies. The significantly greater passive protection afforded by ΔgD-2 persisted after controlling for the total amount of HSV-specific IgG in the transferred serum. The antibodies elicited by rgD-2 had significantly higher neutralizing titers, whereas those elicited by ΔgD-2 had significantly more C1q binding and Fc gamma receptor activation, a surrogate for ADCC function. Together, the findings suggest ADCC is protective in the eye and that nonneutralizing antibodies elicited by ΔgD-2 provide greater protection than neutralizing antibodies elicited by rgD-2 against primary ocular HSV disease. The findings support advancement of vaccines, including ΔgD-2, that elicit polyfunctional antibody responses.IMPORTANCE Herpes simplex virus 1 is the leading cause of infectious corneal blindness in the United States and Europe. Developing vaccines to prevent ocular disease is challenging because the eye is a relatively immune-privileged site. In this study, we compared a single-cycle viral vaccine candidate, which is unique in that it elicits predominantly nonneutralizing antibodies that activate Fc receptors and bind complement, and a glycoprotein D subunit vaccine that elicits neutralizing but not Fc receptor-activating or complement-binding responses. Only the single-cycle vaccine provided both active and passive protection against a lethal ocular challenge. These findings greatly expand our understanding of the types of immune responses needed to protect the eye and will inform future prophylactic and therapeutic strategies.

Cocrystal Structures of Antibody N60-i3 and Antibody JR4 in Complex with gp120 Define More Cluster A Epitopes Involved in Effective Antibody-Dependent Effector Function against HIV-1.

UNLABELLED: Accumulating evidence indicates a role for Fc receptor (FcR)-mediated effector functions of antibodies, including antibody-dependent cell-mediated cytotoxicity (ADCC), in prevention of human immunodeficiency virus type 1 (HIV-1) acquisition and in postinfection control of viremia. Consequently, an understanding of the molecular basis for Env epitopes that constitute effective ADCC targets is of fundamental interest for humoral anti-HIV-1 immunity and for HIV-1 vaccine design. A substantial portion of FcR effector function of potentially protective anti-HIV-1 antibodies is directed toward nonneutralizing, transitional, CD4-inducible (CD4i) epitopes associated with the gp41-reactive region of gp120 (cluster A epitopes). Our previous studies defined the A32-like epitope within the cluster A region and mapped it to the highly conserved and mobile layers 1 and 2 of the gp120 inner domain within the C1-C2 regions of gp120. Here, we elucidate additional cluster A epitope structures, including an A32-like epitope, recognized by human monoclonal antibody (MAb) N60-i3, and a hybrid A32-C11-like epitope, recognized by rhesus macaque MAb JR4. These studies define for the first time a hybrid A32-C11-like epitope and map it to elements of both the A32-like subregion and the seven-layered ß-sheet of the gp41-interactive region of gp120. These studies provide additional evidence that effective antibody-dependent effector function in the cluster A region depends on precise epitope targeting--a combination of epitope footprint and mode of antibody attachment. All together these findings help further an understanding of how cluster A epitopes are targeted by humoral responses. IMPORTANCE: HIV/AIDS has claimed the lives of over 30 million people. Although antiretroviral drugs can control viral replication, no vaccine has yet been developed to prevent the spread of the disease. Studies of natural HIV-1 infection, simian immunodeficiency virus (SIV)- or simian-human immunodeficiency virus (SHIV)-infected nonhuman primates (NHPs), and HIV-1-infected humanized mouse models, passive transfer studies in infants born to HIV-infected mothers, and the RV144 clinical trial have linked FcR-mediated effector functions of anti-HIV-1 antibodies with postinfection control of viremia and/or blocking viral acquisition. With this report we provide additional definition of the molecular determinants for Env antigen engagement which lead to effective antibody-dependent effector function directed to the nonneutralizing CD4-dependent epitopes in the gp41-reactive region of gp120. These findings have important implications for the development of an effective HIV-1 vaccine.

Characterization of humoral responses to soluble trimeric HIV gp140 from a clade A Ugandan field isolate.

Trimeric soluble forms of HIV gp140 envelope glycoproteins represent one of the closest molecular structures compared to native spikes present on intact virus particles. Trimeric soluble gp140 have been generated by several groups and such molecules have been shown to induce antibodies with neutralizing activity against homologous and heterologous viruses. In the present study, we generated a recombinant trimeric soluble gp140, derived from a previously identified Ugandan A-clade HIV field isolate (gp14094UG018). Antibodies elicited in immunized rabbits show a broad binding pattern to HIV envelopes of different clades. An epitope mapping analysis reveals that, on average, the binding is mostly focused on the C1, C2, V3, V5 and C5 regions. Immune sera show neutralization activity to Tier 1 isolates of different clades, demonstrating cross clade neutralizing activity which needs to be further broadened by possible structural modifications of the clade A gp14094UG018. Our results provide a rationale for the design and evaluation of immunogens and the clade A gp14094UG018 shows promising characteristics for potential involvement in an effective HIV vaccine with broad activity.

Effects of adjuvants on IgG subclasses elicited by virus-like particles.

BACKGROUND: Virus-Like Particles (VLPs) represent an efficient strategy to present and deliver conformational antigens to the immune system, inducing both arms of the adaptive immune response. Moreover, their particulate structure surrounded by cell membrane provides an adjuvanted effect to VLP-based immunizations. In the present study, the elicitation of different patterns of IgG subclasses by VLPs, administered in CpG ODN1826 or poly(I:C) adjuvants, has been evaluated in an animal model. RESULTS: Adjuvanted VLPs elicited a higher titer of total specific IgG compared to VLPs alone. Furthermore, while VLPs alone induced a balanced TH2 pattern, VLPs formulated with either adjuvant elicited a TH1-biased IgG subclasses (IgG2a and IgG3), with poly(I:C) more potent than CpG ODN1826. CONCLUSIONS: The results confirmed that adjuvants efficiently improve antigen immunogenicity and represent a suitable strategy to skew the adaptive immune response toward the differentiation of the desired T helper subset, also using VLPs as antigen.

Antibodies Elicited in Response to a Single Cycle Glycoprotein D Deletion Viral Vaccine Candidate Bind C1q and Activate Complement Mediated Neutralization and Cytolysis.

Herpes simplex virus (HSV) prevention is a global health priority but, despite decades of research, there is no effective vaccine. Prior efforts focused on generating glycoprotein D (gD) neutralizing antibodies, but clinical trial outcomes were disappointing. The deletion of gD yields a single-cycle candidate vaccine (∆gD-2) that elicits high titer polyantigenic non-gD antibodies that exhibit little complement-independent neutralization but mediate antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Active or passive immunization with DgD-2 completely protects mice from lethal disease and latency following challenge with clinical isolates of either serotype. The current studies evaluated the role of complement in vaccine-elicited protection. The immune serum from the DgD-2 vaccinated mice exhibited significantly greater C1q binding compared to the serum from the gD protein vaccinated mice with infected cell lysates from either serotype as capture antigens. The C1q-binding antibodies recognized glycoprotein B. This resulted in significantly greater antibody-mediated complement-dependent cytolysis and neutralization. Notably, complete protection was preserved when the DgD-2 immune serum was passively transferred into C1q knockout mice, suggesting that ADCC and ADCP are sufficient in mice. We speculate that the polyfunctional responses elicited by DgD-2 may prove more effective in preventing HSV, compared to the more restrictive responses elicited by adjuvanted gD protein vaccines.

Human immunodeficiency virus type 1 envelope gp120 induces a stop signal and virological synapse formation in noninfected CD4+ T cells.

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.

Induction of Fc-Mediated Effector Functions Against a Stabilized Inner Domain of HIV-1 gp120 Designed to Selectively Harbor the A32 Epitope Region.

Recent clinical trials and studies using nonhuman primates (NHPs) suggest that antibody-mediated protection against HIV-1 will require α-HIV envelope humoral immunity beyond direct neutralization to include Fc-receptor (FcR) mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC). There is also strong evidence indicating that the most potent ADCC response in humans is directed toward transitional non-neutralizing epitopes associated with the gp41-interactive face of gp120, particularly those within the first and second constant (C1-C2) region (A32-like epitopes). These epitopes were shown to be major targets of ADCC responses during natural infection and have been implicated in vaccine-induced protective immunity. Here we describe the immunogenicity of ID2, an immunogen consisting of the inner domain of the clade A/E 93TH057 HIV-1 gp120 expressed independently of the outer domain (OD) and stabilized in the CD4-bound conformation to harbor conformational A32 region epitopes within a minimal structural unit of HIV-1 Env. ID2 induced A32-specific antibody responses in BALB/c mice when injected alone or in the presence of the adjuvants Alum or GLA-SE. Low α-ID2 titers were detected in mice immunized with ID2 alone whereas robust responses were observed with ID2 plus adjuvant, with the greatest ID2 and A32-specific titers observed in the GLA-SE group. Only sera from groups immunized in the presence of GLA-SE were capable of mediating significant ADCC using NKr cells sensitized with recombinant BaL gp120 as targets and human PBMCs as effectors. A neutralization response to a tier 2 virus was not observed. Altogether, our studies demonstrate that ID2 is highly immunogenic and elicits A32-specific ADCC responses in an animal host. The ID2 immunogen has significant translational value as it can be used in challenge studies to evaluate the role of non-neutralizing antibodies directed at the A32 subregion in HIV-1 protection.

Paring Down HIV Env: Design and Crystal Structure of a Stabilized Inner Domain of HIV-1 gp120 Displaying a Major ADCC Target of the A32 Region.

Evidence supports a role of antibody-dependent cellular cytotoxicity (ADCC) toward transitional epitopes in the first and second constant (C1-C2) regions of gp120 (A32-like epitopes) in preventing HIV-1 infection and in vaccine-induced protection. Here, we describe the first successful attempt at isolating the inner domain (ID) of gp120 as an independent molecule that encapsulates the A32-like region within a minimal structural unit of the HIV-1 Env. Through structure-based design, we developed ID2, which consists of the ID expressed independently of the outer domain and stabilized in the CD4-bound conformation by an inter-layer disulfide bond. ID2 expresses C1-C2 epitopes in the context of CD4-triggered full-length gp120 but without any known neutralizing epitope present. Thus, ID2 represents a novel probe for the analysis and/or selective induction of antibody responses to the A32 epitope region. We also present the crystal structure of ID2 complexed with mAb A32, which defines its epitope.

Chemokine receptor interactions with virus-like particles.

Virus-like particles (VLPs) presenting conformational envelope proteins on their surface represent an invaluable tool to study molecular interactions between viruses and cellular receptors/co-receptors, eliminating biological risks associated with working with live native viruses. The availability of target cells expressing specific chemokine receptors facilitates the dissection of specific interactions between human immunodeficiency virus (HIV) viral envelope proteins and these receptors in the laboratory. Here, we describe a method to evaluate HIV-VLP binding to cellular chemokine co-receptors, by carboxyfluorescein succinimidyl ester labeling and cellular uptake.

Developments in virus-like particle-based vaccines for HIV.

Virus-like particles (VLPs) hold great promise for the development of effective and affordable vaccines. VLPs, indeed, are suitable for presentation and efficient delivery to antigen-presenting cells of linear as well as conformational antigens. This will ultimately result in a crosspresentation with both MHC class I and II molecules to prime CD4(+) T-helper and CD8(+) cytotoxic T cells. This review describes an update on the development and use of VLPs as vaccine approaches for HIV.

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.

Immunogenicity of HIV virus-like particles in rhesus macaques by intranasal administration.

Female rhesus macaques were immunized with HIV virus-like particles (HIV-VLPs) or HIV DNA administered as sequential combinations of mucosal (intranasal) and systemic (intramuscular) routes, according to homologous or heterologous prime-boost schedules. The results show that in rhesus macaques only the sequential intranasal and intramuscular administration of HIV-VLPs, and not the intranasal alone, is able to elicit humoral immune response at the systemic as well as the vaginal level.

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.

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

BACKGROUND: The 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. RESULTS: Animals 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. CONCLUSION: Anti-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.

In vivo alteration of humoral responses to HIV-1 envelope glycoprotein gp120 by antibodies to the CD4-binding site of gp120.

The binding of antibodies to the CD4-binding site (CD4bs) of the HIV-1 envelope glycoprotein gp120 has been shown to induce gp120 to undergo conformational changes that can expose and/or shield specific epitopes on gp120. Here, we study alterations in the antigenicity and immunogenicity of gp120 when complexed with human monoclonal antibodies (mAbs) specific for the CD4bs of gp120. The data showed that gp120 bound by anti-CD4bs mAbs had enhanced reactivity with mAbs to the V3 and N-terminal regions, but not with mAb to the C terminus. Moreover, mice immunized with the gp120/anti-CD4bs mAb complexes produced higher titers of gp120-specific serum IgG and IgA than mice immunized with uncomplexed gp120 or other gp120/mAb complexes. Notably, the enhanced antibody production was directed against V3 and correlated with better exposure of V3 on the gp120/anti-CD4bs mAb complexes. The higher antibody reactivity was evident against the homologous V3(LAI) peptide, but not against heterologous V3 peptides. Potent neutralization activity against HIV-1(LAI) was also observed in the sera from mice immunized with gp120/anti-CD4bs mAb complexes, although the sera exhibited poor neutralizing activities against other viruses tested. These results indicate that the anti-CD4bs antibodies alter the antigenicity and immunogenicity of gp120, leading to enhanced production of anti-gp120 antibodies directed particularly against the V3 region.

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.

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.