ABSTRACT
Anti-HIV-1 envelope broadly neutralizing monoclonal antibodies (bNAbs) isolated from memory B cells may not fully represent HIV-1-neutralizing profiles measured in plasma. Accordingly, we characterized near-pan-neutralizing antibodies extracted directly from the plasma of two "elite neutralizers." Circulating anti-gp120 polyclonal antibodies were deconvoluted using proteomics to guide lineage analysis of bone marrow plasma cells. In both subjects, a single lineage of anti-CD4-binding site (CD4bs) antibodies explained the plasma-neutralizing activity. Importantly, members of these lineages potently neutralized 89%-100% of a multi-tier 117 pseudovirus panel, closely matching the specificity and breadth of the circulating antibodies. X-ray crystallographic analysis of one monoclonal, N49P7, suggested a unique ability to bypass the CD4bs Phe43 cavity, while reaching deep into highly conserved residues of Layer 3 of the gp120 inner domain, likely explaining its extreme potency and breadth. Further direct analyses of plasma anti-HIV-1 bNAbs should provide new insights for developing antibody-based antiviral agents and vaccines.
Subject(s)
Antibodies, Neutralizing/immunology , HIV Envelope Protein gp120/immunology , HIV-1/metabolism , Amino Acid Sequence , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/chemistry , Binding Sites , CD4 Antigens/chemistry , CD4 Antigens/metabolism , Crystallography, X-Ray , HIV Antibodies/blood , HIV Antibodies/immunology , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/metabolism , HIV-1/genetics , Humans , Molecular Dynamics Simulation , Protein Binding , Protein Structure, Tertiary , RNA, Viral/blood , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/immunologyABSTRACT
Broadly neutralizing antibodies (bNAbs) are the focus of increasing interest for human immunodeficiency virus type 1 (HIV-1) prevention and treatment. Although several bNAbs are already under clinical evaluation, the development of antibodies with even greater potency and breadth remains a priority. Recently, we reported a novel strategy for improving bNAbs against the CD4-binding site (CD4bs) of gp120 by engraftment of the elongated framework region 3 (FR3) from VRC03, which confers the ability to establish quaternary interactions with a second gp120 protomer. Here, we applied this strategy to a new series of anti-CD4bs bNAbs (N49 lineage) that already possess high potency and breadth. The resultant chimeric antibodies bound the HIV-1 envelope (Env) trimer with a higher affinity than their parental forms. Likewise, their neutralizing capacity against a global panel of HIV-1 Envs was also increased. The introduction of additional modifications further enhanced the neutralization potency. We also tried engrafting the elongated CDR1 of the heavy chain from bNAb 1-18, another highly potent quaternary-binding antibody, onto several VRC01-class bNAbs, but none of them was improved. These findings point to the highly selective requirements for the establishment of quaternary contact with the HIV-1 Env trimer. The improved anti-CD4bs antibodies reported here may provide a helpful complement to current antibody-based protocols for the therapy and prevention of HIV-1 infection.IMPORTANCE Monoclonal antibodies represent one of the most important recent innovations in the fight against infectious diseases. Although potent antibodies can be cloned from infected individuals, various strategies can be employed to improve their activity or pharmacological features. Here, we improved a lineage of very potent antibodies that target the receptor-binding site of HIV-1 by engineering chimeric molecules containing a fragment from a different monoclonal antibody. These engineered antibodies are promising candidates for development of therapeutic or preventive approaches against HIV/AIDS.
Subject(s)
Binding Sites, Antibody , Broadly Neutralizing Antibodies/immunology , CD4 Antigens/metabolism , HIV Antibodies/immunology , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/immunology , HIV-1/immunology , Protein Engineering , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Binding Sites , Binding Sites, Antibody/immunology , Broadly Neutralizing Antibodies/chemistry , Broadly Neutralizing Antibodies/genetics , Broadly Neutralizing Antibodies/therapeutic use , CD4 Antigens/chemistry , Epitopes/chemistry , Epitopes/immunology , HIV Antibodies/chemistry , HIV Antibodies/genetics , HIV Antibodies/therapeutic use , HIV Envelope Protein gp120/metabolism , HIV Infections/prevention & control , HIV Infections/therapy , Humans , Models, Molecular , Mutation , Protein Binding , Protein Multimerization , Protein Subunits/chemistryABSTRACT
BACKGROUND: The chemokine receptor CCR5 is the major coreceptor for HIV-1 cell entry. We previously observed that not all CCR5 mAbs reduce HIV-1 infection, suggesting that only some CCR5 populations are permissive for HIV-1 entry. This study aims to better understand the relevant conformational states of the cellular coreceptor, CCR5, involved in HIV entry. We hypothesized that CCR5 assumes multiple configurations during normal cycling on the plasma membrane, but only particular forms facilitate HIV-1 infection. METHODS: To this end, we quantified different CCR5 populations using six CCR5 monoclonal antibodies (mAbs) with different epitope specificities and visualized them with super-resolution microscopy. We quantified each surface CCR5 population before and after HIV-1 infection. RESULTS: Based on CCR5 conformational changes, down-modulation, and trafficking rates (internalization and recycling kinetics), we were able to distinguish among heterogeneous CCR5 populations and thus which populations might best be targeted to inhibit HIV-1 entry. We assume that a decreased surface presence of a particular CCR5 subpopulation following infection means that it has been internalized due to HIV-1 entry, and that it therefore represents a highly relevant target for future antiviral therapy strategies. Strikingly, this was most true for antibody CTC8, which targets the N-terminal region of CCR5 and blocks viral entry more efficiently than it blocks chemokine binding. CONCLUSIONS: Defining the virus-host interactions responsible for HIV-1 transmission, including specific coreceptor populations capable of establishing de novo infections, is essential for the development of an HIV-1 vaccine. This study hopefully will facilitate further development of inhibitors to block CCR5 usage by HIV-1, as well as inform future HIV-1 vaccine design.
Subject(s)
HIV Infections , HIV-1 , Humans , Receptors, CCR5 , Virus InternalizationABSTRACT
It is clear that antibodies can play a pivotal role in preventing the transmission of HIV-1 and large efforts to identify an effective antibody-based vaccine to quell the epidemic. Shortly after HIV-1 was discovered as the cause of AIDS, the search for epitopes recognized by neutralizing antibodies became the driving strategy for an antibody-based vaccine. Neutralization escape variants were discovered shortly thereafter, and, after almost three decades of investigation, it is now known that autologous neutralizing antibody responses and their selection of neutralization resistant HIV-1 variants can lead to broadly neutralizing antibodies in some infected individuals. This observation drives an intensive effort to identify a vaccine to elicit broadly neutralizing antibodies. In contrast, there has been less systematic study of antibody specificities that must rely mainly or exclusively on other protective mechanisms, although non-human primate (NHP) studies as well as the RV144 vaccine trial indicate that non-neutralizing antibodies can contribute to protection. Here we propose a novel strategy to identify new epitope targets recognized by these antibodies for which viral escape is unlikely or impossible.
Subject(s)
AIDS Vaccines/immunology , Antibodies, Neutralizing/therapeutic use , HIV Antibodies/therapeutic use , HIV Infections/immunology , HIV-1/immunology , Animals , Clinical Trials as Topic , Epitopes/immunology , HIV Antigens/immunology , Humans , Immune Evasion , PrimatesABSTRACT
A complete picture of HIV antigenicity during early replication is needed to elucidate the full range of options for controlling infection. Such information is frequently gained through analyses of isolated viral envelope antigens, host CD4 receptors, and cognate antibodies. However, direct examination of viral particles and virus-cell interactions is now possible via advanced microscopy techniques and reagents. Using such methods, we recently determined that CD4-induced (CD4i) transition state epitopes in the HIV surface antigen, gp120, while not exposed on free particles, rapidly become immunoreactive upon virus-cell binding. Here, we use 3D direct stochastic optical reconstruction microscopy (dSTORM) to show that certain CD4i epitopes specific to transition state structures are exposed across the surface of cell-bound virions, thus explaining their immunoreactivity. Moreover, such structures and their marker epitopes are dispersed to regions of virions distal to CD4 contact. We further show that the appearance and positioning of distal CD4i exposures is partially dependent on Gag maturation and intact matrix-gp41 interactions within the virion. Collectively, these observations provide a unique perspective of HIV during early replication. These features may define unique insights for understanding how humoral responses target virions and for developing related antiviral countermeasures.
Subject(s)
Epitopes/immunology , HIV Envelope Protein gp120/immunology , HIV Infections/virology , HIV-1/immunology , Virion/immunology , Virus Attachment , CD4 Antigens/metabolism , CD4 Lymphocyte Count , Cell Line , Epitopes/chemistry , HIV Antibodies/immunology , HIV Antigens/immunology , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp41/chemistry , HIV Envelope Protein gp41/immunology , HIV Infections/immunology , HIV-1/chemistry , Humans , Virion/chemistry , Virion/metabolismABSTRACT
A guiding principle for HIV vaccine design has been that cellular and humoral immunity work together to provide the strongest degree of efficacy. However, three efficacy trials of Ad5-vectored HIV vaccines showed no protection. Transmission was increased in two of the trials, suggesting that this vaccine strategy elicited CD4+ T-cell responses that provide more targets for infection, attenuating protection or increasing transmission. The degree to which this problem extends to other HIV vaccine candidates is not known. Here, we show that a gp120-CD4 chimeric subunit protein vaccine (full-length single chain) elicits heterologous protection against simian-human immunodeficiency virus (SHIV) or simian immunodeficiency virus (SIV) acquisition in three independent rhesus macaque repeated low-dose rectal challenge studies with SHIV162P3 or SIVmac251. Protection against acquisition was observed with multiple formulations and challenges. In each study, protection correlated with antibody-dependent cellular cytotoxicity specific for CD4-induced epitopes, provided that the concurrent antivaccine T-cell responses were minimal. Protection was lost in instances when T-cell responses were high or when the requisite antibody titers had declined. Our studies suggest that balance between a protective antibody response and antigen-specific T-cell activation is the critical element to vaccine-mediated protection against HIV. Achieving and sustaining such a balance, while enhancing antibody durability, is the major challenge for HIV vaccine development, regardless of the immunogen or vaccine formulation.
Subject(s)
AIDS Vaccines/pharmacology , CD4-Positive T-Lymphocytes/immunology , HIV Antibodies/immunology , HIV Infections/prevention & control , Immunity, Cellular/drug effects , AIDS Vaccines/immunology , Animals , CD4 Antigens/genetics , CD4 Antigens/immunology , CD4 Antigens/pharmacology , CD4-Positive T-Lymphocytes/pathology , Disease Models, Animal , Female , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp120/pharmacology , HIV Infections/immunology , HIV Infections/pathology , Humans , Immunity, Humoral , Macaca mulatta , Male , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacologyABSTRACT
Recently, Oberle et al. published a paper in Retrovirology evaluating the question of whether selection plays a role in HIV transmission. The Oberle study found no obvious genotypic or phenotypic differences between donors and recipients of epidemiologically linked pairs from the Swiss cohort. Thus, Oberle et al. characterized HIV-1 B transmission as largely "stochastic", an imprecise and potentially misleading term. Here, we re-analyzed their data and placed them in the context of transmission data for over 20 other human and animal trials. The present study finds that the transmitted/founder (T/F) viruses from the Swiss cohort show the same non-random genetic signatures conserved in 118 HIV-1, 40 SHIV, and 12 SIV T/F viruses previously published by two independent groups. We provide alternative interpretations of the Swiss cohort data and conclude that the sequences of their donor viruses lacked variability at the specific sites where other studies were able to demonstrate genotypic selection. Oberle et al. observed no phenotypic selection in vitro, so the problem of determining the in vivo phenotypic mechanisms that cause genotypic selection in HIV remains open.
Subject(s)
HIV Infections , HIV-1/genetics , Animals , Genotype , HumansABSTRACT
Newcastle disease virus (NDV) expressing HIV-1 BaL gp160 was evaluated either alone or with monomeric BaL gp120 and BaL SOSIP gp140 protein in a prime-boost combination in guinea pigs to enhance envelope (Env)-specific humoral and mucosal immune responses. We showed that a regimen consisting of an NDV prime followed by a protein boost elicited stronger serum and mucosal Th-1-biased IgG responses and neutralizing antibody responses than NDV-only immunizations. Additionally, these responses were higher after the gp120 than after the SOSIP gp140 protein boost.
Subject(s)
AIDS Vaccines/administration & dosage , AIDS Vaccines/immunology , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp160/immunology , Immunization Schedule , Vaccination/methods , env Gene Products, Human Immunodeficiency Virus/immunology , AIDS Vaccines/genetics , Administration, Intranasal , Animals , Antibodies, Neutralizing/blood , Drug Carriers , Enzyme-Linked Immunosorbent Assay , Female , Genetic Vectors , Guinea Pigs , HIV Antibodies/blood , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp160/genetics , Immunoglobulin G/blood , Injections, Intramuscular , Neutralization Tests , Newcastle disease virus/genetics , Th1 Cells/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , env Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
The HIV-1 envelope glycoprotein, gp120, undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry, which are being increasingly defined at the atomic level using isolated proteins. In comparison, antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells. Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function. Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells. Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events. We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding. This array comprises both neutralizing and non-neutralizing epitopes, the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function. Under non-permissive conditions for viral entry, both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions. Under entry-permissive conditions, epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents. These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step. The elucidation of these epitope exposure patterns during viral entry will help clarify antibody-mediated inhibition of HIV-1 as it is measured in vitro and in vivo.
Subject(s)
Epitopes/chemistry , HIV Antigens/chemistry , HIV Envelope Protein gp120/chemistry , HIV-1/chemistry , Virion/chemistry , Epitopes/genetics , Epitopes/immunology , HIV Antigens/genetics , HIV Antigens/immunology , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , HIV-1/genetics , HIV-1/immunology , HeLa Cells , Humans , Receptors, CCR5/chemistry , Receptors, CCR5/genetics , Receptors, CCR5/immunology , Viral Tropism/genetics , Viral Tropism/immunology , Virion/genetics , Virion/immunologyABSTRACT
The quest for a prophylactic AIDS vaccine is ongoing, but it is now clear that the successful vaccine must elicit protective antibody responses. Accordingly, intense efforts are underway to identify immunogens that elicit these responses. Regardless of the mechanism of antibody-mediated protection, be it neutralization, Fc-mediated effector function, or both, antibody persistence and appropriate T-cell help are significant problems confronting the development of a successful AIDS vaccine. Here, we discuss the evidence illustrating the poor persistence of antibody responses to Env, the envelope glycoprotein of HIV-1, and the related problem of CD4(+) T-cell responses that compromise vaccine efficacy by creating excess cellular targets of HIV-1 infection. Finally, we propose solutions to both problems that are applicable to all Env-based AIDS vaccines regardless of the mechanism of antibody-mediated protection.
Subject(s)
AIDS Vaccines/immunology , CD4-Positive T-Lymphocytes/immunology , HIV Antibodies/immunology , HIV Infections/immunology , HIV-1/immunology , env Gene Products, Human Immunodeficiency Virus/immunology , CD4-Positive T-Lymphocytes/pathology , HIV Infections/pathology , HIV Infections/prevention & control , HumansABSTRACT
The humoral response to human immunodeficiency virus (HIV) remains incompletely understood. In this report, we describe biased λ light chain use during the HIV Env glycoprotein (Env) response in HIV infection and vaccination. We examined HIV Env binding (and neutralization) in the context of light chain use in subjects with acute HIV infection, chronic HIV infection, and among HIV vaccinees. In all populations tested, there was a λ chain bias for HIV Env binding antibodies, compared with other HIV antigens (such as p24) or tetanus toxoid. In subjects with chronic HIV infection, a λ bias was noted for neutralization, with λ antibodies accounting for up to 90% of all neutralization activity observed. This is the first report of antibody function in a human infection being tied to light chain use. In HIV infection, antibodies expressing λ light chains tended to have longer CDRL3s, increased light chain contact with HIV Env, and less hypermutation in the heavy chain, compared with antibodies using the κ light chain. These data also support an evolutionary model for the understanding the various κ to λ light chain ratios observed across species and suggest that the λ light chain bias against HIV provides the host an advantage in developing a more efficient humoral response.
Subject(s)
HIV Antibodies/immunology , HIV Infections/immunology , Immunoglobulin Light Chains/immunology , env Gene Products, Human Immunodeficiency Virus/immunology , Adolescent , Adult , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Cohort Studies , Female , HIV Antibodies/blood , HIV Infections/epidemiology , HIV Infections/virology , HIV-1/immunology , Humans , Immunity, Humoral , Immunoglobulin Light Chains/blood , Male , Middle Aged , Protein Binding , Young AdultABSTRACT
UNLABELLED: Human immunodeficiency virus (HIV) transmission typically results from infection by a single transmitted/founder (T/F) variant. Are T/F variants chosen uniformly at random from the donor pool, or are they selected based on advantageous traits facilitating transmission? Finding evidence for selection during transmission is of particular interest, because it would indicate that phenotypic and/or genetic properties of the viruses might be harnessed as potential vaccine targets or immunotherapies. Here, we systematically evaluated the differences between the Env proteins of simian immunodeficiency virus/simian HIV (SIV/SHIV) stock and T/F variants in search of "signature" sites of transmission. We also surveyed residue preferences in HIV at the SIV/SHIV signature sites. Four sites of gp120 showed significant selection, and an additional two sites showed a similar trend. Therefore, the six sites clearly differentiate T/F viruses from the majority of circulating variants in the stocks. The selection of SIV/SHIV could be inferred reasonably across both vaccinated and unvaccinated subjects, with infections resulting from vaginal, rectal, and intravenous routes of transmission and regardless of viral dosage. The evidence for selection in SIV and SHIV T/F variants is strong and plentiful, and in HIV the evidence is suggestive though commensurate with the availability of suitable data for analysis. Two of the signature residues are completely conserved across the SIV, SHIV, and HIV variants we examined. Five of the signature residues map to the C1 region of gp120 and one to the signal peptide. Our data raise the possibility that C1, while governing the association between gp120 and gp41, modulates transmission efficiency, replicative fitness, and/or host cell tropism at the level of virus-cell attachment and entry. IMPORTANCE: The present study finds significant evidence of selection on gp120 molecules of SIV/SHIV T/F viruses. The data provide ancillary evidence suggesting the same sites are under selection in HIV. Our findings suggest that the signature residues are involved in increasing the transmissibility of infecting viruses; therefore, they are potential targets for developing a vaccine or other protective measures. A recent study identified the same T/F signature motif but interpreted it as an effect of neutralization resistance. Here, we show that the T/F motif has broader functional significance beyond neutralization sensitivity, because it is present in nonimmune subjects. Also, a vaccine regimen popular in animal trials might have increased the transmission of variants with otherwise low transmission fitness. Our observations might explain why many animal vaccine trials have not faithfully predicted outcomes in human vaccine trials and suggest that current practices in vaccine design need to be reexamined accordingly.
Subject(s)
Conserved Sequence , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/metabolism , HIV Infections/transmission , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Simian Acquired Immunodeficiency Syndrome/transmission , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Animals , Female , Genotype , HIV/genetics , HIV/physiology , HIV Infections/virology , Humans , Macaca mulatta , Male , Selection, Genetic , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/physiology , Viral Tropism , Virus Attachment , Virus ReplicationABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/immunology , HIV Antibodies/immunology , HIV Envelope Protein gp120/ultrastructure , HIV Envelope Protein gp41/ultrastructure , HIV-1/immunology , AIDS Vaccines/immunology , Amino Acid Sequence , Animals , Antibody-Dependent Cell Cytotoxicity/immunology , Binding Sites, Antibody/immunology , CD4-Positive T-Lymphocytes/immunology , Crystallography, X-Ray , Epitopes/immunology , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp41/immunology , HIV Infections/immunology , Humans , Immunity, Humoral/immunology , Macaca mulatta/immunology , Molecular Sequence Data , Protein Conformation , Receptors, Fc/immunology , Sequence Alignment , Simian Immunodeficiency Virus/immunology , Viremia/immunology , Viremia/virologyABSTRACT
To evaluate antibody specificities induced by simian immunodeficiency virus (SIV) versus human immunodeficiency virus type 1 (HIV-1) envelope antigens in nonhuman primate (NHP), we profiled binding antibody responses to linear epitopes in NHP studies with HIV-1 or SIV immunogens. We found that, overall, HIV-1 Env IgG responses were dominated by V3, with the notable exception of the responses to the vaccine strain A244 Env that were dominated by V2, whereas the anti-SIVmac239 Env responses were dominated by V2 regardless of the vaccine regimen.
Subject(s)
Antibodies, Viral/immunology , Epitopes/genetics , HIV-1/immunology , Primates/immunology , Simian Immunodeficiency Virus/immunology , Viral Envelope Proteins/immunology , Animals , Antibodies, Viral/genetics , Antibody Specificity , Immunoglobulin G/blood , Microarray Analysis , Species SpecificityABSTRACT
The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain; and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. Thus, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.
Subject(s)
Antibodies, Monoclonal/immunology , CD4 Antigens/metabolism , Epitopes/metabolism , HIV Antibodies/immunology , HIV Envelope Protein gp120/immunology , HIV-1/immunology , Models, Molecular , Antibodies, Monoclonal/chemistry , Antibody Specificity , Antibody-Dependent Cell Cytotoxicity/immunology , Binding Sites/genetics , CD4 Antigens/chemistry , CD4 Antigens/immunology , Crystallography, X-Ray , Enzyme-Linked Immunosorbent Assay , Epitopes/genetics , HIV Antibodies/chemistry , HIV Envelope Protein gp120/genetics , Humans , Neutralization Tests , Protein ConformationABSTRACT
BACKGROUND: In the RV144 trial, the estimated efficacy of a vaccine regimen against human immunodeficiency virus type 1 (HIV-1) was 31.2%. We performed a case-control analysis to identify antibody and cellular immune correlates of infection risk. METHODS: In pilot studies conducted with RV144 blood samples, 17 antibody or cellular assays met prespecified criteria, of which 6 were chosen for primary analysis to determine the roles of T-cell, IgG antibody, and IgA antibody responses in the modulation of infection risk. Assays were performed on samples from 41 vaccinees who became infected and 205 uninfected vaccinees, obtained 2 weeks after final immunization, to evaluate whether immune-response variables predicted HIV-1 infection through 42 months of follow-up. RESULTS: Of six primary variables, two correlated significantly with infection risk: the binding of IgG antibodies to variable regions 1 and 2 (V1V2) of HIV-1 envelope proteins (Env) correlated inversely with the rate of HIV-1 infection (estimated odds ratio, 0.57 per 1-SD increase; P=0.02; q=0.08), and the binding of plasma IgA antibodies to Env correlated directly with the rate of infection (estimated odds ratio, 1.54 per 1-SD increase; P=0.03; q=0.08). Neither low levels of V1V2 antibodies nor high levels of Env-specific IgA antibodies were associated with higher rates of infection than were found in the placebo group. Secondary analyses suggested that Env-specific IgA antibodies may mitigate the effects of potentially protective antibodies. CONCLUSIONS: This immune-correlates study generated the hypotheses that V1V2 antibodies may have contributed to protection against HIV-1 infection, whereas high levels of Env-specific IgA antibodies may have mitigated the effects of protective antibodies. Vaccines that are designed to induce higher levels of V1V2 antibodies and lower levels of Env-specific IgA antibodies than are induced by the RV144 vaccine may have improved efficacy against HIV-1 infection.
Subject(s)
AIDS Vaccines/immunology , HIV Antibodies/blood , HIV Infections/immunology , HIV-1/immunology , Adult , Case-Control Studies , Follow-Up Studies , HIV Infections/prevention & control , Humans , Immunoglobulin A/blood , Multivariate Analysis , Odds Ratio , Regression Analysis , Risk , Treatment OutcomeABSTRACT
The structural flexibility found in human immunodeficiency virus (HIV) envelope glycoproteins creates a complex relationship between antigenicity and sensitivity to antiviral antibodies. The study of this issue in the context of viral particles is particularly problematic as conventional virus capture approaches can perturb antigenicity profiles. Here, we employed a unique analytical system based on fluorescence correlation spectroscopy (FCS), which measures antibody-virion binding with all reactants continuously in solution. Panels of nine anti-envelope monoclonal antibodies (MAbs) and five virus types were used to connect antibody binding profiles with neutralizing activities. Anti-gp120 MAbs against the 2G12 or b12 epitope, which marks functional envelope structures, neutralized viruses expressing CCR5-tropic envelopes and exhibited efficient virion binding in solution. MAbs against CD4-induced (CD4i) epitopes considered hidden on functional envelope structures poorly bound these viruses and were not neutralizing. Anti-gp41 MAb 2F5 was neutralizing despite limited virion binding. Similar antigenicity patterns occurred on CXCR4-tropic viruses, except that anti-CD4i MAbs 17b and 19e were neutralizing despite little or no virion binding. Notably, anti-gp120 MAb PG9 and anti-gp41 MAb F240 bound to both CCR5-tropic and CXCR4-tropic viruses without exerting neutralizing activity. Differences in the virus production system altered the binding efficiencies of some antibodies but did not enhance antigenicity of aberrant gp120 structures. Of all viruses tested, only JRFL pseudoviruses showed a direct relationship between MAb binding efficiency and neutralizing potency. Collectively, these data indicate that the antigenic profiles of free HIV particles generally favor the exposure of functional over aberrant gp120 structures. However, the efficiency of virion-antibody interactions in solution inconsistently predicts neutralizing activity in vitro.
Subject(s)
Antigens, Viral/immunology , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp41/immunology , HIV Infections/virology , HIV-1/immunology , Virion/immunology , Antigens, Viral/chemistry , Antigens, Viral/genetics , Cell Line , Epitope Mapping , HIV Antibodies/immunology , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp41/chemistry , HIV Envelope Protein gp41/genetics , HIV Infections/immunology , HIV-1/chemistry , HIV-1/genetics , Humans , Neutralization Tests , Virion/chemistry , Virion/geneticsABSTRACT
UNLABELLED: The RV144 vaccine trial implicated epitopes in the C1 region of gp120 (A32-like epitopes) as targets of potentially protective antibody-dependent cellular cytotoxicity (ADCC) responses. A32-like epitopes are highly immunogenic, as infected or vaccinated individuals frequently produce antibodies specific for these determinants. Antibody titers, as measured by enzyme-linked immunosorbent assay (ELISA) against these epitopes, however, do not consistently correlate with protection. Here, we report crystal structures of CD4-stabilized gp120 cores complexed with the Fab fragments of two nonneutralizing, A32-like monoclonal antibodies (MAbs), N5-i5 and 2.2c, that compete for antigen binding and have similar antigen-binding affinities yet exhibit a 75-fold difference in ADCC potency. We find that these MAbs recognize overlapping epitopes formed by mobile layers 1 and 2 of the gp120 inner domain, including the C1 and C2 regions, but bind gp120 at different angles via juxtaposed VH and VL contact surfaces. A comparison of structural and immunological data further showed that antibody orientation on bound antigen and the capacity to form multivalent antigen-antibody complexes on target cells were key determinants of ADCC potency, with the latter process having the greater impact. These studies provide atomic-level definition of A32-like epitopes implicated as targets of protective antibodies in RV144. Moreover, these studies establish that epitope structure and mode of antibody binding can dramatically affect the potency of Fc-mediated effector function against HIV-1. These results provide key insights for understanding, refining, and improving the outcome of HIV vaccine trials, in which relevant immune responses are facilitated by A32-like elicited responses. IMPORTANCE: HIV-1 Env is a primary target for antibodies elicited during infection. Although a small number of infected individuals elicit broadly neutralizing antibodies, the bulk of the humoral response consists of antibodies that do not neutralize or do so with limited breadth but may effect protection through Fc receptor-dependent processes, such as antibody-dependent cellular cytotoxicity (ADCC). Understanding these nonneutralizing responses is an important aspect of elucidating the complete spectrum of immune response against HIV-1 infection. With this report, we provide the first atomic-level definition of nonneutralizing CD4-induced epitopes in the N-terminal region of the HIV-1 gp120 (A32-like epitopes). Further, our studies point to the dominant role of precise epitope targeting and mode of antibody attachment in ADCC responses even when largely overlapping epitopes are involved. Such information provides key insights into the mechanisms of Fc-mediated function of antibodies to HIV-1 and will help us understand the outcome of vaccine trials based on humoral immunity.
Subject(s)
Antibody-Dependent Cell Cytotoxicity , HIV Antibodies/immunology , HIV Envelope Protein gp120/immunology , HIV Infections/immunology , HIV-1/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Crystallography, X-Ray , Epitopes/chemistry , Epitopes/immunology , HIV Antibodies/chemistry , HIV Envelope Protein gp120/chemistry , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/immunology , Models, Molecular , Protein Binding , Protein ConformationABSTRACT
T-cell-derived soluble factors that inhibit both X4 and R5 HIV are recognized as important in controlling HIV. Whereas three ß chemokines, regulated-on-activation normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, and MIP-1ß, account for the suppression of R5 HIV by blockade of HIV entry, the major components responsible for the inhibition of X4 HIV strains have not been identified previously. We identify these factors primarily as a mixture of three ß chemokines [macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), and I-309] and two RNases (angiogenin and RNase 4) of lesser potency and show that in a clade B population, some correlate with clinical status and are produced by both CD4(+) and CD8(+) T cells (chemokines, angiogenin) or only by CD8(+) T cells (RNase 4). The antiviral mechanisms of these HIV X4-suppressive factors differ from those of the previously described HIV R5-suppressive ß chemokines.