Zika-specific neutralizing antibodies targeting inter-dimer envelope epitopes.

Zika virus (ZIKV) is an emerging pathogen that causes devastating congenital defects. The overlapping epidemiology and immunologic cross-reactivity between ZIKV and dengue virus (DENV) pose complex challenges to vaccine design, given the potential for antibody-dependent enhancement of disease. Therefore, classification of ZIKV-specific antibody targets is of notable value. From a ZIKV-infected rhesus macaque, we identify ZIKV-reactive B cells and isolate potent neutralizing monoclonal antibodies (mAbs) with no cross-reactivity to DENV. We group these mAbs into four distinct antigenic groups targeting ZIKV-specific cross-protomer epitopes on the envelope glycoprotein. Co-crystal structures of representative mAbs in complex with ZIKV envelope glycoprotein reveal envelope-dimer epitope and unique dimer-dimer epitope targeting. All four specificities are serologically identified in convalescent humans following ZIKV infection, and representative mAbs from all four groups protect against ZIKV replication in mice. These results provide key insights into ZIKV-specific antigenicity and have implications for ZIKV vaccine, diagnostic, and therapeutic development.

Determination of Binding Affinity of Antibodies to HIV-1 Recombinant Envelope Glycoproteins, Pseudoviruses, Infectious Molecular Clones, and Cell-Expressed Trimeric gp160 Using Microscale Thermophoresis.

Developing a preventative vaccine for HIV-1 has been a global priority. The elicitation of broadly neutralizing antibodies (bNAbs) against a broad range of HIV-1 envelopes (Envs) from various strains appears to be a critical requirement for an efficacious HIV-1 vaccine. To understand their ability to neutralize HIV-1, it is important to characterize the binding characteristics of bNAbs. Our work is the first to utilize microscale thermophoresis (MST), a rapid, economical, and flexible in-solution temperature gradient method to quantitatively determine the binding affinities of bNAbs and non-neutralizing monoclonal antibodies (mAbs) to HIV-1 recombinant envelope monomer and trimer proteins of different subtypes, pseudoviruses (PVs), infectious molecular clones (IMCs), and cells expressing the trimer. Our results demonstrate that the binding affinities were subtype-dependent. The bNAbs exhibited a higher affinity to IMCs compared to PVs and recombinant proteins. The bNAbs and mAbs bound with high affinity to native-like gp160 trimers expressed on the surface of CEM cells compared to soluble recombinant proteins. Interesting differences were seen with V2-specific mAbs. Although they recognize linear epitopes, one of the antibodies also bound to the Envs on PVs, IMCs, and a recombinant trimer protein, suggesting that the epitope was not occluded. The identification of epitopes on the envelope surface that can bind to high affinity mAbs could be useful for designing HIV-1 vaccines and for down-selecting vaccine candidates that can induce high affinity antibodies to the HIV-1 envelope in their native conformation.

Decoding human-macaque interspecies differences in Fc-effector functions: The structural basis for CD16-dependent effector function in Rhesus macaques.

Fc mediated effector functions of antibodies play important roles in immunotherapies and vaccine efficacy but assessing those functions in animal models can be challenging due to species differences. Rhesus macaques, Macaca mulatta (Mm) share approximately 93% sequence identity with humans but display important differences in their adaptive immune system that complicates their use in validating therapeutics and vaccines that rely on Fc effector functions. In contrast to humans, macaques only have one low affinity FcγRIII receptor, CD16, which shares a polymorphism at position 158 with human FcγRIIIa with Ile158 and Val158 variants. Here we describe structure-function relationships of the Ile/Val158 polymorphism in Mm FcγRIII. Our data indicate that the affinity of the allelic variants of Mm FcγRIII for the macaque IgG subclasses vary greatly with changes in glycan composition both on the Fc and the receptor. However, unlike the human Phe/Val158 polymorphism in FcγRIIIa, the higher affinity variant corresponds to the larger, more hydrophobic side chain, Ile, even though it is not directly involved in the binding interface. Instead, this side chain appears to modulate glycan-glycan interactions at the Fc/FcγRIII interface. Furthermore, changes in glycan composition on the receptor have a greater effect for the Val158 variant such that with oligomannose type glycans and with glycans only on Asn45 and Asn162, Val158 becomes the variant with higher affinity to Fc. These results have implications not only for the better interpretation of nonhuman primate studies but also for studies performed with human effector cells carrying different FcγRIIIa alleles.

Army liposome formulation containing QS-21 render human monocyte-derived macrophages less permissive to HIV-1 infection by upregulating ABOBEC3A.

Monocyte-derived macrophages (MDM) are highly permissive to HIV-1 infection potentially due to the downregulation of innate factors during the differentiation process. The environmental milieu and innate anti-viral factors which are modulated during macrophage differentiation, have been associated with their increased permissiveness to HIV-1 infection. Here, we demonstrate that the Army Liposome Formulation containing MPLA, and QS-21 (ALFQ) activated MDM that are normally permissive to HIV-1 infection to generate a proinflammatory environment and upregulated anti-viral factors notably APOBEC3A. Induction of APOBEC3A by ALFQ decreased permissiveness to HIV-1 infection, while knockdown of APOBEC3A with APOBEC3AsiRNA resulted in a significant loss in the restriction of HIV-1 infectivity. The liposome formulation ALF55, with identical lipid composition but lacking QS-21 had no effect. Furthermore, the capacity of ALFQ to modulate MDM permissiveness to HIV-1 infection was predominantly mediated by large ALFQ liposomes. Our findings highlight a relationship between innate immune activation, proinflammatory milieu, and upregulation of anti-HIV proteins. Induction of these responses can switch the HIV-1 permissive MDM into a more refractory phenotype.

SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity.

The need for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) next-generation vaccines has been highlighted by the rise of variants of concern (VoCs) and the long-term threat of emerging coronaviruses. Here, we design and characterize four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of the prefusion SARS-CoV-2 spike (S), S1, and receptor-binding domain (RBD). These immunogens induce robust S binding, ACE2 inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2. A spike-ferritin nanoparticle (SpFN) vaccine elicits neutralizing titers (ID50 > 10,000) following a single immunization, whereas RBD-ferritin nanoparticle (RFN) immunogens elicit similar responses after two immunizations and also show durable and potent neutralization against circulating VoCs. Passive transfer of immunoglobulin G (IgG) purified from SpFN- or RFN-immunized mice protects K18-hACE2 transgenic mice from a lethal SARS-CoV-2 challenge. Furthermore, S-domain nanoparticle immunization elicits ACE2-blocking activity and ID50 neutralizing antibody titers >2,000 against SARS-CoV-1, highlighting the broad response elicited by these immunogens.

Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression.

Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53 in vitro and in vivo, representing a viable therapeutic strategy for cancer treatment. Using phage display techniques, we previously identified a potent peptide activator of P53, termed PMI (TSFAEYWNLLSP), with binding affinities for both MDM2 and MDMX in the low nanomolar concentration range. Here we report an ultrahigh affinity, dual-specificity peptide antagonist of MDM2 and MDMX obtained through systematic mutational analysis and additivity-based molecular design. Functional assays of over 100 peptide analogs of PMI using surface plasmon resonance and fluorescence polarization techniques yielded a dodecameric peptide termed PMI-M3 (LTFLEYWAQLMQ) that bound to MDM2 and MDMX with K d values in the low picomolar concentration range as verified by isothermal titration calorimetry. Co-crystal structures of MDM2 and of MDMX in complex with PMI-M3 were solved at 1.65 and 3.0 Å resolution, respectively. Similar to PMI, PMI-M3 occupied the P53-binding pocket of MDM2/MDMX, which was dominated energetically by intermolecular interactions involving Phe3, Tyr6, Trp7, and Leu10. Notable differences in binding between PMI-M3 and PMI were observed at other positions such as Leu4 and Met11 with MDM2, and Leu1 and Met11 with MDMX, collectively contributing to a significantly enhanced binding affinity of PMI-M3 for both proteins. By adding lysine residues to both ends of PMI and PMI-M3 to improve their cellular uptake, we obtained modified peptides termed PMI-2K (KTSFAEYWNLLSPK) and M3-2K (KLTFLEYWAQLMQK). Compared with PMI-2K, M3-2K exhibited significantly improved antitumor activities in vitro and in vivo in a P53-dependent manner. This super-strong peptide inhibitor of the P53-MDM2/MDMX interactions may become, in its own right, a powerful lead compound for anticancer drug development, and can aid molecular design of other classes of P53 activators as well for anticancer therapy.

Structural and genetic convergence of HIV-1 neutralizing antibodies in vaccinated non-human primates.

A primary goal of HIV-1 vaccine development is the consistent elicitation of protective, neutralizing antibodies. While highly similar neutralizing antibodies (nAbs) have been isolated from multiple HIV-infected individuals, it is unclear whether vaccination can consistently elicit highly similar nAbs in genetically diverse primates. Here, we show in three outbred rhesus macaques that immunization with Env elicits a genotypically and phenotypically conserved nAb response. From these vaccinated macaques, we isolated four antibody lineages that had commonalities in immunoglobulin variable, diversity, and joining gene segment usage. Atomic-level structures of the antigen binding fragments of the two most similar antibodies showed nearly identical paratopes. The Env binding modes of each of the four vaccine-induced nAbs were distinct from previously known monoclonal HIV-1 neutralizing antibodies, but were nearly identical to each other. The similarities of these antibodies show that the immune system in outbred primates can respond to HIV-1 Env vaccination with a similar structural and genotypic solution for recognizing a particular neutralizing epitope. These results support rational vaccine design for HIV-1 that aims to reproducibly elicit, in genetically diverse primates, nAbs with specific paratope structures capable of binding conserved epitopes.

SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity.

The need for SARS-CoV-2 next-generation vaccines has been highlighted by the rise of variants of concern (VoC) and the long-term threat of other coronaviruses. Here, we designed and characterized four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of prefusion Spike (S), S1 and RBD. These immunogens induced robust S-binding, ACE2-inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2 in mice. A Spike-ferritin nanoparticle (SpFN) vaccine elicited neutralizing titers more than 20-fold higher than convalescent donor serum, following a single immunization, while RBD-Ferritin nanoparticle (RFN) immunogens elicited similar responses after two immunizations. Passive transfer of IgG purified from SpFN- or RFN-immunized mice protected K18-hACE2 transgenic mice from a lethal SARS-CoV-2 virus challenge. Furthermore, SpFN- and RFN-immunization elicited ACE2 blocking activity and neutralizing ID50 antibody titers >2,000 against SARS-CoV-1, along with high magnitude neutralizing titers against major VoC. These results provide design strategies for pan-coronavirus vaccine development. HIGHLIGHTS: Iterative structure-based design of four Spike-domain Ferritin nanoparticle classes of immunogensSpFN-ALFQ and RFN-ALFQ immunization elicits potent neutralizing activity against SARS-CoV-2, variants of concern, and SARS-CoV-1Passively transferred IgG from immunized C57BL/6 mice protects K18-hACE2 mice from lethal SARS-CoV-2 challenge.

Defining rules governing recognition and Fc-mediated effector functions to the HIV-1 co-receptor binding site.

BACKGROUND: The binding of HIV-1 Envelope glycoproteins (Env) to host receptor CD4 exposes vulnerable conserved epitopes within the co-receptor binding site (CoRBS) which are required for the engagement of either CCR5 or CXCR4 co-receptor to allow HIV-1 entry. Antibodies against this region have been implicated in the protection against HIV acquisition in non-human primate (NHP) challenge studies and found to act synergistically with antibodies of other specificities to deliver effective Fc-mediated effector function against HIV-1-infected cells. Here, we describe the structure and function of N12-i2, an antibody isolated from an HIV-1-infected individual, and show how the unique structural features of this antibody allow for its effective Env recognition and Fc-mediated effector function. RESULTS: N12-i2 binds within the CoRBS utilizing two adjacent sulfo-tyrosines (TYS) for binding, one of which binds to a previously unknown TYS binding pocket formed by gp120 residues of high sequence conservation among HIV-1 strains. Structural alignment with gp120 in complex with the co-receptor CCR5 indicates that the new pocket corresponds to TYS at position 15 of CCR5. In addition, structure-function analysis of N12-i2 and other CoRBS-specific antibodies indicates a link between modes of antibody binding within the CoRBS and Fc-mediated effector activities. The efficiency of antibody-dependent cellular cytotoxicity (ADCC) correlated with both the level of antibody binding and the mode of antibody attachment to the epitope region, specifically with the way the Fc region was oriented relative to the target cell surface. Antibodies with poor Fc access mediated the poorest ADCC whereas those with their Fc region readily accessible for interaction with effector cells mediated the most potent ADCC. CONCLUSION: Our data identify a previously unknown binding site for TYS within the assembled CoRBS of the HIV-1 virus. In addition, our combined structural-modeling-functional analyses provide new insights into mechanisms of Fc-effector function of antibodies against HIV-1, in particular, how antibody binding to Env antigen affects the efficiency of ADCC response.

Antigen-Induced Allosteric Changes in a Human IgG1 Fc Increase Low-Affinity Fcγ Receptor Binding.

Antibody structure couples adaptive and innate immunity via Fab (antigen binding) and Fc (effector) domains that are connected by unique hinge regions. Because antibodies harbor two or more Fab domains, they are capable of crosslinking multi-determinant antigens, which is required for Fc-dependent functions through associative interactions with effector ligands, including C1q and cell surface Fc receptors. The modular nature of antibodies, with distal ligand binding sites for antigen and Fc-ligands, is reminiscent of allosteric proteins, suggesting that allosteric interactions might contribute to Fc-mediated effector functions. This hypothesis has been pursued for over 40 years and remains unresolved. Here, we provide evidence that allosteric interactions between Fab and Fc triggered by antigen binding modulate binding of Fc to low-affinity Fc receptors (FcγR) for a human IgG1. This work opens the path to further dissection of the relative roles of allosteric and associative interactions in Fc-mediated effector functions.

HIV vaccine delayed boosting increases Env variable region 2-specific antibody effector functions.

In the RV144 HIV-1 phase III trial, vaccine efficacy directly correlated with the magnitude of the variable region 2-specific (V2-specific) IgG antibody response, and in the presence of low plasma IgA levels, with the magnitude of plasma antibody-dependent cellular cytotoxicity. Reenrollment of RV144 vaccinees in the RV305 trial offered the opportunity to define the function, maturation, and persistence of vaccine-induced V2-specific and other mAb responses after boosting. We show that the RV144 vaccine regimen induced persistent V2 and other HIV-1 envelope-specific memory B cell clonal lineages that could be identified throughout the approximately 11-year vaccination period. Subsequent boosts increased somatic hypermutation, a critical requirement for antibody affinity maturation. Characterization of 22 vaccine-induced V2-specific mAbs with epitope specificities distinct from previously characterized RV144 V2-specific mAbs CH58 and CH59 found increased in vitro antibody-mediated effector functions. Thus, when inducing non-neutralizing antibodies, one method by which to improve HIV-1 vaccine efficacy may be through late boosting to diversify the V2-specific response to increase the breadth of antibody-mediated anti-HIV-1 effector functions.

Correction: Trinh, H.V., et al. Humoral Response to the HIV-1 Envelope V2 Region in a Thai Early Acute Infection Cohort. Cells 2019, 8, 365.

In the original version of our article [...].

Humoral Response to the HIV-1 Envelope V2 Region in a Thai Early Acute Infection Cohort.

Reduced risk of HIV-1 infection correlated with antibody responses to the envelope variable 1 and 2 regions in the RV144 vaccine trial. To understand the relationship between antibody responses, V2 sequence, and structure, plasma samples (n = 16) from an early acute HIV-1 infection cohort from Thailand infected with CRF01_AE strain were analyzed for binding to V2 peptides by surface plasmon resonance. Five participants with a range of V2 binding responses at week 24 post-infection were further analyzed against a set of four overlapping V2 peptides that were designed based on envelope single-genome amplification. Antibody responses that were relatively consistent over the four segments of the V2 region or a focused response to the C-strand (residues 165-186) of the V2 region were observed. Viral escape in the V2 region resulted in significantly reduced antibody binding. Structural modeling indicated that the C-strand and the sites of viral variation were highly accessible in the open conformation of the HIV-1 Env trimer. V2 residues, 165-186 are preferentially targeted during acute infection. Residues 169-184 were also preferentially targeted by the protective immune response in the RV144 trial, thus emphasizing the importance of these residues for vaccine design.

From Rhesus macaque to human: structural evolutionary pathways for immunoglobulin G subclasses.

The Old World monkey, Rhesus macaque (Macaca mulatta, Mm), is frequently used as a primate model organism in the study of human disease and to test new vaccines/antibody treatments despite diverging before chimpanzees and orangutans. Mm and humans share 93% genome identity with substantial differences in the genes of the adaptive immune system that lead to different functional IgG subclass characteristics, Fcγ receptors expressed on innate immune cells, and biological interactions. These differences put limitations on Mm use as a primary animal model in the study of human disease and to test new vaccines/antibody treatments. Here, we comprehensively analyzed molecular properties of the Fc domain of the four IgG subclasses of Rhesus macaque to describe potential mechanisms for their interactions with effector cell Fc receptors. Our studies revealed less diversity in the overall structure among the Mm IgG Fc, with MmIgG1 Fc being the most structurally like human IgG3, although its CH2 loops and N297 glycan mobility are comparable to human IgG1. Furthermore, the Fcs of Mm IgG3 and 4 lack the structural properties typical for their human orthologues that determine IgG3's reduced interaction with the neonatal receptor and IgG4's ability for Fab-arm exchange and its weaker Fcγ receptor interactions. Taken together, our data indicate that MmIgG1-4 are less structurally divergent than the human IgGs, with only MmIgG1 matching the molecular properties of human IgG1 and 3, the most active IgGs in terms of Fcγ receptor binding and Fc-mediated functions. PDB accession numbers for deposited structures are 6D4E, 6D4I, 6D4M, and 6D4N for MmIgG1 Fc, MmIgG2 Fc, MmIgG3 Fc, and MmIgG4 Fc, respectively.

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.

Dithiocarbamate-inspired side chain stapling chemistry for peptide drug design.

Two major pharmacological hurdles severely limit the widespread use of small peptides as therapeutics: poor proteolytic stability and membrane permeability. Importantly, low aqueous solubility also impedes the development of peptides for clinical use. Various elaborate side chain stapling chemistries have been developed for α-helical peptides to circumvent this problem, with considerable success in spite of inevitable limitations. Here we report a novel peptide stapling strategy based on the dithiocarbamate chemistry linking the side chains of residues Lys(i) and Cys(i + 4) of unprotected peptides and apply it to a series of dodecameric peptide antagonists of the p53-inhibitory oncogenic proteins MDM2 and MDMX. Crystallographic studies of peptide-MDM2/MDMX complexes structurally validated the chemoselectivity of the dithiocarbamate staple bridging Lys and Cys at (i, i + 4) positions. One dithiocarbamate-stapled PMI derivative, DTCPMI, showed a 50-fold stronger binding to MDM2 and MDMX than its linear counterpart. Importantly, in contrast to PMI and its linear derivatives, the DTCPMI peptide actively traversed the cell membrane and killed HCT116 tumor cells in vitro by activating the tumor suppressor protein p53. Compared with other known stapling techniques, our solution-based DTC stapling chemistry is simple, cost-effective, regio-specific and environmentally friendly, promising an important new tool for the development of peptide therapeutics with improved pharmacological properties including aqueous solubility, proteolytic stability and membrane permeability.

Systematic mutational analysis of human neutrophil α-defensin HNP4.

Defensins are a family of cationic antimicrobial peptides of innate immunity with immunomodulatory properties. The prototypic human α-defensins, also known as human neutrophil peptides 1-3 or HNP1-3, are extensively studied for their structure, function and mechanisms of action, yet little is known about HNP4 - the much less abundant "distant cousin" of HNP1-3. Here we report a systematic mutational analysis of HNP4 with respect to its antibacterial activity against E. coli and S. aureus, inhibitory activity against anthrax lethal factor (LF), and binding activity for LF and HIV-1 gp120. Except for nine conserved and structurally important residues (6xCys, 1xArg, 1xGlu and 1xGly), the remaining 24 residues of HNP4 were each individually mutated to Ala. The crystal structures of G23A-HNP4 and T27A-HNP4 were determined, both exhibiting a disulfide-stabilized canonical α-defensin dimer identical to wild-type HNP4. Unlike HNP1-3, HNP4 preferentially killed the Gram-negative bacterium, a property largely attributable to three clustered cationic residues Arg10, Arg11 and Arg15. The cationic cluster was also important for HNP4 killing of S. aureus, inhibition of LF and binding to LF and gp120. However, F26A, while functionally inconsequential for E. coli killing, was far more deleterious than any other mutations. Similarly, N-methylation of Leu20 to destabilize the HNP4 dimer had little effect on E. coli killing, but significantly reduced the ability of HNP4 to kill S. aureus, inhibit LF, and bind to LF and gp120. Our findings unveil the molecular determinants of HNP4 function, completing the atlas of structure and function relationships for all human neutrophil α-defensins.

Identification of Near-Pan-neutralizing Antibodies against HIV-1 by Deconvolution of Plasma Humoral Responses.

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.

Targeting the Late Stage of HIV-1 Entry for Antibody-Dependent Cellular Cytotoxicity: Structural Basis for Env Epitopes in the C11 Region.

Antibodies can have an impact on HIV-1 infection in multiple ways, including antibody-dependent cellular cytotoxicity (ADCC), a correlate of protection observed in the RV144 vaccine trial. One of the most potent ADCC-inducing epitopes on HIV-1 Env is recognized by the C11 antibody. Here, we present the crystal structure, at 2.9 Å resolution, of the C11-like antibody N12-i3, in a quaternary complex with the HIV-1 gp120, a CD4-mimicking peptide M48U1, and an A32-like antibody, N5-i5. Antibody N12-i3 recognizes an epitope centered on the N-terminal "eighth strand" of a critical ß sandwich, which our analysis indicates to be emblematic of a late-entry state, after the gp120 detachment. In prior entry states, this sandwich comprises only seven strands, with the eighth strand instead pairing with a portion of the gp120 C terminus. The conformational gymnastics of HIV-1 gp120 thus includes altered ß-strand pairing, possibly to reduce immunogenicity, although nevertheless still recognized by the human immune system.

Molecular basis for epitope recognition by non-neutralizing anti-gp41 antibody F240.

Antibody-dependent cell-mediated cytotoxicity (ADCC) by non-neutralizing antibodies (nnAbs) specific to the HIV envelope (Env) glycoproteins present at the surface of virus sensitized or infected cells plays a role in the effective adaptive immune response to HIV. Here, we explore the molecular basis for the epitope at the disulfide loop region (DLR) of the principal immunodominant domain of gp41, recognized by the well-known nnAb F240. Our structural studies reveal details of the F240-gp41 interface and describe a structure of DLR that is distinct from known conformations of this region studied in the context of either CD4-unliganded Env trimer or the gp41 peptide in the unbound state. These data coupled with binding and functional analyses indicate that F240 recognizes non-trimeric Env forms which are significantly overexpressed on intact virions but poorly represented at surfaces of cells infected with infectious molecular clones and endogenously-infected CD4 T cells from HIV-1-infected individuals. Furthermore, although we detect ADCC activities of F240 against cells spinoculated with intact virions, our data suggest that these activities result from F240 recognition of gp41 stumps or misfolded Env variants present on virions rather than its ability to recognize functional gp41 transition structures emerging on trimeric Env post CD4 receptor engagement.