Identifying and Assessing Putative Allosteric Sites and Modulators for CXCR4 Predicted through Network Modeling and Site Identification by Ligand Competitive Saturation.

The chemokine receptor CXCR4 is a critical target for the treatment of several cancer types and HIV-1 infections. While orthosteric and allosteric modulators have been developed targeting its extracellular or transmembrane regions, the intramembrane region of CXCR4 may also include allosteric binding sites suitable for the development of allosteric drugs. To investigate this, we apply the Gaussian Network Model (GNM) to the monomeric and dimeric forms of CXCR4 to identify residues essential for its local and global motions located in the hinge regions of the protein. Residue interaction network (RIN) analysis suggests hub residues that participate in allosteric communication throughout the receptor. Mutual residues from the network models reside in regions with a high capacity to alter receptor dynamics upon ligand binding. We then investigate the druggability of these potential allosteric regions using the site identification by ligand competitive saturation (SILCS) approach, revealing two putative allosteric sites on the monomer and three on the homodimer. Two screening campaigns with Glide and SILCS-Monte Carlo docking using FDA-approved drugs suggest 20 putative hit compounds including antifungal drugs, anticancer agents, HIV protease inhibitors, and antimalarial drugs. In vitro assays considering mAB 12G5 and CXCL12 demonstrate both positive and negative allosteric activities of these compounds, supporting our computational approach. However, in vivo functional assays based on the recruitment of ß-arrestin to CXCR4 do not show significant agonism and antagonism at a single compound concentration. The present computational pipeline brings a new perspective to computer-aided drug design by combining conformational dynamics based on network analysis and cosolvent analysis based on the SILCS technology to identify putative allosteric binding sites using CXCR4 as a showcase.

Deficient Generation of Spike-Specific Long-Lived Plasma Cells in the Bone Marrow After Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Generation of a stable long-lived plasma cell (LLPC) population is the sine qua non of durable antibody responses after vaccination or infection. We studied 20 individuals with a prior coronavirus disease 2019 infection and characterized the antibody response using bone marrow aspiration and plasma samples. We noted deficient generation of spike-specific LLPCs in the bone marrow after severe acute respiratory syndrome coronavirus 2 infection. Furthermore, while the regression model explained 98% of the observed variance in anti-tetanus immunoglobulin G levels based on LLPC enzyme-linked immunospot assay, we were unable to fit the same model with anti-spike antibodies, again pointing to the lack of LLPC contribution to circulating anti-spike antibodies.

Two-photon fluorescence lifetime imaging microscopy of NADH metabolism in HIV-1 infected cells and tissues.

Rapid detection of microbial-induced cellular changes during the course of an infection is critical to understanding pathogenesis and immunological homeostasis. In the last two decades, fluorescence imaging has received significant attention for its ability to help characterize microbial induced cellular and tissue changes in in vitro and in vivo settings. However, most of these methods rely on the covalent conjugation of large exogenous probes and detection methods based on intensity-based imaging. Here, we report a quantitative, intrinsic, label-free, and minimally invasive method based on two-photon fluorescence lifetime (FLT) imaging microscopy (2p-FLIM) for imaging 1,4-dihydro-nicotinamide adenine dinucleotide (NADH) metabolism of virally infected cells and tissue sections. To better understand virally induced cellular and tissue changes in metabolism we have used 2p-FLIM to study differences in NADH intensity and fluorescence lifetimes in HIV-1 infected cells and tissues. Differences in NADH fluorescence lifetimes are associated with cellular changes in metabolism and changes in cellular metabolism are associated with HIV-1 infection. NADH is a critical co-enzyme and redox regulator and an essential biomarker in the metabolic processes. Label-free 2p-FLIM application and detection of NADH fluorescence using viral infection systems are in their infancy. In this study, the application of the 2p-FLIM assay and quantitative analyses of HIV-1 infected cells and tissue sections reveal increased fluorescence lifetime and higher enzyme-bound NADH fraction suggesting oxidative phosphorylation (OxPhos) compared to uninfected cells and tissues. 2p-FLIM measurements improve signal to background, fluorescence specificity, provide spatial and temporal resolution of intracellular structures, and thus, are suitable for quantitative studies of cellular functions and tissue morphology. Furthermore, 2p-FLIM allows distinguishing free and bound populations of NADH by their different fluorescence lifetimes within single infected cells. Accordingly, NADH fluorescence measurements of individual single cells should provide necessary insight into the heterogeneity of metabolic activity of infected cells. Implementing 2p-FLIM to viral infection systems measuring NADH fluorescence at the single or subcellular level within a tissue can provide visual evidence, localization, and information in a real-time diagnostic or therapeutic metabolic workflow.

Evidence of variable human Fcγ receptor-Fc affinities across differentially-complexed IgG.

Antibody-mediated effector functions are widely considered to unfold according to an associative model of IgG-Fcγ receptor (FcγR) interactions. The associative model presupposes that Fc receptors cannot discriminate antigen-bound IgG from free IgG in solution and have equivalent affinities for each. Therefore, the clustering of Fcγ receptors (FcγR) in the cell membrane, cross-activation of intracellular signaling domains, and the formation of the immune synapse are all the result of avid interactions between the Fc region of IgG and FcγRs that collectively overcome the individually weak, transient interactions between binding partners. Antibody allostery, specifically conformational allostery, is a competing model in which antigen-bound antibody molecules undergo a physical rearrangement that causes them to stand out from the background of free IgG by virtue of greater FcγR affinity. Various evidence exists in support of this model of antibody allostery, but it remains controversial. We report observations from multiplexed, label-free kinetic experiments in which the affinity values of FcγR were characterized for covalently immobilized, captured, and antigen-bound IgG. Across the strategies tested, receptors had greater affinity for the antigen-bound mode of IgG presentation. This phenomenon was observed across multiple FcγRs and generalized to multiple antigens, antibody specificities, and subclasses. Furthermore, the thermodynamic signatures of FcγR binding to free or immune-complexed IgG in solution differed when measured by an orthogonal label-free method, but the failure to recapitulate the trend in overall affinity leaves open questions as to what additional factors may be at play.

Synthetic Site-Specific Antibody-Ligand Conjugates Promote Asialoglycoprotein Receptor-Mediated Degradation of Extracellular Human PCSK9.

Targeted degradation using cell-specific lysosome targeting receptors is emerging as a new therapeutic strategy for the elimination of disease-associated proteins. The liver-specific human asialoglycoprotein receptor (ASGPR) is a particularly attractive lysosome targeting receptor leveraged for targeted protein degradation (TPD). However, the efficiency of different glycan ligands for ASGPR-mediated lysosomal delivery remains to be further characterized. In this study, we applied a chemoenzymatic Fc glycan remodeling method to construct an array of site-specific antibody-ligand conjugates carrying natural bi- and tri-antennary N-glycans as well as synthetic tri-GalNAc ligands. Alirocumab, an anti-PCSK9 (proprotein convertase subtilisin/kexin type 9) antibody, and cetuximab (an anti-EGFR antibody) were chosen to demonstrate the ASGPR-mediated degradation of extracellular and membrane-associated proteins, respectively. It was found that the nature of the glycan ligands and the length of the spacer in the conjugates are critical for the receptor binding and the receptor-mediated degradation of PCSK9, which blocks low-density lipoprotein receptor (LDLR) function and adversely affects clearance of low-density lipoprotein cholesterol. Interestingly, the antibody-tri-GalNAc conjugates showed a clear hook effect for its binding to ASGPR, while antibody conjugates carrying the natural N-glycans did not. Both the antibody-tri-antennary N-glycan conjugate and the antibody-tri-GalNAc conjugate could significantly decrease extracellular PCSK9, as shown in the cell-based assays. However, the tri-GalNAc conjugate showed a clear hook effect in the receptor-mediated degradation of PCSK9, while the antibody conjugate carrying the natural N-glycans did not. The cetuximab-tri-GalNAc conjugates also showed a similar hook effect on degradation of the membrane-associated protein, epidermal growth factor receptor (EGFR). These results suggest that the two types of ligands may involve a distinct mode of interactions in the receptor binding and target-degradation processes. Interestingly, the alirocumab-tri-GalNAc conjugate was also found to upregulate LDLR levels in comparison with the antibody alone. This study showcases the potential of the targeted degradation strategy against PCSK9 for reducing low-density lipoprotein cholesterol, a risk factor for heart disease and stroke.

Development of an anti-Pseudomonas aeruginosa therapeutic monoclonal antibody WVDC-5244.

The rise of antimicrobial-resistant bacterial infections is a crucial health concern in the 21st century. In particular, antibiotic-resistant Pseudomonas aeruginosa causes difficult-to-treat infections associated with high morbidity and mortality. Unfortunately, the number of effective therapeutic interventions against antimicrobial-resistant P. aeruginosa infections continues to decline. Therefore, discovery and development of alternative treatments are necessary. Here, we present pre-clinical efficacy studies on an anti-P. aeruginosa therapeutic monoclonal antibody. Using hybridoma technology, we generated a monoclonal antibody and characterized its binding to P. aeruginosa in vitro using ELISA and fluorescence correlation spectroscopy. We also characterized its function in vitro and in vivo against P. aeruginosa. The anti-P. aeruginosa antibody (WVDC-5244) bound P. aeruginosa clinical strains of various serotypes in vitro, even in the presence of alginate exopolysaccharide. In addition, WVDC-5244 induced opsonophagocytic killing of P. aeruginosa in vitro in J774.1 murine macrophage, and complement-mediated killing. In a mouse model of acute pneumonia, prophylactic administration of WVDC-5244 resulted in an improvement of clinical disease manifestations and reduction of P. aeruginosa burden in the respiratory tract compared to the control groups. This study provides promising pre-clinical efficacy data on a new monoclonal antibody with therapeutic potential for P. aeruginosa infections.

Maternal transfer of IgA and IgG SARS-CoV-2 specific antibodies transplacentally and via breast milk feeding.

BACKGROUND: Although there have been many studies on antibody responses to SARS-CoV-2 in breast milk, very few have looked at the fate of these in the infant, and whether they are delivered to immunologically relevant sites in infants. METHODS: Mother/infant pairs (mothers who breast milk fed and who were SARS-CoV-2 vaccinated before or after delivery) were recruited for this cross-sectional study. Mother blood, mother breast milk, infant blood, infant nasal specimen, and infant stool was tested for IgA and IgG antibodies against SARS-CoV-2 spike trimer. RESULTS: Thirty-one mother/infant pairs were recruited. Breast milk fed infants acquired systemic anti-spike IgG antibodies only if their mothers were vaccinated antepartum (100% Antepartum; 0% Postpartum; P<0.0001). Breast milk fed infants acquired mucosal anti-spike IgG antibodies (in the nose) only if their mothers were vaccinated antepartum (89% Antepartum; 0% Postpartum; P<0.0001). None of the infants in either group had anti-spike IgA in the blood. Surprisingly, 33% of the infants whose mothers were vaccinated antepartum had high titer anti-spike IgA in the nose (33% Antepartum; 0% Postpartum; P = 0.03). Half-life of maternally transferred plasma IgG antibodies in the Antepartum infant cohort was ~70 days. CONCLUSION: Vaccination antepartum followed by breast milk feeding appears to be the best way to provide systemic and local anti-SARS-CoV-2 antibodies for infants. The presence of high titer SARS-CoV-2-specific IgA in the nose of infants points to the potential importance of breast milk feeding early in life for maternal transfer of mucosal IgA antibodies. Expectant mothers should consider becoming vaccinated antepartum and consider breast milk feeding for optimal transfer of systemic and mucosal antibodies to their infants.

Replacement of Antiretroviral Therapy with HIV Broadly Neutralizing Antibodies to Maximize the Effectiveness of Chemotherapy in HIV Patients with Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most fatal non-AIDS defining cancer in people living with HIV (PWH) on antiretroviral therapy (ART). Treatment of malignancies in PWH requires concomitant cancer therapy and ART, which can lead to potential drug-drug interactions (DDIs) and overlapping toxicities. In this study, we hypothesize that replacement of ART with HIV broadly neutralizing antibodies (bNAbs) during cancer chemotherapy (chemo) may maintain HIV suppression and tumor inhibition while minimizing DDIs and overlapping toxicities. We compared HIV suppression, tumor inhibition, and toxicity between conventional treatment (ART plus chemo) and a new modality (bNAbs plus chemo) in humanized mice. Humanized mice infected with HIVYU2 and xenografted with human NSCLC A549 cells were treated with NSCLC chemo (cisplatin and gemcitabine) and first-line ART (dolutegravir, tenofovir disoproxil difumarate, and emtricitabine) or bNAbs (N49P9.6-FR and PGT 121) at human equivalent drug doses. We monitored plasma HIV RNA, tumor volume, and toxicities over five cycles of chemo. We found that chemo plus ART or bNAbs were equally effective at maintaining suppression of HIV viremia and tumor growth. Comparative analysis showed that mice on ART and chemo had significant reductions in body weight and significant increases in plasma creatinine concentrations compared with mice on bNAbs and chemo, which suggests that a combination of bNAbs and chemo produces less renal toxicity than an ART and chemo combination. These data suggest that bNAb therapy during concomitant chemo may be an improved treatment option over ART for PWH and NSCLC, and possibly other cancers, because bNAbs maintain HIV suppression while minimizing DDIs and toxicities.

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.

Impact of adjuvants on the biophysical and functional characteristics of HIV vaccine-elicited antibodies in humans.

Adjuvants can alter the magnitude, characteristics, and persistence of the humoral response to protein vaccination. HIV vaccination might benefit from tailored adjuvant choice as raising a durable and protective response to vaccination has been exceptionally challenging. Analysis of trials of partially effective HIV vaccines have identified features of the immune response that correlate with decreased risk, including high titers of V1V2-binding IgG and IgG3 responses with low titers of V1V2-binding IgA responses and enhanced Fc effector functions, notably antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). However, there has been limited opportunity to compare the effect of different adjuvants on these activities in humans. Here, samples from the AVEG015 study, a phase 1 trial in which participants (n = 112) were immunized with gp120SF-2 and one of six different adjuvants or combinations thereof were assessed for antibody titer, biophysical features, and diverse effector functions. Three adjuvants, MF59 + MTP-PE, SAF/2, and SAF/2 + MDP, increased the peak magnitude and durability of antigen-specific IgG3, IgA, FcγR-binding responses and ADCP activity, as compared to alum. While multiple adjuvants increased the titer of IgG, IgG3, and IgA responses, none consistently altered the balance of IgG to IgA or IgG3 to IgA. Linear regression analysis identified biophysical features including gp120-specific IgG and FcγR-binding responses that could predict functional activity, and network analysis identified coordinated aspects of the humoral response. These analyses reveal the ability of adjuvants to drive the character and function of the humoral response despite limitations of small sample size and immune variability in this human clinical trial.

Diverse antiviral IgG effector activities are predicted by unique biophysical antibody features.

BACKGROUND: The critical role of antibody Fc-mediated effector functions in immune defense has been widely reported in various viral infections. These effector functions confer cellular responses through engagement with innate immune cells. The precise mechanism(s) by which immunoglobulin G (IgG) Fc domain and cognate receptors may afford protection are poorly understood, however, in the context of HIV/SHIV infections. Many different in vitro assays have been developed and utilized to measure effector functions, but the extent to which these assays capture distinct antibody activities has not been fully elucidated. RESULTS: In this study, six Fc-mediated effector function assays and two biophysical antibody profiling assays were performed on a common set of samples from HIV-1 infected and vaccinated subjects. Biophysical antibody profiles supported robust prediction of diverse IgG effector functions across distinct Fc-mediated effector function assays. While a number of assays showed correlated activities, supervised machine learning models indicated unique antibody features as primary contributing factors to the associated effector functions. Additional experiments established the mechanistic relevance of relationships discovered using this unbiased approach. CONCLUSIONS: In sum, this study provides better resolution on the diversity and complexity of effector function assays, offering a clearer perspective into this family of antibody mechanisms of action to inform future HIV-1 treatment and vaccination strategies.

Safety and immunogenicity of an HIV-1 gp120-CD4 chimeric subunit vaccine in a phase 1a randomized controlled trial.

A major challenge for HIV vaccine development is to raise anti-envelope antibodies capable of recognizing and neutralizing diverse strains of HIV-1. Accordingly, a full length single chain (FLSC) of gp120-CD4 chimeric vaccine construct was designed to present a highly conserved CD4-induced (CD4i) HIV-1 envelope structure that elicits cross-reactive anti-envelope humoral responses and protective immunity in animal models of HIV infection. IHV01 is the FLSC formulated in aluminum phosphate adjuvant. We enrolled 65 healthy adult volunteers in this first-in-human phase 1a randomized, double-blind, placebo-controlled study with three dose-escalating cohorts (75 µg, 150 µg, and 300 µg doses). Intramuscular injections were given on weeks 0, 4, 8, and 24. Participants were followed for an additional 24 weeks after the last immunization. The overall incidence of adverse events (AEs) was not significantly different between vaccinees and controls. The majority (89%) of vaccine-related AE were mild. The most common vaccine-related adverse event was injection site pain. There were no vaccine-related serious AE, discontinuation due to AE, intercurrent HIV infection, or significant decreases in CD4 count. By the final vaccination, all vaccine recipients developed antibodies against IHV01 and demonstrated anti-CD4i epitope antibodies. The elicited antibodies reacted with CD4 non-liganded Env antigens from diverse HIV-1 strains. Antibody-dependent cell-mediated cytotoxicity against heterologous infected cells or gp120 bound to CD4+ cells was evident in all cohorts as were anti-gp120 T-cell responses. IHV01 vaccine was safe, well tolerated, and immunogenic at all doses tested. The vaccine raised broadly reactive humoral responses against conserved CD4i epitopes on gp120 that mediates antiviral functions.

Structure and Fc-Effector Function of Rhesusized Variants of Human Anti-HIV-1 IgG1s.

Passive transfer of monoclonal antibodies (mAbs) of human origin into Non-Human Primates (NHPs), especially those which function predominantly by a Fc-effector mechanism, requires an a priori preparation step, in which the human mAb is reengineered to an equivalent NHP IgG subclass. This can be achieved by changing both the Fc and Fab sequence while simultaneously maintaining the epitope specificity of the parent antibody. This Ab reengineering process, referred to as rhesusization, can be challenging because the simple grafting of the complementarity determining regions (CDRs) into an NHP IgG subclass may impact the functionality of the mAb. Here we describe the successful rhesusization of a set of human mAbs targeting HIV-1 envelope (Env) epitopes involved in potent Fc-effector function against the virus. This set includes a mAb targeting a linear gp120 V1V2 epitope isolated from a RV144 vaccinee, a gp120 conformational epitope within the Cluster A region isolated from a RV305 vaccinated individual, and a linear gp41 epitope within the immunodominant Cys-loop region commonly targeted by most HIV-1 infected individuals. Structural analyses confirm that the rhesusized variants bind their respective Env antigens with almost identical specificity preserving epitope footprints and most antigen-Fab atomic contacts with constant regions folded as in control RM IgG1s. In addition, functional analyses confirm preservation of the Fc effector function of the rhesusized mAbs including the ability to mediate Antibody Dependent Cell-mediated Cytotoxicity (ADCC) and antibody dependent cellular phagocytosis by monocytes (ADCP) and neutrophils (ADNP) with potencies comparable to native macaque antibodies of similar specificity. While the antibodies chosen here are relevant for the examination of the correlates of protection in HIV-1 vaccine trials, the methods used are generally applicable to antibodies for other purposes.

Performance of nucleocapsid and spike-based SARS-CoV-2 serologic assays.

There is an urgent need for an accurate antibody test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have developed 3 ELISA methods, trimer spike IgA, trimer spike IgG, and nucleocapsid IgG, for detecting anti-SARS-CoV-2 antibodies. We evaluated their performance along with four commercial ELISAs, EDI™ Novel Coronavirus COVID-19 ELISA IgG and IgM, Euroimmun Anti-SARS-CoV-2 ELISA IgG and IgA, and one lateral flow assay, DPP® COVID-19 IgM/IgG System (Chembio). Both sensitivity and specificity were evaluated and the probable causes of false-positive reactions were determined. The assays were evaluated using 300 pre-epidemic samples and 100 PCR-confirmed COVID-19 samples. The sensitivities and specificities of the assays were as follows: 90%/100% (in-house trimer spike IgA), 90%/99.3% (in-house trimer spike IgG), 89%/98.3% (in-house nucleocapsid IgG), 73.7%/100% (EDI nucleocapsid IgM), 84.5%/95.1% (EDI nucleocapsid IgG), 95%/93.7% (Euroimmun S1 IgA), 82.8%/99.7% (Euroimmun S1 IgG), 82.0%/91.7% (Chembio nucleocapsid IgM), 92%/93.3% (Chembio nucleocapsid IgG). The presumed causes of false positive results from pre-epidemic samples in commercial and in-house assays were mixed. In some cases, assays lacked reproducibility. In other cases, reactivity was abrogated by competitive inhibition (spiking the sample with the same antigen that was used for coating ELISAs prior to performing the assay), suggesting positive reaction could be attributed to the presence of antibodies against these antigens. In other cases, reactivity was consistently detected but not abrogated by the spiking, suggesting positive reaction was not attributed to the presence of antibodies against these antigens. Overall, there was wide variability in assay performance using our samples, with in-house tests exhibiting the highest combined sensitivity and specificity. The causes of "false positivity" in pre-epidemic samples may be due to plasma antibodies apparently reacting with the corresponding antigen, or spurious reactivity may be directed against non-specific components in the assay system. Identification of these targets will be essential to improving assay performance.

Specificity and Performance of Nucleocapsid and Spike-based SARS-CoV-2 Serologic Assays.

There is an urgent need for an accurate antibody test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this paper, we have developed 3 ELISA methods, trimer spike IgA, trimer spike IgG, and nucleocapsid IgG, for detecting anti-SARS-CoV-2 antibodies. We evaluated their performance in comparison with four commercial ELISAs, EDI™ Novel Coronavirus COVID-19 ELISA IgG and IgM, Euroimmun Anti-SARS-CoV-2 ELISA IgG and IgA, and one lateral flow assay, DPP® COVID-19 IgM/IgG System (Chembio). Both sensitivity and specificity were evaluated and the causes of false-positive reactions were determined. The assays were compared using 300 pre-epidemic samples and 100 PCR-confirmed COVID-19 samples. The sensitivities and specificities of the assays were as follows: 90%/100% (in-house trimer spike IgA), 90%/99.3% (in-house trimer spike IgG), 89%/98.3% (in-house nucleocapsid IgG), 73.7%/100% (EDI nucleocapsid IgM), 84.5%/95.1% (EDI nucleocapsid IgG), 95%/93.7% (Euroimmun S1 IgA), 82.8%/99.7% (Euroimmun S1 IgG), 82.0%/91.7% (Chembio nucleocapsid IgM), 92%/93.3% (Chembio nucleocapsid IgG). The presumed causes of positive signals from pre-epidemic samples in commercial and in-house assays were mixed. In some cases, positivity varied with assay repetition. In other cases, reactivity was abrogated by competitive inhibition (spiking the sample with analyte prior to performing the assay). In other cases, reactivity was consistently detected but not abrogated by analyte spiking. Overall, there was wide variability in assay performance using our samples, with in-house tests exhibiting the highest combined sensitivity and specificity. The causes of "false positivity" in pre-epidemic samples may be due to plasma antibodies apparently reacting with the analyte, or spurious reactivity may be directed against non-specific components in the assay system. Identification of these targets will be essential to improving assay performance.

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.

Recognition Patterns of the C1/C2 Epitopes Involved in Fc-Mediated Response in HIV-1 Natural Infection and the RV114 Vaccine Trial.

Antibodies (Abs) specific for CD4-induced envelope (Env) epitopes within constant region 1 and 2 (C1/C2) were induced in the RV144 vaccine trial, where antibody-dependent cellular cytotoxicity (ADCC) correlated with reduced risk of HIV-1 infection. We combined X-ray crystallography and fluorescence resonance energy transfer-fluorescence correlation spectroscopy to describe the molecular basis for epitopes of seven RV144 Abs and compared them to A32 and C11, C1/C2 Abs induced in HIV infection. Our data indicate that most vaccine Abs recognize the 7-stranded ß-sandwich of gp120, a unique hybrid epitope bridging A32 and C11 binding sites. Although primarily directed at the 7-stranded ß-sandwich, some accommodate the gp120 N terminus in C11-bound 8-stranded conformation and therefore recognize a broader range of CD4-triggered Env conformations. Our data also suggest that Abs of RV144 and RV305, the RV144 follow-up study, although likely initially induced by the ALVAC-HIV prime encoding full-length gp120, matured through boosting with truncated AIDSVAX gp120 variants.IMPORTANCE Antibody-dependent cellular cytotoxicity (ADCC) correlated with a reduced risk of infection from HIV-1 in the RV144 vaccine trial, the only HIV-1 vaccine trial to date to show any efficacy. Antibodies specific for CD4-induced envelope (Env) epitopes within constant region 1 and 2 (cluster A region) were induced in the RV144 trial and their ADCC activities were implicated in the vaccine efficacy. We present structural analyses of the antigen epitope targets of several RV144 antibodies specific for this region and C11, an antibody induced in natural infection, to show what the differences are in epitope specificities, mechanism of antigen recognition, and ADCC activities of antibodies induced by vaccination and during the course of HIV infection. Our data suggest that the truncated AIDSVAX gp120 variants used in the boost of the RV144 regimen may have shaped the vaccine response to this region, which could also have contributed to vaccine efficacy.

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.

Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization.

Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus. We systematically analyzed the antibody repertoire in human survivors and identified a pair of potently neutralizing mAbs that cooperatively bound to the ebolavirus glycoprotein (GP). High-resolution structures revealed that in a two-antibody cocktail, molecular mimicry was a major feature of mAb-GP interactions. Broadly neutralizing mAb rEBOV-520 targeted a conserved epitope on the GP base region. mAb rEBOV-548 bound to a glycan cap epitope, possessed neutralizing and Fc-mediated effector function activities, and potentiated neutralization by rEBOV-520. Remodeling of the glycan cap structures by the cocktail enabled enhanced GP binding and virus neutralization. The cocktail demonstrated resistance to virus escape and protected non-human primates (NHPs) against Ebola virus disease. These data illuminate structural principles of antibody cooperativity with implications for development of antiviral immunotherapeutics.