Conserved antigen structures and antibody-driven variations on foot-and-mouth disease virus serotype A revealed by bovine neutralizing monoclonal antibodies.

Foot-and-mouth disease virus (FMDV) serotype A is antigenically most variable within serotypes. The structures of conserved and variable antigenic sites were not well resolved. Here, a historical A/AF72 strain from A22 lineage and a latest A/GDMM/2013 strain from G2 genotype of Sea97 lineage were respectively used as bait antigen to screen single B cell antibodies from bovine sequentially vaccinated with A/WH/CHA/09 (G1 genotype of Sea97 lineage), A/GDMM/2013 and A/AF72 antigens. Total of 39 strain-specific and 5 broad neutralizing antibodies (bnAbs) were isolated and characterized. Two conserved antigenic sites were revealed by the Cryo-EM structures of FMDV serotype A with two bnAbs W2 and W125. The contact sites with both VH and VL of W125 were closely around icosahedral threefold axis and covered the B-C, E-F, and H-I loops on VP2 and the B-B knob and H-I loop on VP3; while contact sites with only VH of W2 concentrated on B-B knob, B-C and E-F loops on VP3 scattering around the three-fold axis of viral particle. Additional highly conserved epitopes also involved key residues of VP158, VP1147 and both VP272 / VP1147 as determined respectively by bnAb W153, W145 and W151-resistant mutants. Furthermore, the epitopes recognized by 20 strain-specific neutralization antibodies involved the key residues located on VP3 68 for A/AF72 (11/20) and VP3 175 position for A/GDMM/2013 (9/19), respectively, which revealed antigenic variation between different strains of serotype A. Analysis of antibody-driven variations on capsid of two virus strains showed a relatively stable VP2 and more variable VP3 and VP1. This study provided important information on conserve and variable antigen structures to design broad-spectrum molecular vaccine against FMDV serotype A.

Epistasis reduces fitness costs of influenza A virus escape from stem-binding antibodies.

The hemagglutinin (HA) stem region is a major target of universal influenza vaccine efforts owing to the presence of highly conserved epitopes across multiple influenza A virus (IAV) strains and subtypes. To explore the potential impact of vaccine-induced immunity targeting the HA stem, we examined the fitness effects of viral escape from stem-binding broadly neutralizing antibodies (stem-bnAbs). Recombinant viruses containing each individual antibody escape substitution showed diminished replication compared to wild-type virus, indicating that stem-bnAb escape incurred fitness costs. A second-site mutation in the HA head domain (N129D; H1 numbering) reduced the fitness effects observed in primary cell cultures and likely enabled the selection of escape mutations. Functionally, this putative permissive mutation increased HA avidity for its receptor. These results suggest a mechanism of epistasis in IAV, wherein modulating the efficiency of attachment eases evolutionary constraints imposed by the requirement for membrane fusion. Taken together, the data indicate that viral escape from stem-bnAbs is costly but highlights the potential for epistatic interactions to enable evolution within the functionally constrained HA stem domain.

Inactivated genotype 1a, 2a and 3a HCV vaccine candidates induced broadly neutralising antibodies in mice.

OBJECTIVE: A prophylactic vaccine is needed to control the HCV epidemic, with genotypes 1-3 causing >80% of worldwide infections. Vaccine development is hampered by HCV heterogeneity, viral escape including protection of conserved neutralising epitopes and suboptimal efficacy of HCV cell culture systems. We developed cell culture-based inactivated genotype 1-3 HCV vaccine candidates to present natively folded envelope proteins to elicit neutralising antibodies. DESIGN: High-yield genotype 1a, 2a and 3a HCV were developed by serial passage of TNcc, J6cc and DBN3acc in Huh7.5 cells and engineering of acquired mutations detected by next-generation sequencing. Neutralising epitope exposure was determined in cell-based neutralisation assays using human monoclonal antibodies AR3A and AR4A, and polyclonal antibody C211. BALB/c mice were immunised with processed and inactivated genotype 1a, 2a or 3a viruses using AddaVax, a homologue of the licenced adjuvant MF-59. Purified mouse and patient serum IgG were assayed for neutralisation capacity; mouse IgG and immune-sera were assayed for E1/E2 binding. RESULTS: Compared with the original viruses, high-yield viruses had up to ~1000 fold increased infectivity titres (peak titres: 6-7 log10 focus-forming units (FFU)/mL) and up to ~2470 fold increased exposure of conserved neutralising epitopes. Vaccine-induced IgG broadly neutralised genotype 1-6 HCV (EC50: 30-193 µg/mL; mean 71 µg/mL), compared favourably with IgG from chronically infected patients, and bound genotype 1-3 E1/E2; immune-sera endpoint titres reached up to 32 000. CONCLUSION: High-yield genotype 1-3 HCV could be developed as basis for inactivated vaccine candidates inducing broadly neutralising antibodies in mice supporting further preclinical development.

Triggering rare HIV antibodies by vaccination.

Clinical trial shows that an HIV vaccine can elicit rare antibodies, but there is more to do.

Vaccination induces HIV broadly neutralizing antibody precursors in humans.

Broadly neutralizing antibodies (bnAbs) can protect against HIV infection but have not been induced by human vaccination. A key barrier to bnAb induction is vaccine priming of rare bnAb-precursor B cells. In a randomized, double-blind, placebo-controlled phase 1 clinical trial, the HIV vaccine-priming candidate eOD-GT8 60mer adjuvanted with AS01B had a favorable safety profile and induced VRC01-class bnAb precursors in 97% of vaccine recipients with median frequencies reaching 0.1% among immunoglobulin G B cells in blood. bnAb precursors shared properties with bnAbs and gained somatic hypermutation and affinity with the boost. The results establish clinical proof of concept for germline-targeting vaccine priming, support development of boosting regimens to induce bnAbs, and encourage application of the germline-targeting strategy to other targets in HIV and other pathogens.

Long-Term and Low-Level Envelope C2V3 Stimulation by Highly Diverse Virus Isolates Leads to Frequent Development of Broad and Elite Antibody Neutralization in HIV-1-Infected Individuals.

A minority of HIV-1-infected patients produce broadly neutralizing antibodies (bNAbs). Identification of viral and host correlates of bNAb production may help develop vaccines. We aimed to characterize the neutralizing response and viral and host-associated factors in Angola, which has one of the oldest, most dynamic, and most diverse HIV-1 epidemics in the world. Three hundred twenty-two HIV-1-infected adults from Angola were included in this retrospective study. Phylogenetic analysis of C2V3C3 env gene sequences was used for virus subtyping. Env-binding antibody reactivity was tested against polypeptides comprising the C2, V3, and C3 regions. Neutralizing-antibody responses were determined against a reference panel of tier 2 Env pseudoviruses in TZM-bl cells; neutralizing epitope specificities were predicted using ClustVis. All subtypes were found, along with untypeable strains and recombinant forms. Notably, 56% of the patients developed cross neutralizing, broadly neutralizing, or elite neutralizing responses. Broad and elite neutralization was associated with longer infection time, subtype C, lower CD4+ T cell counts, higher age, and higher titer of C2V3C3-specific antibodies relative to failure to develop bNAbs. Neutralizing antibodies targeted the V3-glycan supersite in most patients. V3 and C3 regions were significantly less variable in elite neutralizers than in weak neutralizers and nonneutralizers, suggesting an active role of V3C3-directed bNAbs in controlling HIV-1 replication and diversification. In conclusion, prolonged and low-level envelope V3C3 stimulation by highly diverse and ancestral HIV-1 isolates promotes the frequent elicitation of bNAbs. These results provide important clues for the development of an effective HIV-1 vaccine. IMPORTANCE Studies on neutralization by antibodies and their determinants in HIV-1-infected individuals have mostly been conducted in relatively recent epidemics caused by subtype B and C viruses. Results have suggested that elicitation of broadly neutralizing antibodies (bNAbs) is uncommon. The mechanisms underlying the elicitation of bNAbs are still largely unknown. We performed the first characterization of the plasma neutralizing response in a cohort of HIV-1-infected patients from Angola. Angola is characterized by an old and dynamic epidemic caused by highly diverse HIV-1 variants. Remarkably, more than half of the patients produced bNAbs, mostly targeting the V3-glycan supersite in HIV-1. This was associated with higher age, longer infection time, lower CD4+ T cell counts, subtype C infection, or higher titer of C2V3C3-specific antibodies relative to patients that did not develop bNAbs. These results may help develop the next generation of vaccine candidates for HIV-1.

Complementary Roles of Antibody Heavy and Light Chain Somatic Hypermutation in Conferring Breadth and Potency to the HIV-1-Specific CAP256-VRC26 bNAb Lineage.

Some HIV-infected people develop broadly neutralizing antibodies (bNAbs) that block many diverse, unrelated strains of HIV from infecting target cells and, through passive immunization, protect animals and humans from infection. Therefore, understanding the development of bNAbs and their neutralization can inform the design of an HIV vaccine. Here, we extend our previous studies of the ontogeny of the CAP256-VRC26 V2-targeting bNAb lineage by defining the mutations that confer neutralization to the unmutated common ancestor (CAP256.UCA). Analysis of the sequence of the CAP256.UCA showed that many improbable mutations were located in the third complementarity-determining region of the heavy chain (CDRH3) and the heavy chain framework 3 (FR3). Transferring the CDRH3 from bNAb CAP256.25 (63% breadth and 0.003 µg/mL potency) into the CAP256.UCA introduced breadth and the ability to neutralize emerging viral variants. In addition, we showed that the framework and light chain contributed to potency and that the second CDR of the light chain forms part of the paratope of CAP256.25. Notably, a minimally mutated CAP256 antibody, with 41% of the mutations compared to bNAb CAP256.25, was broader (64% breadth) and more potent (0.39 µg/mL geometric potency) than many unrelated bNAbs. Together, we have identified key regions and mutations that confer breadth and potency in a V2-specific bNAb lineage. These data indicate that immunogens that target affinity maturation to key sites in CAP256-VRC26-like precursors, including the CDRHs and light chain, could rapidly elicit breadth through vaccination. IMPORTANCE A major focus in the search for an HIV vaccine is elucidating the ontogeny of broadly neutralizing antibodies (bNAbs), which prevent HIV infection in vitro and in vivo. The unmutated common ancestors (UCAs) of bNAbs are generally strain specific and acquire breadth through extensive, and sometimes redundant, somatic hypermutation during affinity maturation. We investigated which mutations in the CAP256-VRC26 bNAb lineage conferred neutralization capacity to the UCA. We found that mutations in the antibody heavy and light chains had complementary roles in neutralization breadth and potency, respectively. The heavy chain, particularly the third complementarity-determining region, was responsible for conferring breadth. In addition, previously uninvestigated mutations in the framework also contributed to breadth. Together, approximately half of the mutations in CAP256.25 were necessary for broader and more potent neutralization than many unrelated neutralizing antibodies. Vaccine approaches that promote affinity maturation at key sites could therefore more rapidly produce antibodies with neutralization breadth.

Ultrapotent neutralizing antibodies against SARS-CoV-2 with a high degree of mutation resistance.

Many SARS-CoV-2 neutralizing antibodies (nAbs) lose potency against variants of concern. In this study, we developed 2 strategies to produce mutation-resistant antibodies. First, a yeast library expressing mutant receptor binding domains (RBDs) of the spike protein was utilized to screen for potent nAbs that are least susceptible to viral escape. Among the candidate antibodies, P5-22 displayed ultrahigh potency for virus neutralization as well as an outstanding mutation resistance profile. Additionally, P14-44 and P15-16 were recognized as mutation-resistant antibodies with broad betacoronavirus neutralization properties. P15-16 has only 1 binding hotspot, which is K378 in the RBD of SARS-CoV-2. The crystal structure of the P5-22, P14-44, and RBD ternary complex clarified the unique mechanisms that underlie the excellent mutation resistance profiles of these antibodies. Secondly, polymeric IgG enhanced antibody avidity by eliminating P5-22's only hotspot, residue F486 in the RBD, thereby potently blocking cell entry by mutant viruses. Structural and functional analyses of antibodies screened using both potency assays and the yeast RBD library revealed rare, ultrapotent, mutation-resistant nAbs against SARS-CoV-2.

Computational design of a neutralizing antibody with picomolar binding affinity for all concerning SARS-CoV-2 variants.

Coronavirus disease 2019, caused by SARS-CoV-2, remains an on-going pandemic, partly due to the emergence of variant viruses that can "break-through" the protection of the current vaccines and neutralizing antibodies (nAbs), highlighting the needs for broadly nAbs and next-generation vaccines. We report an antibody that exhibits breadth and potency in binding the receptor-binding domain (RBD) of the virus spike glycoprotein across SARS coronaviruses. Initially, a lead antibody was computationally discovered and crystallographically validated that binds to a highly conserved surface of the RBD of wild-type SARS-CoV-2. Subsequently, through experimental affinity enhancement and computational affinity maturation, it was further developed to bind the RBD of all concerning SARS-CoV-2 variants, SARS-CoV-1 and pangolin coronavirus with pico-molar binding affinities, consistently exhibited strong neutralization activity against wild-type SARS-CoV-2 and the Alpha and Delta variants. These results identify a vulnerable target site on coronaviruses for development of pan-sarbecovirus nAbs and vaccines.

Analysis of antibodies from HCV elite neutralizers identifies genetic determinants of broad neutralization.

The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%-5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates.

Internalization of HIV-1 by Phagocytes Is Increased When Virions Are Opsonized with Multimeric Antibody in the Presence of Complement.

The low abundance of envelope spikes and the inability of IgG to aggregate virions render HIV-1 an inadequate target for antibody-mediated clearance by phagocytes. In an attempt to improve the ability of antibody to mediate the internalization of HIV-1 virions, we generated multimers of the broadly neutralizing HIV-1-specific monoclonal antibody (MAb) VRC01 using site-directed mutagenesis of the Fc segment. We then measured virion internalization using primary human monocytes and neutrophils. We found that, in the absence of complement, immune complexes consisting of HIV-1 virions and VRC01 multimers were slightly more efficiently internalized than were complexes formed with monomeric VRC01. The presence of complement, however, greatly augmented internalization of immune complexes formed with the multimeric MAb but had little impact on monomeric MAb-mediated internalization. Multimerization and the presence of complement overcome the limited ability of monomeric antibody to mediate internalization of HIV-1 virions and may thus provide a therapeutic approach to clearing virus. IMPORTANCE Antibody-mediated internalization of HIV-1 by phagocytes, a potential mechanism for clearing virus, is very inefficient. In an effort to improve viral clearance, we produced a multimeric form of the broadly neutralizing monoclonal antibody VRC01. We found that VRC01 antibody multimers (primarily hexamers) were only slightly more efficient in mediating HIV-1 internalization than was monomeric VRC01. However, the addition of complement resulted in substantially greater internalization of multimer-opsonized virus. In contrast, complement had little if any impact on internalization of monomer-opsonized virus. Therefore, antibody multimerization in combination with complement may overcome the limited ability of monomeric antibody to mediate internalization of HIV-1 virions. Our findings may provide a therapeutic approach to clearing virus.

SOSIP Trimer-Specific Antibodies Isolated from a Simian-Human Immunodeficiency Virus-Infected Monkey with versus without a Pre-blocking Step with gp41.

BG505 SOSIP.664 (hereafter referred to as SOSIP), a stabilized trimeric mimic of the HIV-1 envelope spike resembling the native viral spike, is a useful tool for isolating anti-HIV-1 neutralizing antibodies. We screened long-term SHIV-AD8 infected rhesus monkeys for potency and breadth of serum neutralizing activity against autologous and heterologous viruses: SHIV-AD8, HIV-1 YU2, HIV-1 JR-CSF, and HIV-1 NL4-3. Monkey rh2436 neutralized all viruses tested and showed strong reactivity to the SOSIP trimer, suggesting this was a promising candidate for attempts at monoclonal antibody (MAb) isolation. MAbs were isolated by performing single B-cell sorts from peripheral blood mononuclear cells (PBMC) by FACS using the SOSIP trimer as a probe. An initial round of sorted cells revealed the majority of isolated MAbs were directed to the gp41 external domain portion of the SOSIP trimer and were mostly non-neutralizing against tested isolates. A second sort was performed, introducing a gp41 blocking step prior to PBMC staining and FACS sorting. These isolated MAbs bound SOSIP trimer but were no longer directed to the gp41 external domain portion. A significantly higher proportion of MAbs with neutralizing activity were obtained with this strategy. Our data show this pre-blocking step with gp41 greatly increases the yield of non-gp41-reactive, SOSIP-specific MAbs and increases the likelihood of isolating MAbs with neutralizing activity. IMPORTANCE Recent advancements in the field have focused on the isolation and use of broadly neutralizing antibodies for both prophylaxis and therapy. Finding a useful probe to isolate broad potent neutralizing antibodies while avoiding non-neutralizing antibodies is important. The SOSIP trimer has been shown to be a great tool for this purpose because it binds known broadly neutralizing antibodies. However, the SOSIP trimer can isolate non-neutralizing antibodies as well, including gp41-specific MAbs. Introducing a pre-blocking step with gp41 recombinant protein decreased the percent of gp41-specific antibodies isolated with SOSIP probe, as well as increased the number of neutralizing antibodies isolated. This method can be used as a tool to increase the chances of isolating neutralizing antibodies.

Cell membrane-anchored anti-HIV single-chain antibodies and bifunctional inhibitors targeting the gp41 fusion protein: new strategies for HIV gene therapy.

Emerging studies indicate that infusion of HIV-resistant cells could be an effective strategy to achieve a sterilizing or functional cure. We recently reported that glycosylphosphatidylinositol (GPI)-anchored nanobody or a fusion inhibitory peptide can render modified cells resistant to HIV-1 infection. In this study, we comprehensively characterized a panel of newly isolated HIV-1-neutralizing antibodies as GPI-anchored inhibitors. Fusion genes encoding the single-chain variable fragment (scFv) of 3BNC117, N6, PGT126, PGT128, 10E8, or 35O22 were constructed with a self-inactivating lentiviral vector, and they were efficiently expressed in the lipid raft sites of target cell membrane without affecting the expression of HIV-1 receptors (CD4, CCR5 and CXCR4). Significantly, transduced cells exhibited various degrees of resistance to cell-free HIV-1 infection and cell-associated HIV-1 transmission, as well as viral Env-mediated cell-cell fusion, with the cells modified by GPI-10E8 showing the most potent and broad anti-HIV activity. In mechanism, GPI-10E8 also interfered with the processing of viral Env in transduced cells and attenuated the infectivity of progeny viruses. By genetically linking 10E8 with a fusion inhibitor peptide, we subsequently designed a group of eight bifunctional constructs as cell membrane-based inhibitors, designated CMI01∼CMI08, which rendered cells completely resistant to HIV-1, HIV-2, and simian immunodeficiency virus (SIV). In human CD4+ T cells, GPI-10E8 and its bifunctional derivatives blocked both CCR5- and CXCR4-tropic HIV-1 isolates efficiently, and the modified cells displayed robust survival selection under HIV-1 infection. Therefore, our studies provide new strategies for generating HIV-resistant cells, which can be used alone or with other gene therapy approaches.

Two Distinct Lysosomal Targeting Strategies Afford Trojan Horse Antibodies With Pan-Filovirus Activity.

Multiple agents in the family Filoviridae (filoviruses) are associated with sporadic human outbreaks of highly lethal disease, while others, including several recently identified agents, possess strong zoonotic potential. Although viral glycoprotein (GP)-specific monoclonal antibodies have demonstrated therapeutic utility against filovirus disease, currently FDA-approved molecules lack antiviral breadth. The development of broadly neutralizing antibodies has been challenged by the high sequence divergence among filovirus GPs and the complex GP proteolytic cleavage cascade that accompanies filovirus entry. Despite this variability in the antigenic surface of GP, all filoviruses share a site of vulnerability-the binding site for the universal filovirus entry receptor, Niemann-Pick C1 (NPC1). Unfortunately, this site is shielded in extracellular GP and only uncovered by proteolytic cleavage by host proteases in late endosomes and lysosomes, which are generally inaccessible to antibodies. To overcome this obstacle, we previously developed a 'Trojan horse' therapeutic approach in which engineered bispecific antibodies (bsAbs) coopt viral particles to deliver GP:NPC1 interaction-blocking antibodies to their endo/lysosomal sites of action. This approach afforded broad protection against members of the genus Ebolavirus but could not neutralize more divergent filoviruses. Here, we describe next-generation Trojan horse bsAbs that target the endo/lysosomal GP:NPC1 interface with pan-filovirus breadth by exploiting the conserved and widely expressed host cation-independent mannose-6-phosphate receptor for intracellular delivery. Our work highlights a new avenue for the development of single therapeutics protecting against all known and newly emerging filoviruses.

Hybrid immunity improves B cells and antibodies against SARS-CoV-2 variants.

The emergence of SARS-CoV-2 variants is jeopardizing the effectiveness of current vaccines and limiting the application of monoclonal antibody-based therapy for COVID-19 (refs. 1,2). Here we analysed the memory B cells of five naive and five convalescent people vaccinated with the BNT162b2 mRNA vaccine to investigate the nature of the B cell and antibody response at the single-cell level. Almost 6,000 cells were sorted, over 3,000 cells produced monoclonal antibodies against the spike protein and more than 400 cells neutralized the original SARS-CoV-2 virus first identified in Wuhan, China. The B.1.351 (Beta) and B.1.1.248 (Gamma) variants escaped almost 70% of these antibodies, while a much smaller portion was impacted by the B.1.1.7 (Alpha) and B.1.617.2 (Delta) variants. The overall loss of neutralization was always significantly higher in the antibodies from naive people. In part, this was due to the IGHV2-5;IGHJ4-1 germline, which was found only in people who were convalescent and generated potent and broadly neutralizing antibodies. Our data suggest that people who are seropositive following infection or primary vaccination will produce antibodies with increased potency and breadth and will be able to better control emerging SARS-CoV-2 variants.

Combinations of Single Chain Variable Fragments From HIV Broadly Neutralizing Antibodies Demonstrate High Potency and Breadth.

Broadly neutralizing antibodies (bNAbs) are currently being assessed in clinical trials for their ability to prevent HIV infection. Single chain variable fragments (scFv) of bNAbs have advantages over full antibodies as their smaller size permits improved diffusion into mucosal tissues and facilitates vector-driven gene expression. We have previously shown that scFv of bNAbs individually retain significant breadth and potency. Here we tested combinations of five scFv derived from bNAbs CAP256-VRC26.25 (V2-apex), PGT121 (N332-supersite), 3BNC117 (CD4bs), 8ANC195 (gp120-gp41 interface) and 10E8v4 (MPER). Either two or three scFv were combined in equimolar amounts and tested in the TZM-bl neutralization assay against a multiclade panel of 17 viruses. Experimental IC50 and IC80 data were compared to predicted neutralization titers based on single scFv titers using the Loewe additive and the Bliss-Hill model. Like full-sized antibodies, combinations of scFv showed significantly improved potency and breadth compared to single scFv. Combinations of two or three scFv generally followed an independent action model for breadth and potency with no significant synergy or antagonism observed overall although some exceptions were noted. The Loewe model underestimated potency for some dual and triple combinations while the Bliss-Hill model was better at predicting IC80 titers of triple combinations. Given this, we used the Bliss-Hill model to predict the coverage of scFv against a 45-virus panel at concentrations that correlated with protection in the AMP trials. Using IC80 titers and concentrations of 1µg/mL, there was 93% coverage for one dual scFv combination (3BNC117+10E8v4), and 96% coverage for two of the triple combinations (CAP256.25+3BNC117+10E8v4 and PGT121+3BNC117+10E8v4). Combinations of scFv, therefore, show significantly improved breadth and potency over individual scFv and given their size advantage, have potential for use in passive immunization.

Contribution to HIV Prevention and Treatment by Antibody-Mediated Effector Function and Advances in Broadly Neutralizing Antibody Delivery by Vectored Immunoprophylaxis.

HIV-1 broadly neutralizing antibodies (bNAbs) targeting the viral envelope have shown significant promise in both HIV prevention and viral clearance, including pivotal results against sensitive strains in the recent Antibody Mediated Prevention (AMP) trial. Studies of bNAb passive transfer in infected patients have demonstrated transient reduction of viral load at high concentrations that rebounds as bNAb is cleared from circulation. While neutralization is a crucial component of therapeutic efficacy, numerous studies have demonstrated that bNAbs can also mediate effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent complement deposition (ADCD). These functions have been shown to contribute towards protection in several models of HIV acquisition and in viral clearance during chronic infection, however the role of target epitope in facilitating these functions, as well as the contribution of individual innate functions in protection and viral clearance remain areas of active investigation. Despite their potential, the transient nature of antibody passive transfer limits the widespread use of bNAbs. To overcome this, we and others have demonstrated vectored antibody delivery capable of yielding long-lasting expression of bNAbs in vivo. Two clinical trials have shown that adeno-associated virus (AAV) delivery of bNAbs is safe and capable of sustained bNAb expression for over 18 months following a single intramuscular administration. Here, we review key concepts of effector functions mediated by bNAbs against HIV infection and the potential for vectored immunoprophylaxis as a means of producing bNAbs in patients.

HIV Broadly Neutralizing Antibodies Expressed as IgG3 Preserve Neutralization Potency and Show Improved Fc Effector Function.

The ability of several broadly neutralizing antibodies (bNAbs) to protect against HIV infection is enhanced through Fc receptor binding. Antibody isotype modulates this effect, with IgG3 associated with improved HIV control and vaccine efficacy. We recently showed that an IgG3 variant of bNAb CAP256-VRC26.25 exhibited more potent neutralization and phagocytosis than its IgG1 counterpart. Here, we expanded this analysis to include additional bNAbs targeting all major epitopes. A total of 15 bNAbs were expressed as IgG1 or IgG3, and pairs were assessed for neutralization potency against the multi-subtype global panel of 11 HIV strains. Binding to the neonatal Fc receptor (FcRn) and Fcγ receptors were measured using ELISA and antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis were measured using infectious viruses and global panel Env SOSIP trimers, respectively. IgG3 bNAbs generally showed similar or increased (up to 60 fold) neutralization potency than IgG1 versions, though the effect was virus-specific. This improvement was statistically significant for CAP256-VRC26.25, 35022, PGT135 and CAP255.G3. IgG3 bNAbs also showed significantly improved binding to FcγRIIa which correlated with enhanced phagocytosis of all trimeric Env antigens. Differences in ADCC were epitope-specific, with IgG3 bNAbs to the MPER, CD4 binding site and gp120-gp41 interface showing increased ADCC. We also explored the pH dependence of IgG1 and IgG3 variants for FcRn binding, as this determines the half-life of antibodies. We observed reduced pH dependence, associated with shorter half-lives for IgG3 bNAbs, with κ-light chains. However, IgG3 bNAbs that use λ-light chains showed similar pH dependence to their IgG1 counterparts. This study supports the manipulation of the constant region to improve both the neutralizing and Fc effector activity of bNAbs, and suggests that IgG3 versions of bNAbs may be preferable for passive immunity given their polyfunctionality.

Novel Monoclonal Antibodies and Recombined Antibodies Against Variant SARS-CoV-2.

The mutants resulted from the ongoing SARS-CoV-2 epidemic have showed resistance to antibody neutralization and vaccine-induced immune response. The present study isolated and identified two novel SARS-CoV-2 neutralizing antibodies (nAbs) from convalescent COVID-19 patients. These two nAbs (XG81 and XG83) were then systemically compared with nine nAbs that were reconstructed by using published data, and revealed that, even though these two nAbs shared targeting epitopes on spike protein, they were different from any of the nine nAbs. Compared with XG81, XG83 exhibited a higher RBD binding affinity and neutralization potency against wild-typed pseudovirus, variant pseudoviruses with mutated spike proteins, such as D614G, E484Q, and A475V, as well as the authentic SARS-CoV-2 virus. To explore potential broadly neutralizing antibodies, heavy and light chains from all 18 nAbs (16 published nAbs, XG81 and XG83) were cross-recombined, and some of the functional antibodies were screened and studied for RBD binding affinity, and neutralizing activity against pseudovirus and the authentic SARS-CoV-2 virus. The results demonstrated that several recombined antibodies had a more potent neutralization activity against variant pseudoviruses compared with the originally paired Abs. Taken together, the novel neutralizing antibodies identified in this study are a likely valuable addition to candidate antibody drugs for the development of clinical therapeutic agents against SARS-CoV-2 to minimize mutational escape.

Binding affinity landscapes constrain the evolution of broadly neutralizing anti-influenza antibodies.

Over the past two decades, several broadly neutralizing antibodies (bnAbs) that confer protection against diverse influenza strains have been isolated. Structural and biochemical characterization of these bnAbs has provided molecular insight into how they bind distinct antigens. However, our understanding of the evolutionary pathways leading to bnAbs, and thus how best to elicit them, remains limited. Here, we measure equilibrium dissociation constants of combinatorially complete mutational libraries for two naturally isolated influenza bnAbs (CR9114, 16 heavy-chain mutations; CR6261, 11 heavy-chain mutations), reconstructing all possible evolutionary intermediates back to the unmutated germline sequences. We find that these two libraries exhibit strikingly different patterns of breadth: while many variants of CR6261 display moderate affinity to diverse antigens, those of CR9114 display appreciable affinity only in specific, nested combinations. By examining the extensive pairwise and higher order epistasis between mutations, we find key sites with strong synergistic interactions that are highly similar across antigens for CR6261 and different for CR9114. Together, these features of the binding affinity landscapes strongly favor sequential acquisition of affinity to diverse antigens for CR9114, while the acquisition of breadth to more similar antigens for CR6261 is less constrained. These results, if generalizable to other bnAbs, may explain the molecular basis for the widespread observation that sequential exposure favors greater breadth, and such mechanistic insight will be essential for predicting and eliciting broadly protective immune responses.