A Computational Framework for Determining the Breadth of Antibodies Against Highly Mutable Pathogens.

Highly mutable pathogens pose daunting challenges for antibody design. The usual criteria of high potency and specificity are often insufficient to design antibodies that provide long-lasting protection. This is due, in part, to the ability of the pathogen to rapidly acquire mutations that permit them to evade the designed antibodies. To overcome these limitations, design of antibodies with a larger neutralizing breadth can be pursued. Such broadly neutralizing antibodies (bnAbs) should remain targeted to a specific epitope, yet show robustness against pathogen mutability, thereby neutralizing a higher number of antigens. This is particularly important for highly mutable pathogens, like the influenza virus and the human immunodeficiency virus (HIV). The protocol describes a method for computing the "breadth" of a given antibody, an essential aspect of antibody design.

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.

Enhanced neutralization potency of an identical HIV neutralizing antibody expressed as different isotypes is achieved through genetically distinct mechanisms.

Antibodies with the same variable region can exist as multiple isotypes with varying neutralization potencies, though the mechanism for this is not fully defined. We previously isolated an HIV-directed IgA1 monoclonal antibody (mAb), CAP88-CH06, and showed that IgA1 and IgG3 isotypes of this antibody demonstrated enhanced neutralization compared to IgG1. To explore the mechanism behind this, hinge region and constant heavy chain (CH1) chimeras were constructed between the IgA1, IgG3 and IgG1 mAbs and assessed for neutralization activity, antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC). Hinge chimeras revealed that the increased neutralization potency and phagocytosis of the IgG3 isotype was attributed to its longer hinge region. In contrast, for IgA1, CH1 chimeras showed that this region was responsible both for enhanced neutralization potency and decreased ADCP, though ADCC was not affected. Overall, these data show that the enhanced neutralization potency of CAP88-CH06 IgG3 and IgA1, compared to IgG1, is achieved through distinct mechanisms. Understanding the influence of the hinge and CH1 regions on Fab domain function may provide insights into the engineering of therapeutic antibodies with increased neutralization potency.

Long-primed germinal centres with enduring affinity maturation and clonal migration.

Germinal centres are the engines of antibody evolution. Here, using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal centre B (BGC) cells that last for at least 6 months. A 186-fold increase in BGC cells was present by week 10 compared with conventional immunization. Single-cell transcriptional profiling showed that both light- and dark-zone germinal centre states were sustained. Antibody somatic hypermutation of BGC cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding BGC cells were still 49-fold above baseline at 29 weeks, which suggests that they could remain active for even longer periods of time. High titres of HIV-neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing considerable immunodominance challenges for B cells1,2. Memory B cells generated under these long priming conditions had higher levels of antibody somatic hypermutation, and both memory B cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning more than the 6-month germinal centre period were identified, demonstrating continuous germinal centre activity and selection for at least 191 days with no further antigen exposure. A long-prime, slow-delivery (12 days) immunization approach holds promise for difficult vaccine targets and suggests that patience can have great value for tuning of germinal centres to maximize antibody responses.

In vivo engineered B cells secrete high titers of broadly neutralizing anti-HIV antibodies in mice.

Transplantation of B cells engineered ex vivo to secrete broadly neutralizing antibodies (bNAbs) has shown efficacy in disease models. However, clinical translation of this approach would require specialized medical centers, technically demanding protocols and major histocompatibility complex compatibility of donor cells and recipients. Here we report in vivo B cell engineering using two adeno-associated viral vectors, with one coding for Staphylococcus aureus Cas9 (saCas9) and the other for 3BNC117, an anti-HIV bNAb. After intravenously injecting the vectors into mice, we observe successful editing of B cells leading to memory retention and bNAb secretion at neutralizing titers of up to 6.8 µg ml-1. We observed minimal clustered regularly interspaced palindromic repeats (CRISPR)-Cas9 off-target cleavage as detected by unbiased CHANGE-sequencing analysis, whereas on-target cleavage in undesired tissues is reduced by expressing saCas9 from a B cell-specific promoter. In vivo B cell engineering to express therapeutic antibodies is a safe, potent and scalable method, which may be applicable not only to infectious diseases but also in the treatment of noncommunicable conditions, such as cancer and autoimmune disease.

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.

High-Throughput B Cell Epitope Determination by Next-Generation Sequencing.

Development of novel technologies for the discovery of human monoclonal antibodies has proven invaluable in the fight against infectious diseases. Among the diverse antibody repertoires elicited by infection or vaccination, often only rare antibodies targeting specific epitopes of interest are of potential therapeutic value. Current antibody discovery efforts are capable of identifying B cells specific for a given antigen; however, epitope specificity information is usually only obtained after subsequent monoclonal antibody production and characterization. Here we describe LIBRA-seq with epitope mapping, a next-generation sequencing technology that enables residue-level epitope determination for thousands of single B cells simultaneously. By utilizing an antigen panel of point mutants within the HIV-1 Env glycoprotein, we identified and confirmed antibodies targeting multiple sites of vulnerability on Env, including the CD4-binding site and the V3-glycan site. LIBRA-seq with epitope mapping is an efficient tool for high-throughput identification of antibodies against epitopes of interest on a given antigen target.

Viral resistance to VRC01-like antibodies with mutations in loop D and V5 from an HIV-1 B' subtype infected individual with broadly neutralization activity.

Recently we identified the VRC01-like antibody DRVIA7(A7) from an HIV-1 B' subtype-infected individual (DRVI01) with broad neutralization activity, and almost all viruses from the individual were resistant to both VRC01 and A7 lineage antibodies. Here, we identified and characterized a panel of HIV-1 variants with resistance to VRC01 and A7 using site-directed mutagenesis and swapping amino acid fragments of gp120. Site-directed mutagenesis revealed that E279D/R282K/N460A/T464N of gp120 from DRVI01 produced VRC01-susceptible variants. Multiple mutations significantly increased the neutralization sensitivity to VRC01. Residues N464 located at the tip of the V5 loop were considered irrelevant to the neutralization of VRC01. For DRVI01-derived viruses, the single N464T change fully produced VRC01-resistant variants; conversely, a single T464N mutation generated VRC01-susceptible variants. Alanine scanning revealed that the N464 residue plays a vital role in binding with VRC01. Neutralizing assays against A7 lineage antibodies showed that DRVI01-derived viruses with multiple mutations could be neutralized by A7 lineage antibodies with different neutralizing breadths. Combining the changes in loops D and V5 produced variants that were totally sensitive variants to A7 lineage antibodies.

Deciphering gp120 sequence variation and structural dynamics in HIV neutralization phenotype by molecular dynamics simulations and graph machine learning.

Human immunodeficiency virus (HIV) exploits the sequence variation and structural dynamics of the envelope glycoprotein gp120 to evade the immune attack of neutralization antibodies, contributing to various HIV neutralization phenotypes. Although the HIV neutralization phenotype has been experimentally characterized, the roles of rapid sequence variability and significant structural dynamics of gp120 are not well understood. Here, 45 prefusion gp120 from different HIV strains belong to three tiers of sensitive, moderate, and resistant neutralization phenotype are structurally modeled by homology modeling and then investigated by molecular dynamics (MD) simulations and graph machine learning (ML). Our results show that the structural deviations, population distribution, and conformational flexibility of gp120 are related to the HIV neutralization phenotype. Per-residue dynamics indicate the local regions especially in the second structural elements with high-flexibility, may be responsible for the HIV neutralization phenotype. Moreover, a graph ML model with the attention mechanism was trained to explore inherent representation related to the classification of the HIV neutralization phenotype, further distinguishing the strong related gp120 sequence variation together with structural dynamics in the HIV neutralization phenotype. Our study not only deciphers gp120 sequence variation and structural dynamics in the HIV neutralization phenotype but also explores complex relationships between the sequence, structure, and dynamics of protein by combining MD simulations and ML.

Engineering pan-HIV-1 neutralization potency through multispecific antibody avidity.

Deep mining of B cell repertoires of HIV-1-infected individuals has resulted in the isolation of dozens of HIV-1 broadly neutralizing antibodies (bNAbs). Yet, it remains uncertain whether any such bNAbs alone are sufficiently broad and potent to deploy therapeutically. Here, we engineered HIV-1 bNAbs for their combination on a single multispecific and avid molecule via direct genetic fusion of their Fab fragments to the human apoferritin light chain. The resulting molecule demonstrated a remarkable median IC50 value of 0.0009 µg/mL and 100% neutralization coverage of a broad HIV-1 pseudovirus panel (118 isolates) at a 4 µg/mL cutoff-a 32-fold enhancement in viral neutralization potency compared to a mixture of the corresponding HIV-1 bNAbs. Importantly, Fc incorporation on the molecule and engineering to modulate Fc receptor binding resulted in IgG-like bioavailability in vivo. This robust plug-and-play antibody design is relevant against indications where multispecificity and avidity are leveraged simultaneously to mediate optimal biological activity.

Reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire.

B cells have been engineered ex vivo to express an HIV-1 broadly neutralizing antibody (bNAb). B cell reprograming may be scientifically and therapeutically useful, but current approaches limit B cell repertoire diversity and disrupt the organization of the heavy-chain locus. A more diverse and physiologic B cell repertoire targeting a key HIV-1 epitope could facilitate evaluation of vaccines designed to elicit bNAbs, help identify more potent and bioavailable bNAb variants, or directly enhance viral control in vivo. Here we address the challenges of generating such a repertoire by replacing the heavy-chain CDR3 (HCDR3) regions of primary human B cells. To do so, we identified and utilized an uncharacterized Cas12a ortholog that recognizes PAM motifs present in human JH genes. We also optimized the design of 200 nucleotide homology-directed repair templates (HDRT) by minimizing the required 3'-5' deletion of the HDRT-complementary strand. Using these techniques, we edited primary human B cells to express a hemagglutinin epitope tag and the HCDR3 regions of the bNAbs PG9 and PG16. Those edited with bNAb HCDR3 efficiently bound trimeric HIV-1 antigens, implying they could affinity mature in vivo in response to the same antigens. This approach generates diverse B cell repertoires recognizing a key HIV-1 neutralizing epitope.

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.

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.

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.

A Structural Update of Neutralizing Epitopes on the HIV Envelope, a Moving Target.

Antibodies that can neutralize diverse HIV-1 strains develop in ~10-20% of HIV-1 infected individuals, and their elicitation is a goal of vaccine design. Such antibodies can also serve as therapeutics for those who have already been infected with the virus. Structural characterizations of broadly reactive antibodies in complex with the HIV-1 spike indicate that there are a limited number of sites of vulnerability on the spike. Analysis of their structures can help reveal commonalities that would be useful in vaccine design and provide insights on combinations of antibodies that can be used to minimize the incidence of viral resistance mutations. In this review, we give an update on recent structures determined of the spike in complex with broadly neutralizing antibodies in the context of all epitopes on the HIV-1 spike identified to date.

The light chain of antibodies specific to the V2 region of HIV-1 can determine their function.

We studied the contribution of the light chain to functions of human monoclonal antibodies (mAbs) by measuring the relationships between the rate of mutations and cross-reactivity, binding affinity and neutralization activity. We analyzed 12 mAbs of two clonal families specific to the V2 region of HIV-1 derived from two chronically HIV-1 infected individuals. The clonal mAbs exhibited a range of reactivities, and the clones with superior properties were associated with the rate of mutations and the presence of particular mutated residues in the light chains, but not in the heavy chains. Our observations suggest that for some antibodies, the light chains play a vital role in antibody evolution toward more efficient ones and also suggest the importance of optimal residues rather than the rate of mutations in the variable fragment of the antibody.