Continuous germinal center invasion contributes to the diversity of the immune response.

Antibody responses are characterized by increasing affinity and diversity over time. Affinity maturation occurs in germinal centers by a mechanism that involves repeated cycles of somatic mutation and selection. How antibody responses diversify while also undergoing affinity maturation is not as well understood. Here, we examined germinal center (GC) dynamics by tracking B cell entry, division, somatic mutation, and specificity. Our experiments show that naive B cells continuously enter GCs where they compete for T cell help and undergo clonal expansion. Consistent with late entry, invaders carry fewer mutations but can contribute up to 30% or more of the cells in late-stage germinal centers. Notably, cells entering the germinal center at later stages of the reaction diversify the immune response by expressing receptors that show low affinity to the immunogen. Paradoxically, the affinity threshold for late GC entry is lowered in the presence of high-affinity antibodies.

Induction of HIV Neutralizing Antibody Lineages in Mice with Diverse Precursor Repertoires.

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2(∗)02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages.

Live imaging of SARS-CoV-2 infection in mice reveals that neutralizing antibodies require Fc function for optimal efficacy.

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

Immunization for HIV-1 Broadly Neutralizing Antibodies in Human Ig Knockin Mice.

A subset of individuals infected with HIV-1 develops broadly neutralizing antibodies (bNAbs) that can prevent infection, but it has not yet been possible to elicit these antibodies by immunization. To systematically explore how immunization might be tailored to produce them, we generated mice expressing the predicted germline or mature heavy chains of a potent bNAb to the CD4 binding site (CD4bs) on the HIV-1 envelope glycoprotein (Env). Immunogens specifically designed to activate B cells bearing germline antibodies are required to initiate immune responses, but they do not elicit bNAbs. In contrast, native-like Env trimers fail to activate B cells expressing germline antibodies but elicit bNAbs by selecting for a restricted group of light chains bearing specific somatic mutations that enhance neutralizing activity. The data suggest that vaccination to elicit anti-HIV-1 antibodies will require immunization with a succession of related immunogens.

Structural Repertoire of HIV-1-Neutralizing Antibodies Targeting the CD4 Supersite in 14 Donors.

The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures -8 determined here- of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies.

HIV-1 VRC01 Germline-Targeting Immunogens Select Distinct Epitope-Specific B Cell Receptors.

Activating precursor B cell receptors of HIV-1 broadly neutralizing antibodies requires specifically designed immunogens. Here, we compared the abilities of three such germline-targeting immunogens against the VRC01-class receptors to activate the targeted B cells in transgenic mice expressing the germline VH of the VRC01 antibody but diverse mouse light chains. Immunogen-specific VRC01-like B cells were isolated at different time points after immunization, their VH and VL genes were sequenced, and the corresponding antibodies characterized. VRC01 B cell sub-populations with distinct cross-reactivity properties were activated by each immunogen, and these differences correlated with distinct biophysical and biochemical features of the germline-targeting immunogens. Our study indicates that the design of effective immunogens to activate B cell receptors leading to protective HIV-1 antibodies will require a better understanding of how the biophysical properties of the epitope and its surrounding surface on the germline-targeting immunogen influence its interaction with the available receptor variants in vivo.

Analysis of a SARS-CoV-2-Infected Individual Reveals Development of Potent Neutralizing Antibodies with Limited Somatic Mutation.

Antibody responses develop following SARS-CoV-2 infection, but little is known about their epitope specificities, clonality, binding affinities, epitopes, and neutralizing activity. We isolated B cells specific for the SARS-CoV-2 envelope glycoprotein spike (S) from a COVID-19-infected subject 21 days after the onset of clinical disease. 45 S-specific monoclonal antibodies were generated. They had undergone minimal somatic mutation with limited clonal expansion, and three bound the receptor-binding domain (RBD). Two antibodies neutralized SARS-CoV-2. The most potent antibody bound the RBD and prevented binding to the ACE2 receptor, while the other bound outside the RBD. Thus, most anti-S antibodies that were generated in this patient during the first weeks of COVID-19 infection were non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 S-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive and/or therapeutic potential and can serve as templates for vaccine design.

Inference of the HIV-1 VRC01 Antibody Lineage Unmutated Common Ancestor Reveals Alternative Pathways to Overcome a Key Glycan Barrier.

Elicitation of VRC01-class broadly neutralizing antibodies (bnAbs) is an appealing approach for a preventative HIV-1 vaccine. Despite extensive investigations, strategies to induce VRC01-class bnAbs and overcome the barrier posed by the envelope N276 glycan have not been successful. Here, we inferred a high-probability unmutated common ancestor (UCA) of the VRC01 lineage and reconstructed the stages of lineage maturation. Env immunogens designed on reverted VRC01-class bnAbs bound to VRC01 UCA with affinity sufficient to activate naive B cells. Early mutations defined maturation pathways toward limited or broad neutralization, suggesting that focusing the immune response is likely required to steer B cell maturation toward the development of neutralization breadth. Finally, VRC01 lineage bnAbs with long CDR H3s overcame the HIV-1 N276 glycan barrier without shortening their CDR L1, revealing a solution for broad neutralization in which the heavy chain, not CDR L1, is the determinant to accommodate the N276 glycan.

Short CDRL1 in intermediate VRC01-like mAbs is not sufficient to overcome key glycan barriers on HIV-1 Env.

VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and sometimes accumulate CDRL1 deletions. Such indels may allow VRC01-class antibodies to accommodate the glycans expressed on a conserved N276 N-linked glycosylation site in loop D of the gp120 subunit. These glycans constitute a major obstacle in the development of VRC01-class antibodies, as unmutated antibody forms are unable to accommodate them. Although immunizations of knock-in mice expressing human VRC01-class B-cell receptors (BCRs) with specifically designed Env-derived immunogens lead to the accumulation of somatic mutations in VRC01-class BCRs, CDRL1 deletions are rarely observed, and the elicited antibodies display narrow neutralizing activities. The lack of broad neutralizing potential could be due to the absence of deletions, the lack of appropriate somatic mutations, or both. To address this point, we modified our previously determined prime-boost immunization with a germline-targeting immunogen nanoparticle (426c.Mod.Core), followed by a heterologous core nanoparticle (HxB2.WT.Core), by adding a final boost with a cocktail of various stabilized soluble Env trimers. We isolated VRC01-like antibodies with extensive somatic mutations and, in one case, a seven-amino acid CDRL1 deletion. We generated chimeric antibodies that combine the vaccine-elicited somatic mutations with CDRL1 deletions present in human mature VRC01 bnAbs. We observed that CDRL1 indels did not improve the neutralizing antibody activities. Our study indicates that CDRL1 length by itself is not sufficient for the broadly neutralizing phenotype of this class of antibodies. IMPORTANCE: HIV-1 broadly neutralizing antibodies will be a key component of an effective HIV-1 vaccine, as they prevent viral acquisition. Over the past decade, numerous broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV. Despite an in-depth knowledge of their structures, epitopes, ontogenies, and, in a few rare cases, their maturation pathways during infection, bnAbs have, so far, not been elicited by vaccination. This necessitates the identification of key obstacles that prevent their elicitation by immunization and overcoming them. Here we examined whether CDRL1 shortening is a prerequisite for the broadly neutralizing potential of VRC01-class bnAbs, which bind within the CD4 receptor binding site of Env. Our findings indicate that CDRL1 shortening by itself is important but not sufficient for the acquisition of neutralization breadth, and suggest that particular combinations of amino acid mutations, not elicited so far by vaccination, are most likely required for the development of such a feature.

Immunization expands B cells specific to HIV-1 V3 glycan in mice and macaques.

Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody-envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires.

Common Characteristics of HIV-Neutralizing Antibodies with a Fondness for Sugars.

Broadly neutralizing antibodies targeting quaternary epitopes on the apex of the HIV-1 envelope spike are an attractive vaccine target, yet engineering immunogens that recapitulate such epitopes has proven difficult. In this issue of Immunity, Andrabi and colleagues (2015) identify an exciting new candidate immunogen that could initiate the production of these types of antibodies through vaccination.

Evaluation of Cell-Based and Surrogate SARS-CoV-2 Neutralization Assays.

Determinants of protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using 40 plasma samples from convalescent individuals with mild to moderate coronavirus disease 2019 (COVID-19): four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate enzyme-linked immunosorbent assay (ELISA)-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor human angiotensin converting enzyme 2 (hACE2). Vero cells, Vero E6 cells, HEK293T cells expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND50) geometric mean titers (GMTs) that were highly correlated (Pearson r = 0.81 to 0.89) and ranged within 3.4-fold. The live virus assay and LV pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers, 141 and 178, respectively. ND50 titers positively correlated with plasma IgG targeting SARS-CoV-2 spike protein and RBD (r = 0.63 to 0.89), but moderately correlated with nucleoprotein IgG (r = 0.46 to 0.73). ND80 GMTs mirrored ND50 data and showed similar correlation between assays and with IgG concentrations. The VSV pseudovirus assay and LV pseudovirus assay with HEK293T/hACE2 cells in low- and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms.

Correction: HIV-1 envelope glycan modifications that permit neutralization by germline-reverted VRC01-class broadly neutralizing antibodies.

[This corrects the article DOI: 10.1371/journal.ppat.1007431.].

Anti-HIV-1 B cell responses are dependent on B cell precursor frequency and antigen-binding affinity.

The discovery that humans can produce potent broadly neutralizing antibodies (bNAbs) to several different epitopes on the HIV-1 spike has reinvigorated efforts to develop an antibody-based HIV-1 vaccine. Antibody cloning from single cells revealed that nearly all bNAbs show unusual features that could help explain why it has not been possible to elicit them by traditional vaccination and instead would require a sequence of different immunogens. This idea is supported by experiments with genetically modified immunoglobulin (Ig) knock-in mice. Sequential immunization with a series of specifically designed immunogens was required to shepherd the development of bNAbs. However, knock-in mice contain superphysiologic numbers of bNAb precursor-expressing B cells, and therefore how these results can be translated to a more physiologic setting remains to be determined. Here we make use of adoptive transfer experiments using knock-in B cells that carry a synthetic intermediate in the pathway to anti-HIV-1 bNAb development to examine how the relationship between B cell receptor affinity and precursor frequency affects germinal center (GC) B cell recruitment and clonal expansion. Immunization with soluble HIV-1 antigens can recruit bNAb precursor B cells to the GC when there are as few as 10 such cells per mouse. However, at low precursor frequencies, the extent of clonal expansion is directly proportional to the affinity of the antigen for the B cell receptor, and recruitment to GCs is variable and dependent on recirculation.

Germline-targeting immunogens.

In 2009, Dimitrov's group reported that the inferred germline (iGL) forms of several HIV-1 broadly neutralizing antibodies (bNAbs) did not display measurable binding to a recombinant gp140 Env protein (derived from the dual-tropic 89.6 virus), which was efficiently recognized by the mature (somatically mutated) antibodies. At that time, a small number of bNAbs were available, but in the following years, the implementation of high-throughput B-cell isolation and sequencing assays and of screening methodologies facilitated the isolation of greater numbers of bNAbs from infected subjects. Using these newest bNAbs, and a wide range of diverse recombinant Envs, we and others confirmed the observations made by Dimitrov's group. The results from these studies created a paradigm shift in our collective thinking as to why recombinant Envs are ineffective in eliciting bNAbs and has led to the "germline-targeting" immunization approach. Here we discuss this approach in detail: what has been done so far, the advantages and limitations of the current germline-targeting immunogens and of the animal models used to test them, and we conclude with a few thoughts about future directions in this area of research.

B cell clonal lineage alterations upon recombinant HIV-1 envelope immunization of rhesus macaques.

Broadly neutralizing HIV-1 antibodies (bNAbs) isolated from infected subjects display protective potential in animal models. Their elicitation by immunization is thus highly desirable. The HIV-1 envelope glycoprotein (Env) is the sole viral target of bnAbs, but is also targeted by binding, non-neutralizing antibodies. Env-based immunogens tested so far in various animal species and humans have elicited binding and autologous neutralizing antibodies but not bNAbs (with a few notable exceptions). The underlying reasons for this are not well understood despite intensive efforts to characterize the binding specificities of the elicited antibodies; mostly by employing serologic methodologies and monoclonal antibody isolation and characterization. These approaches provide limited information on the ontogenies and clonal B cell lineages that expand following Env-immunization. Thus, our current understanding on how the expansion of particular B cell lineages by Env may be linked to the development of non-neutralizing antibodies is limited. Here, in addition to serological analysis, we employed high-throughput BCR sequence analysis from the periphery, lymph nodes and bone marrow, as well as B cell- and antibody-isolation and characterization methods, to compare in great detail the B cell and antibody responses elicited in non-human primates by two forms of the clade C HIV Env 426c: one representing the full length extracellular portion of Env while the other lacking the variable domains 1, 2 and 3 and three conserved N-linked glycosylation sites. The two forms were equally immunogenic, but only the latter elicited neutralizing antibodies by stimulating a more restricted expansion of B cells to a narrower set of IGH/IGK/IGL-V genes that represented a small fraction (0.003-0.02%) of total B cells. Our study provides new information on how Env antigenic differences drastically affect the expansion of particular B cell lineages and supports immunogen-design efforts aiming at stimulating the expansion of cells expressing particular B cell receptors.

HIV-1 envelope glycan modifications that permit neutralization by germline-reverted VRC01-class broadly neutralizing antibodies.

Broadly neutralizing antibody (bnAb) induction is a high priority for effective HIV-1 vaccination. VRC01-class bnAbs that target the CD4 binding site (CD4bs) of trimeric HIV-1 envelope (Env) glycoprotein spikes are particularly attractive to elicit because of their extraordinary breadth and potency of neutralization in vitro and their ability to protect against infection in animal models. Glycans bordering the CD4bs impede the binding of germline-reverted forms of VRC01-class bnAbs and therefore constitute a barrier to early events in initiating the correct antibody lineages. Deleting a subset of these glycans permits Env antigen binding but not virus neutralization, suggesting that additional barriers impede germline-reverted VRC01-class antibody binding to functional Env trimers. We investigated the requirements for functional Env trimer engagement of VRC01-class naïve B cell receptors by using virus neutralization and germline-reverted antibodies as surrogates for the interaction. Targeted deletion of a subset of N-glycans bordering the CD4bs, combined with Man5 enrichment of remaining N-linked glycans that are otherwise processed into larger complex-type glycans, rendered HIV-1 426c Env-pseudotyped virus (subtype C, transmitted/founder) highly susceptible to neutralization by near germline forms of VRC01-class bnAbs. Neither glycan modification alone rendered the virus susceptible to neutralization. The potency of neutralization in some cases rivaled the potency of mature VRC01 against wildtype viruses. Neutralization by the germline-reverted antibodies was abrogated by the known VRC01 resistance mutation, D279K. These findings improve our understanding of the restrictions imposed by glycans in eliciting VRC01-class bnAbs and enable a neutralization-based strategy to monitor vaccine-elicited early precursors of this class of bnAbs.

Achieving Potent Autologous Neutralizing Antibody Responses against Tier 2 HIV-1 Viruses by Strategic Selection of Envelope Immunogens.

Advancement in immunogen selection and vaccine design that will rapidly elicit a protective Ab response is considered critical for HIV vaccine protective efficacy. Vaccine-elicited Ab responses must therefore have the capacity to prevent infection by neutralization-resistant phenotypes of transmitted/founder (T/F) viruses that establish infection in humans. Most vaccine candidates to date have been ineffective at generating Abs that neutralize T/F or early variants. In this study, we report that coimmunizing rhesus macaques with HIV-1 gp160 DNA and gp140 trimeric protein selected from native envelope gene sequences (envs) induced neutralizing Abs against Tier 2 autologous viruses expressing cognate envelope (Env). The Env immunogens were selected from envs emerging during the earliest stages of neutralization breadth developing within the first 2 years of infection in two clade B-infected human subjects. Moreover, the IgG responses in macaques emulated the targeting to specific regions of Env known to be associated with autologous and heterologous neutralizing Abs developed within the human subjects. Furthermore, we measured increasing affinity of macaque polyclonal IgG responses over the course of the immunization regimen that correlated with Tier 1 neutralization. In addition, we report firm correlations between Tier 2 autologous neutralization and Tier 1 heterologous neutralization, as well as overall TZM-bl breadth scores. Additionally, the activation of Env-specific follicular helper CD4 T cells in lymphocytes isolated from inguinal lymph nodes of vaccinated macaques correlated with Tier 2 autologous neutralization. These results demonstrate the potential for native Env derived from subjects at the time of neutralization broadening as effective HIV vaccine elements.

HIV-1 single-stranded RNA induces CXCL13 secretion in human monocytes via TLR7 activation and plasmacytoid dendritic cell-derived type I IFN.

Elevated levels of the chemokine CXCL13 have been observed in the plasma of chronically HIV-1-infected subjects and have been correlated with plasma viremia, which in turn has been linked to progressive dysregulation of humoral responses. In this study we sought to identify mechanisms of CXCL13 induction in response to HIV-1 infection. Plasma levels of CXCL13 in HIV-1-infected antiretroviral therapy-naive subjects correlated with viral load and were higher compared with antiretroviral therapy-treated HIV-1-infected and HIV-1-uninfected subjects. To elucidate the relationship between HIV-1 viremia and CXCL13 plasma levels, PBMCs from uninfected donors were stimulated with HIV-1 infectious virions, HIV-1 ssRNA, TLR 7 and 8 agonists, or IFN-α. The cellular sources of CXCL13 were determined by intracellular cytokine staining of cell populations. CXCL13 was produced by monocytes after stimulation with TLR 7 and 8 ligands or HIV-1-derived ssRNA. CXCL13 production by monocytes required TLR7 activation of plasmacytoid dendritic cells and secretion of type I IFN. IFN-α alone was sufficient to induce CXCL13 expression in human monocytes. In sum, we identified a novel mechanism of HIV-1-induced CXCL13 secretion-one caused by TLR7 induction of type I IFN by plasmacytoid dendritic cells and subsequent IFN stimulation of monocytes. Our findings are relevant in understanding how HIV-1 infection leads to immune dysregulation and provide the opportunity to develop and test potential therapeutic interventions.