Characterizing adjuvants' effects at murine immunoglobulin repertoire level.

Generating large-scale, high-fidelity sequencing data is challenging and, furthermore, not much has been done to characterize adjuvants' effects at the repertoire level. Thus, we introduced an IgSeq pipeline that standardized library prep protocols and data analysis functions for accurate repertoire profiling. We then studied systemically effects of CpG and Alum on the Ig heavy chain repertoire using the ovalbumin (OVA) murine model. Ig repertoires of different tissues (spleen and bone marrow) and isotypes (IgG and IgM) were examined and compared in IGHV mutation, gene usage, CDR3 length, clonal diversity, and clonal selection. We found Ig repertoires of different compartments exhibited distinguishable profiles at the non-immunized steady state, and distinctions became more pronounced upon adjuvanted immunizations. Notably, Alum and CpG effects exhibited different tissue- and isotype-preferences. The former led to increased diversity of abundant clones in bone marrow, and the latter promoted the selection of IgG clones in both tissues.

Diversity of B Cell Populations and Ig Repertoire in Human Lungs.

The human lung carries a unique microbiome adapted to the air-filled, mucous-lined environment, the presence of which requires an immune system capable of recognizing harmful populations while preventing reactions toward commensals. B cells in the lung play a key role in pulmonary immunity, generating Ag-specific Abs, as well as cytokine secretion for immune activation and regulation. In this study, we compared B cell subsets in human lungs versus circulating cells by analyzing patient-paired lung and blood samples. We found a significantly smaller pool of CD19+, CD20+ B cells in the lung relative to the blood. CD27+, IgD-, class-switched memory B cells (Bmems) composed a larger proportion of the pool of pulmonary B cells. The residency marker CD69 was also significantly higher in the lung. We also sequenced the Ig V region genes (IgVRGs) of class-switched Bmems that do, or do not, express CD69. We observed the IgVRGs of pulmonary Bmems to be as heavily mutated from the unmutated common ancestor as those in circulation. Furthermore, we found progenies within a quasi-clone can gain or lose CD69 expression, regardless of whether the parent clone expressed the residency marker. Overall, our results show that despite its vascularized nature, human lungs carry a unique proportion of B cell subsets. The IgVRGs of pulmonary Bmems are as diverse as those in blood, and progenies of Bmems retain the ability to gain or lose residency.

An epitope-enriched immunogen expands responses to a conserved viral site.

Pathogens evade host humoral responses by accumulating mutations in surface antigens. While variable, there are conserved regions that cannot mutate without compromising fitness. Antibodies targeting these conserved epitopes are often broadly protective but remain minor components of the repertoire. Rational immunogen design leverages a structural understanding of viral antigens to modulate humoral responses to favor these responses. Here, we report an epitope-enriched immunogen presenting a higher copy number of the influenza hemagglutinin (HA) receptor-binding site (RBS) epitope relative to other B cell epitopes. Immunization in a partially humanized murine model imprinted with an H1 influenza shows H1-specific serum and >99% H1-specific B cells being RBS-directed. Single B cell analyses show a genetically restricted response that structural analysis defines as RBS-directed antibodies engaging the RBS with germline-encoded contacts. These data show how epitope enrichment expands B cell responses toward conserved epitopes and advances immunogen design approaches for next-generation viral vaccines.

Development of Neutralization Breadth against Diverse HIV-1 by Increasing Ab-Ag Interface on V2.

Understanding maturation pathways of broadly neutralizing antibodies (bnAbs) against HIV-1 can be highly informative for HIV-1 vaccine development. A lineage of J038 bnAbs is now obtained from a long-term SHIV-infected macaque. J038 neutralizes 54% of global circulating HIV-1 strains. Its binding induces a unique "up" conformation for one of the V2 loops in the trimeric envelope glycoprotein and is heavily dependent on glycan, which provides nearly half of the binding surface. Their unmutated common ancestor neutralizes the autologous virus. Continuous maturation enhances neutralization potency and breadth of J038 lineage antibodies via expanding antibody-Env contact areas surrounding the core region contacted by germline-encoded residues. Developmental details and recognition features of J038 lineage antibodies revealed here provide a new pathway for elicitation and maturation of V2-targeting bnAbs.

Recapitulation of HIV-1 Env-antibody coevolution in macaques leading to neutralization breadth.

Neutralizing antibodies elicited by HIV-1 coevolve with viral envelope proteins (Env) in distinctive patterns, in some cases acquiring substantial breadth. We report that primary HIV-1 envelope proteins-when expressed by simian-human immunodeficiency viruses in rhesus macaques-elicited patterns of Env-antibody coevolution very similar to those in humans, including conserved immunogenetic, structural, and chemical solutions to epitope recognition and precise Env-amino acid substitutions, insertions, and deletions leading to virus persistence. The structure of one rhesus antibody, capable of neutralizing 49% of a 208-strain panel, revealed a V2 apex mode of recognition like that of human broadly neutralizing antibodies (bNAbs) PGT145 and PCT64-35S. Another rhesus antibody bound the CD4 binding site by CD4 mimicry, mirroring human bNAbs 8ANC131, CH235, and VRC01. Virus-antibody coevolution in macaques can thus recapitulate developmental features of human bNAbs, thereby guiding HIV-1 immunogen design.

Egyptian Rousette IFN-ω Subtypes Elicit Distinct Antiviral Effects and Transcriptional Responses in Conspecific Cells.

Bats host a number of viruses that cause severe disease in humans without experiencing overt symptoms of disease themselves. While the mechanisms underlying this ability to avoid sickness are not known, deep sequencing studies of bat genomes have uncovered genetic adaptations that may have functional importance in the antiviral response of these animals. Egyptian rousette bats (Rousettus aegyptiacus) are the natural reservoir hosts of Marburg virus (MARV). In contrast to humans, these bats do not become sick when infected with MARV. A striking difference to the human genome is that Egyptian rousettes have an expanded repertoire of IFNW genes. To probe the biological implications of this expansion, we synthesized IFN-ω4 and IFN-ω9 proteins and tested their antiviral activity in Egyptian rousette cells. Both IFN-ω4 and IFN-ω9 showed antiviral activity against RNA viruses, including MARV, with IFN-ω9 being more efficient than IFN-ω4. Using RNA-Seq, we examined the transcriptional response induced by each protein. Although the sets of genes induced by the two IFNs were largely overlapping, IFN-ω9 induced a more rapid and intense response than did IFN-ω4. About 13% of genes induced by IFN-ω treatment are not found in the Interferome or other ISG databases, indicating that they may be uniquely IFN-responsive in this bat.

Gain-Scanning for Protein Microarray Assays.

Protein microarrays consist of known proteins spotted onto solid substrates and are used to perform highly multivariate assessments of protein-binding interactions. Human protein arrays are routinely applied to pathogen detection, immune response biomarker profiling, and antibody specificity profiling. Here, we describe and demonstrate a new data processing procedure, gain-scan, in which data were acquired under multiple photomultiplier tube (PMT) settings, followed by data fitting with a power function model to estimate the incident light signals of the array spots. Data acquisition under multiple PMT settings solves the difficulty of determining the single optimal PMT gain setting and allows us to maximize the detection of low-intensity signals while avoiding the saturation of high-intensity ones at the same time. The gain-scan data acquisition and fitting also significantly lower the variances over the detectable range of signals and improve the linear data normalization. The performance of the proposed procedure was verified by analyzing the profiling data of both the human polyclonal serum samples and the monoclonal antibody samples with both technical replicates and biological replicates. We showed that the multigain power function was an appropriate model for describing data acquired under multiple PMT settings. The gain-scan fitting alone or in combination with the linear normalization could effectively reduce the technical variability of the array data and lead to better sample separability and more sensitive differential analysis.

Boosting with AIDSVAX B/E Enhances Env Constant Region 1 and 2 Antibody-Dependent Cellular Cytotoxicity Breadth and Potency.

Induction of protective antibodies is a critical goal of HIV-1 vaccine development. One strategy is to induce nonneutralizing antibodies (NNAbs) that kill virus-infected cells, as these antibody specificities have been implicated in slowing HIV-1 disease progression and in protection. HIV-1 Env constant region 1 and 2 (C1C2) monoclonal antibodies (MAbs) frequently mediate potent antibody-dependent cellular cytotoxicity (ADCC), making them an important vaccine target. Here, we explore the effect of delayed and repetitive boosting of RV144 vaccine recipients with AIDSVAX B/E on the C1C2-specific MAb repertoire. It was found that boosting increased clonal lineage-specific ADCC breadth and potency. A ligand crystal structure of a vaccine-induced broad and potent ADCC-mediating C1C2-specific MAb showed that it bound a highly conserved Env gp120 epitope. Thus, boosting to affinity mature these types of IgG C1C2-specific antibody responses may be one method by which to make an improved HIV vaccine with higher efficacy than that seen in the RV144 trial.IMPORTANCE Over one million people become infected with HIV-1 each year, making the development of an efficacious HIV-1 vaccine an important unmet medical need. The RV144 human HIV-1 vaccine regimen is the only HIV-1 clinical trial to date to demonstrate vaccine efficacy. An area of focus has been on identifying ways by which to improve upon RV144 vaccine efficacy. The RV305 HIV-1 vaccine regimen was a follow-up boost of RV144 vaccine recipients that occurred 6 to 8 years after the conclusion of RV144. Our study focused on the effect of delayed boosting in humans on the vaccine-induced Env constant region 1 and 2 (C1C2)-specific antibody repertoire. It was found that boosting with an HIV-1 Env vaccine increased C1C2-specific antibody-dependent cellular cytotoxicity potency and breadth.

Correction: Neutralization-guided design of HIV-1 envelope trimers with high affinity for the unmutated common ancestor of CH235 lineage CD4bs broadly neutralizing antibodies.

[This corrects the article DOI: 10.1371/journal.ppat.1008026.].

Isolation and Structure of an Antibody that Fully Neutralizes Isolate SIVmac239 Reveals Functional Similarity of SIV and HIV Glycan Shields.

HIV- and SIV-envelope (Env) trimers are both extensively glycosylated, and antibodies identified to date have been unable to fully neutralize SIVmac239. Here, we report the isolation, structure, and glycan interactions of antibody ITS90.03, a monoclonal antibody that completely neutralized the highly neutralization-resistant isolate, SIVmac239. The co-crystal structure of a fully glycosylated SIVmac239-gp120 core in complex with rhesus CD4 and the antigen-binding fragment of ITS90.03 at 2.5-Å resolution revealed that ITS90 recognized an epitope comprised of 45% glycan. SIV-gp120 core, rhesus CD4, and their complex could each be aligned structurally to their human counterparts. The structure revealed that glycans masked most of the SIV Env protein surface, with ITS90 targeting a glycan hole, which is occupied in ∼83% of SIV strains by glycan N238. Overall, the SIV glycan shield appears to functionally resemble its HIV counterpart in coverage of spike, shielding from antibody, and modulation of receptor accessibility.

Neutralization-guided design of HIV-1 envelope trimers with high affinity for the unmutated common ancestor of CH235 lineage CD4bs broadly neutralizing antibodies.

The CD4 binding site (CD4bs) of the HIV-1 envelope glycoprotein is susceptible to multiple lineages of broadly neutralizing antibodies (bnAbs) that are attractive to elicit with vaccines. The CH235 lineage (VH1-46) of CD4bs bnAbs is particularly attractive because the most mature members neutralize 90% of circulating strains, do not possess long HCDR3 regions, and do not contain insertions and deletions that may be difficult to induce. We used virus neutralization to measure the interaction of CH235 unmutated common ancestor (CH235 UCA) with functional Env trimers on infectious virions to guide immunogen design for this bnAb lineage. Two Env mutations were identified, one in loop D (N279K) and another in V5 (G458Y), that acted synergistically to render autologous CH505 transmitted/founder virus susceptible to neutralization by CH235 UCA. Man5-enriched N-glycans provided additional synergy for neutralization. CH235 UCA bound with nanomolar affinity to corresponding soluble native-like Env trimers as candidate immunogens. A cryo-EM structure of CH235 UCA bound to Man5-enriched CH505.N279K.G458Y.SOSIP.664 revealed interactions of the antibody light chain complementarity determining region 3 (CDR L3) with the engineered Env loops D and V5. These results demonstrate that virus neutralization can directly inform vaccine design and suggest a germline targeting and reverse engineering strategy to initiate and mature the CH235 bnAb lineage.

Antibodies to a Conserved Influenza Head Interface Epitope Protect by an IgG Subtype-Dependent Mechanism.

Vaccines to generate durable humoral immunity against antigenically evolving pathogens such as the influenza virus must elicit antibodies that recognize conserved epitopes. Analysis of single memory B cells from immunized human donors has led us to characterize a previously unrecognized epitope of influenza hemagglutinin (HA) that is immunogenic in humans and conserved among influenza subtypes. Structures show that an unrelated antibody from a participant in an experimental infection protocol recognized the epitope as well. IgGs specific for this antigenic determinant do not block viral infection in vitro, but passive administration to mice affords robust IgG subtype-dependent protection against influenza infection. The epitope, occluded in the pre-fusion form of HA, is at the contact surface between HA head domains; reversible molecular "breathing" of the HA trimer can expose the interface to antibody and B cells. Antigens that present this broadly immunogenic HA epitope may be good candidates for inclusion in "universal" flu vaccines.

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.

Intra-seasonal antibody repertoire analysis of a subject immunized with an MF59®-adjuvanted pandemic 2009 H1N1 vaccine.

During the height of the 2009 H1N1 swine-derived influenza pandemic, a clinical trial was conducted in which seven subjects were immunized using a monovalent, MF59®-adjuvanted vaccine, developed from an egg-passaged candidate vaccine virus (CVV), A/California/07/2009 X-181. Whole blood was collected prior to immunization and at 8, 22, and 202 days post-vaccination, and subjects' serological responses were evaluated. Here, we reconstruct and examine the longitudinal, influenza-specific circulating B cell repertoire of one subject in that study. Genotypic analysis of 390 total subject-derived antibodies (Abs) revealed a total of 29 germline genes in use among immunoglobulin heavy chain variable regions (IgHV), with the majority of those sequences isolated representing memory recall responses and two major lineages dominating the early response. In vitro phenotyping showed a diverse set of binding epitopes on the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), many of which are considered subdominant. Strong correlations were found between IgHV germline usage among non-related lineages and both binding epitope and neutralization breadth. Results here highlight the potential for Ab responses to be misdirected to egg-adaptive artifacts on CVVs while simultaneously stressing the ability to mount potent, broadly neutralizing responses to mostly novel antigens via recall of subdominant memory responses, as well as the need for evaluating alternative endpoint assays and anti-NA responses following clinical trials.

Functional Relevance of Improbable Antibody Mutations for HIV Broadly Neutralizing Antibody Development.

HIV-1 broadly neutralizing antibodies (bnAbs) require high levels of activation-induced cytidine deaminase (AID)-catalyzed somatic mutations for optimal neutralization potency. Probable mutations occur at sites of frequent AID activity, while improbable mutations occur where AID activity is infrequent. One bottleneck for induction of bnAbs is the evolution of viral envelopes (Envs) that can select bnAb B cell receptors (BCR) with improbable mutations. Here we define the probability of bnAb mutations and demonstrate the functional significance of key improbable mutations in three bnAb B cell lineages. We show that bnAbs are enriched for improbable mutations, which implies that their elicitation will be critical for successful vaccine induction of potent bnAb B cell lineages. We discuss a mutation-guided vaccine strategy for identification of Envs that can select B cells with BCRs that have key improbable mutations required for bnAb development.

The Egyptian Rousette Genome Reveals Unexpected Features of Bat Antiviral Immunity.

Bats harbor many viruses asymptomatically, including several notorious for causing extreme virulence in humans. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of Rousettus aegyptiacus, a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We found an expanded and diversified KLRC/KLRD family of natural killer cell receptors, MHC class I genes, and type I interferons, which dramatically differ from their functional counterparts in other mammals. Such concerted evolution of key components of bat immunity is strongly suggestive of novel modes of antiviral defense. An evaluation of the theoretical function of these genes suggests that an inhibitory immune state may exist in bats. Based on our findings, we hypothesize that tolerance of viral infection, rather than enhanced potency of antiviral defenses, may be a key mechanism by which bats asymptomatically host viruses that are pathogenic in humans.

Memory B Cells that Cross-React with Group 1 and Group 2 Influenza A Viruses Are Abundant in Adult Human Repertoires.

Human B cell antigen-receptor (BCR) repertoires reflect repeated exposures to evolving influenza viruses; new exposures update the previously generated B cell memory (Bmem) population. Despite structural similarity of hemagglutinins (HAs) from the two groups of influenza A viruses, cross-reacting antibodies (Abs) are uncommon. We analyzed Bmem compartments in three unrelated, adult donors and found frequent cross-group BCRs, both HA-head directed and non-head directed. Members of a clonal lineage from one donor had a BCR structure similar to that of a previously described Ab, encoded by different gene segments. Comparison showed that both Abs contacted the HA receptor-binding site through long heavy-chain third complementarity determining regions. Affinities of the clonal-lineage BCRs for historical influenza-virus HAs from both group 1 and group 2 viruses suggested that serial responses to seasonal influenza exposures had elicited the lineage and driven affinity maturation. We propose that appropriate immunization regimens might elicit a comparably broad response.

Structure and Diversity of the Rhesus Macaque Immunoglobulin Loci through Multiple De Novo Genome Assemblies.

The rhesus macaque is a critically important animal model in biomedical research, most recently playing a key role in the development of vaccines against human immunodeficiency virus-1. Nevertheless, the immunoglobulin (Ig) loci of macaques are as yet incompletely determined and our understanding of differences between human and macaque humoral immunity remains deficient. We completed a high-coverage, high-quality whole genome sequencing and assembly project with a single rhesus macaque of Indian origin, and partial genome assemblies using genomic molecular targeting of the Ig loci in nine other rhesus macaques of Indian origin. These data indicate that the macaque Ig loci are substantially more diverse than those in humans, including greater sequence diversity and copy-number variation between individuals. It appears likely that such copy-number variation even occurs between allelic loci within individuals. Different Ig gene families in the macaque show distinct relationships to the corresponding human gene families and appear to evolve under different mechanisms. These results raise intriguing questions about the evolution of antigen receptors in primates but also have important practical implications for the design and interpretation of biomedical studies.

Reproducibility and Reuse of Adaptive Immune Receptor Repertoire Data.

High-throughput sequencing (HTS) of immunoglobulin (B-cell receptor, antibody) and T-cell receptor repertoires has increased dramatically since the technique was introduced in 2009 (1-3). This experimental approach explores the maturation of the adaptive immune system and its response to antigens, pathogens, and disease conditions in exquisite detail. It holds significant promise for diagnostic and therapy-guiding applications. New technology often spreads rapidly, sometimes more rapidly than the understanding of how to make the products of that technology reliable, reproducible, or usable by others. As complex technologies have developed, scientific communities have come together to adopt common standards, protocols, and policies for generating and sharing data sets, such as the MIAME protocols developed for microarray experiments. The Adaptive Immune Receptor Repertoire (AIRR) Community formed in 2015 to address similar issues for HTS data of immune repertoires. The purpose of this perspective is to provide an overview of the AIRR Community's founding principles and present the progress that the AIRR Community has made in developing standards of practice and data sharing protocols. Finally, and most important, we invite all interested parties to join this effort to facilitate sharing and use of these powerful data sets (join@airr-community.org).

Initiation of HIV neutralizing B cell lineages with sequential envelope immunizations.

A strategy for HIV-1 vaccine development is to define envelope (Env) evolution of broadly neutralizing antibodies (bnAbs) in infection and to recreate those events by vaccination. Here, we report host tolerance mechanisms that limit the development of CD4-binding site (CD4bs), HCDR3-binder bnAbs via sequential HIV-1 Env vaccination. Vaccine-induced macaque CD4bs antibodies neutralize 7% of HIV-1 strains, recognize open Env trimers, and accumulate relatively modest somatic mutations. In naive CD4bs, unmutated common ancestor knock-in mice Env+B cell clones develop anergy and partial deletion at the transitional to mature B cell stage, but become Env- upon receptor editing. In comparison with repetitive Env immunizations, sequential Env administration rescue anergic Env+ (non-edited) precursor B cells. Thus, stepwise immunization initiates CD4bs-bnAb responses, but immune tolerance mechanisms restrict their development, suggesting that sequential immunogen-based vaccine regimens will likely need to incorporate strategies to expand bnAb precursor pools.