A key challenge in B-cell lineage-based vaccine design is understanding the "inducibility" of target neutralizing antibodies-the ability of these antibodies to be elicited by presentation of an immunogen. Induction relies on a combination of stochastic diversification and immunogen-based selection, and as a result is heavily dependent on the probabilistic accessibility of induction pathways. We explored inducibility using a detailed stochastic model of the somatic hypermutation process-which captures the critical "context-dependence" of sequence mutation rates-coupled to a stochastic population model for B-cell clonal maturation. The model is used to calculate inducibilities for a set of critical mutations required by the HIV broadly neutralizing antibody (bnAb) CH235.12. The results provide insight into barriers to the elicitation of HIV bnAbs and explain experimental results observed in mouse models. Our models enable detailed analysis of maturation pathway probabilities, allowing us to identify opportunities for the design of boosting immunogens aimed at elicitation of CH235.12 via a sequential vaccination regimen.

A major obstacle confronting HIV-1 vaccine and cure research is the lack of an outbred animal model for rapid and consistent induction of broadly neutralizing antibodies (bNAbs). We designed an epitope-focused simian-human immunodeficiency virus (SHIV.5MUT) that elicited broad and potent V3-glycan-targeted antibodies within a year of infection in 14 of 22 macaques compared with 0 of 14 control animals. SHIV.5MUT elicited bNAbs by a two-step mechanism, inducing an initial wave of V1-directed antibodies that selected for envelope (Env) glycoprotein variants with shortened, hypoglycosylated V1 loops, which in turn primed V3-glycan bNAb precursors. Rhesus bNAbs were immunogenetically and structurally diverse, closely resembling human V3-glycan bNAbs. Env-bNAb coevolution revealed a diverse repertoire of bNAb precursors and the Env variants that matured them, yielding a molecular blueprint for vaccine design.

We introduce a method for approximating posterior probabilities of phylogenetic trees and reconstructing ancestral sequences under models of sequence evolution with site-dependence, where standard phylogenetic likelihood computations (pruning) fail. Our approach uses a combined data-augmentation and importance sampling scheme. A key advantage of our approach is the ability to leverage existing highly optimized phylogenetic software. We apply our approach to the reconstruction of B cell receptor affinity maturation lineages from high-throughput repertoire sequencing data and evaluate the impact of incorporating site-dependence on the reconstruction accuracy of both trees and ancestral sequences. We show that accounting for context-dependence during inference always improves the estimates of both ancestral sequences and lineage trees on simulated datasets. We also examine the impact of incorporating priors based on VDJ recombination models, and find that they significantly improve ancestral sequence reconstruction in germline-encoded regions, but increase errors in non-templated nucleotides. We propose a modified, piecewise prior to address this demonstrate that it improves empirical reconstruction accuracy. We apply our approach to the analysis of the HIV broadly neutralizing antibodies DH270 and CH235 which are important targets of current vaccine design efforts.

Background: Antibody-dependent cell-mediated cytotoxic (ADCC) response mediated by natural killer (NK) cells correlates with decreased infection risk in studies involving simian immunodeficiency virus (SIV)/simian-human immunodeficiency virus (SHIV), and human immunodeficiency virus (HIV) vaccine candidates. Currently, the heterogeneities of the functional subset of rhesus macaque natural killer (RMNK) cells are under-characterized. Method: We engaged the RMNK cells with ADCC-mediating anti-HIV-1 monoclonal antibodies (ADCCAbs) or anti-CD16 antibodies and used CD107a expression as the surrogate marker for RMNK cells actively involved in ADCC. CD107a+ and CD107a– populations were analyzed individually using single-cell RNA sequencing. Results: Subsets of CD107a+ RMNK cells produced more chemokines than the others, suggesting that these cells not only eliminate infected cells but also provide immunoregulatory signals and potentially curb HIV-1 replication. Crosslinking of Fc gamma receptor IIIa via anti-CD16 antibodies resulted in a significantly higher percentage of degranulating cells than via ADCCAbs. However, the magnitude of degranulation and chemokine production was reduced by 6- to 30-fold. Conclusion: The quality and quantity of receptor engagement are important determinants of achieving an optimal level of the RMNK response.

A major obstacle confronting HIV-1 vaccine and cure research is the lack of an outbred animal model for rapid and consistent induction of broadly neutralizing antibodies (bNAbs). We designed an epitope-focused simian-human immunodeficiency virus (SHIV.5MUT) that elicited broad and potent V3-glycan-targeted antibodies within a year of infection in 14 of 22 macaques compared with 0 of 14 control animals. SHIV.5MUT elicited bNAbs by a novel two-step mechanism, inducing an initial wave of V1-directed antibodies that selected for Envs with shortened, hypoglycosylated V1 loops, which in turn primed V3-glycan bNAb precursors. Rhesus bNAbs were immunogenetically and structurally diverse, closely resembling human V3-glycan bNAbs. Env-bNAb coevolution revealed a diverse repertoire of bNAb precursors and the Env variants that matured them, yielding a molecular blueprint for vaccine design.

SARS-CoV-2 continues to evolve, with new variants emerging that evade pre-existing immunity and limit the efficacy of existing vaccines. One approach toward developing superior, variant-proof vaccines is to engineer immunogens that preferentially elicit antibodies with broad cross-reactivity against SARS-CoV-2 and its variants by targeting conserved epitopes on spike. The inner and outer faces of the receptor binding domain (RBD) are two such conserved regions targeted by antibodies that recognize diverse human and animal coronaviruses. To promote the elicitation of such antibodies by vaccination, we engineered "resurfaced" RBD immunogens that contained mutations at exposed RBD residues outside the target epitopes. In the context of pre-existing immunity, these vaccine candidates aim to disfavor the elicitation of strain-specific antibodies against the immunodominant receptor binding motif (RBM) while boosting the induction of inner and outer face antibodies. The engineered resurfaced RBD immunogens were stable, lacked binding to monoclonal antibodies with limited breadth, and maintained strong interactions with target broadly neutralizing antibodies. When used as vaccines, they limited humoral responses against the RBM as intended. Multimerization on nanoparticles further increased the immunogenicity of the resurfaced RBD immunogens, thus supporting resurfacing as a promising immunogen design approach to rationally shift natural immune responses to develop more protective vaccines.IMPORTANCESARS-CoV-2 is the third coronavirus to cause significant human disease over the last two decades. Despite their success in preventing serious disease, current SARS-CoV-2 vaccines must be updated regularly to match the circulating strains for continued protection. Therefore, it would be advantageous to develop vaccines that protect more broadly against SARS-CoV-2, its variants, and other pre-emergent coronaviruses. This may be achieved by preferentially eliciting antibodies against conserved regions of the spike protein that decorates the virus. Toward this goal, we engineered vaccine candidates to target the conserved inner and outer domains of the Receptor Binding Domain of SARS-CoV-2, by altering the surface of the wild-type protein such that strain-specific antibodies that bind outside these regions are no longer recognized. When used in animals with pre-existing SARS-CoV-2 immunity, these molecules reduce the elicitation of variant-specific antibodies, thus providing a blueprint to alter the natural immunodominance hierarchies of SARS-CoV-2 proteins.

A critical roadblock to HIV vaccine development is the inability to induce B cell lineages of broadly neutralizing antibodies (bnAbs) in humans. In people living with HIV-1, bnAbs take years to develop. The HVTN 133 clinical trial studied a peptide/liposome immunogen targeting B cell lineages of HIV-1 envelope (Env) membrane-proximal external region (MPER) bnAbs (NCT03934541). Here, we report MPER peptide-liposome induction of polyclonal HIV-1 B cell lineages of mature bnAbs and their precursors, the most potent of which neutralized 15% of global tier 2 HIV-1 strains and 35% of clade B strains with lineage initiation after the second immunization. Neutralization was enhanced by vaccine selection of improbable mutations that increased antibody binding to gp41 and lipids. This study demonstrates proof of concept for rapid vaccine induction of human B cell lineages with heterologous neutralizing activity and selection of antibody improbable mutations and outlines a path for successful HIV-1 vaccine development.

Broadly neutralizing antibodies (bNAbs) are rarely elicited during HIV-1 infection. To identify obstacles to bNAb development, we longitudinally studied 122 rhesus macaques infected by one of 16 different simian-human immunodeficiency viruses (SHIVs). We identified V2 apex as the most common bNAb target and a subset of Envs that preferentially elicited these antibodies. In 10 macaques, we delineated Env-antibody coevolution from B cell priming to bNAb development. Antibody phylogenies revealed permissive developmental pathways guided by evolving Envs that contained few mutations in or near the V2 apex C-strand, which were a sensitive indicator of apex-targeted responses. The absence of such mutations reflected a failure in bNAb priming. These results indicate that efficiency of B cell priming, and not complexities in Env-guided affinity maturation, is the primary obstacle to V2 apex bNAb elicitation in SHIV-infected macaques and identify specific HIV-1 Envs to advance as novel vaccine platforms. One sentence summary:B cell priming is the primary bottleneck to HIV-1 V2 apex bNAb elicitation.

BACKGROUND:Sequential multivalent immunizations are used to counter diversity in rapidly mutating viruses. Here, we evaluated the effect of HIV-1 immunogen formats on the binding profile of memory B-cells elicited in two independent Rhesus macaque trials. METHODS:In one trial, female Rhesus macaques were immunized with a multiclade HIV-1 gp120 envelope glycoprotein (Env) cocktail and bled two weeks post final immunization. In another trial, male and female Rhesus macaques were sequentially immunized with clonally-related Env glycoproteins: Four immunogens were administered as non-stabilized gp140 Envs and the fifth as a specially stabilized gp140 Env trimer (SOSIP); animals were bled before and after SOSIP immunization. Immunogen-binding peripheral memory B-cells were sorted and cultured at limiting dilution. Culture supernatants were assessed by ELISA for binding to individual immunogens. RESULTS:In the first trial, 81% (591/734) of B-cells cross-react with multiple Envs and most bind to all immunogens. In the second trial, 81% (331/410) of B-cells isolated before SOSIP administration react with all non-stabilized gp140 Env immunogens and 27% also cross-react with the yet-to-be-administered SOSIP-stabilized Env. However, after SOSIP administration, SOSIP-stabilized trimer-reactive B-cells increase to 86% (219/256) but most (82%) do not cross-react with the preceding immunogens. CONCLUSIONS:Multiclade and sequential regimens before SOSIP-stabilized Env immunization elicited B-cells that converge on shared epitopes. A change in immunogen format results in a divergent B-cell response that vastly fails to engage prior responses. Critically, B-cell priming with non-stabilized Env cannot modify the effect of the epitope immunodominance hierarchy in a SOSIP trimer. These results suggest that a change in immunogen format may cause off-target B-cell engagement, but also that B-cell repriming is possible despite pre-existing immunity.

Henipaviruses, a genus within the Paramyxoviridae family, include the highly virulent Nipah and Hendra viruses that cause reoccurring outbreaks of deadly disease. Recent discoveries of several new Paramyxoviridae species, including the zoonotic Langya virus, have revealed much higher antigenic diversity than currently characterized and prompted the reorganization of these viruses into the Henipavirus and Parahenipavirus genera. Here, to explore the limits of structural and antigenic variation in both genera, collectively referred to here as HNVs, we constructed an expanded, antigenically diverse panel of HNV fusion and attachment glycoproteins from 56 unique HNV strains that better reflects global HNV diversity. We expressed and purified the fusion protein ectodomains and the attachment protein head domains and characterized their biochemical, biophysical and structural properties. We performed immunization experiments in mice leading to the elicitation of antibodies reactive to multiple HNV fusion proteins. Cryo-electron microscopy structures of diverse fusion proteins elucidated molecular determinants of differential pre-fusion state metastability and higher order contacts. A crystal structure of the Gamak virus attachment head domain revealed an additional domain added to the conserved 6-bladed, β-propeller fold. Taken together, these studies expand the known structural and antigenic limits of the HNVs, reveal new cross-reactive epitopes within both genera and provide foundational data for the development of broadly reactive countermeasures.

Elicitation of HIV broadly neutralizing antibodies (bnAbs) by vaccination first requires the activation of diverse precursors, followed by successive boosts that guide these responses to enhanced breadth through the acquisition of somatic mutations. Because HIV bnAbs contain mutations in their B cell receptors (BCRs) that are rarely generated during conventional B cell maturation, HIV vaccine immunogens must robustly engage and expand B cells with BCRs that contain these improbable mutations. Here, we engineered an immunogen that activates diverse precursors of an HIV V3-glycan bnAb and promotes their acquisition of a functionally critical improbable mutation. This immunogen was validated biochemically, structurally, and in three different humanized immunoglobulin mouse models that were designed to test HIV immunogens. These results provide a blueprint for rationally designing priming immunogens that explicitly target the elicitation of antibodies with functional yet improbable mutations.

Although most broadly neutralizing antibodies (bNAbs) specific for the CD4-binding site (CD4-BS) of HIV-1 interact with a single gp120 protomer, a few mimic the quaternary binding mode of CD4, making contact with a second protomer through elongated heavy chain framework 3 (FRH3) or complementarity-determining region 1 (CDRH1) loops. Here, we show that a CDRH3-dominated anti-CD4-BS bNAb, CH103, establishes quaternary interaction despite regular-length FRH3 and CDRH1. This quaternary interaction is critical for neutralization and is primarily mediated by two FRH3 acidic residues that were sequentially acquired and subjected to strong positive selection during CH103 maturation. Cryoelectron microscopy (cryo-EM) structures confirmed the role of the two FRH3 acidic residues in mediating quaternary contact and demonstrated that CH103 reaches the adjacent gp120 protomer by virtue of its unique angle of approach. Thus, the acquisition of quaternary interaction may constitute a key step in the lineage maturation of a broad and potent HIV-1 neutralizing antibody.

The thymus is critical for the development and selection of T cells with a diverse range of non-self-reactive antigen receptors. Both the thymus and circulating T cells can be damaged by acute exposure to ionizing radiation, leading to dose-dependent lymphopenia, a temporarily increased risk of infection that can be life-threatening, and long-term disruptions in T cell homeostasis and function. Currently, there are no biomedical countermeasures available to prevent radiation-induced T cell lymphopenia or other T cell defects caused by radiation. Therefore, preclinical models of radiation-induced thymic injury are necessary for testing countermeasures. Adult mice and non-human primates (NHP) that are subjected to whole-body or thorax irradiation are suitable models for this purpose. However, findings from these models may not directly apply to juveniles, given the significant changes in thymus size and function during childhood. To address this, we characterized the effects of 10 Gy whole-thorax irradiation on the thymus of pediatric rhesus macaque NHPs. Computed tomography (CT) assessments of thymic density and volume were used as in vivo indicators of thymic injury, but they did not correlate with the changes in thymic weight observed 19 weeks after irradiation. Histopathological staining revealed that whole-thorax irradiation caused disruption of thymic architecture, evident four months post-irradiation in some animals. Molecular analyses showed that radiation led to a decrease in thymic output, reduced diversity of T cell antigen receptors, and an over-representation of certain receptor sequences indicative of extensive clonal expansion. Overall, this work demonstrates the usefulness of the NHP whole-thorax irradiation model-commonly employed in lung radiobiology research-in studying radiation-induced thymic injury in children and in developing medical countermeasures.

We previously identified in non-small cell lung cancer (NSCLC) patients an autoantibody to complement factor H (CFH) that is associated with non-metastatic disease and longer time to progression in patients with stage I disease. A recombinant human antibody, GT103, was cloned from single B cells isolated from patients with the autoantibody. GT103 inhibits tumor growth and establishes an antitumor microenvironment. The anti-CFH autoantibody and GT103 recognize the epitope PIDNGDIT within the SCR19 domain of CFH. Here, we asked if this autoantibody could have originally arisen as a humoral response to a similar epitope in a viral protein from a prior infection. Homologous viral peptides with high sequence identity to the core PIDNGDIT epitope sequence were identified and synthesized. NSCLC patient plasma containing anti-CFH autoantibodies were assayed by ELISA against these peptides. GT103 was assayed on a 4345-peptide pathogen microarray. Epitopes similar to the GT103 epitope are present in several viruses, including human metapneumovirus-1 (HMPV-1) that contains a sequence within attachment glycoprotein G that differs by one amino acid. Anti-CFH autoantibodies in NSCLC patient plasma weakly bound to an HMPV-1 peptide containing the epitope. GT103 cross-reacted with multiple viral epitopes on a peptide microarray, with the top hits being peptides in the human endogenous retrovirus-K polymerase (HERV-K pol) protein and measles hemagglutinin glycoprotein. GT103 bound the viral HMPV-1, HERV-K pol, and measles epitope peptides but with lower affinity compared to the GT103 epitope peptide. These findings suggest that memory B cells against a viral target could have affinity matured to produce an antibody that recognizes a similar epitope on tumor cells and exhibits antitumor properties.

Current vaccine strategies to elicit broadly neutralizing antibodies (bnAbs) against HIV-1 generally propose complex, multi-boost immunization regimens. In rhesus macaques, SHIV infection has been observed to rapidly drive the development of some classes of bnAbs that share structural similarities with those in humans. Here, we generated a knockin mouse model with B cells bearing the unmutated common ancestor (UCA) of the V2 apex-targeted bnAb lineage, V033-a. A single immunization of mice with a germline-targeting native-like trimer was sufficient to recapitulate the ontogeny of the mature rhesus bnAb in knockin mice-including rare, disfavored somatic mutations-leading to the induction of antibodies that exhibited potent neutralization against both autologous and heterologous tier 2 viruses. A boost with Env escape mutant trimers further improved breadth and potency, and cryo-EM structure revealed the structural basis for heterologous neutralization breadth. Non-human primate and mouse models can thus combine with structure to serve as a platform for identifying and confirming immunogens that streamline HIV-vaccination regimens.

Current seasonal influenza virus vaccines induce responses primarily against immunodominant but highly plastic epitopes in the globular head of the hemagglutinin (HA) glycoprotein. Because of viral antigenic drift at these sites, vaccines need to be updated and readministered annually. To increase the breadth of influenza vaccine-mediated protection, we developed an antigenically complex mixture of recombinant HAs designed to redirect immune responses to more conserved domains of the protein. Vaccine-induced antibodies were disproportionally redistributed to the more conserved stalk of the HA without hindering, and in some cases improving, antibody responses against the head domain. These improved responses led to increased protection against homologous and heterologous viral challenges in both mice and ferrets compared with conventional vaccine approaches. Thus, antigenically complex protein mixtures can at least partially overcome HA head domain antigenic immunodominance and may represent a step toward a more universal influenza vaccine.

The details of the pediatric immune system that supports induction of antibodies capable of neutralizing geographically-diverse or heterologous HIV-1 is currently unclear. Here we explore the pediatric immune environment in neonatal macaque undergoing Simian-HIV infection. Simian-HIV infection of 11 pairs of therapy-naive dams and infant rhesus macaques for 24 months results in heterologous HIV-1 neutralizing antibodies in 64% of young macaques compared to 18% of adult macaques. Heterologous HIV-1 neutralizing antibodies emerge by 12 months post-infection in young macaques, in association with lower expression of immunosuppressive genes, fewer germinal center CD4 + T regulatory cells, and a lower ratio of CD4 + T follicular regulatory to helper cells. Antibodies from peripheral blood B cells in two young macaques following SHIV infection neutralize 13% of 119 heterologous HIV-1 strains and map to regions of canonical broadly neutralizing antibody epitopes on the envelope surface protein. Here we show that pediatric immunity to SHIV infection in a macaque model may inform vaccine strategies to induce effective HIV-1 neutralizing antibodies in infants and children prior to viral exposure. The specifics of the pediatric immune response that gives rise to antibodies capable of neutralising diverse HIV-1 strains is not fully understood. Here the authors characterise the immune environment of Simian-HIV infected paediatric macaques and link to antibody neutralisation induction.

Fcγ receptors (FcγRs) are membrane-bound glycoproteins that bind to the fragment crystallizable (Fc) constant regions of IgG antibodies. Interactions between IgG immune complexes and FcγRs can initiate signal transduction that mediates important components of the immune response including activation of immune cells for clearance of opsonized pathogens or infected host cells. In humans, many studies have identified associations between FcγR gene polymorphisms and risk of infection, or progression of disease, suggesting a gene-level impact on FcγR-dependent immune responses. Rhesus macaques are an important translational model for most human health interventions, yet little is known about the breadth of rhesus macaque FcγR genetic diversity. This lack of knowledge prevents evaluation of the impact of FcγR polymorphisms on outcomes of preclinical studies performed in rhesus macaques. In this study we used long-read RNA sequencing to define the genetic diversity of FcγRs in 206 Indian-origin Rhesus macaques, Macaca mulatta. We describe the frequency of single nucleotide polymorphisms, insertions, deletions, frame-shift mutations, and isoforms. We also index the identified diversity using predicted and known rhesus macaque FcγR and Fc-FcγR structures. Future studies that define the functional significance of this genetic diversity will facilitate a better understanding of the correlation between human and macaque FcγR biology that is needed for effective translation of studies with antibody-mediated outcomes performed in rhesus macaques.

Adolescents are a growing population of people living with HIV. The period between weaning and sexual debut presents a low-risk window for HIV acquisition, making early childhood an ideal time for implementing an immunization regimen. Because the elicitation of broadly neutralizing antibodies (bnAbs) is critical for an effective HIV vaccine, our goal was to assess the ability of a bnAb B cell lineage-designed HIV envelope SOSIP (protein stabilized by a disulfide bond between gp120-gp41-named "SOS"-and an isoleucine-to-proline point mutation-named "IP"-at residue 559) to induce precursor CD4 binding site (CD4bs)-targeting bnAbs in early life. Infant rhesus macaques received either a BG505 SOSIP, based on the infant BG505 transmitted/founder virus, or the CD4bs germ line-targeting BG505 SOSIP GT1.1 (n = 5 per group). Although both strategies induced durable, high-magnitude plasma autologous virus neutralization responses, only GT1.1-immunized infants (n = 3 of 5) exhibited VRC01-like CD4bs bnAb precursor development. Thus, a multidose immunization regimen with bnAb lineage-designed SOSIPs shows promise for inducing early B cell responses with the potential to mature into protective HIV bnAbs before sexual debut.