During lymphocyte development, V(D)J recombination assembles antigen receptor genes from component V, D, and J gene segments. These gene segments are flanked by a recombination signal sequence (RSS), which serves as the binding site for the recombination machinery. The murine Jbeta2.6 gene segment is a recombinationally inactive pseudogene, but examination of its RSS reveals no obvious reason for its failure to recombine. Mutagenesis of the Jbeta2.6 RSS demonstrates that the sequences of the heptamer, nonamer, and spacer are all important. Strikingly, changes solely in the spacer sequence can result in dramatic differences in the level of recombination. The subsequent analysis of a library of more than 4,000 spacer variants revealed that spacer residues of particular functional importance are correlated with their degree of conservation. Biochemical assays indicate distinct cooperation between the spacer and heptamer/nonamer along each step of the reaction pathway. The results suggest that the spacer serves not only to ensure the appropriate distance between the heptamer and nonamer but also regulates RSS activity by providing additional RAG:RSS interaction surfaces. We conclude that while RSSs are defined by a "digital" requirement for absolutely conserved nucleotides, the quality of RSS function is determined in an "analog" manner by numerous complex interactions between the RAG proteins and the less-well conserved nucleotides in the heptamer, the nonamer, and, importantly, the spacer. Those modulatory effects are accurately predicted by a new computational algorithm for "RSS information content." The interplay between such binary and multiplicative modes of interactions provides a general model for analyzing protein-DNA interactions in various biological systems.

The membrane proximal external region (MPER) of HIV-1 gp41 has several features that make it an attractive antibody-based vaccine target, but eliciting an effective gp41 MPER-specific protective antibody response remains elusive. One fundamental issue is whether the failure to make gp41 MPER-specific broadly neutralizing antibodies like 2F5 and 4E10 is due to structural constraints with the gp41 MPER, or alternatively, if gp41 MPER epitope-specific B cells are lost to immunological tolerance. An equally important question is how B cells interact with, and respond to, the gp41 MPER epitope, including whether they engage this epitope in a non-canonical manner i.e., by non-paratopic recognition via B cell receptors (BCR). To begin understanding how B cells engage the gp41 MPER, we characterized B cell-gp41 MPER interactions in BALB/c and C57BL/6 mice. Surprisingly, we found that a significant (approximately 7%) fraction of splenic B cells from BALB/c, but not C57BL/6 mice, bound the gp41 MPER via their BCRs. This strain-specific binding was concentrated in IgM(hi) subsets, including marginal zone and peritoneal B1 B cells, and correlated with enriched fractions (approximately 15%) of gp41 MPER-specific IgM secreted by in vitro-activated splenic B cells. Analysis of Igh(a) (BALB/c) and Igh(b) (C57BL/6) congenic mice demonstrated that gp41 MPER binding was controlled by determinants of the Igh(a) locus. Mapping of MPER gp41 interactions with IgM(a) identified MPER residues distinct from those to which mAb 2F5 binds and demonstrated the requirement of Fc C(H) regions. Importantly, gp41 MPER ligation produced detectable BCR-proximal signaling events, suggesting that interactions between gp41 MPER and IgM(a) determinants may elicit partial B cell activation. These data suggest that low avidity, non-paratopic interactions between the gp41 MPER and membrane Ig on naïve B cells may interfere with or divert bnAb responses.

The cohesin ring holds newly replicated sister chromatids together until their separation at anaphase. Initiation of sister chromatid cohesion depends on a separate complex, Scc2(NIPBL)/Scc4(Mau2) (Scc2/4), which loads cohesin onto DNA and determines its localization across the genome. Proper cohesin loading is essential for cell division, and partial defects cause chromosome missegregation and aberrant transcriptional regulation, leading to severe developmental defects in multicellular organisms. We present here a crystal structure showing the interaction between Scc2 and Scc4. Scc4 is a TPR array that envelops an extended Scc2 peptide. Using budding yeast, we demonstrate that a conserved patch on the surface of Scc4 is required to recruit Scc2/4 to centromeres and to build pericentromeric cohesion. These findings reveal the role of Scc4 in determining the localization of cohesin loading and establish a molecular basis for Scc2/4 recruitment to centromeres.

Kinetochores, multisubunit protein assemblies, connect chromosomes to spindle microtubules to promote chromosome segregation. The 10-subunit KMN assembly (comprising KNL1, MIS12, and NDC80 complexes, designated KNL1C, MIS12C, and NDC80C) binds microtubules and regulates mitotic checkpoint function through NDC80C and KNL1C, respectively. MIS12C, on the other hand, connects the KMN to the chromosome-proximal domain of the kinetochore through a direct interaction with CENP-C. The structural basis for this crucial bridging function of MIS12C is unknown. Here, we report crystal structures of human MIS12C associated with a fragment of CENP-C and unveil the role of Aurora B kinase in the regulation of this interaction. The structure of MIS12:CENP-C complements previously determined high-resolution structures of functional regions of NDC80C and KNL1C and allows us to build a near-complete structural model of the KMN assembly. Our work illuminates the structural organization of essential chromosome segregation machinery that is conserved in most eukaryotes.

Establishment of humoral immunity against pathogens is dependent on events that occur in the germinal center and the subsequent induction of high-affinity neutralizing antibodies. Quantitative assays that allow monitoring of affinity maturation and duration of antibody responses can provide useful information regarding the efficacy of vaccines and adjuvants. Using an anthrax protective antigen (rPA) and alum model antigen/adjuvant system, we describe a methodology for monitoring antigen-specific serum antibody concentration and avidity by surface plasmon resonance during primary and secondary immune responses. Our analyses showed that following a priming dose in mice, rPA-specific antibody concentration and avidity increases over time and reaches a maximal response in about six weeks, but gradually declines in the absence of antigenic boost. Germinal center reactions were observed early with maximal development achieved during the primary response, which coincided with peak antibody avidity responses to primary immunization. Boosting with antigen resulted in a rapid increase in rPA-specific antibody concentration and five-fold increase in avidity, which was not dependent on sustained GC development. The described methodology couples surface plasmon resonance-based plasma avidity measurements with germinal center analysis and provides a novel way to monitor humoral responses that can play a role in facilitating vaccine and adjuvant development.

One of the key phenomena in the adaptive immune response to infection and immunization is affinity maturation, during which antibody genes are mutated and selected, typically resulting in a substantial increase in binding affinity to the eliciting antigen. Advances in technology on several fronts have made it possible to clone large numbers of heavy-chain light-chain pairs from individual B cells and thereby identify whole sets of clonally related antibodies. These collections could provide the information necessary to reconstruct their own history - the sequence of changes introduced into the lineage during the development of the clone - and to study affinity maturation in detail. But the success of such a program depends entirely on accurately inferring the founding ancestor and the other unobserved intermediates. Given a set of clonally related immunoglobulin V-region genes, the method described here allows one to compute the posterior distribution over their possible ancestors, thereby giving a thorough accounting of the uncertainty inherent in the reconstruction. I demonstrate the application of this method on heavy-chain and light-chain clones, assess the reliability of the inference, and discuss the sources of uncertainty.

Maternal vaccination to induce anti-HIV immune factors in breast milk is a potential intervention to prevent postnatal HIV-1 mother-to-child transmission (MTCT). We previously demonstrated that immunization of lactating rhesus monkeys with a modified vaccinia Ankara (MVA) prime/intramuscular (i.m.) protein boost regimen induced functional IgG responses in milk, while MVA prime/intranasal (i.n.) boost induced robust milk Env-specific IgA responses. Yet, recent studies have suggested that prevention of postnatal MTCT may require both Env-specific IgA and functional IgG responses in milk. Thus, to investigate whether both responses could be elicited by a combined systemic/mucosal immunization strategy, animals previously immunized with the MVA prime/i.n. boost regimen received an i.n./i.m. combined C.1086 gp120 boost. Remarkably, high-magnitude Env-specific IgA responses were observed in milk, surpassing those in plasma. Furthermore, 29% of vaccine-elicited Env-specific B cells isolated from breast milk were IgA isotype, in stark contrast to the overwhelming predominance of IgG isotype Env-specific B cells in breast milk of chronically HIV-infected women. A clonal relationship was identified between Env-specific blood and breast milk B cells, suggesting trafficking of that cell population between the two compartments. Furthermore, IgA and IgG monoclonal antibodies isolated from Env-specific breast milk B cells demonstrated diverse Env epitope specificities and multiple effector functions, including tier 1 neutralization, antibody-dependent cellular cytotoxicity (ADCC), infected cell binding, and inhibition of viral attachment to epithelial cells. Thus, maternal i.n./i.m. combined immunization is a novel strategy to enhance protective Env-specific IgA in milk, which is subsequently transferred to the infant via breastfeeding.

Germinal center (GC) B cells evolve toward increased affinity by a Darwinian process that has been studied primarily in genetically restricted, hapten-specific responses. We explored the population dynamics of genetically diverse GC responses to two complex antigens-Bacillus anthracis protective antigen and influenza hemagglutinin-in which B cells competed both intra- and interclonally for distinct epitopes. Preferred VH rearrangements among antigen-binding, naive B cells were similarly abundant in early GCs but, unlike responses to haptens, clonal diversity increased in GC B cells as early "winners" were replaced by rarer, high-affinity clones. Despite affinity maturation, inter- and intraclonal avidities varied greatly, and half of GC B cells did not bind the immunogen but nonetheless exhibited biased VH use, V(D)J mutation, and clonal expansion comparable to antigen-binding cells. GC reactions to complex antigens permit a range of specificities and affinities, with potential advantages for broad protection.

The HIV-1 gp41 envelope (Env) membrane proximal external region (MPER) is an important vaccine target that in rare subjects can elicit neutralizing antibodies. One mechanism proposed for rarity of MPER neutralizing antibody generation is lack of reverted unmutated ancestor (putative naive B cell receptor) antibody reactivity with HIV-1 envelope. We have studied the effect of partial deglycosylation under non-denaturing (native) conditions on gp140 Env antigenicity for MPER neutralizing antibodies and their reverted unmutated ancestor antibodies. We found that native deglycosylation of clade B JRFL gp140 as well as group M consensus gp140 Env CON-S selectively increased the reactivity of Env with the broad neutralizing human mAbs, 2F5 and 4E10. Whereas fully glycosylated gp140 Env either did not bind (JRFL), or weakly bound (CON-S), 2F5 and 4E10 reverted unmutated ancestors, natively deglycosylated JRFL and CON-S gp140 Envs did bind well to these putative mimics of naive B cell receptors. These data predict that partially deglycoslated Env would bind better than fully glycosylated Env to gp41-specific naïve B cells with improved immunogenicity. In this regard, immunization of rhesus macaques demonstrated enhanced immunogenicity of the 2F5 MPER epitope on deglyosylated JRFL gp140 compared to glycosylated JRFL gp140. Thus, the lack of 2F5 and 4E10 reverted unmutated ancestor binding to gp140 Env may not always be due to lack of unmutated ancestor antibody reactivity with gp41 peptide epitopes, but rather, may be due to glycan interference of binding of unmutated ancestor antibodies of broad neutralizing mAb to Env gp41.

Epitope-focused approaches for selective clonal induction of broadly neutralizing antibodies (bnAbs) inform most current vaccine strategies for influenza virus and other rapidly evolving pathogens. The two conserved epitopes on the influenza hemagglutinin (HA) - the "stem" and the receptor-binding site (RBS) on the "head" - are the focus of the current "universal" influenza vaccine development efforts. Because stem-directed serum bnAbs are much less abundant than head-directed ones, we hypothesized that the HA stem bnAbs may be autoreactive and thus eliminated through the mechanisms of self-tolerance. We compared autoreactivity profiles of a set of stem and head-directed bnAbs. Most of the stem bnAbs we examined bound autoantigens; several showed staining of HEp-2 cells. A smaller proportion of the head-directed bnAbs were polyreactive. Gene usage did not correlate with autoreactivity. We suggest that complex foreign antigens may often have surface patches resembling some host epitope; our results indicate that HA stem epitopes resemble a host epitope more frequently than does the RBS.

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.

We present a technology to screen millions of B cells for natively paired human antibody repertoires. Libraries of natively paired, variable region heavy and light (VH:VL) amplicons are expressed in a yeast display platform that is optimized for human Fab surface expression. Using our method we identify HIV-1 broadly neutralizing antibodies (bNAbs) from an HIV-1 slow progressor and high-affinity neutralizing antibodies against Ebola virus glycoprotein and influenza hemagglutinin.

HIV-1 envelope (Env) mimetics are candidate components of prophylactic vaccines and potential therapeutics. Here we use a synthetic V3-glycopeptide ("Man9-V3") for structural studies of an HIV Env third variable loop (V3)-glycan directed, broadly neutralizing antibody (bnAb) lineage ("DH270"), to visualize the epitope on Env and to study how affinity maturation of the lineage proceeded. Unlike many previous V3 mimetics, Man9-V3 encompasses two key features of the V3 region recognized by V3-glycan bnAbs-the conserved GDIR motif and the N332 glycan. In our structure of an antibody fragment of a lineage member, DH270.6, in complex with the V3 glycopeptide, the conformation of the antibody-bound glycopeptide conforms closely to that of the corresponding segment in an intact HIV-1 Env trimer. An additional structure identifies roles for two critical mutations in the development of breadth. The results suggest a strategy for use of a V3 glycopeptide as a vaccine immunogen.

A paradigm shifting study demonstrated that induction of MHC class E and II-restricted CD8+ T cells was associated with the clearance of SIV infection in rhesus macaques. Another recent study highlighted the presence of HIV-1-specific class II-restricted CD8+ T cells in HIV-1 patients who naturally control infection (virus controllers; VCs). However, questions regarding class II-restricted CD8+ T cells ontogeny, distribution across different HIV-1 disease states and their role in viral control remain unclear. In this study, we investigated the distribution and anti-viral properties of HLA-DRB1*0701 and DQB1*0501 class II-restricted CD8+ T cells in different HIV-1 patient cohorts; and whether class II-restricted CD8+ T cells represent a unique T cell subset. We show that memory class II-restricted CD8+ T cell responses were more often detectable in VCs than in chronically infected patients, but not in healthy seronegative donors. We also demonstrate that VC CD8+ T cells inhibit virus replication in both a class I- and class II-dependent manner, and that in two VC patients the class II-restricted CD8+ T cells with an anti-viral gene signature expressed both CD4+ and CD8+ T cell lineage-specific genes. These data demonstrated that anti-viral memory class II-restricted CD8+ T cells with hybrid CD4+ and CD8+ features are present during natural HIV-1 infection.

Structure-based vaccine design depends on extensive structural analyses of antigen-antibody complexes.Single-particle electron cryomicroscopy (cryoEM) can circumvent some of the problems of x-ray crystallography as a pipeline for obtaining the required structures. We have examined the potential of single-particle cryoEM for determining the structure of influenza-virus hemagglutinin (HA):single-chain variable-domain fragment complexes, by studying a complex we failed to crystallize in pursuing an extended project on the human immune response to influenza vaccines.The result shows that a combination of cryoEM and molecular modeling can yield details of the antigen-antibody interface, although small variation in the twist of the rod-likeHA trimer limited the overall resolution to about 4.5Å.Comparison of principal 3D classes suggests ways to modify the HA trimer to overcome this limitation. A closely related antibody from the same donor did yield crystals when bound with the same HA, giving us an independent validation of the cryoEM results.The two structures also augment our understanding of receptor-binding site recognition by antibodies that neutralize a wide range of influenza-virus variants.

Antibodies are key immune effectors that confer protection against pathogenic threats. The nature and longevity of the antibody response to SARS-CoV-2 infection are not well defined. We charted longitudinal antibody responses to SARS-CoV-2 in 92 subjects after symptomatic COVID-19. Antibody responses to SARS-CoV-2 are unimodally distributed over a broad range, with symptom severity correlating directly with virus-specific antibody magnitude. Seventy-six subjects followed longitudinally to ∼100 days demonstrated marked heterogeneity in antibody duration dynamics. Virus-specific IgG decayed substantially in most individuals, whereas a distinct subset had stable or increasing antibody levels in the same time frame despite similar initial antibody magnitudes. These individuals with increasing responses recovered rapidly from symptomatic COVID-19 disease, harbored increased somatic mutations in virus-specific memory B cell antibody genes, and had persistent higher frequencies of previously activated CD4+ T cells. These findings illuminate an efficient immune phenotype that connects symptom clearance speed to differential antibody durability dynamics.

Rab11 recycling endosomes are involved in immunological synaptic functions, but the roles of Rab11 family-interacting protein 5 (Rab11Fip5), one of the Rab11 effectors, in the immune system remain obscure. Our previous study demonstrated that RAB11FIP5 transcripts are significantly elevated in PBMCs from HIV-1-infected individuals, making broadly HIV-1-neutralizing Abs compared with those without broadly neutralizing Abs; however, the role of Rab11FiP5 in immune functions remains unclear. In this study, a RAB11FIP5 gene knockout (RAB11FIP5 -/-) mouse model was employed to study the role of Rab11Fip5 in immune responses. RAB11FIP5 -/- mice exhibited no perturbation in lymphoid tissue cell subsets, and Rab11Fip5 was not required for serum Ab induction following HIV-1 envelope immunization, Ab transcytosis to mucosal sites, or survival after influenza challenge. However, differences were observed in multiple transcripts, including cytokine genes, in lymphocyte subsets from envelope-immunized RAB11FIP5 -/- versus control mice. These included alterations in several genes in NK cells that mirrored observations in NKs from HIV-infected humans expressing less RAB11FIP5, although Rab11Fip5 was dispensable for NK cell cytolytic activity. Notably, immunized RAB11FIP5 -/- mice had lower IL4 expression in CD4+ T follicular helper cells and showed lower TNF expression in CD8+ T cells. Likewise, TNF-α production by human CD8+ T cells correlated with PBMC RAB11FIP5 expression. These observations in RAB11FIP5 -/- mice suggest a role for Rab11Fip5 in regulating cytokine responses.

The global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made the development of a vaccine a top biomedical priority. In this study, we developed a series of DNA vaccine candidates expressing different forms of the SARS-CoV-2 spike (S) protein and evaluated them in 35 rhesus macaques. Vaccinated animals developed humoral and cellular immune responses, including neutralizing antibody titers at levels comparable to those found in convalescent humans and macaques infected with SARS-CoV-2. After vaccination, all animals were challenged with SARS-CoV-2, and the vaccine encoding the full-length S protein resulted in >3.1 and >3.7 log10 reductions in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, as compared with viral loads in sham controls. Vaccine-elicited neutralizing antibody titers correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate vaccine protection against SARS-CoV-2 in nonhuman primates.

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.

Eliciting protective titers of HIV-1 broadly neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development, but current vaccine strategies have yet to induce bnAbs in humans. Many bnAbs isolated from HIV-1-infected individuals are encoded by immunoglobulin gene rearrangments with infrequent naive B cell precursors and with unusual genetic features that may be subject to host regulatory control. Here, we administer antibodies targeting immune cell regulatory receptors CTLA-4, PD-1 or OX40 along with HIV envelope (Env) vaccines to rhesus macaques and bnAb immunoglobulin knock-in (KI) mice expressing diverse precursors of CD4 binding site HIV-1 bnAbs. CTLA-4 blockade augments HIV-1 Env antibody responses in macaques, and in a bnAb-precursor mouse model, CTLA-4 blocking or OX40 agonist antibodies increase germinal center B and T follicular helper cells and plasma neutralizing antibodies. Thus, modulation of CTLA-4 or OX40 immune checkpoints during vaccination can promote germinal center activity and enhance HIV-1 Env antibody responses.