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.

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.

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.

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.

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.

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.

With the advent of DNA hybridization microarrays comes the remarkable ability, in principle, to simultaneously monitor the expression levels of thousands of genes. The quantitative comparison of two or more microarrays can reveal, for example, the distinct patterns of gene expression that define different cellular phenotypes or the genes induced in the cellular response to insult or changing environmental conditions. Normalization of the measured intensities is a prerequisite of such comparisons, and indeed, of any statistical analysis, yet insufficient attention has been paid to its systematic study. The most straightforward normalization techniques in use rest on the implicit assumption of linear response between true expression level and output intensity. We find that these assumptions are not generally met, and that these simple methods can be improved.

Development of strategies for induction of HIV-1 broadly neutralizing antibodies (bnAbs) by vaccines is a priority. Determining the steps of bnAb induction in HIV-1-infected individuals who make bnAbs is a key strategy for immunogen design. Here, we study the B cell response in a bnAb-producing individual and report cooperation between two B cell lineages to drive bnAb development. We isolated a virus-neutralizing antibody lineage that targeted an envelope region (loop D) and selected virus escape mutants that resulted in both enhanced bnAb lineage envelope binding and escape mutant neutralization-traits associated with increased B cell antigen drive. Thus, in this individual, two B cell lineages cooperated to induce the development of bnAbs. Design of vaccine immunogens that simultaneously drive both helper and broadly neutralizing B cell lineages may be important for vaccine-induced recapitulation of events that transpire during the maturation of neutralizing antibodies in HIV-1-infected individuals.

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.

Human monoclonal antibody CH58 isolated from an RV144 vaccinee binds at Lys169 of the HIV-1 Env gp120 V2 region, a site of vaccine-induced immune pressure. CH58 neutralizes HIV-1 CRF_01 AE strain 92TH023 and mediates ADCC against CD4 + T cell targets infected with CRF_01 AE tier 2 virus. CH58 and other antibodies that bind to a gp120 V2 epitope have a second light chain complementarity determining region (LCDR2) bearing a glutamic acid, aspartic acid (ED) motif involved in forming salt bridges with polar, basic side amino acid side chains in V2. In an effort to learn how V2 responses develop, we determined the crystal structures of the CH58-UA antibody unliganded and bound to V2 peptide. The structures showed an LCDR2 structurally pre-conformed from germline to interact with V2 residue Lys169. LCDR3 was subject to conformational selection through the affinity maturation process. Kinetic analyses demonstrate that only a few contacts were responsible for a 2000-fold increase in KD through maturation, and this effect was predominantly due to an improvement in off-rate. This study shows that preconformation and preconfiguration can work in concert to produce antibodies with desired immunogenic properties.

Developing predictive animal models to assess how candidate vaccines and infection influence the ontogenies of Envelope (Env)-specific antibodies is critical for the development of an HIV vaccine. Here we use two nonhuman primate models to compare the roles of antigen persistence, diversity and innate immunity. We perform longitudinal analyses of HIV Env-specific B-cell receptor responses to SHIV(AD8) infection and Env protein vaccination with eight different adjuvants. A subset of the SHIV(AD8)-infected animals with higher viral loads and greater Env diversity show increased neutralization associated with increasing somatic hypermutation (SHM) levels over time. The use of adjuvants results in increased ELISA titres but does not affect the mean SHM levels or CDR H3 lengths. Our study shows how the ontogeny of Env-specific B cells can be tracked, and provides insights into the requirements for developing neutralizing antibodies that should facilitate translation to human vaccine studies.

Affinity maturation, the process in which somatic hypermutation and positive selection generate antibodies with increasing affinity for an antigen, is pivotal in acquired humoral immunity. We have studied the mechanism of affinity gain in a human B-cell lineage in which two main maturation pathways, diverging from a common ancestor, lead to three mature antibodies that neutralize a broad range of H1 influenza viruses. Previous work showed that increased affinity in the mature antibodies derives primarily from stabilization of the CDR H3 loop in the antigen-binding conformation. We have now used molecular dynamics simulations and existing crystal structures to identify potentially key maturation mutations, and we have characterized their effects on the CDR H3 loop and on antigen binding using further simulations and experimental affinity measurements, respectively. In the two maturation pathways, different contacts between light and heavy chains stabilize the CDR H3 loop. As few as two single-site mutations in each pathway can confer substantial loop stability, but none of them confers experimentally detectable stability on its own. Our results support models of the germinal center reaction in which two or more mutations can occur without concomitant selection and show how divergent pathways have yielded functionally equivalent antibodies.

Most HIV-1-specific neutralizing antibodies isolated to date exhibit unusual characteristics that complicate their elicitation. Neutralizing antibodies that target the V1V2 apex of the HIV-1 envelope (Env) trimer feature unusually long protruding loops, which enable them to penetrate the HIV-1 glycan shield. As antibodies with loops of requisite length are created through uncommon recombination events, an alternative mode of apex binding has been sought. Here, we isolated a lineage of Env apex-directed neutralizing antibodies, N90-VRC38.01-11, by using virus-like particles and conformationally stabilized Env trimers as B cell probes. A crystal structure of N90-VRC38.01 with a scaffolded V1V2 revealed a binding mode involving side-chain-to-side-chain interactions that reduced the distance the antibody loop must traverse the glycan shield, thereby facilitating V1V2 binding via a non-protruding loop. The N90-VRC38 lineage thus identifies a solution for V1V2-apex binding that provides a more conventional B cell pathway for vaccine design.

During the recent H1N1 influenza pandemic, excess morbidity and mortality was seen in young but not older adults suggesting that prior infection with influenza strains may have protected older subjects. In contrast, a history of recent seasonal trivalent vaccine in younger adults was not associated with protection.

Antibodies that neutralize autologous transmitted/founder (TF) HIV occur in most HIV-infected individuals and can evolve to neutralization breadth. Autologous neutralizing antibodies (nAbs) against neutralization-resistant (Tier-2) viruses are rarely induced by vaccination. Whereas broadly neutralizing antibody (bnAb)-HIV-Envelope structures have been defined, the structures of autologous nAbs have not. Here, we show that immunization with TF mutant Envs gp140 oligomers induced high-titer, V5-dependent plasma neutralization for a Tier-2 autologous TF evolved mutant virus. Structural analysis of autologous nAb DH427 revealed binding to V5, demonstrating the source of narrow nAb specificity and explaining the failure to acquire breadth. Thus, oligomeric TF Envs can elicit autologous nAbs to Tier-2 HIVs, but induction of bnAbs will require targeting of precursors of B cell lineages that can mature to heterologous neutralization.

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.

Broadly neutralizing antibodies are not commonly produced in HIV-1 infected individuals nor by experimental HIV-1 vaccines. When these antibodies do occur, it is important to be able to isolate and characterize them to provide clues for vaccine design. CAP206 is a South African subtype C HIV-1-infected individual previously shown to have broadly neutralizing plasma antibodies targeting the envelope gp41 distal membrane proximal external region (MPER). We have now used a fluoresceinated peptide tetramer antigen with specific cell sorting to isolate a human neutralizing monoclonal antibody (mAb) against the HIV-1 envelope gp41 MPER. The isolated recombinant mAb, CAP206-CH12, utilized a portion of the distal MPER (HXB2 amino acid residues, 673-680) and neutralized a subset of HIV-1 pseudoviruses sensitive to CAP206 plasma antibodies. Interestingly, this mAb was polyreactive and used the same germ-line variable heavy (V(H)1-69) and variable kappa light chain (V(K)3-20) gene families as the prototype broadly neutralizing anti-MPER mAb, 4E10 (residues 672-680). These data indicate that there are multiple immunogenic targets in the C-terminus of the MPER of HIV-1 gp41 envelope and suggests that gp41 neutralizing epitopes may interact with a restricted set of naive B cells during HIV-1 infection.

In HIV-1, the ability to mount antibody responses to conserved, neutralizing epitopes is critical for protection. Here we have studied the light chain usage of human and rhesus macaque antibodies targeted to a dominant region of the HIV-1 envelope second variable (V2) region involving lysine (K) 169, the site of immune pressure in the RV144 vaccine efficacy trial. We found that humans and rhesus macaques used orthologous lambda variable gene segments encoding a glutamic acid-aspartic acid (ED) motif for K169 recognition. Structure determination of an unmutated ancestor antibody demonstrated that the V2 binding site was preconfigured for ED motif-mediated recognition prior to maturation. Thus, light chain usage for recognition of the site of immune pressure in the RV144 trial is highly conserved across species. These data indicate that the HIV-1 K169-recognizing ED motif has persisted over the diversification between rhesus macaques and humans, suggesting an evolutionary advantage of this antibody recognition mode.

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.