Slow Delivery Immunization Enhances HIV Neutralizing Antibody and Germinal Center Responses via Modulation of Immunodominance.

Conventional immunization strategies will likely be insufficient for the development of a broadly neutralizing antibody (bnAb) vaccine for HIV or other difficult pathogens because of the immunological hurdles posed, including B cell immunodominance and germinal center (GC) quantity and quality. We found that two independent methods of slow delivery immunization of rhesus monkeys (RMs) resulted in more robust T follicular helper (TFH) cell responses and GC B cells with improved Env-binding, tracked by longitudinal fine needle aspirates. Improved GCs correlated with the development of >20-fold higher titers of autologous nAbs. Using a new RM genomic immunoglobulin locus reference, we identified differential IgV gene use between immunization modalities. Ab mapping demonstrated targeting of immunodominant non-neutralizing epitopes by conventional bolus-immunized animals, whereas slow delivery-immunized animals targeted a more diverse set of epitopes. Thus, alternative immunization strategies can enhance nAb development by altering GCs and modulating the immunodominance of non-neutralizing epitopes.

Antibodies from primary humoral responses modulate the recruitment of naive B cells during secondary responses.

Vaccines generate high-affinity antibodies by recruiting antigen-specific B cells to germinal centers (GCs), but the mechanisms governing the recruitment to GCs on secondary challenges remain unclear. Here, using preclinical SARS-CoV and HIV mouse models, we demonstrated that the antibodies elicited during primary humoral responses shaped the naive B cell recruitment to GCs during secondary exposures. The antibodies from primary responses could either enhance or, conversely, restrict the GC participation of naive B cells: broad-binding, low-affinity, and low-titer antibodies enhanced recruitment, whereas, by contrast, the high titers of high-affinity, mono-epitope-specific antibodies attenuated cognate naive B cell recruitment. Thus, the directionality and intensity of that effect was determined by antibody concentration, affinity, and epitope specificity. Circulating antibodies can, therefore, be important determinants of antigen immunogenicity. Future vaccines may need to overcome-or could, alternatively, leverage-the effects of circulating primary antibodies on subsequent naive B cell recruitment.

Vaccine-Induced Protection from Homologous Tier 2 SHIV Challenge in Nonhuman Primates Depends on Serum-Neutralizing Antibody Titers.

Passive administration of HIV neutralizing antibodies (nAbs) can protect macaques from hard-to-neutralize (tier 2) chimeric simian-human immunodeficiency virus (SHIV) challenge. However, conditions for nAb-mediated protection after vaccination have not been established. Here, we selected groups of 6 rhesus macaques with either high or low serum nAb titers from a total of 78 animals immunized with recombinant native-like (SOSIP) Env trimers. Repeat intrarectal challenge with homologous tier 2 SHIVBG505 led to rapid infection in unimmunized and low-titer animals. High-titer animals, however, demonstrated protection that was gradually lost as nAb titers waned over time. An autologous serum ID50 nAb titer of ∼1:500 afforded more than 90% protection from medium-dose SHIV infection. In contrast, antibody-dependent cellular cytotoxicity and T cell activity did not correlate with protection. Therefore, Env protein-based vaccination strategies can protect against hard-to-neutralize SHIV challenge in rhesus macaques by inducing tier 2 nAbs, provided appropriate neutralizing titers can be reached and maintained.

Electron-Microscopy-Based Epitope Mapping Defines Specificities of Polyclonal Antibodies Elicited during HIV-1 BG505 Envelope Trimer Immunization.

Characterizing polyclonal antibody responses via currently available methods is inherently complex and difficult. Mapping epitopes in an immune response is typically incomplete, which creates a barrier to fully understanding the humoral response to antigens and hinders rational vaccine design efforts. Here, we describe a method of characterizing polyclonal responses by using electron microscopy, and we applied this method to the immunization of rabbits with an HIV-1 envelope glycoprotein vaccine candidate, BG505 SOSIP.664. We detected known epitopes within the polyclonal sera and revealed how antibody responses evolved during the prime-boosting strategy to ultimately result in a neutralizing antibody response. We uncovered previously unidentified epitopes, including an epitope proximal to one recognized by human broadly neutralizing antibodies as well as potentially distracting non-neutralizing epitopes. Our method provides an efficient and semiquantitative map of epitopes that are targeted in a polyclonal antibody response and should be of widespread utility in vaccine and infection studies.

Mapping the immunogenic landscape of near-native HIV-1 envelope trimers in non-human primates.

The induction of broad and potent immunity by vaccines is the key focus of research efforts aimed at protecting against HIV-1 infection. Soluble native-like HIV-1 envelope glycoproteins have shown promise as vaccine candidates as they can induce potent autologous neutralizing responses in rabbits and non-human primates. In this study, monoclonal antibodies were isolated and characterized from rhesus macaques immunized with the BG505 SOSIP.664 trimer to better understand vaccine-induced antibody responses. Our studies reveal a diverse landscape of antibodies recognizing immunodominant strain-specific epitopes and non-neutralizing neo-epitopes. Additionally, we isolated a subset of mAbs against an epitope cluster at the gp120-gp41 interface that recognize the highly conserved fusion peptide and the glycan at position 88 and have characteristics akin to several human-derived broadly neutralizing antibodies.

Neutralizing Antibody Induction by HIV-1 Envelope Glycoprotein SOSIP Trimers on Iron Oxide Nanoparticles May Be Impaired by Mannose Binding Lectin.

We covalently attached human immunodeficiency virus type 1 (HIV-1) Env SOSIP trimers to iron oxide nanoparticles (IO-NPs) to create a particulate immunogen for neutralizing antibody (NAb) induction. The attached trimers, ∼20 per particle, retained native-like antigenicity, judged by reactivity with NAbs and non-NAbs. Bivalent (BG505 and B41) trimer IO-NPs were made, as were IO-NPs displaying B41 trimers carrying a PADRE T-cell helper epitope (TCHE). We immunized mice with B41 soluble or IO-NP trimers after PADRE peptide priming. After two immunizations, IO-NP presentation and the TCHE tag independently and substantially increased anti-trimer antibody responses, but titer differences waned after two further doses. Notable and unexpected findings were that autologous NAbs to the N289 glycan hole epitope were consistently induced in mice given soluble but not IO-NP trimers. Various recombinant mannose binding lectins (MBLs) and MBLs in sera of both murine and human origin bound to soluble and IO-NP trimers. MBL binding occluded the autologous NAb epitope on the B41 IO-NP trimers, which may contribute to its poor immunogenicity. The exposure of a subset of broadly active NAb epitopes was also impaired by MBL binding, which could have substantial implications for the utility of trimer-bearing nanoparticles in general and perhaps also for soluble Env proteins.IMPORTANCE Recombinant trimeric SOSIP proteins are vaccine components intended to induce neutralizing antibodies (NAbs) that prevent cells from infection by human immunodeficiency virus type 1 (HIV-1). A way to increase the strength of antibody responses to these proteins is to present them on the surface of nanoparticles (NPs). We chemically attached about 20 SOSIP trimers to NPs made of iron oxide (IO). The resulting IO-NP trimers had appropriate properties when we studied them in the laboratory but, unexpectedly, were less able to induce NAbs than nonattached trimers when used to immunize mice. We found that mannose binding lectins, proteins naturally present in the serum of mice and other animals, bound strongly to the soluble and IO-NP trimers, blocking access to antibody epitopes in a way that may impede the development of NAb responses. These findings should influence how trimer-bearing NPs of various designs are made and used.

Time-course, negative-stain electron microscopy-based analysis for investigating protein-protein interactions at the single-molecule level.

Several biophysical approaches are available to study protein-protein interactions. Most approaches are conducted in bulk solution, and are therefore limited to an average measurement of the ensemble of molecular interactions. Here, we show how single-particle EM can enrich our understanding of protein-protein interactions at the single-molecule level and potentially capture states that are unobservable with ensemble methods because they are below the limit of detection or not conducted on an appropriate time scale. Using the HIV-1 envelope glycoprotein (Env) and its interaction with receptor CD4-binding site neutralizing antibodies as a model system, we both corroborate ensemble kinetics-derived parameters and demonstrate how time-course EM can further dissect stoichiometric states of complexes that are not readily observable with other methods. Visualization of the kinetics and stoichiometry of Env-antibody complexes demonstrated the applicability of our approach to qualitatively and semi-quantitatively differentiate two highly similar neutralizing antibodies. Furthermore, implementation of machine-learning techniques for sorting class averages of these complexes into discrete subclasses of particles helped reduce human bias. Our data provide proof of concept that single-particle EM can be used to generate a "visual" kinetic profile that should be amenable to studying many other protein-protein interactions, is relatively simple and complementary to well-established biophysical approaches. Moreover, our method provides critical insights into broadly neutralizing antibody recognition of Env, which may inform vaccine immunogen design and immunotherapeutic development.

Priming antibody responses to the fusion peptide in rhesus macaques.

Immunodominance of antibodies targeting non-neutralizing epitopes and the high level of somatic hypermutation within germinal centers (GCs) required for most HIV broadly neutralizing antibodies (bnAbs) are major impediments to the development of an effective HIV vaccine. Rational protein vaccine design and non-conventional immunization strategies are potential avenues to overcome these hurdles. Here, we report using implantable osmotic pumps to continuously deliver a series of epitope-targeted immunogens to rhesus macaques over the course of six months to prime and elicit antibody responses against the conserved fusion peptide (FP). GC responses and antibody specificities were tracked longitudinally using lymph node fine-needle aspirates and electron microscopy polyclonal epitope mapping (EMPEM), respectively, to show antibody responses to the FP/N611 glycan hole region were primed, although exhibited limited neutralization breadth. Application of cryoEMPEM delineated key residues for on-target and off-target responses that can drive the next round of structure-based vaccine design.

Engineering upper hinge improves stability and effector function of a human IgG1.

Upper hinge is vulnerable to radical attacks that result in breakage of the heavy-light chain linkage and cleavage of the hinge of an IgG1. To further explore mechanisms responsible for the radical induced hinge degradation, nine mutants were designed to determine the roles that the upper hinge Asp and His play in the radical reactions. The observation that none of these substitutions could inhibit the breakage of the heavy-light chain linkage suggests that the breakage may result from electron transfer from Cys(231) directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His(229)-mediated hinge cleavage. On the other hand, the substitution of His(229) with Tyr showed promising advantages over the native antibody and other substitutions in improving the stability and function of the IgG1. This substitution inhibited the hinge cleavage by 98% and suggests that the redox active nature of Tyr did not enable it to replicate the ability of His to facilitate radical induced degradation. We propose that the lower redox potential of Tyr, a residue that may be the ultimate sink for oxidizing equivalents in proteins, is responsible for the inhibition. More importantly, the substitution increased the antibody's binding to FcγRIII receptors by 2-3-fold, and improved ADCC activity by 2-fold, while maintaining a similar pharmacokinetic profile with respect to the wild type. Implications of these observations for antibody engineering and development are discussed.

Dose response of running on blood biomarkers of wellness in generally healthy individuals.

Exercise is effective toward delaying or preventing chronic disease, with a large body of evidence supporting its effectiveness. However, less is known about the specific healthspan-promoting effects of exercise on blood biomarkers in the disease-free population. In this work, we examine 23,237 generally healthy individuals who self-report varying weekly running volumes and compare them to 4,428 generally healthy sedentary individuals, as well as 82 professional endurance runners. We estimate the significance of differences among blood biomarkers for groups of increasing running levels using analysis of variance (ANOVA), adjusting for age, gender, and BMI. We attempt and add insight to our observational dataset analysis via two-sample Mendelian randomization (2S-MR) using large independent datasets. We find that self-reported running volume associates with biomarker signatures of improved wellness, with some serum markers apparently being principally modified by BMI, whereas others show a dose-effect with respect to running volume. We further detect hints of sexually dimorphic serum responses in oxygen transport and hormonal traits, and we also observe a tendency toward pronounced modifications in magnesium status in professional endurance athletes. Thus, our results further characterize blood biomarkers of exercise and metabolic health, particularly regarding dose-effect relationships, and better inform personalized advice for training and performance.

Focusing antibody responses to the fusion peptide in rhesus macaques.

Immunodominance of antibodies targeting non-neutralizing epitopes and the high level of somatic hypermutation within germinal centers (GCs) required for most HIV broadly neutralizing antibodies (bnAbs) are major impediments to the development of an effective HIV vaccine. Rational protein vaccine design and non-conventional immunization strategies are potential avenues to overcome these hurdles. Here, we report using implantable osmotic pumps to continuously deliver a series of epitope-targeted immunogens to rhesus macaques over the course of six months to elicit immune responses against the conserved fusion peptide. Antibody specificities and GC responses were tracked longitudinally using electron microscopy polyclonal epitope mapping (EMPEM) and lymph node fine-needle aspirates, respectively. Application of cryoEMPEM delineated key residues for on-target and off-target responses that can drive the next round of structure-based vaccine design.

From structure to sequence: Antibody discovery using cryoEM.

One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions, and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next-generation sequencing of immune repertoires, we were able to specifically identify clonal family members, synthesize the monoclonal antibodies, and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design.

Gut Microbiota-Informed Precision Nutrition in the Generally Healthy Individual: Are We There Yet?

Since next generation sequencing facilitated high-throughput and cost-efficient genomics analyses, the human gut metagenome has become an emerging frontier to explore toward precision nutrition. Significant progress has been made in identifying gut microbial features associated with a wide spectrum of human disease. However, other than a few microbiome-disease relations, there is a dearth of confirmed causal inferences, particularly in generally healthy populations. The relatively high unexplained variability in microbiome compositions in this group warrants caution in applying this complex biomarker toward precision nutrition, because our understanding of what constitutes a healthy microbiome is still rudimentary. Although gut microbiota harbor integrated environmental and host-specific information with the potential to facilitate personalized nutritional and lifestyle advice, these data cannot yet be confidently interpreted toward precise recommendations. Thus, nutritional advice for generally healthy individuals based on personal microbiome composition analysis might not yet be appropriate unless accompanied by established blood and physiological biomarkers.

Mapping Polyclonal Antibody Responses in Non-human Primates Vaccinated with HIV Env Trimer Subunit Vaccines.

Rational immunogen design aims to focus antibody responses to vulnerable sites on primary antigens. Given the size of these antigens, there is, however, potential for eliciting unwanted, off-target responses. Here, we use our electron microscopy polyclonal epitope mapping approach to describe the antibody specificities elicited by immunization of non-human primates with soluble HIV envelope trimers and subsequent repeated viral challenge. An increased diversity of epitopes recognized and the approach angle by which these antibodies bind constitute a hallmark of the humoral response in most protected animals. We also show that fusion peptide-specific antibodies are likely responsible for some neutralization breadth. Moreover, cryoelectron microscopy (cryo-EM) analysis of a fully protected animal reveals a high degree of clonality within a subset of putatively neutralizing antibodies, enabling a detailed molecular description of the antibody paratope. Our results provide important insights into the immune response against a vaccine candidate that entered into clinical trials in 2019.

Mapping Neutralizing Antibody Epitope Specificities to an HIV Env Trimer in Immunized and in Infected Rhesus Macaques.

BG505 SOSIP is a well-characterized near-native recombinant HIV Envelope (Env) trimer that holds promise as part of a sequential HIV immunogen regimen to induce broadly neutralizing antibodies (bnAbs). Rhesus macaques are considered the most appropriate pre-clinical animal model for monitoring antibody (Ab) responses. Accordingly, we report here the isolation of 45 BG505 autologous neutralizing antibodies (nAbs) with multiple specificities from SOSIP-immunized and BG505 SHIV-infected rhesus macaques. We associate the most potent neutralization with two epitopes: the C3/V5 and V1/V3 regions. We show that all of the nAbs bind in close proximity to known bnAb epitopes and might therefore sterically hinder elicitation of bnAbs. We also identify a "public clonotype" that targets the immunodominant C3/V5 nAb epitope, which suggests that common antibody rearrangements might help determine humoral responses to Env immunogens. The results highlight important considerations for vaccine design in anticipation of results of the BG505 SOSIP trimer in clinical trials.

HIV envelope trimer-elicited autologous neutralizing antibodies bind a region overlapping the N332 glycan supersite.

To date, immunization studies of rabbits with the BG505 SOSIP.664 HIV envelope glycoprotein trimers have revealed the 241/289 glycan hole as the dominant neutralizing antibody epitope. Here, we isolated monoclonal antibodies from a rabbit that did not exhibit glycan hole-dependent autologous serum neutralization. The antibodies did not compete with a previously isolated glycan hole-specific antibody but did compete with N332 glycan supersite broadly neutralizing antibodies. A 3.5-Å cryoEM structure of one of the antibodies in complex with the BG505 SOSIP.v5.2 trimer demonstrated that while the epitope recognized overlapped the N332 glycan supersite by contacting the GDIR motif at the base of V3, primary contacts were located in the variable V1 loop. These data suggest that strain-specific responses to V1 may interfere with broadly neutralizing responses to the N332 glycan supersite and vaccine immunogens may require engineering to minimize these off-target responses or steer them toward a more desirable pathway.

Longitudinal analysis of biomarker data from a personalized nutrition platform in healthy subjects.

The trend toward personalized approaches to health and medicine has resulted in a need to collect high-dimensional datasets on individuals from a wide variety of populations, in order to generate customized intervention strategies. However, it is not always clear whether insights derived from studies in patient populations or in controlled trial settings are transferable to individuals in the general population. To address this issue, a longitudinal analysis was conducted on blood biomarker data from 1032 generally healthy individuals who used an automated, web-based personalized nutrition and lifestyle platform. The study had two main aims: to analyze correlations between biomarkers for biological insights, and to characterize the effectiveness of the platform in improving biomarker levels. First, a biomarker correlation network was constructed to generate biological hypotheses that are relevant to researchers and, potentially, to users of personalized wellness tools. The correlation network revealed expected patterns, such as the established relationships between blood lipid levels, as well as novel insights, such as a connection between neutrophil and triglyceride concentrations that has been suggested as a relevant indicator of cardiovascular risk. Next, biomarker changes during platform use were assessed, showing a trend toward normalcy for most biomarkers in those participants whose values were out of the clinically normal range at baseline. Finally, associations were found between the selection of specific interventions and corresponding biomarker changes, suggesting directions for future study.

Publisher Correction: Longitudinal analysis of biomarker data from a personalized nutrition platform in healthy subjects.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Select host cell proteins coelute with monoclonal antibodies in protein A chromatography.

The most significant factor contributing to the presence of host cell protein (HCP) impurities in Protein A chromatography eluates is their association with the product monoclonal antibodies (mAbs) has been reported previously, and it has been suggested that more efficacious column washes may be developed by targeting the disruption of the mAbs-HCP interaction. However, characterization of this interaction is not straight forward as it is likely to involve multiple proteins and/or types of interaction. This work is an attempt to begin to understand the contribution of HCP subpopulations and/or mAb interaction propensity to the variability in HCP levels in the Protein A eluate. We performed a flowthrough (FT) recycling study with product respiking using two antibody molecules of apparently different HCP interaction propensities. In each case, the ELISA assay showed depletion of select subpopulations of HCP in Protein A eluates in subsequent column runs, while the feedstock HCP in the FTs remained unchanged from its native harvested cell culture fluid (HCCF) levels. In a separate study, the final FT from each molecule's recycling study was cross-spiked with various mAbs. In this case, Protein A eluate levels remained low for all but two molecules which were known as having high apparent HCP interaction propensity. The results of these studies suggest that mAbs may preferentially bind to select subsets of HCPs, and the degree of interaction and/or identity of the associated HCPs may vary depending on the mAb.