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.
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•Slow delivery immunization enhances HIV neutralizing antibody development in monkeys•Slow delivery immunization alters immunodominance of the responding B cells•Weekly longitudinal germinal center (GC) B and TFH analyses provides new GC insights•High-resolution rhesus immunoglobulin locus genomic reference sequence
An integrated immunological, bioinformatic and imaging approach demonstrates how slow delivery immunization enhances neutralizing antibody and germinal center reactions over conventional strategies in response to HIV Env protein immunization in non-human primates.
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.
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.
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•Electron microscopy polyclonal epitope mapping of immunized and challenged macaques•Diversity of epitopes and specific angles of approach are hallmarks of protection•Neutralization breadth is due in part to fusion peptide-specific antibodies•Cryo-EM analysis of a protected animal details a putatively neutralizing paratope
Nogal et al. use electron microscopy polyclonal epitope mapping of BG505 Env-immunized and matched SHIVBG505-challenged non-human primates to identify hallmarks of protection. Additionally, cryo-EM polyclonal analysis of a fully protected animal reveals a high degree of clonality, allowing detailed characterization of a putative neutralizing paratope.
We report a case study of an IgG1 with a unique basic charge variant profile caused by C-terminal proline amidation on either one or two heavy chains. The proline amidation was sensitive to copper ...ion concentration in the production media during cell culture: the higher the Cu
2+
ion concentration, the higher the level of proline amidation detected. This conclusion was supported by the analysis of samples that revealed direct correlation between the proline amidation level observed from peptide maps and the level of basic peaks measured by imaged capillary isoelectric focusing and a pH gradient ion-exchange chromatography method. The importance of these observations to therapeutic antibody production is discussed.
Environmental heat stress creates a detriment to the welfare and performance in broiler chickens. While there are some dietary mineral and vitamin supplements that mitigate this condition, a rapid, ...plasma-based detection method would improve management response and broaden the scientific understanding of heat stress. A total of 960 broilers were used to determine the effect of heat stress and dietary electrolyte balance on blood biochemistry. Sex sorted chicks were allocated to 48 pens with 20 chicks per pen creating 6 treatments (3 diets x 2 house environments) with eight replicates and fed one of three dietary treatments: a control containing primarily sodium chloride (NaCl), a heat stress formulation containing bicarbonate (NaHCO
3
), or heat stress fortified with 200 ppm vitamin C and E (NaHCO
3
Fortified). Birds were housed in two different temperature-controlled environments either a thermoneutral (Control) or heat stressed (Heat Stress) environment. At day 28, 35 and 42 venous blood was collected and analyzed using rapid detection methods followed by post-mortem veterinary evaluations. Performance was measured at weekly intervals. Mortality was significantly higher in broilers exposed to heat stress as compared to thermoneutral, while broilers that received dietary sodium chloride also had higher mortality than bicarbonate fed birds. Heat stress significantly impacted potassium, hematocrit, uric acid, total protein, globulin, hematocrit, lymphocytes, sodium, and glucose. This study demonstrates that blood biochemistry of broiler chickens is influenced by dietary intervention and changing environmental conditions. This pattern suggests a blood biomarker footprint of sub-optimal nutrition or poor environmental conditions that may provide valuable information into physiological changes in response to dietary electrolytes, vitamins, and heat stress. Furthermore, this footprint may potentiate the development of diagnostic tools, combining biomarkers to determine nutrition and health status of individual broiler flocks, for nutritionists, veterinarians, and live production managers to manage flocks for environmental, humane, and productive purposes.
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.
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•Mapped polyclonal epitopes of HIV-Env-immunized rabbits via electron microscopy•Followed development and specificity of neutralizing antibodies over time•Identified a class of antibodies binding between the HIV Env trimer blades•Neutralizing antibody class displays highly convergent structural features
Comprehensive epitope mapping is fundamental to understanding immune responses. Bianchi et al. applied electron microscopy to study polyclonal antibody responses in HIV-Env-immunized rabbits. Different antibody specificities were identified and followed over time, revealing the source of neutralization, a distinct class of antibodies, and a highly convergent structural evolution of the neutralizing antibodies.
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.
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.
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•HIV Env trimer immunization protects from autologous tier 2 challenge in macaques•Neutralizing antibody (nAb) titers correlate with protection and peak viremia•T cell and ADCC functions do not correlate with protection•nAb titers of ∼1:500 (pseudovirus)/∼1:30 (live virus) required for ∼90% protection
Vaccine-induced immunity to HIV is a major research focus, but relevant correlates of protection remain controversial. Here, Pauthner et al. study protection from hard-to-neutralize SHIVBG505 challenge after immunization with native-like BG505 Envelope trimers in nonhuman primates and identify neutralizing antibodies, but not T cell or ADCC activity, as correlates of protection.
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.
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•Circulating antibodies affect the recruitment of naive B cells to germinal centers (GCs)•Broad, low-affinity responses enhance naive B cells in GCs on secondary challenge•High-titer, high-affinity, epitope-focused antibodies block cognate naive B cells•Blocking can be overcome by the administration of excess antigen
Using preclinical models for HIV and SARS-CoV, Tas et al. show that circulating antibodies from primary responses affect which naive B cells participate in germinal centers after secondary challenges. The directionality and intensity of this influence is determined by the epitope specificity of the primary response and by the affinity and concentration of the circulating antibody.
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