Recombinant MVA-prime elicits neutralizing antibody responses by inducing antigen-specific B cells in the germinal center.

The RV144 HIV-1 vaccine trial has been the only clinical trial to date that has shown any degree of efficacy and associated with the presence of vaccine-elicited HIV-1 envelope-specific binding antibody and CD4+ T-cell responses. This trial also showed that a vector-prime protein boost combined vaccine strategy was better than when used alone. Here we have studied three different priming vectors-plasmid DNA, recombinant MVA, and recombinant VSV, all encoding clade C transmitted/founder Env 1086 C gp140, for priming three groups of six non-human primates each, followed by a protein boost with adjuvanted 1086 C gp120 protein. Our data showed that MVA-priming favors the development of higher antibody binding titers and neutralizing activity compared with other vectors. Analyses of the draining lymph nodes revealed that MVA-prime induced increased germinal center reactivity characterized by higher frequencies of germinal center (PNA <sup>hi</sup> ) B cells, higher frequencies of antigen-specific B-cell responses as well as an increased frequency of the highly differentiated (ICOS <sup>hi</sup> CD150 <sup>lo</sup> ) Tfh-cell subset

Application of B cell immortalization for the isolation of antibodies and B cell clones from vaccine and infection settings

The isolation and characterization of neutralizing antibodies from infection and vaccine settings informs future vaccine design, and methodologies that streamline the isolation of antibodies and the generation of B cell clones are of great interest. Retroviral transduction to express Bcl-6 and Bcl-xL and transform primary B cells has been shown to promote long-term B cell survival and antibody secretion in vitro, and can be used to isolate antibodies from memory B cells. However, application of this methodology to B cell subsets from different tissues and B cells from chronically infected individuals has not been well characterized. Here, we characterize Bcl-6/Bcl-xL B cell immortalization across multiple tissue types and B cell subsets in healthy and HIV-1 infected individuals, as well as individuals recovering from malaria. In healthy individuals, naïve and memory B cell subsets from PBMCs and tonsil tissue transformed with similar efficiencies, and displayed similar characteristics with respect to their longevity and immunoglobulin secretion. In HIV-1-viremic individuals or in individuals with recent malaria infections, the exhausted CD27-CD21- memory B cells transformed with lower efficiency, but the transformed B cells expanded and secreted IgG with similar efficiency. Importantly, we show that this methodology can be used to isolate broadly neutralizing antibodies from HIV-infected individuals. Overall, we demonstrate that Bcl-6/Bcl-xL B cell immortalization can be used to isolate antibodies and generate B cell clones from different B cell populations, albeit with varying efficiencies

Ultrapotent antibodies against diverse and highly transmissible SARS-CoV-2 variants

IC80 1.5 to 34.5 nanograms per milliliter). We define the structural and functional determinants of binding for all four VOC-targeting antibodies and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting their potential in mitigating resistance development.The emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identified four receptor binding domain-targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 23 variants, including the B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, and B.1.617 VOCs. Two antibodies are ultrapotent, with subnanomolar neutralization titers [half-maximal inhibitory concentration (IC50) 0.3 to 11.1 nanograms per millilite

Quantitative Multiplexed Imaging Analysis Reveals a Strong Association between Immunogen-Specific B Cell Responses and Tonsillar Germinal Center Immune Dynamics in Children after Influenza Vaccination

Generation of Ag-specific humoral responses requires the orchestrated development and function of highly specialized immune cells in secondary lymphoid organs. We used a multiparametric approach combining flow cytometry, confocal microscopy, and histocytometry to analyze, for the first time to our knowledge in children, tonsils from seasonal influenza-vaccinated children. We used these novel imaging assays to address the mucosal immune dynamics in tonsils investigating the spatial positioning, frequency, and phenotype of immune cells after vaccination. Vaccination was associated with a significantly higher frequency of follicular helper CD4 T cells compared with the unvaccinated control group. The imaging analysis revealed that potential suppressor (FOXP3(hi)) CD4 T cells are mainly located in extrafollicular areas. Furthermore, a significantly reduced frequency of both follicular and extrafollicular FOXP3(hi) CD4 T cells was found in the vaccine group compared with the control group. Levels of circulating CXCL13 were higher in those vaccinated compared with controls, mirroring an increased germinal center reactivity in the tonsils. Notably, a strong correlation was found between the frequency of tonsillar T follicular helper cells and tonsillar Ag-specific Ab-secreting cells. These data demonstrate that influenza vaccination promotes the prevalence of relevant immune cells in tonsillar follicles and support the use of tonsils as lymphoid sites for the study of germinal center reactions after vaccination in children

Loss of Circulating CD4 T Cells with B Cell Helper Function during Chronic HIV Infection

The interaction between follicular T helper cells (TFH) and B cells in the lymph nodes and spleen has a major impact on the development of antigen-specific B cell responses during infection or vaccination. Recent studies described a functional equivalent of these cells among circulating CD4 T cells, referred to as peripheral TFH cells. Here, we characterize the phenotype and in vitro B cell helper activity of peripheral TFH populations, as well as the effect of HIV infection on these populations. In co-culture experiments we confirmed CXCR5+ cells from HIV-uninfected donors provide help to B cells and more specifically, we identified a CCR7(high)CXCR5(high)CCR6(high)PD-1(high) CD4 T cell population that secretes IL-21 and enhances isotype-switched immunoglobulin production. This population is significantly decreased in treatment-naïve, HIV-infected individuals and can be recovered after anti-retroviral therapy. We found impaired immunoglobulin production in co-cultures from HIV-infected individuals and found no correlation between the frequency of peripheral TFH cells and memory B cells, or with neutralization activity in untreated HIV infection in our cohort. Furthermore, we found that within the peripheral TFH population, the expression level of TFH-associated genes more closely resembles a memory, non-TFH population, as opposed to a TFH population. Overall, our data identify a heterogeneous population of circulating CD4 T cells that provides in vitro help to B cells, and challenges the origin of these cells as memory TFH cells

Soluble Prefusion Closed DS-SOSIP.664-Env Trimers of Diverse HIV-1 Strains

Summary: The elicitation of autologous neutralizing responses by immunization with HIV-1 envelope (Env) trimers conformationally stabilized in a prefusion closed state has generated considerable interest in the HIV-1 vaccine field. However, soluble prefusion closed Env trimers have been produced from only a handful of HIV-1 strains, limiting their utility as vaccine antigens and B cell probes. Here, we report the engineering from 81 HIV-1 strains of soluble, fully cleaved, prefusion Env trimers with appropriate antigenicity. We used a 96-well expression-screening format to assess the ability of artificial disulfides and Ile559Pro substitution (DS-SOSIP) to produce soluble cleaved-Env trimers; from 180 Env strains, 20 yielded prefusion closed trimers. We also created chimeras, by utilizing structure-based design to incorporate select regions from the well-behaved BG505 strain; from 180 Env strains, 78 DS-SOSIP-stabilized chimeras, including 61 additional strains, yielded prefusion closed trimers. Structure-based design thus enables the production of prefusion closed HIV-1-Env trimers from dozens of diverse strains. : Joyce et al. use a structure-based chimeric strategy to produce prefusion closed Env trimers from 81 strains of HIV-1 encompassing all major clades. These soluble Env trimers should have utility as B cell probes and HIV-1 immunogens, thereby enabling both antibody identification and vaccine development. Keywords: conformational stabilization, envelope trimer, HIV-1 vaccine, prefusion closed trimer, structure-based desig

Safety and pharmacokinetics of the Fc-modified HIV-1 human monoclonal antibody VRC01LS: A Phase 1 open-label clinical trial in healthy adults

<div><p>Background</p><p>VRC01 is a human broadly neutralizing monoclonal antibody (bnMAb) against the CD4-binding site of the HIV-1 envelope glycoprotein (Env) that is currently being evaluated in a Phase IIb adult HIV-1 prevention efficacy trial. VRC01LS is a modified version of VRC01, designed for extended serum half-life by increased binding affinity to the neonatal Fc receptor.</p><p>Methods and findings</p><p>This Phase I dose-escalation study of VRC01LS in HIV-negative healthy adults was conducted by the Vaccine Research Center (VRC) at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD). The age range of the study volunteers was 21–50 years; 51% of study volunteers were male and 49% were female. Primary objectives were safety and tolerability of VRC01LS intravenous (IV) infusions at 5, 20, and 40 mg/kg infused once, 20 mg/kg given three times at 12-week intervals, and subcutaneous (SC) delivery at 5 mg/kg delivered once, or three times at 12-week intervals. Secondary objectives were pharmacokinetics (PK), serum neutralization activity, and development of antidrug antibodies. Enrollment began on November 16, 2015, and concluded on August 23, 2017. This report describes the safety data for the first 37 volunteers who received administrations of VRC01LS. There were no serious adverse events (SAEs) or dose-limiting toxicities. Mild malaise and myalgia were the most common adverse events (AEs). There were six AEs assessed as possibly related to VRC01LS administration, and all were mild in severity and resolved during the study. PK data were modeled based on the first dose of VRC01LS in the first 25 volunteers to complete their schedule of evaluations. The mean (±SD) serum concentration 12 weeks after one IV administration of 20 mg/kg or 40 mg/kg were 180 ± 43 μg/mL (<i>n</i> = 7) and 326 ± 35 μg/mL (<i>n</i> = 5), respectively. The mean (±SD) serum concentration 12 weeks after one IV and SC administration of 5 mg/kg were 40 ± 3 μg/mL (<i>n</i> = 2) and 25 ± 5 μg/mL (<i>n</i> = 9), respectively. Over the 5–40 mg/kg IV dose range (<i>n</i> = 16), the clearance was 36 ± 8 mL/d with an elimination half-life of 71 ± 18 days. VRC01LS retained its expected neutralizing activity in serum, and anti-VRC01 antibody responses were not detected. Potential limitations of this study include the small sample size typical of Phase I trials and the need to further describe the PK properties of VRC01LS administered on multiple occasions.</p><p>Conclusions</p><p>The human bnMAb VRC01LS was safe and well tolerated when delivered intravenously or subcutaneously. The half-life was more than 4-fold greater when compared to wild-type VRC01 historical data. The reduced clearance and extended half-life may make it possible to achieve therapeutic levels with less frequent and lower-dose administrations. This would potentially lower the costs of manufacturing and improve the practicality of using passively administered monoclonal antibodies (mAbs) for the prevention of HIV-1 infection.</p><p>Trial registration</p><p>ClinicalTrials.gov <a target="_blank">NCT02599896</a></p></div

Soluble prefusion-closed HIV-envelope trimers with glycan-covered bases

Summary: Soluble HIV-1-envelope (Env) trimers elicit immune responses that target their solvent-exposed protein bases, the result of removing these trimers from their native membrane-bound context. To assess whether glycosylation could limit these base responses, we introduced sequons encoding potential N-linked glycosylation sites (PNGSs) into base-proximal regions. Expression and antigenic analyses indicated trimers bearing six-introduced PNGSs to have reduced base recognition. Cryo-EM analysis revealed trimers with introduced PNGSs to be prone to disassembly and introduced PNGS to be disordered. Protein-base and glycan-base trimers induced reciprocally symmetric ELISA responses, in which only a small fraction of the antibody response to glycan-base trimers recognized protein-base trimers and vice versa. EM polyclonal epitope mapping revealed glycan-base trimers –even those that were stable biochemically– to elicit antibodies that recognized disassembled trimers. Introduced glycans can thus mask the protein base but their introduction may yield neo-epitopes that dominate the immune response

Measurement of antibody serum concentration (μg/mL).

<p>(A) Serum VRC01LS concentrations (colored plots) are shown from first measurement through week 24 after a single administration. The infusion dose and route are as specified in the legend. All values are the mean of duplicate samples run in different wells within the same plate. Previously published VRC01 concentrations based on historical data (black plots) after administration at weeks 0 and 4 are shown for comparison. (B) Geometric mean serum VRC01LS concentrations per group over time. The dotted line at 10 μg/mL on each graph is shown as a reference value. IV, intravenous; SC, subcutaneous.</p