Search

Eliciting potent and broadly neutralizing antibodies (bnAbs) is a major goal in HIV-1 vaccine development. Here, we describe how germline-targeting immunogen BG505 SOSIP germline trimer 1.1 (GT1.1), generated through structure-based design, engages a diverse range of VRC01-class bnAb precursors. A single immunization with GT1.1 expands CD4 binding site (CD4bs)-specific VRC01-class B cells in knock-in mice and drives VRC01-class maturation. In nonhuman primates (NHPs), GT1.1 primes CD4bs-specific neutralizing serum responses. Selected monoclonal antibodies (mAbs) isolated from GT1.1-immunized NHPs neutralize fully glycosylated BG505 virus. Two mAbs, 12C11 and 21N13, neutralize subsets of diverse heterologous neutralization-resistant viruses. High-resolution structures revealed that 21N13 targets the same conserved residues in the CD4bs as VRC01-class and CH235-class bnAbs despite its low sequence similarity (~40%), whereas mAb 12C11 binds predominantly through its heavy chain complementarity-determining region 3. These preclinical data underpin the ongoing evaluation of GT1.1 in a phase 1 clinical trial in healthy volunteers.

Many HIV-1 vaccines are designed to elicit neutralizing antibodies, and pre-clinical testing is often carried out in rhesus macaques (RMs). We have therefore adapted a method of B cell immortalization for use with RM B cells. In this system, RM B cells are activated with CD40 ligand and RM IL-21 before transduction with a retroviral vector encoding Bcl-6, Bcl-xL, and green fluorescent protein. Importantly, RM B cells from lymph nodes are more effectively immortalized by this method than B cells from PBMC, a difference not seen in humans. We suggest the discrepancy between these two tissues is due to increased expression of CD40 on RM lymph node B cells. Immortalized RM B cells expand long-term, undergo minimal somatic hypermutation, express surface B cell receptor, and secrete antibodies into culture. This allows for the identification of cells based on antigen specificity and/or functional assays. Here, we show the characterization of this system and its application for the isolation of HIV-1 neutralizing antibodies from a SHIV.CH505-infected animal, both with and without antigen probe. Taken together, we show that Bcl-6/xL immortalization is a valuable and flexible tool for antibody discovery in RMs, but with important distinctions from application of the system in human cells., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Broadly neutralizing antibody (bNAb) CAP256-VRC26.25 (abbreviated CAP256LS), a human IgGI monoclonal antibody targeting the V1V2 site of the HIV-1 envelope, has demonstrated high therapeutic potential as a broadly neutralizing monoclonal antibody against HIV-1. During the process development, a heavy chain fragmentation (clipping) was observed, that led to a relative potency reduction. In this report, we highlighted a series of process and product mitigation strategies deployed to advance this product. We have detailed how analytical characterization tools, especially the microchip reduced capillary gel electrophoresis (CGE-SDS), played a pivotal role in identifying the development issues and in providing measurements to guide implementation of mitigation strategies., (Published 2022. This article is a U.S. Government work and is in the public domain in the USA.)

A closed prefusion conformation or an open (non-prefusion) conformational state of a protein vaccine candidate molecule can determine if it effectively elucidates a desired immunity. A quick and reliable method to monitor conformational state is important during vaccine development. In addition to our existing immunoassays, we have developed a unique physicochemical approach using size-exclusion chromatography to assess binding between antibody and the structurally desired antigen protein. Through the bound monoclonal antibody protein vaccine peak shift in the size-exclusion chromatography profile, this method determines the percent closed (prefusion) conformation present in a sample. Since only the closed prefusion conformation binds to the specific antibody, the population of the closed versus the open conformation of the vaccine molecule can be monitored without the need for a reference calibrator. This new method can be applied broadly to vaccine development, as well as for antibody selection during antibody drug discovery. The mAb CAP256V2LS (250 µg/mL) specific to prefusion conformation was mixed with HIV trimer (250 µg/mL) at 2:1 volume ratio, incubated at 37 °C for 30 mins and injected onto HPLC column. The percent of non-prefusion conformation was calculated based on ratio of peak area of unbound trimer and total area of control trimer sample (without mAb)., (Published by Elsevier B.V.)

Combination antiretroviral therapy (cART) controls but does not eradicate HIV infection; HIV persistence is the principal obstacle to curing infections. The proportion of defective proviruses increases during cART, but the dynamics of this process are not well understood, and a quantitative analysis of how the proviral landscape is reshaped after cART is initiated is critical to understanding how HIV persists. Here, we studied longitudinal samples from HIV infected individuals undergoing long term cART using multiplexed Droplet Digital PCR (ddPCR) approaches to quantify the proportion of deleted proviruses in lymphocytes. In most individuals undergoing cART, HIV proviruses that contain gag are lost more quickly than those that lack gag . Increases in the fraction of gag -deleted proviruses occurred only after 1-2 years of therapy, suggesting that the immune system, and/or toxicity of viral re-activation helps to gradually shape the proviral landscape. After 10-15 years on therapy, there were as many as 3.5-5 times more proviruses in which gag was deleted or highly defective than those containing intact gag . We developed a provirus-specific ddPCR approach to quantify individual clones. Investigation of a clone of cells containing a deleted HIV provirus integrated in the HORMAD2 gene revealed that the cells underwent a massive expansion shortly after cART was initiated until the clone, which was primarily in effector memory cells, dominated the population of proviruses for over 6 years. The expansion of this HIV-infected clone had substantial effects on the overall proviral population., Competing Interests: The authors declare no conflict of interest.

The enteric species F human adenovirus types 40 and 41 (HAdV-40 and -41) are the third most common cause of infantile gastroenteritis in the world. Knowledge about HAdV-40 and -41 cellular infection is assumed to be fundamentally different from that of other HAdVs since HAdV-40 and -41 penton bases lack the αV-integrin-interacting RGD motif. This motif is used by other HAdVs mainly for internalization and endosomal escape. We hypothesised that the penton bases of HAdV-40 and -41 interact with integrins independently of the RGD motif. HAdV-41 transduction of a library of rodent cells expressing specific human integrin subunits pointed to the use of laminin-binding α2-, α3- and α6-containing integrins as well as other integrins as candidate co-receptors. Specific laminins prevented internalisation and infection, and recombinant, soluble HAdV-41 penton base proteins prevented infection of human intestinal HT-29 cells. Surface plasmon resonance analysis demonstrated that HAdV-40 and -41 penton base proteins bind to α6-containing integrins with an affinity similar to that of previously characterised penton base:integrin interactions. With these results, we propose that laminin-binding integrins are co-receptors for HAdV-40 and -41.

HIV infection changes the lymph node (LN) tissue architecture, potentially impairing the immunologic response to antigenic challenge. The tissue-resident immune cell dynamics in virologically suppressed HIV+ patients on combination antiretroviral therapy (cART) are not clear. We obtained LN biopsies before and 10 to 14 days after trivalent seasonal influenza immunization from healthy controls (HCs) and HIV+ volunteers on cART to investigate CD4+ T follicular helper (Tfh) and B cell dynamics by flow cytometry and quantitative imaging analysis. Prior to vaccination, compared with those in HCs, HIV+ LNs exhibited an altered follicular architecture, but harbored higher numbers of Tfh cells and increased IgG+ follicular memory B cells. Moreover, Tfh cell numbers were dependent upon preservation of the follicular dendritic cell (FDC) network and were predictive of the magnitude of the vaccine-induced IgG responses. Interestingly, postvaccination LN samples in HIV+ participants had significantly (P = 0.0179) reduced Tfh cell numbers compared with prevaccination samples, without evidence for peripheral Tfh (pTfh) cell reduction. We conclude that influenza vaccination alters the cellularity of draining LNs of HIV+ persons in conjunction with development of antigen-specific humoral responses. The underlying mechanism of Tfh cell decline warrants further investigation, as it could bear implications for the rational design of HIV vaccines.

RSV is a major cause of severe lower respiratory infection in infants and young children. With no vaccine yet available, it is important to clarify mechanisms of disease pathogenesis. Since indoleamine-2,3-dioxygenase (IDO) is an immunomodulatory enzyme and is upregulated with RSV infection, we studied it in vivo during infection of BALB/c mice and in vitro in A549 cells. RSV infection upregulated IDO transcripts in vivo and in vitro. IDO siRNA decreased IDO transcripts ~2 fold compared to control siRNA after RSV infection but this decrease did not affect RSV replication. In the presence of IFN-γ, siRNA-induced a decrease in IDO expression that was associated with an increase in virus replication and increased levels of IL-6, IL-8, CXCL10 and CCL4. Thus, our results show IDO is upregulated with RSV infection and this upregulation likely participates with IFN-γ in inhibition of virus replication and suppression of some host cell responses to infection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In this paper, we describe development of a high-throughput, highly sensitive method based on Lab Chip CGE-SDS platform for purity determination and characterization of virus-like particle (VLP) vaccines. A capillary gel electrophoresis approach requiring about 41 s per sample for analysis and demonstrating sensitivity to protein initial concentrations as low as 20 μg/mL, this method has been used previously to evaluate monoclonal antibodies, but this application for lot release assay of VLPs using this platform is unique. The method was qualified and shown to be accurate for the quantitation of VLP purity. Assay repeatability was confirmed to be less than 2% relative standard deviation of the mean (% RSD) with interday precision less than 2% RSD. The assay can evaluate purified VLPs in a concentration range of 20-249 μg/mL for VEE and 20-250 μg/mL for EEE and WEE VLPs., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Flow cytometry has been the premier tool for single cell analysis since its invention in the 1960s. It has maintained this position through steady advances in technology and applications, becoming the main force behind interrogating the complexities of the immune system. Technology development was a three-pronged effort, including the hardware, reagents, and analysis algorithms to allow measurement of as many as 20 independent parameters on each cell, at tens of thousands of cells per second. In the coming years, cytometry technology will integrate with other techniques, such as transcriptomics, metabolomics, and so forth. Ongoing efforts are aimed at algorithms to analyse these aggregated datasaets over large numbers of samples. Here we review the development efforts heralding the next stage of flow cytometry., (Published by Elsevier Inc.)

Equine influenza A (H3N8) virus infection is a leading cause of respiratory disease in horses, resulting in widespread morbidity and economic losses. As with influenza in other species, equine influenza strains continuously mutate, often requiring the development of new vaccines. Current inactivated (killed) vaccines, while efficacious, only offer limited protection against diverse subtypes and require frequent boosts. Research into new vaccine technologies, including gene-based vaccines, aims to increase the neutralization potency, breadth, and duration of protective immunity. Here, we demonstrate that a DNA vaccine expressing the hemagglutinin protein of equine H3N8 influenza virus generates homologous and heterologous immune responses, and protects against clinical disease and viral replication by homologous H3N8 virus in horses. Furthermore, we demonstrate that needle-free delivery is as efficient and effective as conventional parenteral injection using a needle and syringe. These findings suggest that DNA vaccines offer a safe, effective, and promising alternative approach for veterinary vaccines against equine influenza., (Published by Elsevier Ltd.)

Following respiratory syncytial virus (RSV) challenge, mice immunized with RSV G or with formalin-inactivated RSV (FI-RSV) exhibit severe disease associated with type 2 cytokine production and pulmonary eosinophilia. This has led to the proposal that the presence of RSV G is the factor in FI-RSV that induces disease-enhancing T-cell responses. Therefore, we evaluated the role of RSV G and its immunodominant region in the induction of aberrant immune responses during FI-RSV immunization. BALB/c mice were immunized with FI preparations of wild-type (wt) RSV or recombinant RSV (rRSV) containing deletions of (i) the entire G gene, (ii) the region of the G gene encoding amino acids 187 to 197 of the immunodominant region, or (iii) the entire SH gene. After challenge, illness, RSV titers, cytokine levels, and pulmonary eosinophilia were measured. Peak RSV titers postchallenge were significantly greater in mice immunized with FI preparations of the deletion viruses than in those immunized with FI-rRSV wt, suggesting that the absence of G or SH in FI-RSV reduced its protective efficacy. Deletion of G or its epitope did not reduce illness, cytokine production, or eosinophilia relative to that in mice immunized with FI-rRSV wt. While cytokine levels and eosinophilia were similar, illness was reduced in mice immunized with SH-deleted FI-RSV. These data suggest that G-specific immune responses may be important for vaccine-induced protection and are not solely the basis for FI-RSV vaccine-enhanced illness. These data suggest that the method of RSV antigen delivery, rather than the protein composition, influences the phenotype of the induced immune responses and that RSV G should not necessarily be excluded from potential vaccine strategies.