Antibody Responses against Xenotropic Murine Leukemia Virus-Related Virus Envelope in a Murine Model

Xenotropic murine leukemia virus-related virus (XMRV) was recently discovered to be the first human gammaretrovirus that is associated with chronic fatigue syndrome and prostate cancer (PC). Although a mechanism for XMRV carcinogenesis is yet to be established, this virus belongs to the family of gammaretroviruses well known for their ability to induce cancer in the infected hosts. Since its original identification XMRV has been detected in several independent investigations; however, at this time significant controversy remains regarding reports of XMRV detection/prevalence in other cohorts and cell type/tissue distribution. The potential risk of human infection, coupled with the lack of knowledge about the basic biology of XMRV, warrants further research, including investigation of adaptive immune responses. To study immunogenicity in vivo, we vaccinated mice with a combination of recombinant vectors expressing codon-optimized sequences of XMRV gag and env genes and virus-like particles (VLP) that had the size and morphology of live infectious XMRV.Immunization elicited Env-specific binding and neutralizing antibodies (NAb) against XMRV in mice. The peak titers for ELISA-binding antibodies and NAb were 1:1024 and 1:464, respectively; however, high ELISA-binding and NAb titers were not sustained and persisted for less than three weeks after immunizations.Vaccine-induced XMRV Env antibody titers were transiently high, but their duration was short. The relatively rapid diminution in antibody levels may in part explain the differing prevalences reported for XMRV in various prostate cancer and chronic fatigue syndrome cohorts. The low level of immunogenicity observed in the present study may be characteristic of a natural XMRV infection in humans

Proximity Ligation Assays for In Situ Detection of Innate Immune Activation: Focus on In Vitro-Transcribed mRNA

International audienceThe characterization of innate immune activation is crucial for vaccine and therapeutic development, including RNA-based vaccines, a promising approach. Current measurement methods quantify type I interferon and inflammatory cytokine production, but they do not allow for the isolation of individual pathways, do not provide kinetic activation or spatial information within tissues, and cannot be translated into clinical studies. Here we demonstrated the use of proximity ligation assays (PLAs) to detect pattern recognition receptor (PRR) activation in cells and in tissue samples. First, we validated PLA's sensitivity and specificity using well-characterized soluble agonists. Next, we characterized PRR activation from in vitro-transcribed (IVT) mRNAs, as well as the effect of sequence and base modifications in vitro. Finally, we established the measurement of PRR activation in tissue sections via PLA upon IVT mRNA intramuscular (i.m.) injection in mice. Overall, our results indicate that PLA is a valuable, versatile , and sensitive tool to monitor PRR activation for vaccine, adjuvant, and therapeutic screening

Visualization of early events in mRNA vaccine delivery in non-human primates via PET–CT and near-infrared imaging

These authors contributed equally : Kevin E. Lindsay, Sushma M. Bhosle and Chiara Zurla.International audienceVisualization of the spatio-temporal trafficking of vaccines after their delivery would help evaluate the efficacy of candidate formulations and aid their rational design for preclinical and translational studies. Here, we show that a dual radionuclide-near-infrared probe allows for quantitative, longitudinal and non-invasive monitoring, via positron emission tomography-computed tomography and near-infrared imaging of cynomolgus macaques, of the trafficking dynamics to draining lymph nodes of a model messenger RNA vaccine labelled with the probe. After intramuscular administration of the vaccine to the monkeys, we observed the dynamics of the mRNA vaccine at the injection site and in the draining lymph nodes, performed cellular analyses of the involved tissues using flow cytometry and identified through immunofluorescence that professional antigen-presenting cells are the primary cells containing the injected mRNA and encoding the antigen. This approach may reveal spatio-temporal determinants of vaccine efficacy in preclinical and translational studies employing large mammals

Duplex One-Step RT-qPCR Assays for Simultaneous Detection of Genomic and Subgenomic RNAs of SARS-CoV-2 Variants

A hallmark of severe acute respiratory syndrome virus (SARS-CoV-2) replication is the discontinuous transcription of open reading frames (ORFs) encoding structural virus proteins. Real-time reverse transcription PCR (RT-qPCR) assays in previous publications used either single or multiplex assays for SARS-CoV-2 genomic RNA detection and a singleplex approach for subgenomic RNA detection. Although multiplex approaches often target multiple genomic RNA segments, an assay that concurrently detects genomic and subgenomic targets has been lacking. To bridge this gap, we developed two duplex one-step RT-qPCR assays that detect SARS-CoV-2 genomic ORF1a and either subgenomic spike or subgenomic ORF3a RNAs. All primers and probes for our assays were designed to bind to variants of SARS-CoV-2. In this study, our assays successfully detected SARS-CoV-2 Washington strain and delta variant isolates at various time points during the course of live virus infection in vitro. The ability to quantify subgenomic SARS-CoV-2 RNA is important, as it may indicate the presence of active replication, particularly in samples collected longitudinally. Furthermore, specific detection of genomic and subgenomic RNAs simultaneously in a single reaction increases assay efficiency, potentially leading to expedited lucidity about viral replication and pathogenesis of any variant of SARS-CoV-2

Characterization of antibodies purified from immune and control mouse sera.

<p>Total immunoglobulin pool was affinity purified from immune or control sera collected at the peak time point of neutralizing activity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018272#pone-0018272-g003" target="_blank">figure 3B</a>). The ELISA-binding (A) and NAb (B) activities were then measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018272#pone-0018272-g003" target="_blank">Figure 3</a> and showed that immunization elicited an immune response leading to the production of anti-XMRV immunoglobulins.</p