Abstract In the present study, the recognition of epitope variants of influenza A viruses by human CTL was investigated. To this end, human CD8+ CTL clones, specific for natural variants of the HLA-B*3501-restricted epitope in the nucleoprotein (NP418–426), were generated. As determined in 51Cr release assays and by flow cytometry with HLA-B*3501-peptide tetrameric complexes, CTL clones were found to be specific for epitopes within one subtype or cross-reactive with heterosubtypic variants of the epitope. Using eight natural variants of the epitope, positions in the 9-mer important for T cell recognition and involved in escape from CTL immunity were identified and visualized using multidimensional scaling. It was shown that positions 4 and 5 in the 9-mer epitope were important determinants of T cell specificity. The in vivo existence of CD8+ cells cross-reactive with homo- and heterosubtypic variants of the epitope was further confirmed using polyclonal T cell populations obtained after stimulation of PBMC with different influenza A viruses. Based on the observed recognition patterns of the clonal and polyclonal T cell populations and serology, it is hypothesized that consecutive infections with influenza viruses containing different variants of the epitope select for cross-reactive T cells in vivo.
ABSTRACT We previously described a nasally delivered monovalent adenoviral-vectored SARS- CoV-2 vaccine (ChAd-SARS-CoV-2-S, targeting Wuhan-1 spike [S]; iNCOVACC®) that is currently used in India as a primary or booster immunization. Here, we updated the mucosal vaccine for Omicron variants by creating ChAd-SARS-CoV-2-BA.5-S, which encodes for a pre- fusion and surface-stabilized S protein of the BA.5 strain, and then tested monovalent and bivalent vaccines for efficacy against circulating variants including BQ.1.1 and XBB.1.5. Whereas monovalent ChAd-vectored vaccines effectively induced systemic and mucosal antibody responses against matched strains, the bivalent ChAd-vectored vaccine elicited greater breadth. However, serum neutralizing antibody responses induced by both monovalent and bivalent vaccines were poor against the antigenically distant XBB.1.5 Omicron strain and did not protect in passive transfer experiments. Nonetheless, nasally delivered bivalent ChAd- vectored vaccines induced robust antibody and spike-specific memory T cell responses in the respiratory mucosa, and conferred protection against WA1/2020 D614G and Omicron variants BQ.1.1 and XBB.1.5 in the upper and lower respiratory tracts of both mice and hamsters. Our data suggest that a nasally delivered bivalent adenoviral-vectored vaccine induces protective mucosal and systemic immunity against historical and emerging SARS-CoV-2 strains without requiring high levels of serum neutralizing antibody.
Airborne transmission via aerosols is a dominant route for the transmission of respiratory pathogens, including avian H5N1 influenza A virus and E. coli bacteria. Rapid and direct detection of respiratory pathogen aerosols has been a long-standing technical challenge. Herein, we develop a novel label-free capacitive biosensor using an interlocked Prussian blue (PB)/graphene oxide (GO) network on a screen-printed carbon electrode (SPCE) for direct detection of avian H5N1 and E. coli. A single-step electro-co-deposition process grows GO branches on the SPCE surface, while the PB nanocrystals simultaneously decorate around the GO branches, resulting in an ultrasensitive capacitive response at nanofarad levels. We tested the biosensor for H5N1 concentrations from 2.0 viral RNA copies/mL to 1.6 × 105 viral RNA copies/mL, with a limit of detection (LoD) of 56 viral RNA copies/mL. We tested it on E. coli for concentrations ranging from 2.0 bacterial cells/mL to 1.8 × 104 bacterial cells/mL, with a LoD of 5 bacterial cells/mL. The detection times for both pathogens were under 5 min. When integrated with a custom-built wet cyclone bioaerosol sampler, our biosensor could detect and quasi-quantitatively estimate H5N1 and E. coli concentrations in air with spatial resolutions of 93 viral RNA copies/m3 and 8 bacterial cells/m3, respectively. The quasi-quantification method, based on dilution and binary detection (positive/negative), achieved an overall accuracy of >90% for pathogen-laden aerosol samples. This biosensor is adaptable for multiplexed detection of other respiratory pathogens, making it a versatile tool for real-time airborne pathogen monitoring and risk assessment.
In vitro studies have demonstrated positive effects of bioactive compounds on several functions of the immune system. In the present study, 25 of such compounds were tested for their immune modulating properties on influenza virus specific human B‐ and T‐cell responses in vitro . One of these compounds, N‐acetyl‐ l ‐cysteine was shown to increase influenza virus specific lymphocyte proliferation and interferon(IFN)‐γ production at a concentration of 1.0 mmol/l. Furthermore, N‐acetyl‐ l ‐cysteine was found to enhance a specific activity of two influenza specific CD8 + cytotoxic T‐lymphocyte clones directed towards HLA‐A*0201 and HLA‐B*2705 restricted epitopes. A second compound, chlorogenic acid, was shown to enhance antigen specific proliferation of lymphocytes in three out of four donors, at concentrations of 10–50 µmol/l. Neither of the two compounds exhibited a positive effect on the production of influenza virus specific antibodies by human peripheral blood mononuclear cells in vitro .
Objective To determine whether maternal influenza virus infection in the second and third trimesters of pregnancy results in transplacental transmission of infection, maternal auto‐antibody production or an increase in complications of pregnancy. Design Case‐control cohort study. Population Study and control cohorts were derived from 3975 women who were consecutively delivered at two Nottingham teaching hospitals between May 1993 and July 1994. A complete set of three sera was available for 1659 women. Methods Paired maternal ante‐ and postnatal sera were screened for a rise in anti‐influenza virus antibody titre by single radial haemolysis and haemagglutination inhibition. Routine obstetric data collected during and after pregnancy were retrieved from the Nottingham obstetric database. Cord samples were tested for the presence of IgM anti‐influenza antibodies, and postnatal infant sera were tested for the persistence of influenza‐virus specific IgG. Paired antenatal and postnatal sera were tested against a standard range of auto‐antigens by immunofluorescence. Main outcome measures Classification of women as having definite serological evidence of an influenza virus infection in pregnancy (cases) or as controls. Results Intercurrent influenza virus infections were identified in 182/1659 (11.0%) pregnancies. None of 138 cord sera from maternal influenza cases was positive for influenza A virus specific IgM. IgG anti‐influenza antibodies did not persist in any of 12 infant sera taken at age 6–12 months. Six of 172 postnatal maternal sera from cases of influenza were positive for auto‐antibodies. In all cases the corresponding antenatal serum was also positive for the same auto‐antibody. There were no significant differences in pregnancy outcome measures between cases and controls. Overall, there were significantly more complications of pregnancy in the cases versus the controls, but no single type of complication achieved statistical significance. Influenza infection in the second and third trimesters of pregnancy is a relatively common event. We found no evidence for transplacental transmission of influenza virus or auto‐antibody production in pregnancies complicated by influenza infections. There was an increase in the complications of pregnancy in our influenza cohort.
Abstract The stem-loop II motif (s2m) is an RNA element present in viruses from divergent viral families, including astroviruses and coronaviruses, but its functional significance is unknown. We created deletions or substitutions of the s2m in astrovirus VA1 (VA1), classic human astrovirus 1 (HAstV1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For VA1, recombinant virus could not be rescued upon partial deletion of the s2m or substitutions of G-C base pairs. Compensatory substitutions that restored the G-C base-pair enabled recovery of VA1. For HAstV1, a partial deletion of the s2m resulted in decreased viral titers compared to wild-type virus, and reduced activity in a replicon system. In contrast, deletion or mutation of the SARS-CoV-2 s2m had no effect on the ability to rescue the virus, growth in vitro , or growth in Syrian hamsters. Our study demonstrates the importance of the s2m is virus-dependent.
Tissue-resident memory CD8+ T cells (TRMs) confer rapid protection and immunity against viral infections. Many viruses have evolved mechanisms to inhibit MHCI presentation in order to evade CD8+ T cells, suggesting that these mechanisms may also apply to TRM-mediated protection. However, the effects of viral MHCI inhibition on the function and generation of TRMs is unclear. Herein, we demonstrate that viral MHCI inhibition reduces the abundance of CD4+ and CD8+ TRMs, but its effects on the local microenvironment compensate to promote antigen-specific CD8+ TRM formation. Unexpectedly, local cognate antigen enhances CD8+ TRM development even in the context of viral MHCI inhibition and CD8+ T cell evasion, strongly suggesting a role for in situ cross-presentation in local antigen-driven TRM differentiation. However, local cognate antigen is not required for CD8+ TRM maintenance. We also show that viral MHCI inhibition efficiently evades CD8+ TRM effector functions. These findings indicate that viral evasion of MHCI antigen presentation has consequences on the development and response of antiviral TRMs.
ABSTRACT Bourbon virus (BRBV) is an emerging pathogen that can cause severe and fatal disease in humans. BRBV is vectored by Amblyomma americanum (lone star ticks), which are widely distributed across the central, southern, and eastern United States. Wildlife species are potentially important for the maintenance and transmission of BRBV, but little is known about which species are involved, and what other factors play a role in the exposure to BRBV. To assess the exposure risk to BRBV among wildlife in the St. Louis area, we collected sera from 98 individuals, representing 6 different mammalian species from two locations in St. Louis County: Tyson Research Center (TRC) and WildCare Park (WCP) from fall 2021 to spring 2023. The sera were used in a BRBV neutralization assay to detect neutralizing antibodies and RT-qPCR for viral RNA analysis. We also sampled and compared the abundance of A. americanum ticks at the two locations and modeled which factors influenced BRBV seropositivity across species. In TRC, we observed a high rate of seropositivity in raccoons ( Procyon lotor , 23/25), and white-tailed deer ( Odocoileus virginianus , 18/27), but a low rate in opossums ( Didelphis virginiana , 1/18). Neutralizing antibodies were also detected in sampled TRC bobcats ( Lynx rufus , 4/4), coyotes ( Canis latrans , 3/3), and a red fox ( Vulpes vulpes , 1/1). The virological analysis identified BRBV RNA in one of the coyote serum samples. In contrast to TRC, all sera screened from WCP were negative for BRBV-specific neutralizing antibodies, and significantly fewer ticks were collected at WCP (31) compared to TRC (2,316). Collectively, these findings suggest that BRBV circulates in multiple wildlife species in the St. Louis area and that tick density and host community composition may be important factors in BRBV ecology.
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