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The analysis of T-cell responses in HIV-1-infected controllers may contribute to a better understanding of the protective components of the immune system. Here, we analyzed the HIV-1-specific T-cell response in a 59-year-old HIV-1-infected controller, infected for at least seven years, who presented with low viral loads ranging from <20 copies/mL to 200 copies/mL and normal CD4 counts of >800 cells/µL. In γ-IFN-ELISpot assays using freshly isolated PBMCs, he displayed a very strong polyclonal T-cell response to eight epitopes in Gag, Nef and Rev; with the dominant responses directed against the HLA-B*57-epitope AISPRTLNAW and against a so-far-unknown epitope within Rev. Further analyses using peptide-stimulated T-cell lines in γ-IFN-ELISpot assays delineated the peptide RQRQIRSI (Rev-RI8) as a newly defined HLA-B*52-restricted epitope located within a functionally important region of Rev. Peptide-stimulation assays in 15 HLA-B*52-positive HIV-1-infected subjects, including the controller, demonstrated recognition of the Rev-RI8 epitope in 6/15 subjects. CD4 counts before the start of antiviral therapy were significantly higher in subjects with recognition of the Rev-RI8 epitope. Targeting of the Rev-RI8 epitope in Rev by CTL could contribute to the positive association of HLA-B*52 with a more favorable course of HIV-1-infection. [ABSTRACT FROM AUTHOR]

High-throughput multiplexed assays are needed to simplify detection of Helicobacter species in experimental infection and routine health monitoring of laboratory mice. Therefore, fluorescent bead-based hybridization assays for Helicobacter sp. DNA and serology were developed. Multiplex PCR amplicons (H. hepaticus, H. bilis, H. typhlonius, H. pylori, H. muridarum, H. pullorum, H. cinaedi, H. heilmanii, C. jejuni) and antibodies against H. pylori, H. hepaticus, H. bilis were assessed in naturally and experimentally infected mice, and results compared to conventional PCR. Species-specific and sensitive detection of seven Helicobacter spp. <100 copies/PCR, and of two species <1000 copies/PCR was successfully established in the Helicobacter multiplex DNA finder. The novel assay was highly comparable with conventional PCR (kappa = 0.98, 95%CI: 0.94–1.00). Antibody detection of H. hepaticus and H. bilis showed low sensitivity (71% and 62%, respectively) and cross-reactivity in H. typhlonius-infected mice. Infection experiments showed that antibodies develop earliest two weeks after DNA detection in feces. In conclusion, detection of Helicobacter antibodies showed low sensitivity depending on the timing relative to infection. However, Helicobacter multiplex DNA finder is a sensitive and specific high-throughput assay applicable in routine health monitoring for laboratory animals. [ABSTRACT FROM AUTHOR]

Only limited data are available regarding the immunogenicity of the BNT162b2 mRNA vaccine in HIV-1+ patients. Therefore, we investigated the humoral immune response after BNT162b2-mRNA vaccination or SARS-CoV-2 infection in HIV-1+ patients on antiretroviral therapy compared to HIV-1-uninfected subjects. Serum and saliva samples were analysed by SARS-CoV-2 spike-specific IgG and IgA ELISAs and a surrogate neutralization assay. While all subjects developed anti-spike IgG and IgA and neutralizing antibodies in serum after two doses of BNT162b2 mRNA vaccine, the HIV-1+ subjects displayed significantly lower neutralizing capacity and anti-spike IgA in serum compared to HIV-1-uninfected subjects. Serum levels of anti-spike IgG and neutralizing activity were significantly higher in vaccinees compared to SARS-CoV-2 convalescents irrespective of HIV-1 status. Among SARS-CoV-2 convalescents, there was no significant difference in spike-specific antibody response between HIV-1+ and uninfected subjects. In saliva, anti-spike IgG and IgA antibodies were detected both in vaccinees and convalescents, albeit at lower frequencies compared to the serum and only rarely with detectable neutralizing activity. In summary, our study demonstrates that the BNT162b2 mRNA vaccine induces SARS-CoV-2-specific antibodies in HIV-1-infected patients on antiretroviral therapy, however, lower vaccine induced neutralization activity indicates a lower functionality of the humoral vaccine response in HIV-1+ patients. [ABSTRACT FROM AUTHOR]

The Flavivirus genus includes a number of important viruses that are pathogenic to humans and animals and are responsible for outbreaks across the globe. Integrins, a family of heterodimeric transmembrane molecules expressed in all nucleated cells mediate critical functions of cell physiology and cell cycle. Integrins were previously postulated to be involved in flavivirus entry and to modulate flavivirus replication efficiency. In the present study, mouse embryonic fibroblasts (MEF), lacking the expression of αVβ3 integrin (MEF-αVβ3−/−), were infected with four different flaviviruses, namely yellow fever virus (YFV), West Nile virus (WNV), Usutu virus (USUV) and Langat virus (LGTV). The effects of the αVβ3 integrin absence in double-knockout MEF-αVβ3−/− on flavivirus binding, internalization and replication were compared to the respective wild-type cells. Binding to the cell surface for all four flaviviruses was not affected by the ablation of αVβ3 integrin, whereas internalization of USUV and WNV was slightly affected by the loss of αVβ3 integrin expression. Most interestingly, the deletion of αVβ3 integrin strongly impaired replication of all flaviviruses with a reduction of up to 99% on virus yields and a strong reduction on flavivirus anti-genome RNA synthesis. In conclusion, our results demonstrate that αVβ3 integrin expression in flavivirus-susceptible cell lines enhances the flavivirus replication. [ABSTRACT FROM AUTHOR]

In times of spreading multidrug-resistant bacteria, species identification and decontamination of cell cultures can be challenging. Here, we describe a mobile cell culture contaminant with "black dot"-like microscopic appearance in newly established irreplaceable hybridoma cell lines and its identification. Using 16S rRNA gene sequencing, species-specific PCRs, whole genome sequencing (WGS), and MALDI-TOF mass spectrometry, the contaminant was identified as the ubiquitous environmental and clinically relevant Gram-negative bacterium Ralstonia insidiosa (R. insidiosa), a strong biofilm producer. Further characterizations by transmission electron microscopy (TEM) and biochemical API test were not conclusive. Whole genome sequencing of our R. insidiosa isolate revealed numerous drug-resistance determinants. Genome-wide comparison to other Ralstonia species could not unambiguously designate our isolate to R. insidiosa (<95% average nucleotide identity) suggesting a potential novel species or subspecies, closely related to R. insidiosa and R. pickettii. After determining the antibiotic susceptibility profile, the hybridoma cell culture was successfully decontaminated with ciprofloxacin without affecting antibody production. [ABSTRACT FROM AUTHOR]

The Flavivirus genus includes a number of important viruses that are pathogenic to humans and animals and are responsible for outbreaks across the globe. Integrins, a family of heterodimeric transmembrane molecules expressed in all nucleated cells mediate critical functions of cell physiology and cell cycle. Integrins were previously postulated to be involved in flavivirus entry and to modulate flavivirus replication efficiency. In the present study, mouse embryonic fibroblasts (MEF), lacking the expression of αVβ3 integrin (MEF-αVβ3 -/- ), were infected with four different flaviviruses, namely yellow fever virus (YFV), West Nile virus (WNV), Usutu virus (USUV) and Langat virus (LGTV). The effects of the αVβ3 integrin absence in double-knockout MEF-αVβ3 -/- on flavivirus binding, internalization and replication were compared to the respective wild-type cells. Binding to the cell surface for all four flaviviruses was not affected by the ablation of αVβ3 integrin, whereas internalization of USUV and WNV was slightly affected by the loss of αVβ3 integrin expression. Most interestingly, the deletion of αVβ3 integrin strongly impaired replication of all flaviviruses with a reduction of up to 99% on virus yields and a strong reduction on flavivirus anti-genome RNA synthesis. In conclusion, our results demonstrate that αVβ3 integrin expression in flavivirus-susceptible cell lines enhances the flavivirus replication.