Xenoantigen-Dependent Complement-Mediated Neutralization of Lymphocytic Choriomeningitis Virus Glycoprotein-Pseudotyped Vesicular Stomatitis Virus in Human Serum.

Neutralization by antibodies and complement limits the effective dose and thus the therapeutic efficacy of oncolytic viruses after systemic application. We and others previously showed that pseudotyping of oncolytic rhabdoviruses such as maraba virus and vesicular stomatitis virus (VSV) with the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) results in only a weak induction of neutralizing antibodies. Moreover, LCMV-GP-pseudotyped VSV (VSV-GP) was significantly more stable in normal human serum (NHS) than VSV. Here, we demonstrate that depending on the cell line used for virus production, VSV-GP showed different complement sensitivities in nonimmune NHS. The NHS-mediated titer reduction of VSV-GP was dependent on activation of the classical complement pathway, mainly by natural IgM antibodies against xenoantigens such as galactose-α-(1,3)-galactose (α-Gal) or N-glycolylneuraminic acid (Neu5Gc) expressed on nonhuman production cell lines. VSV-GP produced on human cell lines was stable in NHS. However, VSV-GP generated in transduced human cells expressing α-Gal became sensitive to NHS. Furthermore, GP-specific antibodies induced complement-mediated neutralization of VSV-GP independently of the producer cell line, suggesting that complement regulatory proteins potentially acquired by the virus during the budding process are not sufficient to rescue the virus from antibody-dependent complement-mediated lysis. Thus, our study points to the importance of a careful selection of cell lines for viral vector production for clinical use.IMPORTANCE Systemic application aims to deliver oncolytic viruses to tumors as well as to metastatic lesions. However, we found that xenoantigens incorporated onto the viral surface from nonhuman production cell lines are recognized by natural antibodies in human serum and that the virus is thereby inactivated by complement lysis. Hence, to maximize the effective dose, careful selection of cell lines for virus production is crucial.

Oncolytic virotherapy enhances the efficacy of a cancer vaccine by modulating the tumor microenvironment.

The efficacy of cancer vaccines has been limited by the immunosuppressive tumor microenvironment, which can be alleviated by immune checkpoint inhibitor (ICI) therapy. Here, we tested if oncolytic viruses (OVs), similar to ICI, can also synergize with cancer vaccines by modulating the tumor microenvironment. VSV-GP, a chimeric vesicular stomatitis virus (VSV) pseudotyped with the glycoprotein (GP) of the lymphocytic choriomeningitis virus, is a promising new OV candidate. Here, we show that in mouse B16-OVA melanoma, combination treatment of VSV-GP with an ovalbumin (OVA) peptide-loaded dendritic cell (DC) vaccine (DCVacc) significantly enhanced survival over the single agent therapies, although both DCVacc and DCVacc/VSV-GP treatments induced comparable levels of OVA-specific CD8 T cell responses. Virus replication was minimal so that direct viral oncolysis in B16-OVA did not contribute to this synergism. The strong therapeutic effect of the DCVacc/VSV-GP combination treatment was associated with high numbers of tumor-infiltrating, highly activated T cells and the relative reduction of regulatory T cells in treated and contra-lateral nontreated tumors. Accordingly, depletion of CD8 T cells but not natural killer cells abrogated the therapeutic effect of DCVacc/VSV-GP supporting the crucial role of CD8 T cells. In addition, a drastic increase in several proinflammatory cytokines was observed in VSV-GP-treated tumors. Taken together, OVs, similar to ICI, have the potential to markedly increase the efficacy of cancer vaccines by alleviating local immune suppression in the tumor microenvironment.

Characterization of the Anti-Viral and Vaccine-Specific CD8+ T Cell Composition upon Treatment with the Cancer Vaccine VSV-GP.

Numerous factors influence the magnitude and effector phenotype of vaccine-induced CD8+ T cells, thereby potentially impacting treatment efficacy. Here, we investigate the effect of vaccination dose, route of immunization, presence of a target antigen-expressing tumor, and heterologous prime-boost with peptide vaccine partner following vaccination with antigen-armed VSV-GP. Our results indicate that a higher vaccine dose increases antigen-specific CD8+ T cell proportions while altering the phenotype. The intravenous route induces the highest proportion of antigen-specific CD8+ T cells together with the lowest anti-viral response followed by the intraperitoneal, intramuscular, and subcutaneous routes. Moreover, the presence of a B16-OVA tumor serves as pre-prime, thereby increasing OVA-specific CD8+ T cells upon vaccination and thus altering the ratio of anti-tumor versus anti-viral CD8+ T cells. Interestingly, tumor-specific CD8+ T cells exhibit a different phenotype compared to bystander anti-viral CD8+ T cells. Finally, the heterologous combination of peptide and viral vaccine elicits the highest proportion of antigen-specific CD8+ T cells in the tumor and tumor-draining lymph nodes. In summary, we provide a basic immune characterization of various factors that affect anti-viral and vaccine target-specific CD8+ T cell proportions and phenotypes, thereby enhancing our vaccinology knowledge for future vaccine regimen designs.

Prediction and validation of murine MHC class I epitopes of the recombinant virus VSV-GP.

Oncolytic viruses are currently tested as a novel platform for cancer therapy. These viruses preferentially replicate in and kill malignant cells. Due to their microbial origin, treatment with oncolytic viruses naturally results in anti-viral responses and general immune activation. Consequently, the oncolytic virus treatment also induces anti-viral T cells. Since these can constitute the dominant activated T cell pool, monitoring of the anti-viral T cell response may aid in better understanding of the immune responses post oncolytic virotherapy. This study aimed to identify the anti-viral T cells raised by VSV-GP virotherapy in C57BL/6J mice, one of the most widely used models for preclinical studies. VSV-GP is a novel oncolytic agent that recently entered a clinical phase I study. To identify the VSV-GP epitopes to which mouse anti-viral T cells react, we used a multilevel adapted bioinformatics viral epitope prediction approach based on the tools netMHCpan, MHCflurry and netMHCstabPan, which are commonly used in neoepitope identification. Predicted viral epitopes were ranked based on consensus binding strength categories, predicted stability, and dissimilarity to the mouse proteome. The top ranked epitopes were selected and included in the peptide candidate matrix in order to use a matrix deconvolution approach. Using ELISpot, we showed which viral epitopes presented on C57BL/6J mouse MHC-I alleles H2-Db and H2-Kb trigger IFN-γ secretion due to T cell activation. Furthermore, we validated these findings using an intracellular cytokine staining. Collectively, identification of the VSV-GP T cell epitopes enables monitoring of the full range of anti-viral T cell responses upon VSV-GP virotherapy in future studies with preclinical mouse models to more comprehensively delineate anti-viral from anti-tumor T cell responses. These findings also support the development of novel VSV-GP variants expressing immunomodulatory transgenes and can improve the assessment of anti-viral immunity in preclinical models.

Differential infection of murine and human dendritic cell subsets by oncolytic vesicular stomatitis virus variants.

Oncolytic viruses (OVs) can eradicate tumor cells and elicit antitumor immunity. VSV-GP, a chimeric vesicular stomatitis virus (VSV) with the glycoprotein (GP) of the lymphocytic choriomeningitis virus, is a promising new OV candidate. However, the interaction of VSV-GP with host immune cells is not fully understood. Dendritic cells (DCs) are essential for inducing efficient antitumor immunity. Thus, we aimed to investigate the interaction of VSV-GP with different murine and human DCs subsets in direct comparison to the less cytopathic variant VSV-dM51-GP and wild type VSV. Immature murine bone marrow-derived DCs (BMDCs) were equally infected and killed by VSV and VSV-GP. Human monocyte-derived DCs (moDCs) were more permissive to VSV. Interestingly, VSV-dM51-GP induced maturation instead of killing in both BMDCs and moDCs as well as a pronounced release of pro-inflammatory cytokines. Importantly, matured BMDCs and moDCs were no longer susceptible to VSV-GP infection. Mouse splenic conventional DC type 1 (cDC1) could be infected ex vivo by VSV and VSV-GP to a higher extent than cDC2. Systemic infection of mice with VSV-GP and VSV-dM51-GP resulted in strong activation of cDCs despite low infection rates in spleen and tumor tissue. Human blood cDC1 were equally infected by VSV and VSV-GP, whereas cDC2 showed preferential infection with VSV. Our study demonstrated differential DC infection, activation, and cytokine production after the treatment with VSV and VSV-GP variants among species and subsets, which should be taken into account when investigating immunological mechanisms of oncolytic virotherapy in mouse models and human clinical trials.

High SARS-CoV-2 seroprevalence in children and adults in the Austrian ski resort of Ischgl.

BACKGROUND: In early March 2020, a SARS-CoV-2 outbreak in the ski resort Ischgl in Austria initiated the spread of SARS-CoV-2 throughout Austria and Northern Europe. METHODS: Between April 21st and 27th 2020, a cross-sectional epidemiologic study targeting the full population of Ischgl (n = 1867), of which 79% could be included (n = 1473, incl. 214 children), was performed. For each individual, the study involved a SARS-CoV-2 PCR, antibody testing and structured questionnaires. A mathematical model was used to help understand the influence of the determined seroprevalence on virus transmission. RESULTS: The seroprevalence was 42.4% (95% confidence interval (CI) 39.8-44.7). Individuals under 18 showed a significantly lower seroprevalence of 27.1% (95% CI 21.3-33.6) than adults (45%; 95% CI 42.2-47.7; OR of 0.455, 95% CI 0.356-0.682, p < 0.001). Of the seropositive individuals, 83.7% had not been diagnosed to have had SARS-CoV-2 infection previously. The clinical course was generally mild. Over the previous two months, two COVID-19-related deaths had been recorded, corresponding to an infection fatality rate of 0.25% (95% CI 0.03-0.91). Only 8 (0.5 %) individuals were newly diagnosed to be infected with SARS-CoV-2 during this study. CONCLUSIONS: Ischgl was hit early and hard by SARS-CoV-2 leading to a high local seroprevalence of 42.4%, which was lower in individuals below the age of 18 than in adults. Mathematical modeling suggests that a drastic decline of newly infected individuals in Ischgl by the end of April occurred due to the dual impact from the non-pharmacological interventions and a high immunization of the Ischgl population.

Persistence of immunity to SARS-CoV-2 over time in the ski resort Ischgl.

Background In early March 2020, a SARS-CoV-2 outbreak in the ski resort Ischgl in Austria triggered the spread of SARS-CoV-2 throughout Austria and Northern Europe. In a previous study, we found that the seroprevalence in the adult population of Ischgl had reached 45% by the end of April, representing an exceptionally high level of local seropositivity in Europe. We performed a follow-up study in Ischgl, which is the first to show persistence of immunity and protection against SARS-CoV-2 and some of its variants at a community level. Methods Of the 1259 adults that participated in the baseline study, 801 have been included in the follow-up in November 2020. The study involved the analysis of binding and neutralizing antibodies and T cell responses. In addition, the incidence of SARS-CoV-2 and its variants in Ischgl was compared to the incidence in similar municipalities in Tyrol until April 2021. Findings For the 801 individuals that participated in both studies, the seroprevalence declined from 51.4% (95% confidence interval (CI) 47.9-54.9) to 45.4% (95% CI 42.0-49.0). Median antibody concentrations dropped considerably (5.345, 95% CI 4.833 - 6.123 to 2.298, 95% CI 2.141 - 2.527) but antibody avidity increased (17.02, 95% CI 16.49 - 17.94 to 42.46, 95% CI 41.06 - 46.26). Only one person had lost detectable antibodies and T cell responses. In parallel to this persistent immunity, we observed that Ischgl was relatively spared, compared to similar municipalities, from the prominent second COVID-19 wave that hit Austria in November 2020. In addition, we used sequencing data to show that the local immunity acquired from wild-type infections also helped to curb infections from variants of SARS-CoV-2 which spread in Austria since January 2021. Interpretation The relatively high level of seroprevalence (40-45%) in Ischgl persisted and might have been associated with the observed protection of Ischgl residents against virus infection during the second COVID-19 wave as well as against variant spread in 2021. Funding Funding was provided by the government of Tyrol and the FWF Austrian Science Fund.

Immune transcriptomes of highly exposed SARS-CoV-2 asymptomatic seropositive versus seronegative individuals from the Ischgl community.

SARS-CoV-2 infection ranges from asymptomatic to severe with lingering symptomatology in some. This prompted investigation of whether or not asymptomatic disease results in measurable immune activation post-infection. Immune activation following asymptomatic SARS-CoV-2 infection was characterized through a comparative investigation of the immune cell transcriptomes from 43 asymptomatic seropositive and 52 highly exposed seronegative individuals from the same community 4-6 weeks following a superspreading event. Few of the 95 individuals had underlying health issues. One seropositive individual reported Cystic Fibrosis and one individual reported Incontinentia pigmenti. No evidence of immune activation was found in asymptomatic seropositive individuals with the exception of the Cystic Fibrosis patient. There were no statistically significant differences in immune transcriptomes between asymptomatic seropositive and highly exposed seronegative individuals. Four positive controls, mildly symptomatic seropositive individuals whose blood was examined 3 weeks following infection, showed immune activation. Negative controls were four seronegative individuals from neighboring communities without COVID-19. All individuals remained in their usual state of health through a five-month follow-up after sample collection. In summary, whole blood transcriptomes identified individual immune profiles within a community population and showed that asymptomatic infection within a super-spreading event was not associated with enduring immunological activation.

Immune transcriptomes of highly exposed SARS-CoV-2 asymptomatic seropositive versus seronegative individuals from the Ischgl community.

To investigate prevalence of ongoing activation of inflammation following asymptomatic SARS-CoV-2 infection we characterized immune cell transcriptomes from 43 asymptomatic seropositive and 52 highly exposed seronegative individuals with few underlying health issues following a community superspreading event. Four mildly symptomatic seropositive individuals examined three weeks after infection as positive controls demonstrated immunological activation. Approximately four to six weeks following the event, the two asymptomatic groups showed no significant differences. Two seropositive patients with underlying genetic disease impacting immunological activation were included (Cystic Fibrosis (CF), Nuclear factor-kappa B Essential Modulator (NEMO) deficiency). CF, but not NEMO, associated with significant immune transcriptome differences including some associated with severe SARS-CoV-2 infection (IL1B, IL17A, respective receptors). All subjects remained in their usual state of health from event through five-month follow-up. Here, asymptomatic infection resolved without evidence of prolonged immunological activation. Inclusion of subjects with underlying genetic disease illustrated the pathophysiological importance of context on impact of immunological response.

Immune transcriptomes of highly exposed SARS-CoV-2 asymptomatic seropositive versus seronegative individuals from the Ischgl community.

To investigate prevalence of ongoing activation of inflammation following asymptomatic SARS-CoV-2 infection we characterized immune cell transcriptomes from 43 asymptomatic seropositive and 52 highly exposed seronegative individuals with few underlying health issues following a community superspreading event. Four mildly symptomatic seropositive individuals examined three weeks after infection as positive controls demonstrated immunological activation. Approximately four to six weeks following the event, the two asymptomatic groups showed no significant differences. Two seropositive patients with underlying genetic disease impacting immunological activation were included (Cystic Fibrosis (CF), Nuclear factor-kappa B Essential Modulator (NEMO) deficiency). CF, but not NEMO, associated with significant immune transcriptome differences including some associated with severe SARS-CoV-2 infection (IL1B, IL17A, respective receptors). All subjects remained in their usual state of health from event through five-month follow-up. Here, asymptomatic infection resolved without evidence of prolonged immunological activation. Inclusion of subjects with underlying genetic disease illustrated the pathophysiological importance of context on impact of immunological response.