Therapeutic Efficacy of a VSV-GP-based Human Papilloma Virus Vaccine in a Murine Cancer Model.

Human papilloma virus (HPV) infections are associated with almost all cervical cancers and to a lower extend also with anogenital or oropharyngeal cancers. HPV proteins expressed in HPV-associated tumors are attractive antigens for cancer vaccination strategies as self-tolerance, which is associated with most endogenous tumor-associated antigens, does not need to be overcome. In this study, we generated a live attenuated cancer vaccine based on the chimeric vesicular stomatitis virus VSV-GP, which has previously proven to be a potent vaccine vector and oncolytic virus. Genes at an earlier position in the genome more to the 3' end are expressed stronger compared to genes located further downstream. By inserting an HPV16-derived antigen cassette consisting of E2, E6 and E7 into VSV-GP either at first (HPVp1) or fifth (HPVp5) position in VSV-GP's genome we aimed to analyze the effect of vaccine antigen position and consequently expression level on viral fitness, immunogenicity, and anti-tumoral efficacy in a syngeneic mouse tumor model. HPVp1 expressed higher amounts of HPV antigens compared to HPVp5 in vitro but had a slightly delayed replication kinetic which overall translated into increased HPV-specific T cell responses upon vaccination of mice. Immunization with both vectors protected mice in prophylactic and in therapeutic TC-1 tumor models with HPVp1 being more effective in the prophylactic setting. Taken together, VSV-GP is a promising candidate as therapeutic HPV vaccine and first position of the vaccine antigen in a VSV-derived vector seems to be superior to fifth position.

Seroprevalence of SARS-CoV-2 infection in the Tyrolean district of Schwaz at the time of the rapid mass vaccination in March 2021 following B.1.351-variant outbreak.

In order to curb the rapid dissemination of the B.1.351 variant of SARS-CoV-2 in the district of Schwaz and beyond, the EU allocated additional vaccine doses at the beginning of March 2021 to implement a rapid mass vaccination of the population (16+). The aim of our study was to determine the seroprevalence of SARS-CoV-2 among the adult population in the district of Schwaz at the time of the implementation. Data on previous history of infections, symptoms and immunization status were collected using a structured questionnaire. Blood samples were used to determine SARS-CoV-2 specific anti-spike, anti-nucleocapsid and neutralizing antibodies. We recruited 2,474 individuals with a median age (IQR) of 42 (31-54) years. Using the official data on distribution of age and sex, we found a standardized prevalence of undocumented infections at 15.0% (95% CI: 13.2-16.7). Taken together with the officially documented infections, we estimated that 24.0% (95% CI: 22.5-25.6) of the adult population had prior SARS-CoV-2 infection. Hence, the proportion of undocumented infections identified by our study was 55.8% (95% CI: 52.7-58.5). With a vaccination coverage of 10% among the adults population at that time, we imply that a minimum of two-thirds of the target popuation was susceptible to the circulating threat when this unique campaign started.

ADAM10 and ADAM17 promote SARS-CoV-2 cell entry and spike protein-mediated lung cell fusion.

The severe-acute-respiratory-syndrome-coronavirus-2 (SARS-CoV-2) is the causative agent of COVID-19, but host cell factors contributing to COVID-19 pathogenesis remain only partly understood. We identify the host metalloprotease ADAM17 as a facilitator of SARS-CoV-2 cell entry and the metalloprotease ADAM10 as a host factor required for lung cell syncytia formation, a hallmark of COVID-19 pathology. ADAM10 and ADAM17, which are broadly expressed in the human lung, cleave the SARS-CoV-2 spike protein (S) in vitro, indicating that ADAM10 and ADAM17 contribute to the priming of S, an essential step for viral entry and cell fusion. ADAM protease-targeted inhibitors severely impair lung cell infection by the SARS-CoV-2 variants of concern alpha, beta, delta, and omicron and also reduce SARS-CoV-2 infection of primary human lung cells in a TMPRSS2 protease-independent manner. Our study establishes ADAM10 and ADAM17 as host cell factors for viral entry and syncytia formation and defines both proteases as potential targets for antiviral drug development.

Heterologous ChAdOx1/BNT162b2 vaccination induces stronger immune response than homologous ChAdOx1 vaccination: The pragmatic, multi-center, three-arm, partially randomized HEVACC trial.

BACKGROUND: Several COVID-19 vaccines have been approved. The mRNA vaccine from Pfizer/BioNTech (Comirnaty, BNT162b2; BNT) and the vector vaccine from AstraZeneca (Vaxzevria, ChAdOx1; AZ) have been widely used. mRNA vaccines induce high antibody and T cell responses, also to SARS-CoV-2 variants, but are costlier and less stable than the slightly less effective vector vaccines. For vector vaccines, heterologous vaccination schedules have generally proven more effective than homologous schedules. METHODS: In the HEVACC three-arm, single-blinded, adaptive design study (ClinicalTrials.gov Identifier: NCT04907331), participants between 18 and 65 years with no prior history of SARS-CoV-2 infection and a first dose of AZ or BNT were included. The AZ/AZ and the AZ/BNT arms were randomized (in a 1:1 ratio stratified by sex and trial site) and single-blinded, the third arm (BNT/BNT) was observational. We compared the reactogenicity between the study arms and hypothesized that immunogenicity was higher for the heterologous AZ/BNT compared to the homologous AZ/AZ regimen using neutralizing antibody titers as primary endpoint. FINDINGS: This interim analysis was conducted after 234 participants had been randomized and 254 immunized (N=109 AZ/AZ, N=115 AZ/BNZ, N=30 BNT/BNT). Heterologous AZ/BNT vaccination was well tolerated without study-related severe adverse events. Neutralizing antibody titers on day 30 were statistically significant higher in the AZ/BNT and the BNT/BNT groups than in the AZ/AZ group, for B.1.617.2 (Delta) AZ/AZ median reciprocal titer 75.9 (99.9% CI 58.0 - 132.5), AZ/BNT 571.5 (99.9% CI 396.6 - 733.1), and BNT/BNT 404.5 (99.9% CI 68.3 - 1024). Similarly, the frequency and multifunctionality of spike-specific T cell responses was comparable between the AZ/BNT and the BNT/BNT groups, but lower in the AZ/AZ vaccinees. INTERPRETATION: This study clearly shows the immunogenicity and safety of heterologous AZ/BNT vaccination and encourages further studies on heterologous vaccination schedules. FUNDING: This work was supported by the Medical University of Innsbruck, and partially funded by NIAID contracts No. 75N9301900065, 75N93021C00016, and 75N93019C00051.

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.

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.

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.

Characterization of Immune Responses to SARS-CoV-2 and Other Human Pathogenic Coronaviruses Using a Multiplex Bead-Based Immunoassay.

Serological assays that simultaneously detect antibodies to multiple targets of SARS-CoV-2 and to other structurally related coronaviruses provide a holistic picture of antibody response patterns. Well-validated multiplex immunoassays are scarce. Here, we evaluated the performance of an 11-plex serological assay capable of detecting antibodies directed to four antigenic targets of SARS-CoV-2 and to S1 proteins of other human pathogenic coronaviruses. We used 620 well-characterized sera (n = 458 seropositive and n = 110 seronegative for SARS-CoV-2 in the pre-SARS-CoV-2 era and n = 52 seronegative for SARS-CoV-2 in the era of SARS-CoV-2) as positive and negative standards. We calculated the sensitivity, specificity, as well as positive and negative predictive values, including a 95% confidence interval. The difference in mean fluorescence intensity (95% CI) was used to assess a potential cross-reaction between antibodies to SARS-CoV-2 and the other coronaviruses. The sensitivity (95% CI) of detecting anti-SARS-CoV-2 antibodies to four antigenic targets ranged from 83.4% (76.7-86.7) to 93.7% (91.0-95.7) and the specificity from 98.2% (93.6-99.8) to 100% (96.7-100). We observed no obvious cross-reaction between anti-SARS-CoV-2 antibodies and antibodies to the other coronaviruses except for SARS-CoV-1. The high sensitivity and specificity warrant a reliable utilization of the assay in population-based seroprevalence surveys or vaccine efficacy studies.

Marked Increase in Avidity of SARS-CoV-2 Antibodies 7-8 Months After Infection Is Not Diminished in Old Age.

The kinetics of immunoglobulin G (IgG) avidity maturation during severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection obtained from 217 participants of the Ischgl cohort, Austria, was studied 0.5-1.5 months (baseline) and 7-8 months (follow-up) after infection. The IgG avidity assay, using a modified IgG enzyme-linked immunosorbent assay (ELISA) and 5.5 M urea, revealed that old age does not diminish the increase in avidity, detected in all participants positive at both time points, from 18% to 42%. High avidity was associated with a marked residual neutralization capacity in 97.2.% of participants (211/217), which was even higher in the older age group, revealing an important role of avidity assays as easy and cheap surrogate tests for assessing the maturation of the immune system conveying potential protection against further SARS-CoV-2 infections without necessitating expensive and laborious neutralization assays.

Comparison of Four SARS-CoV-2 Neutralization Assays.

Neutralizing antibodies are a major correlate of protection for many viruses including the novel coronavirus SARS-CoV-2. Thus, vaccine candidates should potently induce neutralizing antibodies to render effective protection from infection. A variety of in vitro assays for the detection of SARS-CoV-2 neutralizing antibodies has been described. However, validation of the different assays against each other is important to allow comparison of different studies. Here, we compared four different SARS-CoV-2 neutralization assays using the same set of patient samples. Two assays used replication competent SARS-CoV-2, a focus forming assay and a TCID50-based assay, while the other two assays used replication defective lentiviral or vesicular stomatitis virus (VSV)-based particles pseudotyped with SARS-CoV-2 spike. All assays were robust and produced highly reproducible neutralization titers. Titers of neutralizing antibodies correlated well between the different assays and with the titers of SARS-CoV-2 S-protein binding antibodies detected in an ELISA. Our study showed that commonly used SARS-CoV-2 neutralization assays are robust and that results obtained with different assays are comparable.

Fcγ Receptor Type I (CD64)-Mediated Impairment of the Capacity of Dendritic Cells to Activate Specific CD8 T Cells by IgG-opsonized Friend Virus.

Dendritic cells (DCs) express Fcγ receptors (FcγRs) for the binding immune complexes (ICs) consisting of IgG and antigens (Ags). IC⁻FcγR interactions have been demonstrated to enhance activation and antigen-presenting functions of DCs. Utilizing Friend virus (FV), an oncogenic mouse retrovirus, we investigated the effect of IgG-opsonization of retroviral particles on the infection of DCs and the subsequent presentation of viral antigens by DCs to virus-specific CD8 T cells. We found that opsonization by virus-specific non-neutralizing IgG abrogated DC infection and as a consequence significantly reduced the capacity of DCs to activate virus-specific CD8 T cells. Effects of IgG-opsonization were mediated by the high-affinity FcγR type I, CD64, expressed on DCs. Our results suggest that different opsonization patterns on the retroviral surface modulate infection and antigen-presenting functions of DCs, whereby, in contrast to complement, IgG reduces the capacity of DCs to activate cytotoxic T cell (CTL) responses.