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Due to the widespread of the COVID-19 pandemic, the SARS-CoV-2 genome is evolving in diverse human populations. Several studies already reported different strains and an increase in the mutation rate. Particularly, mutations in SARS-CoV-2 spike-glycoprotein are of great interest as it mediates infection in human and recently approved mRNA vaccines are designed to induce immune responses against it. We analyzed 1,036,030 SARS-CoV-2 genome assemblies and 30,806 NGS datasets from GISAID and European Nucleotide Archive (ENA) focusing on non-synonymous mutations in the spike protein. Only around 2.5% of the samples contained the wild-type spike protein with no variation from the reference. Among the spike protein mutants, we confirmed a low mutation rate exhibiting less than 10 non-synonymous mutations in 99.6% of the analyzed sequences, but the mean and median number of spike protein mutations per sample increased over time. 5,472 distinct variants were found in total. The majority of the observed variants were recurrent, but only 21 and 14 recurrent variants were found in at least 1% of the mutant genome assemblies and NGS samples, respectively. Further, we found high-confidence subclonal variants in about 2.6% of the NGS data sets with mutant spike protein, which might indicate co-infection with various SARS-CoV-2 strains and/or intra-host evolution. Lastly, some variants might have an effect on antibody binding or T-cell recognition. These findings demonstrate the continuous importance of monitoring SARS-CoV-2 sequences for an early detection of variants that require adaptations in preventive and therapeutic strategies. [ABSTRACT FROM AUTHOR]

Murine cytomegalovirus (mCMV) codes for MHC class-I trafficking modulators m04/gp34, m06/gp48, and m152/gp40. By interacting with the MHC class-Iα chain, these proteins disconnect peptide-loaded MHC class-I (pMHC-I) complexes from the constitutive vesicular flow to the cell surface. Based on the assumption that all three inhibit antigen presentation, and thus the recognition of infected cells by CD8 T cells, they were referred to as "immunoevasins." Improved antigen presentation mediated by m04 in the presence of m152 after infection with deletion mutant mCMV-Δm06W, compared to mCMV-Δm04m06 expressing only m152, led us to propose renaming these molecules "viral regulators of antigen presentation" (vRAP) to account for both negative and positive functions. In accordance with a positive function, m04-pMHC-I complexes were found to be displayed on the cell surface, where they are primarily known as ligands for Ly49 family natural killer (NK) cell receptors. Besides the established role of m04 in NK cell silencing or activation, an anti-immunoevasive function by activation of CD8 T cells is conceivable, because the binding site of m04 to MHC class-Iα appears not to mask the peptide binding site for T-cell receptor recognition. However, functional evidence was based on mCMV-Δm06W, a virus of recently doubted authenticity. Here we show that mCMV-Δm06W actually represents a mixture of an authentic m06 deletion mutant and a mutant with an accidental additional deletion of a genome region encompassing also gene m152. Reanalysis of previously published experiments for the authentic mutant in the mixture confirms the previously concluded positive vRAP function of m04. [ABSTRACT FROM AUTHOR]

Antigen-encoding, lipoplex-formulated RNA (RNA-LPX) enables systemic delivery to lymphoid compartments and selective expression in resident antigen-presenting cells. We report here that the rejection of CT26 tumors, mediated by local radiotherapy (LRT), is further augmented in a CD8+ T cell-dependent manner by an RNA-LPX vaccine that encodes CD4+ T cell-recognized neoantigens (CD4 neoantigen vaccine). Whereas CD8+ T cells induced by LRT alone were primarily directed against the immunodominant gp70 antigen, mice treated with LRT plus the CD4 neoantigen vaccine rejected gp70-negative tumors and were protected from rechallenge with these tumors, indicating a potent poly-antigenic CD8+ T cell response and T cell memory. In the spleens of CD4 neoantigen-vaccinated mice, we found a high number of activated, poly-functional, Th1-like CD4+ T cells against ME1, the immunodominant CD4 neoantigen within the poly-neoantigen vaccine. LRT itself strongly increased CD8+ T cell numbers and clonal expansion. However, tumor infiltrates of mice treated with CD4 neoantigen vaccine/LRT, as compared to LRT alone, displayed a higher fraction of activated gp70-specific CD8+ T cells, lower PD-1/LAG-3 expression and contained ME1-specific IFNγ+ CD4+ T cells capable of providing cognate help. CD4 neoantigen vaccine/LRT treatment followed by anti-CTLA-4 antibody therapy further enhanced the efficacy with complete remission of gp70-negative CT26 tumors and survival of all mice. Our data highlight the power of combining synergistic modes of action and warrants further exploration of the presented treatment schema. [ABSTRACT FROM AUTHOR]

HPV16 infections are associated with a variety of cancers and there is compelling evidence that the transforming activity of HPV16 critically depends on the expression of the viral oncoproteins E6 and E7. Therapeutic cancer vaccines capable of generating durable and specific immunity against these HPV16 antigens hold great promise to achieve long-term disease control. Here we show in mice that HPV16 E7 RNA-LPX, an intravenously administered cancer vaccine based on immuno-pharmacologically optimized antigen-encoding mRNA, efficiently primes and expands antigen-specific effector and memory CD8+ T cells. HPV-positive TC-1 and C3 tumors of immunized mice are heavily infiltrated with activated immune cells and HPV16-specific T cells and are polarized towards a proinflammatory, cytotoxic and less immune-suppressive contexture. E7 RNA-LPX immunization mediates complete and durable remission of progressing tumors. Circulating memory T cells are highly cytotoxic and protect from tumor rechallenge. Moreover, E7 RNA-LPX immunization sensitizes anti-PD-L1 refractory tumors to checkpoint blockade. In conclusion, our data highlight the potential of HPV16 RNA-LPX for the treatment of HPV-driven cancers. [ABSTRACT FROM AUTHOR]

The ability to control autoreactive T cells without inducing systemic immune suppression is the major goal for treatment of autoimmune diseases. The key challenge is the safe and efficient delivery of pharmaceutically well-defined antigens in a noninflammatory context. Here, we show that systemic delivery of nanoparticle-formulated 1 methylpseudouridine-modified messenger RNA (m1Y mRNA) coding for disease-related autoantigens results in antigen presentation on splenic CD11c+ antigen-presenting cells in the absence of costimulatory signals. In several mouse models of multiple sclerosis, the disease is suppressed by treatment with such m1Y mRNA. The treatment effect is associated with a reduction of effector T cells and the development of regulatory T cell (Treg cell) populations. Notably, these Treg cells execute strong bystander immunosuppression and thus improve disease induced by cognate and noncognate autoantigens. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR)–T cells have shown efficacy in patients with B cell malignancies. Yet, their application for solid tumors has challenges that include limited cancer-specific targets and nonpersistence of adoptively transferred CAR-T cells. Here, we introduce the developmentally regulated tight junction protein claudin 6 (CLDN6) as a CAR target in solid tumors and a strategy to overcome inefficient CAR-T cell stimulation in vivo. We demonstrate that a nanoparticulate RNA vaccine, designed for body-wide delivery of the CAR antigen into lymphoid compartments, stimulates adoptively transferred CAR-T cells. Presentation of the natively folded target on resident antigen-presenting cells promotes cognate and selective expansion of CAR-T cells. Improved engraftment of CAR-T cells and regression of large tumors in difficult-to-treat mouse models was achieved at subtherapeutic CAR-T cell doses. [ABSTRACT FROM AUTHOR]