Heterologous priming with the ChAdOx1 nCoV-19 vector vaccine followed by boosting with a messenger RNA vaccine (BNT162b2 or mRNA-1273) is currently recommended in Germany, although data on immunogenicity and reactogenicity are not available. In this observational study we show that, in healthy adult individuals (n = 96), the heterologous vaccine regimen induced spike-specific IgG, neutralizing antibodies and spike-specific CD4 T cells, the levels of which which were significantly higher than after homologous vector vaccine boost (n = 55) and higher or comparable in magnitude to homologous mRNA vaccine regimens (n = 62). Moreover, spike-specific CD8 T cell levels after heterologous vaccination were significantly higher than after both homologous regimens. Spike-specific T cells were predominantly polyfunctional with largely overlapping cytokine-producing phenotypes in all three regimens. Recipients of both the homologous vector regimen and the heterologous vector/mRNA combination reported greater reactogenicity following the priming vector vaccination, whereas heterologous boosting was well tolerated and comparable to homologous mRNA boosting. Taken together, heterologous vector/mRNA boosting induces strong humoral and cellular immune responses with acceptable reactogenicity profiles.

Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species. Herpesvirus proteins interact with each other in a complex manner throughout the infectious cycle. This is probably best exemplified in the process where a large number of viral proteins come together to form new viral particles which are subsequently released from the infected cell. A more detailed understanding of how viral proteins interact with each other might assist the development of drugs which may inhibit these interactions and consequently block viral replication. Here we present three genome-wide studies of protein-protein interactions in the herpesviruses herpes simplex virus I, murine cytomegalovirus and Epstein-Barr virus. Altogether we identified 735 interactions in the three viruses, most of which have not previously been reported. By combining these studies with our previously published studies for Kaposi's sarcoma-associated herpesvirus and varicella-zoster virus we were able to perform a comparative analysis of interactions in five related viral species. We observed that a high proportion of interactions were conserved between the different species, despite a low degree of sequence conservation. This implies that by comparing interaction data, we were able to increase the coverage of our viral networks and thus obtain a better and more complete picture of interactions between herpesviral proteins.

Knowledge on the immunogenicity of vector-based and mRNA-vaccines in solid organ transplant recipients is limited. Therefore, SARS-CoV-2–specific T cells and antibodies were analyzed in 40 transplant recipients and 70 controls after homologous or heterologous vaccine-regimens. Plasmablasts and SARS-CoV-2–specific CD4 and CD8 T cells were quantified using flow cytometry. Specific antibodies were analyzed by ELISA and neutralization assay. The two vaccine types differed after the first vaccination, as IgG and neutralizing activity were more pronounced after mRNA priming (p = .0001 each), whereas CD4 and CD8 T cell levels were higher after vector priming (p = .009; p = .0001). All regimens were well tolerated, and SARS-CoV-2–specific antibodies and/or T cells after second vaccination were induced in 100% of controls and 70.6% of transplant recipients. Although antibody and T cell levels were lower in patients, heterologous vaccination led to the most pronounced induction of antibodies and CD4 T cells. Plasmablast numbers were significantly higher in controls and correlated with SARS-CoV-2–specific IgG- and T cell levels. While antibodies were only detected in 35.3% of patients, cellular immunity was more frequently found (64.7%) indicating that assessment of antibodies is insufficient to identify COVID-19-vaccine responders. In conclusion, heterologous vaccination seems promising in transplant recipients, and combined analysis of humoral and cellular immunity improves the identification of responders among immunocompromised individuals.

BACKGROUND: Patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) differ in the severity of disease. We hypothesized that characteristics of SARS-CoV-2–specific immunity correlate with disease severity. METHODS: In this study, SARS-CoV-2–specific T cells and antibodies were characterized in uninfected controls and patients with different coronavirus disease 2019 (COVID-19) disease severity. SARS-CoV-2–specific T cells were flow cytometrically quantified after stimulation with SARS-CoV-2 peptide pools and analyzed for expression of cytokines (IFN-γ, IL-2, and TNF-α) and markers for activation, proliferation, and functional anergy. SARS-CoV-2–specific IgG and IgA antibodies were quantified using ELISA. Moreover, global characteristics of lymphocyte subpopulations were compared between patient groups and uninfected controls. RESULTS: Despite severe lymphopenia affecting all major lymphocyte subpopulations, patients with severe disease mounted significantly higher levels of SARS-CoV-2–specific T cells as compared with convalescent individuals. SARS-CoV-2–specific CD4 ⁺ T cells dominated over CD8 ⁺ T cells and closely correlated with the number of plasmablasts and SARS-CoV-2–specific IgA and IgG levels. Unlike in convalescent patients, SARS-CoV-2–specific T cells in patients with severe disease showed marked alterations in phenotypical and functional properties, which also extended to CD4 ⁺ and CD8 ⁺ T cells in general. CONCLUSION: Given the strong induction of specific immunity to control viral replication in patients with severe disease, the functionally altered characteristics may result from the need for contraction of specific and general immunity to counteract excessive immunopathology in the lung. FUNDING: The study was supported by institutional funds to MS and in part by grants of Saarland University, the State of Saarland, and the Rolf M. Schwiete Stiftung.

Transplant patients are at increased risk of viral complications due to impaired control of viral replication, resulting from HLA mismatching between graft and host and the immunosuppression needed to avert alloimmune reactions. In the past decade, quantitative viral load measurements have become widely available to identify patients at risk and to inform treatment decisions with respect to immunosuppressive drugs and antiviral therapies. Because viral loads are viewed as the result of viral replication and virus‐specific immune control, virus‐specific T cell monitoring has been explored to optimize management of adenovirus, BK polyomavirus and cytomegalovirus (“ABC”) in transplant patients. Although most studies are descriptive using different technologies, the overall results show that the quantity and quality of virus‐specific T cells inversely correlate with viral replication, whereby strong cellular immune responses are associated with containment of viral replication. The key obstacles to the introduction of assays for virus‐specific T cells into clinical practice is the definition of reliable cutoffs for clinical decision making, the poor negative predictive value of some assays, and the absence of interventional trials justifying changes of antiviral treatment or immunosuppression. More clinical research is needed using optimized assays and targets before standardization and commutability can be envisaged as achieved for viral load testing.

BK polyomavirus (BKPyV) infection is widespread and typically asymptomatic during childhood, but may cause nephropathy in kidney transplant recipients. However, there is only limited knowledge on BKPyV‐specific immunity in children and adults, and its role in BKPyV‐replication and disease posttransplant. We therefore characterized BKPyV‐specific immunity from 122 immunocompetent individuals (1–84 years), 38 adult kidney recipients with (n = 14) and without BKPyV‐associated complications (n = 24), and 25 hemodialysis (HD) patients. Blood samples were stimulated with overlapping peptides of BKPyV large‐T antigen and VP1 followed by flow‐cytometric analysis of activated CD4 T cells expressing interferon‐γ, IL‐2 and tumor necrosis factor‐α. Antibody‐levels were determined using enzyme‐linked immunosorbent assay. Both BKPyV‐IgG levels and BKPyV‐specific CD4 T cell frequencies were age‐dependent (p = 0.0059) with maximum levels between 20 and 30 years (0.042%, interquartile range 0.05%). Transplant recipients showed a significantly higher BKPyV‐specific T cell prevalence (57.9%) compared to age‐matched controls (21.7%) or HD patients (28%, p = 0.017). Clinically relevant BKPyV‐replication was associated with elevated frequencies of BKPyV‐specific T cells (p = 0.0002), but decreased percentage of cells expressing multiple cytokines (p = 0.009). In conclusion, BKPyV‐specific cellular immunity reflects phases of active BKPyV‐replication either after primary infection in childhood or during reactivation after transplantation. Combined analysis of BKPyV‐specific T cell functionality and viral loads may improve individual risk assessment.