Highlights d T cell responses recognize at least 30-40 epitopes in each donor d Immunodominance is correlated with HLA binding d Immunodominant regions for CD4 + T cells have minimal overlap with antibody epitopes d CD8 + T cell responses depend on the repertoire of HLA class I alleles

SUMMARYT cells are involved in control of SARS-CoV-2 infection. To establish the patterns of immunodominance of different SARS-CoV-2 antigens, and precisely measure virus-specific CD4+ and CD8+ T cells, we studied epitope-specific T cell responses of approximately 100 convalescent COVID-19 cases. The SARS-CoV-2 proteome was probed using 1,925 peptides spanning the entire genome, ensuring an unbiased coverage of HLA alleles for class II responses. For HLA class I, we studied an additional 5,600 predicted binding epitopes for 28 prominent HLA class I alleles, accounting for wide global coverage. We identified several hundred HLA-restricted SARS-CoV-2-derived epitopes. Distinct patterns of immunodominance were observed, which differed for CD4+ T cells, CD8+ T cells, and antibodies. The class I and class II epitopes were combined into new epitope megapools to facilitate identification and quantification of SARS-CoV-2-specific CD4+ and CD8+ T cells.

Members of the flavivirus genus share a high level of sequence similarity and often circulate in the same geographical regions. However, whether T cells induced by one viral species cross-react with other related flaviviruses has not been globally addressed. In this study, we tested pools of epitopes derived from dengue (DENV), Zika (ZIKV), Japanese encephalitis (JEV), West Nile (WNV), and yellow fever (YFV) viruses by intracellular cytokine staining (ICS) using peripheral blood mononuclear cells (PBMCs) of individuals naturally exposed to DENV or immunized with DENV (TV005) or YF17D vaccine. CD8 T cell responses recognized epitopes from multiple flaviviruses; however, the magnitude of cross-reactive responses was consistently severalfold lower than those to the autologous epitope pools and was associated with lower expression of activation markers such as CD40L, CD69, and CD137. Next, we characterized the antigen sensitivity of short-term T cell lines (TCL) representing 29 different individual epitope/donor combinations. TCL derived from DENV monovalent vaccinees induced CD8 and CD4 T cells that cross-reacted within the DENV serocomplex but were consistently associated with >100-fold-lower antigen sensitivity for most other flaviviruses, with no cross-recognition of YFV-derived peptides. CD8 and CD4 TCL from YF17D vaccinees were associated with very limited cross-reactivity with any other flaviviruses and in five out of eight cases >1,000-fold-lower antigen sensitivity. Overall, our data suggest limited cross-reactivity for both CD4 and CD8 T cell responses between flaviviruses and have implications for understanding immunity elicited by natural infection and strategies to develop live attenuated vaccines against flaviviral species. IMPORTANCE The envelope (E) protein is the dominant target of neutralizing antibodies for dengue virus (DENV) and yellow fever virus (YFV). Accordingly, several DENV vaccine constructs use the E protein in a live attenuated vaccine format, utilizing a backbone derived from a heterologous flavivirus (such as YF) as a delivery vector. This backbone comprises the nonstructural (NS) and capsid (C) antigens, which are dominant targets of T cell responses. Here, we demonstrate that cross-reactivity at the level of T cell responses among different flaviviruses is very limited, despite high levels of sequence homology. Thus, the use of heterologous flavivirus species as a live attenuated vaccine vector is not likely to generate optimal T cell responses and might thus impair vaccine performance.

T cell responses induced by attenuated flavivirus vaccination are specific and show limited cross-1 reactivity with other flavivirus species. 2 Abstract word count: 247 24 Manuscript word count: 6692 25 Abstract 26Members of the flavivirus genus share a high level of sequence similarity and often circulate in 27 the same geographical regions. However, whether T cells induced by one viral species cross-28 react with other related flaviviruses has not been globally addressed. Here, we tested pools of 29 epitopes derived from dengue (DENV), zika (ZIKV), Japanese Encephalitis (JEV), West Nile 30 (WNV), and yellow fever (YFV) viruses by Intracellular Cytokine Staining (ICS) using PBMCs 31 of individuals naturally exposed to DENV or immunized with DENV (TV005) or YF17D 32vaccines. CD8 T cell responses recognized epitopes from multiple flaviviruses, however, the 33 magnitude of cross-reactive responses was consistently several-fold lower than those to the 34 autologous epitope pools, and associated with lower expression of activation markers such as 35 CD40L, CD69, and CD137. Next, we characterized the antigen sensitivity of short-term T cell 36 lines (TCL) representing twenty-nine different individual epitope/donor combinations. TCL 37 derived from DENV monovalent vaccinees induced CD8 and CD4 T cells that cross-reacted 38 within the DENV serocomplex but were consistently associated with more than 100-fold lower 39 antigen sensitivity for most other flaviviruses, with no cross-recognition of YFV derived 40 peptides. CD8 and CD4 TCL from YF17D vaccinees were associated with very limited cross-41 reactivity with any other flaviviruses, and in five out of eight cases more than 1000-fold lower 42 antigen sensitivity. Overall, our data suggest limited cross-reactivity for both CD4 and CD8 T 43 cell responses between flaviviruses and has implications for understanding immunity elicited by 44 natural infection, and strategies to develop live attenuated vaccines against flaviviral species. 45 Importance 46The envelope (E) protein is the dominant target of neutralizing antibodies for dengue virus 47 (DENV) and yellow fever virus (YFV). Accordingly, several DENV vaccine constructs use the E 48 protein in a live attenuated vaccine format, utilizing a backbone derived from a heterologous 49 flavivirus (such as YF) as a delivery vector. This backbone comprises the non-structural (NS) 50 and capsid (C) antigens which are dominant targets of T cell responses. Here, we demonstrate 51 that cross-reactivity at the level of T cell responses amongst different flaviviruses is very limited, 52 despite high levels of sequence homology. Thus, the use of heterologous flavivirus species as a 53 live attenuated vaccine vector is not likely to generate optimal T cell responses, and might thus 54 impair vaccine performance. 55 Introduction 56Flavivirus infections can cause a wide variety of clinical manifestations and complications 57 in humans, ranging from undifferentiated fever, vascular leak syndrome, encephalitis and death. 58Because of their...