Highlights d Flaviviridae infection alters m 6 A modification of specific cellular mRNAs d Innate immune and ER stress signaling contribute to altered m 6 A modification d Gain of m 6 A regulates RIOK3 translation, and loss of m 6 A influences CIRBP splicing d m 6 A-altered mRNAs encode factors that affect Flaviviridae infection

BackgroundDengue is one of the fastest spreading vector-borne diseases, caused by four antigenically distinct dengue viruses (DENVs). Antibodies against DENVs are responsible for both protection as well as pathogenesis. A vaccine that is safe for and efficacious in all people irrespective of their age and domicile is still an unmet need. It is becoming increasingly apparent that vaccine design must eliminate epitopes implicated in the induction of infection-enhancing antibodies.Methodology/principal findingsWe report a Pichia pastoris-expressed dengue immunogen, DSV4, based on DENV envelope protein domain III (EDIII), which contains well-characterized serotype-specific and cross-reactive epitopes. In natural infection, <10% of the total neutralizing antibody response is EDIII-directed. Yet, this is a functionally relevant domain which interacts with the host cell surface receptor. DSV4 was designed by in-frame fusion of EDIII of all four DENV serotypes and hepatitis B surface (S) antigen and co-expressed with unfused S antigen to form mosaic virus-like particles (VLPs). These VLPs displayed EDIIIs of all four DENV serotypes based on probing with a battery of serotype-specific anti-EDIII monoclonal antibodies. The DSV4 VLPs were highly immunogenic, inducing potent and durable neutralizing antibodies against all four DENV serotypes encompassing multiple genotypes, in mice and macaques. DSV4-induced murine antibodies suppressed viremia in AG129 mice and conferred protection against lethal DENV-4 virus challenge. Further, neither murine nor macaque anti-DSV4 antibodies promoted mortality or inflammatory cytokine production when passively transferred and tested in an in vivo dengue disease enhancement model of AG129 mice.Conclusions/significanceDirecting the immune response to a non-immunodominant but functionally relevant serotype-specific dengue epitope of the four DENV serotypes, displayed on a VLP platform, can help minimize the risk of inducing disease-enhancing antibodies while eliciting effective tetravalent seroconversion. DSV4 has a significant potential to emerge as a safe, efficacious and inexpensive subunit dengue vaccine candidate.

Despite many years of research, a dengue vaccine is not available, and the more advanced live attenuated vaccine candidate in clinical trials requires multiple immunizations with long interdose periods and provides low protective efficacy. Here, we report important contributions to the development of a second-generation dengue vaccine. First, we demonstrate that a nonpropagating vaccine vector based on Venezuelan equine encephalitis virus replicon particles (VRP) expressing two configurations of dengue virus E antigen (subviral particles [prME] and soluble E dimers [E85]) successfully immunized and protected macaques against dengue virus, while antivector antibodies did not interfere with a booster immunization. Second, compared to prME-VRP, E85-VRP induced neutralizing antibodies faster, to higher titers, and with improved protective efficacy. Third, this study is the first to map antigenic domains and specificities targeted by vaccination versus natural infection, revealing that, unlike prME-VRP and live virus, E85-VRP induced only serotype-specific antibodies, which predominantly targeted EDIII, suggesting a protective mechanism different from that induced by live virus and possibly live attenuated vaccines. Fourth, a tetravalent E85-VRP dengue vaccine induced a simultaneous and protective response to all 4 serotypes after 2 doses given 6 weeks apart. Balanced responses and protection in macaques provided further support for exploring the immunogenicity and safety of this vaccine candidate in humans.

35The RNA modification N6-methyladenosine (m 6 A) can modulate mRNA fate and thus 36 affect many biological processes. We analyzed m 6 A modification across the transcriptome 37 following infection by dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and 38 hepatitis C virus (HCV). We found that infection by these viruses in the Flaviviridae family alters 39 m 6 A modification of specific cellular transcripts, including RIOK3 and CIRBP. During viral 40 infection, the addition of m 6 A to RIOK3 promotes its translation, while loss of m 6 A in CIRBP 41 promotes alternative splicing. Importantly, we found that activation of innate immune sensing or 42 the endoplasmic reticulum (ER) stress response by viral infection contributes to the changes in 43 m 6 A modification in RIOK3 and CIRBP, respectively. Further, several transcripts with infection-44 altered m 6 A profiles, including RIOK3 and CIRBP, encode proteins that influence DENV, ZIKV, 45 and HCV infection. Overall, this work reveals that cellular signaling pathways activated during 46 viral infection lead to alterations in m 6 A modification of host mRNAs to regulate infection. 47 48 Introduction 49

Key Points Question What is the durability of humoral and cellular immune responses in individuals who originally received the BNT162b2 vaccine and were boosted with Ad26.COV2.S or BNT162b2? Findings In this cohort study of 68 adults, both Ad26.COV2.S and BNT162b2 were associated with increased humoral and cellular immune responses. Boosting with Ad26.COV2.S was associated with durable antibody and T-cell responses for at least 4 months. Meaning A heterologous mix-and-match vaccine strategy was associated with durable antibody and T-cell responses against the SARS-CoV-2 Omicron variant.