KEYWORDS coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), nucleocapsid protein, vaccine D uring the current coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there has been an unprecedented level of global collaboration that has led to a rapid characterization of SARS-CoV-2 (1). Its sequence shares 79.6% identity to SARS-CoV (1, 2), the infectious virus that caused an epidemic in 2003 (2, 3). SARS-CoV-2 has a single-stranded, plus-sense, RNA genome of approximately 30 kb, which includes five major open reading frames encoding nonstructural replicase polyproteins and structural proteins (1), namely, spike (S) (4-6), envelope (E), membrane (M), and nucleocapsid (N) (7), and they are in the same order and of approximately the same sizes as those in SARS-CoV. The SARS-CoV-2 S protein is being used as the leading target antigen in vaccine development (8, 9). However, the complex molecular details of viral entry may lead to complications with the vaccine response, similar to those seen with HIV type 1 (HIV-1) Env protein vaccine efforts (10). The SARS-CoV-2 S gene has 76% amino acid similarity to the SARS-CoV S gene (11), and nonsynonymous mutations developed in the S protein as the SARS-CoV epidemic progressed (12, 13). In contrast, the N gene is more conserved and stable, with 90% amino acid homology and fewer mutations over time (2, 3, 11, 14-16). N proteins of many coronaviruses are highly immunogenic and are expressed abundantly during infection (17). High levels of IgG antibodies against N have been detected in sera from SARS patients (18), and the N protein is a representative antigen for the T-cell response in a vaccine setting, inducing SARS-specific T-cell proliferation and cytotoxic activity (19, 20). We have already shown that the middle or C-terminal region of the SARS-CoV N protein is important for eliciting antibodies against SARS-CoV during the immune response (21-23). New reports have additionally shown that the crystal structure of the SARS-CoV-2 nucleocapsid protein is similar to those of previously described coronavirus N proteins, but their surface electrostatic potential characteristics are distinct (7). Sheikh et al. studied the factors influencing N gene variations among 13 coronaviruses and how these affect virus-host relationships, reporting a high AT% and low GC% in the nucleotide contents of SARS coronavirus (24). In this issue, Cong et al. (17) used a mouse hepatitis virus (MHV) model to show that the viral nucleocapsid (N) protein contributes to forming helical ribonucleoproteins during the packaging of the RNA genome, regulating viral RNA synthesis during replication and transcription and modulating metabolism in infected subjects. This study complements others that have shown N to have multiple functions (25). It is becoming more evident just how critical this protein is for multiple steps of the viral life cycle. These reports offer important and timely insights relevant ...

Coxsackievirus A16 (CV-A16), CV-A6, and enterovirus D68 (EV-D68) belong to the Picornaviridae family and are major causes of hand, foot, and mouth disease (HFMD) and pediatric respiratory disease worldwide. The biological characteristics of these viruses, especially their interplay with the host innate immune system, have not been well investigated. In this study, we discovered that the 3C pro proteins from CV-A16, CV-A6, and EV-D68 bind melanoma differentiation-associated gene 5 (MDA5) and inhibit its interaction with MAVS. Consequently, MDA5-triggered type I interferon (IFN) signaling in the retinoic acid-inducible gene I-like receptor (RLR) pathway was blocked by the CV-A16, CV-A6, and EV-D68 3C pro proteins. Furthermore, the CV-A16, CV-A6, and EV-D68 3C pro proteins all cleave transforming growth factor ␤-activated kinase 1 (TAK1), resulting in the inhibition of NF-B activation, a host response also critical for Toll-like receptor (TLR)-mediated signaling. Thus, our data demonstrate that circulating HFMD-associated CV-A16 and CV-A6, as well as severe respiratory disease-associated EV-D68, have developed novel mechanisms to subvert host innate immune responses by targeting key factors in the RLR and TLR pathways. Blocking the ability of 3C pro proteins from diverse enteroviruses and coxsackieviruses to interfere with type I IFN induction should restore IFN antiviral function, offering a potential novel antiviral strategy.IMPORTANCE CV-A16, CV-A6, and EV-D68 are emerging pathogens associated with hand, foot, and mouth disease and pediatric respiratory disease worldwide. The pathogenic mechanisms of these viruses are largely unknown. Here we demonstrate that the CV-A16, CV-A6, and EV-D68 3C pro proteins block MDA5-triggered type I IFN induction. The 3C pro proteins of these viruses bind MDA5 and inhibit its interaction with MAVS. In addition, the CV-A16, CV-A6, and EV-D68 3C pro proteins cleave TAK1 to inhibit the NF-B response. Thus, our data demonstrate that circulating HFMDassociated CV-A16 and CV-A6, as well as severe respiratory disease-associated EV-D68, have developed a mechanism to subvert host innate immune responses by simultaneously targeting key factors in the RLR and TLR pathways. These findings indicate the potential merit of targeting the CV-A16, CV-A6, and EV-D68 3C pro proteins as an antiviral strategy.KEYWORDS MDA5, TAK1, MAVS, 3C protease, CV-A16, CV-A6, EV-D68, HFMD, innate immune response

BackgroundAlthough therapeutic cancer vaccines have been mostly disappointing in the clinic, the advent of novel immunotherapies and the future promise of neoantigen-based therapies have created the need for new vaccine modalities that can easily adapt to current and future developments in cancer immunotherapy. One such novel platform is a DNA vaccine fusing the chemokine Macrophage Inflammatory Protein-3α (MIP-3α) to an antigen, here melanoma antigen gp100. Previous published work has indicated that MIP-3α targets nascent peptides to immature dendritic cells, leading to processing by class I and II MHC pathways. This platform has shown enhanced efficacy in prophylactic melanoma and therapeutic lymphoma model systems.MethodsThe B16F10 melanoma syngeneic mouse model system was utilized, with a standard therapeutic protocol: challenge with lethal dose of B16F10 cells (5 × 104) on day 0 and then vaccinate by intramuscular electroporation with 50 μg plasmid on days three, 10, and 17. Efficacy was assessed by analysis of tumor burden, tumor growth, and mouse survival, using the statistical tests ANOVA, mixed effects regression, and log-rank, respectively. Immunogenicity was assessed by ELISA and flow cytometric methods, including intracellular cytokine staining to assess vaccine-specific T-cell responses, all tested by ANOVA.ResultsWe demonstrate that the addition of MIP3α to gp100 significantly enhances systemic anti-gp100 immunological parameters. Further, chemokine-fusion vaccine therapy significantly reduces tumor burden, slows tumor growth, and enhances mouse overall survival compared to antigen-only, irrelevant-antigen, and mock vaccines, with efficacy mediated by both CD4+ and CD8+ effector T cells. Antigen-only, irrelevant-antigen, and chemokine-fusion vaccines elicit significantly higher and similar CD4+ and CD8+ tumor-infiltrating lymphocyte (TIL) levels compared to mock vaccine. However, vaccine-specific CD8+ TILs are significantly higher in the chemokine-fusion vaccine group, indicating that the critical step induced by the fusion vaccine construct is the enhancement of vaccine-specific T-cell effectors.ConclusionsThe current study shows that fusion of MIP3α to melanoma antigen gp100 enhances the immunogenicity and efficacy of a DNA vaccine in a therapeutic B16F10 mouse melanoma model. This study analyzes an adaptable and easily produced MIP3α-antigen modular vaccine platform that could lend itself to a variety of functionalities, including combination treatments and neoantigen vaccination in the pursuit of personalized cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-016-0189-y) contains supplementary material, which is available to authorized users.

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