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Dengue virus (DENV) is the most common mosquito-borne flavivirus, and it affects millions of people globally every year. Currently, there are no approved drugs for the treatment of dengue infection. By screening a natural product library, we identified a novel compound, cyclovirobuxine D (Cvb D), that displays anti-DENV activity. Cvb D inhibits DENV replication in vitro in a dose-dependent manner and protects suckling mice against lethal DENV infection. Mechanistically, Cvb D regulates the expression of genes related to the cellular cholesterol pathway. As a result, Cvb D increases cellular cholesterol synthesis and accumulation, activates mTOR, and inhibits viral-dependent autophagy. Cvb D does not suppress autophagy initiation but impedes the nuclear translocation of the lysosome transcription factor TFEB. In addition, Cvb D restricts the replication of other positive-strand RNA viruses such as Zika virus and Coxsackievirus B3. We speculate that Cvb D could be a broad-spectrum antiviral drug candidate for use against positive-strand RNA viruses that require autophagy for optimal replication.

In this study, we investigated drug resistance levels in human immunodeficiency virus (HIV)-1-infected patients in Suzhou by retrospectively analyzing this property and the characteristics of circulating HIV-1 strains collected from 2009 to 2014. A total of 261 HIV-1-positive plasma samples, confirmed by the Suzhou CDC, were collected and evaluated to detect HIV-1 drug resistance genotypes using an in-house method. The pol gene fragment was amplified, and its nucleic acid sequence was determined by Sanger sequencing. Drug resistance mutations were then analyzed using the Stanford University HIV resistance database ( https://hivdb.stanford.edu ). A total of 216 pol gene fragments were amplified and sequenced with 16.7% (36/216) of sequences revealing these mutations. The drug resistance rates of protease, nucleoside reverse transcriptase, and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were 4/36 (11.1%), 2/36 (5.6%), and 30/36 (83.3%), respectively. Five surveillance drug resistance mutations were found in 36 sequences, of which, three were found among specimens of men who have sex with men. Potential low-level resistance accounted for 33% of amino acid mutations associated with NNRTIs. Two of the mutations, M230L and L100I, which confer a high level of resistance efavirenz (EFV) and nevirapine (NVP) used as NNRTIs for first-line antiretroviral therapy (ART), were detected in this study. Therefore, when HIV-1 patients in Suzhou are administered fist-line ART, much attention should be paid to the status of these mutations that cause resistance to EVP, EFV, and NVP.

Cathelicidins have been shown to effectively inhibit flavivirus replication in vitro. However, the effects of mouse and human endogenous cathelicidins on flavivirus infection in vivo are rarely known. We herein found that mouse endogenous cathelicidin CRAMP was significantly up-regulated upon Zika virus (ZIKV) infection. CRAMP deficiency markedly exacerbated ZIKV replication in testis, and aggravated ZIKV-induced testicular damage and spermatic damage in mice, indicating that endogenous cathelicidin is required for protection against ZIKV-caused male infertility in mice. In vitro antiviral assay showed that both mouse cathelidin CRAMP and human cathelicidin LL-37 obviously reduced ZIKV-caused cytopathic effect and inhibited ZIKV replication in Vero cells. Antiviral mechanism revealed that they both directly inactivated ZIKV virons by binding to ZIKV virons and inducing the leakage of ZIKV genomic RNA, consequently inactivated ZIKV virons. In vivo antiviral assay indicated that both of them effectively inhibited ZIKV replication in C57BL/6J and IFNα/β receptor-deficient (Ifnar1

ZIKV infection can cause other severe neurological disorders, such as Guillain-Barre syndrome. Currently, more than 70 countries have reported Zika virus (ZIKV) infections, making it a global public health issue. However, there is no clinically approved vaccine available. Flaviviruses often show antigenic cross-reactivity, which can be beneficial and result in cross-protection. However, humoral cross-reactivity can also exacerbate disease via antibody-dependent enhancement (ADE). The prM-E proteins have been the primary targets of most ZIKV vaccine candidates. Therefore, to increase safety, it is necessary to investigate the use of protective ZIKV antigen for vaccine development as compared with prM-E or E protein. The capsid protein plays a crucial role in Flaviviridae biology, with a report indicating that a dengue virus vaccine engineered with a capsid protein alone produced neutralizing-antibody independent immunity and significantly reduced viral loads in the brains of challenged monkeys. In the present study, a recombinant vesicular stomatitis virus (VSV)-based vaccine carrying the ZIKV capsid protein (VSV-Capsid) was generated. VSV-capsid vaccination induced strong humoral immune response as well as cellular immune response compared with E protein based vaccine (VSV-E260-425). More importantly, the protective role was found in mice with VSV-capsid vaccination upon ZIKV infection. The viral RNA is significantly reduced in spinal cord, brain and testis of these immunized mice. Our findings demonstrated that the ZIKV capsid protein was an effective antigen in VSV vector-based delivery for ZIKV vaccine design.

It is unclear how the type I IFN response is regulated by mycobacterial determinants. Here, we characterized the previously unreported role of M. tuberculosis MmsA in immunological regulation of type I IFN response by targeting the central adaptor STING in the DNA sensing pathway. We identified STING-interacting MmsA by coimmunoprecipitation-mass spectrometry-based (IP-MS) proteomic analysis and showed MmsA interacting with STING and autophagy receptor p62 via its N terminus and C terminus, respectively. We also showed that MmsA downregulated type I IFN by promoting p62-mediated STING degradation. Moreover, the MmsA mutant R138W is potentially associated with the virulence of M. tuberculosis clinical strains owing to the modulation of STING protein. Our results provide novel insights into the regulatory mechanism of type I IFN response manipulated by mycobacterial MmsA and the additional cross talk between autophagy and STING in M. tuberculosis infection, wherein a protein from microbial pathogens induces autophagic degradation of host innate immune molecules., Type I interferon (IFN) plays an important role in Mycobacterium tuberculosis persistence and disease pathogenesis. M. tuberculosis has evolved a number of mechanisms to evade host immune surveillance. However, it is unclear how the type I IFN response is tightly regulated by the M. tuberculosis determinants. Stimulator of interferon genes (STING) is an essential adaptor for type I IFN production triggered by M. tuberculosis genomic DNA or cyclic dinucleotides upon infection. To investigate how the type I IFN response is regulated by M. tuberculosis determinants, immunoprecipitation-mass spectrometry-based (IP-MS) proteomic analysis was performed to screen proteins interacting with STING in the context of M. tuberculosis infection. Among the many predicted candidates interacting with STING, the M. tuberculosis coding protein Rv0753c (MmsA) was identified. We confirmed that MmsA binds and colocalizes with STING, and the N-terminal regions of MmsA (amino acids [aa] 1 to 251) and STING (aa 1 TO 190) are responsible for MmsA-STING interaction. Type I IFN production was impaired with exogenous expression of MmsA in RAW264.7 cells. MmsA inhibited the STING-TBK1-IRF3 pathway, as evidenced by reduced STING levelS and subsequent IRF3 activation. Furthermore, MmsA facilitated p62-mediated STING autophagic degradation by binding p62 with its C terminus (aa 252 to 455), which may account for the negative regulation of M. tuberculosis MmsA in STING-mediated type I IFN production. Additionally, the M. tuberculosis mmsA R138W mutation, detected in a hypervirulent clinical isolate, enhanced the degradation of STING, implying the important relevance of MmsA in disease outcome. Together, we report a novel mechanism where M. tuberculosis MmsA serves as an antagonist of type I IFN response by targeting STING with p62-mediated autophagic degradation.

Tuberculosis (TB) remains a major health problem worldwide, and the development of effective vaccines is urgently needed. Vaccination strategies based on heterologous prime-boost protocols using Mycobacterium bovis bacillus Calmette-Guerin (BCG) as primer and modified vaccinia virus Ankara strain expressing the mycobacterial antigen Ag85A (MVA85A) as booster may increase the protective efficacy of BCG. In addition, vaccination with the recombinant viral vaccine vesicular stomatitis virus (VSV)-846 (Rv3615c, Mtb10.4, and Rv2660c) can elicit a remarkable T-cell-mediated immune response and provide an effective long-term protection after the BCG challenge. In this study, we used VSV-846 to boost BCG and evaluated its immunogenicity in BALB/c mice. In this prime-boost approach, boosting with VSV-846 significantly enhanced IFN-γ CD4 T cell responses, which are crucial for anti-TB immune responses. Moreover, VSV-846 boosting significantly reduced pathology compared with mock vaccination, and decreased the bacterial loads in lung tissues compared with BCG or VSV-846 vaccination alone. The analysis of vaccine-induced immunity identified that polyfunctional T cells might contribute to the enhanced protection by VSV-846 boosting. This study proved that viral booster VSV-846 in mice improved the protection against mycobacteria infection, which could be helpful in designing an efficient vaccination strategy against TB in humans.

Background Coxsackievirus (CVB3) infection is the most common cause of viral myocarditis (VMC) characterized by viral infection and myocardial inflammation. ADAR1, the interferon (IFN)-inducible adenosine deaminase acting on RNA, has been reported to be functional in various viruses. Recent studies have demonstrated that ADAR1 holds an antiviral role or promotes viral replication depending on virus type. Objectives This study aims to investigate whether or not ADAR1 affects CVB3-induced VMC. Methods We generated an acute VMC mouse model by CVB3 infection. ADAR1 expression was manipulated by in vivo polyethyleneimine-mediated ADAR1 up/down-regulation plasmid delivery. Results Our study indicated that ADAR1 was up-regulated after CVB3 infection. ADAR1 down-regulation in the early stage of viral infection ameliorated CVB3-induced VMC. In this stage, viral replication was a key point to initiate inflammatory response. ADAR1 may affect inflammation mainly through viral replication as shown by the elevated IFN-β and decreased viral load with ADAR1 down-regulation. However, when the inflammatory response was established in the middle–late stage of viral infection, ADAR1 down-regulation aggravated disease progression. In this stage, Western blot analysis indicated that ADAR1 may directly influence inflammatory response through PKR and NF-κB signaling. Conclusion We demonstrated that ADAR1 exhibited double-edged effects during the early or middle–late stage of CVB3-induced VMC. Our findings may provide new insights into the therapeutic treatments of VMC.

Outbreaks of viral infections are a global health burden. Although type I interferon (IFN-I) exerts broad-spectrum antiviral effects, its antiviral efficacy in host cells is largely restricted by viruses. How the antiviral efficacy of IFN-I can be improved remains to be explored. Here, we identified the ADP-ribosyltransferase poly(ADP-ribose) polymerase family member 11 (PARP11) as a potent regulator of IFN-I antiviral efficacy. PARP11 does not restrict IFN-I production induced by vesicular stomatitis virus or Sendai virus but inhibits the strength of IFN-I-activated signalling. Mechanistically, PARP11 mono-ADP-ribosylates the ubiquitin E3 ligase β-transducin repeat-containing protein (β-TrCP). Mono-ADP-ribosylation of β-TrCP promotes IFNα/β receptor subunit 1 (IFNAR1) ubiquitination and degradation. Moreover, PARP11 expression is upregulated by virus infections, including vesicular stomatitis virus, herpes simplex virus-1 and influenza A virus, thus promoting ADP-ribosylation-mediated viral evasion. We further highlight the potential for repurposing clinical ADP-ribosylation inhibitors. We found that rucaparib can target PARP11 to stabilize IFNAR1 and therefore exhibits efficient enhancement of IFN-I signalling and the host antiviral response. Consequently, rucaparib renders mice more resistant to viral infection. Our study updates the understanding of how β-TrCP regulates its substrates and may provide a druggable target for improving IFN antiviral efficacy.

The recurrent Coxsackievirus B3 (CVB3) infection is the most important cause of intractable myocarditis which often leads to chronic myocarditis and even dilated cardiomyopathy. Therefore, enhanced DNA vaccines capable of memory CD8 T cells are essential for long-lasting immunological protection against CVB3 infection. In this study, absent in melanoma 2 (AIM2) was used as an adjuvant to enhance the induction of memory CD8 T cells elicited by VP1 (viral capsid protein 1) vaccine. Mice were intramuscularly injected with 50 μg AIM2 plasmid and equal amount of VP1 plasmid (pAIM2/pVP1) vaccine 4 times at 2 week-intervals. We observed that the protection of pAIM2/pVP1 vaccine against CVB3 challenge was evidenced by significantly improved cardiac function, reduced myocardial injuries, and increased survival rate when compared with immunization with pVP1. Co-immunization with pAIM2/pVP1 robustly augmented T lymphocytes proliferation and CVB3-specific cytotoxic T lymphocyte responses. Importantly, 16 weeks after the last immunization, pAIM2/pVP1 co-immunization significantly enhanced the expression of Bcl-6, SOCS3, and Sca-1 which are critical for memory CD8 T cells as compared with pVP1 immunization. Notably, CD8 T cells that are likely vaccine-induced memory T cells were responsible for the protective efficacy of pAIM2/pVP1 vaccine by abolition of a CD8 T cell immune response following a lethal dose of CVB3 infection. Our results indicate that AIM2-adjuvanted vaccine could be a potential and promising approach to promote a long-lasting protection against CVB3-induced myocarditis.

Tuberculosis (TB) remains a serious health problem worldwide, and an urgent need exists to improve or replace the available vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG). Most vaccination protocols adapt two or three doses to induce long-term lasting immunity. Our previous study showed that the naked DNA encoding the triple-antigen fusion TFP846 (Rv3615c-Mtb10.4-Rv2660c) induced robust T cellular immune responses accompanying four inoculations against mycobacteria infection. However, a number of compliance issues exist in some areas lacking the appropriate medical infrastructure with multiple administrations. In this study, a novel vesicular stomatitis virus expressing TFP846 (VSV-846) was developed and the immune responses elicited by VSV-846 were evaluated. We observed that intranasal delivery of VSV-846 induced a potent antigen-specific T cell response following a single dose and VSV-846 efficiently controlled bacterial growth to levels ~10-fold lower than that observed in the mock group 6 weeks post-infection in BCG-infected mice. Importantly, mice immunized with VSV-846 provided long-term protection against mycobacteria infection compared with those receiving p846 or BCG immunization. Increased memory T cells were also observed in the spleens of VSV-846-vaccinated mice, which could be a potential mechanism associated with long-term protective immune response. These findings supported the use of VSV as an antigen delivery vector with the potential for TB vaccine development.

Highlights • Immunization with VSV-based vaccine is able to generate strong mucosal immunity. • VSV-based vaccine can induce significant higher polyfunctional T cells. • VSV-based vaccine significantly alleviates CVB3-induced viral myocarditis., Recombinant vesicular stomatitis virus (VSV) is widely used as a vaccine platform. However, the capacity of VSV-based vaccines to induce mucosal immunity has not been fully investigated. In the present study, a recombinant VSV expressing coxsackievirus B3 (CVB3) major immunogen VP1 has been generated and the immune protection elicited by VSV-VP1 was evaluated. We demonstrated that intranasal delivery of VSV-VP1 can induce a potent antigen-specific mucosal immune response as well as a systemic immune response, particularly the induction of polyfunctional T cells. Importantly, mice immunized with VSV-VP1 were better protected against CVB3-induced viral myocarditis than those receiving a chitosan-formulated DNA vaccine. Increased dendritic cell (DC) maturation in the mesenteric lymph node (MLN) was observed in the mice vaccinated with VSV-VP1, which could be a potential mechanism for the protective immune response. These findings support VSV as a viral delivery vector that can induce robust mucosal immunity that should be considered for further vaccine development.

Interleukin (IL)-35 is a new member of the IL-12 cytokine family. The suppressive role of IL-35 in the immune response to parasitic and bacterial infections and in autoimmunity has been demonstrated in terms of its anti-inflammatory properties. However, the functional role of IL-35 in viral myocarditis has not been investigated. In this study, IL-35 expression was measured in heart tissues with coxsackievirus B3 (CVB3)-induced myocarditis. It was significantly reduced in the late stage of viral infection and correlated negatively with disease severity. To examine the therapeutic role of IL-35 in viral myocarditis, an IL-35-expressing plasmid (pIL-35-FC) was packaged with polyethyleneimine and delivered intraperitoneally to BALB/c male mice before and after CVB3 infection. The severity of myocarditis was assessed 7 days after infection. The in vivo delivery of IL-35 significantly ameliorated the severity of viral myocarditis, reflected in an increased survival rate and increased bodyweights, and reduced serum creatine kinase (CK) and CK-MB activities, cardiac pathological scores, and viral replication. We also show that the overexpression of IL-35 reduced splenic Th17 cells and Th17-related proinflammatory cytokines in heart tissues. In conclusion, our data indicate that IL-35 effectively protects the myocardium from the pathogenesis of CVB3-induced viral myocarditis, which may be attributable to reduced Th17 production. This suggests that supplementation with IL-35 could be a novel therapeutic treatment for viral myocarditis.

The purpose of this study was to identify protein-protein interactions among the components of the occlusion-derived virus (ODV) of Helicoverpa armigera nucleopolyhedrovirus (HearNPV), a group II alphabaculovirus in the family Baculoviridae. To achieve this, 39 selected genes of potential ODV structural proteins were cloned and expressed in the Gal4 yeast two-hybrid (Y2H) system. The direct-cross Y2H assays identified 22 interactions comprising 13 binary interactions [HA9-ODV-EC43, ODV-E56-38K, ODV-E56-PIF3, LEF3-helicase, LEF3-alkaline nuclease (AN), GP41-38K, GP41-HA90, 38K-PIF3, 38K-PIF2, VP80-HA100, ODV-E66-PIF3, ODV-E66-PIF2 and PIF3-PIF2] and nine self-associations (IE1, HA44, LEF3, HA66, GP41, CG30, 38K, PIF3 and P24). Five of these interactions - LEF3-helicase and LEF3-AN, and the self-associations of IE1, LEF3 and 38K - have been reported previously in Autographa californica multiple nucleopolyhedrovirus. As HA44 and HA100 were two newly identified ODV proteins of group II viruses, their interactions were further confirmed. The self-association of HA44 was verified with a His pull-down assay and the interaction of VP80-HA100 was confirmed by a co-immunoprecipitation assay. A summary of the protein-protein interactions of baculoviruses reported so far, comprising 68 interactions with 45 viral proteins and five host proteins, is presented, which will facilitate our understanding of the molecular mechanisms of baculovirus infection.

Monocytes are critical precursors of dendritic cells and macrophages, which play an important role in the pathogenesis of human immunodeficiency virus type 1 (HIV-1). HIV-1 postentry infection is blocked in undifferentiated monocytes in vitro, while the underlying mechanisms are not fully understood. HIV-1 Tat-mediated transactivation of the viral long terminal repeat (LTR) promoter is essential for HIV-1 transcription. Two critical cellular cofactors of HIV-1 Tat, cyclin T1 (CycT1) and cyclin-dependent kinase 9 (CDK9), are required for LTR-directed HIV-1 transcription. In addition to the previously identified restrictions in early viral life cycle, we find that HIV-1 gene expression is impaired in undifferentiated primary monocytes. Transfection of monocytes by nucleofection with HIV-1 proviral DNA could not produce infectious HIV-1. The lack of Tat transactivation of the LTR promoter correlated with the impaired HIV-1 gene expression in monocytes. Interestingly, heterokaryons between primary monocytes and a human embryonic kidney cell line restored Tat transactivation of LTR, suggesting that monocytes lack cellular factors required for Tat transactivation. CycT1 protein was undetectable in freshly isolated monocytes and induced in monocyte-differentiated macrophages, while the expression of CDK9 remained constant. Transient expression of CycT1 in undifferentiated monocytes could not rescue Tat transactivation, suggesting that CycT1 is not the only limiting factor of HIV-1 infection in monocytes. Furthermore, monocyte differentiation into macrophages appeared to enhance the phosphorylation of CDK9, which correlated with significantly increased HIV-1 infection in macrophages. Our results provide new insights into HIV-1 infection and regulation in primary monocytes and viral pathogenesis.

Human immunodeficiency virus type 1 (HIV-1) enters dendritic cells (DCs) through endocytosis and viral receptor-mediated fusion. Although endocytosis-mediated HIV-1 entry can generate productive infection in certain cell types, including human monocyte-derived macrophages, productive HIV-1 infection in DCs appears to be dependent on fusion-mediated viral entry. It remains to be defined whether endocytosed HIV-1 in DCs can initiate productive infection. Using HIV-1 infection and cellular fractionation assays to measure productive viral infection and entry, here we show that HIV-1 enters monocyte-derived DCs predominately through endocytosis; however, endocytosed HIV-1 cannot initiate productive HIV-1 infection in DCs. In contrast, productive HIV-1 infection in DCs requires fusion-mediated viral entry. Together, these results provide functional evidence in understanding HIV-1 cis-infection of DCs, suggesting that different pathways of HIV-1 entry into DCs determine the outcome of viral infection.

Dendritic cells (DCs) transmit human immunodeficiency virus type 1 (HIV-1) to CD4+T cells through thetrans- andcis-infection pathways; however, little is known about the relative efficiencies of these pathways and whether they are interdependent. Here we comparecis- andtrans-infections of HIV-1 mediated by immature DCs (iDCs) and mature DCs (mDCs), using replication-competent and single-cycle HIV-1. Monocyte-derived iDCs were differentiated into various types of mDCs by lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-α), and CD40 ligand (CD40L). iDCs and CD40L-induced mDCs were susceptible to HIV-1 infection and mediated efficient viral transmission to CD4+T cells. Although HIV-1cis-infection was partially restricted in TNF-α-induced mDCs and profoundly blocked in LPS-induced mDCs, these cells efficiently promoted HIV-1trans-infection of CD4+T cells. The postentry restriction of HIV-1 infection in LPS-induced mDCs was identified at the levels of reverse transcription and postintegration, using real-time PCR quantification of viral DNA and integration. Furthermore, nucleofection of DCs with HIV-1 proviral DNA confirmed that impaired gene expression of LPS-induced mDCs was responsible for the postentry restriction of HIV-1 infection. Our results suggest that various DC subsets in vivo may differentially contribute to HIV-1 dissemination via dissociablecis- andtrans-infections.

Baculovirus P10 protein is a small conserved protein and is expressed as bundles of filaments in the host cell during the late phase of virus infection. So far the published results on the domain responsible for filament structural formation have been contradictory. Electron microscopy revealed that the C-terminus basic region was involved in filament structural formation in the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) [van Oers, M.M., Flipsen, J.T., Reusken, C.B., Sliwinsky, E.L., Vlak, J.M., 1993. Functional domains of the p10 protein of Autographa californica nuclear polyhedorsis virus. J. Gen. Virol. 74, 563–574.]. While in the Helicoverpa armigera nucleopolyhedrovirus (HearNPV), the heptad repeats region but not the C-terminus domain was proven to be responsible for filament formation [Dong, C., Li, D., Long, G., Deng, F., Wang, H., Hu, Z., 2005. Identification of functional domains required for HearNPV P10 filament formation. Virology 338, 112–120.]. In this manuscript, fluorescence confocal microscopy was applied to study AcMNPV P10 filament formation. A set of plasmids containing different P10 structural domains fused with a fluorescent protein were constructed and transfected into Sf-9 cells. The data indicated that the heptad repeats region, but not the proline-rich region or the C-terminus basic region, is essential for AcMNPV P10 filament formation. Co-transfection of P10s tagged with different fluorescent revealed that P10s with defective heptad repeats region could not interact with intact heptad repeats region or even full-length P10s to form filament structure. Within the heptad repeats region, deletion of the three amino acids spacing of AcMNPV P10 appeared to have no significant impact on the formation of filament structures, but the content of the heptad repeats region appeared to play a role in the morphology of the filaments.

Baculovirus encoded P10 form fibrillar structures in infected cells. We have tried to identify the functional domains for the P10 filament formation by green fluorescence protein (GFP) tag. The p10 gene of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) was the subject of these studies. Different lengths of HearNPV p10 gene were constructed with GFP fused in frame to the C-terminus. The constructs were transfected into insect and mammalian cells and observed by confocal microscopy. The results indicated that the first N-terminal 66 amino acids, which form the complete coiled-coil domain, were necessary for the aggregation and formation of filament structures of HearNPV P10. The proline-rich region and the C-terminal positively charged amino acids were not necessary for the formation of fibrillar structure but had some impact on the shape of the fibrillar structures. No other baculoviral proteins were needed for the formation of P10 filament structures in transfected cells.

Coxsackievirus B3 (CVB3) infection can cause acute myocarditis and chronic myocarditis, leading to dilated cardiomyopathy (DCM) with no effective therapeutic strategy. Therefore, we investigated the potential of absent in melanoma 2 (AIM2) to enhance the therapeutic efficacy of DNA vaccine against CVB3-induced chronic myocarditis. Mice were infected with CVB3 and then intranasally immunized with chitosan-pcDNA3.1 (mock), chitosan-pAIM2 (CS-pAIM2), chitosan-pVP1 (CS-pVP1), or chitosan-pAIM2 plus chitosan-pVP1 (CS-pAIM2/CS-pVP1) at 7, 21, and 35 d. Therapeutic efficacies of various vaccines were evaluated at day 56 d. Compared with CS-pVP1 immunization, CS-pAIM2/CS-pVP1 co-immunization significantly increased survival rate, improved cardiac function, as well as decreased myocardial injury and fibrosis, this result indicated that CVB3-induced chronic myocarditis was alleviated. CVB3-specific T lymphocyte proliferation and cytotoxic T lymphocyte responses of the CS-pAIM2/CS-pVP1 co-immunization group were also increased. More interestingly, CS-pAIM2/CS-pVP1 co-immunization could facilitate CVB3-specific multifunctional CD8 + T cell induction in the intestinal mucosa, and this induction was closely correlated with myocardial scores, this result indicated that CS-pAIM2/CS-pVP1 vaccine exhibits therapeutic efficacy by enhancing multifunctional CD8 + T cells. This study may represent a novel therapy for CVB3-induced chronic myocarditis.

Chronic hepatitis B virus (HBV) infection causes liver cirrhosis and hepatocellular carcinoma and remains a serious health problem worldwide. Covalently closed circular DNA (cccDNA) in the liver cell nucleus sustains HBV infection. Major treatments for HBV infection include the use of interferon-α and nucleotide analogs, but they cannot eradicate cccDNA. As a novel tool for genome editing, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system developed from bacteria can be used to accurately and efficiently engineer and modify genomic DNA. In this study, the CRISPR/Cas9 system was used to target the HBV genome and efficiently inhibit HBV infection. We synthesized four single-guide RNAs (sgRNAs) targeting the conserved regions of HBV. The expression of these sgRNAS with Cas9 reduced the viral production in Huh7 cells as well as in HBV-replication cell HepG2.2.15. We further demonstrated that CRISPR/Cas9 direct cleavage and cleavage-mediated mutagenesis occurred in HBV cccDNA of transfected cells. In the new mouse model carrying HBV cccDNA, injection of sgRNA–Cas9 plasmids via rapid tail vein resulted in the low level of cccDNA and HBV protein. In conclusion, the designed CRISPR/Cas9 system can accurately and efficiently target HBV cccDNA and inhibit HBV replication. This system may be used as a novel therapeutic strategy against chronic HBV infection.

Development of effective anti-tuberculosis (TB) vaccines is one of the important steps to improve control of TB. Cell-mediated immune response significantly affects the control of M. tuberculosis infection. Thus, vaccines able to elicit strong cellular immune response hold special advantages against TB. In this study, three well-defined mycobacterial antigens (Rv3615c, Mtb10.4 [Rv0228], and Rv2660c) were engineered as a novel triple-antigen fusion DNA vaccine p846. The p846 vaccine consists of a high density of CD4(+) and CD8(+) T-cell epitopes. Intramuscular immunization of p846 induced robust T cells mediated immune response comparable to that of bacillus Calmette-Guérin (BCG) vaccination but more effective than that of individual antigen vaccination. After mycobacterial challenge, p846 immunization decreased bacterial burden at least 15-fold compared with individual antigen-based vaccination. Notably, the lungs of mice immunized with p846 exhibited fewer inflammatory cell infiltrates and less damage than those of control group mice. Our data demonstrate that the potential of p846 vaccine to protect against TB and the feasibility of this design strategy for further TB vaccine development.