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Development of vaccines against highly pathogenic avian influenza virus H5N1 subtypes posing a pandemic threat remains a priority. Limitations in manufacturing capacity and production time of conventional inactivated vaccines highlight the need for additional approaches. We conducted two double-blind, placebo-controlled phase 1 studies involving a total of 103 healthy adults who received two intramuscular injections of Vaxfectin ®-adjuvanted plasmid DNA vaccine or placebo 21 days apart. Vaccine cohorts received either a monovalent vaccine containing an A/Vietnam/1203/04 H5 hemagglutinin-encoding plasmid or a trivalent vaccine with plasmids encoding H5, NP, and M2 proteins in doses from 0.1 to 1 mg of DNA/injection. All doses were well tolerated without vaccine-related serious adverse events or discontinuations. In the monovalent cohorts, hemagglutination inhibition (HI) titers of ≥40 and 4-fold rises from baseline were achieved in 47–67% of subjects and H5-specific T-cell responses in 75–100%. Trivalent cohorts had lower HI response rates (≤20%), but 72% of subjects achieved T-cell and/or antibody responses to one or more antigens. Vaxfectin ®-adjuvanted monovalent H5 DNA vaccines were well tolerated and induced HI response rates and titers in the reported range of inactivated protein-based H5 vaccines, suggesting that adjuvanted DNA vaccines with rapid vaccine production could be useful for pandemic control.

Ebola virus (EBOV) is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG) responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP), Nucleoprotein (NP), and matrix Viral Protein (VP)40 and VP35). For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms) and the late memory phase (7-12 years post-infection). We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects). We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP1 subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods.

Introduction: A lack of access to hepatitis C virus (HCV) diagnostics is a significant barrier to achieving the World Health Organization 2030 global elimination goal. HCV core antigen (HCVcAg) quantification and dried blood spot (DBS) are appealing alternatives to conventional HCV serology and nucleic acid testing (NAT) for resource‐constraint settings, particularly in difficult‐to‐reach populations. We assessed the accuracy of serum and DBS HCVcAg testing in people who inject drugs in Tanzania using HCV NAT as a reference. Method: Between May and July 2015, consecutive HCV‐seropositive patients enrolled in the local opioid substitution treatment centre were invited to participate in the study. All had HCV RNA detection (Roche Molecular Systems, Pleasanton, CA, USA), genotyping (NS5B gene phylogenetic analysis) and HCVcAg on blood samples and DBS (Architect assay; Abbott Diagnostics, Chicago, IL, USA). Results: Out of 153 HCV‐seropositive individuals, 65 (42.5%) and 15 (9.8%) were co‐infected with HIV (41 (63%) were on anti‐retroviral therapy (ARVs)) and hepatitis B respectively. In total, 116 were viraemic, median viral load of 5.7 (Interquartile range (IQR); 4.0–6.3) log iU/ml (75 (68.2%) were genotype 1a, 35 (31.8%) genotype 4a). The median alanine transaminase (ALT) (iU/l), aspartate transaminase (AST) (iU/l) and gamma‐glutamyl transferase (GGT) (iU/l) were 35 (IQR; 23–51), 46 (32–57) and 69 (35–151) respectively. For the quantification of HCV RNA, serum HCVcAg had a sensitivity at 99.1% and a specificity at 94.1%, with an area under the receiver operating curve (AUROC) at 0.99 (95% CI 0.98–1.00). DBS HCVcAg had a sensitivity of 76.1% and a specificity of 97.3%, with an AUROC of 0.87 (95% CI 0.83–0.92). HCVcAg performance did not differ by HIV co‐infection or HCV genotype. Conclusions: Our study suggests that HCVcAg testing in serum is an excellent alternative to HCV polymerase chain reaction in Africa. Although HCVcAg detection and quantification in DBS has a reduced sensitivity, its specificity and accuracy are good and it could therefore be used for scaling up HCV testing and care in resource‐limited African settings.

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.

Hepatitis B virus (HBV) contains a partially double-stranded DNA genome. During infection, its replication is mediated by reverse transcription (RT) of an RNA intermediate termed pregenomic RNA (pgRNA) within core particles in the cytoplasm. An epsilon structural element located in the 5′ end of the pgRNA primes the RT activity. We have previously identified the N6-methyladenosine (m⁶A)–modified DRACH motif at 1905 to 1909 nucleotides in the epsilon structure that affects myriad functions of the viral life cycle. In this study, we investigated the functional role of m⁶A modification of the 5′ ε (epsilon) structural element of the HBV pgRNA in the nucleocapsid assembly. Using the m⁶A sitemutant in the HBV 5′ epsilon, we present evidence that m⁶A methylation of 5′ epsilon is necessary for its encapsidation. The m⁶A modification of 5′ epsilon increased the efficiency of viral RNA packaging, whereas the m⁶A of 3′ epsilon is dispensable for encapsidation. Similarly, depletion of methyltransferases (METTL3/14) decreased pgRNA and viral DNA levels within the core particles. Furthermore, the m⁶A modification at 5′ epsilon of HBV pgRNA promoted the interaction with core proteins, whereas the 5′ epsilon m⁶A site–mutated pgRNA failed to interact. HBV polymerase interaction with 5′ epsilon was independent of m⁶A modification of 5′ epsilon. This study highlights yet another pivotal role of m⁶A modification in dictating the key events of the HBV life cycle and provides avenues for investigating RNA–protein interactions in various biological processes, including viral RNA genome encapsidation in the context of m⁶A modification.

Background. Influenza A viruses cause occasional pandemics and frequent epidemics. Licensed influenza vaccines that induce high antibody titers to the highly polymorphic viral surface antigen hemagglutinin must be reformulated and readministered annually. A vaccine providing protective immunity to the highly conserved internal antigens could provide longer-lasting protection against multiple influenza subtypes. Methods. We prepared a Modified Vaccinia virus Ankara (MVA) vector encoding nucleoprotein and matrix protein 1 (MVA−NP+M1) and conducted a phase I clinical trial in healthy adults. Results. The vaccine was generally safe and well tolerated, with significantly fewer local side effects after intramuscular rather than intradermal administration. Systemic side effects increased at the higher dose in both frequency and severity, with 5 out of 8 volunteers experiencing severe nausea/vomiting, malaise, or rigors. Ex vivo T-cell responses to NP and M1 measured by IFN-γ ELISPOT assay were significantly increased after vaccination (prevaccination median of 123 spot-forming units/million peripheral blood mononuclear cells, postvaccination peak response median 339, 443, and 1443 in low-dose intradermal, low-dose intramuscular, and high-dose intramuscular groups, respectively), and the majority of the antigen-specific T cells were CD8⁺. Conclusions. We conclude that the vaccine was both safe and remarkably immunogenic, leading to frequencies of responding T cells that appear to be much higher than those induced by any other influenza vaccination approach. Further studies will be required to find the optimum dose and to assess whether the increased T-cell response to conserved influenza proteins results in protection from influenza disease.

Current hemagglutinin (HA)-based seasonal influenza vaccines induce vaccine strain-specific neutralizing antibodies that usually fail to provide protection against mismatched circulating viruses. Inclusion in the vaccine of highly conserved internal proteins such as the nucleoprotein (NP) and the matrix protein 1 (M1) was shown previously to increase vaccine efficacy by eliciting cross-reactive T-cells. However, appropriate delivery systems are required for efficient priming of T-cell responses. In this study, we demonstrated that administration of novel self-amplifying mRNA (SAM®) vectors expressing influenza NP (SAM(NP)), M1 (SAM(M1)), and NP and M1 (SAM(M1-NP)) delivered with lipid nanoparticles (LNP) induced robust polyfunctional CD4 T helper 1 cells, while NP-containing SAM also induced cytotoxic CD8 T cells. Robust expansions of central memory (TCM) and effector memory (TEM) CD4 and CD8 T cells were also measured. An enhanced recruitment of NP-specific cytotoxic CD8 T cells was observed in the lungs of SAM(NP)-immunized mice after influenza infection that paralleled with reduced lung viral titers and pathology, and increased survival after homologous and heterosubtypic influenza challenge. Finally, we demonstrated for the first time that the co-administration of RNA (SAM(M1-NP)) and protein (monovalent inactivated influenza vaccine (MIIV)) was feasible, induced simultaneously NP-, M1- and HA-specific T cells and HA-specific neutralizing antibodies, and enhanced MIIV efficacy against a heterologous challenge. In conclusion, systemic administration of SAM vectors expressing conserved internal influenza antigens induced protective immune responses in mice, supporting the SAM® platform as another promising strategy for the development of broad-spectrum universal influenza vaccines.

Here, we used a biologically contained Ebola virus system to characterize the spatio-temporal distribution of Ebola virus proteins and RNA during virus replication. We found that viral nucleoprotein (NP), the polymerase cofactor VP35, the major matrix protein VP40, the transcription activator VP30 and the minor matrix protein VP24 were distributed in cytoplasmic inclusions. These inclusions enlarged near the nucleus, became smaller pieces and subsequently localized near the plasma membrane. GP was distributed in the cytoplasm and transported to the plasma membrane independent of the other viral proteins. We also found that viral RNA synthesis occurred within the inclusions. Newly synthesized negative-sense RNA was distributed inside the inclusions, whereas positive-sense RNA was distributed both inside and outside. These findings provide useful insights into Ebola virus replication.

Goatpox and sheeppox are highly contagious and economically important viral diseases of small ruminants. Due to the risk they pose to animal health, livestock production, and international trade, capripoxviruses are a considerable threat to the livestock economy. In this study, we expressed two core proteins (A4L and A12L) and one extracellular enveloped virion protein (A33R) of goatpox virus in a baculovirus expression vector system and evaluated their use as diagnostic antigens in ELISA. Full-length A4L, A12L, and A33R genes of the GTPV Uttarkashi strain were amplified, cloned into the pFastBac HT A donor vector, and introduced into DH10Bac cells containing a baculovirus shuttle vector plasmid to generate recombinant bacmids. The recombinant baculoviruses were produced in Sf-21 cells by transfection, and proteins were expressed in TN5 insect cells. The recombinant proteins were analysed by SDS-PAGE and confirmed by western blot, with expected sizes of ~30 kDa, ~31 kDa, and ~32 kDa for A4L, A12L, and A33R, respectively. The recombinant proteins were purified, and the immunoreactivity of the purified proteins was confirmed by western blot using anti-GTPV serum. The antigenic specificity of the expressed proteins as diagnostic antigens was evaluated by testing their reactivity with infected, vaccinated, and negative GTPV/SPPV serum in indirect ELISA, and the A33R-based indirect ELISA was optimized. The diagnostic sensitivity and specificity of the A33R-based indirect ELISA were found to be of 89% and 94% for goats and 98% and 91%, for sheep, respectively. No cross-reactivity was observed with other related viruses. The recombinant-A33R-based indirect ELISA developed in the present study shows that it has potential for the detection of antibodies in GTPV and SPPV infected/vaccinated animals.

Hepatitis B virus (HBV) capsid or core protein (HBc) contains an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a short linker peptide. HBc plays a critical role in virtually every step of viral replication, which is further modulated by dynamic phosphorylation and dephosphorylation of its CTD. While several cellular kinases have been identified that mediate HBc CTD phosphorylation, there is little information on the cellular phosphatases that mediate CTD dephosphorylation. Herein, a consensus binding motif for the protein phosphatase 2A (PP2A) regulatory subunit B56 was recognized within the HBc linker peptide. Mutations within this motif designed to block or enhance B56 binding showed pleiotropic effects on CTD phosphorylation state as well as on viral RNA packaging, reverse transcription, and virion secretion. Furthermore, linker mutations affected the HBV nuclear episome (the covalently closed circular or CCC DNA) differentially during intracellular amplification vs. infection. The effects of linker mutations on CTD phosphorylation state varied with different phosphorylation sites and were only partially consistent with the linker motif serving to recruit PP2A-B56, specifically, to dephosphorylate CTD, suggesting that multiple phosphatases and/or kinases may be recruited to modulate CTD (de)phosphorylation. Furthermore, pharmacological inhibition of PP2A could decrease HBc CTD dephosphorylation and increase the nuclear HBV episome. These results thus strongly implicate the HBc linker in recruiting PP2A and other host factors to regulate multiple stages of HBV replication.