Transcriptome of human neuroblastoma SH-SY5Y cells in response to 2B protein of enterovirus-A71.

Infection with enterovirus-A71 (EV-A71) can cause hand-foot-mouth disease associated with fatal neurological complications. The host response to EV-A71 has not yet been fully elucidated, thus, hampering the development of a precise therapeutic approach. A nonstructural 2B protein of EV-A71 has been reported to involve with calcium dysregulation and apoptosis induction in human neuroblastoma SH-SY5Y cells. However, the molecular mechanism has not been delineated. To address this, comprehensive study of the gene expression from SH-SY5Y cells transfected with EV-A71 2B was carried out by RNA sequencing and transcriptomic analysis. It was found that the signature of the upregulated genes of SH-SY5Y cells expressing EV-A71 2B involved the Ca2+-related signaling pathways participating gene expression, inflammatory response, apoptosis, and long-term potentiation of the neuron. Protein-protein interaction network analysis revealed that the products encoded by CCL2, RELB, BIRC3, and TNFRSF9 were the most significant hub proteins in the network. It indicated that EV-A71 2B protein might play a role in immunopathogenesis of the central nervous system (CNS) which probably associated with the non-canonical NF-κB pathway. The data suggest that transcriptomic profiling can provide novel information source for studying the neuropathogenesis of EV-A71 infection leading to development of an effective therapeutic measure for CNS complications.

Development of a novel NS1 competitive enzyme-linked immunosorbent assay for the early detection of Zika virus infection.

Zika virus (ZIKV) is a flavivirus that has emerged as a global health threat after the 2015 outbreak in the Americas, where devastating congenital defects were documented. There are currently no vaccines to prevent ZIKV infections nor commercially available clinical diagnostic tests demonstrated to identify ZIKV without cross-reactive interference of related flaviviruses. Early diagnosis is critical when treating symptomatic patients and in preventing ZIKV transmission. In this context, the development of sensitive and accurate diagnostic methods are urgently needed for the detection of ZIKV acute infection. The aim of this study consisted of obtaining monoclonal antibodies (mAbs) against denatured monomeric ZIKV Nonstructural protein 1 (ZNS1), a useful diagnostic marker for flavivirus early detection, in order to develop a highly specific and sensitive ZNS1 indirect competitive ELISA (icELISA). The production of hybridomas secreting ZNS1 mAbs was carried out through immunizations with denatured monomeric ZNS1. We selected 1F5 and 6E2 hybridoma clones, which recognized the heat-denatured ZNS1 hexameric form by indirect ELISA. Cross-reaction studies indicated that these mAbs specifically bind to a ZNS1 linear epitope, and that they do not cross-react with the NS1 protein from other related flaviviruses. The 1F5 mAb enabled the development of a sensitive and reproducible icELISA to detect and quantify small amounts of ZNS1 disease marker in heat-denatured human sera. Here, we establish a reliable 1F5 based-icELISA that constitutes a promising diagnostic tool for control strategies and the prevention of ZIKV propagation.

Role of Chikungunya nsP3 in Regulating G3BP1 Activity, Stress Granule Formation and Drug Efficacy.

BACKGROUND: Ras-GTPase activating protein SH3-domain-binding proteins (G3BP) are a small family of RNA-binding proteins implicated in regulating gene expression. Changes in expression of G3BPs are correlated to several cancers including thyroid, colon, pancreatic and breast cancer. G3BPs are important regulators of stress granule (SG) formation and function. SG are ribonucleoprotein (RNP) particles that respond to cellular stresses to triage mRNA resulting in transcripts being selectively degraded, stored or translated resulting in a change of gene expression which confers a survival response to the cell. These changes in gene expression contribute to the development of drug resistance. Many RNA viruses, including Chikungunya (and potentially Coronavirus), dismantle SG so that the cell cannot respond to the viral infection. Non-structural protein 3 (nsP3), from the Chikungunya virus, has been shown to translocate G3BP away from SG. Interestingly in cancer cells, the formation of SG is correlated to drug-resistance and blocking SG formation has been shown to reestablish the efficacy of the anticancer drug bortezomib. METHODS: Chikungunya nsP3 was transfected into breast cancer cell lines T47D and MCF7 to disrupt SG formation. Changes in the cytotoxicity of bortezomib were measured. RESULTS: Bortezomib cytotoxicity in breast cancer cell lines changed with a 22 fold decrease in its IC50 for T47D and a 7 fold decrease for MCF7 cells. CONCLUSIONS: Chikungunya nsP3 disrupts SG formation. As a result, it increases the cytotoxicity of the FDA approved drug, bortezomib. In addition, the increased cytotoxicity appears to correlate to improved bortezomib selectivity when compared to control cell lines.

Combination of three adjuvants enhances the immunogenicity of a recombinant protein containing the CTL epitopes of non-structural proteins of hepatitis C virus.

Hepatitis C virus (HCV) can cause chronic infection and evade the immune response. The generation and maintenance of an effective T-cell response is important for immune-mediated control of HCV infection. The purpose of this study was to obtain recombinant mosaic proteins containing the cytotoxic T lymphocyte (CTL) epitopes of HCV fused with different adjuvants and analyse their immunogenicity. A recombinant polyepitope protein comprising HLA-A2-restricted CTL epitopes of the NS3, NS4ab and NS5a proteins of HCV was designed. Adjuvant compounds, the T-helper (Th) epitope PADRE, lipopeptide from Neisseria meningiditis and interleukin 2 (IL-2) were included in the fusion proteins. Three proteins differing in their adjuvant content were expressed in Escherichia coli and purified. The purified proteins formed nanosized particles. The proteins were characterized by their ability to cause proliferation of spleen cells, induce expression of cytokine genes and production of interferon gamma by T lymphocytes of immunized mice. The obtained recombinant vaccine proteins effectively stimulate dendritic cells, which in turn specifically activate Th1 and Th2 lymphocytes. Adjuvant components act additively to enhance the stimulation of dendritic cells and polarize them in the direction of Th1 lymphocyte activation. Analysis of spleen cell proliferation, expression of Th1 and Th2 cytokines and production of interferon gamma by lymphocytes of immunized mice after specific stimulation in vitro revealed that recombinant protein comprising CTL epitopes of HCV, Th epitope PADRE, lipoprotein and IL-2 induced the highest response of T-lymphocytes.

Simultaneous use of natural adjuvants and cell penetrating peptides improves HCV NS3 antigen-specific immune responses.

To improve an effective hepatitis C virus (HCV) therapeutic vaccine, induction of a strong and long term HCV antigen-specific immune response is an important parameter. HCV non-structural protein 3 (NS3) has antigenic properties and plays a major role in viral clearance. In this study, DNA constructs encoding HCV NS3 and heat shock protein 27 (Hsp27)-NS3 genes, and the recombinant (r) NS3 and rHsp27-NS3 proteins complexed with HR9 and Cady-2 cell penetrating peptides (CPPs) were utilized to evaluate antibody, cytokine and Granzyme B secretion in mice. Herein, the formation of NS3 and Hsp27-NS3 DNA/ HR9 CPP complexes were revealed by gel retardation assay and protection against DNase and protease. Cady-2 peptide was used to form the nanoparticles with rNS3 and rHsp27-NS3 proteins. The size and charge of the nanoparticles were confirmed by SEM and Zetasizer instruments. Next, in vitro transfection of the nanoparticles was assessed by flow cytometry and western blotting. Finally, humoral and cellular immune responses were evaluated using different modalities in mice. Our data showed that HR9 and Cady-2 could form stable nanoparticles with DNA and proteins, respectively and enhance their delivery into HEK-293 T cells in a non-covalent approach. Furthermore, the heterologous Hsp27-NS3 DNA + HR9 prime/rHsp27-NS3+Cady-2 protein boost elicited a higher Th1 cellular immune response with a predominant IgG2a, IgG2b, IFN-γ profile and strong Granzyme B secretion than those induced by other groups. Briefly, the combination of a natural adjuvant (Hsp27) and CPPs (HR9 and Cady-2) could significantly stimulate effective immune responses as a promising approach for development of HCV therapeutic vaccines.

Bovine serum albumin nanoparticles induce histopathological changes and inflammatory cell recruitment in the skin of treated mice.

Albumin is a natural, biocompatible, biodegradable and nontoxic polymer and due to these features, nanoparticles made of albumin are a good system for drug or antigen delivery. Polymeric nanoparticles are being widely explored as new vaccines platforms due to the capacity of those nanoparticles to prime the immune system by providing sustained release of the antigen after injection. Biodegradable nanoparticles associated with proteins represent a promising method for in vivo delivery of vaccines. In our previous studies, bovine serum albumin nanoparticles (BSA-NPs) were identified as a promising system for in vivo delivery of microbial antigens. The aim of this work was to show the effect of BSA-NPs on skin after nanoparticles administration. The pro-inflammatory activity of BSA-NPs was evaluated using in vivo models. BSA-NPs are easily uptake by macrophagic RAW 264.7 and BHK-21 cells without any significant cytotoxicity. Histological examination of skin sections from BSA-NPs-treated mice revealed intense cellular infiltration, increased skin thickness, follicular hypertrophy, vascular congestion and marked collagenesis. Mice immunized with recombinant non-structural protein 1 (rNS1) from Dengue virus 1 and BSA-NPs showed a high seroconversion rate if compared to animals immunized only with rNS1. Therefore, the effect of BSA-NPs on skin after BSA-NPs administration has a biotechnological relevance to the rational design of vaccine formulations based on albumin nanocarriers. However in the next years future studies should be carried out to best characterize the effect of BSA-NPs on dendritic cells and establish the role of these nanoparticles as a new vaccine platform for infectious diseases or cancer.

Design and testing of a highly conserved human rotavirus VP8* immunogenic peptide with potential for vaccine development.

Rotavirus infection of young children particularly below five years of age resulting in severe diarhoea, is the cause of a large number of infant deaths all over the world, more so in developing countries like India. Vaccines developed against this infection in the last two decades have shown mixed results with some of them leading to complications. Oral vaccines have not been very effective in India. Significant diversity has been found in circulating virus strains in India. Development of a vaccine against diverse genetic variants of the different strains would go a long way in reducing the incidence of infection in developing countries. Success of such a vaccine would depend to a large extent on the antigenic peptide to be used in antibody production. The non-glycosylated protein VP4 on the surface capsid of the virus is important in rota viral immunogenicity and the major antigenic site(s) responsible for neutralization of the virus via VP4 is in the VP8* subunit of VP4. It is necessary that the peptide should be very specific and a peptide sequence which would stimulate both the T and B immunogenic cells would provide maximum protection against the virus. Advanced computational techniques and existing databases of sequences of the VP4 protein of rotavirus help in identification of such specific sequences. Using an in silico approach we have identified a highly conserved VP8* subunit of the VP4 surface protein of rotavirus which shows both T and B cell processivity and is also non-allergenic. This sub-unit could be used in in vivo models for induction of antibodies.

Development of Monoclonal Antibodies Specifically Recognizing the Nonstructural Protein 12 of Type 2 Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens that has caused tremendous economic losses to the swine industry worldwide. Although extensive research has been focused on PRRSV, little is known about the structure and biological functions of individual nonstructural viral proteins, especially the nonstructural protein 12 (Nsp12). In this study, we generated and identified the monoclonal antibody (mAb) against PRRSV Nsp12. Six strains of hybridoma cells named 2B10, 2B12, 5E1, 5G6, 5E7, and 8B2 secreting anti-Nsp12 mAbs were obtained by the hybridoma technique. All the mAbs were specifically reacted with PRRSV by indirect immunofluorescence assay and four of them (2B12, 5E1, 5G6, and 5E7) were specifically reacted by Western blot. Furthermore, the 5E7 specifically recognized multiple type 2 PRRSV strains, including highly pathogenic and classical PRRSV strains, but not type 1 PRRSV strain. Taken together, the mAbs against Nsp12 provide a valuable tool to specifically recognize type 2 PRRSV as a diagnostic reagent and study the biological function of Nsp12 in the future.

Attenuation and efficacy of live-attenuated Rift Valley fever virus vaccine candidates in non-human primates.

Rift Valley fever virus (RVFV) is an important mosquito-borne veterinary and human pathogen that has caused large outbreaks of severe disease throughout Africa and the Arabian Peninsula. Currently, no licensed vaccine or therapeutics exists to treat this potentially deadly disease. The explosive nature of RVFV outbreaks and the severe consequences of its accidental or intentional introduction into RVFV-free areas provide the impetus for the development of novel vaccine candidates for use in both livestock and humans. Rationally designed vaccine candidates using reverse genetics have been used to develop deletion mutants of two known RVFV virulence factors, the NSs and NSm genes. These recombinant viruses were demonstrated to be protective and immunogenic in rats, mice, and sheep, without producing clinical illness in these animals. Here, we expand upon those findings and evaluate the single deletion mutant (ΔNSs rRVFV) and double deletion mutant (ΔNSs-ΔNSm rRVFV) vaccine candidates in the common marmoset (Callithrix jacchus), a non-human primate (NHP) model resembling severe human RVF disease. We demonstrate that both the ΔNSs and ΔNSs-ΔNSm rRVFV vaccine candidates were found to be safe and immunogenic in the current study. The vaccinated animals received a single dose of vaccine that led to the development of a robust antibody response. No vaccine-induced adverse reactions, signs of clinical illness or infectious virus were detected in the vaccinated marmosets. All vaccinated animals that were subsequently challenged with RVFV were protected against viremia and liver disease. In summary, our results provide the basis for further development of the ΔNSs and ΔNSs-ΔNSm rRVFV as safe and effective human RVFV vaccines for this significant public health threat.

12 Weeks of a Ribavirin-Free Sofosbuvir and Nonstructural Protein 5A Inhibitor Regimen Is Enough to Treat Recurrence of Hepatitis C After Liver Transplantation.

Sofosbuvir (SOF) combined with nonstructural protein 5A (NS5A) inhibitors has demonstrated its efficacy in treating a recurrence of hepatitis C virus (HCV) after liver transplantation (LT). However, the duration of treatment and need for ribavirin (RBV) remain unclear in this population. Our aim was to determine whether LT recipients could be treated with an SOF + NS5A inhibitor-based regimen without RBV for 12 weeks post-LT. Between October 2013 and December 2015, 699 LT recipients experiencing an HCV recurrence were enrolled in the multicenter ANRS CO23 CUPILT cohort. We selected patients receiving SOF and NS5A inhibitor ± RBV and followed for at least 12 weeks after treatment discontinuation. The primary efficacy endpoint was a sustained virological response 12 weeks after the end of treatment (SVR12). Among these 699 patients, 512 fulfilled the inclusion criteria. Their main characteristics were: 70.1% genotype 1, 18.2% genotype 3, 21.1% cirrhosis, and 34.4% previously treated patients. We identified four groups of patients according to their treatment and duration: SOF + NS5A without RBV for 12 (156 patients) or 24 (239 patients) weeks; SOF + NS5A + RBV for 12 (47 patients) or 24 (70 patients) weeks. SVR12 values reached 94.9%, 97.9%, 95.7%, and 92.9%, respectively (P = 0.14). Only 20 patients experienced a treatment failure. Under multivariate analysis, factors such as fibrosis stage, previous treatment, HCV genotype, and baseline HCV viral load did not influence SVR12 rates in the four groups (P = 0.21). Hematological adverse events (AEs) were more common in the RBV group: anemia (P < 0.0001) and blood transfusion (P = 0.0001). CONCLUSION: SOF + NS5A inhibitors without RBV for 12 weeks constituted reliable therapy for recurrent HCV post-LT with an excellent SVR12 whatever the fibrosis stage, HCV genotype, and previous HCV treatment. (Hepatology 2018; 00:000-000).

Progress toward an enhanced vaccine: Eight marked attenuated viruses to porcine reproductive and respiratory disease virus.

Recombinant viruses of strain Ingelvac® PRRS porcine reproductive and respiratory syndrome virus (PRRSV) modified live virus vaccine were produced with two individual small in-frame deletions in nonstructural protein 2 (nsp2; Δ23 and Δ87) and also the same deletions supplanted with foreign tags (Δ23-V5, Δ23-FLAG, Δ23-S, Δ87-V5, Δ87-FLAG, Δ87-S). The viruses, but one (Δ87-FLAG), were stable for 10 passages and showed minimal effects on in vitro growth. Northern hybridization showed that the Δ23-tagged probe detected intracellular viral genome RNA as well as shorter RNAs that may represent heteroclite species, while the Δ87-tagged probe detected predominantly only genome length RNAs. When the tagged viruses were used to probe nsp2 protein in infected cells, perinuclear localization similar to native nsp2 was seen. Dual infection of Δ23-S and Δ87-S viruses allowed some discrimination of individual tagged nsp2 protein, facilitating future research. The mutants could potentially also be used to differentiate infected from vaccinated animals.

Immunization of Mice by Rotavirus NSP4-VP6 Fusion Protein Elicited Stronger Responses Compared to VP6 Alone.

Due to the limitations and safety issues of the two currently approved live attenuated rotavirus (RV) vaccines "RotaTeq and Rotarix," studies on nonreplicating sources of RV vaccines and search for proper RV antigens are actively carried out. The adjuvant activity of NSP4 and highly immunogenic properties of RV VP6 protein prompted us to consider the construction of a NSP4112-175-VP6 fusion protein and to assess the anti-VP6 IgG, IgA, and IgG subclass responses induced by Escherichia coli-derived NSP4-VP6 fusion protein compared to that of VP6 protein with/without formulation in Montanide ISA 50V2 (M50) in BALB/c mice. Results indicated to the proper expression of the fused NSP4-VP6 and VP6 proteins in E. coli. Intraperitoneal immunization by M50 formulated NSP4-VP6 fusion protein (M5+NSP4-VP6) induced the highest titration of VP6-specific IgG and IgA responses compared to the other groups. Indeed, the presence of NSP4 resulted to the induction of stronger humoral immune responses against the fused protein compared to that elicited by administration of VP6 protein alone (with/without M50 formulation), implying the adjuvant properties of NSP4 for the fused protein. Moreover, the "M50+NSP4-VP6" formulation induced higher serum IgG2a titers than IgG1 and increased Interferon-γ levels, despite unchanged interleukin-4 amounts compared to other groups, indicating Th1-oriented responses with a possible role of NSP4. In conclusion, this study further highlights the potentiality of NSP4-VP6 fusion protein as an efficient and cost-effective immunogen in the field of RV vaccine development.

Canine Parvovirus ns1 gene and Chicken Anemia vp3 gene induce partial oncolysis of Canine Transmissible Venereal Tumor.

The oncolytic effect of Canine Parvovirus ns1 gene and Chicken Anemia vp3 gene in naturally occurring cases of Canine Transmissible Venereal Tumor (CTVT) is being reported. Dogs suffering from CTVT (N = 18) were systematically randomized into three groups viz. A, B, and C (n = 6). Animals of the groups A, B, and C received 100 µg of the ns1 gene, vp3 gene, and ns1 + vp3 gene combination, respectively, for three weeks intratumorally at weekly intervals; results were normalized against base values before commencement of therapy and after complete remission that were taken as negative and positive controls, respectively. Initiation of oncolytic gene therapy arrested the further progression of the tumor but most of the animals in the study underwent incomplete remission, indicating incomplete activity of ns1 and vp3 genes. The oncolytic effect of the treatments was in the order ns1 > vp3 > ns1 + vp3. Oncolysis was accompanied by decreased mitotic index and AgNOR count, and increased TUNEL positive cells and CD4+ lymphocyte counts. Our findings show that Canine Parvovirus ns1 may eventually find an important role as an oncolytic agent.

Canine influenza viruses with modified NS1 proteins for the development of live-attenuated vaccines.

Canine Influenza Virus (CIV) H3N8 is the causative agent of canine influenza, a common and contagious respiratory disease of dogs. Currently, only inactivated influenza vaccines (IIVs) are available for the prevention of CIV H3N8. However, live-attenuated influenza vaccines (LAIVs) are known to provide better immunogenicity and protection efficacy than IIVs. Influenza NS1 is a virulence factor that offers an attractive target for the preparation of attenuated viruses as LAIVs. Here we generated recombinant H3N8 CIVs containing truncated or a deleted NS1 protein to test their potential as LAIVs. All recombinant viruses were attenuated in mice and showed reduced replication in cultured canine tracheal explants, but were able to confer complete protection against challenge with wild-type CIV H3N8 after a single intranasal immunization. Immunogenicity and protection efficacy was better than that observed with an IIV. This is the first description of a LAIV for the prevention of H3N8 CIV in dogs.

Differential humoral and cellular immunity induced by vaccination using plasmid DNA and protein recombinant expressing the NS3 protein of dengue virus type 3.

BACKGROUND: The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serine-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work we evaluated the potential of the NS3 (protease domain) as a protective antigen by comparing the administration of a recombinant protein versus a DNA vaccine in the mouse model. RESULTS: BALB/c mice were immunized with the recombinant protein NS3-DEN3 via intraperitoneal and with plasmid pcDNA3/NS3-DEN3 intramuscularly and the immune response was evaluated. The activity of T lymphocytes was analyzed by the MTT assay, and cells of mice immunized with the recombinant protein showed no activity when stimulated with the homologous protein. However, cells from mice immunized with DNA, responded to stimulation with the recombinant protein. When the expression (RT-PCR) and cytokine production (ELISA) was evaluated in the splenocytes, different behavior depending on the type of immunization was observed, splenocytes of mice immunized with the recombinant protein expressed cytokines such as IL-4, IL-10 and produced high concentrations of IL-1, IL-6 and TNFα. Splenocytes from mice immunized with DNA expressed IL-2 and IFNγ and did not produce IL-6. In addition, immunization with the recombinant protein induced the production of antibodies that are detected up to a dilution 1:3200 by ELISA and Western blot assays, however, the serum of mice immunized with DNA presented no detectable antibody titers. CONCLUSION: The results obtained in this study show that administration of pcDNA3/NS3-DEN3 induces a favorable response in the activation of T lymphocytes with low production of specific antibodies against NS3-DEN3.

Polymeric hepatitis C virus non-structural protein 5A nanocapsules induce intrahepatic antigen-specific immune responses.

Targeting antigen combined with adjuvants to hepatic antigen-presenting cells (APCs) is essential for the induction of intrahepatic T cellular immunity controlling and resolving viral infections of the liver. Intravenous injection of antigen-loaded nanoparticles is a promising approach for the delivery of antigens to liver APCs. Accordingly, polymeric nanocapsules (NCs) synthesized exclusively of hepatitis C virus non-structural protein 5A (NS5A) and the adjuvant monophosphoryl lipid A (MPLA) adsorbed to the nanocapsule surface were developed. Aim of the present study was the evaluation of the in vitro and in vivo behavior of MPLA-functionalized NS5A-NCs regarding the interaction with liver dendritic cells (DCs) and the potential to induce intrahepatic immune responses in a mouse model. Maturation of DCs was significantly increased by application of NS5A+MPLA-NCs compared to non-functionalized NS5A-NCs promoting a vigorous expression of CD40, CD80, CD86 and a strong secretion of the Th1-related cytokine IL-12. NS5A-NCs were preferentially deposited in DCs and Kupffer cells residing in the liver after intravenous administration. Immunization with NS5A-NCs induced intrahepatic antigen-specific CD4(+) T cellular immune responses determined by the secretion of IFNγ and IL-2. Furthermore, supplementation with MPLA induced significant levels of NS5A-specific antibodies. The application of polymeric nanocapsules synthesized exclusively out of antigen avoids the risk of unintended side effects caused by additional carrier substances. Functionalization with adjuvants like MPLA and the efficient targeting to liver-resident APCs inherits the potential for application of antigen nanocapsules in further vaccination approaches against pathogens affecting the liver.

Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen.

Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses.

Analysis of the temperature sensitivity of Japanese rubella vaccine strain TO-336.vac and its effect on immunogenicity in the guinea pig.

The marker of Japanese domestic rubella vaccines is their lack of immunogenicity in guinea pigs. This has long been thought to be related to the temperature sensitivity of the viruses, but supporting evidence has not been described. In this study, we generated infectious clones of TO-336.vac, a Japanese domestic vaccine, TO-336.GMK5, the parental virus of TO-336.vac, and their mutants, and determined the molecular bases of their temperature sensitivity and immunogenicity in guinea pigs. The results revealed that Ser(1159) in the non-structural protein-coding region was responsible for the temperature sensitivity of TO-336.vac dominantly, while the structural protein-coding region affected the temperature sensitivity subordinately. The findings further suggested that the temperature sensitivity of TO-336.vac affected the antibody induction in guinea pigs after subcutaneous inoculation.

Priming with two DNA vaccines expressing hepatitis C virus NS3 protein targeting dendritic cells elicits superior heterologous protective potential in mice.

Development an effective vaccine may offer an alternative preventive and therapeutic strategy against HCV infection. DNA vaccination has been shown to induce robust humoral and cellular immunity and overcome many problems associated with conventional vaccines. In this study, mice were primed with either conventional pVRC-based or suicidal pSC-based DNA vaccines carrying DEC-205-targeted NS3 antigen (DEC-NS3) and boosted with type 5 adenoviral vectors encoding the partial NS3 and core antigens (C44P). The prime boost regimen induced a marked increase in antigen-specific humoral and T-cell responses in comparison with either rAd5-based vaccines or DEC-205-targeted DNA immunization in isolation. The protective effect against heterogeneous challenge was correlated with high levels of anti-NS3 IgG and T-cell-mediated immunity against NS3 peptides. Moreover, priming with a suicidal DNA vaccine (pSC-DEC-NS3), which elicited increased TNF-α-producing CD4+ and CD8+ T-cells against NS3-2 peptides (aa 1245-1461), after boosting, showed increased heterogeneous protective potential compared with priming with a conventional DNA vaccine (pVRC-DEC-NS3). In conclusion, a suicidal DNA vector (pSC-DEC-NS3) expressing DEC-205-targeted NS3 combined with boosting using an rAd5-based HCV vaccine (rAd5-C44P) is a good candidate for a safe and effective vaccine against HCV infection.

Development of a dual-protective live attenuated vaccine against H5N1 and H9N2 avian influenza viruses by modifying the NS1 gene.

An increasing number of outbreaks of avian influenza H5N1 and H9N2 viruses in poultry have caused serious economic losses and raised concerns for human health due to the risk of zoonotic transmission. However, licensed H5N1 and H9N2 vaccines for animals and humans have not been developed. Thus, to develop a dual H5N1 and H9N2 live-attenuated influenza vaccine (LAIV), the HA and NA genes from a virulent mouse-adapted avian H5N2 (A/WB/Korea/ma81/06) virus and a recently isolated chicken H9N2 (A/CK/Korea/116/06) virus, respectively, were introduced into the A/Puerto Rico/8/34 backbone expressing truncated NS1 proteins (NS1-73, NS1-86, NS1-101, NS1-122) but still possessing a full-length NS gene. Two H5N2/NS1-LAIV viruses (H5N2/NS1-86 and H5N2/NS1-101) were highly attenuated compared with the full-length and remaining H5N2/NS-LAIV viruses in a mouse model. Furthermore, viruses containing NS1 modifications were found to induce more IFN-ß activation than viruses with full-length NS1 proteins and were correspondingly attenuated in mice. Intranasal vaccination with a single dose (10(4.0) PFU/ml) of these viruses completely protected mice from a lethal challenge with the homologous A/WB/Korea/ma81/06 (H5N2), heterologous highly pathogenic A/EM/Korea/W149/06 (H5N1), and heterosubtypic highly virulent mouse-adapted H9N2 viruses. This study clearly demonstrates that the modified H5N2/NS1-LAIV viruses attenuated through the introduction of mutations in the NS1 coding region display characteristics that are desirable for live attenuated vaccines and hold potential as vaccine candidates for mammalian hosts.