Yao Deng

Origin and Possible Genetic Recombination of the Middle East Respiratory Syndrome Coronavirus from the First Imported Case in China: Phylogenetics and Coalescence Analysis

ABSTRACT The Middle East respiratory syndrome coronavirus (MERS-CoV) causes a severe acute respiratory tract infection with a high fatality rate in humans. Coronaviruses are capable of infecting multiple species and can evolve rapidly through recombination events. Here, we report the complete genomic sequence analysis of a MERS-CoV strain imported to China from South Korea. The imported virus, provisionally named ChinaGD01, belongs to group 3 in clade B in the whole-genome phylogenetic tree and also has a similar tree topology structure in the open reading frame 1a and -b (ORF1ab) gene segment but clusters with group 5 of clade B in the tree constructed using the S gene. Genetic recombination analysis and lineage-specific single-nucleotide polymorphism (SNP) comparison suggest that the imported virus is a recombinant comprising group 3 and group 5 elements. The time-resolved phylogenetic estimation indicates that the recombination event likely occurred in the second half of 2014. Genetic recombination events between group 3 and group 5 of clade B may have implications for the transmissibility of the virus. IMPORTANCE The recent outbreak of MERS-CoV in South Korea has attracted global media attention due to the speed of spread and onward transmission. Here, we present the complete genome of the first imported MERS-CoV case in China and demonstrate genetic recombination events between group 3 and group 5 of clade B that may have implications for the transmissibility of MERS-CoV. The recent outbreak of MERS-CoV in South Korea has attracted global media attention due to the speed of spread and onward transmission. Here, we present the complete genome of the first imported MERS-CoV case in China and demonstrate genetic recombination events between group 3 and group 5 of clade B that may have implications for the transmissibility of MERS-CoV.

Systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector‐based vaccines carrying the spike protein of Middle East respiratory syndrome coronavirus

An ideal vaccine against mucosal pathogens such as Middle East respiratory syndrome coronavirus (MERS‐CoV) should confer sustained, protective immunity at both systemic and mucosal levels. Here, we evaluated the in vivo systemic and mucosal antigen‐specific immune responses induced by a single intramuscular or intragastric administration of recombinant adenoviral type 5 (Ad5) or type 41 (Ad41) ‐based vaccines expressing the MERS‐CoV spike (S) protein. Intragastric administration of either Ad5‐S or Ad41‐S induced antigen‐specific IgG and neutralizing antibody in serum; however, antigen‐specific T‐cell responses were not detected. In contrast, after a single intramuscular dose of Ad5‐S or Ad41‐S, functional antigen‐specific T‐cell responses were elicited in the spleen and pulmonary lymphocytes of the mice, which persisted for several months. Both rAd‐based vaccines administered intramuscularly induced systemic humoral immune responses (neutralizing IgG antibodies). Our results show that a single dose of Ad5‐S‐ or Ad41‐S‐based vaccines represents an appealing strategy for the control of MERS‐CoV infection and transmission.

Tailoring Subunit Vaccine Immunity with Adjuvant Combinations and Delivery Routes Using the Middle East Respiratory Coronavirus (MERS-CoV) Receptor-Binding Domain as an Antigen

The development of an effective vaccine is critical for prevention of a Middle East respiratory syndrome coronavirus (MERS-CoV) pandemic. Some studies have indicated the receptor-binding domain (RBD) protein of MERS-CoV spike (S) is a good candidate antigen for a MERS-CoV subunit vaccine. However, highly purified proteins are typically not inherently immunogenic. We hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. Therefore, the immunogenicity of a novel MERS-CoV RBD-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. Different vaccination regimens were compared in BALB/c mice immunized 3 times intramuscularly (i.m.) with a vaccine containing 10 µg of recombinant MERS-CoV RBD in combination with either aluminium hydroxide (alum) alone, alum and polyriboinosinic acid (poly I:C) or alum and cysteine-phosphate-guanine (CpG) oligodeoxynucleotides (ODN). The immune responses of mice vaccinated with RBD, incomplete Freund’s adjuvant (IFA) and CpG ODN by a subcutaneous (s.c.) route were also investigated. We evaluated the induction of RBD-specific humoral immunity (total IgG and neutralizing antibodies) and cellular immunity (ELISpot assay for IFN-γ spot-forming cells and splenocyte cytokine production). Our findings indicated that the combination of alum and CpG ODN optimized the development of RBD-specific humoral and cellular immunity following subunit vaccination. Interestingly, robust RBD-specific antibody and T-cell responses were induced in mice immunized with the rRBD protein in combination with IFA and CpG ODN, but low level of neutralizing antibodies were elicited. Our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. The vaccination regimen used in this study is promising and could improve the protection offered by the MERS-CoV subunit vaccine by eliciting effective humoral and cellular immune responses.

Recombinant Receptor Binding Domain Protein Induces Partial Protective Immunity in Rhesus Macaques Against Middle East Respiratory Syndrome Coronavirus Challenge

Abstract Background Development an effective vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) is urgent and limited information is available on vaccination in nonhuman primate (NHP) model. We herein report of evaluating a recombinant receptor-binding domain (rRBD) protein vaccine in a rhesus macaque model. Methods Nine monkeys were randomly assigned to high-dose, low-dose and mock groups,which were immunized with different doses of rRBD plus alum adjuvant or adjuvant alone at different time points (0, 8, 25weeks). Immunological analysis was conducted after each immunisation. Monkeys were challenged with MERS-CoV at 14days after the final immunisation followed by observation for clinical signs and chest X-rays. Nasal, oropharyngeal and rectal swabs were also collected for analyses. Monkeys were euthanized 3days after challenge and multiple specimens from tissues were collected for pathological, virological and immunological tests. Conclusion Robust and sustained immunological responses (including neutralisation antibody) were elicited by the rRBD vaccination. Besides, rRBD vaccination alleviated pneumonia with evidence of reduced tissue impairment and clinical manifestation in monkeys. Furthermore, the rRBD vaccine decreased viral load of lung, trachea and oropharyngeal swabs of monkeys. These data in NHP paves a way for further development of an effective human vaccine against MERS-CoV infection.

Poly(I:C)/Alum Mixed Adjuvant Priming Enhances HBV Subunit Vaccine-Induced Immunity in Mice When Combined with Recombinant Adenoviral-Based HBV Vaccine Boosting

Background Virus-specific cellular immune responses play a critical role in virus clearance during acute or chronic HBV infection. Currently, the commercially available HBV vaccine is combined with alum adjuvant, which stimulates mainly Th2 immune responses. Therefore, development of new therapeutic HBV vaccine adjuvants and immune strategies that also promote Th1 and CTL responses is urgently needed. Methodology/Principal findings To improve the immunity induced by the novel HBSS1 HBV vaccine, we evaluated the ability of adjuvants, including alum, CpG and polyriboinosinic polyribocytidylic acid [poly(I:C)], to enhance the response when boosted with the recombinant adenoviral vector vaccine rAdSS1. The immune responses to different adjuvant combinations were assessed in C57BL/6 mice by enzyme-linked immunosorbent assay (ELISA), ELISpot and cytokine release assays. Among the combinations tested, a HBV protein particle vaccine with CpG/alum and poly(I:C)/alum priming combinations accelerated specific seroconversion and produced high antibody (anti-PreS1, anti-S antibody) titres with a Th1 bias. After boosting with recombinant adenoviral vector vaccine rAdSS1, both groups produced a strong multi-antigen (S and PreS1)-specific cellular immune response. HBSS1 immunisation with poly(I:C)/alum priming also generated high-level CD4+ and CD8+ T cell responses in terms of Th1 cytokines (IFN-γand IL-2). Conclusions The protein-vaccine HBSS1 with mixed poly(I:C)/alum adjuvant priming, followed by a rAdSS1 vaccine boost, maximises specific antibody and Th1-biased cellular immune responses. This regime might prove useful in the development of HBV therapeutic vaccines. Furthermore, this promising strategy might be applied to vaccines against other persistent infections, such as human immunodeficiency virus and tuberculosis.

Complete Genome Sequence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) from the First Imported MERS-CoV Case in China

ABSTRACT On 26 May 2015, an imported Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in Guangdong Province, China, and found to be closely related to the MERS-CoV strain prevalent in South Korea. The full genome of the ChinaGD01 strain was sequenced and analyzed to investigate the epidemiology and evolution of MERS-CoV circulating in South Korea and China.

Optimisation of Prime–Boost Immunization in Mice Using Novel Protein-Based and Recombinant Vaccinia (Tiantan)-Based HBV Vaccine

Background A therapeutic vaccine for chronic hepatitis B virus (HBV) infection that enhances virus-specific cellular immune responses is urgently needed. The “prime–boost” regimen is a widely used vaccine strategy against many persistence infections. However, few reports have addressed this strategy applying for HBV therapeutic vaccine development. Methodology/Principal Findings To develop an effective HBV therapeutic vaccine, we constructed a recombinant vaccinia virus (Tiantan) containing the S+PreS1 fusion antigen (RVJSS1) combined with the HBV particle-like subunit vaccine HBVSS1 to explore the most effective prime–boost regimen against HBV. The immune responses to different prime–boost regimens were assessed in C57BL/C mice by ELISA, ELISpot assay and Intracellular cytokine staining analysis. Among the combinations tested, an HBV protein particle vaccine priming and recombinant vaccinia virus boosting strategy accelerated specific seroconversion and produced high antibody (anti-PreS1, anti-S antibody) titres as well as the strongest multi-antigen (PreS1, and S)-specific cellular immune response. HBSS1 protein prime/RVJSS1 boost immunization was also generated more significant level of both CD4+ and CD8+ T cell responses for Th1 cytokines (TNF-α and IFN-γ). Conclusions The HBSS1 protein-vaccine prime plus RVJSS1 vector boost elicits specific antibody as well as CD4 and CD8 cells secreting Th1-like cytokines, and these immune responses may be important parameters for the future HBV therapeutic vaccines.

Signal peptide replacements enhance expression and secretion of hepatitis C virus envelope glycoproteins

A large number of researches focused on glycoproteins E1 and E2 of hepatitis C virus (HCV) aimed at the development of anti-HCV vaccines and inhibitors. Enhancement of E1/E2 expression and secretion is critical for the characterization of these glycoproteins and thus for subunit vaccine development. In this study, we designed and synthesized three signal peptide sequences based on online programs SignalP, TargetP, and PSORT, then removed and replaced the signal peptide preceding E1/E2 by overlapping the polymerase chain reaction method. We assessed the effect of this alteration on E1/E2 expression and secretion in mammalian cells, using western blot analysis, dot blot, and Galanthus nivalis agglutinin lectin capture enzyme immunoassay. Replacing the peptides preceding E1 and E2 with the signal peptides of the tissue plasminogen activator and Gaussia luciferase resulted in maximum enhancement of E1/E2 expression and secretion of E1 in mammalian cells, without altering glycosylation. Such an advance would help to facilitate both the research of E1/E2 biology and the development of an effective HCV subunit vaccine. The strategy used in this study could be applied to the expression and production of other glycoproteins in mammalian cell line-based systems.

A recombinant DNA and vaccinia virus prime-boost regimen induces potent long-term T-cell responses to HCV in BALB/c mice.

Abstract To explore the best prime–boost regimen and evaluate the T-cellular response memory against HCV, we constructed two DNA vaccine candidates (pVRC-CE1E2 and pAAV-CE1E2) and two recombinant viruses (rTTV-E1E2 and rAAV-E1E2) and then assessed the immune response to different prime–boost patterns in BALB/c mice. The rTTV-E1E2 boosted the immune response to HCV DNA vaccine prime significantly, and the inverted terminal repeat sequence harboring DNA construct PAAV-CE1E2 was the best prime agent in this study. Our study provides new information for both the prime–boost regimen and long-term T-cell response for HCV vaccine development.

Lentiviral backbone-based hepatitis B virus replicon-mediated transfer favours the establishment of persistent hepatitis B virus infection in mice after hydrodynamic injection.

Establishment of a non-transgenic mouse model of persistent hepatitis B virus (HBV) infection is urgently needed. In this study, we constructed novel lentiviral-transfer plasmids containing HBV replicon DNA (pCS-HBV1.3, containing a 1.3-fold-overlength genome of HBV) and employed hydrodynamic injection (HDI) to develop an HBV-persistent mouse model. We explored the impact of host (different mouse strains, BALB/c and C57BL/6), gender, and the plasmid backbone on persistent HBV in mice. Our data showed that HBV antigenaemia (HBsAg, HBeAg) and HBV DNA persisted for >56days post-injection, while the appearance of anti-HBs antibody in the serum was only found among <30% of female C57BL/6 mice injected with pCS-HBV1.3. Moreover, HBcAg and HBV DNA were also detected in the liver of HDI mice. Compared with previous AAV-backbone based HBV replicon DNA transfer, we found that the HDI transfer with the lentiviral vector-based HBV replicon (pCS-HBV1.3) in this study resulted in a significantly higher level of HBV DNA transfer in the liver and longer persistence of HBV DNA and antigenaemia in the serum. Furthermore, we also showed that immunization of HBV replicon transfer mice with the novel HBSS1-based vaccines was able to overcome tolerance against HBV in mice and induces robust immunity (humoral as well as T-cell responses), followed by the clearance of the HBV viremia. We concluded that lentiviral backbone-based transfer vectors more readily establish persistent HBV infection in mouse models via HDI, providing a new tool useful for the study of HBV infection and immune-based therapies.