A flagellin-adjuvanted PED subunit vaccine improved protective efficiency against PEDV variant challenge in pigs

PEDV variants, causing severe diarrhea in neonatal suckling piglets with a mortality rate up to 100%, have being epidemic since late 2010 in china. To meet the pressing need of safe and cost-efficient PED maternal vaccines against PEDV variant, we vaccinated growing piglets with a flagellin-adjuvanted PED vaccine rSF-COE-3D by injection at Houhai acupoint. The vaccination not only enhanced the antibody responses of serum IgG/IgA, mucosal IgA and serum neutralizing antibody, but also improved the production of IFN-γ and IL-4. Moreover, rSF-COE-3D could provide a better protection against the challenge of a high pathogenic PEDV variant, with less diarrhea pigs, less pigs with detectable PEDV shed, lower rank values of feces and less apparent lesions and inflammation in duodenum of the PEDV infected pigs. The improved protective efficiency of rSF-COE-3D compared with COE was mostly benefited from the enhanced production of serum IgA, mucosal IgA in feces and serum neutralizing antibody titers. Taken together, our data suggest that rSF-COE-3D would be a novel efficient PED vaccine.     

Virus-like particle vaccine with B-cell epitope from porcine epidemic diarrhea virus (PEDV) incorporated into hepatitis B virus core capsid provides clinical alleviation against PEDV in neonatal piglets through lactogenic immunity

Porcine epidemic diarrhea virus (PEDV) has had a negative economic impact on the global swine industry for decades since its first emergence in the 1970s in Europe. In 2013, PEDV emerged for the first time in the United States, causing immense economic losses to the swine industry. Efforts to protect U.S. swine herds from PEDV infection and limit PEDV transmission through vaccination had only limited success so far. Following the previous success in our virus-like particle (VLP) based vaccine in mouse model, in this study we determined the immunogenicity and protective efficacy of a VLP-based vaccine containing B-cell epitope ⁷⁴⁸YSNIGVCK⁷⁵⁵ from the spike protein of PEDV incorporated into the hepatitis B virus core capsid (HBcAg), in a comprehensive pregnant gilt vaccination and piglet challenge model. The results showed that the vaccine was able to induce significantly higher virus neutralization response in gilt milk, and provide alleviation of clinical signs for piglets experimentally infected with PEDV. Piglets from pregnant gilt that was vaccinated with the VLP vaccine had faster recovery from the clinical disease, less small intestinal lesions, and higher survival rate at 10 days post-challenge (DPC).     

Cell attenuated porcine epidemic diarrhea virus strain Zhejiang08 provides effective immune protection attributed to dendritic cell stimulation

Since 2010, the porcine epidemic diarrhea coronavirus (PEDV) has caused significant damage to the global pork industry. However, classical PEDV vaccine strains only provide limited protection against emerging strains. In this study, we successfully isolated and attenuated the PEDV epidemic strain Zhejiang08, which was characterized by good cell adaptation and high-titer production 48 h post infection in Vero E6 cells. The attenuated virus induced a high level of virus-specific neutralizing antibodies until 120 days after immunization in piglets and provided complete protection when challenged with an emerging virus strain on day 14 post immunization. Moreover, the capability to activate dendritic cells (DCs) of this isolate was identified. Higher expression levels of IL-12 and IFN-γ were recorded in DCs after treatment with Zhejiang08 for 24 h. Furthermore, genome sequencing and phylogenetic analysis revealed high homology between the main antigen epitopes of Zhejiang08 and PEDV pandemic isolates following 2011. Combining the glycosylation site prediction results and their distribution within the spatial structure of the S protein, led to the conclusion that the observed more effective host immune response of Zhejiang08 compared to CV777 was possibly associated with a lack of the potential glycosylation site in the 296 amino acids of the S protein. In summary, we illustrated that the attenuated virus represents a promising vaccine candidate.     

Comparative genome analysis and molecular epidemiology of the reemerging porcine epidemic diarrhea virus strains isolated in Korea

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, is an enveloped, positive-sense, single-stranded RNA virus, which causes severe diarrhea and dehydration in suckling pigs. We detected three PEDV strains from ten small intestine samples from piglets with acute diarrhea and we determined the complete genome sequences of the reemerging Korean PEDV field isolates, except for the noncoding regions from both ends. The complete genome sequences of the strains were identical or almost identical (one synonymous single-nucleotide polymorphism (SNP) in the ORF1a/1b genomic sequence). Interestingly, comparative genome analysis of recent Korean PEDV isolates and other strains revealed that the complete genome sequences of recent Korean strains were almost identical (99.9%) to those of the US PEDV strains isolated in 2013. These results suggest that the three reemerging Korean strains are distinct from previous endemic Korean PEDV strains and has been recently introduced into Korea from oversea with high likelihood.     

Genetic diversity of ORF3 and spike genes of porcine epidemic diarrhea virus in Thailand

Porcine epidemic diarrhea virus (PEDV) has become endemic in the Thai swine industry, causing economic losses and repeated outbreaks since its first emergence in 2007. In the present study, 69 Thai PEDV isolates were obtained from 50 swine herds across Thailand during the period 2008–2012. Both partial and complete nucleotide sequences of the spike (S) glycoprotein and the nucleotide sequences of ORF3 genes were determined to investigate the genetic diversity and molecular epidemiology of Thai PEDV. Based on the analysis of the partial S glycoprotein genes, the Thai PEDV isolates were clustered into 2 groups related to Korean and Chinese field isolates. The results for the complete spike genes, however, demonstrated that both groups were grouped in the same cluster. Interestingly, both groups of Thai PEDV isolates had a 4-aa (GENQ) insertion between positions 55 and 56, a 1-aa insertion between positions 135 and 136, and a 2-aa deletion between positions 155 and 156, making them identical to the Korean KNU series and isolates responsible for outbreaks in China in recent years. In addition to the complete S sequences, the ORF3 gene analyses suggested that the isolates responsible for outbreaks in Thailand are not vaccine related. The results of this study suggest that the PEDV isolates responsible for outbreaks in Thailand since its emergence represent a variant of PEDV that was previously reported in China and Korea.     

New variants of porcine epidemic diarrhea virus, china, 2011

In 2011, porcine epidemic diarrhea virus (PEDV) infection rates rose substantially in vaccinated swine herds. To determine the distribution profile of PEDV outbreak strains, we sequenced the full-length spike gene from samples from 9 farms where animals exhibited severe diarrhea and mortality rates were high. Three new PEDV variants were identified.     

A novel subunit vaccine co-expressing GM-CSF and PCV2b Cap protein enhances protective immunity against porcine circovirus type 2 in piglets

Porcine circovirus type 2 (PCV2) causes porcine circovirus-associated disease. Capsid (Cap) protein of PCV2 is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. GM-CSF is an immune adjuvant that enhances responses to vaccines. In this study, recombinant baculoviruses Ac-Cap and Ac-Cap-GM-CSF expressing the Cap protein alone and co-expressing the Cap protein and porcine GM-CSF, respectively, were constructed successfully. The target proteins were analyzed by western blotting and IFA. Further, these proteins were confirmed by electron microscopy, which showed that Cap proteins could self-assemble into virus-like particles having diameters of 17–25 nm. Animal experiments showed that pigs immunized with Cap-GM-CSF subunit vaccine showed significantly higher levels of PCV2-specific antibodies and neutralizing antibodies than pigs immunized with the Cap subunit vaccine and a commercial vaccine (Ingelvac CircoFLEX; P < 0.05). After PCV2 wild strain challenged, Pigs receiving the Cap-GM-CSF subunit vaccine showed significantly higher average daily weight gain after wild-type PCV2 challenge than pigs receiving the other three vaccines (P < 0.05). None of PCV2 DNA was detected in all immunized animals, except control animals immunized with phosphate-buffered saline. These results indicated that GM-CSF was a powerful immunoadjuvant for PCV2 subunit vaccines because it enhanced humoral immune response and improved immune protection against PCV2 infection in pigs. Thus, the novel Cap-GM-CSF subunit vaccine has the potential to be used as an effective and safe vaccine candidate against PCV2 infection.     

A candidate multi-epitope vaccine against porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae induces robust humoral and cellular response in mice

Porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae (M. hyopneumoniae, Mhp) are two of the most common pathogens involved in the porcine respiratory disease complex (PRDC) resulting in significant economic losses worldwide. Vaccination is the most effective approach to disease prevention. Since PRRSV and Mhp co-infections are very common, an efficient dual vaccine against these pathogens is required for the global swine industry. Compared with traditional vaccines, multi-epitope vaccines have several advantages, they are comparatively easy to produce and construct, are chemically stable, and do not have an infectious potential. In this study, to develop a safe and effective vaccine, B cell and T cell epitopes of PRRSV-GP5, PRRSV-M, Mhp-P46, and Mhp-P65 protein had been screened to construct a recombinant epitope protein rEP-PM that has good hydrophilicity, strong antigenicity, and high surface accessibility, and each epitope is independent and complete. After immunization in mice, rEP-PM could induce the production of high levels of antibodies, and it had good immunoreactivity with anti-rEP-PM, anti-PRRSV, and anti-Mhp antibodies. The anti-rEP-PM antibody specifically recognizes proteins from PRRSV and Mhp. Moreover, rEP-PM induced a Th1-dominant cellular immune response in mice. Our results showed that the rEP-PM protein could be a potential candidate for the development of a safe and effective multi-epitope peptide combined vaccine to control PRRSV and Mhp infections.     

Multivalent DNA vaccine induces protective immune responses and enhanced resistance against Cryptosporidium parvum infection

The aim of this work was to evaluate efficiency as well as the type of immune response, Th1 or Th2, induced by multivalent DNA vaccinations in C57BL/6 interleukin-12p40 (IL-12p40) knockout (KO) mice. A recombinant pVAX-15-23 plasmid DNA was constructed by inserting surface glycoprotein (cp15- and p23)-encoding DNA into the pVAX1 expression vector. Various parameters including antibody and cytokine responses, proliferation assay and oocyst shedding were used to evaluate the type of immune response and the level of protection against challenge infection. Obtained results indicated that plasmid pVAX-15-23 induced strong protective immune response against C. parvum characterized by dominance of IgG2a, high level of INF-γ and lower level of the oocysts shedding after challenge infection. Moreover, co-immunization with the multivalent DNA and pMEM12R plasmid encoding IL-12 can further enhance these responses compared with the multivalent DNA alone. The obtained results suggest that multivalent pVAX-15-23 DNA vaccine may be a candidate as a generic approach to C. parvum immunization applicable to clinical practice.     

Immunogenicity study of plant-made oral subunit vaccine against porcine reproductive and respiratory syndrome virus (PRRSV)

Currently, killed-virus and modified-live PRRSV vaccines are used to control porcine reproductive and respiratory syndrome disease (PRRS). However, very limited efficacy of killed-virus vaccines and serious safety concerns for modified-live virus vaccines demand the development of novel PRRSV vaccines. In this report, we investigated the possibility of using transgenic plants as a cost-effective and scalable system for production and delivery of a viral protein as an oral subunit vaccine against PRRSV. Corn calli were genetically engineered to produce PRRSV viral envelope-associated M protein. Both serum and intestine mucosal antigen-specific antibodies were induced by oral administration of the transgenic plant tissues to mice. In addition, serum and mucosal antibodies showed virus neutralization activity. The neutralization antibody titers after the final boost reached 6.7 in serum and 3.7 in fecal extracts, respectively. A PRRSV-specific IFN-γ response was also detected in splenocytes of vaccinated animals. These results demonstrate that transgenic corn plants are an efficient subunit vaccine production and oral delivery system for generation of both systemic and mucosal immune responses against PRRSV.     

Vaccination with a modified-live bovine viral diarrhea virus (BVDV) type 1a vaccine completely protected calves against challenge with BVDV type 1b strains

Vaccination plays a significant role in the control of bovine viral diarrhea virus (BVDV) infection and spread. Recent studies revealed that type 1b is the predominant BVDV type 1 subgenotype, representing more than 75% of field isolates of BVDV-1. However, nearly all current, commercially available BVDV type 1 vaccines contain BVDV-1a strains. Previous studies have indicated that anti-BVDV sera, induced by BVDV-1a viruses, show less neutralization activity to BVDV-1b isolates than type 1a. Therefore, it is critically important to evaluate BVDV-1a vaccines in their ability to prevent BVDV-1b infection in calves. In current studies, calves were vaccinated subcutaneously, intradermally or intranasally with a single dose of a multivalent, modified-live viral vaccine containing a BVDV-1a strain, and were challenged with differing BVDV-1b strains to determine the efficacy and duration of immunity of the vaccine against these heterologous virus strains. Vaccinated calves, in all administration routes, were protected from respiratory disease caused by the BVDV-1b viruses, as indicated by significantly fewer clinical signs, lower rectal temperatures, reduced viral shedding and greater white blood cell counts than non-vaccinated control animals. The BVDV-1a vaccine elicited efficacious protection in calves against each BVDV-1b challenge strain, with a duration of immunity of at least 6 months.     

Recombinant gp90 protein expressed in Pichia pastoris induces a protective immune response against reticuloendotheliosis virus in chickens

Reticuloendotheliosis virus (REV) causes an oncogenic, immunosuppressive and runting syndrome in multiple avian hosts worldwide. In this study, the gp90 protein of REV was secretory expressed in Pichia pastoris with high production level and good antigenicity. To fully utilize the expression potential of the P. pastoris expression system, a panel of Pichia clones carrying increasing copies of the gp90 expression cassette was created using an in vitro multimerization approach and the effects of gene dosage on gp90 expression were investigated. Results demonstrated that an increase in gp90 copy number can significantly improve the yields of gp90 protein. Following expression and scale-up, the gp90 protein production level could reach up to 400mg/L, and the protein could be detected by gp90-specific monoclonal antibody. Investigations of its vaccine efficacy demonstrated that the recombinant gp90 protein was able to induce sustained high levels of antibodies against REV as being detected by ELISA and virus neutralizing test. Furthermore, immunization of chickens with the recombinant gp90 vaccine fully protected the animals from viremia after REV infection. Overall, the yeast-expressed gp90 protein retains good immunogenicity and could be used as a potential subunit vaccine candidate for REV prevention.     

Cholera toxin-B (ctxB) antigen expressing Salmonella Typhimurium polyvalent vaccine exerts protective immune response against Vibrio cholerae infection

Live attenuated vaccines are cost effective approach for preventing a broad range of infectious diseases, and thus are of great interest. However, immune-defects can predispose the patient to infections by the vaccine candidate itself. So far, few live vaccine candidates have been designed specifically for immune compromised individuals. Recently, we reported a new Salmonella Typhimurium Z234-vaccine strain (Periaswamy et al., PLoS ONE 2012;7:e45433), which was specifically attenuated in the NADPH-oxidase deficient host. In the present study, the Z234-vaccine strain was further engineered to express heterologous antigen (Vibrio cholerae toxin antigen subunit-B, i.e. CtxB) with the intention of creating a vector for simultaneous protection against Cholera and Salmonellosis. The primary aim of this study was to ensure the expression of CtxB antigen by the recombinant vaccine strain Z234-pMS101. The antigen CtxB was expressed through Z234 as a fusion protein with N-terminal signal sequence of Salmonella outer protein (SopE), an effector protein from Salmonella under the control of SopE promoter. The CtxB-expressing plasmid construct pMS101 (pM968-pSopE-ctxB) was found to be stable both in vitro and in vivo. In an oral mouse infection model, the vaccine strain Z234-pMS101 efficiently colonized the host gut. The extent of protection was confirmed after challenging the immunized hosts with live V. cholerae. Vaccinated mice showed reduced gut colonization by V. cholerae. Further assessment of immunological parameters supported the possibility of conferring effective immune response by Z234-pMS101 vaccine strain. Overall, the Z234-pMS101 vaccine strain showed potential as a promising polyvalent vaccine candidate to protect against S. Typhimurium and V. cholerae infection simultaneously.     

Intranasal administration of a flagellin-adjuvanted inactivated influenza vaccine enhances mucosal immune responses to protect mice against lethal infection

The influenza virus, a mucosal pathogen that infects the respiratory tract, is a major global health issue. There have been attempts to mucosally administer inactivated influenza vaccines to induce both mucosal and systemic immune responses. However, mucosally administered inactivated influenza vaccine has low immunogenicity, which is partially due to the lack of an effective mucosal adjuvant. The development of a safe and effective mucosal adjuvant is a prerequisite to the practical use of a mucosal inactivated influenza vaccine. We have previously demonstrated that a bacterial flagellin, Vibrio vulnificus FlaB, when mixed with antigen and administered intranasally, exerts a strong mucosal adjuvant activity by stimulating the Toll-like receptor 5 (TLR5). In this study, we tested whether the FlaB protein could serve as an effective mucosal adjuvant for an inactivated trivalent influenza vaccine (TIV) manufactured for humans; in a murine vaccination model, this vaccine consists of A/Brisbane/59/07 (H1N1 subtype), A/Uruguay/716/07 (H3N2 subtype), and B/Florida/4/06 (B type). Intranasal co-administration of the TIV with FlaB induced prominent humoral responses as demonstrated by high influenza-specific IgA levels in both the mucosal secretions and serum and significant specific IgG induction in the systemic compartment. The FlaB protein significantly potentiated influenza-specific cytokine production by draining lymph node cells and splenocytes. The FlaB mucosal adjuvant conferred excellent protection against a lethal challenge with a live virulent virus with high hemagglutination inhibition (HAI) antibody (Ab) titers. The FlaB did not accumulate in the olfactory nerve and epithelium, guaranteeing against a retrograde uptake into the central nervous system. These results suggest that FlaB can be used as a promising mucosal adjuvant for nasal inactivated influenza vaccine development.     

Electroporation enhances protective immune response of a DNA vaccine against Japanese encephalitis in mice and pigs

Japanese encephalitis virus (JEV) is a pathogenic cause of Japanese Encephalitis (JE), which is a zoonotic disease transmitted by mosquitoes and amplified by pigs. Infection of JEV may lead to severe neurological sequelae, even death in humans and reproductive disorders in pigs. Vaccination is the only way to control JEV infection in humans. For pigs play important role in the JEV transmission cycle, developing a new veterinary vaccine is considered as a useful strategy for cutting off the transmission route of JEV. We have previously reported that DNA vaccine pCAG-JME, expressing prM-E proteins of JEV, is effective in mice through intramuscular injection (IM). However, the poor immunogenicity, due to low expression of immunogen, is the major obstacle for the development of DNA vaccine in large animals. In the present study, therefore, we immunized mice and pigs with pCAG-JME intramuscularly accompanied with electroporation (EP) stimulation, the attractive gene delivery approach. As compared with IM, EP-mediated vaccination markedly increased the expression of immunogen in the injection site and induced a dose- and time-dependent immune response. 100% survival rate was observed in groups vaccinated with doses ranged from 10 to 100μg, indicating that 10μg of DNA with EP for individual was enough for inducing effective protection in mice. Surprisingly, survival rate and end-point titers of anti-JEV antibodies were higher in mice even at lower dose of DNA (5μg) than that in mice inoculated 100μg through IM. Notably, the prM-E antigens also induced high antibody response in pig, while the neutralizing antibody titer achieved 1:320. Our results suggested that EP-mediated DNA immunization might act as an effective strategy against JEV, at least in pig, and that EP has a potential application prospect in DNA vaccination.     

VennVax, a DNA-prime, peptide-boost multi-T-cell epitope poxvirus vaccine, induces protective immunity against vaccinia infection by T cell response alone

The potential for smallpox to be disseminated in a bioterror attack has prompted development of new, safer smallpox vaccination strategies. We designed and evaluated immunogenicity and efficacy of a T-cell epitope vaccine based on conserved and antigenic vaccinia/variola sequences, identified using bioinformatics and immunological methods. Vaccination in HLA transgenic mice using a DNA-prime/peptide-boost strategy elicited significant T cell responses to multiple epitopes. No antibody response pre-challenge was observed, neither against whole vaccinia antigens nor vaccine epitope peptides. Remarkably, 100% of vaccinated mice survived lethal vaccinia challenge, demonstrating that protective immunity to vaccinia does not require B cell priming.     

Glycoprotein E2 of classical swine fever virus expressed by baculovirus induces the protective immune responses in rabbits

Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious and devastating disease that affects the pig industry worldwide. The glycoprotein E2 of CSFV is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. Several CSFV genotypes, including 1.1, 2.1, 2.2, and 2.3, have been identified in Mainland China. The glycoprotein E2 of genotypes 1.1 and 2.1 was expressed by using a baculovirus system and tested for its protective immunity in rabbits to develop novel CSF vaccines that elicit a broad immune response. Twenty CSFV seronegative rabbits were randomly divided into five groups. Each rabbit was intramuscularly immunized with E2 of genotypes 1.1 (CSFV-1.1E2), 2.1 (CSFV-2.1E2), or their combination (CSFV-1.1 + 2.1E2). A commercial CSF vaccine (C-strain) and phosphate-buffered saline (PBS) were used as positive or negative controls, respectively. All animals were challenged with CSFV C-strain at 4 weeks and then boosted with the same dose. All rabbits inoculated with CSFV-1.1E2, CSFV-2.1E2, and CSFV-1.1 + 2.1E2 elicited high levels of ELISA antibody, neutralizing antibody, and lymphocyte proliferative responses to CSFV. The rabbits inoculated with CSFV-1.1E2 and CSFV-1.1 + 2.1E2 received complete protection against CSFV C-strain. Two of the four rabbits vaccinated with CSFV-2.1E2 were completely protected. These results demonstrate that CSFV-1.1E2 and CSFV-1.1 + 2.1E2 not only elicit humoral and cell-mediated immune responses but also confer complete protection against CSFV C-strain in rabbits. Therefore, CSFV-1.1E2 and CSFV-1.1 + 2.1E2 are promising candidate subunit vaccines against CSF.     

Single mucosal immunization of recombinant adenovirus-based vaccine expressing F1 protein fragment induces protective mucosal immunity against respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV. The fusion (F) protein of RSV is a potentially important target for protective antiviral immune responses. Here, we studied the immune responses elicited by recombinant replication-deficient adenovirus (rAd)-based vaccines expressing the soluble F1 fragment of F protein (amino acids 155–524) in murine model. The expression of secreted F1 fragment by rAd was significantly increased by codon optimization. Strong mucosal IgA response was induced by single intranasal immunization of codon-optimized vaccine, rAd/F1co, but not by rAd/F1wt. A single intranasal immunization with rAd/F1co provided potent protection against subsequent RSV challenge. Interestingly, neither serum Ig nor T-cell response directed to F protein was detected in the rAd/F1co-immune mice, suggesting that protective immunity by rAd/F1co is mainly mediated through mucosal IgA induction. Indeed, co-delivery of cholera toxin B subunit significantly enhanced mucosal IgA responses by the optimized vaccine, which correlates with protective efficacy. Taken together, our data demonstrate that a single intranasal administration of rAd/F1co is sufficient for the protection and represents a promising prophylactic vaccination regimen against RSV infection.