Candidate peptide vaccine induced protection against classical swine fever virus

Former investigations demonstrated that the envelope glycoprotein E2 could protect pigs from classical swine fever virus (CSFV). Based on these findings, we prepared synthetic peptide vaccine using E2 N-terminal antigenic units B/C and hoped to induce protective activity against lethal challenge of virulent CSFV strain Shimen. Five overlapped peptides sequence-covering amino acids 693-777 on E2 of Shimen were synthesized and then conjugated with bovine serum albumin (BSA), respectively. In the vaccination course, the candidate peptide vaccines in combination (multi-peptide vaccine (MPV)) were applied for immunization of pigs (n=10) and induced strong antibody response against CSFV. It is subsequently demonstrated that this peptide vaccine could provide immunized pigs complete protection against lethal CSFV challenge as C-strain does, while all non-immunized pigs in negative control group manifested obvious typical symptoms and died during the second and third weeks after viral challenge. In order to confirm the neutralizing activity of the polyclonal antibodies induced by MPV, neutralization assay were carried out on rabbits. The live C-strain alone could ordinarily induce typical fever on rabbits. The typical fever of rabbits induced by the live C-strain could be inhibited by pre-incubation with the anti-sera (dilution 1:4 and 1:16) induced by MPV, but not inhibited by pre-incubation with the same anti-sera from which the antibodies against five peptides were removed by peptide-specific affinity chromatography, which indicates that these peptide-specific antibodies in the anti-sera induced by MPV provided protective activity against CSFV. Our finding provides a new way to develop marker vaccine against CSFV.     

An E2 sub-unit marker vaccine does not prevent horizontal or vertical transmission of classical swine fever virus

An experimental infection with classical swine fever (CSF) virus in E2 sub-unit marker vaccine vaccinated gilts was conducted in order to evaluate the effect of vaccination on virus transmission and course of the disease. Therefore, clinical signs as well as horizontal and vertical virus transmission were monitored in two inoculated, non-vaccinated and 10 vaccinated conventional gilts, housed in individual sow boxes. Within 10 days post-inoculation, all vaccinated gilts became infected. Depending on the definition of the infectious period, two different estimates of R0 were calculated (R0=14.8 and 3.3), both significantly larger than 1 (P<0.01). In three out of the eight vaccinated pregnant gilts vertical virus transmission occurred, resulting in infected offspring. Based on the results of this experiment, it can be concluded that double vaccination with an E2 sub-unit marker vaccine only protects pregnant gilts from the clinical course of the disease but does not prevent horizontal nor vertical spread of the CSF virus.     

Evaluation of genetic vaccine against classical swine fever

Classical swine fever is important diseases affecting pigs. It results in great losses in their population and in limitations in the commercial international trade of pigs. The aim of the study was the preparation of the genetic vaccine against CSF and the estimation of its safety, protection value and immunogenicity. Clinical observations, body temperature and the immune response (haematological and FACS analyses) were monitored. Pigs vaccinated with the DNA vaccine were protected from the challenge, however, 2 days fever > 40 degrees C was registered. Slight activity of B and T cells was noted in those animals.     

Spying the neutralizing epitopes on E2 N-terminal by candidate epitope-vaccines against classical swine fever virus

Our previous study proved that the N-terminal (aa693-711) of glycoprotein E2 contained sequential neutralizing epitopes. In this study, four candidate epitope-vaccines (EVs) were separately prepared and evaluated. Among them, epitope-vaccine EV-BC1a (BC1a: aa693-699) induced high level of epitope-specific neutralizing antibodies and exhibited similar protective capability with that induced by Chinese vaccine strain (C-strain). These results confirmed CKEDYRY (aa693-699) as a principal sequential neutralizing epitope on E2 N-terminal. Moreover, these findings also indicate that epitope-vaccine is a potent candidate strategy for marker vaccine against classical swine fever virus (CSFV).     

DNA-mediated protection against classical swine fever virus

Four eukaryotic expression plasmids containing the entire E2 gene sequence of classical swine fever virus (CSFV) were constructed: (a) pcDST, with 5' signal and 3' transmembrane sequences; (b) pcDSW, with 5' signal sequence only; (c) pcDWT, with transmembrane sequences only; and (d) pcDWW, containing the E2 gene alone. All four plasmids were readily transfected into BHK-21 cells, with pcDST and pcDSW resulting in secretion of E2 antigen. The latter two plasmids were also shown to induce a humoral immune response against CSFV in mice when administered intramuscularly, but no immune responses were detected with either pcDWT or pcDWW. The antibody level elicited by pcDSW was higher than that induced by pcDST. When pcDSW was used to immunize rabbits and pigs, both species were shown to be protected from challenge with virulent CSFV (hog cholera lapinized virus for rabbits and Shimen strain for pigs).     

A single dose of the novel chimeric subunit vaccine E2-CD154 confers early full protection against classical swine fever virus

Classical swine fever is an economically important, highly contagious disease of swine worldwide. Subunit vaccines are a suitable alternative for the control of classical swine fever. However, such vaccines have as the main drawback the relatively long period of time required to induce a protective response, which hampers their use under outbreak conditions. In this work, a lentivirus-based gene delivery system is used to obtain a stable recombinant HEK 293 cell line for the expression of E2-CSFV antigen fused to porcine CD154 as immunostimulant molecule. The E2-CD154 chimeric protein was secreted into the medium by HEK293 cells in a concentration around 50 mg/L in suspension culture conditions using spinner bottles. The E2-CD154 immunized animals were able to overcome the challenge with a high virulent CSF virus strain performed 7 days after a unique dose of the vaccine without clinical manifestations of the disease. Specific anti-CSFV neutralizing antibodies and IFN-γ were induced 8 days after challenge equivalent to 14 days post-vaccination. The present work constitutes the first report of a subunit vaccine able to confer complete protection by the end of the first week after a single vaccination. These results suggest that the E2-CD154 antigen could be potentially used under outbreak conditions to stop CSFV spread and for eradication programs in CSF enzootic areas.     

An experimental infection with classical swine fever in E2 sub-unit marker-vaccine vaccinated and in non-vaccinated pigs

The clinical and virological protection induced by an E2 sub-unit marker-vaccine against Classical Swine Fever (CSF) was examined during an experimental infection in vaccinated and non-vaccinated pigs. Forty-five pigs were equally distributed over three adjacent pens of an isolation unit, there was only indirect (airborne) contact between pigs in the different pens. In pen 3 all pigs were vaccinated twice with 4 weeks interval. Pigs in pens 1 and 2 were not vaccinated. Two weeks after booster vaccination, one randomly selected pig in the middle pen was experimentally inoculated with CSF virus. After the initial virus spread in the infected pen, all pigs in the non-vaccinated adjacent pen were infected. In the vaccinated pen, seven out of 14 pigs became infected during the experiment. Survival analysis showed that virus transmission by direct and indirect contact was significantly (p<0.001) delayed in vaccinated pigs as compared to non-vaccinated pigs. In the non-vaccinated pens over 40% of the pigs died and typical clinical signs were noticed. In the vaccinated pen no mortality and no clinical symptoms were observed. Although double vaccination with an E2 sub-unit marker-vaccine was able to prevent the clinical course of the disease it was unable to prevent infection through indirect contact. This finding combined with the slow serological response after vaccination will complicate the possible use of the vaccine in emergency vaccination programmes.     

The swine CD81 enhances E2-based DNA vaccination against classical swine fever

Classical swine fever (CSF) is a highly contagious and economically important viral disease that affects the pig industry worldwide. The glycoprotein E2 of CSFV can induce neutralizing antibodies and protective immunity, and is widely used for novel vaccine development. The objective of this study was to explore whether a tetraspanin molecule CD81 could improve the immune responses of an E2-based DNA vaccine. Plasmids pVAX-CD81, pVAX-E2 and pVAX-CD81-E2 were constructed and the expression of target proteins was confirmed in BHK-21 cells by indirect immunofluorescence assay. BALB/c mice were divided into 5 groups and immunized with different plasmids (pVAX-E2, pVAX-CD81-E2, pVAX-E2 + pVAX-CD81, pVAX-CD81 and PBS) three times with two weeks interval. The results showed that the introduction of CD81 promoted higher humoral and cellular immune responses than E2 expression alone (P < 0.05). In addition, immunization with pVAX-CD81-E2 induced stronger immune responses than pVAX-E2 + pVAX-CD81. Furthermore, four groups of pigs were immunized with pVAX-E2, pVAX-CD81-E2, pVAX-CD81 and PBS, respectively. Humoral and cellular immune responses detection showed similar results with those in mice. Compared to pVAX-E2, pVAX-CD81-E2 induced higher titers of neutralizing antibodies after viral challenge and conferred stronger protection. These results confirmed the capacity of swine CD81 enhancing the humoral and cellular responses with an adjuvant effect on CSFV DNA vaccine. This is the first report demonstrating the adjuvant effect of CD81 to enhance the DNA vaccination for swine pathogen.     

Candidate multi-peptide-vaccine against classical swine fever virus induced potent immunity with serological marker

Our previous study proposed a protective multi-peptide-vaccine (MPV) with Freund's adjuvant against classical swine fever virus (CSFV). In this study, another candidate MPV, using aluminum adjuvant, was further examined. All immunized pigs kept healthy during the experimental period, while the control group rapidly showed clinical symptoms and died. Moreover, anti-sera from MPV-immunized pigs could interact with peptides involved in the MPV, in contrast to anti-sera from non-immunized or infected ones. This property permits MPV-immunized pigs to be easily differentiated from infected ones with simple serological method. Therefore, this new MPV is suitable to act as a candidate marker vaccine against CSFV.     

Yeast expressed classical swine fever E2 subunit vaccine candidate provides complete protection against lethal challenge infection and prevents horizontal virus transmission

Classical swine fever (CSF) caused by the classical swine fever virus (CSFV) is a highly contagious swine disease resulting in large economical losses worldwide. The viral envelope glycoprotein E(rns) and E2 are major targets for eliciting antibodies against CSFV in infected animals. A Pichia pastoris yeast expressed E2 protein (yE2) has been shown to induce a protective immune response against CSFV challenge. The purpose of this study is to determine the optimal dose of yE2 and its efficacy on the prevention of virus horizontal transmission. A yeast-expressed E(rns) (yE(rns)) protein was also included to evaluate its immunogenicity. The yE(rns) vaccinated pigs seroconverted to CSFV-E(rns)-specific antibody but no neutralizing antibody was detected and none survived after challenge infection, suggesting yE(rns) and yE2 retain correct immunogenicity but only the yE2 is able to induce a protective immune response. All three doses of yE2 (200, 300, and 400μg) could elicit high titers of neutralizing antibodies and protective responses after challenge. The yE2/200 group demonstrated a mild fever response but recovered soon, and none of the yE2/300 and yE2/400 pigs became febrile. The optimal dose of yE2 was recommended to be 300μg of the total amount of secreted proteins. In addition, the yE2 vaccine could cross-protect from all three genotypes of viruses. Further, the yE2 vaccine efficacy in preventing virus horizontal transmission was evaluated by cohabitation of unimmunized sentinels 3 days after challenge infection. All the sentinel pigs were alive and had no clinical symptoms confirming yE2 vaccine could confer a protective immune response and prevent horizontal transmission of CSFV.     

Vaccination with a single dose of a recombinant porcine adenovirus expressing the classical swine fever virus gp55 (E2) gene protects pigs against classical swine fever

A recombinant porcine adenovirus (rPAV) with the gp55 (E2) gene from the classical swine fever virus (CSFV) 'Weybridge' strain inserted into the right hand end of the PAV serotype 3 (PAV3) genome was constructed. Expression of gp55 was directed by the major late promoter and tri-partite leader sequences located and cloned from PAV3. No compensatory deletions of PAV DNA sequences were made. Vaccination of outbred pigs with a single dose of the recombinant virus (rPAV-gp55) resulted in complete protection from lethal challenge with CSFV. No adverse clinical signs were observed in vaccinated animals following administration of rPAV-gp55 and following challenge, no clinical signs of CSF were observed prior to, or at, post mortem. The insert made into the rPAV increased the genome length to 106.8% of wild type and therefore exceeded the expected maximum insert size for a stable recombinant by almost 2%. Thus rPAV-gp55 contains the largest stable insertion made into a non-deleted Mastadeno virus recombinant so far reported.     

Comprehensive evaluation of the adenovirus/alphavirus-replicon chimeric vector-based vaccine rAdV-SFV-E2 against classical swine fever

Classical swine fever (CSF) is an economically important, highly contagious swine disease caused by classical swine fever virus (CSFV). Marker vaccines and companion serological diagnostic tests are thought to be a promising strategy for future control and eradication of CSF. Previously, we have demonstrated that an adenovirus-vectored Semliki forest virus replicon construct expressing the E2 glycoprotein from CSFV, rAdV-SFV-E2, induced sterile immunity against a lethal CSFV challenge. In this study, we further evaluated the vaccine with respect to its safety, number and dose of immunization, and effects of maternally derived antibodies, re-immunization of the vaccine or co-administration with pseudorabies vaccine on the vaccine efficacy. The results showed that: (1) the vaccine was safe for mice, rabbits and pigs; (2) two immunizations with a dose as low as 6.25 × 10⁵ TCID₅₀ or a single immunization with a dose of 10⁷ TCID₅₀ rAdV-SFV-E2 provided complete protection against a lethal CSFV challenge; (3) maternally derived antibodies had no inhibitory effects on the efficacy of the vaccine; (4) the vaccine did not induce interfering anti-vector immunity; and (5) co-administration of rAdV-SFV-E2 with a live pseudorabies vaccine induced antibodies and protection indistinguishable from immunization with either vaccine administered alone. Taken together, the chimeric vaccine represents a promising marker vaccine candidate for control and eradication of CSF.     

A Semliki Forest virus replicon vectored DNA vaccine expressing the E2 glycoprotein of classical swine fever virus protects pigs from lethal challenge

Classical swine fever virus (CSFV) causes significant losses in pig industry in many countries in Asia and Europe. The E2 glycoprotein of CSFV is the main target for neutralizing antibodies. Recently, the replicon of alphaviruses, such as Semliki Forest virus (SFV), has been developed as replicative expression vectors for gene delivery. In this study, we constructed a plasmid DNA based on SFV replicon encoding the E2 glycoprotein of CSFV and evaluated its efficacy in rabbits and pigs. The results showed that the animals immunized with the DNA vaccine developed CSFV-specific neutralizing antibodies and were protected from virulent or lethal challenge. This demonstrates that the SFV replicon-derived DNA vaccine can be a potential marker vaccine against CSFV infections.     

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.     

A novel alphavirus replicon-vectored vaccine delivered by adenovirus induces sterile immunity against classical swine fever

Low efficacy of gene-based vaccines due to inefficient gene delivery and expression has been major bottleneck of their applications. Efforts have been made to improve the efficacy, such as gene gun and electroporation, but the strategies are difficult to put into practical use. In this study, we developed and evaluated an adenovirus-delivered, alphavirus replicon-vectored vaccine (chimeric vector-based vaccine) expressing the E2 gene of classical swine fever virus (CSFV) (rAdV-SFV-E2). Rabbits immunized with rAdV-SFV-E2 developed CSFV-specific antibodies as early as 9 days and as long as 189 days and completely protected from challenge with C-strain. Pigs immunized with rAdV-SFV-E2 (n = 5) developed robust humoral and cell-mediated responses to CSFV and were completely protected from subsequent lethal CSFV infection clinically and virologically. The level of immunity and protection induced by rAdV-SFV-E2 was comparable to that provided by the currently used live attenuated vaccine, C-strain. In contrast, both the conventional alphavirus replicon-vectored vaccine pSFV1CS-E2 and conventional adenovirus-vectored vaccine rAdV-E2 provided incomplete protection. The chimeric vector-based vaccine represents the first gene-based vaccine that is able to confer sterile immunity and complete protection against CSFV. The new-concept vaccination strategy may also be valuable in vaccine development against other pathogens.     

Hypervariable antigenic region 1 of classical swine fever virus E2 protein impacts antibody neutralization

Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies and confers protection against CSFV infection. There are three hypervariable antigenic regions (HAR1, HAR2 and HAR3) of E2 that are different between the group 1 vaccine C-strain and group 2 clinical isolates. This study was aimed to characterize the antigenic epitope region recognized by monoclonal antibody 4F4 (mAb-4F4) that is present in the group 2 field isolate HZ1-08, but not in the C-strain, and examine its impact on neutralization titers when antisera from different recombinant viruses were cross-examined. Indirect ELISA with C-strain E2-based chimeric proteins carrying the three HAR regions showed that the mAb-4F4 bound to HAR1 from HZ1-08 E2, but not to HAR2 or HAR3, indicating that the specific epitope is located in the HAR1 region. Of the 6 major residues differences between C-strain and field isolates, Glu713 in the HAR1 region of strain HZ1-08 is critical for mAb-4F4 binding either at the recombinant protein level or using intact recombinant viruses carrying single mutations. C-strain-based recombinant viruses carrying the most antigenic part of E2 or HAR1 from strain HZ1-08 remained non-pathogenic to pigs and induced good antibody responses. By cross-neutralization assay, we observed that the anti-C-strain serum lost most of its neutralization capacity to RecC-HZ-E2 and QZ-14 (subgroup 2.1d field isolate in 2014), and vice versa. More importantly, the RecC-HAR1 virus remained competent in neutralizing ReC-HZ-E2 and QZ-14 strains without compromising the neutralization capability to the recombinant C-strain. Thus, we propose that chimeric C-strain carrying the HAR1 region of field isolates is a good vaccine candidate for classical swine fever.     

Novel marker vaccines against classical swine fever

Classical swine fever (CSF) is one of the most devastating epizootic diseases of pigs worldwide. For eradication and control purposes, CSF vaccination is an important tool, and efficacious and safe attenuated vaccines have been available for many decades (for example, the C-strain vaccines). In addition to administering them parenterally, live attenuated vaccines are also administered orally for the control and eradication of CSF in wild boar populations. However, antibodies against live attenuated vaccines do not allow to differentiate infected from vaccinated animals (DIVA principle) and the mechanism responsible for attenuation is not known. Only a few years ago the first DIVA vaccines based on baculovirus-expressed E2 glycoprotein have been put on the market [Hulst MM, Westra DF, Wensvoort G, Moormann RJ. Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera. J Virol 1993;67(9):5435-42]. However, these subunit E2 marker vaccines are less efficient and more than one parenteral application is necessary. Furthermore, oral vaccination is not possible. Taking these disadvantages into account, the development of novel CSF vaccines has been focussed on five different strategies, mainly based on genetically engineered constructs: (1) immunogenic CSFV peptides, (2) DNA vaccines, (3) viral vectors expressing CSFV proteins, (4) chimeric pestiviruses, and (5) trans-complemented deleted CSFV genomes (replicons).     

Efficient priming against classical swine fever with a safe glycoprotein E2 expressing Orf virus recombinant (ORFV VrV-E2)

An increasing demand in livestock animal husbandry for intervention or emergency vaccination strategies requires a rapid onset of protection linked to prevention of infectious agent spread. Using the new recombinant parapoxvirus (PPV) Orf virus (ORFV) as a vaccine expressing the CSFV E2 glycoprotein we demonstrate that a single intra-muscular application confers solid protection. In the prime only concept, multi-site application of the vector vaccine turned out to be superior to single-site application as no pyrexia occurred after virulent CSFV challenge and CSFV neutralizing serum antibodies regularly were detectable before challenge. Vector virus vaccinated swine were able to cope with the lymphocyte and in particular B-cell depression in peripheral blood after challenge showing no clinical signs and no viremia. Early after challenge CSFV-specific IFN gamma production (IFN-gamma) and high neutralizing serum antibody titers clearly differentiated na?ve from vaccinated and protected animals. After CSFV challenge neutralizing serum antibodies titers in vector vaccinated swine were significantly higher than those in sera from live attenuated vaccine primed animals. Horizontal challenge virus transmission was prevented under strict sentinel isolation before mingling but not in next-door stables separated by a wooden barrier at the day of challenge. The presence of CSFV-specific pre-challenge serum antibodies although in low titers is a good prognostic parameter for solid protection after ORFV vector vaccination even when a significant CSFV-specific IFN-gamma production was not detectable before challenge. A heterologous prime-boost regimen as a combination of prime with baculovirus-expressed glycoprotein E2 followed by boost with the parapoxvirus vector turned out to be a better immune stimulant than a homologous prime/boost with the modified live CSFV vaccine. A similar beneficial effect became evident when the challenge infection mimicked the booster vaccination after a single PPV vector prime.     

Quadruple antigenic epitope peptide producing immune protection against classical swine fever virus

Research on epitope-based vaccines is a current focus in the development of new vaccines against classical swine fever virus (CSFV). The present study aimed to engineer a quadruple antigenic epitope peptide of the CSFV immunogen E2 glycoprotein by splice overlap extension (SOE) PCR, expressed in E. coli fused with glutathione S-transferase (GST), and named rGST-4E. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that purified rGST-4E had an excellent immunoreactivity with swine anti-CSFV serum and rabbit anti-E2 serum. Animal vaccination trials showed that the rGST-4E was more immunogenic than mono-epitope peptide and was able to produce effective immune protection in rabbits against challenge with hog cholera lapinized virus, and in pigs against challenge with virulent CSFV. These data show that the recombinant repeated epitope peptide could be considered a potential epitope-based vaccine for prevention of the disease.     

Assessment of classical swine fever diagnostics and vaccine performance

Rapid and accurate diagnosis is of the utmost importance in the control of epizootic diseases such as classical swine fever (CSF), and efficacious vaccination can be used as a supporting tool. While most of the recently developed CSF vaccines and diagnostic kits are mostly validated according to World Organisation for Animal Health (OIE) standards, not all of the well-established traditional vaccines and diagnostic tests were subject to these validation procedures and requirements. In this report, data were compiled on performance and validation of CSF diagnostic tests and vaccines. In addition, current strategies for differentiating infected from vaccinated animals are reviewed, as is information on the control of CSF in wildlife. Evaluation data on diagnostic tests were kindly provided by National Reference Laboratories for CSF in various European countries.