Joan Tarradas

Interferon-gamma induction correlates with protection by DNA vaccine expressing E2 glycoprotein against classical swine fever virus infection in domestic pigs

Classical swine fever (CSF) is a highly contagious viral infection affecting domestic and wild pigs. For classical swine fever virus (CSFV), immunization with plasmids expressing different versions of glycoprotein E2 has proven an effective way to induce protection. Previously, we have also shown that immunization with DNA vaccine expressing glycoprotein E2 (DNA-E2) induced specific T helper cell responses in the absence of neutralizing antibodies. However, the role of T cell responses in protection against CSFV is largely unknown. Here we have extended these studies to deeply characterize the role of T cell responses by a DNA-E2 and their correlation with protection against CSFV infection. Thus, pigs vaccinated with the DNA vaccine induced a strong cellular immune response, characterized by the specific induction IFN-gamma expressing T cells after vaccination without any detectable levels of CSFV neutralizing antibodies. Constant levels of CSFV-specific IFN-gamma producing cells observed from the beginning of the infection until 7 days after challenge in vaccinated animals might contribute to early control of CSFV replication, at least until neutralizing antibodies are developed. Severe clinical signs of the disease, including high titers of viremia, pyrexia and virus spread to different organs, were recorded in the non-vaccinated challenged animals, in comparison to the vaccinated animals where only one animal showed mild clinical signs and a short peak of viremia. Lack of complete protection in this animal correlated with a delay on the induction of neutralizing antibodies, detectable only from day 11 post-CSFV challenge. Conversely, the rest of the pigs within the group developed neutralizing antibodies as early as at day two post-challenge, correlating with sterile protection. Finally, an inverse correlation seemed to exist between early induction of IFN-alpha and the protection observed, while IL-10 seemed to be differentially regulated in vaccinated and non-vaccinated animals. Our results support the relevance of the induction of a strong T cellular response to confer a solid protection upon DNA vaccination against CSFV. Further experiments are needed to be done in order to clarify the key cytokines playing a role in CSFV-protection and to obtain emergency vaccines capable to confer robust and fast protection.

Partial protection against classical swine fever virus elicited by dendrimeric vaccine-candidate peptides in domestic pigs.

We report the immunogenicity of three dendrimeric peptide vaccine candidates for classical swine fever virus (CSFV). Each dendrimeric construct contained four copies of a B-cell epitope from the E2 glycoprotein of CSFV [construct 1: E2 (694-712); 2: E2 (712-727); 3: E2 (829-842)] joined to a T-cell epitope from the NS3 protein (residues 1446-1460). Intramuscular immunization of domestic pigs with the different constructs significantly reduced the clinical score after lethal challenge with CSFV. In contrast, control pigs developed severe clinical signs of the disease. All pigs vaccinated with construct 1, containing a B-cell epitope from the E2 B-C domain, developed an antibody response that recognized not only the original dendrimeric immunogen but also its constituting E2 epitope in linear form, albeit no neutralizing antibodies were detected prior to viral challenge. Two of these pigs were partially protected, which associated with the induction of IFN-γ producing cells and of neutralizing antibodies upon challenge. Interestingly, the serological response elicited by construct 1 lacked antibodies to E2 A domain, used as infection markers. The dendrimeric approach could therefore provide a basis for the development of CSFV marker (DIVA) vaccines, and contribute to a better understanding of the immune responses against CSFV.

Peptide vaccine candidates against classical swine fever virus: T cell and neutralizing antibody responses of dendrimers displaying E2 and NS2–3 epitopes

Three peptide‐based systems integrating B and T antigenic sites of CSFV and displaying the B epitopes in fourfold presentation have been designed and produced, and shown to bring about significant enhancements in immunogenicity over the peptides in monomeric form. Of the different strategies tested for producing the dendrimeric constructs, stepwise SPPS using 3,6‐dioxaoctanoic acid as flexible, PEG‐like spacer units at the branching points is clearly advantageous, in particular over ligation in solution. The constructs have been used for immunization of domestic pigs, in order to evaluate the protective response induced by each peptide constructs, and to characterize the B‐ and T‐cell response against CSFV in the natural host. Copyright

Development and validation of a novel SYBR Green real-time RT-PCR assay for the detection of classical swine fever virus evaluated on different real-time PCR platforms.

Classical swine fever is a highly contagious viral disease that causes significant economic losses in pig production on a global scale. The rapid dissemination of the virus and the variability of the clinical signs merit the development of swift and accurate classical swine fever virus (CSFV) detection methods, which can assist in disease control. The development and evaluation of a novel quantitative real-time RT-PCR assay for CSFV detection, based on SYBR Green coupled to melting curve analysis, is described. The analytical and diagnostic performances of the method using two real-time PCR instruments were compared. The assay was specific and detected the major genotypes of CSFV. The limit of detection in cell culture medium and serum was 0.1 TCID50/reaction, while in tissue homogenate for both platforms, it was 1 TCID50/reaction. The limit of detection was 1, 10 and 10² gene copies/μL when nuclease-free water, serum and tissue homogenate, respectively, were used as sample matrices for both instruments. The analysis of 108 tissue homogenate and serum samples from animals infected with CSFV naturally and experimentally and non-infected animals showed that the assay provided a highly sensitive and specific method for classical swine fever.

The impact of CSFV on the immune response to control infection.

The severity of the acute form of CSF is responsible for the high mortality rate and has been the subject of many studies. Nevertheless, some animals are likely to develop a mild, chronic, or unapparent form of the disease. Paradoxically, this clinical form of the disease has not been well studied, especially regarding its pathogenesis. In this study, we investigated the infection in domestic pigs that is caused by the CSFV Cat01 strain, which is responsible for the 2001-2002 CSFV outbreak in Catalonia, Spain, and which caused mild and nonspecific clinical signs compared to the infection that is caused by another CSFV strain that is responsible for inducing severe clinical symptoms of disease. We assessed the impact of the CSFV infection in the immune system of domestic pigs, mainly on the kinetics of different cytokines, such as IFN-α (innate immunity) and IFN-γ (adaptive immune response), during the first weeks after infection. In addition, we evaluated the impact on the induction of the humoral response and its relation to the course of infection and the RNA CSFV viral load. The IFN-α levels in the serum samples from the pigs that developed a milder form of the CSF disease (infected with Cat01 strain) were lower than those that were detected in the pig with severe clinical CSF signs (Margarita strain). After infection with Cat01 strain, the IFN-γ levels in response to CSFV were detected in addition to the humoral response. Interestingly, in the serum samples of these animals, we detected the lowest load of CSFV RNA. Similarly, the lowest viral load levels were detected in the tonsils of these pigs. Both the T cells and the humoral response that were generated in most of the pigs that were infected with strain Cat01 may be related to the protection in the symptom progression of CSF against this viral strain. These results explain the antiviral role of IFN-γ in the absence of an antibody response. Likewise, these results corroborate the relevance and relationship that exists between the intensity of the T cell response and the protection against CSFV replication. Additionally, these results also explain how the failure to induce optimal levels of humoral and cellular responses after CSFV infection promotes the spread and persistence of the virus.

Phylogenetic networks to study the origin and evolution of porcine circovirus type 2 (PCV2) in Cuba.

Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of postweaning multisystemic wasting syndrome (PMWS), which is considered one of the most economically important swine diseases worldwide. In this study, a comparison between methodologies based on classical phylogenetic trees and networks to infer the origin of PCV2 in Cuba was performed. In addition, the mechanisms supporting the genetic variability of Cuban PCV2 populations were investigated. A retrospective study, using pig sera collected in Cuba from 1993 to 2004, to evaluate the presence of PCV2 genome and PCV2-specific antibodies was also conducted and revealed a lack of evidence of PCV2 infection in Cuban swine from years 1993 to 2004. A total of 24 complete Cuban PCV2 sequences collected between 2005 and 2009 from different regions of the country were analyzed. Three classical methods of phylogenetic analysis, namely Neighbour-Joining, Maximum Parsimony and Bayesian Inference, as well as haplotype network construction, were used. Whereas the classical phylogenetic trees suggested different origins for the Cuban PCV2 strains, the haplotype network revealed a direct connection between all the Cuban sequences in agreement with the obtained epidemiological and viral sequence data. Moreover, the importation of pigs carried out in 2005 from the Quebec-Ontario region, Canada, seems to be the most likely origin of PCV2 in Cuba. Likewise, the genetic variability of Cuban PCV2 sequences was supported by geographic segregation and positive selection pressure with estimated rates of nucleotide substitution on the order of 3.12×10(-3) and 6.57×10(-3) substitutions/site/year, which are closer to those reported for RNA viruses.

Increased viral load and prevalence of Torque teno sus virus 2 (TTSuV2) in pigs experimentally infected with classical swine fever virus (CSFV).

Torque teno sus viruses (TTSuVs) are considered non-pathogenic viruses, although lately they have been linked to porcine circovirus diseases, mainly with post weaning multisystemic wasting syndrome (PMWS). These associations point out a possible pathogenic role of TTSuVs or, alternatively, that TTSuV replication is up-regulated under disease conditions. In order to further explore the association of TTSuVs with disease occurrence, TTSuVs prevalence and viral load were assessed before and after an experimental infection with a highly pathogenic classical swine fever (CSF) virus (CSFV) isolate. Serum samples from 56 animals were analyzed by means of a real time quantitative PCR (qPCR) for TTSuV1 and TTSuV2 before and after (between 6 and 13 days post-inoculation) the CSFV challenge. Based on the post-infection clinical evolution and immune response against CSFV, the animals were divided into two groups: group I, with protecting immunity against CSFV and no clinical signs at the day of necropsy, and group II, with no detectable immune response against CSFV and moderate to severe clinical signs. TTSuVs qPCR results indicated that TTSuV2 and not TTSuV1 load in serum increased significantly after challenge with CSFV in the group of pigs with clinical signs, specifically in those with a moderate course of the disease. Therefore, this study emphasizes the different behaviour of both TTSuVs, as already found in the PMWS background, and further supports the association of TTSuV2 with disease occurrence.

A T-cell epitope on NS3 non-structural protein enhances the B and T cell responses elicited by dendrimeric constructions against CSFV in domestic pigs

It has been recently reported by our group that dendrimeric constructs combining B- and T-cell epitopes from classical swine fever virus (CSFV) provided partial protection against experimental infection. This research evaluated four newly designed constructions while taking into account our previous work, including the direct implication that a T-cell epitope from the NS3 protein contributes to the generation of the immune response against CSFV. To this end, the dendrimeric constructions, including either this NS3 T-cell epitope alone or two different B-cell epitopes without this T-cell epitope, were used to immunise pigs. Thus, construct 1, containing the NS3 T-cell epitope and four copies of a previously described B-cell epitope, significantly reduced the clinical scores and RNA viral loads after challenge relative to the control group. In three out of six animals in this group, vaccination achieved partial protection and was associated with IFN-gamma producing-cells and neutralising antibodies. In contrast, the pigs immunised with construct 2, again with four copies of the B epitope of construct 1 but lacking the T-cell motif, developed more severe clinical signs. Finally, the additional constructs 3 and 4 included four copies of a B epitope that was different from the epitope used in constructs 1 and 2 with or without the abovementioned NS3 T-cell epitope, respectively. Pigs immunised with these latter constructs developed low levels of peptide-specific antibodies that correlated with equally low levels of cellular responses, an absence of neutralising antibodies and a lack of protection. Even so, the clinical scores in the first week after the challenge were less severe for animals vaccinated with construct 3 than for those given construct 4. Our results confirm the relevant role of the B-cell epitope in residues 694-712 of the glycoprotein E2 (which is used in both constructs 1 and 2) for protection against CSFV, as well as the appropriateness of the newly used NS3 peptide as a specific T-cell epitope in domestic pigs.

Immunization with DNA vaccines containing porcine reproductive and respiratory syndrome virus open reading frames 5, 6, and 7 may be related to the exacerbation of clinical disease after an experimental challenge.

Pigs were immunized with DNA plasmids containing different open reading frames (ORFs) of a porcine reproductive and respiratory syndrome virus (PRRSV) genotype I strain. One group was injected with three inoculations of ORF7, a second group was immunized with three inoculations of plasmids containing ORF5 and ORF6, and a third group was kept as controls. Later, +21 days after the last inoculation, animals were challenged with the homologous strain. After the challenge, PRRSV-specific interferon (IFN)-γ-secreting cells and anti-PRRSV IgG antibodies developed faster in DNA vaccinated pigs (p<0.05). However, DNA-immunized pigs showed an exacerbation of the disease compared to the unvaccinated challenged pigs. The data suggest that previous immunization with DNA vaccines against glycoprotein 5 and/or matrix protein of PRRSV, as well as nucleoprotein but to a lesser degree, could result in an exacerbation of the clinical course in terms of fever upon challenge.

Immunomodulatory effect of swine CCL20 chemokine in DNA vaccination against CSFV

The objective of this work was to explore whether a plasmid expressing CCL20 chemokine could improve the immune response against CSFV in co-administration with a DNA vaccine expressing the E2 protein. The immunization of pigs with the DNA vaccine formulation, that contains swine CCL20 chemokine, resulted in the homogenous induction of detectable levels of CSFV antibodies at 36 days after the first injection. Remarkably, immunized animals with E2 DNA vaccine in co-administration with the plasmid containing swine CCL20 developed high titers of neutralizing antibodies against homologous and heterologous CSFV strains and were totally protected upon a lethal viral challenge (sterilizing protection). Our results confirm the role of CCL20 to increase antibody-mediated responses. At the same time suggest the ability of CCL20 to enhance the T helper cell response associated with the induction of neutralizing antibodies against CSFV in pigs previously reported. Systemic replication of virulent CSFV in vivo during the acute phase of infection induces type I IFN. Lower average values of IFN alpha were detected in the serum of pigs immunized with pE2 and pCCL20 at 3 days after challenge. The levels of IFN-alpha detected in pigs immunized with pE2 and principally in non-vaccinated challenged animals can be related to viral load in serum at 3 and 7 days post infection and the clinical signs observed. Our results emphasized the capacity of swine CCL20 chemokine to enhance cellular, humoral and anti viral response with an adjuvant effect in the immune response elicited by E2-DNA vaccination against CSFV. To our knowledge, this is the first report demonstrating the adjuvant effect of swine CCL20 to effectively enhance the potential of DNA vaccine in the immune induction and protection against virus challenge in swine infection model.