DNA Vaccination Partially Protects against African Swine Fever Virus Lethal Challenge in the Absence of Antibodies

Abstract

The lack of available vaccines against African swine fever virus (ASFV) means that the evaluation of new immunization strategies is required. Here we show that fusion of the extracellular domain of the ASFV Hemagglutinin (sHA) to p54 and p30, two immunodominant structural viral antigens, exponentially improved both the humoral and the cellular responses induced in pigs after DNA immunization. However, immunization with the resulting plasmid (pCMV-sHAPQ) did not confer protection against lethal challenge with the virulent E75 ASFV-strain. Due to the fact that CD8+ T-cell responses are emerging as key components for ASFV protection, we designed a new plasmid construct, pCMV-UbsHAPQ, encoding the three viral determinants above mentioned (sHA, p54 and p30) fused to ubiquitin, aiming to improve Class I antigen presentation and to enhance the CTL responses induced. As expected, immunization with pCMV-UbsHAPQ induced specific T-cell responses in the absence of antibodies and, more important, protected a proportion of immunized-pigs from lethal challenge with ASFV. In contrast with control pigs, survivor animals showed a peak of CD8+ T-cells at day 3 post-infection, coinciding with the absence of viremia at this time point. Finally, an in silico prediction of CTL peptides has allowed the identification of two SLA I-restricted 9-mer peptides within the hemagglutinin of the virus, capable of in vitro stimulating the specific secretion of IFNγ when using PBMCs from survivor pigs. Our results confirm the relevance of T-cell responses in protection against ASF and open new expectations for the future development of more efficient recombinant vaccines against this disease.

Expression Library Immunization Can Confer Protection against Lethal Challenge with African Swine Fever Virus

“…In this report, we present clear evidence demonstrating the protective capability of the ASFV Ublib , a DNA library encoding short restriction fragments from the ASFV genome as fusions with ubiquitin to increase their proteasomal degradation and to enhance the induction of specific CTL responses (36,37). These results confirm and extend those recently obtained by immunizing with an individual plasmid encoding three ASFV antigens in frame with ubiquitin (18). The presence of ubiquitin in the vaccine was determinant, since vaccination with ASFV library DNA bearing ASFV genome fragments as fusions with the extracellular domain of hemagglutinin, ASFV sHAlib , failed to induce protection against ASFV lethal challenge (data not shown), perhaps due to the presence of low, albeit detectable, exacerbating anti-ASFV antibodies, as has been described before (18).…”

“…These results confirm and extend those recently obtained by immunizing with an individual plasmid encoding three ASFV antigens in frame with ubiquitin (18). The presence of ubiquitin in the vaccine was determinant, since vaccination with ASFV library DNA bearing ASFV genome fragments as fusions with the extracellular domain of hemagglutinin, ASFV sHAlib , failed to induce protection against ASFV lethal challenge (data not shown), perhaps due to the presence of low, albeit detectable, exacerbating anti-ASFV antibodies, as has been described before (18). The fact that the partial protection provided by the ASFV Ublib was obtained in the absence of antibodies seems to give strength to this hypothesis and points out once more the relevance of CD8 ϩ T-cell responses in protection against ASFV.…”

“…ϩ T-cell responses in the surviving pigs; the vaccines were targeted mainly against two specific 9-mer peptides located within the hemagglutinin antigen (18). These results confirmed the key role that specific CD8 ϩ T cells can play in the partial protection conferred by our DNA vaccines.…”

“…Development of T-cell immune responses to ASFV was analyzed by a gamma interferon (IFN-␥) enzyme-linked immunosorbent spot (ELISPOT) assay as described previously (17,18). Briefly, peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation with Histopaque 1077 (Sigma-Aldrich, Madrid, Spain).…”

“…Thus, immunization with baculovirus-expressed recombinant p54 and p30 ASFV proteins (13,14), with the viral hemagglutinin (15), or with a combination of p54, p30, p72, and p22 (16) has yielded different protective outcomes, also depending on the ASFV strain used for the challenge. These studies have, more recently, been extended to the field of immunization with DNA (17,18). Interestingly, the outcome of the immune response and, consequently, the level of protection afforded by the DNA vaccines dramatically changed depending on the plasmid version used.…”