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Valdeolmos, Spain

Gallardo C.,CISA INIA | Salguero F.J.,CISA INIA | Borrego B.,CISA INIA | Accensi F.,Autonomous University of Barcelona | And 2 more authors.

One of the main criticisms to DNA vaccines is the poor immunogenicity that they confer on occasions, at least in large animals. Confirming this theory, immunization with plasmid DNA encoding two African swine fever virus genes in frame (pCMV-PQ), failed in inducing detectable immune responses in pigs, while it was successful in mice. Aiming to improve the immune responses induced in swine, a new plasmid was constructed, encoding the viral genes fused in frame with a single chain variable fragment of an antibody specific for a swine leukocyte antigen II (pCMV-APCH1PQ). Our results clearly demonstrate that targeting antigens to antigen professional cells exponentially enhanced the immune response induced in pigs, albeit that the DNA vaccine was not able to confer protection against lethal viral challenge. Indeed, a viremia exacerbation was observed in each of the pigs that received the pCMV-APCH1PQ plasmid, this correlating with the presence of non-neutralizing antibodies and antigen-specific SLA II-restricted T-cells. The implications of our discoveries for the development of future vaccines against African swine fever virus and other swine pathogens are discussed. © 2011 Elsevier Ltd. Source

Beck C.,French National Institute for Agricultural Research | Jimenez-Clavero M.A.,CISA INIA | Leblond A.,VetAgroSup | Leblond A.,French National Institute for Agricultural Research | And 7 more authors.
International Journal of Environmental Research and Public Health

In Europe, many flaviviruses are endemic (West Nile, Usutu, tick-borne encephalitis viruses) or occasionally imported (dengue, yellow fever viruses). Due to the temporal and geographical co-circulation of flaviviruses in Europe, flavivirus differentiation by diagnostic tests is crucial in the adaptation of surveillance and control efforts. Serological diagnosis of flavivirus infections is complicated by the antigenic similarities among the Flavivirus genus. Indeed, most flavivirus antibodies are directed against the highly immunogenic envelope protein, which contains both flavivirus cross-reactive and virus-specific epitopes. Serological assay results should thus be interpreted with care and confirmed by comparative neutralization tests using a panel of viruses known to circulate in Europe. However, antibody cross-reactivity could be advantageous in efforts to control emerging flaviviruses because it ensures partial cross-protection. In contrast, it might also facilitate subsequent diseases, through a phenomenon called antibody-dependent enhancement mainly described for dengue virus infections. Here, we review the serological methods commonly used in WNV diagnosis and surveillance in Europe. By examining past and current epidemiological situations in different European countries, we present the challenges involved in interpreting flavivirus serological tests and setting up appropriate surveillance programs; we also address the consequences of flavivirus circulation and vaccination for host immunity. Source

Argilaguet J.M.,University Pompeu Fabra | Perez-Martin E.,U.S. Department of Agriculture | Gallardo C.,CISA INIA | Accensi F.,Autonomous University of Barcelona | Reche P.A.,University Computense Of Madrid Ucm

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. © 2012 Argilaguet et al. Source

Gomez-Laguna J.,University of Cordoba, Spain | Salguero F.J.,CISA INIA | Barranco I.,University of Cordoba, Spain | Pallares F.J.,University of Murcia | And 3 more authors.
Journal of Comparative Pathology

Porcine reproductive and respiratory syndrome (PRRS) is caused by a virus that predominantly replicates in alveolar macrophages. The aim of the present study was to characterize the production of cytokines by subpopulations of pulmonary macrophages in pigs infected by the PRRS virus (PRRSV). Expression of interleukin (IL) 1α, IL-6 and tumour necrosis factor (TNF)-α correlated with the severity of pulmonary pathology and the numbers of pulmonary macrophages. Significant correlations were observed between PRRSV infection and the expression of IL-10, between the expression of IL-12p40 and interferon (IFN)-γ, and between the expression of TNF-α and IFN-γ. These findings suggest that PRRSV modulates the immune response by the up-regulation of IL-10, which may in turn reduce expression of cytokines involved in viral clearance (e.g. IFN-α, IFN-γ, IL-12p40 and TNF-α). The results also suggest that expression of IFN-γ is stimulated by IL-12p40 and TNF-α, but not by IFN-α. All of these cytokines were expressed mainly by septal macrophages with weaker expression by alveolar macrophages, lymphocytes and neutrophils. There appears to be differential activation of septal and alveolar macrophages in PRRSV infection, with septal macrophages being the major source of cytokines. © 2009 Elsevier Ltd. Source

Gomez-Laguna J.,University of Cordoba, Spain | Salguero F.J.,CISA INIA | Salguero F.J.,Veterinary Laboratories Agency | Pallares F.J.,University of Murcia | And 6 more authors.
Comparative Immunology, Microbiology and Infectious Diseases

This study was focused on the changes observed in the serum concentration of haptoglobin (Hp), C-reactive protein (CRP), serum amyloid A (SAA) and Pig-major acute protein (Pig-MAP), during experimental porcine reproductive and respiratory syndrome virus (PRRSV) infection and in their relationship with the expression of interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). Hp and Pig-MAP serum levels were increased at 10. dpi, but CRP and SAA showed a delayed and highly variable increase. All three proinflammatory cytokines were poorly expressed, and only a mild increase in IL-1β was observed at 7. dpi. The increased expression of Hp coincided with the light enhancement observed in both IL-6 and TNF-α, and might be related with an increased expression of IL-10. The low expression of TNF-α might point to a possible mechanism of viral evasion of host-immune response. This issue and the delayed expression of CRP and SAA should be taken into account in future studies about modulation of the immune response by PRRSV infection. © 2009 Elsevier Ltd. Source

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