De la caracterització genètica al desenvolupament de la vacuna de la malaltia de Glässer

"Polinucleòtids d’Haemophilus parasuis i el seu ús" és una patent del CReSA (WO/2007/039070) que ha permès la selecció de nous antígens candidats per a  la vacunació contra la malaltia de Glässer. Aquests antígens es varen seleccionar mitjançant un enfocament de vacunologia inversa.

Haemophilus parasuis és l’agent etiològic de la malaltia de Glässer, una malaltia reemergent porcina caracteritzada per poliserositis fibrinosa, poliartritis i meningitis. Una de les característiques d’Haemophilus parasuis és la seva alta variació antigènica. S’han descrit quinze serotips i existeixen moltes soques que no pertanyen a cap dels serotips descrits. També hi ha diferències en la virulència demostrada en la inoculació experimental de diferents soques, que van des de les altament virulentes a les no patògenes. Això podria estar relacionat amb la existència d’un llinatge no virulent d’Haemophilus parasuis, comensal del tracte respiratori superior dels porcs. El control de la malaltia de Glässer mitjançant la vacunació s’ha intentat mitjançant l’ús de bactèries inactivades (bacterines). No obstant, aquestes vacunes tenen un ús limitat per la falta de protecció creuada entres les soques.

Investigadors del CReSA han utilitzat un enfoc de la vacunologia inversa per a determinar nous antígens protectors. Aquesta metodologia es basa en la seqüenciació del genoma complet i l’ús d’anàlisis bioinformàtics per a identificar nous candidats vacunals, basant-se en la predicció de que es troben exposats en la membrana externa o que siguin proteïnes segregades. Posteriorment, els candidats són clonats i produïts com a proteïnes recombinants. Les proteïnes recombinants s’utilitzen per a provar la seva immunogenicitat i capacitat protectora in vivo. Cal afegir, que els gens d’aquests nous antígens s’han utilitzat per a desenvolupar diferents eines moleculars per a millorar el diagnòstic d’Haemophilus parasuis.

Quatre publicacions en revistes científiques resumeixen el treball realitzat pels investigadors del CReSA per a definir nous antígens que podrien ajudar a millorar el diagnòstic i les vacunes actuals enfront d’Haemophilus parasuis.

Pina S, Olvera A, Barceló A, Bensaid A. Trimeric autotransporters of Haemophilus parasuis: generation of an extensive passenger domain repertoire specific for pathogenic strains. J Bacteriol. 2009 Jan;191(2):576-87.
This first work describes the identification of the first antigen candidates, the virulence-associated trimeric autotransporters (vtaA). In this study the whole genome of the highly virulent strain Nagasaki was sequenced. This allowed the in silico characterization of 13 trimeric autotransporters as assessed by the presence of a YadA C-terminal translocator domain. Their function was predicted to be related to adhesion of the bacteria to host cells, since the predicted structure of these proteins possess motifs commonly found in adhesins, hemagglutinins and invasins. In addition, they have various copies of collagen-like repeats in the middle of the proteins. The phylogenetic analysis of the translocator domains classified the vtaA into three groups. Then the genome comparison using microarray hybridization of the Nagasaki strain with different strains revealed that group 1 vtaA genes were divergent among virulent and non-virulent strains. This initial study allowed the development of different applications of the VtaA in diagnostics and vaccination.

Olvera A, Pina S, Pérez-Simó M, Oliveira S, Bensaid A. Virulence-associated trimeric autotransporters of Haemophilus parasuis are antigenic proteins expressed in vivo. Vet Res. 2010 May-Jun;41(3):26.
Trimeric autotransporters are good vaccine candidates. For this reason the antigenic capacity of VtaA was evaluated. Fifteen recombinant VtaA passenger domains were produced as recombinant proteins and used to screen H. parasuis immune sera by immunoblotting. After infection of 4 animals with a subclinical dose of H. parasuis Nagasaki, an IgG mediated antibody response against 6 of the 13 VtaA of the Nagasaki strain was detected. IgA production against VtaA was detected in only one animal. Antibody cross-reaction with two of the corresponding proteins of strain HP1319 was also detected. No antibodies against VtaA were detected in the sera of animals immunized with a bacterin of the Nagasaki strain. Taken together, these results indicate that the 6 VtaA were expressed during infection, inducing an antibody response. However, they are poorly expressed in the in vitro conditions used to produce the bacterin. Thus, VtaA are good immunogen candidates that could be used to improve the antigenicity of H. parasuis bacterines.

Olvera A, Pina S, Pérez-Simó M, Aragón V, Segalés J, Bensaid A. Immunogenicity and protection against Haemophilus parasuis infection after vaccination with recombinant virulence associated trimeric autotransporters (VtaA). Vaccine. 2011 Mar 24;29(15):2797-802.
After the description of the antigenicity of VtaA their immunogenic and protective capacity was evaluated. In this study 6 VtaA were produced as recombinant proteins and used to immunize snatch-farrowed, colostrum-deprived piglets. An in house developed ELISA demonstrated that immunized animals developed specific anti-VtaA systemic and mucosal antibodies. The protective capacity of the anti-VtaA antibodies was evaluated by the inoculation of 3×108 or 6×106 colony forming units (CFU) of the highly virulent strain Nagasaki. While non-vaccinated animals died in the first 2 days of the trial, vaccinated animals had a delayed course of disease and 33 or 57%, respectively, of the animals survived the lethal challenge. The protection achieved with the recombinant VtaA supports their potential as candidates to improve future vaccine formulations against H. parasuis.

Olvera A, Pina S, Macedo N, Oliveira S, Aragon V, Bensaid A. Identification of potentially virulent strains of Haemophilus parasuis using a multiplex PCR for virulence-associated autotransporters (vtaA). Vet J. 2011 Jan 17. [Epub ahead of  print].
In this final study CReSA researchers evaluated by PCR the presence of the three groups of VtaA in 157 isolates. Group 3 vtaA genes were demonstrated in all H. parasuis isolates tested. Group 1 vtaA genes were associated with virulent strains: 96% of the group 1 negative isolates were isolated from nasal passages of healthy animals, whereas no group 1 negative isolates were isolated from cases of Glässer's disease. There was an association between absence of group 1 vtaA, sensitivity to phagocytosis and serum and classification of isolates into nasal cluster C by multilocus sequence typing. A multiplex PCR was developed for diagnosis of H. parasuis at the species level (group 3 vtaA positive) and to differentiate non-virulent strains (group 1 vtaA negative). When applied to field samples, the PCR confirmed a high prevalence of H. parasuis in conventionally farmed pigs and demonstrated that almost half of the animals carried potentially virulent strains.

Per a contactar amb el responsable de la patent "Polinucleòtids d’Haemophilus parasuis i el seu ús ":

Dr. Albert Moisés Bensaid
Investigador responsable del Subprograma de Recerca “Malalties Transfrontereres “
Correu electrònic: albert.bensaid@cresa.uab.cat
Telèfon: +34 93 581 45 58
Fax: +34 93 581 44 90
Edifici CReSA. Campus UAB
08193 Bellaterra (Barcelona) Espanya

 

 

 

 

 

 

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