Optimized FaeG Expression and a Thermolabile Enterotoxin DNA Adjuvant Enhance Priming of an Intestinal Immune Response by an FaeG DNA Vaccine in Pigs

Abstract

One of the problems hindering the development of DNA vaccines is the relatively low immunogenicity often seen in humans and large animals compared to that in mice. In the present study, we tried to enhance the immunogenicity of a pcDNA1/faeG19 DNA vaccine in pigs by optimizing the FaeG expression plasmid and by coadministration of the plasmid vectors encoding the A and B subunits of the Escherichia coli thermolabile enterotoxin (LT). The insertion of a Kozak sequence and optimization of vector (cellular localization and expression) and both vector and codon usage were all shown to enhance in vitro FaeG expression compared to that of pcDNA1/faeG19. Subsequently, pcDNA1/faeG19 and the vector-optimized and the vector-codon-optimized construct were tested for their immunogenicity in pigs. In line with the in vitro results, antibody responses were better induced with increasing expression. The LT vectors additionally enhanced the antibody response, although not significantly, and were necessary to induce an F4-specific cellular response. These vectors were also added because LT has been described to direct the systemic response towards a mucosal immunoglobulin A (IgA) response in mice. Here, however, the intradermal FaeG DNA prime-oral F4 boost immunization resulted in a mainly systemic IgG response, with only a marginal but significant reduction in F4 ؉ E. coli fecal excretion when the piglets were primed with pWRGFaeGopt and pWRGFaeGopt with the LT vectors.Intestinal infections with enterotoxigenic Escherichia coli (ETEC) remain problematic for humans, pigs, and calves. The bacteria possess adhesins which allow them to colonize the small intestine and to produce enterotoxins which act locally on enterocytes. These adhesins bind to specific receptors on the enterocyte brush borders, and the absence of these receptors renders the animal resistant to bacterial colonization and consequently to ETEC-induced diarrhea (27). In pigs, F4 (K88) fimbriae are the best-characterized adhesins (37). They are long proteinaceous appendages composed mainly of several hundreds of identical adhesive subunits called FaeG (22).Our laboratory developed a challenge model in pigs in which new vaccination strategies against ETEC can be tested. With this model, it was demonstrated that the oral administration of F4 fimbriae to weaned F4R ϩ pigs led to protection against a subsequent F4 ϩ ETEC infection (35). In practice, a protective mucosal immune response often needs to be induced in the presence of maternal antibodies.Several studies have shown that DNA vaccines, in contrast to conventional vaccines, can successfully prime immune responses in the presence of maternal antibodies, pointing out their potential use in young animals (3). Our previous pcDNA1/faeG19 DNA vaccine, however, was only marginally immunogenic in pigs (42). This is in agreement with other studies in large animals and humans, suggesting poor immunogenicity of DNA vaccines in these species (20,39). Several approaches have been explored to enhance the potency of DNA vaccin...

Comparison of Five Expression Vectors for the Ha Gene in Constructing a DNA Vaccine for H6N2 Influenza Virus in Chickens

“…Some DNA vaccination studies indicate that there was a direct correlation between the level of protein expression in vitro and immunogenicity in mice [34], pigs [30] and chickens [5] [8]. In the present study, although the various HA-expressing DNA construct vaccines did not induce significant antibody responses, there was some enhanced HA antibody responses and some reduction in virus shedding after virus challenge with these vaccines.…”

Comparison of immune responses in parenteral FaeG DNA primed pigs boosted orally with F4 protein or reimmunized with the DNA vaccine

“…Furthermore, addition of plasmid vectors encoding the LTA and LTB subunits to the FaeG DNA vaccine enhanced the IgG response, but did not result in the sIgA response that is desired to completely prevent an F4 + ETEC infection. Nevertheless, a significant reduction was obtained in the amount of F4 + ETEC excreted as well as in the duration of faecal F4 + ETEC excretion (Melkebeek et al, 2007). These data suggest that the systemic DNA immunization is weak in priming mucosal responses.…”

“…immunization with pWRGFaeGopt or pWRGFaeGopt in combination with the LT vectors as well as the priming of an F4-specific intestinal mucosal IgG response by i.d. immunization with these vectors followed by an oral F4 boost as seen in a previous study (Melkebeek et al, 2007). Furthermore, we wanted to evaluate if the immunomodulating adjuvant vitD 3 could enhance the mucosal priming by the FaeG DNA vaccine and/or modulate the induced response towards an IgA response.…”

“…The pWRGFaeGopt vaccine was constructed as previously described (Melkebeek et al, 2007). This construct consists of a codon optimized faeG cloned into the pWRG7079 vector behind a tPA signal sequence, allowing the extracellular secretion of the encoded FaeG.…”

Evaluation of attenuated Salmonella choleraesuis-mediated inhibin recombinant DNA vaccine in rats

“…Because of their convenience, stability, and cost, a variety of DNA vaccines encoding the inhibin α (1-32) gene or a fusion with the S gene of the hepatitis B surface antigen have been developed (Han et al, 2008;Wang et al, 2012), and the results showed that an inhibin DNA vaccine was a promising tool for improving animal fertility. However, DNA vaccines face several problems such as low-uptake efficiency (Greenland et al, 2007), dose-dependence (Huang et al, 2008), low immunogenicity in humans and large animals (Melkebeek et al, 2007), and, particularly, antibiotic-resistance-based plasmid selection systems (Galen et al, 2010), making it important to develop an alternative approach for inhibin DNA vaccination.…”