Recombinant Secreted Antigens from Mycoplasma hyopneumoniae Delivered as a Cocktail Vaccine Enhance the Immune Response of Mice

Abstract

bMycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), which is a respiratory disease responsible for huge economic losses in the pig industry worldwide. The commercially available vaccines provide only partial protection and are expensive. Thus, the development of alternatives for the prophylaxis of EP is critical for improving pig health. The use of multiple antigens in the same immunization may represent a promising alternative. In the present study, seven secreted proteins of M. hyopneumoniae were cloned, expressed in Escherichia coli, and evaluated for antigenicity using serum from naturally and experimentally infected pigs. In addition, the immunogenicity of the seven recombinant proteins delivered individually or in protein cocktail vaccines was evaluated in mice. In Western blot assays and enzyme-linked immunosorbent assays, most of the recombinant proteins evaluated were recognized by convalescent-phase serum from the animals, indicating that they are expressed during the infectious process. The recombinant proteins were also immunogenic, and most induced a mixed IgG1/ IgG2a humoral immune response. The use of these proteins in a cocktail vaccine formulation enhanced the immune response compared to their use as antigens delivered individually, providing evidence of the efficacy of the multiple-antigen administration strategy for the induction of an immune response against M. hyopneumoniae. P orcine enzootic pneumonia (EP) is a chronic respiratory disease with a high incidence in pig farms worldwide. Mycoplasma hyopneumoniae, the etiological agent of this disease, colonizes and destroys the main mechanism of innate immunity of the pig respiratory system-the respiratory ciliated epithelium-predisposing the pigs to secondary and opportunistic infections (1). EP prophylaxis comprises the use of antibiotics, management procedures, and vaccination, which is considered the most efficient control measure (2). The commercially available vaccines consist of inactivated whole cells (bacterins) and have high production costs, mainly due to the fastidious growth of this microorganism. In addition, these vaccines do not eliminate M. hyopneumoniae from infected pig herds (1, 3, 4). Thus, efforts have been directed toward the search for new prophylaxis strategies against EP that do not include the use of bacterins.M. hyopneumoniae does not penetrate host cells, and thus it is believed that the pathogenesis of this microorganism is mediated by a complex and multifactorial process that involves components of the cell membrane and secreted proteins, many of which are still unidentified (5). Data generated from the sequencing of four isolates of M. hyopneumoniae (7448, 168, J, and 232) and the comparative genomic and proteomic analyses of pathogenic strains (7448 and 232) and a nonpathogenic strain (J) of M. hyopneumoniae (6,7,8,9) have allowed the identification of coding sequences (CDS) from secreted antigenic proteins and/or proteins involved in the pathogenicity of the microorganism. Som...

Antimicrobial susceptibility and genetic profile of Mycoplasma hyopneumoniae isolates from Brazil

“…Virginio et al [23] demonstrated that mnuA induced a strong humoral immune response when IgG was tested in the murine model; it was shown to be a strong immunogenic antigen as it significantly stimulated high levels of interferon-gamma and increased the amount of interleukin-10. When evaluating the antigenicity of proteins secreted by M. hyopneumoniae, mhp418 was recognized in serum from swine that were naturally infected by the agent [24]. The obtained results, with the presence of isolates from the same batch of animals belonging to four different genetic groups, demonstrated the possible genetic diversity of M. hyopneumoniae strains within the same batch, farm, city, and region, as has been also observed in other genetic diversity studies [20,25].…”

Antigenicity, Immunogenicity and Protective Efficacy of Three Proteins Expressed in the Promastigote and Amastigote Stages of Leishmania infantum against Visceral Leishmaniasis

“…Vaccine studies concerning leishmaniasis call for the critical evaluation of the parasitological and immunological parameters in murine models [ 13 , 28 ]. To properly evaluate new antigens, it is important to optimize their dose and route of administration [ 39 ]. The present study aimed to evaluate a polyproteins vaccine composed by three different antigens, based on their potential to induce a more effective protective immunity against murine VL.…”

Genetic variation of Mycoplasma hyopneumoniae from Brazilian field samples

“…The mhp0418 gene encodes a hypothetical protein expressed during Mhp infection [24], however, mhp0418 was found at low frequency in this study. When we compared the detection frequency of mhp0418 in samples from both regions studied, the gene was significantly more frequently found in the AP samples (p < 0.05) (Table 2).…”

“…The mhp0272 gene encodes a P97 paralog located on the Mhp surface [24]. Our results demonstrated that mhp0272 had a significantly higher frequency ( p < 0.05) in the ZM samples, compared to the AP samples.…”

“…High variation in the detection of the selected genes was noted in the PCR results (Table 2), suggesting a high genetic diversity in Mhp field strains. Some of these genes encode hypothetical proteins ( mhp0443 , mhp0660 , mhp0487 , and mhp0418 ) whose function has not yet been clarified, but they are expressed during in vivo infection caused by Mhp [24, 25]. Some hypothetical proteins ( mhp0443 , mhp0660 , and mhp0487 ) showed a high frequency of detection (93.8, 98.4, and 92.2%, respectively), which suggests that they could play a significant role in Mhp infection, even if their functions have not yet been determined.…”