Veridiana Gomes Virginio

A set of recombinant antigens from Echinococcus granulosus with potential for use in the immunodiagnosis of human cystic hydatid disease

Several recombinant clones expressing antigens from Echinococcus granulosus were isolated previously from a parasite cDNA library using cystic hydatid disease (CHD) patients’ sera or rabbit hyperimmune antiserum against a lipoproteic fraction from bovine cyst fluid. Six of these antigens were expressed in Escherichia coli and the purified recombinant proteins were tested in enzyme‐linked immunosorbent assay (ELISA) for specific IgG with a panel of sera from patients with surgically confirmed (n = 58) or immunologically diagnosed (n = 71) CHD. Sera from clinically normal individuals (n = 203) and sera from individuals with other helminthic infections (n = 65) were assayed for the assessment of specificity. A cut‐off value was determined by receiver‐operating‐characteristic plots for each antigen. A recombinant antigen B subunit (AgB8/2) presented the highest sensitivity (93·1%), considering the group of sera from patients with CHD surgically confirmed, and specificity (99·5%) and is proposed as the basis for an immunodiagnostic test. The other recombinant antigens tested presented sensitivities between 58·6% and 89·7%, and three of them were considered of complementary value. In subclass‐specific ELISA, different IgG isotypes showed dominance in the response for each of the recombinant antigens. There was a clear predominance of IgG4 response for all antigens tested, indicating that this would be the subclass of choice to be assessed for these recombinant proteins.

Excretory/secretory products from in vitro-cultured Echinococcus granulosus protoscoleces

Cystic hydatid disease (CHD) is caused by infection with Echinococcus granulosus metacestodes and affects humans and livestock. Proteins secreted or excreted by protoscoleces, pre-adult worms found in the metacestode, are thought to play fundamental roles in the host-parasite relationship. In this work, we performed an LC-MS/MS proteomic analysis of the excretory-secretory products obtained from the first 48 h of an in vitro culture of the protoscoleces. We identified 32 proteins, including 18 that were never detected previously in metacestode proteomic studies. Among the novel identified excretory-secretory products are antigenic proteins, such as EG19 and P-29 and a calpain protease. We also identified other important protoscolex excretory-secretory products, such as thioredoxin peroxidase and 14-3-3 proteins, which are potentially involved in evasion mechanisms adopted by parasites to establish infection. Several intracellular proteins were found in the excretory-secretory products, revealing a set of identified proteins not previously thought to be exposed at the host-parasite interface. Additionally, immunological analyses established the antigenic profiles of the newly identified excretory-secretory products and revealed, for the first time, the in vitro secretion of the B antigen by protoscoleces. Considering that the excretory-secretory products obtained in vitro might reflect the products released and exposed to the host in vivo, our results provide valuable information on parasite survival strategies in adverse host environments and on the molecular mechanisms underpinning CHD immunopathology.

New insights on the biology of swine respiratory tract mycoplasmas from a comparative genome analysis

BackgroundMycoplasma hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis live in swine respiratory tracts. M. flocculare, a commensal bacterium, is genetically closely related to M. hyopneumoniae, the causative agent of enzootic porcine pneumonia. M. hyorhinis is also pathogenic, causing polyserositis and arthritis. In this work, we present the genome sequences of M. flocculare and M. hyopneumoniae strain 7422, and we compare these genomes with the genomes of other M. hyoponeumoniae strain and to the a M. hyorhinis genome. These analyses were performed to identify possible characteristics that may help to explain the different behaviors of these species in swine respiratory tracts.ResultsThe overall genome organization of three species was analyzed, revealing that the ORF clusters (OCs) differ considerably and that inversions and rearrangements are common. Although M. flocculare and M. hyopneumoniae display a high degree of similarity with respect to the gene content, only some genomic regions display considerable synteny. Genes encoding proteins that may be involved in host-cell adhesion in M. hyopneumoniae and M. flocculare display differences in genomic structure and organization. Some genes encoding adhesins of the P97 family are absent in M. flocculare and some contain sequence differences or lack of domains that are considered to be important for adhesion to host cells. The phylogenetic relationship of the three species was confirmed by a phylogenomic approach. The set of genes involved in metabolism, especially in the uptake of precursors for nucleic acids synthesis and nucleotide metabolism, display some differences in copy number and the presence/absence in the three species.ConclusionsThe comparative analyses of three mycoplasma species that inhabit the swine respiratory tract facilitated the identification of some characteristics that may be related to their different behaviors. M. hyopneumoniae and M. flocculare display many differences that may help to explain why one species is pathogenic and the other is considered to be commensal. However, it was not possible to identify specific virulence determinant factors that could explain the differences in the pathogenicity of the analyzed species. The M. hyorhinis genome contains differences in some components involved in metabolism and evasion of the host’s immune system that may contribute to its growth aggressiveness. Several horizontal gene transfer events were identified. The phylogenomic analysis places M. hyopneumoniae, M. flocculare and M. hyorhinis in the hyopneumoniae clade.

Effects of protoscoleces and AgB from Echinococcus granulosus on human neutrophils: possible implications on the parasite’s immune evasion mechanisms

The factors affecting the innate susceptibility to Echinococcus granulosus infections are largely unknown. We assessed the interaction of healthy human neutrophils with protoscoleces (PSC) and antigen B (AgB) of E. granulosus by analysis of CD11b upregulation and H2O2 production by flow cytometry. PSC induced neutrophil activation, but their viability was not affected. In contrast, no effects were observed with AgB in both assays. Neutrophil-enriched fractions were also incubated with PSC or AgB, and interleukin 8 (IL-8) production was measured by ELISA. Significant increment in IL-8 production was detected only in supernatants from neutrophil-enriched fractions cultured with PSC. The possible effect of a prior incubation with AgB on the phorbol myristate acetate-induced activation was also evaluated. No changes were observed in CD11b expression, but the H2O2 production was significantly reduced in platelet-activating factor (PAF)-primed neutrophils. These results suggest a possible AgB-mediated mechanism of evasion of the host immune response, which would operate upon events of spillage of the fertile hydatid cyst content.

Immune responses elicited by Mycoplasma hyopneumoniae recombinant antigens and DNA constructs with potential for use in vaccination against porcine enzootic pneumonia.

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (PEP) and causes major economic losses to the pig industry worldwide. Commercially available vaccines provide only partial protection and are relatively expensive. In this study, we assessed the humoral and cellular immune responses to three recombinant antigens of M. hyopneumoniae. Immune responses to selected domains of the P46, HSP70 and MnuA antigens (P46102-253, HSP70212-601 and MnuA182-378), delivered as recombinant subunit or DNA vaccines, were evaluated in BALB/c mice. All purified recombinant antigens and two DNA vaccines, pcDNA3.1(+)/HSP70212-601 and pcDNA3.1(+)/MnuA182-378, elicited a strong humoral immune response, indicated by high IgG levels in the serum. The cellular immune response was assessed by detection of IFN-γ, IL-10 and IL-4 in splenocyte culture supernatants. The recombinant subunit and DNA vaccines induced Th1-polarized immune responses, as evidenced by increased levels of IFN-γ. All recombinant subunit vaccines and the pcDNA3.1(+)/MnuA182-378 vaccine also induced the secretion of IL-10, a Th2-type cytokine, in large quantities. The mixed Th1/Th2-type response may elicit an effective immune response against M. hyopneumoniae, suggesting that P46102-253, HSP70212-601 and MnuA182-378 are potential novel and promising targets for the development of vaccines against PEP.

Mycoplasma hyopneumoniae and Mycoplasma flocculare differential domains from orthologous surface proteins induce distinct cellular immune responses in mice

Mycoplasma hyopneumoniae and Mycoplasma flocculare are two genetically close species found in the swine respiratory tract. Despite their similarities, while M. hyopneumoniae is the causative agent of porcine enzootic pneumonia, M. flocculare is a commensal bacterium. Genomic and transcriptional comparative analyses so far failed to explain the difference in pathogenicity between these two species. We then hypothesized that such difference might be, at least in part, explained by amino acid sequence and immunological or functional differences between ortholog surface proteins. In line with that, it was verified that approximately 85% of the ortholog surface proteins from M. hyopneumoniae 7448 and M. flocculare present one or more differential domains. To experimentally assess possible immunological implications of this kind of difference, the extracellular differential domains from one pair of orthologous surface proteins (MHP7448_0612, from M. hyopneumoniae, and MF_00357, from M. flocculare) were expressed in E. coli and used to immunize mice. The recombinant polypeptides (rMHP61267-169 and rMF35767-196, respectively) induced distinct cellular immune responses. While, rMHP61267-169 induced both Th1 and Th2 responses, rMF35767-196 induced just an early pro-inflammatory response. These results indicate that immunological properties determined by differential domains in orthologous surface protein might play a role in pathogenicity, contributing to elicit specific and differential immune responses against each species.

Assessment of the adjuvant activity of mesoporous silica nanoparticles in recombinant Mycoplasma hyopneumoniae antigen vaccines

The adjuvant potential of two mesoporous silica nanoparticles (MSNs), SBa-15 and SBa-16, was assessed in combination with a recombinant HSP70 surface polypeptide domain from Mycoplasma hyopneumoniae, the etiological agent of porcine enzootic pneumonia (PEP). The recombinant antigen (HSP70212-600), previously shown as immunogenic in formulation with classic adjuvants, was used to immunize BALB/c mice in combination with SBa-15 or SBa-16 MSNs, and the effects obtained with these formulations were compared to those obtained with alum, the adjuvant traditionally used in anti-PEP bacterins. The HSP70212-600 + SBa-15 vaccine elicited a strong humoral immune response, with high serum total IgG levels, comparable to those obtained using HSP70212-600 + alum. The HSP70212-600 + SBa-16 vaccine elicited a moderate humoral immune response, with lower levels of total IgG. The cellular immune response was assessed by the detection of IFN-γ, IL-4 and IL-10 in splenocyte culture supernatants. The HSP70212-600 + SBa-15 vaccine increased IFN-γ, IL-4 and IL-10 levels, while no stimulation was detected with the HSP70212-600 + SBa-16 vaccine. The HSP70212-600 + SBa-15 vaccine induced a mixed Th1/Th2-type response, with an additional IL-10 mediated anti-inflammatory effect, both of relevance for an anti-PEP vaccine. Alum adjuvant controls stimulated an unspecific cellular immune response, with similar levels of cytokines detected in mice immunized either with HSP70212-600 + alum or with the adjuvant alone. The better humoral and cellular immune responses elicited in mice indicated that SBa-15 has adjuvant potential, and can be considered as an alternative to the use of alum in veterinary vaccines. The use of SBa-15 with HSP70212-600 is also promising as a potential anti-PEP subunit vaccine formulation.

Evaluation of the immunodiagnostic potential of a recombinant surface protein domain from Acanthamoeba castellanii

Acanthamoeba spp. are free-living protists widely distributed in environment, able to cause keratitis, encephalitis and skin lesions in humans and animals. Acanthamoeba spp. exist in two forms: an infective trophozoite and a dormant cyst. Several factors contribute to the pathogenesis of Acanthamoeba spp. The parasite adhesion to the host cell is the primary step for infection and is mediated by a mannose binding-protein, expressed in the surface and considered the main pathogenicity factor in Acanthamoeba spp. So far, there was no evidence of another surface protein of Acanthamoeba spp. relevant for host invasion or infection by these organisms. The aims of this study were to identify and characterize an Acanthamoeba castellanii surface protein and to evaluate its diagnostic potential. In silico predictions of surface proteins allowed to identify the A. castellanii calreticulin as a possible surface antigen. The coding sequence of a predicted extracellular domain of A. castellanii calreticulin was cloned by in vivo homologous recombination and the recombinant polypeptide (AcCRT29-130) was produced. Its immunodiagnostic potential was assessed in a recombinant antigen-based ELISA with sera from experimentally infected rats that developed keratitis and encephalitis, and sera from patients with encephalitis. The AcCRT29-130 was significantly more recognized by sera from encephalitis infected rats in comparison with the non-infected controls. Human sera from encephalitis patients, however presented no significant response. These results showed the AcCRT29-130 potential for A. castellanii infection immunodiagnosis in animals, with further studies being required for assessment of its use for human infections.

Orthologous surface proteins from Mycoplasma hyopneumoniae and Mycoplasma flocculare: in silico comparison and heterologous expression of differential extracellular domains

Background Mycoplasma hyopneumoniae and Mycoplasma flocculare are two closely related mycoplasma species often found in the porcine respiratory tract[1]. However, M. hyopenumoniae is pathogenic, being the causative agent of enzootic pneumonia, while M. flocculare is a commensal bacterium. Enzootic pneumonia is a contagious respiratory disease characterized by chronic cough, growth retardation, low mortality, and a high morbidity. It causes significative economic losses in pig industry worldwide. Some of main interactions between the host and the bacteria are mediated by surface proteins, and a comparison of surface proteins between these two mycoplasmas species can lead to identification of determinants of pathogenicity or commensalism.

Pro-apoptotic effect of a Mycoplasma hyopneumoniae putative type I signal peptidase on PK(15) swine cells

Mycoplasma hyopneumoniae is an economically significant swine pathogen that causes porcine enzootic pneumonia (PEP). Important processes for swine infection by M. hyopneumoniae depend on cell surface proteins, many of which are secreted by secretion pathways not completely elucidated so far. A putative type I signal peptidase (SPase I), a possible component of a putative Sec-dependent pathway, was annotated as a product of the sipS gene in the pathogenic M. hyopneumoniae 7448 genome. This M. hyopneumoniae putative SPase I (MhSPase I) displays only 14% and 23% of sequence identity/similarity to Escherichia coli bona fide SPase I, and, in complementation assays performed with a conditional E. coli SPase I mutant, only a partial restoration of growth was achieved with the heterologous expression of a recombinant MhSPase I (rMhSPase I). Considering the putative surface location of MhSPase I and its previously demonstrated capacity to induce a strong humoral response, we then assessed its potential to elicit a cellular and possible immunomodulatory response. In assays for immunogenicity assessment, rMhSPase I unexpectedly showed a cytotoxic effect on murine splenocytes. This cytotoxic effect was further confirmed using the swine epithelial PK(15) cell line in MTT and annexin V-flow cytometry assays, which showed that rMhSPase I induces apoptosis in a dose dependent-way. It was also demonstrated that this pro-apoptotic effect of rMhSPase I involves activation of a caspase-3 cascade. The potential relevance of the rMhSPase I pro-apoptotic effect for M. hyopneumoniae-host interactions in the context of PEP is discussed.