Oral immunization of mice with recombinant Lactococcus lactis expressing Cu,Zn superoxide dismutase of Brucella abortus triggers protective immunity.

Brucella infections mainly occur through mucosal surfaces. Thus, the development of mucosal administered vaccines could be instrumental for the control of brucellosis. Here, we evaluated the usefulness of recombinant Lactococcus lactis secreting Brucella abortus Cu-Zn superoxide dismutase (SOD) as oral antigen delivery system, when administered alone or in combination with L. lactis expressing IL-12. To this end, mice were vaccinated by oral route with L. lactis NZ9000 transformed with pSEC derivatives encoding for SOD (pSEC:SOD) and IL-12 (pSEC:scIL-12). In animals receiving L. lactis pSEC:SOD alone, anti-SOD-specific IgM antibodies were detected in sera at day 28 post-vaccination, together with an IgG2a dominated IgG response. SOD-specific sIgA was also detected in nasal and bronchoalveolar lavages. In addition, T-cell-proliferative responses upon re-stimulation with either recombinant SOD or crude Brucella protein extracts were observed up to 6 months after the last boost, suggesting the induction of long term memory. Vaccinated animals were also protected against challenge with the virulent B. abortus 2308 strain. Responses were mildly improved when L. lactis pSEC:SOD was co-administered with L. lactis pSEC:scIL-12. These results indicated that vaccines based on lactococci-derived live carriers are promising interventions against B. abortus infections.

Cloning, expression and immunogenicity of the translation initiation factor 3 homologue of Brucella abortus.

The infC gene of Brucella abortus encoding the translation initiation factor 3 (IF3) was cloned, sequenced and expressed in Escherichia coli. The amino acid sequence analysis predicted a product with 74-80% identity with the IF3 proteins from Mesorhizobium loti, Sinorhizobium meliloti, Aurantimona sp. and Mesorhizobium sp. This protein also show 54% amino acid sequence identity with the E. coli IF3, sharing most of the residues which were described as responsible for the biological activity of this protein. Since we have previously reported the immunoprotective capacity of this Brucella protein, we stimulated lymphoid cells from animals immunized with purified recombinant Brucella IF3 protein "in vitro" with this antigen. The lymphocytes were able to mount a strong proliferative response with concomitant production of gamma interferon, but without the secretion of either IL-4 or antibodies. Thus, immunization with the Brucella recombinant IF3 protein promotes a TH-1 polarized response, allowing us to propose it as a promising candidate antigen for the development of subunit vaccines against Brucella.

Vaccination with recombinant Semliki Forest virus particles expressing translation initiation factor 3 of Brucella abortus induces protective immunity in BALB/c mice.

Recombinant replicons of Semliki Forest virus (SFV) can be used to induce high-level, transient expression of heterologous proteins in vivo. We constructed infectious but replication-deficient SFV particles carrying recombinant RNA encoding the Brucella abortus translation initiation factor 3 (IF3). The recombinant SFV particles (SFV-IF3 particles) were then evaluated for their ability to induce immune responses and to protect BALB/c mice against a challenge with B. abortus 2308 following vaccination. Animals inoculated with SFV-IF3 developed IF3-specific IgM antibodies at day 14 post-immunization. In vitro stimulation of splenocytes from vaccinated mice with either recombinant IF3 (rIF3) or crude Brucella protein extracts resulted in a T-cell proliferative response and induction of interferon gamma secretion, but not interleukin-4. In addition, mice immunized with SFV-IF3 exhibited a significant level of resistance against challenge with the virulent B. abortus strain 2308 (P<0.01). These findings indicate that an SFV-based vector carrying RNA encoding Brucella IF3 has potential for use as a vaccine to induce protection against B. abortus infections.

The influence of mast cell mediators on migration of SW756 cervical carcinoma cells.

The role of mast cell mediators on cervical cancer cell migration was assessed using an in vitro assay of scratch wound healing onto monolayers of HPV18-positive cervical carcinoma cells (SW756). Migration of SW756 cells was accelerated by co-culture with the mast cell line LAD2. This effect was inhibited by the H1R antagonist pyrilamine and the cannabinoid agonists 2-arachidonylglycerol (2AG) and Win 55,212-2. Therefore, the specific effects of histamine and cannabinoids on SW756 migration and LAD2 activation were analyzed. Histamine added to the in vitro assay of scratch wound healing either increased or inhibited SW756 migration rate by acting either on H1R or H4R, respectively. Cannabinoids acted on CB1 receptors to inhibit SW756 migration. Supernatants from SW756 cells stimulated LAD2 cell degranulation, which in turn was inhibited by cannabinoids acting via CB2 receptors. RT-PCR showed that SW756 expressed mRNA for CB1, CB2, H1R, H2R, and H4R. On the other hand, LAD2 expressed mRNA for all four HRs and CB2. The results suggest that mast cells could be contributing to cervical cancer cell invasion and spreading by the release of histamine and cannabinoids. Therefore, therapeutic modulation of specific mast cell mediators may be beneficial for cervical cancer treatment.

Evaluation of Brucella abortus DNA and RNA vaccines expressing Cu-Zn superoxide dismutase (SOD) gene in cattle.

This study was conducted to evaluate the immunogenicity of a DNA or RNA vaccines encoding Brucella abortus Cu-Zn superoxide dismutase (SOD) in cattle. Intramuscular injection of plasmid DNA carrying Brucella SOD gene (pcDNA-SOD) into animals elicited both humoral and cellular immune responses. Animals injected with pcDNA-SOD developed SOD IgG antibody with predominance of immunoglobulin G1 (IgG1) isotype over IgG2. In addition, the DNA vaccine elicited a specific T-cell-proliferative response. Furthermore, intraperitoneal injection of cattle with recombinant Semliki Forest virus particles carrying recombinant RNA encoding SOD (SFV-SOD) did not lead to the induction of SOD IgG 1 or 2 antibody, but induced specific T-cell activation. Both vaccines were able to induce a non-significant secretion of gamma interferon and did not induce the secretion of IL-4 or tumor necrosis factor (TNF)-alpha. These results suggest that SOD gene in a genetic vaccine formulation (DNA or RNA) might be of potential us as a vaccine to induce cell-mediated immunity in cattle. To our knowledge, this is the first study to evaluate a genetic vaccine against Brucella in cattle.

Vaccination with live Escherichia coli expressing Brucella abortus Cu/Zn superoxide-dismutase: II. Induction of specific CD8+ cytotoxic lymphocytes and sensitized CD4+ IFN-gamma-producing cell.

Previously we reported that immunization with Escherichia coli DH5alpha-expressing Brucella abortus Cu/Zn superoxide dismutase [E. coli (pBSSOD)] induces a protective immune response in BALB/c mice. Here we studied the type of immune defense that the recombinant E. coli induces in mice using as our experimental model Brucella superoxide dismutase Cu/Zn presented by J744.A1 to sensitized lymphocytes as the target of specific lysis or as cytokine inductors. The results indicate that E. coli carrying the Cu/Zn gene was able to induce specific cytotoxic T cells, mainly from CD8(+) subpopulation and IFN-gamma-producing cells belonging in their vast majority to the CD4(+) subpopulation.

Evaluation of Brucella abortus DNA vaccine by expression of Cu-Zn superoxide dismutase antigen fused to IL-2.

The Cu-Zn superoxide dismutase (SOD) antigen of Brucella abortus was previously identified to be a T cell antigen which induces both proliferation of and gamma interferon (IFN-gamma) secretion by T cells from infected mice. In an earlier study, we demonstrated that intramuscular injection of mice with a plasmid DNA carrying the gene for SOD leads to the development of significant protection against B. abortus challenge. It has been reported that the antigen-specific immune responses generated by a DNA vaccine can be enhanced by co-delivery of certain cytokine genes. In this study, we evaluated the effect of delivering IL-2 on the efficacy of SOD DNA vaccine by generating a plasmid (pSecTag-SOD-IL2) that codes for a secretory fusion protein of SOD and IL-2. Another plasmid (pSecTag-SOD) that codes for only SOD as a secretory protein was used for comparison. BALB/c mice injected intramuscularly with pSecTag-SOD or pSecTag-SOD-IL2, but not the control plasmid pSecTag, developed SOD-specific antibody and T cell immune responses. Upon in vitro stimulation with recombinant SOD (rSOD) antigen, T cells from mice immunized with pSecTag-SOD-IL2, in comparison with those from mice immunized with pSecTag-SOD, exhibited a lower proliferation response but produced significantly higher concentrations of IFN-gamma. Both DNA vaccines, however, induced similar levels of SOD-specific antibodies and cytotoxic T cell response. Although mice immunized with pSecTag-SOD-IL2 showed increased resistance to challenge with B. abortus virulent strain 2308, this increase was not statistically significant from that of pSecTag-SOD vaccinated mice. These results suggest that a SOD DNA vaccine fused to IL2 did not improve protection efficacy.

An RNA vaccine based on recombinant Semliki Forest virus particles expressing the Cu,Zn superoxide dismutase protein of Brucella abortus induces protective immunity in BALB/c mice.

We constructed infectious but replication-deficient Semliki Forest virus (SFV) particles carrying recombinant RNA encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). The recombinant SFV particles (SFV-SOD particles) were then evaluated for their ability to induce a T-cell immune response and to protect BALB/c mice against a challenge with B. abortus 2308. Intraperitoneal injection of mice with recombinant SFV-SOD particles did not lead to the induction of SOD-specific antibodies, at least until week 6 after immunization (the end of the experiment). In vitro stimulation of splenocytes from the vaccinated mice with either recombinant Cu,Zn SOD (rSOD) or crude Brucella protein resulted in a T-cell proliferative response and the induction of gamma interferon secretion but not interleukin-4. In addition, the splenocytes exhibited significant levels of cytotoxic T-lymphocyte activity against Brucella-infected cells. The SFV-SOD particles, but not the control virus particles, induced a significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308. These findings indicated that an SFV-based vector carrying the SOD gene has potential for use as a vaccine to induce resistance against B. abortus infections.

Intraspleen delivery of a DNA vaccine coding for superoxide dismutase (SOD) of Brucella abortus induces SOD-specific CD4+ and CD8+ T cells.

In the development of vaccines capable of providing immunity against brucellosis, Cu-Zn superoxide dismutase (SOD) has been demonstrated to be one of the protective immunogens of Brucella abortus. In an earlier study, we provided strong evidence that intramuscular injection with a plasmid DNA carrying the SOD gene (pcDNA-SOD) was able to induce a protective immune response. The present study was designed to characterize T-cell immune responses after an intraspleen (i.s.) vaccination of BALB/c mice with pcDNA-SOD. Animals vaccinated with pcDNA-SOD did not develop SOD-specific antibodies, at least until week 4 after immunization (the end of the experiment), and in vitro stimulation of their splenocytes with either recombinant Cu-Zn SOD or crude Brucella protein induced the secretion of gamma interferon (IFN-gamma), but not interleukin-4, and elicited the induction of cytotoxic-T-lymphocyte activity. Upon analyzing the SOD-specific T-cell responses, the pcDNA-SOD vaccination was found to be stimulating both CD4(+)- and CD8(+)-T-cell populations. However, only the CD4(+) population was able to produce IFN-gamma and only the CD8(+) population was able to induce cytotoxic activity. Nevertheless, although i.s. route vaccination induces a significant level of protection in BALB/c mice against challenge with the virulent B. abortus strain 2308, vaccination by the intramuscular route with a similar amount of plasmid DNA does not protect. Based on these results, we conclude that i.s. immunization with pcDNA-SOD vaccine efficiently induced a Th1 type of immune response and a protective response that could be related to IFN-gamma production and cytotoxic activity against infected cells by SOD-specific CD4(+) and CD8(+) T cells, respectively.

Neonatal Campylobacter coli hemorrhagic enteritis and bacteraemia

Um caso de campylobacteriose neonatal com enterite hemorrágica e bacteremia produzido por Campylobacter coli é apresentado. A mãe, proveniente de uma região rural, apresentou durante a gravidez, três episódios de diarréia autolimitada. A infeccão no recém nascido provavelmente foi adqüirida durante o parto. Os altos níveis séricos de immunoglobulinas específicas poderiam explicar a escassa sintomatologia, apesar da demorada prescricão do tratamento com gentamicina.

A DNA vaccine encoding Cu,Zn superoxide dismutase of Brucella abortus induces protective immunity in BALB/c mice.

This study was conducted to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). Intramuscular injection of plasmid DNA carrying the SOD gene (pcDNA-SOD) into BALB/c mice elicited both humoral and cellular immune responses. Animals injected with pcDNA-SOD developed SOD-specific antibodies which exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the DNA vaccine elicited a T-cell-proliferative response and also induced the production of gamma interferon, but not interleukin-10 (IL-10) or IL-4, upon restimulation with either recombinant SOD or crude Brucella protein, suggesting the induction of a typical T-helper-1-dominated immune response in mice. The pcDNA-SOD (but not the control vector) induced a strong, significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308; the level of protection was similar to the one induced by B. abortus vaccine strain RB51. Altogether, these data suggest that pcDNA-SOD is a good candidate for use in future studies of vaccination against brucellosis.

Identification and partial characterisation of a new protective antigen of Brucella abortus.

Two novel Brucella abortus proteins were isolated from B. abortus strain RB51 and their immunological properties were determined. These proteins precipitated in the 40-60% saturated concentration range of ammonium sulphate and had a molecular mass of 32.2 kDa and 22.9 kDa, respectively. Both were able to induce a strong in-vitro blast transformation in lymphoid cells obtained from mice previously sensitised with a crude brucella protein extract. The protection studies showed that the 22.9-kDa protein used as a protective immunogen was as effective as the live B. abortus RB51 vaccine but the 32.2-kDa protein had a poor protective effect under similar conditions. The amino-terminal sequence of the 22.9-kDa and 32.2-kDa proteins was determined and analysed in a database. The lack of homology with other known B. abortus proteins indicated that both proteins were novel antigens.

Participacion de epitopes flagelares de Campylobacter jejuni en la adhesion celular / Partipation of Campylobacter jejuni flagellar epitopes in cellular adhesion

Utilizando una cepa flagelada (052) y una aflagelada (T-1), se estudió la participación de epítopes flagelares de C. jejuni en la adhesión a células HEp-2 in vitro. La cepa 052 presentó una capacidad de adhesion significativamente mayor que la cepa T-1. Cuando los ensayos de adhesión fueron realizados en presencia de anticuerpos monoclonales dirigidos contra epítopes flagelares, la capacidad de adhesión de la cepa 052 experimentó una inhibición que fluctuó entre 64,3 y 92,9 por ciento. Mediante una prueba de ELISA se comprobó que estos anticuerpos monoclonales eran específicos y estaban dirigidos exclusivamente contra epítopes flagelares de la cepa 052, no reaccionando con la cepa T-1. Estos resultados demuestran que epítopes flagelares de C. jejuni estarían participando en el proceso de adhesión, sugiriendo la intervención del flagelo en la instalación del proceso infeccioso.

Participacion de epitopes flagelares de Campylobacter jejuni en la adhesion celular / Partipation of Campylobacter jejuni flagellar epitopes in cellular adhesion

Utilizando una cepa flagelada (052) y una aflagelada (T-1), se estudió la participación de epítopes flagelares de C. jejuni en la adhesión a células HEp-2 in vitro. La cepa 052 presentó una capacidad de adhesion significativamente mayor que la cepa T-1. Cuando los ensayos de adhesión fueron realizados en presencia de anticuerpos monoclonales dirigidos contra epítopes flagelares, la capacidad de adhesión de la cepa 052 experimentó una inhibición que fluctuó entre 64,3 y 92,9 por ciento. Mediante una prueba de ELISA se comprobó que estos anticuerpos monoclonales eran específicos y estaban dirigidos exclusivamente contra epítopes flagelares de la cepa 052, no reaccionando con la cepa T-1. Estos resultados demuestran que epítopes flagelares de C. jejuni estarían participando en el proceso de adhesión, sugiriendo la intervención del flagelo en la instalación del proceso infeccioso. (AU)