This can be advantageous considering that TNF- is a prototypical proinflammatory cytokine [38] whose overproduction is associated with the occurrence of host tissue injury [39]. IL-4, an essential cytokine for generation of Th2 responses [40], is not produced by the antigen-stimulated spleen cells of vaccinated mice. vaccinated mice, compared with the non-vaccinated ones. The IL-10 content and the mRNA transcription level of TGF- are also higher in the organs of immunized mice. A greater incidence of CD4+ and CD8+ T cells and B lymphocytes is verified in vaccinated mice. However, there is no difference between vaccinated and non-vaccinated mice in terms of the frequency of CD4+CD25+Foxp3+ T cells. Finally, we show that the vaccination confers a long-term protection against infection. Altogether, these data indicate that the oral vaccination of mice with Typhimurium expressing VapA induces specific and long-lasting humoral and cellular responses against the pathogen, which are appropriately regulated and allow tissue integrity after challenge. Introduction is able to infect, survive, and multiply inside the host cells, mainly in alveolar macrophages [5]. The infection begins through inhalation of bacteria from the soil or dust and can result in a severe disease, characterized by chronic pyogranulomatous pneumonia and lung abscesses in both foals and humans. Extrapulmonary lesions may also occur [1]. Although the pathogenic mechanisms of remain largely unknown, there is evidence that virulent strains contain a large 85- to 90-kb plasmid bearing a 27.5-kb pathogenicity island that encodes, among others, nine genes of the virulence-associated protein (vap) family [6], [7]. One member of this family is VapA, a highly immunogenic 15C17 kDa protein that is abundantly expressed on the bacterial surface [6], [8] and plays a crucial role in pathogen growth Rabbit Polyclonal to MOBKL2A/B inside macrophages as well as disease development [9], [10]. Furthermore, VapA is thought to be important in generating immunity against pneumonia is the administration of specific hyperimmune plasma [13], which can provide positive effects [14] but is expensive, labor-intensive, and not universally effective Medroxyprogesterone [15], [16]. Therefore, an effective vaccine suitable for large-scale administration is greatly needed for the prevention of rhodococcal infection. To protect host against rhodoccocosis, a vaccine may need to stimulate both cell-mediated and humoral immunity [14]. Data obtained from immune adult horses and deepened by studies in the murine model of rhodococcosis indicate that resistance to is mainly mediated by T-lymphocyte and depends on IFN- production [14], [17]C[19]. In recent years, several studies have demonstrated the feasibility of using attenuated Gram-positive and Gram-negative intracellular bacteria as live vectors for the oral delivery of recombinant vaccine antigens [20], [21]. Several Typhimurium strains submitted to Medroxyprogesterone attenuation procedures lost their pathogenicity but remained invasive and are used as live vectors for delivery of foreign antigens. These strains are able to induce protective mucosal, humoral, and systemic immune responses against bacteria, viruses, and parasites in a variety of animal models [22], [23]. When used as oral vehicle, they invade enterocytes of the small intestine, including the M cells of the Peyer’s patches, before disseminating to the mesenteric lymph nodes and through the reticuloendothelial system to deep tissues, such as the liver and spleen. Both antibody and cellular specific responses to recombinant antigens expressed by strains have been detected after immunization of mice via mucosal surfaces [24], [25]. The response includes the production of specific secretory immunoglobulins [25], [26]. We have previously reported that oral vaccination of mice with an attenuated Typhimurium vaccine strain expressing the VapA protein confers safety against virulent illness. Materials and Methods Experimental Animals Each experimental or control group consisted of five BALB/c mice, which were housed under specific-pathogen-free conditions in the Animal Research Facilities of the Medical School of Ribeir?o Preto-USP. All animals utilized for the experiments were female, at 6 to 8 8 wk of age. The Ethics Committee on Animal Research of the University or college of S?o Paulo approved all the methods performed in the studies described here. Bacterial Strain and Growth Conditions The Typhimurium 3987 attenuated strain [28] was used previously by us for manifestation of the VapA antigen [27]. Bacterial strains were cultivated in Luria Broth (LB) medium, inside a rotary shaker at 250 rpm, at 37C. For the preparation of bacterial suspensions for administration in mice, overnight ethnicities of the Typhimurium 3987 strains were precipitated by centrifugation (3000g; 15 min), and the pellet was re-suspended in phosphate-buffered saline (PBS) to a final cell denseness of 1-51010 CFU/mL. CFU ideals were determined by plating dilutions of the bacterial suspension onto MacConkey agar plates. The virulent strain of ATCC33701 was kindly provided by Dr. Shinji Takai (University Medroxyprogesterone or college of Kitasato, Towada, Japan). was inoculated into 250 mL mind heart Medroxyprogesterone infusion broth (BHI, Oxoid, Hampshire, England). Cultures were incubated inside a rotary shaker at 100 rpm, 37C,.