Immunization with Single Oral Dose of Alginate-Encapsulated BCG Elicits Effective and Long-Lasting Mucosal Immune Responses.

Effective vaccination against pathogens, which enter the body through mucosal surfaces, requires the induction of both mucosal and systemic immune responses. Here, mucosal as well as systemic immune responses in the lung and spleen of BALB/c mice which were orally vaccinated with a single dose of alginate-encapsulated bacille Calmette-Guerin (BCG) were evaluated. Twenty weeks after immunization, the vaccinated mice were challenged intranasally with BCG. Twelve weeks after immunization and 5 weeks after challenge, the immune responses were evaluated. Moreover, immune responses were compared with those of mice that were vaccinated with free BCG by subcutaneous (sc) and oral routes. Twelve weeks after the immunization, serum IgG level was higher in the sc-immunized mice, while serum IgA level was higher in the orally immunized mice with encapsulated BCG. Significant productions of both IgG and IgA were only detected in lungs of mice orally immunized with encapsulated BCG. Proliferative and delayed-type hypersensitivity responses and IFN-γ production were significantly higher in mice immunized orally with encapsulated BCG, compared to mice immunized orally with free BCG. After challenge, the levels of IFN-γ were comparable between sc-immunized mice with free BCG and orally immunized with encapsulated BCG; however, significantly less IL-4 was detected in mice which had received encapsulated BCG via oral route. Moreover, significant control of the bacilli growth in the lung of the immunized mice after intranasal challenge with BCG was documented in mice vaccinated with encapsulated BCG. These results suggest that oral immunization with alginate-encapsulated BCG is an effective mean of inducing mucosal and systemic specific immune responses.

Distinct strains of Leishmania major induce different cytokine mRNA expression in draining lymph node of BALB/c mice.

Four genotypically distinct strains of L. major collected from persons residing in different endemic areas of cutaneous leishmaniasis in Iran were evaluated in BALB/c mice. Parasite virulence was evaluated by measuring the parasite burden in the lymph nodes. Immunogenicity of the strains was assessed by analysis of cytokines mRNA expression levels in popliteal lymph nodes of the mice in early (3, 16, 40 h) and late (week 1, W3, W5 and W8) time periods after infection. The expression of cytokines mRNA, namely Ifng, Il2,Il4,Il10 and Il12, was quantitated by real-time PCR. The lowest and the highest parasite loads were induced by Damghan (2·15 × 107) and Shiraz (9·59 × 109) strains, respectively. Moreover, Damghan strain elicited higher expression levels of Ifng and Il2 mRNA and the highest ratio of Ifng/Il4 mRNA expression compared with the other strains at 40 h and 8 weeks post-infection. The results indicate that the inoculation of BALB/c mice with different strains induced high diversity in parasite burden and cytokines gene expression. Amongst the four strains, Damghan strain showed the lowest parasite load and the highest tendency to induce expression of Th1 cytokines gene and might be considered as a safe and immunogenic strain.

CD26 expression on CD4+T cells in patients with cutaneous leishmaniasis.

Surrogate marker(s) of protection in human leishmaniasis is not well defined. In this study, T helper 1 (Th1) and Th2 cytokine profiles and CD26 expression on CD4(+) T cells in peripheral blood mononuclear cells of patients with healing or non-healing forms of cutaneous leishmaniasis (CL) stimulated with Leishmania antigens were assessed. The level of interferon (IFN)-gamma production was significantly higher in patients with healing or non-healing forms of CL than in healthy controls, but it was not significantly different between the two patient groups. The level of interleukin-5 production was significantly higher in patients with the non-healing form of CL than in the two other groups. There was a significant increase in the level of CD26 expression on CD4(+) T cells in patients with healing (P < 0.001) or non-healing (P = 0.025) forms of CL compared with the control group, but no significant difference was seen between the two patient groups. A weak positive correlation was seen between IFN-gamma production and CD26 expression on CD4(+) T cells of patients with the healing form of lesion (r = 0.54, P = 0.008), but this correlation was not observed in patients with the non-healing form of CL (r = 0.53, P = 0.078). Surface CD26 is not correlated with the clinical manifestation of CL or IFN-gamma production. Therefore, CD26 is not a surrogate marker for IFN-gamma production in CL.

Correlation between Plasma Interleukin-3, the α/ß Globin Ratio, and Globin mRNA Stability.

Background. Globin chain synthesis (GCS) analysis is used in the diagnosis of thalassemia. However, the wide reference range limits its use as a decisive diagnostic tool. It has been shown that α and ß globin mRNA increase through stimulation of cells by interleukin-3 (IL-3). Therefore, this study investigates the relationship between plasma IL-3 and the ß/α globin ratio. Methods. Blood samples were collected from 32 healthy participants on two occasions one month apart. GCS analysis, real-time PCR, and ELISA tests were conducted to determine the ß/α globin ratio, globin mRNA expression and stability rate, and IL-3 levels. Results. On the basis of IL-3 levels, the participants were divided in two groups. One group included participants who showed a significant increase in IL-3 as indicated by a significant rise in mean values of α, ß, and γ globin mRNA, α and ß globin, RBC, and hemoglobin. The other group included participants who showed no difference in IL-3 levels with no significant variations in the above-mentioned parameters. Conclusion. The results of this study indicate that IL-3 has an equivalent positive effect on α and ß globin chain synthesis. Therefore, IL-3 levels do not explain the wide reference range of the α/ß globin ratio.

Validation of an in-vitro method for Hepatitis B vaccine potency assay: specification setting.

AIM: Production batches of the Hepatitis B vaccine should be tested by the National Control Laboratory (NCL) before being released to the market, in terms of their potency. This can be done either by means of the mouse immunogenicity (in-vivo) method, which is a time-consuming and labor intensive process, or by an in-vitro method with acceptable analytical performance and with specifications determined based on the results obtained from testing some production batches of the vaccine with proven efficacy. Here we report the feasibility of using and validation of a commercial enzyme-linked immunosorbent assay (ELISA) kit replacing the manufacturer's method and setting of different specification for potency of the particular vaccine. METHODS: For the in-vitro potency assay of the Hepavax-Gene®, produced by Berna Biotech Korea Corp, a commercial ELISA kit for hepatitis B surface antigen (HBsAg) quantitation (Hepanostika® HBsAg Ultra from Biomerieux) was used to determine the relative potency. Validation parameters were evaluated following the International Conference on Harmonization (ICH) guidelines. Specification of the vaccine potency was determined based on the results generated by the commercial ELISA kit. Some batches were tested by in-vivo method as well. RESULTS: It was confirmed that the ELISA kit, when used for vaccine potency testing, meets the criteria for accuracy (80% to 110% recovery), precision (repeatability, with a CV% less than 5%; and intermediate precision, with a CV% less than 10%) and Linearity (r2> 98%), as well as being able to detect HBsAg specifically. Specification of the in-vitro method was also determined as having a relative potency of >50%. CONCLUSION: The Hepanostika® HBsAg Ultra kit from Biomerieux can be used to determine the relative potency of the Hepavax-Gene® Hep B vaccine as an alternative to the manufacturer's method and with different specifications.

Soluble CD26/CD30 levels in visceral leishmaniasis: markers of disease activity.

Leishmania infantum is the causative agent of zoonotic visceral leishmaniasis (VL). If untreated the disease could be fatal; however, in some cases the infection can run a subclinical course. In subclinical infections a Th1-response predominates, while Th2-responses and/or probably Treg cells are related to unfavourable outcome of the disease in active VL. In the present study we determined the levels of soluble (s) CD26 and CD30 co-stimulatory molecules in sera from patients with active VL, asymptomatic individuals and healthy volunteers. Results showed a significant difference in both sCD26 and sCD30 between infected cases and normal individuals (P < or = 0.001). However, there was no significant difference in sCD26 levels between asymptomatic cases and patients, although the difference was not significant. sCD30 levels were significantly higher in VL patients than asymptomatic cases (P < or = 0.001). These findings suggest a possible association between sCD26 and sCD30 levels and the clinical manifestation of L. infantum infection.

Stabilizing effects of calcium alginate microspheres on Mycobacterium bovis BCG intended for oral vaccination.

In this study, alginate microspheres containing BCG were prepared at a diameter of approximately 10 microm by emulsification-internal gelation of an alginate-BCG solution dispersed in olive oil using a high rate speed stirrer. The stability of BCG was assayed at 4 degrees C showing that the encapsulated BCG was more stable than free BCG at least for 5 weeks; however, BCG in sodium alginate solution was not stable at all. On the other hand, the studies using media with different pH (1.2, 4.4, 6.2, 6.8 and 7.5) have demonstrated that the alginate microspheres are stable in acidic medium for upto 1.5 h without any sign of disintegration. Moreover, BCG incorporated in alginate microspheres demonstrated an almost 9-fold increase in viable bacilli in simulated gastric fluid (SGF) after 1.5 h in comparison with free BCG.

Recombinant interleukin-1 promotes leishmaniasis in susceptible mice.

Interleukin-1 exacerbates leishmanial lesions in Leishmania major-infected BALB/c mice. Indomethacin can modulate the effect of IL-1, so at least part of the IL-1 effect on disease progression is due to the induction of prostaglandin synthesis.

Comparison of the immune profile of nonhealing cutaneous Leishmaniasis patients with those with active lesions and those who have recovered from infection.

Th1-type cellular immune responses play a critical role in protection against infection with Leishmania parasites, whereas activation of Th2-type cells results in progressive disease. Cutaneous leishmaniasis caused by Leishmania major is often a self-healing disease; however, persistent nonhealing forms are also known. In the present study, we have described cell-mediated immune responses in nonhealing patients by measuring T-cell proliferation, cytokine production, and phenotypic characterization of these cells. The responses were compared with those of patients with active lesions, patients who had recovered from infection, and healthy controls. Peripheral blood mononuclear cells from patients with active lesions and recovered donors proliferated vigorously and produced Th1-type cytokine when stimulated with L. major antigens, whereas in nonhealing patients the proliferative responses were significantly lower and showed a Th2-type response to Leishmania antigens. Interleukin-10 (IL-10) production was not a feature of L. major stimulation. Flow cytometric analysis revealed that L. major antigen induced proliferation of the CD4-positive population and that these cells were the major source of gamma interferon and IL-4. These results show a distinct dichotomy in the cytokine response to L. major infection.