Vaccination of mice with a combination of BCG and killed Leishmania promastigotes reduces acute Trypanosoma cruzi infection by promoting an IFN-gamma response

The combination of BCG with killed Leishmania promastigotes, demonstrated to be efficient in the cure of patients suffering American cutaneous leishmaniasis and in the induction of a long-term immune response in healthy vaccinated volunteers, was tested in BALB/c mice infected with Trypanosoma cruzi, in comparison to BCG or Leishmania alone, and a vehicle (PBS) control. BCG-Leishmania vaccination, applied intra-peritoneally 10 and 3 days before T. cruzi trypomastigote inoculation, prolonged the survival, and reduced blood parasitaemia of infected animals. Proliferation studies indicated that splenocytes of mice vaccinated with BCG-Leishmania and harvested in the acute phase of T. cruzi infection displayed stimulation indices higher than cells from PBS-treated mice when stimulated with PHA mitogen, PPD, Leishmania or T. cruzi antigens. Injections of a monoclonal antibody able to neutralise IFN-gamma into BCG-Leishmania vaccinated mice increased parasitaemia to levels similar to those of control animals (treated with PBS) and reversed the beneficial effect of vaccination on the proliferative response to T. cruzi antigen. These results show that vaccination of mice with BCG plus killed Leishmania promastigotes delayed acute T. cruzi infection, stimulated a T-cell response to T. cruzi antigen and promoted IFN-gamma production.     

Acute Trypanosoma cruzi infection: IL-12, IL-18, TNF, sTNFR and NO in T. rangeli-vaccinated mice

We have developed an experimental model of vaccination against the infection with the protozoa Trypanosoma cruzi, the agent of Chagas disease in Latin America. Vaccination was performed with Trypanosoma rangeli, a non-pathogenic protozoa sharing many antigens with T. cruzi. It strongly protected BALB/c mice, sharply reducing parasitaemia and mortality rate of the acute T. cruzi infection. The aim of the present work was to complete our previous study on the production of IFN-gamma and IL-10 in this vaccination model by investigating the production of IL-12p35 and p40, IL-18, TNF, TNF soluble receptors (sTNFR), and nitric oxide (NO), factors known to play a key role in the outcome of T. cruzi infection. We show that the protection obtained against the acute T. cruzi infection was surprisingly associated with reduced circulating levels of IL-18 and NO, whereas the release of IL-12p40 was enhanced in comparison to non-vaccinated infected animals. IL-12p35 remained undetectable in infected animals, vaccinated or not. The balance between sTNFR and TNF suggested a decrease of TNF bioactivity in vaccinated mice. These results show that the protection induced by the vaccination with T. rangeli against a challenging infection with T. cruzi is not associated with the strong type 1 immune response usually involved in the control of intracellular pathogens, particularly questioning the protective role of NO during the acute phase of T. cruzi infection.     

Successful vaccination against Leishmania donovani infection in Indian langur using alum-precipitated autoclaved Leishmania major with BCG

Autoclaved Leishmania major (ALM) along with BCG, presently undergoing phase II clinical trial by WHO for its vaccine potential against cutaneous leishmaniasis, has been successfully evaluated in single and triple dose schedules against L. donovani in Indian langurs (Presbytis entellus). Encouraged with the results, another formulation alum-precipitated ALM (provided by WHO) along with BCG has been evaluated in this system. Eight monkeys were vaccinated with alum-precipitated ALM + BCG (1 mg of each per animal) while four were kept as unvaccinated controls. All were challenged with 100 x 10(6) amastigotes i.v. on day 60 post vaccination. Parasitic assessment in splenic tissue was performed on day 45, 90 and 180 p.c. Initially, seven of the eight vaccinated monkeys developed infection (two to six amastigotes per 1000 cell nuclei), which resolved by day 180 p.c., while the eighth monkey had a parasite burden of 14 amastigotes per 1000 cell nuclei on day 45 p.c. and died on day 130 p.c. On the other hand, there was progressive infection in unvaccinated control animals and three out of four died between days 110 and 120 p.c., and one monkey, which had low parasite burden, died on day 178 p.c. Prior to challenge, there was an initial rise in antileishmanaial antibodies in the vaccinated group compared to the unvaccinated control group, which later came down to normal level, while it remained higher in the unvaccinated control group. An increasing pattern of antigen-specific proliferative responses and interferon-gamma level to the two antigens--autoclaved L. donovani (ALD) and ALM--was observed in vaccinated monkeys throughout the experiment. There was a good correlation between parasite burden and IFN-gamma level on days 90 and 180 p.c., indicating IFN-gamma response as a sensitive parameter of immune status. The findings suggest alum-precipitated ALM+BCG as a potential vaccine against visceral leishmaniasis and warrants clinical trials.     

Immunization with Leishmania infantum released proteins confers partial protection against parasite infection with a predominant Th1 specific immune response

In this study, protective effect and immune response elicited by protein fractions LiRic1 (>75 kDa) and LiRic2 (<37 kDa) released by Leishmania infantum promastigotes were analysed in challenged BALB/c mice. Viable parasites were quantified in spleen and isolated CD4(+) and CD8(+) T cells were stimulated for evaluation of proliferative response and cytokine production. Immunization triggered 50.4-66.9% of parasite reduction. Stimulated CD4(+) T cells from challenged animals revealed high proliferation. IL-12 and IFN-gamma were released by CD4(+) T cells whereas IL-4 and IL-10 were impaired. LiRic1 and LiRic2 immunization gave partial protection and a CD4(+) Th1 response. LiRic2 generated IL-12 by CD8(+) T cells pointing to its participation in protective response. These results encourage further research on the development of a vaccine that provides long-lasting protection against zoonotic visceral leishmaniasis.     

A combined DNA vaccine enhances protective immunity against Mycobacterium tuberculosis and Brucella abortus in the presence of an IL-12 expression vector

We examined the immunogenicity and protective efficacy of a combined DNA vaccine that included six genes encoding immunodominant antigens from Mycobacterium tuberculosis and Brucella abortus. The IL-12 adjuvant system was used for immunization in combination with the combined DNA vaccine (DNA-IL-12(+)). Mice immunized with DNA-IL-12(+) had significantly reduced CFU counts for M. tuberculosis and B. abortus in lung and spleen, respectively (P<0.001), and DNA-IL-12(+) elicited better protection than the combined DNA vaccine alone (DNA-IL-12(-)) or with the positive control groups after challenge with a virulent M. tuberculosis strain and B. abortus 2308 infection. The DNA-IL-12(+) group had stronger antigen-specific IFN-gamma ELISPOT activities and higher levels of antigen-specific CD4(+) and CD8(+) T cell responses than either the DNA-IL-12(-) or positive control groups. Likewise, antigen-specific IgG titers were also much higher than in other immunized groups. Moreover, DNA-IL-12(+) gave a stronger IgG2a-skewed response than did DNA-IL-12(-). In addition, its mean concentrations of IFN-gamma and IL-2 were about 2.5- to 4.5-fold higher than those observed in the DNA-IL-12(-)-treated mice, and were significantly higher than control groups (P<0.01 or P<0.001), whereas IL-4 and IL-10 secretion were lower. These results suggest that IL-12 acts as an adjuvant to enhance protective immunity against M. tuberculosis and B. abortus through the induction of stronger Th1-associated immune responses. This is the first report to show that a single combined DNA vaccine protects animals against two infectious diseases.     

Recombinant M. smegmatis vaccine targeted delivering IL-12/GLS into macrophages can induce specific cellular immunity against M. tuberculosis in BALB/c mice

In the present study, we constructed a viable therapeutic vaccine of recombinant M. smegmatis mediated IL-12/GLS (granulysin) gene transfer into murine macrophages to exert the immunotherapy effects on the Mycobacterium tuberculosis infection. We tested this recombinant therapeutic vaccine in an in vivo study to determine its capability of stimulating host specific immune responses against M. tuberculosis. BALB/c mice intranasally immunized with the therapeutic vaccine developed an efficient Th1 protective immune response against M. tuberculosis which was equal to that of the BCG strain. Inoculation intranasally with this viable vaccine induced high level of serum IFN-gamma, IL-12 and IgG2a. The viable vaccine was capable of inducing purified protein derivative (PPD) antigen-specific splenocytes proliferation and IFN-gamma production from T cells in spleens of the immunized mice. In addition, intranasally inoculation with the viable vaccine can induce PPD antigen-specific sIgA production in the broncho-alveolar lavage fluid (BALF) of the immunized mice. No change of IL-4 level was found in all groups. The therapeutic mechanism of this viable vaccine against M. tuberculosis infection observed here appeared to be a result of the specific Th1 immune response activated by mycobacterium antigen from M. smegmatis and the expression of sIL-12/GLS in alveolar macrophages via the M. smegmatis-mediated gene transfer method. This research demonstrates that the therapeutic gene can be introduced into a host by viable mycobacteria works to induce the host specific immune response against M. tuberculosis infection in vivo. Since this therapeutic vaccine can strongly induce specific Th1 responses against M. tuberculosis in BALB/c mice and has no obviously harmfulness to the host simultaneously, the recombinant vaccine might be a potential candidate therapeutic vaccine against tuberculosis.     

Immunochemotherapy of persistent post-kala-azar dermal leishmaniasis: a novel approach to treatment

Post-kala-azar dermal leishmaniasis (PKDL) is a recognized dermatosis that follows successful treatment of visceral leishmaniasis in the Sudan. This randomized and double-blind study aimed to assess safety, immunogenicity and curative potentials of a novel immunochemotherapy regimen in patients with persistent PKDL. Following informed consent, 30 patients were randomized to receive alum-precipitated autoclaved Leishmania major (Alum/ALM) vaccine+Bacille Calmette-Guérin (BCG) and sodium stibogluconate (SSG) or vaccine diluent and SSG. The SSG+Alum/ALM+BCG proved safe with minimal local adverse events. In the SSG+vaccine group, 87% of the patients were cured by day 60 compared with 53% in the SSG alone group (SSG+vaccine efficacy=71%, 95% CI for risk ratio 0.7-1.16). On day 90 of follow-up there were two relapses in the SSG alone arm and none in the SSG+vaccine arm. Pre-treatment cytokines showed high IFN-gamma or high IFN-gamma/IL-10 levels and leishmanin skin test (LST) non-reactivity, while healing/clinical improvement were associated with LST reactivity and low IFN-gamma levels in both study groups (P=0.004). In conclusion, SSG+Alum/ALM+BCG is safe and immunogenic with significant healing potentials in persistent PKDL lesions. Immunochemotherapy probably augmented IFN-gamma production, which induced healing. Leishmanin skin reactivity is a good surrogate marker of cure in persistent PKDL lesions.     

Characterization of M. Tuberculosis-derived IL-12-inducing material by alveolar macrophages

We have investigated the substance derived from Mycobacterium tuberculosis (Mtb) that induces interleukin (IL)-12 production by alveolar macrophages (AMs) in vitro. The cytosol fraction of live Mtb H37Rv induced IL-12 production by AMs in a dose-dependent manner. The addition of interferon-gamma (IFN-gamma) augmented IL-12 production. IL-12-inducing activity by AMs (termed as surely active keeping rescue antigen, SAKRA) was purified by gel filtration and ion exchange column chromatography, and the molecular weight of SAKRA was estimated by gel filtration to be more than 700 kDa. SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting of SAKRA using rabbit anti-SAKRA antibody suggested that SAKRA is composed with several low molecular weight proteins. Amino acids sequence analysis of several bands after SDS-PAGE suggested that SAKRA is a part of ribosomes. RT-PCR showed that SAKRA induced not only expression of IL-12 p40 mRNA, but expression of tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS) mRNA at least 6 h after stimulation, suggesting that SAKRA activates the bactericidal activity of macrophages. To investigate the potential use of SAKRA as a vaccine against tuberculosis, SAKRA was administered to BALB/c mouse that had been immunized with BCG for 18 months, and mouse were infected with Mtb H37Rv via a respiratory route. Replication of Mtb in lungs and spleens was examined 6 weeks after infection. Administration of SAKRA to BCG-vaccinated mice significantly reduced the numbers of Mtb in lungs and spleens as compared with BCG-vaccinated control mice. Taken together, these results suggest that SAKRA is one of the Mtb-derived immunomodulatory substances which induce IL-12 production during infection and also increases mycobactericidal activities of macrophages, and that SAKRA may be a promising new vaccine candidate against tuberculosis.     

Oral immunization using live Lactococcus lactis co-expressing LACK and IL-12 protects BALB/c mice against Leishmania major infection

Leishmaniasis is a parasitic disease affecting over 12 million individuals worldwide. Current treatments are laborious, expensive, cause severe side effects, and emerging drug resistance has been reported. While vaccination is the most cost-effective means to control infectious diseases there is no human vaccine currently available against Leishmania infections. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used for the expression and delivery of biologically active molecules, such as antigens and cytokines, in mice and humans. In this study, we report the generation of L. lactis(alr-) strains solely expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall or co-expressing mouse IL-12. We show that oral immunization using live L. lactis, secreting both LACK and IL-12 was the only regimen that partially protected BALB/c mice against subsequent Leishmania major challenge. This highlights the importance of temporal and physical proximity of the delivered antigen and adjuvant for optimal immune priming by oral immunization since co-administration of L. lactis strains independently expressing secLACK and secIL-12 did not induce protective immunity. Protected animals displayed a delay in footpad swelling, which correlated with a significant reduction of parasite burden. Immunization with the L. lactis strain secreting both LACK and IL-12 induced an antigen-specific mucosal immune response and a LACK-specific T(H)1 immune response in splenocytes and mesenteric lymph node cells. Further, protection in immunized animals correlated with a strong Leishmania-specific T(H)1 immune response post-challenge, detectable in splenocytes and lymph node cells draining the site of infection. This report demonstrates the use of L. lactis as an oral live vaccine against L. major infection in susceptible BALB/c mice. The vaccine strains generated in this study provide the basis for the development of an inexpensive and safe oral live vaccine against the human parasite Leishmania.     

Life-long systemic protection in mice vaccinated with L. major and adenovirus IL-12 vector requires active infection, macrophages and intact lymph nodes

Immunization with soluble leishmanial antigen (SLA) in IFA plus Ad5IL-12 vector induced protection confined to the immunized footpad in BALB/c mice. However, animals that controlled a primary infection with a Leishmania major challenge in the same immunized footpad, became resistant to subsequent contralateral rechallenges due to expansion of IFN-gamma secreting cells. This systemic immunity could be disrupted either by macrophage depletion during immunization or by lymphadenectomy after challenge. We show that this procedure does not interfere with tissue-compartmentalized protection, since lymphadenectomized and splenectomized animals were resistant to rechallenges performed in the immunized footpads. Our results indicate that SLA-Ad5IL-12 vector priming requires macrophages to generate systemic protection. Furthermore, a previously undescribed lymphoid organ-independent, protective immune response is contained within the tissue microenvironment of the immunized/challenged footpad. These results have important implications for vaccine design against leishmanial and mycobacterial infections and diseases caused by intracellular pathogens.     

Intranasal delivery of naked DNA encoding the LACK antigen leads to protective immunity against visceral leishmaniasis in mice

We previously showed that intranasal (i.n.) vaccination with pCIneo plasmid encoding the leishmanial LACK gene (pCIneo-LACK) induces long-lasting protective immunity against cutaneous leishmaniasis in mice. In this work, we proposed to investigate whether the efficacy of i.n. pCIneo-LACK is extensive to visceral leishmaniasis. BALB/c mice received two i.n. doses of 30 microg pCIneo-LACK prior to intravenous (i.v.) infection with Leishmania chagasi. Vaccinated mice developed significantly lower parasite burden in the liver and spleen than control mice receiving empty pCIneo or saline. The spleen cells of vaccinated mice produced significantly increased IFN-gamma and IL-4 concomitant with decreased IL-10 production during infection. Serum levels of specific IgG were elevated whereas TNF-alpha were decreased as compared with controls. These results show that the practical needle-free i.n. pCIneo-LACK vaccine displays potential broad-spectrum activity against leishmaniasis.     

Gene gun-based co-immunization of merozoite surface protein-1 cDNA with IL-12 expression plasmid confers protection against lethal Plasmodium yoelii in A/J mice

The carboxyl-terminal region of the merozoite surface protein-1 (MSP1) is a leading candidate for a vaccine against malaria in the erythrocytic stage. In this study, we investigated the utility of interleukin-12 (IL-12) cDNA as an adjuvant for malaria DNA vaccine in a mouse challenge model. We found that co-immunization of expression plasmids encoding a C-terminal 15-kDa fragment of MSP1 (MSP1-15) with the IL-12 gene using a gene gun significantly increased the protective immunity against malaria as compared with MSP1-15 DNA immunization alone. Co-immunization of IL-12 DNA potentiated MSP1-15-specific T helper (Th)1-type immune responses as evaluated by in vivo antibody (Ab) responses and in vitro cytokine profiles. After the Plasmodium yoelii challenge, mice immunized with MSP1-15 plus IL-12 DNA showed a higher level of interferon gamma (IFN-gamma) production than did other groups of mice. In vivo neutralization of IFN-gamma or depletion of CD4(+) T cells completely abolished this protective immunity. Macrophages, but not nitric oxide (NO), were found to play an important role in this effector mechanism. The sera from mice in which the infection had been cleared by the vaccination showed strong protection against P. yoelii infection. Thus, in addition to cellular immune responses, Abs against parasites induced in the course of infection are essential for protection against P. yoelii. The results indicate that combined vaccination with DNA encoding antigenic peptides plus IL-12 DNA provides a strategy for improving the prophylactic efficacy of a vaccine for malaria infection.     

DNA vaccine using hemagglutinating virus of Japan-liposome encapsulating combination encoding mycobacterial heat shock protein 65 and interleukin-12 confers protection against Mycobacterium tuberculosis by T cell activation

We investigated the immunogenicity and protective efficacy of DNA vaccine combinations expressing mycobacterial heat shock protein 65 (Hsp65) and interleukin-12 (IL-12) using gene gun bombardment and the hemagglutinating virus of Japan (HVJ)-liposome method. A mouse IL-12 expression vector (mIL-12 DNA) encoding single-chain IL-12 proteins comprised of p40 and p35 subunits were constructed. In a mouse model, a single gene gun vaccination with the combination of Hsp65 DNA and mIL-12 DNA provided a remarkably high degree of protection against challenge with virulent Mycobacterium tuberculosis; bacterial numbers were 100-fold lower in the lungs compared to BCG-vaccinated mice. To explore the clinical use of the DNA vaccines, we evaluated HVJ-liposome encapsulated Hsp65 DNA and mIL-12DNA (Hsp65 + mIL-12/HVJ). The HVJ-liposome method improved the protective efficacy of the Hsp65 DNA vaccine compared to gene gun vaccination. Hsp65 + mIL-12/HVJ induced CD8+ cytotoxic T lymphocyte activity against Hsp65 antigen. Most importantly, Hsp65+mIL-12/HVJ vaccination resulted in a greater degree of protection than that evoked by BCG. This protective efficacy was associated with the emergence of IFN-gamma-secreting T cells and activation of proliferative T cells and cytokines (IFN-gamma and IL-2) production upon stimulation with Hsp65 and antigens from M. tuberculosis. These results suggest that Hsp65 + IL-12/HVJ could be a promising candidate for a new tuberculosis DNA vaccine, which is superior to BCG vaccine.     

IL-12-mediated increases in protection elicited by pneumococcal and meningococcal conjugate vaccines

Interleukin-12 (IL-12) may be a beneficial adjuvant for augmenting vaccine efficacy against encapsulated bacteria such as Streptococcus pneumoniae and Neisseria meningitidis since it can stimulate production of interferon-gamma (IFN-gamma) and secretion of antibody isotypes that are efficient at mediating complement fixation and opsonophagocytosis. In this study, we demonstrate the ability of IL-12 to enhance murine antibody responses, particularly IgG2a levels, to both pneumococcal and meningococcal conjugate vaccines. Transfer of immune serum from mice immunized with the meningococcal conjugate vaccine and IL-12 resulted in increased survival times, whereas transfer of serum from mice immunized with the pneumococcal conjugate and IL-12 resulted in protection from death upon bacterial challenge. Although treatment with vaccine and IL-12 increased levels of IFN-gamma mRNA, IL-12-mediated enhancement of antibody responses still occurred in IFN-gamma(-/-) mice. The results demonstrate the effectiveness of IL-12 as an adjuvant for polysaccharide conjugate vaccines, especially the pneumococcal conjugate vaccine.     

A mutant of Escherichia coli enterotoxin inducing a specific Thl-type of T cells to varicella-zoster vaccine enhances the production of IL-12 by IFNgamma-stimulated macrophages

A mutant of Escherichia coli enterotoxin induces specific Thl-type T cells to varicella-zoster vaccine. The mutant increased IL-12p40, TNFalpha and nitric oxide production by IFNgamma-stimulated bone marrow macrophages but cholera toxin did not. Anti-TNFalpha antibodies blocked its stimulation of IL-12p40 production but iNOS inhibitor did not. IL-12p40 and IL-12p35 production was stimulated at the level of mRNA formation by the mutant. Cholera toxin suppressed IL-12beta1 expression by spleen T cells stimulated with anti-CD3 antibodies but the mutant did not. These findings indicate that the mutant may induce Thl-type response to the vaccine through its IL-12 and TNFalpha induction by macrophages.     

Robust gammadelta+ T cell expansion in infants immunized at birth with BCG vaccine

Cell-mediated immune responses to BCG vaccine were evaluated in 7-month-old infants vaccinated with intradermal combined BCG and Hepatitis B or intradermal BCG and intramuscular Hepatitis B at birth. Peripheral blood mononuclear cell cultures from both groups showed CD4(+), CD8(+) and remarkable gammadelta(+) T cell BCG-specific proliferation, without significant differences. Also, IL-10, IL-12, IFN-gamma and TNF-alpha concentrations in culture supernatants, measured by ELISA, were similar. The results suggested that the combined BCG and Hepatitis B vaccine was as immunogenic as BCG separated from Hepatitis B vaccine.     

A mixed Th1/Th2 response elicited by a liposomal formulation of Leishmania vaccine instructs Th1 responses and resistance to Leishmania donovani in susceptible BALB/c mice

In this study, we have developed a vaccine with Leishmania donovani promastigote membrane antigens (leishmanial antigens (LAg)) encapsulated in a liposome carrier formulated with distearyol (DSPC, transition temperature (Tc) = 54 degrees C) derivative of l-alpha-phosphatidyl choline, for immunizing BALB/c mice against progressive visceral leishmaniasis. This formulation could limit hepatosplenomegaly to almost normal levels and conferred strong levels of protection in both liver and spleen against challenge infection. Immunization with liposomal LAg activated peritoneal macrophages for enhanced leishmanicidal activity in association with NO production, and induced antibody as well as T-cell mediated immune responses. Production of both IFN-gamma and IL-4 by splenic T cells, and serum IgG1 and IgG2a, suggest induction of a mixed Th1/Th2 response following immunization. Experimental challenge corresponded with elevated DTH, and mitogen and antigen specific cellular responses. Increased production of NO and IFN-gamma by spleen cells, and down regulation of IL-4, demonstrate that an initial stimulation of a mixed Th1/Th2 response by vaccination instructs Th1 responses and resistance against a progressive infection by L. donovani.     

Leishmania donovani: identification of stimulatory soluble antigenic proteins using cured human and hamster lymphocytes for their prophylactic potential against visceral leishmaniasis

Most of the studies for the identification of prophylactic antigens that elicit T cell responses were concentrated on membrane proteins of Leishmania donovani. This study was taken up to assess L. donovani soluble promastigote antigens for their ability to stimulate proliferation of peripheral blood mononuclear cells (PBMCs) from cured visceral leishmaniasis (VL) patients, endemic and non-endemic controls and lymphocytes/peritoneal macrophages of cured hamsters. The soluble protein was subjected to sequential precipitation with saturated ammonium sulphate (20%, 40%, 60% and 80%), of which largely 80% fractioned protein showed significant cellular responses in cured patients and hamsters. This fraction was further fractionated into five sub fractions by preparative SDS-PAGE and subjected to re-evaluation for their ability to induce cellular responses. Out of these, only F2 sub fraction belonging to the MW of 97.4-68 kDa stimulated remarkable lymphoproliferative and IFN-gamma responses in cured VL patients and in endemic controls. Similarly, significant lymphoproliferative responses and nitric oxide production were also noticed in cured Leishmania infected animals indicating an element of uniformity in responses between hamster and human. F2 sub fraction, when evaluated for its prophylactic efficacy with BCG against L. donovani challenge in hamster exhibited significant parasite inhibition in spleen (71.1%; p<0.001) and liver (68.2%; p<0.001) as compared to their unvaccinated counterpart. The vaccinated animals showed significant lymphoproliferative response and nitric oxide production but leishmania specific IgG level were suppressed. The results indicate the presence of immunostimulatory and protective molecules in F2 sub fraction which may further be exploited for the development of a vaccine against VL, hitherto an unrealized goal.     

Recombinant DNA vaccine of the early secreted antigen ESAT-6 by Mycobacterium tuberculosis and Flt3 ligand enhanced the cell-mediated immunity in mice

Cell-mediated immune (CMI) responses are crucial in the protection against tuberculosis. 6-kDa early secretary antigenic target (ESAT-6) is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. Flt3 ligand (FL) is a growth factor of dendritic cell (DC), which, as a powerful antigen presenting cell (APC), is essential for CMI response. In this study, we constructed a recombinant DNA vaccine encoding ESAT-6 and FL. The recombinants were identified by restriction enzyme digestion and sequence analysis. Subsequently, the recombinants were transfected into glomerular mesangial cells (GMCs) of rat. The expressed proteins were detected by Western blot. C57BL/6 female mice were immunized three times with the recombinant plasmids. The results showed that immunization with pIRES-ESAT-6 plasmid induced an obvious T-cell response compared with controls (mice immunized with PBS, pIRES or BCG). However, mice immunized with pIRES-ESAT-6-FL presented a more stronger T helper 1 (Th1)-biased response, accompanied by higher levels of lymphocytes proliferation, elevated production of Th1 cytokines (IFN-gamma and IL-2) by spleen cells, as well as increased specific antibody in sera, together with lower levels of Th2 cytokines (IL-4 and IL-10). Our results suggested that EAST-6 was a useful vaccine candidate and FL might be a powerful adjuvant, which could effectively promote T cell-mediated immune response.     

Poor immunogenicity of BCG in helminth infected population is associated with increased in vitro TGF-beta production

The only vaccine available against tuberculosis (TB), BCG, so effective in experimental animal models, has been under scrutiny for a long time owing to its variable efficacy against pulmonary tuberculosis in adults. In this study, we evaluated whether anti-helminthic therapy prior to BCG vaccination could increase the immunogenicity of BCG vaccination in helminth infected population. We recruited volunteers with evidence of prior mycobacterial infection and who were asymptomatic carriers of helminths. The subjects were randomized to receive either anti-helminthic drugs or placebo. Three months later, BCG vaccination was administered to volunteers. Mycobacterial antigen-specific cytokine responses were assessed 2 months after vaccination. The results show that peripheral blood mononuclear cells obtained from the placebo group were found to have a lower frequency of IFN-gamma (129 vs 191, p=0.03) and IL-12 (149 vs 243, p=0.013) producing cells per 2 x 10(5) PBMC (peripheral blood mononuclear cells) when stimulated in vitro with a mycobacterial antigen mixture (purified protein derivative (PPD)) compared to those from the dewormed group. On the other hand the placebo group had higher frequency of TGF-beta producing cells in response to PPD (152 vs 81.3, p=0.002) or the T cell mitogen concanavalin A (Con A) (210 vs 157, p=0.03). However, no detectable IL-4 or IL-5 producing cells were observed when cells were stimulated with PPD. Comparable numbers of both cytokine producing cells were induced in both groups upon stimulation with concanavalin A (IL-4 217 vs 191, p=0.08) and IL-5 (131 vs 103, p=0.14). The data presented here demonstrate that chronic worm infection reduces the immunogenicity of BCG in humans and this was associated with increased TGF-beta production but not with enhanced Th2 immune response.