Keratinase Production by Three Bacillus spp. Using Feather Meal and Whole Feather as Substrate in a Submerged Fermentation.

Three Bacillus species (B. subtilis LFB-FIOCRUZ 1270, B. subtilis LFB-FIOCRUZ 1273, and B. licheniformis LFB-FIOCRUZ 1274), isolated from the poultry industry, were evaluated for keratinase production using feathers or feather meal as the sole carbon and nitrogen sources in a submerged fermentation. The three Bacillus spp. produced extracellular keratinases and peptidases after 7 days. Feather meal was the best substrate for keratinase and peptidase production in B. subtilis 1273, with 412 U/mL and 463 U/ml. The three strains were able to degrade feather meal (62-75%) and feather (40-95%) producing 3.9-4.4 mg/ml of soluble protein in feather meal medium and 1.9-3.3 mg/ml when feather medium was used. The three strains produced serine peptidases with keratinase and gelatinase activity. B. subtilis 1273 was the strain which exhibited the highest enzymatic activity.

Cross-protective efficacy of a prophylactic Leishmania donovani DNA vaccine against visceral and cutaneous murine leishmaniasis.

The fucose-mannose ligand (FML) complex of Leishmania donovani is a promising vaccine candidate against murine and canine visceral leishmaniasis, and its main component is a 36-kDa nucleoside hydrolase (NH36). In this study, we tested the immune response and protection induced by the purified FML, the recombinant NH36 (rNH36), and NH36 DNA vaccines against the agents of visceral (L. chagasi) and cutaneous (L. mexicana) leishmaniasis in BALB/c mice. Mice developed weak humoral response to the vaccines alone, except for those immunized with FML. However, all three vaccine groups presented elevated immunoglobulin G (IgG), IgG1, and IgG2a levels after infection with L. chagasi, whereas no differences were observed between vaccine and control groups after infection with L. mexicana. A strong intradermal reaction to L. donovani and L. mexicana antigens was observed in mice immunized with rNH36 or FML, whereas mice immunized with NH36 DNA only reacted against L. donovani antigens. Experimental infection of immunized mice demonstrated that FML and rNH36 induced significant protection against L. chagasi infection with reductions in parasite loads of 79%. FML also conferred partial protection against L. mexicana infection. The best protection was observed in mice immunized with the VR1012-NH36 DNA vaccine, which induced an 88% reduction in L. chagasi parasite load and a 65% reduction in L. mexicana lesion size. Fluorescence-activated cell sorting analysis indicated the DNA vaccine induced a two- to fivefold increase in gamma interferon-producing CD4(+) T cells, indicating a Th1-type immune response. Our results showed that the NH36 DNA vaccine induced a strong immunoprotection against visceral and cutaneous leishmaniasis, suggesting that this DNA vaccine represents a very good candidate for use against several Leishmania species.

Effective immunotherapy against canine visceral leishmaniasis with the FML-vaccine.

The potential effect of the fucose mannose ligand (FML)-vaccine on immunotherapy of canine visceral leishmaniasis was assayed on five mongrel dogs experimentally infected with Leishmania donovani and on 21 Leishmania chagasi naturally infected dogs when seropositive to FML but completely asymptomatic. The clinical signs of the experimentally infected, symptomatic dogs only disappeared after the complete vaccination. Protection was obtained in 3/5 animals that remained asymptomatic, IDR positive and parasite free, 1 year after infection. Furthermore, the asymptomatic, FML-vaccine treated dogs showed stable anti-FML IgG1 levels, increasing IgG2 levels and 79-95% of positive DTH response, during the whole experiment. Twenty-two months after complete vaccination, no obits due to visceral leishmaniasis were recorded and 90% of these dogs were still asymptomatic, healthy and parasite free. On the other hand, 37% (17/46 dogs) kala-azar obits were recorded in a control group that received no treatment during the same period, and that was FML-seropositive and asymtpomatic at the beginning of the assay. Our results indicate that the FML-vaccine was effective in the immunotherapy against visceral leishmaniasis of asymptomatic infected dogs. Normal proportions of CD4 and CD21 lymphocytes were detected in PBMC by FACS analysis, in dogs submitted to immunotherapy, suggesting their non-infectious condition. All animals showed as well significantly increased percents of CD8 lymphocytes as expected for Quillaja saponin (QuilA) vaccine treatments.

Immunotherapy against murine experimental visceral leishmaniasis with the FML-vaccine.

The fucose mannose ligand (Leishmania donovani FML)-saponin vaccine has earlier shown its immunoprophylactic potential against visceral leishmaniasis in the CB hamster (87.7% of parasite load reduction), Balb/c (84.4%) and Swiss albino mouse (85-93%) models. In this investigation its specific immunotherapeutic efficacy against L. donovani infection in Balb/c mice was studied. The effects of vaccine treatment on the humoral response, delayed type of hypersensitivity to promastigote lysate (DTH), cytokine levels in sera and reduction of the liver parasitic load of L. donovani infected mice, were examined. The types and subtypes of anti-FML antibodies increased significantly in the vaccinees over the saline and saponin controls. As expected for a saponin vaccine, the highest ratios were found in relation to IgG1, IgG2a and IgG2b (4.4, 5 and 2.5, respectively). The DTH response and the in vitro ganglion cell proliferative response against FML antigen were also significantly higher than controls (P<0.005). Concomitantly, an impressive and specific decrease of liver parasitic burden was detected only in vaccine-treated animals (94.7%). Our results indicate that the therapeutic FML-vaccine has a potent effect on modulation of the murine infection leading to the reduction of parasitic load and signs of disease, being a new potential tool in the therapy and control of visceral leishmaniasis.

IgG1/IgG2 antibody dichotomy in sera of vaccinated or naturally infected dogs with visceral leishmaniosis.

Canine antibody IgG, IgG1 and IgG2 anti-FML responses were investigated in dogs vaccinated with the fucose-mannose ligand (FML)-vaccine of Leishmania donovani and in dogs with naturally acquired visceral leishmaniosis. While similar levels of total IgG antibodies were seen in the seropositive naturally infected dogs and in vaccinees, significant differences between the groups were found regarding their IgG1/IgG2 anti-FML antibody composition (P<0.005). Higher IgG1 absorbencies were seen in infected dogs, while the IgG2 subtype was predominant in pre-immune sera, and in vaccinated animals, both after the first and the third dose (P<0.005). The average ratio between IgG1/IgG2 was then 1.124 for infected animals and 0.733 for FML-vaccinees. Also, a significant increase in IgG2 antibodies was observed from the first to the third vaccine injection (P<0.005). In the infected dogs, a high correlation between their IgG absorbance (Abs) values and the number of symptoms (P=0.017) was disclosed. Thus, the analysis of IgG subclasses disclosed a dichotomous response to visceral leishmaniosis: IgG1 associated to natural infection and IgG2 associated to a humoral response subsequent to the FML-vaccine treatment. An IgG1/IgG2>or=1 would characterize the sera of visceral leishmaniasis infected animals evoluting towards the overt disease while ratios

The FML vaccine (fucose-mannose ligand) protects hamsters from experimental kala-azar

Visceral leishmaniais or kala-azar, is a chronic and frequently lethal disease, caused by Leishmania donovani. Clinical signs include malaise, hepatosplenomegaly, hypergammaglobulinemia, fever, cachexia and progressive suppresion of the cellular immune response. Only few studies on prophylactic immunization against this disease have been understaken, mostly with crude antigens, and no vaccine against kala-azar is yet available. In previous studies, we have isolated the Fucose-Mannose Ligand (FML) of L. donovani that strongly and specifically inhibits the in vitro infection of macrophages by promastigotes and amastigotes. The FML behaves as a pontent immunogen for rabbits and mice, and is specifically recognized by kala-azar patient sera. The protective pontential of FML on kala-azar was now analyzed in the CB-hamster model. We studied the efect of three intraperitoneal weekly doses of FML (100 mg) in saponin (100 mg), folowed by an intracardiac injection of 107 amastigotes. Saponin- and saline-treated controls were also included. Protection was highly significant regarding the enhancement of anti-FML antibodies titers, the splenocyte proliferative response, and the intradermal delayed hypersensitivity reaction to antigen, as well as the decrease of the parasite burden in spleen and of splenomegaly. Protection to kala-azar was due to specific FML antigenic properties, since the results obtained by saponin alone were significantly different. We conclude that the use of FML and saponin as a vaccine reduced the disease impact and retarded its onset.