Immunogenicity of HLA-DR1 and HLA-A2 peptides derived from Leishmania major Gp63 in golden hamsters.

AIMS: This study aimed to evaluate the toxicity and humoral and cellular immune response of three heterologous vaccines against Leishmania infantum, yet containing synthetic peptides from Leishmania major in the experimental model in hamsters. METHODS AND RESULTS: Through bioinformatics analyses, two Leishmania major Gp63 peptides were predicted and selected for vaccine formulations. Hamsters were divided into four groups, with each group receiving doses of three vaccine formulations containing HLA-DR1 or HLA-A2 peptides plus MontanideTM or both associated with the adjuvant. The animals received three vaccine doses and were evaluated for toxicity after each dose, in addition to being analysed for the production of antibodies and lymphoproliferation on day 211 after the last vaccine dose. Peptides predicted in association with oily adjuvant induced a humoral response and strong lymphoproliferation to Leishmania infantum antigen-specific stimulation.

Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom.

Loxosceles spiders' venom comprises a complex mixture of biologically active toxins, mostly consisting of low molecular mass components (2-40 kDa). Amongst, isoforms of astacin-like metalloproteases were identified through transcriptome and proteome analyses. Only LALP1 (Loxosceles Astacin-Like protease 1) has been characterized. Herein, we characterized LALP3 as a novel recombinant astacin-like metalloprotease isoform from Loxosceles intermedia venom. LALP3 cDNA was cloned in pET-SUMO vector, and its soluble heterologous expression was performed using a SUMO tag added to LALP3 to achieve solubility in Escherichia coli SHuffle T7 Express LysY cells, which express the disulfide bond isomerase DsbC. Protein purification was conducted by Ni-NTA Agarose resin and assayed for purity by SDS-PAGE under reducing conditions. Immunoblotting analyses were performed with specific antibodies recognizing LALP1 and whole venom. Western blotting showed linear epitopes from recombinant LALP3 that cross-reacted with LALP1, and dot blotting revealed conformational epitopes with native venom astacins. Mass spectrometry analysis revealed that the recombinant expressed protein is an astacin-like metalloprotease from L. intermedia venom. Furthermore, molecular modeling of LALP3 revealed that this isoform contains the zinc binding and Met-turn motifs, forming the active site, as has been observed in astacins. These data confirmed that LALP3, which was successfully obtained by heterologous expression using a prokaryote system, is a new astacin-like metalloprotease isoform present in L. intermedia venom.

Potentiating effects of MPL on DSPC bearing cationic liposomes promote recombinant GP63 vaccine efficacy: high immunogenicity and protection.

BACKGROUND: Vaccines that activate strong specific Th1-predominant immune responses are critically needed for many intracellular pathogens, including Leishmania. The requirement for sustained and efficient vaccination against leishmaniasis is to formulate the best combination of immunopotentiating adjuvant with the stable antigen (Ag) delivery system. The aim of the present study is to evaluate the effectiveness of an immunomodulator on liposomal Ag through subcutaneous (s.c.) route of immunization, and its usefulness during prime/boost against visceral leishmaniasis (VL) in BALB/c mice. METHODOLOGY/PRINCIPAL FINDINGS: Towards this goal, we formulated recombinant GP63 (rGP63)-based vaccines either with monophosphoryl lipid A-trehalose dicorynomycolate (MPL-TDM) or entrapped within cationic liposomes or both. Combinatorial administration of liposomes with MPL-TDM during prime confers activation of dendritic cells, and induces an early robust T cell response. To investigate whether the combined formulation is required for optimum immune response during boost as well, we chose to evaluate the vaccine efficacy in mice primed with combined adjuvant system followed by boosting with either rGP63 alone, in association with MPL-TDM, liposomes or both. We provide evidences that the presence of either liposomal rGP63 or combined formulations during boost is necessary for effective Th1 immune responses (IFN-γ, IL-12, NO) before challenge infection. However, boosting with MPL-TDM in conjugation with liposomal rGP63 resulted in a greater number of IFN-γ producing effector T cells, significantly higher levels of splenocyte proliferation, and Th1 responses compared to mice boosted with liposomal rGP63, after virulent Leishmania donovani (L. donovani) challenge. Moreover, combined formulations offered superior protection against intracellular amastigote replication in macrophages in vitro, and hepatic and splenic parasite load in vivo. CONCLUSION: Our results define the immunopotentiating effect of MPL-TDM on protein Ag encapsulated in a controlled release system against experimental VL.

Immunization with a ZmpB-based protein vaccine could protect against pneumococcal diseases in mice.

Zinc metalloprotease B (ZmpB) is present in all isolated pneumococcal strains and contributes to the pathogenesis of pneumococcal infection. In this study, recombinant ZmpB was cloned and expressed in Escherichia coli. The expression of ZmpB by different pneumococcal strains was detectable by Western blotting with antisera raised to recombinant ZmpB. Flow cytometry analysis demonstrated that anti-ZmpB polyclonal antibodies could bind to the cell surface of the pneumococcal strains analyzed. Both recombinant ZmpB protein and anti-ZmpB polyclonal antibodies significantly inhibited the adhesion of Streptococcus pneumoniae D39 to A549 cells. In mouse models, mucosal immunization with recombinant ZmpB could significantly reduce pneumococcal lung colonization caused by S. pneumoniae serotypes 19F and 14 and significantly increase mice survival times following invasive pneumococcal challenge with different pneumococcal strains, including serotypes 2, 3, 6B, and 14. Furthermore, intraperitoneal immunization with recombinant ZmpB in combination with the recombinant pneumolysin mutant (DeltaA146 Ply) and heat shock protein 40 (DnaJ) could enhance the protection against pneumococcal infection compared to protection provided by single-protein antigens. Passive immunization with hyperimmune antisera against these three antigens also demonstrated that the combination of three hyperimmune antisera could provide better protection than single antisera. Taken together, our results suggest that ZmpB is a good candidate pneumococcal vaccine antigen.

gp63 in stable cationic liposomes confers sustained vaccine immunity to susceptible BALB/c mice infected with Leishmania donovani.

Visceral leishmaniasis is deadly if not treated, and development of a vaccine with long-term immunity remains a challenge. In this study, we showed that cationic distearoyl phosphatidylcholine (DSPC) liposomes, when used as vaccine adjuvant with the immunodominant 63-kDa glycoprotein (gp63) of Leishmania donovani promastigotes, induced significant protection against progressive visceral leishmaniasis in susceptible BALB/c mice. gp63 used without adjuvant elicited partial protection but in association with liposomes exhibited marked resistance in both the livers and spleens of the mice challenged 10 days after the last vaccination. The protective efficacy of liposomal gp63 vaccination was dose dependent, with 2.5 mug of protein showing optimal protection. The immunity conferred by this vaccine formulation was durable, as mice challenged 12 weeks after immunization were still protected, and the infection was controlled for at least 3 months postchallenge. Production of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) by splenic T cells, and of serum immunoglobulin G1 (IgG1) and IgG2a following immunization, suggested that a mixed Th1/Th2 response had been induced following immunization. However, control of disease progression and parasitic burden in mice vaccinated with gp63 in cationic DSPC liposomes was associated with enhancement of antigen-specific IFN-gamma and downregulation of IL-4, demonstrating a Th1 bias. Long-term immunity elicited by this vaccine corresponded to, in addition to the presence of antigen-specific Th1, CD8+ T-cell responses. Our results demonstrated that stable cationic liposomes containing gp63 acted as a potent adjuvant for protein antigen to induce long-term protection against L. donovani that represents an alternative to DNA vaccination.

Immune response and protection assay of recombinant major surface glycoprotein of Leishmania (rgp63) reconstituted with liposomes in BALB/c mice.

In this study the ability of recombinant gp63 entrapped in liposomes to induce immune response and protection against L. major infection in susceptible BALB/c mice was studied. Liposomes containing rgp63 (Lip-rgp63) were prepared from egg lecithin and cholesterol using detergent solubilization method. Immunization of BALB/c mice with rgp63 alone conferred a partial protection while entrapment of rgp63 in liposomes significantly increased the rate of protection (P<0.05). The parasite burden of spleen in mice challenged with L. major was significantly (p<0.001) lower in group of mice immunized with rgp63 alone or Lip-rgp63, however, the least parasite burden was seen in Lip-rgp63 group. Both rgp63 alone and Lip-rgp63 elicited significant delayed-type hypersensitivity (DTH) response compared to controls (p<0.01), however, the DTH response of PBS-rgp63 was less than the Lip-rgp63. Titration of anti-Leishmania IgG isotypes (IgG2a/IgG1) showed a preferential Th1 type of immune response only in mice immunized with Lip-rgp63. The results indicate that liposomes might be used as a suitable immunoadjuvant for development of Leishmania vaccine.

Transmission blocking vaccine studies in leishmaniasis: II. Effect of immunisation using Leishmania major derived 63 kilodalton glycoprotein, lipophosphoglycan and whole parasite antigens on the course of L. major infection in BALB/c mice.

BACKGROUND: Safe, effective and inexpensive vaccines may be the most practical tool for control of any form of leishmaniasis. Leishmaniasis produces a state of pre-immunition which is the underlying mechanism for prolonged immunity to re-infection. Low doses of parasites has been shown to be able to induce protection in mice. It is not known, however, how immune sera from a susceptible host immunised with Leishmania-derived antigens when taken in by the sandfly affects the development and the subsequent transmission of the parasite to naive hosts. OBJECTIVE: To monitor the course of disease in BALB/c mice following challenge using L. major infected P. duboscqi which had previously fed on immunised mice. METHODS: BALB/c mice were immunised adequately with Leishmania major-derived antigens namely, crude whole parasite (WPA), recombinant 63 kilodalton glycoprotein (rgp63), lipophosphoglycan (LPG) and a cocktail composed of rgp63 plus LPG antigens. Laboratory reared Phlebotomus duboscqi sandflies, the natural vector for L. major were later allowed to feed on immunised animals, interrupted and allowed to continue feeding on infected animals for an equal amount of time until they became fully engorged. The sandflies were maintained on apples as a carbohydrate source in an insectary maintained at a temperature of 25 degrees C and 80% relative humidity. On the seventh day these sandflies were used to infect naive BALB/c mice and the course of infection followed for a period of at least three months. RESULTS: Mice infected using sandflies which had previously fed on WPA or rgp63-immunized mice showed disease exacerbation as the infection progressed, whereas those infected using sandflies which had previously fed on LPG-immunised mice had the least lesion sizes compared to control mice infected using sandflies which had fed on saline immunised mice (p < 0.05). CONCLUSIONS: Results from this study indicate that the course of L. major infection in BALB/c mice was dependent on the infective dose of parasites transmitted by the sandflies. Results from this study suggests that sub-infective doses of the parasite from sandflies previously fed on animals immunised with Leishmania-derived antigens needs to be evaluated for their potential in vaccine development against Leishmania infections.

Transmission blocking vaccine studies in leishmaniasis: I. Lipophosphoglycan is a promising transmission blocking vaccine molecule against cutaneous leishmaniasis.

BACKGROUND: New strategies for control of leishmaniasis is needed as chemotherapy using antimonial drugs is prolonged, expensive, associated with side effects and relapses. Vector control has limitations and a vaccine which may be the best approach is not available. OBJECTIVES: To assess the level of inhibition of promastigote development and gut morphology in infected Phlebotomus duboscqi sandflies fed on different groups of BALB/c mice immunised with rgp63, lipophosglycan (LPG) or their cocktail and whole parasite antigens prepared from L. major culture-derived promastigotes. METHODS: BALB/c mice were immunised adequately with Leishmania major-derived antigens namely, crude whole parasite (WPA), recombinant 63 kilodalton glycoprotein (rgp63), LPG and a cocktail composed of rgp63 plus LPG antigens. Laboratory reared Phlebotomus duboscqi sandflies, the natural vector for L. major were later allowed to feed on immunised animals, interrupted and allowed to continue feeding on infected animals for an equal amount of time until they became fully engorged. The sandflies were maintained on apples as a carbohydrate source in an insectary maintained at a temperature of 25 degrees C and 80% relative humidity. Some of the sandflies were dissected on days 2, 4 and 6 after feeding and observed using the light and the transmission electron microscopy for any changes in their gut morphology. The remaining sandflies were all dissected on the sixth day post-feeding and examined for procyclics, nectomonads, haptomonads and metacyclic promastigote forms of Leishmania. RESULTS: Sandflies which had previously fed on WPA, LPG plus rgp63 cocktail and LPG-immunised mice showed the lowest infection rates compared to control sandflies fed on saline immunised mice (p < 0.05). A significant number of procyclic promastigotes, the first developmental form of the parasite in culture as well as in the sandfly was observed in sandflies which fed on LPG-immunised mice (p < 0.05). The dominant parasite form in sandflies which fed on rgp63 or LPG-immunised mice was the nectomonad form but very few of the infective metacyclic forms (p < 0.05). Control sandflies fed on saline immunised or infected mice alone displayed a normal pattern of parasite development up to the metacyclic stage. Studies showed that two possible mechanisms through which immune sera from immunised mice may cause inhibition of parasite development is by exflagellation of nectomonad forms and degeneration of the sandfly midgut epithelium as revealed by light and electron microscopy studies respectively. CONCLUSIONS: This study has shown that immune-mediated transmission blocking may be applied to Leishmania infections. Based on observation of the procyclic promastigotes, the dominance of the nectomonad forms, low infectivity rates in sandflies fed on LPG-immunised mice, we concluded that LPG stands out to be a promising transmission blocking vaccine candidate in leishmaniasis.

Fragilysin, the enterotoxin from Bacteroides fragilis, enhances the serum antibody response to antigen co-administered by the intranasal route.

Fragilysin, an extracellular zinc metalloprotease produced by enterotoxigenic strains of the anaerobic bacterium Bacteroides fragilis, disrupts the paracellular barrier by cleavage of the intercellular proteins between epithelial cells resulting in fluid secretion. Intranasal immunization of mice with fragilysin and co-administered ovalbumin (Ova) resulted in an Ova-specific serum IgG response that was over 18000-fold higher than Ova alone, as well as detectable levels of serum IgA. Serum IgG titers were comparable with those seen when whole cholera toxin was used as the adjuvant, although the responses obtained with fragilysin showed more variability between mice. Metalloproteases to which fragilysin is structurally related were ineffective as mucosal adjuvants. Our results and similar studies with enterotoxins that affect the paracellular barrier suggest that alteration of mucosal permeability may play an important role in the mechanisms of adjuvanticity.

Identification and characterization of hydra metalloproteinase 2 (HMP2): a meprin-like astacin metalloproteinase that functions in foot morphogenesis.

Several members of the newly emerging astacin metalloproteinase family have been shown to function in a variety of biological events, including cell differentiation and morphogenesis during both embryonic development and adult tissue differentiation. We have characterized a new astacin proteinase, hydra metalloproteinase 2 (HMP2) from the Cnidarian, Hydra vulgaris. HMP2 is translated from a single mRNA of 1.7 kb that contains a 1488 bp open reading frame encoding a putative protein product of 496 amino acids. The overall structure of HMP2 most closely resembles that of meprins, a subgroup of astacin metalloproteinases. The presence of a transient signal peptide and a putative prosequence indicates that HMP2 is a secreted protein that requires post-translational processing. The mature HMP2 starts with an astacin proteinase domain that contains a zinc binding motif characteristic of the astacin family. Its COOH terminus is composed of two potential protein-protein interaction domains: an "MAM" domain (named after meprins, A-5 protein and receptor protein tyrosine phosphatase mu) that is only present in meprin-like astacin proteinases; and a unique C-terminal domain (TH domain) that is also present in another hydra metalloproteinase, HMP1, in Podocoryne metalloproteinase 1 (PMP1) of jellyfish and in toxins of sea anemone. The spatial expression pattern of HMP2 was determined by both mRNA whole-mount in situ hybridization and immunofluorescence studies. Both morphological techniques indicated that HMP2 is expressed only by the cells in the endodermal layer of the body column of hydra. While the highest level of HMP2 mRNA expression was observed at the junction between the body column and the foot process, immunofluorescence studies indicated that HMP2 protein was present as far apically as the base of the tentacles. In situ analysis also indicated expression of HMP2 during regeneration of the foot process. To test whether the higher levels of HMP2 mRNA expression at the basal pole related to processes underlying foot morphogenesis, antisense studies were conducted. Using a specialized technique named localized electroporation (LEP), antisense constructs to HMP2 were locally introduced into the endodermal layer of cells at the basal pole of polyps and foot regeneration was initiated and monitored. Treatment with antisense to HMP2 inhibited foot regeneration as compared to mismatch and sense controls. These functional studies in combination with the fact that HMP2 protein was expressed not only at the junction between the body column and the foot process, but also as far apically as the base of the tentacles, suggest that this meprin-class metalloproteinase may be multifunctional in hydra.

Long-term protection of mice against Leishmania major with a synthetic peptide vaccine.

A single synthetic T cell epitope (PT3), obtained from the histidine zinc-binding region of the metalloprotease gp63, was employed in a vaccine trial using two virulent strains of L. major. When a single subcutaneous injection of PT3 was delivered with the Thl stimulating adjuvant poloxamer 407, BALB/c mice were protected for at least 10 months against the disease. Vaccinated mice were largely free of lesions on termination of the experiment. Protection was similar for both L. major MRHP/SU/59 Neals and L. major WHOM/IR/-/173 strains which manifest different disease sequelae. Thus, these data provide evidence that a single subcutaneous injection of a single synthetic T cell epitope is sufficient to provide long-lasting protection against two highly virulent strains of L. major in BALB/c mice.

Cloning of murine membrane-type-1-matrix metalloproteinase (MT-1-MMP) and its metanephric developmental regulation with respect to MMP-2 and its inhibitor.

BACKGROUND: Extracellular matrix macromolecules regulate morphogenesis of embryonic organs, and are developmentally regulated. Their expression and turnover is regulated by matrix metalloproteinases (MMPs). Recently, an epithelial cell "membrane" associated metalloproteinase (MT-1-MMP) has been identified that acts as an activator of a "secreted" MMP-2, and is produced by mesenchymal fibroblasts. The activity of MMP-2 is inhibited by a "soluble" tissue inhibitor of MMP-2, TIMP-2. The role of MT-1-MMP in renal development is unknown. METHODS: MT-1-MMP was cloned from embryonic mouse kidney cDNA library, and its spatio-temporal distribution during development in the context of MMP-2 and tissue inhibitor of metalloproteinase-2 (TIMP-2) was studied. RESULTS: The cloned MT-1-MMP exhibited approximately 86% nucleotide sequence homology with human MT-1-MMP, and had a catalytic domain and a zinc binding site preceded by a RRKR furin recognition motif. A approximately 4.5 Kb MT-1-MMP mRNA transcript was detected, and its expression was developmentally regulated. A parallel developmental regulation of MMP-2 mRNA expression was also observed. TIMP-2 expression was also developmentally regulated, but lagged behind MT-1-MMP and MMP-2. By in situ hybridization, MT-1-MMP mRNA was seen to be confined to ureteric bud epithelia, and was absent in the mesenchyme, while MMP-2 was confined to the mesenchyme. MT-1-MMP protein expression was seen on ureteric bud epithelia, induced mesenchyme and nascent nephrons, and it was highest during mid gestation. Similar spatio-temporal expressions of MMP-2 and TIMP-2 proteins were observed. CONCLUSIONS: mRNAs of MT-MMP-1 and MMP-2 are expressed in the respective epithelial and mesenchymal compartments, while their proteins are co-expressed in the epithelia suggest that MT-1-MMP and MMP-2, in conjunction with TIMP-2, may be involved in paracrine/juxtacrine epithelial:mesenchymal interactions during metanephrogenesis.

Matrix metalloproteinase MMP-19 (RASI-1) is expressed on the surface of activated peripheral blood mononuclear cells and is detected as an autoantigen in rheumatoid arthritis.

In order to characterize the autoimmune response participating in the pathogenesis of rheumatoid arthritis (RA) a cDNA expression library constructed from mRNAs which had been isolated from the inflamed synovium of an RA patients was screened with autologous IgG autoantibodies. This led to the identification of gene rasi-1 which encodes a protein showing sequence identity with the zinc-binding matrix metalloproteinase MMP-19. MMP-19 is detected on the surface of activated PBMCs, TH1 lymphocytes, and Jurkat T lymphoma cells. It exhibits gelatinolytic activity and is recognized by autoantibodies in 26% and, respectively, 33% of sera collected from RA patients and systemic lupus erythematosus (SLE) patients. The novel autoantigen MMP-19 thus could play a role in the pathological processes participating in RA-associated joint tissue destruction.