Molecular vaccines against parasites

Prophylactic vaccines can be expected to be one of the major practical outputs of parasitology research. Various groups within Australia have pursued the vaccine objective for several years, with particular emphasis on blood-stage falciparum malaria in man, intestinal helminths of sheep and cattle, cutaneous myiasis (blowfly strike) in sheep, cysticercosis in sheep and cattle, bovine babesiosis, and cattle ticks. Other vaccine programmes are concerned with giardiasis, filariasis, toxoplasmosis, fascioliasis, coccidiosis in poultry, cutaneous leishmaniasis and schistosomiasis japonica. For many years, the only available vaccine against a parasite in Australia has been the attenuated Babesia bovis vaccine produced by the Tick Fever Research Centre of the Queensland Department of Primary Industries. Strategies for achieving molecular vaccines are generally similar within the various research groups. They involve analysis of the immunology and immunochemistry of a model or in-vitro system; development of functional monoclonal antibodies; analysis of antibody specificities in clinically and/or functionally defined polyclonal sera; screening of cDNA or genomic expression libraries; peptide synthesis; identification of an appropriate vaccination schedule involving adjuvants or new recombinant DNA-based antigen delivery systems. Outlined below are five of the major vaccine programmes.     

Antigenic differences among Leishmania amazonensis isolates and their relationship with distinct clinical forms of the disease.

Immunoblot analysis was used to investigate antigenic differences among clinical isolates of Leishmania amazonensis and their role in the etiology of the disease. Western blots of promastigote homogenates were analyzed with either monoclonal antibodies (MAbs) specific for the L. mexicana complex (M-4, M-6, M-9, and M-11) or polyclonal sera from L. amazonensis infected patients with the various forms of clinical disease. In the case of the MAbs, no significant variation was observed among the strains of L. amazonensis, isolated from cases of cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), visceral leishmaniasis (VL) or post kala-azar dermal leishmaniasis (PKDL), in either the relative mobility (Mr) or the quantitative amount (intensity) of the antigenic determinants. In the case of the sera of the infected patients, the patterns of antigenic reactivity of these strains revealed that, despite showing the presence of shared antigens, differences were observed between some of the antigenic components of the various isolates of L. amazonensis that were recognized by a single serum. Differences were also demonstrated between the antigenic determinants of a single isolate of L. amazonensis that were recognized by the different patients' sera. No apparent association was consistently found, however, between the Mr components identified in these isolates and the clinical form of the disease or the geographical area of isolation. In addition, the spectrum of antigens recognized by the sera from patients with the same clinical form were not identical; although in some instances, similar Mr antigens were shared.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of antibodies directed against O-acetylated sialic acids in visceral leishmaniasis: its diagnostic and prognostic role

A significantly increased O-acetylated sialic acid (O-AcSA) binding fraction was purified from serum of visceral leishmaniasis (VL) patients by affinity chromatography on immobilized bovine submaxillary mucin (BSM) and found to be immunoglobulin in origin. The serodiagnostic and prognostic potential of BSM as a capture antigen was established by ELISA with no cross reactivity with coendemic diseases like malaria, tuberculosis, leprosy, chagas disease and cutaneous leishmaniasis; however, a strong cross reactivity was present with trypanosomiasis patients. In 56 clinically diagnosed VL patients, the BSM-ELISA was compared with diagnosis by microscopy using Giemsa stained tissue smears and direct ELISA using crude parasite antigen (parasite-ELISA); 49/56(87.5%) and 5/56(9.0%) were positive and negative respectively by all 3 methods. The BSM-ELISA failed to diagnose 2/56(3.5%) patients which were biopsy and parasite-ELISA positive. The prognostic potential of the BSM-ELISA in 18 longitudinally monitored VL patients before and after conventional antimonial treatment showed a significant decrease in anti O-AcSA titres in drug responsive patients whereas anti O-AcSA levels persisted in drug unresponsive patients. The IgG subclass distribution of antibodies directed against O-AcSA showed increased IgG2 levels in VL patients as compared to healthy controls. The BSM-based ELISA holds great promise as a serodiagnostic and prognostic assay for VL.     

Characterization of the immune response to Leishmania infantum recombinant antigens

Leishmaniases have a high prevalence in tropical countries. In order to improve existing diagnostic systems based on total Leishmania proteins, and to identify antigen candidates for vaccine development, an intensive search for the identification of antigens was performed using molecular biology techniques. In this study, the immune response to three L. infantum recombinant antigens was evaluated. Upon stimulation with KMP11, mononuclear cells from leishmaniasis patients produced high levels of IL-10, while a predominant IFN-gamma production could be observed in cultures stimulated with H2A and soluble Leishmania antigen. All the recombinant antigens induced very little IL-5. KMP11 decreased IFN-gamma production by 48% in cultures of peripheral blood mononuclear cells from cutaneous leishmaniasis patients who had been stimulated with soluble Leishmania antigen. Furthermore, antibodies to KMP11 were detected in the sera from all patients with visceral leishmaniasis and in the majority of the sera from patients with cutaneous leishmaniasis or individuals with asymptomatic L. chagasi infection. Thus, KMP11 is recognized by cells and sera of patients with different clinical forms of leishmaniasis, and KMP11, through IL-10 production, proved to be a potent antigen in modulating type 1 immune response.     

Immunoenzyme assay using micro Dot on nitrocellulose (Dot-ELISA) in the diagnosis of Chagas' disease. I. Comparative study of 2 antigenic preparations of Trypanosoma cruzi

Using the Dot-ELISA technique, two antigenic preparations of Trypanosoma cruzi epimastigote forms have been compared for the diagnosis of Chagas' disease: (1) The cytoplasmic fraction (cytoplasmic antigen) and (2) whole formalin fixed epimastigotes (integral antigen). There was been used sera from 95 chagasic patients with chronic cardiomyopathy, positive conventional serology and either positive or negative xenodiagnosis; 74 subjects with negative conventional serology, and either clinically normal or presenting cardiomyopathy; 74 patients with different diseases including syphilis, toxoplasmosis, leishmaniasis or autoantibodies such as rheumatoid factor and antinuclear antibodies. By defining the diagnostic titers (cut off): 1:512 for cytoplasmic antigen and 1:128 for the integral antigen, a sensitivity of 100% has been obtained with both antigenic preparations, being the specificity of 96% for the former and 100% for the latter when leishmaniasis sera were not included. A comparative study with conventional serology was carried out using 147 sera from a Laboratory of Chagas' diagnosis; Dot-ELISA with cytoplasmic antigen showed co-positivity index of 1.0, co-negativity 0.989 and efficiency of 0.993, and Dot-ELISA with integral antigen 1.0, 0.979 and 0.986 respectively. According to this evaluation, Dot-ELISA using whole formalin fixed epimastigotes might be a practical alternative for the serological diagnosis of Chagas' disease.     

Serological differentiation of acute and chronic schistosomiasis using Schistosoma mansoni recombinant protein RP26

We obtained a recombinant protein encoded by Schistosoma mansoni gene which was able to differentiate acute from chronic schistosomiasis when applied as antigen in enzyme-linked immunosorbent assay (ELISA). A cDNA clone encoding a 26 kDa recombinant protein (RP26) was selected by screening of an adult worm S. mansoni λZAP expression library with rabbit sera produced against PIII, an adult worm protein fraction already known to possess protective and immunomodulating effects. The clone cDNA presented 99% identity with S. mansoni Sm22.3 gene. We assayed IgG reactivity of sera from 18 patients with acute, 25 patients with chronic S. mansoni infection and 20 uninfected donors with RP26 in ELISA. Our results showed that 89% of sera were positive in acute schistosomiasis group, and only 26% in chronic group, without false-positive reactions in uninfected group. In mice the immune response to RP26 increased up to week 9 after infection and then diminished. We proposed that production of antibodies binding to RP26 stopped at the chronic stage of disease. The testing of sera from eight other parasitic infections with RP26 revealed no positive reactions in majority of sera. However, we observed low positive reaction in sera from 20% of leishmaniasis patients. Our results indicate that a recombinant protein RP26 can be used as immunodiagnostic reagent for detection of acute phase of schistosomiasis mansoni.     

Production and Evaluation of Toxoplasma gondii Recombinant GRA7 for Serodiagnosis of Human Infections

The precise diagnosis of the acute toxoplasmosis in pregnant women and immunocompromsied patients has critical importance. Most of the commercially available assays use the whole Toxoplasma soluble extract as the antigen. However, the assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the lack of a purified standardized antigen. The aim of this study was production and evaluation of the usefulness of the recombinant Toxoplasma gondii GRA7 antigen for the serodiagnosis of Toxoplasma gondii IgM and IgG by ELISA. A total of 70 T. gondii IgM positive sera, 74 T. gondii IgG positive sera, and 60 sera from subjects who were not infected with T. gondii were examined. These sera were shown different absorbance values in ELISA test. To control the specificity of the rGRA7 other parasitic diseases, for example, echinococcosis, malaria, leishmaniasis, fascioliasis, and strongyloidiasis were tested of which none showed positive results. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (com ELISA) were 89% and 90%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 96% and 90%, respectively. The results obtained here show that this antigen is useful for diagnostic purposes.     

Indirect immunofluorescence test in New World leishmaniasis: serological and clinical relationship

The indirect immunofluorescence test (IF) for anti-Leishmania antibodies (IgG and IgM) was performed with sera from the following groups of individuals: 214 cutaneous leishmaniasis patients, 28 healthy subjects with positive Montenegro's skin test (MST), 29 healthy subjects with negative MST and 16 visceral leishmaniasis patients. The first four groups came from a suburban area of Rio de Janeiro (Jacarepaguá) where cutaneous leishmaniasis caused by Leishmania braziliensis braziliensis is endemic. It was observed that IF-IgM titers were significantly higher amongst the cutaneous leishmaniasis patients with less than four months of disease as compared to those with longer periods and that IF-IgG titers were significantly higher in patients with multiple lesions as compared to those with single lesions. The visceral leishmaniasis patients had IF-IgG titers significantly higher than those from cutaneous leishmaniasis patients. A group of 28 individuals selected amongst the 214 cutaneous leishmaniasis patients had their IF-titers (IgG and IgM) compared to those of the two control groups of healthy subjects from the endemic area, respectively with positive and negative MST. Significantly higher titers of IF-IgG and IF-IgM were found in the group with active disease. The same group of patients showed IF-IgG titers significantly lower at the end of the antimonial therapy than those observed during this treatment.     

Lipid and lipopolysaccharide-like antigens of Leishmania promastigotes

Extraction of whole promastigotes of Leishmania tropica major and L. donovani with a mixture of hexane and isopropanol (3:2) yielded three fractions containing immunological activity: lipids, where the activity was determined by radioimmunoassay; a lipopolysaccharide-like (LPS-like), water-soluble precipitate, where activity was determined both by radioimmunoassay and double gel diffusion, and the phenol: water extract of the lipid-free promastigotes, where activity was followed by double gel diffusion. The use of a solid state, lipid-based radioimmunoassay for detection of leishmanial antigens provided a sensitive measure of their activity with a considerable degree of species and serotype specificity. We found antibodies to leishmanial lipids in sera from immunized rabbits, convalescent mice, and human patients with confirmed cases of cutaneous leishmaniasis or kala azar. There was very little activity in normal human or animal sera. Analysis by SDS-polyacrylamide gel electrophoresis of fractions from promastigotes surface-labeled with galactose oxidase and sodium borotritiate and preliminary immunochemical characterization of the LPS-like antigen showed that it contained galactose, but otherwise differed immunologically and chemically from excreted factor (EF), the best characterized leishmanial antigen.     

Value of a dipstick based on recombinant RK39 antigen for differential diagnosis of American visceral leishmaniasis from other sympatric endemic diseases in Venezuela.

A laboratory trial using recombinant rK39 dipsticks for differential diagnosis of American visceral leishmaniasis (AVL) from other sympatric endemic diseases which share similar clinic features (Chagas disease, malaria, schistosomiasis and toxoplasmosis) was conducted in Venezuela. The 100% specificity of the test previously obtained in other countries was confirmed. The use of this test at the primary health care level in Venezuela for a rapid diagnosis of active AVL cases, which may avoid deaths, is recommended.     

Production and standardization of Paracoccidioides brasiliensis, Histoplasma capsulatum and Aspergillus fumigatus antigens to be used in immunodiagnosis. Comparison between immunodiffusion and immunoelectrophoresis

Soluble antigens (Ag) from Paracoccidioides brasiliensis, Histoplasma capsulatum and Aspergillus fumigatus were prepared and standardized by double immunodiffusion (DID) and immunoelectroosmophoresis (IEOP). No difference in sensitivity was observed between the two techniques; 100% of standard patient sera were positive with P. brasiliensis and A. fumigatus Ag and 83.3% were positive with H. capsulatum Ag. The specificity of the tests was verified testing 96 sera from patients with paracoccidioidomycosis, histoplasmosis, systemic candidiasis, sporotrichosis, tuberculosis, lung cancer, visceral or cutaneous leishmaniasis and 18 sera from healthy individuals. All the three antigens were 100% specific with the DID (using the identification pattern indicated by the confluence of test serum with standard serum precipitin lines as a positive criterium). However in the IEOP, the specificity varied with each Ag. Positive reactions with P. brasiliensis Ag were observed in 16.7% of histoplasmosis sera and in 10% of cutaneous leishmaniasis sera. On the other hand 31.8% of paracoccidioidomycosis and 10% of cutaneous leishmaniasis sera reacted with H. capsulatum Ag. The high sensitivity and specificity of the DID test, its easy reproducibility and low cost, led us to consider it highly appropriate as a routine procedure for the screening of patients with respiratory infections.     

Humoral immune responses among mucosal and cutaneous leishmaniasis patients caused by Leishmania braziliensis

Mucosal leishmaniasis is arguably the most morbid sequelae of cutaneous leishmaniasis. The importance of early diagnosis for effective therapy, coupled with the difficulty of diagnosing the disease parasitologically, prompted this investigation of humoral immune markers of mucosal disease. Promastigote soluble antigens of Leishmania braziliensis, isolated from cutaneous and mucosal lesions, were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis; antigens were identified by immunoblotting with parasite-specific IgG antibody-positive sera of patients with mucosal disease (n = 18) and cutaneous disease (n = 23). For antigens of the cutaneous parasite WR 2095, mucosal sera generally reacted intensely to antigens of 75, 66, and 45 kDa and weakly to 48-50-kDa antigens, whereas cutaneous sera generally detected weakly the first 3 antigens and intensely the latter doublet. The data suggest that the transition from the cutaneous antigenic profile to a mucosal antigenic profile could be used to predict mucosal disease in approximately half of mucosal patients. An additional finding was that antibodies present in the sera of patients with mucosal disease labeled a 66-kDa peptide of normal human lip mucosa more intensely than did cutaneous sera. Autoimmune processes stimulated by the reaction of IgG, originally directed against the 66-kDa of L. braziliensis, to the 66-kDa antigen of mucosal tissue may contribute to the clinical presentation of mucosal leishmaniasis.     

Antigenic heterogeneity in patients with reactions in borderline leprosy.

Fifteen patients with borderline leprosy who developed "reversal" reactions were studied from the inception of treatment. Thirteen showed an appreciable increase in lymphocyte transformation (LT) when preparations of Mycobacterium leprae were used as antigen. The LT responses to either "whole" or "sonicated" preparations of the bacillus in these 15 patients and in nine others also in reaction correlated with the clinical presentation. Those with skin disease predominating in the reaction showed an appreciable increase in LT when whole M leprae was used as antigen. Those with nerve disease predominating showed an increase with sonicated M leprae. In those with both skin and nerve disease there was an increase with both antigen preparations. The ratios of the LT test results (whole to sonicated M leprae) showed highly significant differences between the three groups.     

Specificity of the rapid rK39 antigen-based immunochromatographic test Kalazar Detect(r) in patients with cutaneous leishmaniasis in Brazil

The aim of this study was to evaluate the specificity of a rapid immunochromatographic test that was developed to detect antibodies against the rK39 antigen for the diagnosis of visceral leishmaniasis (VL). This evaluation was performed using sera from patients with a confirmed diagnosis of active cutaneous leishmaniasis. The sera from 272 patients with a confirmed diagnosis of localised cutaneous leishmaniasis (CL) who resided in an area endemic for Leishmania braziliensis in Brazil were obtained before the initiation of antileishmanial treatment. Kalazar Detect^(r)(InBios, Seattle, WA) recombinant K39 antigen-based immunochromatographic strips were used according to the manufacturer''s instructions. The test results were evaluated independently by two examiners in sequential order. The positive controls for the test included five serum samples from five patients with parasitologically confirmed diagnosis of VL caused by Leishmania infantum in Brazil. Overall, 100% of the samples obtained from patients with CL were negative, confirming the absence of a serological cross-reaction for individuals with cutaneous disease when these patients were evaluated using the rapid test. The lack of a cross-reaction in patients who were infected by parasites of the same genus highlights the specificity of the rK39 antigen for the diagnosis of VL in areas with the sympatric circulation of L. braziliensis and L. infantum.     

Cell mediated immunity in American cutaneous and mucosal leishmaniasis

Cellular immune responses were studied in 35 Brazilian patients with either active cutaneous leishmaniasis (CL), active mucosal leishmaniasis (ML), or healed cutaneous leishmaniasis. The mean age and duration of illness in the two groups were as follows: 14 CL patients, age 28 +/- 13 yr, disease 5 +/- mo; and 16 ML patients, age 34 +/- 15 yr, disease 86 +/- 117 mo. Patients with CL and ML responded well to leishmania antigen in blastogenesis assays. However, the response of ML patients was over three times greater than the response of CL patients. There was a significant correlation between the magnitude of the lymphoproliferative response and the duration of disease activity. There were no significant differences between CL and ML patients in terms of the following parameters: lymphoproliferative responsiveness to mitogens (phytohemagglutinin, concanavalin A, and pokeweed mitogen) and peripheral blood lymphocyte subpopulations (T and B cells, oKT8+ and OKT4+ cells, OKT4:OKT8 ratio). Peripheral blood mononuclear cells from ML patients also generated interferon-gamma containing lymphokine in response to stimulation with leishmania antigen. This lymphokine was capable of inducing macrophages from ML patients to inhibit the intracellular multiplication of leishmania in vitro. These studies have determined that the parameters of lymphocyte and macrophage functions evaluated in ML and CL patients are comparable, except for an enhanced lymphoproliferative response, with leishmania antigen in ML patients. This later finding may be a function of the long duration of active disease in this population and unrelated to the pathogenesis of their mucosal lesions.     

Evaluation of antibody responses in American visceral leishmaniasis by ELISA and immunoblot

American visceral leishmaniasis (AVL) is an important disease among children of northeast Brazil. In order to characterize antibody responses during AVL, sera of hospitalized patients were analyzed by ELISA and Western blot using a Leishmania chagasi antigen preparation. The ELISA was positive (absorbance greater than or equal to 0.196) at a serum dilution of 1:1024 in all patients at presentation, and fell to ward control levels over the following year. Only one of 72 control subjects tested positive, and that donor had a sibling with AVL. Immunoblots of the patients' sera recognized multiple bands, the most frequent of which were at approximately 116 kDa, 70 kDa, and 26 kDa. Less frequently observed were bands at approximately 93 kDa, 74 kDa, 62 kDa, 46 kDa and 32 kDa. The ELISA responses and patterns of banding were distinctive for AVL, and could be used to differentiate patients with AVL from those with Chagas' disease or cutaneous leishmaniasis. Sera from six AVL patients followed for up to six weeks after treatment identified no new bands. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of surface iodinated parasite proteins showed one major band and four minor bands, whereas SDS-PAGE of biotinylated parasite proteins revealed a banding pattern similar to those of patient sera. AVL appears to produce characteristic immunoblot patterns which can be used along with a sensitive screening ELISA to diagnose AVL.     

Serum neopterin concentrations during treatment of leishmaniasis: useful as test of cure?

Neopterin, a product of gamma-interferon-activated macrophages, was measured in sera from 28 patients (12 patients with cutaneous leishmaniasis and 16 patients with visceral leishmaniasis) to determine the utility as a marker of disease activity and therapeutic efficacy. Patients originated from Kenya (n=5) and from the Academic Medical Center, Amsterdam, The Netherlands (n=23). In seven patients follow-up sera after treatment were available. Two patients at the time of diagnosis of visceral leishmaniasis were co-infected with HIV. The 12 patients with cutaneous leishmaniasis had serum neopterin levels below the upper limit of the normal range. All 16 patients with visceral leishmaniasis had elevated levels of serum neopterin before treatment. In six out of seven patients with visceral leishmaniasis followed during treatment neopterin levels decreased to values below the upper limit of the normal range (10 nmol l(-1)). Sequential measurements of serum neopterin levels may be useful for monitoring therapeutic efficacy in patients with visceral leishmaniasis.     

Identification and characterisation of a Leishmania donovani antigen belonging to the 70-kDa heat-shock protein family

A Leishmania donovani promastigote cDNA library was screened with serum obtained from a patient infected with visceral leishmaniasis. Sequence analysis of a clone obtained from this library revealed that the 600-bp insert corresponded to the carboxy-terminal region of an antigen related to the 70-kDa heat-shock protein family. The full-length sequence of the corresponding gene (1959 nucleotides) was determined after isolation of genomic clones. Genes encoding the antigen are present on a single chromosome as a series of approximately twelve 3.7-kb direct tandem repeats. The antigen can be identified as a 70-kDa heat-shock cognate protein by virtue of its molecular mass, sequence and constitutive expression during heat shock. It is expressed at all stages of the parasite life-cycle. Antibodies against the lambda gt11 fusion protein were detected in more than 50% of serum samples obtained from patients with visceral leishmaniasis, but were not detected in sera from patients with cutaneous leishmaniasis or Chagas' disease.     

The serodiagnosis of human hydatid disease: 2. Additional studies on selected sera using indirect haemagglutination (IHA), enzyme linked immunosorbent assay (ELISA) and defined antigen substrate spheres (DASS).

Sixty-one serum samples selected on the basis of reactivity in the complement fixation (CF) and latex agglutination (LA) test, were further examined for sensitivity and specificity by indirect haemagglutination (IHA), enzyme linked immunosorbent assay (ELISA) and defined antigen substrate spheres (DASS). Twenty sera from healthy Europeans and 48 samples from patients with either schistosomiasis or trichinosis were also tested. Comparable levels of sensitivity were found between the CF and LA positive sera and IHA, ELISA and DASS. Of the CF positive LA negative group of sera, many were positive by DASS but only a few reacted in IHA and ELISA. Some cross reactivity was also observed in the schistosomiasis sera tested by IHA and ELISA.     

IgG anti-IgE autoantibodies in visceral leishmaniasis

Procedures for IgG depletion in visceral leishmaniasis (VL) and schistosomiasis sera using Sepharose-protein G beads also deplete IgE. In this study, the presence of IgG anti-IgE autoantibodies in sera from patients with VL (n = 10), and hepatic-intestinal schistosomiasis (n = 10) and from healthy individuals (n = 10) was investigated. A sandwich ELISA using goat IgG anti-human IgE to capture serum IgE and goat anti-human IgG peroxidase conjugate to demonstrate the binding of IgG to the IgE captured was performed. VL sera had higher titers (p < 0.05) of IgG anti-IgE autoantibodies (OD = 2.01 +/- 0.43) than sera from healthy individuals (OD = 1.35 +/- 0.16) or persons infected with Schistosoma mansoni (OD = 1.34 +/- 0.18). The immunoblotting carried out with eluates from Sepharose-protein G beads used to deplete IgG from these sera and goat anti-human IgE peroxidase conjugate, showed a similar pattern of bands, predominating the 75 kDa epsilon-heavy chain and also polypeptides resulting from physiological enzymatic digestion of IgE. A frequent additional band immediately above 75 kDa was observed only in VL sera.