Glutathione S-transferases of 28kDa as major vaccine candidates against schistosomiasis

For the development of vaccine strategies to generate efficient protection against chronic infections such as parasitic diseases, and more precisely schistosomiasis, controlling pathology could be more relevant than controlling the infection itself. Such strategies, motivated by the need for a cost-effective complement to existing control measures, should focus on parasite molecules involved in fecundity, because in metazoan parasite infections pathology is usually linked to the output of viable eggs. In numerous animal models, vaccination with glutathione S-transferases of 28kDa has been shown to generate an immune response strongly limiting the worm fecundity, in addition to the reduction of the parasite burden. Recent data on acquired immunity directed to 28GST in infected human populations, and new development to draw adapted vaccine formulations, are presented.

Schistosomiasis vaccine development: progress and prospects

The undisputed, worldwide success of chemotherapy notwithstanding, schistosomiasis continues to defy control efforts in as much rapid reinfection demands repeated treatment, sometimes as often as once a year. There is thus a need for a complementary tool with effect for the longer term, notably a vaccine. International efforts in this direction have been ongoing for several decades but, until the recombinant DNA techniques were introduced, antigen production remained an unsurmountable bottleneck. Although animal experiments have been highly productive and are still much needed, they probably do not reflect the human situation adequately and real progress can not be expected until more is known about human immune responses to schistosome infection. It is well-known that irradiated cercariae consistently produce high levels of protection in experimental animals but, for various reasons, this proof of principle cannot be directly exploited. Research has instead been focussed on the identification and testing of specific schistosome antigens. This work has been quite successful and is already at the stage where clinical trials are called for. Preliminary results from coordinated in vitro laboratory and field epidemiological studies regarding the protective potential of several antigens support the initiation of such trials. A series of meetings, organized earlier this year in Cairo, Egypt, reviewed recent progress, selecteded suitable vaccine candidates and made firm recommendations for future action including pledging support for large-scale production according to good manufacturing practice (GMP) and Phase I trials. Scientists at the American Centers for Disease Control and Prevention (CDC) have drawn up a detailed research plan. The major financial support will come from USAID, Cairo, which has established a scientific advisory group of Egyptian scientists and representatives from current and previous international donors such as WHO, NIAID, the European Union and the Edna McConnell Clark Foundation.

Induction of protective immunity against Schistosoma mansoni infection by antigens purified from PIII, a fraction of adult worm, associated to the downregulation of granuloma formation

This study was performed in order to define Schistosoma mansoni antigens able to function as modulator agents in BALB/c mice granulomatous hypersensitivity to parasite egg. The antigens P-24, P-35 and P-97 were purified by affinity chromatography from a fraction of S. mansoni adult worm antigenic preparation, denominated PIII, involved in the inhibition of granulomatous response to eggs. Immunization of mice with these antigens, in the presence of Corynebacterium parvum and Al(OH)3 as adjuvant, induced a significant protection degree against challenge infection, as observed by the decrease on worm burden recovered from portal system. In vitro blastogenesis assays revealed that purified antigens were able to induce significant proliferation of spleen cells from S. mansoni-infected mice. This protection was correlated to significant decrease in granuloma size induced by PIII. From these results, we concluded that PIII preparation contains antigens capable of mediating protective anti-parasite immunity and down-regulating granulomatous hypersensitivity to S. mansoni eggs.

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

Antibodies anti-Chlamydia pneumoniae and anti-Mycoplasma pneumoniae in patients with negative serology for hantavirus. Retrospective study

The seroprevalence of Chlamydia pneumoniae and Mycoplasma pneumoniae in hantavirus seronegative patients, who had symptoms and signs compatible with pneumonia was established. For this purpose we used the indirect fluorescent antibody test. Titers ³ 1:16 for C. pneumoniae and M. pneumoniae were found in 8.6% and 17.1% of the serum, respectively, showing evidence of recent or current infection.

Intradermal vaccination of adults with three low doses (2 µg) of recombinant hepatitis B vaccine. II. Persistence of immunity and induction of immunologic memory

Of the 110 dentists who had presented seroconversion 50 days after the intradermal application of three 2 µg doses of the Belgian recombinant vaccine against hepatitis B (HB), administered eight years before at an interval of one month between the 1st and 2nd doses and of five months between the 2nd and 3rd doses, 51 were included for the assessment of the persistence of immunity. None of the dentists had hepatitis or had received HB vaccine during this period. All subjects were submitted to serological tests for the detection of the following markers of hepatitis B virus (HBV) infection: HBsAg, anti-HBc, HBeAg, anti-HBe, and anti-HBs, with no HBsAg, anti-HBc, HBeAg or anti-HBe being detected. A microparticle enzyme immunoassay (MEIA) revealed the presence of anti-HBs at protective titers (> 10 mIU/ml) in 42 dentists (82.4%), with the anti-HBs titer being higher than 100 mIU/ml in 36 of them (70.6%) (good responders), between 10 and 100 mIU/ml in 6 (11.8%) (poor responders), and lower than 10 mIU/ml in 9 (17.6%) (non-responders). According to clinical data and serological tests, none of the dentists had presented disease or latent HBV infection during the eight years following the first vaccination. A 2 µg booster dose was administered intradermally to eight dentists with anti-HBs titers lower than 10 mIU/ml (non-responders) and to six dentists with titers ranging from 10 to 100 mIU/ml (poor responders); the determination of anti-HBs one month later demonstrated the occurrence of seroconversion in the eight non-responders and an increase in anti-HBs titer in the six poor responders. In summary, the present results demonstrated the prolonged persistence of protection against HBV infection and the development of immunologic memory provided by vaccination against HB - with intra-dermal application of three 2 µg doses of the Belgian recombinant vaccine at 0, 1, and 6 months - carried out eight years before in 51 dentists.

Infection of Calomys callosus (Rodentia Cricetidae) with strains of different Trypanosoma cruzi biodemes: pathogenicity, histotropism, and fibrosis induction

The influence of different Trypanosoma cruzi biodemes on the evolution of the infection and on the histopathological lesions of the heart and skeletal muscles, during the experimental infection of Calomys callosus, was investigated. Three groups of C. callosus were infected, respectively, with parasite strains representative of three different Biodemes: Type I (Y strain), Type II (21 SF strain), and Type III (Colombian strain). For each group, normal C. callosus were also used as controls. Marked differences have been detected in the responses of C. callosus to the infection with the three strains in this model. The strains Types I and II (Y and 21 SF) determined moderate lesions, mostly in the myocardium, with low parasitism, a rapid course, and total regression of the lesions by the 60th day of infection. Differently, Type III strain (Colombian), was more pathogenic for C. callosus and induced necrotic-inflammatory lesions in skeletal muscles and myocardium, in correspondence to intracellular parasitism. Proliferation of fibroblasts and amorphous matrix deposits, followed by interstitial fibrosis were present. Progressive regression of the inflammatory changes and collagen deposits occurred spontaneously. The progression and regression of both inflammation and fibrosis induced by the Colombian strain were further submitted to quantitative evaluation by morphometry. Results of the morphometric studies presented good correlation with the histopathological findings. The results confirm the importance of the different biodemes in the determination of tissue lesions and the peculiarities of response of C. callosus to infection with T. cruzi.

Vaccination with antioxidant enzymes confers protective immunity against challenge infection with Schistosoma mansoni

Schistosoma mansoni, an intravascular parasite, lives in a hostile environment in close contact with host humoral and cellular cytotoxic factors. To establish itself in the host, the parasite has evolved a number of immune evasion mechanisms, such as antioxidant enzymes. Our laboratory has demonstrated that the expression of antioxidant enzymes is developmentally regulated, with the highest levels present in the adult worm, the stage least susceptible to immune elimination, and the lowest levels in the larval stages, the most susceptible to immune elimination. Vaccination of mice with naked DNA constructs containing Cu/Zn cytosolic superoxide dismutase (CT-SOD), signal-peptide containing SOD or glutathione peroxidase (GPX) showed significant levels of protection compared to a control group. We have further shown that vaccination with SmCT-SOD but not SmGPX results in elimination of adult worms. Anti-oxidant enzyme vaccine candidates offer an advance over existing vaccine strategies that all seem to target the larval developmental stages in that they target adult worms and thus may have therapeutic as well as prophylactic value. To eliminate the potential for cross-reactivity of SmCT-SOD with human superoxide dismutase, we identified parasite-specific epitope-containing peptides. Our results serve as a basis for developing a subunit vaccine against schistosomiasis.

Identification of immunodominant epitopes of Schistosoma mansoni vaccine candidate antigens using human T cells

Paramyosin and Sm14 are two of the six antigens selected by the World Health Organization as candidates to compose a subunit vaccine against schistosomiasis. Both antigens are recognized by individuals naturally resistant to Schistosoma mansoni infection and induced protective immunity in the murine model. Three Sm14 epitopes and eleven paramyosin epitopes were selected by their ability to bind to different HLA-DR molecules using the TEPITOPE computer program, and these peptides were synthetically produced. The cellular recognition of Sm14 and paramyosin epitopes by peripheral blood mononuclear cells of individuals living in endemic area for schistosomiasis was tested by T cell proliferation assay. Among all Sm14 and paramyosin epitopes studied, Sm14-3 was preferentially recognized by individuals naturally resistant to S. mansoni infection while Para-5 was preferentially recognized by individuals resistant to reinfection. These two peptides represent promising antigens to be used in an experimental vaccine against schistosomiasis, since their preferential recognition by resistant individuals suggest their involvement in the induction of protective immunity.

Seroepidemiology of hepatitis B virus infection and high rate of response to hepatitis B virus Butang®vaccine in adolescents from low income families in Central Brazil

In order to evaluate the seroepidemiology and response to Butang® vaccine in adolescents from low income families in Central Brazil, blood samples of 664 adolescents were tested for hepatitis B surface antigen (HBsAg), hepatitis B core antibody (anti-HBc), and hepatitis B surface antibody (anti-HBs) markers, and multiple logistical regression analysis was carried out to determine variables associated with hepatitis B virus (HBV) infection markers. further, three 20 µg butang® vaccine doses were offered to all susceptible individuals (n = 304). Among those who accepted them (n = 182), the seroresponse was evaluated in 170 individuals by quantitative anti-HBs. an overall hbv prevalence of 5.9% was found: four adolescents were HBsAg positive, 24 were anti-HBc, anti-HBs-reactive, and 11 were anti-HBc only. The analyse of risk factors showed that age 16-19 years, place of birth outside Goiás, school B and body piercing were statistically associated with HBV infection markers (p < 0.05). All 170 adolescents responded to butang®, and a geometric mean titer (gmt) of 4344 mui/ml was obtained. these results reinforce the importance of hepatitis b vaccine in adolescents despite of the hbv regional endemicity, and suggest that three doses of 20 µg of the butang® should guarantee protective anti-hbs levels to individuals at a critical time for hepatitis b acquiring such as latter adolescence and adulthood.

Seropositivity for hepatitis B virus, vaccination coverage, and vaccine response in dentists from Campo Grande, Mato Grosso do Sul, Brazil

This study investigated the seropositivity for hepatitis B virus (HBV), the vaccination index, and the vaccine response index in dentists from Campo Grande, MS. Blood samples from 474 dentists (63.7% women and 36.3% men), with a mean age of 38.5 ± 10.5 years were analyzed by enzyme-linked immunosorbent assay to detect the serological markers: HBsAg, anti-HBs, and anti-HBc. The HBsAg positive samples were tested for anti-HBc IgM, HBeAg, and anti-HBe. A total of 51 (10.8%) dentists showed seropositivity for HBV. Three (0.6%) were HBsAg/anti-HBc/anti-HBe positive, 43 (9.1%) were anti-HBc/anti-HBs positive, and 5 (1.1%) had only anti-HBc. Viral DNA was detected by polymerase chain reaction in 9 (17.6%) out of 51 HBV seropositive samples. A vaccination index of 96.6% (458/474) was observed, although 73.1% (335/458)completed the three-dose schedule. Excluding 46 HBV seropositive individuals from 458 that reported vaccination, 412 were analyzed for vaccine response index. It was observed that 74.5% (307/412) were anti-HBs positive; this percentage increased to 79.1% when three doses were administered. The results showed a high vaccination index and a good rate of vaccine response; however, the failure in completing the three-dose schedule and the occurrence of HBV infection reinforce the need for more effective prevention strategies.

Schistosoma mansoni major egg antigen Smp40: molecular modeling and potential immunoreactivity for anti-pathology vaccine development

The pathogenesis of Schistosoma mansoni infection is largely determined by host T-cell mediated immune responses such as the granulomatous response to tissue deposited eggs and subsequent fibrosis. The major egg antigens have a valuable role in desensitizing the CD4+ Th cells that mediate granuloma formation, which may prevent or ameliorate clinical signs of schistosomiasis.S. mansoni major egg antigen Smp40 was expressed and completely purified. It was found that the expressed Smp40 reacts specifically with anti-Smp40 monoclonal antibody in Western blotting. Three-dimensional structure was elucidated based on the similarity of Smp40 with the small heat shock protein coded in the protein database as 1SHS as a template in the molecular modeling. It was figured out that the C-terminal of the Smp40 protein (residues 130 onward) contains two alpha crystallin domains. The fold consists of eight beta strands sandwiched in two sheets forming Greek key. The purified Smp40 was used for in vitro stimulation of peripheral blood mononuclear cells from patients infected with S. mansoni using phytohemagglutinin mitogen as a positive control. The obtained results showed that there is no statistical difference in interferon-g, interleukin (IL)-4 and IL-13 levels obtained with Smp40 stimulation compared with the control group (P > 0.05 for each). On the other hand, there were significant differences after Smp40 stimulation in IL-5 (P = 0.006) and IL-10 levels (P < 0.001) compared with the control group. Gaining the knowledge by reviewing the literature, it was found that the overall pattern of cytokine profile obtained with Smp40 stimulation is reported to be associated with reduced collagen deposition, decreased fibrosis, and granuloma formation inhibition. This may reflect its future prospect as a leading anti-pathology schistosomal vaccine candidate.

Morphological studies in a model for dengue-2 virus infection in mice

One of the main difficulties in studying dengue virus infection in humans and in developing a vaccine is the absence of a suitable animal model which develops the full spectrum of dengue fever, dengue haemorrhagic fever, and dengue shock syndrome. It is our proposal to present morphological aspects of an animal model which shows many similarities with the dengue infection in humans. BALB/c mice were intraperitoneally infected with non-neuroadapted dengue virus serotype 2 (DENV-2). Histopathological and morphometrical analyses of liver tissue revealed focal alterations along the infection, reaching wide-ranging portal and centrolobular veins congestion and sinusoidal cell death. Additional ultrastructural observations demonstrated multifocal endothelial injury, platelet recruitment, and alterated hepatocytes. Dengue virus antigen was detected in hepatocytes and in the capillar endothelium of the central lobular vein area. Liver function tests showed high levels of aspartate transaminase and alanine transaminase enzyme activity. Lung tissue showed interstitial pneumonia and mononuclear cells, interseptal oedema, hyperplasia, and hypertrophy of the bronchiolar epithelial cells. DENV-2 led to a transient inflammatory process, but caused focal alterations of the blood-exchange barrier. Viremia was observed from 2nd to 11th day p.i. by isolation of DENV-2 in C6/36 mosquito cell line inoculated with the supernatant of macerated liver, lung, kidney, and cerebellum tissues of the infected mice.

Leishmania (Viannia) lainsoni: occurrence of intracellular promastigote forms in vivo and in vitro

Experimental chronic (45-day-old) skin lesion in hamster hind foot induced by Leishmania (Viannia) lainsoni infection showed the presence of promastigote forms in the tissue, inside parasitophorous vacuoles, as assessed by transmission electron microscopy. Experimental in vitro interaction (24 and 48 h) between Leishmania (V.)lainsoni and J774-G8 macrophage cells also demonstrated the same profile. This morphological aspect is unusual, since in this parasite genus only amastigote forms have been described as the resistant and obligate intracellular forms.

Aluminum hydroxide associated to Schistosoma mansoni 22.6 kDa protein abrogates partial protection against experimental infection but not alter interleukin-10 production

The need to develop a vaccine against schistosomiasis led several researches and our group to investigate proteins from Schistosoma mansoni as vaccine candidates. Sm22.6 is a protein from S. mansoni that shows high identity with Sj22.6 and Sh22.6 (79 and 91%, respectively). These proteins are associated with high levels of IgE and protection to reinfection. Previously, we have shown that Sm22.6 induced a partial protection of 34.5% when used together with Freund's adjuvant and produced a Th0 type of immune response with interferon-g and interleukin-4. In this work, mice were immunized with Sm22.6 alone or with aluminum hydroxide adjuvant and high levels of IgG, IgG1, and IgG2a were measured. Unfortunately, no protection was detected. Since IL-10 is a modulating cytokine in schistosomiasis, we also observed a high level of this molecule in splenocytes of vaccinated mice. In conclusion, we did not observe the adjuvant effect of aluminum hydroxide associated with rSm22.6 in protective immunity.