Development of a vaccine strategy against human and bovine schistosomiasis: background and update

Schistosomiasis is a chronic and debilitating parasitic disease that affects over 200 million people throughout the world and causes about 500,000 deaths annually. Two specific characteristics of schistosome infection are of primordial importance to the development of a vaccine: schistosomes do not multiply within the tissues of their definitive hosts (unlike protozoan parasites) and a partial non-sterilizing immunity can have a marked effect on the incidence of pathology and on disease transmission. Since viable eggs are the cause of disease pathology, a reduction in worm fecundity whether or not accompanied by a reduction in parasite burden is a sufficient goal for vaccine induced immunity. We originally showed that IgE antibodies played in experimental models a pivotal role for the development of protective immunity. These laboratory findings have been now confirmed in human populations. Following the molecular cloning and expression of a protein 28 kDa protein of Schistosoma mansoni and its identification as a glutathion S-transferase, immunization experiments have been undertaken in several animal species (rats, mice, baboons). Together with a significant reduction in parasite burden, vaccination with Sm28 GST was recently shown to reduce significantly parasite fecundity and egg viability leading to a decrease in liver pathology. Whereas IgE antibodies were shown to be correlated with protection against infection, IgA antibodies have been identified as one of the factors affecting egg laying and viability. In human populations, a close association was found between IgA antibody production to Sm28 GST and the decrease of egg output. The use of appropriate monoclonal antibody probes has allowed the demonstration that the inhibition of parasite fecundity following immunization was related to the inhibition of enzymatic activity of the molecule. Epitope mapping of Sm28 GST has indicated the prominent role of the N and C terminal domains. Immunization with the corresponding synthetic peptides was followed by a decrease of 70% of parasite fecundity and egg viability. As a preliminary step towards phase I human trials, vaccination experiments have been performed in cattle, a natural model for Schistosoma bovis. Vaccination of calves with the S. bovis GST has led to a reduction of ever 80% of egg output and tissue egg count. Significant levels of protection were also observed in goats after immunization with the recombinant S. bovis GST. Increasing evidence of the participation of IgA antibodies in protective immunity has prompted us toward the development of mucosal immunization. Preliminary results indicate that significant levels of protection can be achieved following oral immunization with live attenuated vectors or liposomes. These studies seem to represent a promising approach towards the future development of a vaccine strategy against one of major human parasitic diseases.

Acute respiratory infections in children living in two low income communities of Rio de Janeiro, Brazil

Community studies of non-hospitalized children are essential to obtain a more thorough understanding of acute respiratory infections (ARI) and provide important information for public health authorities. This study identified a total ARI incidence rate (IR) of 4.5 per 100 child-weeks at risk and 0.78 for lower respiratory tract infections (LRI). Disease duration averaged less than one week and produced a total time ill with ARI of 5.8% and for LRI 1.2%. No clear seasonal variation was observed, the sex-specific IR showed a higher proportion of boys becoming ill with ARI and LRI and the peak age-specific IR occurred in infants of 6-11 months. Correlation with risk factors of the child (breastfeeding, vaccination, diarrheal disease, undernourishment) and the environment (crowding, living conditions, maternal age and education) showed marginal increases in the rate ratios, making it difficult to propose clear-cuts targets for action to lower the ARI and LRI morbidity. The importance of an integral maternal-child health care program and public education in the early recognition of LRI is discussed.

Tuberculin Skin Test and CD4+/CD8+ T Cell Counts in Adults Infected with the Human Immunodeficiency Virus in Medellín, Colombia

To evaluate the effect of BCG vaccination and T lymphocyte subpopulations on the reactivity to the tuberculin skin test, 113 asymtomatic HIV+ individuals were tuberculin tested by intradermal injection of 5TU of purified protein derivative and the levels of circulating lymphocyte (CD3, CD4 and CD8) subpopulations determined by indirect immunofluorescence. Ninety-two percent of the subjects included in the study were males. The mean age of the group was 32.1±7.4 years. Sixty-two percent presented a BCG scar. However, only 22% exhibited positive tuberculin reactions (³5mm) irrespective of the presence of the BCG scar. Tuberculin positive individuals exhibited higher CD4+ cell counts (p=0.004) and CD4+/CD8+ ratios (p=0.006) than tuberculin negative (<5mm) HIV+ individuals. The number of individuals with positive tuberculin reactions was significantly higher in subjects with more than 500 CD4+ lymphocytes/ml (p=0.02) or CD4+/CD8+ ratios ³1.12 (p=0.002). These results suggest that a prior BCG vaccination does not influence the reactivity to the tuberculin skin test in HIV+ asymptomatic individuals and that the number of CD4+ lymphocytes and the CD4+/CD8+ ratio positively correlate with the tuberculin reactivity

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.

A dhfr-ts- Leishmania major Knockout Mutant Cross-protects against Leishmania amazonensis

E10-5A3 is a dhfr-ts- Leishmania major double knockout auxotrophic shown previously to induce substantial protection against virulent L. major infection in both genetically susceptible and resistant mice. We investigated the capacity of dhfr-ts- to protect against heterologous infection by L. amazonensis. The degree of protection was evaluated by immunization of BALB/c or C57BL/6 mice with E10-5A3, followed by L. amazonensis challenge. Whether immunized by subcutaneous (SC) or intravenous (IV) inoculation, susceptible and resistant mice displayed a partial degree of protection against challenge with virulent L. amazonensis. SC-immunized BALB/c mice developed lesions 40 to 65% smaller than non immunized mice, while IV immunization led to protection ranging from 40 to 75% in four out of six experiments compared to non immunized animals. The resistant C57BL/6 mice displayed comparable degrees of protection, 57% by SC and 49% by IV immunization. Results are encouraging as it has been previously difficult to obtain protection by SC vaccination against Leishmania, the preferred route for human immunization.

Immune response in cattle vaccinated against rabies

In order to determine the best type of rabies vaccine to use as a booster, 78 serological samples from singly vaccinated cattle were analyzed by counterimmunoelectrophoresis technique. The animals were divided into several groups, received the first vaccine dose with modified live virus vaccine (ERA strain) and were revaccinated with inactivated virus or modified live virus vaccines. Boosters were given at 2, 4, 8, 12 and 16 weeks following first vaccination. Results showed high titres in the cases of booster with inactivated vaccine. In all cases, however, detectable antibody titres declined quickly.

Is there a role for autoimmunity in immune protection against malaria?

Much remains to be known about the mechanisms involved in protective immunity against malaria and the way it is acquired. This is probably the reason why, in spite of so much progress, it has not yet been possible to develop an anti-malaria vaccine able to induce parasite specific antibodies (Ab) and/or T-cells. It has been considered in the early 80s that the induction of efficient protection against the blood stage forms of Plasmodium falciparum would not be possible without simultaneously eliciting an autoimmune (AI) response against erythrocytes, even at the price of inducing an AI pathology. Despite the description of the reciprocal relationship, i.e. the protective effect of malaria on the development of AI diseases - demonstrated since 1970 - no effort has been made to verify the possible involvement of the AI response in protection against malaria. With this end in view - and in the light of the knowledge acquired in autoimmunity and the existence of the so called "natural" (not associated with pathology) autoantibodies - we propose to examine the hypothesis that the participation of the AI response (not necessarily restricted to autologous erythrocyte antigens) in the immune protection against malaria is possible or even necessary.

Protective CD8+ T cell responses against the pre-erythrocytic stages of malaria parasites: an overview

CD8+ T cells have been implicated as critical effector cells in protection against the pre-erythrocytic stage of malaria in mice and humans following irradiated sporozoite immunization. Immunization experiments in animal models by several investigators have suggested different strategies for vaccination against malaria and many of the targets from liver stage malaria antigens have been shown to be immunogenic and to protect mice from the sporozoite challenge. Several prime/boost protocols with replicating vectors, such as vaccinia/influenza, with non-replicating vectors, such as recombinant particles derived from yeast transposon (Ty-particles) and modified vaccinia virus Ankara, and DNA, significantly enhanced CD8+ T cell immunogenicity and also the protective efficacy against the circumsporosoite protein of Plasmodium berghei and P. yeti. Based on these experimental results the development of a CD8+ T cell inducing vaccine has moved forward from epitope identification to planning stages of safety and immunogenicity trials of candidate vaccines.

The early use of yellow fever virus strain 17D for vaccine production in Brazil - a review

The use of yellow fever (YF) virus 17D strain for vaccine production adapted in Brazil since its introduction in 1937 was reviewed. This was possible due to the availability of official records of vaccine production. The retrieved data highlight the simultaneous use of several serially passaged 17D substrain viruses for both inocula and vaccine preparation that allowed uninterrupted production. Substitution of these substrain viruses became possible with the experience gained during quality control and human vaccination. Post-vaccinal complications in humans and the failure of some viruses in quality control tests (neurovirulence for monkeys) indicated that variables needed to be reduced during vaccine production, leading to the development of the seed lot system. The 17DD substrain, still used today, was the most frequently used substrain and the most reliable in terms of safety and efficacy. For this reason, it is possible to derive an infectious cDNA clone of this substrain combined with production in cell culture that could be used to direct the expression of heterologous antigens and lead to the development of new live vaccines.

Autotransplant of spleen tissue in children with schistosomiasis: evaluation of splenic function after splenosis

Autotransplantation of spleen tissue has been done, in the past ten years, in children with schistosomiasis mansoni with bleeding varices. The purposes of this investigation were: (1) to study the morphology and function of the remnant spleen tissue; (2) to quantify the production of tuftsin; and (3) to assess the immune response to pneomococcal vaccine of these patients. Twenty three children, who underwent splenectomy and autologous implantation of spleen tissue into the greater omentum were included in this investigation. The average postoperative follow-up is five years. Splenosis was proved by colloid liver-spleen scans. Search for Howell-Jolly bodies assessed the filtration function. Tuftsin and the titer of pneumococcal antibodies were quantified by ELISA. Splenosis was evident in all children; however, it was insufficient in two. Howell-Jolly bodies were found only in these two patients. The mean tuftsin serum concentration (335.0 ± 29.8 ng/ml) was inside the normal range. The immune response to pneumococcal vaccination was adequate in 15 patients; intermediate in four; and inadequate in four. From the results the following conclusions can be drawn: splenosis was efficient in maintaining the filtration splenic function in more than 90% and produced tuftsin inside the range of normality. It also provided the immunologic splenic response to pneumococcal vaccination in 65% of the patients of this series.

r-Sm14 - pRSETA efficacy in experimental animals

Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega). The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system.

Additives and Protein-DNA Combinations Modulate the Humoral Immune Response Elicited by a Hepatitis C Virus Core-encoding Plasmid in Mice

Humoral and cellular immune responses are currently induced against hepatitis C virus (HCV) core following vaccination with core-encoding plasmids. However, the anti-core antibody response is frequently weak or transient. In this paper, we evaluated the effect of different additives and DNA-protein combinations on the anti-core antibody response. BALB/c mice were intramuscularly injected with an expression plasmid (pIDKCo), encoding a C-terminal truncated variant of the HCV core protein, alone or combined with CaCl2, PEG 6000, Freund's adjuvant, sonicated calf thymus DNA and a recombinant core protein (Co.120). Mixture of pIDKCo with PEG 6000 and Freund's adjuvant accelerated the development of the anti-core Ab response. Combination with PEG 6000 also induced a bias to IgG2a subclass predominance among anti-core antibodies. The kinetics, IgG2a/IgG1 ratio and epitope specificity of the anti-core antibody response elicited by Co.120 alone or combined with pIDKCo was different regarding that induced by the pIDKCo alone. Our data indicate that the antibody response induced following DNA immunization can be modified by formulation strategies.

Detection and Characterization of Rotavirus G and P Types from Children Participating in a Rotavirus Vaccine Trial in Belém, Brazil

This study sought the characterization of rotaviruses in a trial with a tetravalent rhesus-human rotavirus vaccine in Belém, Brazil in children who received three doses of vaccine or placebo in the 1st, 3rd and 5th months of life. Rotavirus electropherotypes, subgroups, G serotypes, G, [P] and [P],G genotypes were determined in 93.3%, 95.9%, 93.3%, 73.3%, 95.5% and 92.2% of isolates, respectively. Serotypes G1, G2 and G4 were detected in 58.9%, 30% and 4.4% of the cases, respectively. Rotavirus genotype G5 was detected for the first time in Northern region in 4.4% of the infections. Rotavirus genotypes P[8], P[4], P[6] and P[8+6] were detected in 54.5%, 26.7%, 12.2%, and 2.2% of the cases, respectively. The predominant genotypes were P[8],G1 and P[4],G2 with 53% and 26.6% of the infections, respectively. Unusual strains accounted for 20.5% including P[4],G1, P[6],G1, P[6],G4, P[6],G5, P[8],G2, P[8],G5. Mixed infections involving P[8+6],G2 and P[8+6],G1 were also noted. The neonatal P[6] strains associated with diarrhea were detected among children aged 9-24 months. To our knowledge, this study represents the first in Brazil to analyse, on molecular basis, rotavirus genotypes from children participating in a rotavirus vaccine trial. These results are of potential importance regarding future rotavirus vaccination strategies in Brazil.

Comparison between the Counter Immunoelectrophoresis Test and Mouse Neutralization Test for the Detection of Antibodies against Rabies Virus in Dog Sera

The detection of rabies antibodies is extremely valuable for epidemiological studies, determination of immune status in man, animals, and for the diagnosis of the disease. Several serological procedures have been described for this purpose. The present study reports a comparison between counterimmunoelectrophoresis test (CIET) and mouse neutralization test (MNT) in the detection of antibodies against rabies virus from 212 serum samples of vaccinated dogs. The agreement between both techniques was 79.7% and a significative association was demonstrated. The correlation coefficients between MNT and the CIET titers was determined considering 88 samples showing positive results in both techniques [CIET = 2 and MNT = 5 (0.13 IU/ml)] and resulted r² = 0.7926 (p < 0.001). The performance of CIET system was technically simple, cheap and rapid, and thereby it could be useful for serological monitoring of dog vaccination campaigns as well as for individual analysis.

Hepatitis A Outbreak in a Public School in Rio de Janeiro, Brazil

From June 1 to July 1 1999, an outbreak involving 25 cases of hepatitis A occurred in a public school in Rio de Janeiro, Brazil. Since these cases were notified to the State Health Department, the National Reference Center for Hepatitis Viruses (CNRHV) was required to investigate the extent of hepatitis A virus (HAV) dissemination. Blood samples from all students were tested for IgM and total anti-HAV antibodies using a commercial enzyme-linked immunoassay (ELISA). At the same time, a questionnaire was completed in order to identify possible risk factors for HAV infection. The environmental investigation showed that there was no fecal contamination of the water supply. The epidemiological investigation demonstrated that almost 50% of this population was susceptible to HAV infection and probably person-to-person transmission was the principal mode of virus dissemination. In this situation, a massive vaccination campaign could control the HAV infection.