Seroprevalence of viral hepatitis in riverine communities from the Western Region of the Brazilian Amazon Basin

The western region of the Brazilian Amazon Basin has long been shown to be a highly endemic area for hepatitis B and hepatitis D viruses. Data concerning the prevalence of hepatitis C and E viruses in this region are still scarce. In this study we investigated the presence of hepatitis A, B, C, D and E viruses infection in communities that live along the Purus and Acre rivers in the states of Acre and Amazonas within the Amazon Basin. A total of 349 blood samples were collected and tested for hepatitis A-E serological markers (antibodies and/or antigens) using commercial enzyme linked immunosorbent assays. Anti-HCV positive sera were further assayed by an immunoblot. HBsAg positive sera were subtyped by immunodifusion. The overall prevalence for hepatitis A, B, C, and E were 93.7%, 66.1%, 1.7%, and 4%, respectively. A very high prevalence of delta hepatitis (66.6%) was found among HBsAg positive subjects. Hepatitis A, B and D viruses were shown to be largely disseminated in this population, while hepatitis C and E viruses infection presented low prevalence rates in this region. The analysis of risk factors for HBV infection demonstrated that transmission was closely associated with sexual activity.

Immunogenicity test of tetanus component in adsorbed vaccines by toxin binding inhibition test

Samples from 20 lots of diphtheria-tetanus (adult use dT) vaccine and from 20 lots of diphtheria-tetanus-pertussis (DTP) vaccine were used to standardize and validate the in vitro toxin binding inhibition (ToBI) test for the immunogenicity test of the tetanus component. The levels of tetanus antitoxin obtained by ToBI test were compared to those obtained using the toxin neutralization (TN) test in mice routinely employed to perform the quality control of the tetanus component in adsorbed vaccines. The results ranged from 1.8 to 3.5 IU/ml for dT and 2 to 4 IU/ml for DTP by ToBI test and 1.4 to 3 IU/ml for dT and 1.8 to 3.5 IU/ml for DTP by TN in mice. These results were significantly correlated. From this study, it is concluded that the ToBI test is an alternative to the in vivo neutralization procedure in the immunogenicity test of the tetanus component in adsorbed vaccines. A substantial refinement and a reduction in use of animals can be achieved.

Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease

We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guérin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79% in attenuated parasite-vaccinated monkeys, versus 75% in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-g) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and naïve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application.

Antigenic community between Schistosoma mansoni and Biomphalaria glabrata: on the search of candidate antigens for vaccines

We have previously confirmed the presence of common antigens between Schistosoma mansoni and its vector, Biomphalaria glabrata. Cross-reactive antigens may be important as possible candidates for vaccine and diagnosis of schistosomiasis. Sera from outbred mice immunized with a soluble Biomphalaria glabrata antigen (SBgA) of non-infected B. glabrata snails recognized molecules of SBgA itself and S. mansoni AWA by Western blot. Recognition of several molecules of the SBgA were inhibited by pre-incubation with AWA (16, 30, 36, 60 and 155 kDa). The only specific molecule of AWA, inhibited by SBgA, was a 120 kDa protein. In order to determine which epitopes of SBgA were glycoproteins, the antigen was treated with sodium metaperiodate and compared with non-treated antigen. Molecules of 140, 60 and 24 kDa in the SBgA appear to be glycoproteins. Possible protective effects of the SBgA were evaluated immunizing outbred mice in two different experiments using Freund's Adjuvant. In the first one (12 mice/group), we obtained a significant level of protection (46%) in the total worm load, with a high variability in worm recovery. In the second experiment (22 mice/group), no significant protection was observed, neither in worm load nor in egg production per female. Our results suggest that SBgA constitutes a rich source of candidate antigens for diagnosis and prophylactic studies.

Protective immunity of single and multi-antigen DNA vaccines against schistosomiasis

We evaluated the usefulness of the combination of three plasmids encoding tegumental (pECL and pSM14) and muscular (pIRV5) antigens of the Schistosoma mansoni on improving the protective immunity over the use of a single antigen as DNA vaccines. Female BALB/c mice were inoculated twice with 25 µg DNA plasmid within two weeks interval. The challenge was performed with 80 cercarias of a regional isolate of S. mansoni (SLM) one week after the last immunization. Six weeks after challenge, all mice were perfused for worm load determination. The following groups were analyzed: saline; empty vector; monovalent formulations of pECL; pSM14 and pIRV5 and also double combinations of pECL/pIRV5 and pIRV5/pSM14 and a triple combination of pECL/pIRV5/pSM14. The protection was expressed as a percentage of worm loads in each group compared with the saline group. The results obtained were 41% (p < 0.05); 52% (p < 0.05); 51% (p < 0.05); 48% (p < 0.05); 55% (p < 0.05); 45% (p < 0.05); 65% (p < 0.05) for each group respectively.

Multicenter study on the immunogenicity and safety of two recombinant vaccines against hepatitis B

The immunogenicity and safety of a new recombinant hepatitis B vaccine from the Instituto Butantan (Butang®) were evaluated in a multicenter, double-blind, prospective equivalence study in three centers in Brazil. Engerix B® was the standard vaccine. A total of 3937 subjects were recruited and 2754 (70%) met all protocol criteria at the end of the study. All the subjects were considered healthy and denied having received hepatitis B vaccine before the study. Study subjects who adhered to the protocol were newborn infants (566), children 1 to 10 years old (484), adolescents from 11 to 19 years (740), adults from 20 to 30 years (568), and adults from 31 to 40 years (396). Vaccine was administered in three doses on the schedule 0, 1, and 6 months (newborn infants, adolescents, and adults) or 0, 1, and 7 months (children). Vaccine dose was intramuscular 10 µg (infants, children, and adolescents) or 20 µg (adults). Percent seroprotection (assumed when anti-HBs titers were > 10mIU/ml) and geometric mean titer (mIU/ml) were: newborn infants, 93.7% and 351.1 (Butang®) and 97.5% and 1530.6 (Engerix B®); children, 100% and 3600.0 (Butang®) and 97.7% and 2753.1 (Engerix B®); adolescents, 95.1% and 746.3 (Butang®) and 96% and 1284.3 (Engerix B®); adults 20-30 years old, 91.8% and 453.5 (Butang®) and 95.5% and 1369.0 (Engerix B®); and adults 31-40 years old, 79.8% and 122.7 (Butang®) and 92.4% and 686.2 (Engerix B®). There were no severe adverse events following either vaccine. The study concluded that Butang® was equivalent to Engerix B® in children, and less immunogenic but acceptable for use in newborn infants, adolescents, and young adults.

From genomes to vaccines via the proteome

An effective vaccine against schistosomiasis mansoni would be a valuable control tool and the high levels of protection elicited in rodents and primates by radiation-attenuated cercariae provide proof of principle. A major obstacle to vaccine development is the difficulty of identifying the antigens that mediate protection, not least because of the size of the genome at 280Mb DNA encoding 14,000 to 20,000 genes. The technologies collectively called proteomics, including 2D electrophoresis, liquid chromatography and mass spectrometry, now permit any protein to be identified provided there is extensive DNA data, and preferably a genome sequence. Applied to soluble (cytosolic) proteins from schistosomes, proteomics reveals the great similarity in composition between life cycle stages, with several WHO vaccine candidates amongst the most abundant constituents. The proteomic approach has been successfully applied to identify the secretions used by cercaria to penetrate host skin, the gut secretions of adult worms and the proteins exposed on the tegument surface. Soluble proteins can also be separated by 2D electrophoresis before western blotting to identify the full range of antigenic targets present in a parasite preparation. The next step is to discover which target proteins represent the weak points in the worm's defences.

Evaluation of respiratory model employing conventional NIH mice to access the immunity induced by cellular and acellular pertussis vaccines

The increasing number of pertussis cases reported on the last twenty years and the existence of new acellular vaccines reinforce the need of research for experimental models to assure the quality of available pertussis vaccines. In this study, allotments of whole-cell and acellular pertussis vaccines were tested through the Intranasal Challenge Model (INM) using conventional NIH mice. The results have been compared to those achieved by the "Gold standard" Intracerebral Challenge Model (ICM). In contrast to ICM, INM results did not show intralaboratorial variations. Statistical analysis by Anova and Ancova tests revealed that the INM presented reproducibility and allowed identification and separation of different products, including three-component and four-component accellular pertussis vaccines. INM revealed differences between pertussis vaccines. INM provides lower distress to the mice allowing the reduction of mice number including the possibility of using conventional mice (less expensive) under non-aseptic environment. Thus, INM may be used as an alternative method of verifying the consistence of allotment production, including acellular pertussis vaccines.

Schistosome vaccines: a critical appraisal

An effective schistosome vaccine is a desirable control tool but progress towards that goal has been slow. Protective immunity has been difficult to demonstrate in humans, particularly children, so no routes to a vaccine have emerged from that source. The concept of concomitant immunity appeared to offer a paradigm for a vaccine operating against incoming larvae in the skin but did not yield the expected dividends. The mining of crude parasite extracts, the use of monoclonal antibodies and protein selection based on immunogenicity produced a panel of vaccine candidates, mostly of cytoplasmic origin. However, none of these performed well in independent rodent trials, but glutathione-S-transferease from Schistosoma haematobium is currently undergoing clinical trials as an anti-fecundity vaccine. The sequencing of the S. mansoni transcriptome and genome and the development of proteomic and microarray technologies has dramatically improved the possibilities for identifying novel vaccine candidates, particularly proteins secreted from or exposed at the surface of schistosomula and adult worms. These discoveries are leading to a new round of protein expression and protection experiments that will enable us to evaluate systematically all the major targets available for immune intervention. Only then will we know if schistosomes have an Achilles' heel.

Comparison between morphological and staining characteristics of live and dead eggs of Schistosoma mansoni

Schistosoma mansoni eggs are classified, according to morphological characteristics, as follows: viable mature and immature eggs; dead mature and immature eggs, shells and granulomas. The scope of this study was to compare the staining characteristics of different morphological types of eggs in the presence of fluorescent labels and vital dyes, aiming at differentiating live and dead eggs. The eggs were obtained from the intestines of infected mice, and put into saline 0.85%. The fluorescent labels were Hoechst 33258 and Acridine Orange + Ethidium Bromide and vital dyes (Trypan Blue 0.4% and Neutral Red 1%). When labelled with the probe Hoechst 33258, some immature eggs, morphologically considered viable, presented fluorescence (a staining characteristic detected only in dead eggs); mature eggs did not present fluorescence, and the other types of dead eggs, morphologically defined, showed fluorescence. As far as Acridine Orange + Ethidium Bromide are concerned, either the eggs considered to be live, or the dead ones, presented staining with green color, and only the hatched and motionless miracidium was stained with an orange color. Trypan Blue was not able to stain the eggs, considered to be dead but only dead miracidia which had emerged out of the shell. Neutral Red stained both live and dead eggs. Only the fluorescent Hoechst 33258 can be considered a useful tool for differentiation between dead and live eggs.

Comparison of two commercial formulations of Bacillus thuringiensis var. israelensis for the control of Anopheles aquasalis (Diptera: Culicidae) at three salt concentrations

Anopheles aquasalis larvae are salt water tolerant, preferring concentrations between 10 and 20 parts per thousand (ppt). The larvicidal efficacy of two formulations of Bacillus thuringiensis var. israelensis (Vectobac-12AS® and Bactivec®), was investigated against An. aquasalis at salinities of 0, 10, and 20 ppt. A probit analysis was used to calculate the lethal concentrations (LC50 and LC95) for each product at each salinity. The LC50 and LC95 were higher for Bactivec® than Vectobac-12AS®, and for Bactivec®, the LC50 and LC95 increased with salinity. Vectobac-12AS® should thus be preferred to Bactivec® for An. aquasalis control, especially in saline breeding habitats.

Wild dengue virus types 1, 2 and 3 viremia in rhesus monkeys

Among the flaviviruses, dengue, with its four serotypes, has spread throughout the tropics. The most advanced vaccines developed so far include live attenuated viruses, which have been tested in humans but none has been licensed. Preclinical testing of dengue vaccine candidates is performed initially in mice and in nonhuman primates. In the latter the main criteria used to assay protection are neutralizing antibodies elicited by the vaccine candidate and the magnitude and duration of peripheral viremia upon challenge of previously immunized animals. Towards the identification of wild-type viruses that could be used in challenge experiments a total of 31 rhesus monkeys were inoculated subcutaneously of wild dengue types 1, 2, and 3 viruses. The viremia caused by the different viruses was variable but it was possible to identify dengue viruses useful as challenge strains.

The utility of rhesus monkey (Macaca mulatta) and other non-human primate models for preclinical testing of Leishmania candidate vaccines

Leishmaniasis causes significant morbidity and mortality, constituting an important global health problem for which there are few effective drugs. Given the urgent need to identify a safe and effective Leishmania vaccine to help prevent the two million new cases of human leishmaniasis worldwide each year, all reasonable efforts to achieve this goal should be made. This includes the use of animal models that are as close to leishmanial infection in humans as is practical and feasible. Old world monkey species (macaques, baboons, mandrills etc.) have the closest evolutionary relatedness to humans among the approachable animal models. The Asian rhesus macaques (Macaca mulatta) are quite susceptible to leishmanial infection, develop a human-like disease, exhibit antibodies to Leishmania and parasite-specific T-cell mediated immune responses both in vivo and in vitro, and can be protected effectively by vaccination. Results from macaque vaccine studies could also prove useful in guiding the design of human vaccine trials. This review summarizes our current knowledge on this topic and proposes potential approaches that may result in the more effective use of the macaque model to maximize its potential to help the development of an effective vaccine for human leishmaniasis.

Veterinary vaccines against Toxoplasma gondii

Toxoplasma gondii has a very wide intermediate host range and is thought to be able to infect all warm blooded animals. The parasite causes a spectrum of different diseases and clinical symptoms within the intermediate hosts and following infection most animals develop adaptive humoral and cell-mediated immune responses. The development of protective immunity to T. gondii following natural infection in many host species has led researchers to look at vaccination as a strategy to control disease, parasite multiplication and establishment in animal hosts. A range of different veterinary vaccines are required to help control T. gondii infection which include vaccines to prevent congenital toxoplasmosis, reduce or eliminate tissue cysts in meat producing animals and to prevent oocyst shedding in cats. In this paper we will discuss some of the history, challenges and progress in the development of veterinary vaccines against T. gondii.