Human IgG responses against the N-terminal region of Merozoite Surface Protein 1 of Plasmodium vivax

The complete primary structure of the gene encoding the Merozoite Surface Protein 1 of Plasmodium vivax (PvMSP-1) revealed the existence of interspecies conserved regions among the analogous proteins of other Plasmodia species. Here, three DNA recombinant clones expressing 50, 200 and 500 amino acids from the N-terminal region of the PvMSP-1 protein were used on ELISA and protein immunoblotting assays to look at the IgG antibody responses of malaria patients from the Brasilian amazon region of Rondônia. The results showed the existance of P. vivax and P. falciparum IgG antibodies directed against PvMSP-1 antigenic determinants expressed in the clones containing the first 200 and the following 500 amino acids of the molecule, but not within the one expressing the most N-terminal 50 amino acids. Interestingly, there was no correlation between the levels of these IgG antibodies and the previous number of malaria infections.

Protection of aouts monkeys after immunization with recombinant antigens of Plasmodium falciparum

The genus Aotus spp. (owl monkey) is one of the WHO recommended experimental models for Plasmodium falciparum blood stage infection, especially relevant for vaccination studies with asexual blood stage antigens of this parasite. For several immunization trials with purified recombinant merozoite/schizont antigens, the susceptible Aouts kenotypes II, III, IV and VI were immunized with Escherichia coli derived fusion proteins containg partial sequences of the proteins MSAI (merozoite surface antigen I), SERP (serine-strech protein) and HRPII (histidine alanine rich protein II) as well as with a group of recombinant antigens obtained by an antiserum raised against a protective 41 kD protein band. The subcutaneous application (3x) of the antigen preparations was carried out in intact animals followed by splenectomy prior to challange, in order to increase the susceptibility of the experimental hosts to the parasite. A partial sequence of HRPII, the combination of three different fusion proteins of the 41 kD group and mixture of two sequences of SERP in the presence of the modified Al(OH)3 adjuvant conferred significant protection against a challange infection with P. falciparum blood stages (2-5 x 10 (elevado a sexta potência) i. RBC). Monkey immunized with the MS2-fusion protein carrying the N-terminal part of the 195 kD precursor of the major merozoite surface antigens induced only marginal protection showing some correlation between antibody titer and degree of parasitaemia. Based on the protective capacity of these recombinant antigens we have expressed two hybrid proteins (MS2/SERP/HRPII and SERP/MSAI/HRPII) in E. coli containing selected partial sequences of SERP, HRPII and MSAI. Antibodies raised against both hybrid proteins in rabbits and Aotus monkeys recognize the corresponding schizont polypeptides. In two independent immunization trials using 13 animals (age 7 months to 3 years) we could show that immunization of Aotus monkeys with either of the two hybrid proteins administered in an oil-based well tolerated formulation protected the animals frm a severe experimental P. falciparum (strain Palo Alto) infection.

Efficience of human Plasmodium falciparum malaria vaccine candidates in Aotus lemurinus monkeys

The protective efficacy of several recombinat and a synthetic Plasmodium falciparum protein was assessed in Aoutus monkeys. The rp41 aldolase, the 190L fragment of the MSA-1 protein and fusion 190L-CS. T3 protein containg the CS. T3 helper "universal epitope were emulsified in Freund's adjuvants and injected 3 times in groups of 4-5 monkeys each one. The synthetic polymer Spf (66)30 also emulsified in Freund's adjuvants was injected 6 times. Control groups for both experiments were immunized with saline solution in the same adjuvant following the same schedules. Serology for malaria specific antibodies showed seroconversion in monkeys immunized with the recombinant proteins but not in those immunized with the polymer nor in the controls. Challenge was performed with the 10 (elevado a quinta potência) parasites from the P. falciparum FVO isolate. Neither rp41 nor SPf (66)30 induced protection, whereas 190L induced significant delay of parasitemia. The fusion of the CS. T3 epitope to 190L significantly increased is protective capacity.

Prevalence of human papillomavirus DNA in female cervical lesions from Rio de Janeiro, Brazil

A hundred-sixty paraffin-embedded specimens from female cervical lesions were examined for human papillomavirus (HPV) types 6, 11, 16 and 18 infections by non-isotopic in situ hybridization. The data were compared with histologic diagnosis. Eighty-eight (55) biopsies contained HPV DNA sequences. In low grade cervical intraepithelial neoplasias (CIN I), HPV infection was detected in 78.7 of the cases, the benign HPV 6 was the most prevalent type. HPV DNA was detected in 58 of CIN II and CIN III cases and in 41.8 of squamous cell carcinomas (SCC). Histologically normal women presented 20 of HPV infection. Oncogenic HPV was found in 10 of these cases, what may indicate a higher risk of developing CINs and cancer. Twenty-five percent of the infected tissues contained mixed infections. HPV 16 was the most common type infecting the cervix and its prevalence raised significantly with the severity of the lesions, pointing its role in cancer pathogenesis. White women presented twice the cervical lesions of mulatto and African origin women, although HPV infection rates were nearly the same for the three groups (approximately 50). Our results showed that HPV typing by in situ hybridization is a useful tool for distinguishing between low and high risk cervical lesions. Further studies are required to elucidate risk factors associated with HPV infection and progression to malignancy in Brazilian population.

Aspects of immunity for the AMA-1 family of molecules in humans and non-human primates malarias

The apical membrane antigen (AMA-1) family of malaria merozoite proteins is characterised by a high degree of inter-species conservation. Evidence that the protein (PK66/AMA-1) from the simian parasite Plasmodium knowlesi was protective in rhesus monkeys suggested that the 83kDa P. falciparum equivalent (PF83/AMA-1) should be investigated for protective effects in humans. Here we briefly review pertinent comparative data, and describe the use of an eukaryotic full length recombinant PF83/AMA-1 molecule to develop a sensitive ELISA for the determination of serological responses in endemic populations. The assay has revealed surprisingly high levels of humoral response to this quantitatively minor antigen. We also show that PK66/AMA-1 inhibitory mAb's are active against merozoites subsequent to release from schizont-infected red cells, further implicating AMA-1 molecules in red cell invasion.

Chimpanzees and supporting models in the study of malaria pre-erythrocytic stages

Chimpanzees are being used in the study of immune response to Plasmodium falciparum malaria pre-erythrocytic stages (MPES). Responses induced by immunisation with recombinant/synthetic antigens and by irradiated sporozoites are being evaluated in a model system that is phylogenetically close to humans and that is amenable to limited manipulation not possible in humans. The value of chimpanzees for the in-depth study of immunological mechanisms at work in MPES-induced protection are discussed. A total number of 7 chimpanzees have been used to evaluate the immune response to recombinant antigens, and 5 have been challenged with large numbers of sporozoites, followed by surgical liver-wedge resection, in order to generate infected liver tissue for histological and immunological studies. As a complementary model, SCID mice carrying live, transplanted human and primate hepatocytes have been inoculated with sporozoites and infection of transplanted cells has been monitored by histological and immunological methods. In ongoing experiments chimpanzees are being immunised with MPES-derived lipopeptides that have been shown to overcome MHC restriction in mice, and with irradiated sporozoites.

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.

Vaccination against schistosomiasis and fascioliasis with the new recombinant antigen Sm14: potential basis of a multi-valent anti-helminth vaccine?

Molecular cloning of components of protective antigenic preparations have suggested that related parasite fatty acid binding proteins could form the basis of the well documented protective, immune cross reactivity between the parasitic trematode worms Fasciola hepatica and Schistosoma mansoni. We have now confirmed the cross protective potential of parasite fatty acid binding proteins and suggest that it may be possible to produce a single vaccine that would be effective against at least two parasites, F. hepatica and S. mansoni of veterinary and human importance respectively.

Human papillomavirus infection and cervical cancer in Brazil: a retrospective study

Two hundred and thirty paraffin-embedded biopsies obtained from female cervical lesions were tested for the presence of human papillomavirus (HPV) types 6/11,16/18 and 31/33/35 DNA using non-isotopic in situ hybridization. Specimens were classified according to the Bethesda System in low grade squamous intraepithelial lesion (LSIL), high grade SIL (HSIL) and squamous cell carcinoma (SCC). HPV prevalence ranged from 92.5% in LSIL to 68.5% in SCC. Benign types were prevalent in LSILs while oncogenic types infected predominantly HSILs and SCC. HPV infection showed to be age-dependent, but no significant relation to race has been detected. Patients were analyzed through a five-year period: 20.7% of the lesions spontaneously regressed while 48.9% persisted and 30.4% progressed to carcinoma. Patients submitted to treatment showed a 19.4% recurrence rate. High risk types were present in 78.6% (CrudeOR 13.8, P=0.0003) of the progressive lesions, and in 73.7% of the recurrent SILs (COR 19.3, P=0.0000001). Possible co-factors have also been evaluated: history of other sexually transmitted diseases showed to be positively related either to progression (Adjusted OR 13.0, P=0.0002) or to recurrence (AOR 17.2, P=0.0002) while oral contraceptive use and tobacco smoking were not significantly related to them (P>0.1). Association of two or more co-factors also proved to be related to both progression and recurrence, indicating that they may interact with HPV infection in order to increase the risk of developing malignant lesions.

Serotypes of Vibrio cholerae Non-O1 Isolated from Water Supplies for Human Consumption in Campeche, México and their Antibiotic Susceptibility Pattern

The presence of Vibrio cholerae non-O1 in water supplies for human consumption in the city of Campeche and rural locality of Bécal was investigated. V. cholerae non-O1 was detected in 5.9% of the samples obtained in deep pools of Campeche. Studies conducted in Bécal and neighbourhood of Morelos in Campeche indicated that collected samples harbored V. cholerae non-O1 in 31.5% and 8.7% respectively. There was a particular pattern of distribution of V. cholerae non-O1 serotypes among different studied regions. Accordingly, V. cholerae non-O1 serotype O14 predominated in the deep pools of Campeche and together with V. cholerae non-O1, O155 were preferentially founds in samples taken from intradomiciliary faucets in the neighbourhood of Morelos. Samples from Bécal predominantly presented the serotype O112. 60% and 53.8% of all studied strains of V. cholerae non-O1 proved to be resistant to ampicillin and carbenicillin. 3.1%, 7.7% and 6.2% presented resistant to doxycycline, trimethoprim-sulfamethoxazole and erythromycin respectively. The study showed the necessity of performing a strong epidemiologic surveillance for emergence and distribution of V. cholerae non-O1

Molecular Epidemiology of Human Polyomavirus JC in the Biaka Pygmies and Bantu of Central Africa

Polyomavirus JC (JCV) is ubiquitous in humans and causes a chronic demyelinating disease of the central nervous system , progressive multifocal leukoencephalopathy which is common in AIDS. JCV is excreted in urine of 30-70% of adults worldwide. Based on sequence analysis of JCV complete genomes or fragments thereof, JCV can be classified into geographically derived genotypes. Types 1 and 2 are of European and Asian origin respectively while Types 3 and 6 are African in origin. Type 4, a possible recombinant of European and African genotypes (1 and 3) is common in the USA. To delineate the JCV genotypes in an aboriginal African population, random urine samples were collected from the Biaka Pygmies and Bantu from the Central African Republic. There were 43 males and 25 females aged 4-55 years, with an average age of 26 years. After PCR amplification of JCV in urine, products were directly cycle sequenced. Five of 23 Pygmy adults (22%) and four of 20 Bantu adults (20%) were positive for JC viruria. DNA sequence analysis revealed JCV Type 3 (two), Type 6 (two) and one Type 1 variant in Biaka Pygmies. All the Bantu strains were Type 6. Type 3 and 6 strains of JCV are the predominant strains in central Africa. The presence of multiple subtypes of JCV in Biaka Pygmies may be a result of extensive interactions of Pygmies with their African tribal neighbors during their itinerant movements in the equatorial forest.

Evaluation of DNA Recombinant Methodologies for the Diagnosis of Plasmodium falciparum and their Comparison with the Microscopy Assay

Since 1984, DNA tests based on the highly repeated subtelomeric sequences of Plasmodium falciparum (rep 20) have been frequently used in malaria diagnosis. Rep 20 is very specific for this parasite, and is made of 21 bp units, organized in repeated blocks with direct and inverted orientation. Based in this particular organization, we selected a unique consensus oligonucleotide (pf-21) to drive a PCR reaction coupled to hybridization to non-radioactive labeled probes. The pf-21 unique oligo PCR (pf-21-I) assay produced DNA amplification fingerprints when was applied on purified P. falciparum DNA samples (Brazil and Colombia), as well as in patient's blood samples from a large area of Venezuela. The performance of the Pf-21-I assay was compared against Giemsa stained thick blood smears from samples collected at a malaria endemic area of the Bolívar State, Venezuela, at the field station of Malariología in Tumeremo. Coupled to non-radioactive hybridization the pf-21-I performed better than the traditional microscopic method with a r=1.7:1. In the case of mixed infections the r value of P. falciparum detection increased to 2.5:1. The increased diagnostic sensitivity of the test produced with this homologous oligonucleotide could provide an alternative to the epidemiological diagnosis of P. falciparum being currently used in Venezuela endemic areas, where low parasitemia levels and asymptomatic malaria are frequent. In addition, the DNA fingerprint could be tested in molecular population studies

Frequency of CCR5 Delta-32 Mutation in Human Immunodeficiency Virus (HIV)-seropositive and HIV-exposed Seronegative Individuals and in General Population of Medellin, Colombia

Repeated exposure to human immunodeficiency virus (HIV) does not always result in seroconversion. Modifications in coreceptors for HIV entrance to target cells are one of the factors that block the infection. We studied the frequency of Delta-32 mutation in ccr5 gene in Medellin, Colombia. Two hundred and eighteen individuals distributed in three different groups were analyzed for Delta-32 mutation in ccr5 gene by polymerase chain reaction (PCR): 29 HIV seropositive (SP), 39 exposed seronegative (ESN) and 150 individuals as a general population sample (GPS). The frequency of the Delta-32 mutant allele was 3.8% for ESN, 2.7% for GPS and 1.7% for SP. Only one homozygous mutant genotype (Delta-32/Delta-32) was found among the ESN (2.6%). The heterozygous genotype (ccr5/Delta-32) was found in eight GPS (5.3%), in one SP (3.4%) and in one ESN (2.6%). The differences in the allelic and genotypic frequencies among the three groups were not statistically significant. A comparison between the expected and the observed genotypic frequencies showed that these frequencies were significantly different for the ESN group, which indirectly suggests a protective effect of the mutant genotype (Delta-32/Delta-32). Since this mutant genotype explained the resistance of infection in only one of our ESN persons, different mechanisms of protection must be playing a more important role in this population.

Risk Factors for tuberculosis among human immunodeficiency virus-infected persons. A case-control study in Belo Horizonte, Minas Gerais, Brazil (1985-1996)

The objective of this study was to identify tuberculosis risk factors and possible surrogate markers among human immunodeficiency virus (HIV)-infected persons. A retrospective case-control study was carried out at the HIV outpatient clinic of the Universidade Federal de Minas Gerais in Belo Horizonte. We reviewed the demographic, social-economical and medical data of 477 HIV-infected individuals evaluated from 1985 to 1996. The variables were submitted to an univariate and stratified analysis. Aids related complex (ARC), past history of pneumonia, past history of hospitalization, CD4 count and no antiretroviral use were identified as possible effect modifiers and confounding variables, and were submitted to logistic regression analysis by the stepwise method. ARC had an odds ratio (OR) of 3.5 (CI 95% - 1.2-10.8) for tuberculosis development. Past history of pneumonia (OR 1.7 - CI 95% 0.6-5.2) and the CD4 count (OR 0.4 - CI 0.2-1.2) had no statistical significance. These results show that ARC is an important clinical surrogate for tuberculosis in HIV-infected patients. Despite the need of confirmation in future studies, these results suggest that the ideal moment for tuberculosis chemoprophylaxis could be previous to the introduction of antiretroviral treatment or even just after the diagnosis of HIV infection.

Chagas disease and human migration

Human Chagas disease is a purely accidental occurrence. As humans came into contact with the natural foci of infection might then have become infected as a single addition to the already extensive host range of Trypanosoma cruzi that includes other primates. Thus began a process of adaptation and domiciliation to human habitations through which the vectors had direct access to abundant food as well as protection from climatic changes and predators. Our work deals with the extraction and specific amplification by polymerase chain reaction of T. cruzi DNA obtained from mummified human tissues and the positive diagnosis of Chagas disease in a series of 4,000-year-old Pre-Hispanic human mummies from the northern coast of Chile. The area has been inhabited at least for 7,000 years, first by hunters, fishers and gatherers, and then gradually by more permanent settlements. The studied specimens belonged to the Chinchorro culture, a people inhabiting the area now occupied by the modern city of Arica. These were essentially fishers with a complex religious ideology, which accounts for the preservation of their dead in the way of mummified bodies, further enhanced by the extremely dry conditions of the desert. Chinchorro mummies are, perhaps, the oldest preserved bodies known to date.