Killer cell immunoglobulin-like receptor (KIR) gene diversity in a population naturally exposed to malaria in Porto Velho, Northern Brazil.

Killer cell immunoglobulin-like receptors (KIR) are expressed mainly in natural killer cells and specifically recognize human leukocyte antigen (HLA) class I molecules. The repertoire of KIR genes and KIR-HLA pairs is known to play a key role in the susceptibilities to and the outcomes of several diseases, including malaria. The aim of this study was to investigate the distribution of KIR genes, KIR genotypes and KIR-HLA pair combinations in a population naturally exposed to malaria from Brazilian Amazon. All 16 KIR genes investigated were present in the studied population. Overall, 46 KIR genotypes were defined. The two most common genotypes in the Porto Velho communities, genotypes 1 and 2, were present at similar frequencies as in the Americas. Principal component analysis based on the frequencies of the KIR genes placed the Porto Velho population closer to the Venezuela Mestizos, USA California hispanic and Brazil Paraná Mixed in terms of KIR gene frequencies. This analysis highlights the multi-ethnic profile of the Porto Velho population. Most of the individuals were found to have at least one inhibitory KIR-HLA pair. Seventy-five KIR-HLA pair combinations were identified. The KIR-2DL2/3_HLA-C1, KIR3DL1_HLA-Bw4 and KIR2DL1_HLA-C2 pairs were the most common. There was no association between KIR genes, KIR genotypes or KIR-HLA pair combinations and malaria susceptibility in the studied population. This is the first report on the distribution of KIR and known HLA ligands in the Porto Velho population. Taken together, these results should provide baseline information that will be relevant to population evolutionary history, malaria and other diseases studies in populations of the Brazilian Amazon.

B cell epitope mapping and characterization of naturally acquired antibodies to the Plasmodium vivax merozoite surface protein-3α (PvMSP-3α) in malaria exposed individuals from Brazilian Amazon.

The Plasmodium vivax Merozoite Surface Protein-3α (PvMSP-3α) is considered as a potential vaccine candidate. However, the detailed investigations of the type of immune responses induced in naturally exposed populations are necessary. Therefore, we aim to characterize the naturally induced antibody to PvMSP-3α in 282 individuals with different levels of exposure to malaria infections residents in Brazilian Amazon. PvMSP3 specific antibodies (IgA, IgG and IgG subclass) to five recombinant proteins and the epitope mapping by Spot-synthesis technique to full-protein sequence of amino acids (15aa sequence with overlapping sequence of 9aa) were performed. Our results indicates that PvMSP3 is highly immunogenic in naturally exposed populations, where 78% of studied individuals present IgG immune response against the full-length recombinant protein (PVMSP3-FL) and IgG subclass profile was similar to all five recombinant proteins studied with a high predominance of IgG1 and IgG3. We also observe that IgG and subclass levels against PvMSP3 are associated with malaria exposure. The PvMSP3 epitope mapping by Spot-synthesis shows a natural recognition of at least 15 antigenic determinants, located mainly in the two blocks of repeats, confirming the high immunogenicity of this region. In conclusion, PvMSP-3α is immunogenic in naturally exposed individuals to malaria infections and that antibodies to PvMSP3 are induced to several B cell epitopes. The presence of PvMSP3 cytophilic antibodies (IgG1 and IgG3), suggests that this mechanism could also occur in P. vivax.

Promiscuous T-cell epitopes of Plasmodium merozoite surface protein 9 (PvMSP9) induces IFN-gamma and IL-4 responses in individuals naturally exposed to malaria in the Brazilian Amazon.

Plasmodium vivax merozoite surface protein (PvMSP9) stimulates both cellular and humoral immune responses in individuals who are naturally infected by this parasite species. To identify immunodominant human T-cell epitopes in PvMSP9, we used the MHC class II binding peptide prediction algorithm ProPred. Eleven synthetic peptides representing predicted putative promiscuous T-cell epitopes were tested in IFN-gamma and IL-4 ELISPOT assays using peripheral blood mononuclear cells (PBMC) derived from 142 individuals from Rondonia State, Brazil who had been naturally exposed to P. vivax infections. To determine whether the predicted epitopes are preferentially recognized in the context of multiple alleles, MHC Class II typing of the cohort was also performed. Five synthetic peptides elicited robust cellular responses, and the overall frequencies of IFN-gamma and IL-4 responders to at least one of the promiscuous peptides were 62% and 46%, respectively. The frequencies of IFN-gamma and IL-4 responders to each peptide were not associated with a particular HLA-DRB1 allelic group since most of the peptides induced a response in individuals of 12 out of 13 studied allelic groups. The prediction of promiscuous epitopes using ProPred led to the identification of immunodominant epitopes recognized by PBMC from a significant proportion of a genetically heterogeneous population exposed to malaria infections. The combination of several such T-cell epitopes in a vaccine construct may increase the frequency of responders and the overall efficacy of subunit vaccines in genetically distinct populations.

Cellular and antibody responses to the Plasmodium falciparum heat shock protein Pf72/HSP70 during and after acute malaria in individuals from an endemic area of Brazil.

Proliferative and antibody responses to three synthetic peptides corresponding to Pf72/ HSP70 were followed-up in acute malaria patients from an endemic area of Brazil. In vitro lymphocyte responsiveness to all peptides was relatively low and short-lived and there was a considerable variation in the frequency and magnitude of the individual lymphoproliferative response to the peptides at different periods after the onset of infection. Although 96% of the patients had IgG antibodies to crude Plasmodium falciparum asexual blood stage antigens, specific IgG antibody responses to the peptides varied from 12.5 to 40% according to the tested peptides. No significant difference was observed in the proliferative or antibody responses to the peptides between individuals that remained parasitemic after treatment and those that recovered from malaria infection. The different frequencies of proliferative responses in peripheral blood T cells on different occasions after the onset of their infection show that, in order to be informative, evaluation of the in vitro cellular immune response to peptides requires longitudinal studies in which each individual is tested repeatedly.

Immune response and lack of immune response to Plasmodium falciparum P126 antigen and its amino-terminal repeat in malaria-infected humans.

A parasitophorous vacuole protein of Plasmodium falciparum, p126, is a potential candidate for a malaria vaccine. Its N-terminal region, composed of six repeats of eight amino acids, appears to be involved in the induction of protective immunity against P. falciparum challenge in monkeys. This study evaluated the immune response to p126 and to its N-terminal region (Nt47) in patients (n = 45) living in a malaria-endemic area of Brazil (Colina, Porto Velho, Rondonia). Cellular proliferative responses against Nt47 were low and infrequent. The study of the humoral immune response demonstrated that 95% of the patients had detectable anti-p126 antibodies and 77% had anti-Nt47 antibodies. Analysis of the antibody isotypes specific for Nt47 revealed that all four IgG subclasses were present and individuals with higher levels of anti-Nt47 cytophilic IgG antibody (IgG1 + IgG3/IgG2 + IgG4) had significantly lower parasitemia levels, suggesting that antibodies to the N-terminal region of the p126 protein may contribute to acquisition of immunity to P. falciparum malaria.

Induction of antibodies against the Plasmodium falciparum p126 antigen in non-responder H-2b and partial-responder H-2d mice using synthetic peptides.

The p126 Plasmodium falciparum antigen is processed into two fragments, p50 and p73, the latter one containing the subfragments p47 and p18 when the schizonts rupture. An absence of antibody response against the p126 antigen has been reported recently in H-2b mice and limited to the p73 processed fragment in H-2d mice. Synthetic peptides corresponding to various domains of the molecule have been used to immunize mice in order to overcome the absence of an immune response. Synthetic peptides corresponding to the N-terminus of p50 or p18 as well as to the C-terminus of p47 were unable to induce anti-peptide antibodies when injected carrier-free or coupled to ovalbumin. Synthetic peptides corresponding to the C-terminus of p18 or composed of 6 or 9 serines were able to induce anti-peptide antibodies when injected coupled to a carrier protein. However, none of these antibodies was able to recognize the native p126 molecule. Various synthetic peptides corresponding to the 6-octapeptide [Nt47 (6 x 8)] or the 4-octapeptide [Nt47(4 x 8)] repeat sequence localized at the N-terminus of the p47 have also been used to immunize mice. No antibodies were generated using a carrier-free [Nt47(6 x 8)-Cys]2 or [Nt47 (4 x 8)-Cys]2 peptide, an octameric multiple antigen peptide construct [Nt47(6 x 8)]-MAP or the [Nt47(6 x 8)] coupled to one or two palmitic acids. In contrast, [Nt47(6 x 8)]-Cys coupled to either tetanus toxoid (TT) or ovalbumin (OVA) and [Nt47(4 x 8)]-Cys coupled to OVA induced antibodies against the synthetic peptide and the native p126 molecule in both H-2d and H-2b mice. A multiple antigen peptide construct [Nt47(4 x 8)-MSP-3b]-MAP containing 4 [Nt47(4 x 8)] and 4 [MSP-3b] also induced antibodies against the synthetic peptide [Nt47(4 x 8)-Cys]2 and the native p126 molecule in both H-2d and H-2b mice.

Can software be used to predict antigenic regions in Plasmodium falciparum peptides?

We compared the antigenicity of p126 Plasmodium falciparum peptides with predicted antigenic regions identified using the methods described by Garnier et al. (1978) and Chou & Fasman (1974). For this purpose nine different P. falciparum peptides were synthesized in accordance with the deduced amino acid sequence of the p126 gene, and their reactivity was tested using an enzyme linked immunosorbent assay against sera from individuals with a natural malaria infection. Both predictive methods gave similar antigenic-index scores, however, a comparison of these predictive results with data obtained by ELISA showed that the probability of a correct prediction was only around 45% for both cases. Thus, in our view computer software could not be used in isolation for screening purposes, and other parameters must also be taken into account when using such software to assess antigenicity.

Malaria diagnosis: standardization of a polymerase chain reaction for the detection of Plasmodium falciparum parasites in individuals with low-grade parasitemia.

In Brazil, no study has been done concerning the detection of malaria parasites by polymerase chain reaction (PCR) related to the diagnosis of Plasmodium falciparum malaria. In the present report we describe a highly sensitive methodology for malaria diagnosis using a nested PCR method based on amplification of the p126 P. falciparum gene detected by simple ethidium bromide staining. The P. falciparum Palo Alto strain (culture samples) was serially diluted in blood from an uninfected donor to a final level of parasitemia corresponding to 10(-8)% and was processed for PCR amplification. In each of these dilutions a parasitological examination was performed to compare the sensitivity with that of PCR amplification. Blood samples (field samples) were obtained from 51 malarious patients with positive thick blood smears (TBS) who were living in endemic regions of the Brazilian Amazon. They corresponded to 42 P. falciparum and 9 P. vivax cases, with parasitemia levels ranging from only 16 to 20,200 parasites/microliter for P. falciparum disease and from 114 to 11,000 parasites/microliter for P. vivax malaria. We demonstrate that the use of nested PCR allows the detection of 0.005 parasites/microliter without the use of radioactive material. The use of a 1-ml sample volume and the organic DNA extraction method should be suitable in blood banks and for the evaluation of patients during and after drug treatment.

The art of parasite survival.

Parasites develop and survive in an environment which is often hostile to them. When facing aggressive conditions parasites are able to use various and complex strategies. Echinococcus granulosus, Toxocara canis, Pneumocystis carinii, Entamoeba or Toxoplasma gondii are able to seclude from the environment when stressed by surrounding (immunologic or non-immunologic) aggressive factors. Specific antigens which exert a functional activity during a short period of time appear to be concealed from the immune attack at this crucial moment. This is the case for rhoptry or dense granule antigens of Plasmodium or Toxoplasma sporozoa involved in the formation of the parasitophorous vacuole which are released in a space perfectly isolated from the outside and therefore from antibodies. Some parasites like Schistosoma mansoni or Trypanosoma brucei reveal an amazing opportunistic behavior when they use cytokines of host origin induced by the infectious process for their own development. Leishmania, Toxoplasma and Trypanosoma cruzi are able to invade immunologically competent macrophages and to avoid the triggering of killing mechanisms of these cells. Parasites also take advantage of the genetic restriction of the immune response and it has been observed for Plasmodia that some high molecular weight antigens are unable to induce an immune response in particular strains of mice. Parasite receptors involved in the invasion of host cells by parasites can function in the presence of antibodies which can explain the failure of vaccination attempts targeting this type of molecules. Among the mechanisms developed by parasites to resist to drugs it appears that transmembrane transporters described in many protozoa or helminth parasites could play a role. Moreover, the description of parasite-specific enzymes able to protect them against the damaging effects of oxygen radicals suggests that parasites are potentially able to develop a resistance phenomenon against drugs acting via an oxidative burst.

The art of parasite survival

Parasites develop and survive in an environment which is often hostile to them. When facing aggressive conditions parasites are able to use various and complex strategies. Echinococcus granulosus, Toxocara canis, Pneumocystis carinii, Entamoeba or Toxoplasma gandii are able to seclude from the environment when stressed by surrounding (immunologic or non-immunologic) agressive factors. Specific antigens which exert a functional activity during a short period of time appear to be concealed from the immune attack at this crucial moment. This is the case for rhoptry or dense granule antigens of Plasmodium or Toxoplasma sporozoa involved in the formation of the parasitophorous vacuole which are released in a space perfectly isolated from the outside and therefore from antibodies. Some parasites like Schistosoma mansoni or Trypanosoma brucei reveal an amazing opportunistic behavior when they use cytokines of host origin induced by the infectious process for their own development. Leishmania, Toxoplasma and Trypanosoma cruzi are able to invade immunologically competent macrophages and to avoid the triggering of killing mechanisms of these cells. Parasites also take advantage of the genetic restriction of the immune response and it has been observed for Plasmodia that some high molecular weight antigens are unable to induce an immune response in particular strains of mice. Parasite receptors involved in the invasion of host cells by parasites can function in the presence of antibodies which can explain the failure of vaccination attempts targeting this type of molecules. Among the mechanisms developed by parasites to resist to drugs it appears that transmembrane transporters described in many protozoa or helminth parasites could play a role. Moreover, the description of parasite-specific enzymes able to protect them against the damaging effects of oxygen radicals suggests that parasites are potentially able to develop a resistance phenomenon against drugs acting via an oxidative burst

H-2b restriction of the immune response to the p126 Plasmodium falciparum antigen.

Inbred BALB/c (H-2d), CBA (H-2k) and C57B1/6 (H-2b) mice immunized with Plasmodium falciparum schizonts or culture supernates develop antibodies of different antigenic specificities. It has been observed that C57B1/6 mice were unable to produce detectable antibodies against the p126 antigen (native molecule and p73 or p50 processed fragments) compared with other inbred mice. Similar results were obtained using BALB congenic mice with a lack of p126 antibody response in H-2b mice, while H-2d and H-2k mice produced antibodies against the p126. Lymphocyte proliferation assays performed by incubation of spleen cells with immunopurified p126 were positive for immunized BALB/c (H-2d) and congenic H-2d or H-2k mice. On the other hand, no lymphocyte stimulation was observed with either C57B1/6 (H-2b) or congenic H-2b mice. These results suggest an MHC restriction of the immune response against the entire p126 (found in schizonts) and its p73 and p50 naturally processed fragments (found in culture supernates).

Immunogenicity and antigenicity of the N-term repeat amino acid sequence of the Plasmodium falciparum P126 antigen.

The P126 protein, a parasitophorus vacuole antigen of Plasmodium falciparum has been shown to induce protective immunity in Saimiri and Aotus monkeys. In the present work we investigated its immunogenicity. Our results suggest that the N-term of P126 is poorly immunogenic and the antibody response against the P126 could be under a MHC restricted control in C57BL/6(H-2b) mice, which could be problematic in terms of a use of the P126 in a vaccine program. However, we observed that a synthetic peptide, copying the 6 octapeptide repeat corresponding to the N-term of the P126, induces an antibody response to the native molecule in C57BL/6 non-responder mice. Moreover, the vaccine-P126 recombinant induced antibodies against the N-term of the molecule in rabbits while the unprocessed P126 did not.

Polyclonal B-lymphocyte stimulation in human malaria and its association with ongoing parasitemia.

To study the polyclonal B-lymphocyte stimulation or activation (PBA) phenomenon in human malaria, the numbers of immunoglobulin (G, A, and M)-secreting cells in the peripheral blood, serum levels of immunoglobulins, and the presence of antibodies directed against the DNA-autoantigen were evaluated in Plasmodium falciparum- and P. vivax-infected individuals. Individuals chronically exposed to the risk of infection or non-immune subjects who contracted malaria during first visits to endemic areas were studied. Numbers of immunoglobulin-secreting cells (IgSC) were increased dependent upon ongoing parasitemia. Levels of IgG and the anti-DNA activity were also augmented in malarious individuals from the endemic area. Study of the kinetics of PBA done in treated patients showed that PBA decreased during treatment and disappeared 5-15 days after the start of chemotherapy.

Polyclonal B cell activation in paracoccidioidomycosis. Polyclonal activation in paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is often associated with hypergammaglobulinemia and increased serum levels of circulating immune complexes (CIC). In order to investigate whether polyclonal B lymphocyte activation (PBA) is a current process in PCM, we measured the numbers of IgG secreting cells (IgG SC) in the peripheral blood of 16 patients and of 8 healthy controls. The numbers of IgG SC were found to be significantly elevated in PCM patients. We also observed increased serum levels of IgG, IgA and CIC. These data reflect an activation of B lymphocytes in PCM patients.

Can malaria-associated polyclonal B-lymphocyte activation interfere with the development of anti-sporozoite specific immunity?

To study the relevance of polyclonal B cell activation (PBA) associated with malaria in the development of specific anti-sporozoite immunity, we used a reverse haemolytic plaque assay and an immunoradiometric assay employing the synthetic peptide (NANP)3, the main epitope of the circumsporozoite (CS) protein of Plasmodium falciparum, to assess respectively the degree of activation of IgG and IgM secreting cells and the level of anti-sporozoite antibodies in 95 subjects with malaria and 21 non-infected individuals. A positive correlation was observed between the anti-(NANP)3 antibody levels and the number of past attacks of malaria but not between the former and the age of individuals or the number of months of residence in the endemic region. Individuals with high numbers of IgG or IgM secreting cells (SC) had lower levels of anti-(NANP)3 antibodies; those with levels of antibodies above the mean for malaria-infected individuals had lower numbers of IgGSC and higher haematocrit and haemoglobin values. These data show the existence of a negative relationship between malaria-induced PBA and anti-sporozoite immunity, and it is suggested that either PBA blocks the development of anti-sporozoite immunity or, alternatively, the latter protects individuals against malaria and malaria-associated PBA.