Evaluating seroprevalence to circumsporozoite protein to estimate exposure to three species of Plasmodium in the Brazilian Amazon

Background:Brazil has seen a great decline in malaria and the country is moving towards elimination.However,for eventual elimination,the control program needs efficient tools in order to monitor malaria exposure and transmission.In this study,we aimed to evaluate whether seroprevalence to the circumsporozoite protein(CSP)is a good tool for monitoring the exposure to and/or evaluating the burden and distribution of Plasmodium species in the Brazilian Amazon.Methods:Cross-sectional surveys were conducted in a rural area of Porto Velho,Rondônia state.Parasite infection was detected by microscopy and polymerase chain reaction.Antibodies to the sporozoite CSP repeats of Plasmodium vivax,P.falciparum,and P.malariae(PvCS,PfCS,and PmCS)were detected using the enzyme-linked immunosorbent assay technique.Human leukocyte antigen(HLA)-DRB1 and DQB1 genes were typed using Luminex®xMAP®technology.Results:The prevalence of immunoglobulin G against P.vivax CSP peptide(62%)was higher than P.falciparum(49%)and P.malariae(46%)CSP peptide.Most of the studied individuals had antibodies to at least one of the three peptides(72%),34%had antibodies to all three peptides and 28%were non-responders.Although the majority of the population was not infected at the time of the survey,74.3%of parasite-negative individuals had antibodies to at least one of the CSPs.Importantly,among individuals carrying the haplotypes DRB1*04~DQB1*03,there was a significantly higher frequency of PfCS responders,and DRB1*16~DQB1*03 haplotype for PvCS and PfCS responders.In contrast,HLA-DRB1*01 and HLA-DQB1*05 allelic groups were associated with a lack of antibodies to P.vivax and P.falciparum CSP repeats,and the haplotype DRB1*01~DQB1*05 was also associated with non-responders,including non-responders to P.malariae.Conclusions:Our results show that in low transmission settings,naturally acquired antibody responses against the CSP repeats of P.vivax,P.falciparum,and P.malariae in a single cross-sectional study may not represent a valuable marker for monitoring recent malaria exposure,especially in an area with a high prevalence of P.vivax.Furthermore,HLA class II molecules play an important role in antibody response and require further study with a larger sample size.It will be of interest to consider HLA analysis when using serosurveillance to monitor malaria exposure among genetically diverse populations.

Immune responses to the expressed products of the CSP antigen gene of Plasmodi um falciparum southern China isolate FCC1/HN in Hela cell

Objective To construct a eukaryotic expression system with pcDNA3-PfCSP/Hela for the Circumsporozoite protein (CSP) gene of Plasmodium falciparum (P. falciparum), to observe the immune responses in BALB/c mice induced by the expressed proteins. Methods The recombinant plasmid pcDNA3-PfCSP was transformed into the Hela cell line. The expressed protein was isolated and analyzed by using SDS-PAGE and used for immunization of BALB/c mice by subcutaneous, intravenous, and intraperitoneal adminstration. Enzyme-linked immunosorbent assay(ELISA), Dot-ELISA, Western blot, T lymphocyte proliferation test, natural killer cell(NKC) activity assay, and CD4+ and CD8+ T cell detection were used for observation of humoral and cellular immune responses. Results Immune sera strongly reacted with the expressed protein, antibody titer was up to 1∶6400 as detected by ELISA. Western blot analysis revealed a specific band at 38.3?Kda. When the spleen cells of normal and immunized BALB/c mice were specifically stimulated with expressed protein, the optical densities were 0.12±0.03 and 0.34±0.04, respectively. The latter were significantly higher than the former (P<0.01). We used the MTT colorimetric assay to measure NKC activity of mice spleen. The results showed that the NKC activity of immunized BALB/c mice was remarkably higher than that of the controls (P<0.05). CD4+ and CD8+ T cells were detected by using monoclonal antibody immunofluorescence methods. The results showed that the percentage of CD4+ and CD8+ T cells of immunized group were significantly higher than that of control group (P<0.05).Conclusions The humoral and cell-mediated immune responses and elevated NKC activity to products made with a eukaryotic expression system could be specifically detected in BALB/c mice. These findings indicate that the expressed protein could enhance the immune function in mice.