Characterization of immunoglobulin G antibodies to Plasmodium falciparum sporozoite surface antigen MB2 in malaria exposed individuals

<p>Abstract</p> <p>Background</p> <p>MB2 protein is a sporozoite surface antigen on the human malaria parasite <it>Plasmodium falciparum</it>. MB2 was identified by screening a <it>P. falciparum </it>sporozoite cDNA expression library using immune sera from a protected donor immunized via the bites of <it>P. falciparum</it>-infected irradiated mosquitoes. It is not known whether natural exposure to <it>P. falciparum </it>also induces the anti-MB2 response and if this response differs from that in protected individuals immunized via the bites of <it>P. falciparum </it>infected irradiated mosquitoes. The anti-MB2 antibody response may be part of a robust protective response against the sporozoite.</p> <p>Methods</p> <p>Fragments of polypeptide regions of MB2 were constructed as recombinant fusions sandwiched between glutathione S-transferase and a hexa histidine tag for bacterial expression. The hexa histidine tag affinity purified proteins were used to immunize rabbits and the polyclonal sera evaluated in an <it>in vitro </it>inhibition of sporozoite invasion assay. The proteins were also used in immunoblots with sera from a limited number of donors immunized via the bites of <it>P. falciparum </it>infected irradiated mosquitoes and plasma and serum obtained from naturally exposed individuals in Kenya.</p> <p>Results</p> <p>Rabbit polyclonal antibodies targeting the non-repeat region of the basic domain of MB2 inhibited sporozoites entry into HepG2-A16 cells <it>in vitro</it>. Analysis of serum from five human volunteers that were immunized via the bites of <it>P. falciparum </it>infected irradiated mosquitoes that developed immunity and were completely protected against subsequent challenge with non-irradiated parasite also had detectable levels of antibody against MB2 basic domain. In contrast, in three volunteers not protected, anti-MB2 antibodies were below the level of detection. Sera from protected volunteers preferentially recognized a non-repeat region of the basic domain of MB2, whereas plasma from naturally-infected individuals also had antibodies that recognize regions of MB2 that contain a repeat motif in immunoblots. Sequence analysis of eleven field isolates and four laboratory strains showed that these antigenic regions of the basic domain of the <it>MB2 </it>gene are highly conserved in parasites obtained from different parts of the world. Moreover, anti-MB2 antibodies also were detected in the plasma of 83% of the individuals living in a malaria endemic area of Kenya (n = 41).</p> <p>Conclusion</p> <p>A preliminary analysis of the human humoral response against MB2 indicates that it may be an additional highly conserved target for immune intervention at the pre-erythrocytic stage of <it>P. falciparum </it>life cycle.</p

‘I can die today, I can die tomorrow’: lay perceptions of sickle cell disease in Kumasi, Ghana at a point of transition

Objective. To describe the lay meanings of sickle cell disease (SCD) in the Ashanti region of Ghana

Detection of PIGO-Deficient Cells Using Proaerolysin: A Valuable Tool to Investigate Mechanisms of Mutagenesis in the DT40 Cell System

While isogenic DT40 cell lines deficient in DNA repair pathways are a great tool to understand the DNA damage response to genotoxic agents by a comparison of cell toxicity in mutants and parental DT40 cells, no convenient mutation assay for mutagens currently exists for this reverse-genetic system. Here we establish a proaerolysin (PA) selection-based mutation assay in DT40 cells to identify glycosylphosphatidylinositol (GPI)-anchor deficient cells. Using PA, we detected an increase in the number of PA-resistant DT40 cells exposed to MMS for 24 hours followed by a 5-day period of phenotype expression. GPI anchor synthesis is catalyzed by a series of phosphatidylinositol glycan complementation groups (PIGs). The PIG-O gene is on the sex chromosome (Chromosome Z) in chicken cells and is critical for GPI anchor synthesis at the intermediate step. Among all the mutations detected in the sequence levels observed in DT40 cells exposed to MMS at 100 µM, we identified that ∼55% of the mutations are located at A:T sites with a high frequency of A to T transversion mutations. In contrast, we observed no transition mutations out of 18 mutations. This novel assay for DT40 cells provides a valuable tool to investigate the mode of action of mutations caused by reactive agents using a series of isogenic mutant DT40 cells

A Role for Fetal Hemoglobin and Maternal Immune IgG in Infant Resistance to Plasmodium falciparum Malaria

In Africa, infant susceptibility to Plasmodium falciparum malaria increases substantially as fetal hemoglobin (HbF) and maternal immune IgG disappear from circulation. During the first few months of life, however, resistance to malaria is evidenced by extremely low parasitemias, the absence of fever, and the almost complete lack of severe disease. This resistance has previously been attributed in part to poor parasite growth in HbF-containing red blood cells (RBCs). A specific role for maternal immune IgG in infant resistance to malaria has been hypothesized but not yet identified.We found that P. falciparum parasites invade and develop normally in fetal (cord blood, CB) RBCs, which contain up to 95% HbF. However, these parasitized CB RBCs are impaired in their binding to human microvascular endothelial cells (MVECs), monocytes, and nonparasitized RBCs--cytoadherence interactions that have been implicated in the development of high parasite densities and the symptoms of malaria. Abnormal display of the parasite's cytoadherence antigen P. falciparum erythrocyte membrane protein-1 (PfEMP-1) on CB RBCs accounts for these findings and is reminiscent of that on HbC and HbS RBCs. IgG purified from the plasma of immune Malian adults almost completely abolishes the adherence of parasitized CB RBCs to MVECs.Our data suggest a model of malaria protection in which HbF and maternal IgG act cooperatively to impair the cytoadherence of parasitized RBCs in the first few months of life. In highly malarious areas of Africa, an infant's contemporaneous expression of HbC or HbS and development of an immune IgG repertoire may effectively reconstitute the waning protective effects of HbF and maternal immune IgG, thereby extending the malaria resistance of infancy into early childhood

Molecular Characterization of HIV-1 CRF01_AE in Mekong Delta, Vietnam, and Impact of T-Cell Epitope Mutations on HLA Recognition (ANRS 12159)

To date, 11 HIV-1 subtypes and 48 circulating recombinant forms have been described worldwide. The underlying reason why their distribution is so heterogeneous is not clear. Host genetic factors could partly explain this distribution. The aim of this study was to describe HIV-1 strains circulating in an unexplored area of Mekong Delta, Vietnam, and to assess the impact of optimal epitope mutations on HLA binding.We recruited 125 chronically antiretroviral-naive HIV-1-infected subjects from five cities in the Mekong Delta. We performed high-resolution DNA typing of HLA class I alleles, sequencing of Gag and RT-Prot genes and phylogenetic analysis of the strains. Epitope mutations were analyzed in patients bearing the HLA allele restricting the studied epitope. Optimal wild-type epitopes from the Los Alamos database were used as reference. T-cell epitope recognition was predicted using the immune epitope database tool according to three different scores involved in antigen processing (TAP and proteasome scores) and HLA binding (MHC score). with a Vietnamese specificity held by two different haplotypes. The percentage of homology between Mekong and B consensus HIV-1 sequences was above 85%. Divergent epitopes had TAP and proteasome scores comparable with wild-type epitopes. MHC scores were significantly lower in divergent epitopes with a mean of 2.4 (±0.9) versus 2 (±0.7) in non-divergent ones (p<0.0001).Our study confirms the wide predominance of CRF01_AE in the Mekong Delta where patients harbor a specific HLA pattern. Moreover, it demonstrates the lower MHC binding affinity among divergent epitopes. This weak immune pressure combined with a narrow genetic diversity favors immune escape and could explain why CRF01_AE is still predominant in Vietnam, particularly in the Mekong area

Haptoglobin and Sickle Cell Polymorphisms and Risk of Active Trachoma in Gambian Children

BACKGROUND: Susceptibility and resistance to trachoma, the leading infectious cause of blindness, have been associated with a range of host genetic factors. In vitro studies of the causative organism, Chlamydia trachomatis, demonstrate that iron availability regulates its growth, suggesting that host genes involved in regulating iron status and/or availability may modulate the risk of trachoma. The objective was to investigate whether haptoglobin (Hp) haplotypes constructed from the functional polymorphism (Hp1/Hp2) plus the functional promoter SNPs -61A-C (rs5471) and -101C-G (rs5470), or sickle cell trait (HbAS, rs334) were associated with risk of active trachoma when stratified by age and sex, in rural Gambian children. METHODOLOGY AND PRINCIPAL FINDINGS: In two cross sectional surveys of children aged 6-78 months (n = 836), the prevalence of the clinical signs of active trachoma was 21.4%. Within boys, haplotype E (-101G, -61A, Hp1), containing the variant allele of the -101C-G promoter SNP, was associated with a two-fold increased risk of active trachoma (OR = 2.0 [1.17-3.44]). Within girls, an opposite association was non-significant (OR = 0.58 [0.32-1.04]; P = 0.07) and the interaction by sex was statistically significant (P = 0.001). There was no association between trachoma and HbAS. CONCLUSIONS: These data indicate that genetic variation in Hp may affect susceptibility to active trachoma differentially by sex in The Gambia

Intra- and Inter-clade Cross-reactivity by HIV-1 Gag Specific T-Cells Reveals Exclusive and Commonly Targeted Regions: Implications for Current Vaccine Trials

The genetic diversity of HIV-1 across the globe is a major challenge for developing an HIV vaccine. To facilitate immunogen design, it is important to characterize clusters of commonly targeted T-cell epitopes across different HIV clades. To address this, we examined 39 HIV-1 clade C infected individuals for IFN-γ Gag-specific T-cell responses using five sets of overlapping peptides, two sets matching clade C vaccine candidates derived from strains from South Africa and China, and three peptide sets corresponding to consensus clades A, B, and D sequences. The magnitude and breadth of T-cell responses against the two clade C peptide sets did not differ, however clade C peptides were preferentially recognized compared to the other peptide sets. A total of 84 peptides were recognized, of which 19 were exclusively from clade C, 8 exclusively from clade B, one peptide each from A and D and 17 were commonly recognized by clade A, B, C and D. The entropy of the exclusively recognized peptides was significantly higher than that of commonly recognized peptides (p = 0.0128) and the median peptide processing scores were significantly higher for the peptide variants recognized versus those not recognized (p = 0.0001). Consistent with these results, the predicted Major Histocompatibility Complex Class I IC50 values were significantly lower for the recognized peptide variants compared to those not recognized in the ELISPOT assay (p<0.0001), suggesting that peptide variation between clades, resulting in lack of cross-clade recognition, has been shaped by host immune selection pressure. Overall, our study shows that clade C infected individuals recognize clade C peptides with greater frequency and higher magnitude than other clades, and that a selection of highly conserved epitope regions within Gag are commonly recognized and give rise to cross-clade reactivities