Chiaka I. Anumudu

The Human Immune Response to Plasmodium falciparum Includes Both Antibodies That Inhibit Merozoite Surface Protein 1 Secondary Processing and Blocking Antibodies

ABSTRACT Malaria merozoite surface protein 1 (MSP1) is cleaved in an essential step during erythrocyte invasion. The responses of children to natural malaria infection included antibodies that inhibit this cleavage and others that block the binding of these inhibitory antibodies. There was no correlation between the titer of the antibody to the 19-kDa fragment of MSP1 and its inhibitory activity. These findings have implications for the design of MSP1-based vaccines.

Developmental Regulation of Genes encoding Universal stress proteins in Schistosoma mansoni

The draft nuclear genome sequence of the snail-transmitted, dimorphic, parasitic, platyhelminth Schistosoma mansoni revealed eight genes encoding proteins that contain the Universal Stress Protein (USP) domain. Schistosoma mansoni is a causative agent of human schistosomiasis, a severe and debilitating Neglected Tropical Disease (NTD) of poverty, which is endemic in at least 76 countries. The availability of the genome sequences of Schistosoma species presents opportunities for bioinformatics and genomics analyses of associated gene families that could be targets for understanding schistosomiasis ecology, intervention, prevention and control. Proteins with the USP domain are known to provide bacteria, archaea, fungi, protists and plants with the ability to respond to diverse environmental stresses. In this research investigation, the functional annotations of the USP genes and predicted nucleotide and protein sequences were initially verified. Subsequently, sequence clusters and distinctive features of the sequences were determined. A total of twelve ligand binding sites were predicted based on alignment to the ATP-binding universal stress protein from Methanocaldococcus jannaschii. In addition, six USP sequences showed the presence of ATP-binding motif residues indicating that they may be regulated by ATP. Public domain gene expression data and RT-PCR assays confirmed that all the S. mansoni USP genes were transcribed in at least one of the developmental life cycle stages of the helminth. Six of these genes were up-regulated in the miracidium, a free-swimming stage that is critical for transmission to the snail intermediate host. It is possible that during the intra-snail stages, S. mansoni gene transcripts for universal stress proteins are low abundant and are induced to perform specialized functions triggered by environmental stressors such as oxidative stress due to hydrogen peroxide that is present in the snail hemocytes. This report serves to catalyze the formation of a network of researchers to understand the function and regulation of the universal stress proteins encoded in genomes of schistosomes and their snail intermediate hosts.

A cross-sectional study on urogenital schistosomiasis in children; haematuria and proteinuria as diagnostic indicators in an endemic rural area of Nigeria

BACKGROUND Rapid and accurate diagnosis is necessary for the management of schistosomiasis in endemic areas. OBJECTIVE To assess the burden of urogenital schistosomiasis and the diagnostic efficiency of morbidity indicators of the disease in an endemic rural community of Nigeria. METHODS A cross-sectional school-based study was conducted. Urine samples of 487 pupils were screened microscopically for S. haematobium and tested for haematuria and proteinuria using chemical reagent strips. RESULTS The prevalence and intensity of infection were 57.1% and 45.0 eggs/10 mL urine respectively. Prevalence of infection in male (54.1%) and female (60.3%) individuals showed no significant variation (P>0.05). However, prevalence of infection was age dependent with those in age groups 3-5 and 12-14 years having the least and highest prevalence of infection respectively (P<0.05). Microhaematuria and proteinuria varied significantly with ages of the pupils with least (14.0, 40.0%) and highest (60.0, 80.0%) prevalence recorded in age groups 3-5 and 15-19 years respectively (P<0.05). Proteinuria showed higher sensitivity (80.3%) compared to microhaematuria (73.3%). CONCLUSION Schistosomiasis is highly endemic in the study area and the use of microhaematuria and proteinuria for mapping the infected population prior treatment could be adopted.

Angiotensin II type 1 receptor A1166C gene polymorphism and essential hypertension in Calabar and Uyo cities, Nigeria.

OBJECTIVES: The angiotensin II protein is a vasoconstrictor that exerts most of its influence through the angiotensin II type 1 receptor (AT1R). Inconsistent association between the A1166C polymorphism of the AT1R gene and hypertension has been reported among various populations but not among the peoples of Calabar and Uyo. This study was designed to determine the frequency of the A1166C polymorphism of the AT1R gene and its association with hypertension in a sample population of Calabar and Uyo. MATERIALS AND METHODS: A population-based case control design consisting of total of 1224 participants, 612 each of patients and controls were randomly recruited from hypertension clinics and the general population. Genotyping of the A1166C allele of the AT1R gene to identify variants was performed using polymerase chain reaction and restriction enzyme digestion. Multiple regressions were applied to test whether the A1166 genotypes were predictors of hypertension. RESULTS: 99% of the study population had the wild type AA genotype, and 1% was AC heterozygous carriers of the A1166C polymorphism. CONCLUSION: The A1166C polymorphism was not a predictor of hypertension in the sample population of Calabar and Uyo.

Antibody specificities of children living in a malaria endemic area to inhibitory and blocking epitopes on MSP-119 of Plasmodium falciparum

Merozoite surface protein-1(19) (MSP-1(19)) specific antibodies which include processing inhibitory, blocking and neutral antibodies have been identified in individuals exposed to Plasmodium falciparum. Here we intend to look at the effect of single and multiple amino acid substitutions of MSP-1(19) on the recognition by polyclonal antibodies from children living in Igbo-Ora, Nigeria. This would provide us with information on the possibility of eliciting mainly processing inhibitory antibodies with a recombinant MSP-1(19) vaccine. Blood was collected from children in the rainy season and binding of anti-MSP-1(19) antibodies to modified mutants of MSP-1(19) was analysed by ELISA. The MSP-1(19) mutant proteins with single substitutions at positions 22 (Leu-->Arg), 43 (Glu-->Leu) and 53 (Asn-->Arg) and the MSP-1(19) mutant protein with multiple substitutions at positions 27+31+34+43 (Glu-->Tyr, Leu-->Arg, Tyr-->Ser, Glu-->Leu); which had inhibitory epitopes; had the highest recognition. Children recognised both sets of mutants with different age groups having different recognition levels. The percentage of malaria positive individuals (32-80%) with antibodies that bound to the mutants MSP-1(19) containing epitopes that recognise only processing inhibitory and not blocking antibodies, were significantly different from those with antibodies that did not bind to these mutants (21-28%). The amino acid substitutions that abolished the binding of blocking antibodies without affecting the binding of inhibitory antibodies are of particular interest in the design of MSP-1(19) based malaria vaccines. Although these MSP-1(19) mutants have not been found in natural population, their recognition by polyclonal antibodies from humans naturally infected with malaria is very promising for the future use of MSP-1(19) mutants in the design of a malaria vaccine.

The microbiome in urogenital schistosomiasis and induced bladder pathologies

Background Human schistosomiasis is a highly prevalent neglected tropical disease (NTD) caused by Schistosoma species. Research on the molecular mechanisms influencing the outcomes of bladder infection by Schistosoma haematobium is urgently needed to develop new diagnostics, therapeutics and infection prevention strategies. The objective of the research study was to determine the microbiome features and changes in urine during urogenital schistosomiasis and induced bladder pathologies. Methodology Seventy participants from Eggua, southwestern Nigeria provided morning urine samples and were screened for urogenital schistosomiasis infection and bladder pathologies in a cross-sectional study. Highthroughput NGS sequencing was carried out, targeting the 16S V3 region. Filtered reads were processed and analyzed in a bioinformatics pipeline. Principal findings The study participants (36 males and 34 females, between ages 15 and 65) were categorized into four groups according to status of schistosomiasis infection and bladder pathology. Data analytics of the next-generation sequencing reads revealed that Proteobacteria and Firmicutes dominated and had influence on microbiome structure of both non-infected persons and persons with urogenital schistosomiasis. Furthermore, gender and age influenced taxa abundance independent of infection or bladder pathology. Several taxa distinguished urogenital schistosomiasis induced bladder pathologies from urogenital schistosomiasis infection alone and from healthy persons, including known immune-stimulatory taxa such as Fusobacterium, Sphingobacterium and Enterococcus. Some of these significant taxa, especially Sphingobacterium were projected as markers of infection, while several genera including potentially beneficial taxa such as Trabulsiella and Weissella, were markers of the non-infected. Finally, expected changes in protein functional categories were observed to relate to cellular maintenance and lipid metabolism. Conclusion The urinary microbiome is a factor to be considered in developing biomarkers, diagnostic tools, and new treatment for urogenital schistosomiasis and induced bladder pathologies.

Fine specificity of anti-MSP119 antibodies and multiplicity of Plasmodium falciparum Merozoite Surface Protein 1 types in individuals in Nigeria with sub-microscopic infection

BackgroundThe absence of antibodies specific for the 19 kDa C-terminal domain of merozoite surface protein 1 (MSP119) has been associated with high-density malaria parasitaemia in African populations. The hypothesis that a high prevalence and/or level of anti-MSP119 antibodies that may inhibit erythrocyte invasion would be present in apparently healthy individuals who harbour a sub-microscopic malaria infection was tested in this study.MethodsPlasma samples were collected from residents in a region in Nigeria hyperendemic for malaria, who had no detectable parasitaemia by microscopy. Using a competition-based enzyme-linked-immunosorbent assay with two invasion-inhibitory monoclonal antibodies (mAbs) 12.10 and 12.8, the levels and prevalence of specific antibodies were measured. The minimum multiplicity of infection was determined using PCR. The prevalence of anaemia was also measured.ResultsPlasma samples from 85% of individuals contained antibodies that bound to MSP119. The inhibition of mAb 12.10 binding was strongly correlated with the prevalence (Spearman correlation test, p < 0.0001) and mean titre of anti-MSP119 antibodies (Spearman correlation test, p < 0.001) in the samples. Comparing samples from individuals with multiple infection (group M) and single infection (Group S), group M contained a higher (p = 0.04) prevalence of anti-MSP119 antibodies that competed with mAb 12.10. Using a logistic regression model, it was found that the presence of antibodies competitive with mAb 12.10 was affected negatively by anaemia (p = 0.0016) and positively by the carriage of multiple parasite genotypes (p = 0.04).ConclusionsIn the search for correlates of protection against malaria, which will be essential to evaluate clinical trials of malaria vaccines based on MSP1, this study examines some potential assays and the factors that need to taken into account during their evaluation, using samples from individuals naturally exposed to malaria infection.

Cellular responses to modified Plasmodium falciparum MSP119 antigens in individuals previously exposed to natural malaria infection

BackgroundMSP1 processing-inhibitory antibodies bind to epitopes on the 19 kDa C-terminal region of the Plasmodium falciparum merozoite surface protein 1 (MSP119), inhibiting erythrocyte invasion. Blocking antibodies also bind to this antigen but prevent inhibitory antibodies binding, allowing invasion to proceed. Recombinant MSP119 had been modified previously to allow inhibitory but not blocking antibodies to continue to bind. Immunization with these modified proteins, therefore, has the potential to induce more effective protective antibodies. However, it was unclear whether the modification of MSP119 would affect critical T-cell responses to epitopes in this antigen.MethodsThe cellular responses to wild-type MSP119 and a panel of modified MSP119 antigens were measured using an in-vitro assay for two groups of individuals: the first were malaria-naïve and the second had been naturally exposed to Plasmodium falciparum infection. The cellular responses to the modified proteins were examined using cells from malaria-exposed infants and adults.ResultsInterestingly, stimulation indices (SI) for responses induced by some of the modified proteins were at least two-fold higher than those elicited by the wild-type MSP119. A protein with four amino acid substitutions (Glu27→Tyr, Leu31→Arg, Tyr34→Ser and Glu43→Leu) had the highest stimulation index (SI up to 360) and induced large responses in 64% of the samples that had significant cellular responses to the modified proteins.ConclusionThis study suggests that specific MSP119 variants that have been engineered to improve their antigenicity for inhibitory antibodies, retain T-cell epitopes and the ability to induce cellular responses. These proteins are candidates for the development of MSP1-based malaria vaccines.

Genetic diversity of Plasmodium falciparum isolates from naturally infected children in north-central Nigeria using the merozoite surface protein-2 as molecular marker

OBJECTIVE To characterize the genetic diversity of Plasmodium falciparum (P. falciparum) field isolates in children from Lafia, North-central Nigeria, using the highly polymorphic P. falciparum merozoite surface protein 2 (MSP-2) gene as molecular marker. METHODS Three hundred and twenty children were enrolled into the study between 2005 and 2006. These included 140 children who presented with uncomplicated malaria at the Dalhatu Araf Specialist Hospital, Lafia and another 180 children from the study area with asymptomatic infection. DNA was extracted from blood spot on filter paper and MSP-2 genes were genotyped using allele-specific nested PCR in order to analyze the genetic diversity of parasite isolates. RESULTS A total of 31 and 34 distinct MSP-2 alleles were identified in the asymptomatic and uncomplicated malaria groups respectively. No difference was found between the multiplicity of infection in the asymptomatic group and that of the uncomplicated malaria group (P>0.05). However, isolates of the FC27 allele type were dominant in the asymptomatic group whereas isolates of the 3D7 allele type were dominant in the uncomplicated malaria group. CONCLUSIONS This study showed a high genetic diversity of P. falciparum isolates in North-central Nigeria and is comparable to reports from similar areas with high malaria transmission intensity.

Acquisition, maintenance and adaptation of invasion inhibitory antibodies against Plasmodium falciparum invasion ligands involved in immune evasion

Erythrocyte-binding antigens (EBAs) and P. falciparum reticulocyte-binding homologue proteins (PfRhs) are two important protein families that can vary in expression and utilization by P. falciparum to evade inhibitory antibodies. We evaluated antibodies at repeated time-points among individuals living in an endemic region in Nigeria over almost one year against these vaccine candidates. Antibody levels against EBA140, EBA175, EBA181, PfRh2, PfRh4, and MSP2, were measured by ELISA. We also used parasites with disrupted EBA140, EBA175 and EBA181 genes to show that all these were targets of invasion inhibitory antibodies. However, antigenic targets of inhibitory antibodies were not stable and changed substantially over time in most individuals, independent of age. Antibodies levels measured by ELISA also varied within and between individuals over time and the antibodies against EBA181, PfRh2 and MSP2 declined more rapidly in younger individuals (≤15 years) compared with older (>15). The breadth of high antibody responses over time was more influenced by age than by the frequency of infection. High antibody levels were associated with a more stable invasion inhibitory response, which could indicate that during the long process of formation of immunity, many changes not only in levels but also in functional responses are needed. This is an important finding in understanding natural immunity against malaria, which is essential for making an efficacious vaccine.