Roseangela I. Nwuba

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.

Purification and characterisation of an extracellularly released protease of Trypanosoma brucei

Abstract Thrombocytopaenia, or platelet aggregation, is a serious complication of African trypanosomiasis. The biochemical basis is not clearly known. Proteases are known potent inducers of blood coagulation and platelet aggregation, and unknown factors released by Trypanosoma brucei have been shown to induce platelet aggregation. In attempts to define the biochemical mechanisms involved in thrombocytopaenia we purified and characterised a major proteolytic enzyme released extracellularly by T. brucei. Actively motile trypanosomes released proteins into the medium (phosphate/saline/glucose, pH 8.0) in which the organisms were incubated in vitro. The Mr of the released polypeptides ranged from 15 to >200 kDa, amongst which are proteases. One of the major protein bands, a 250 kDa protease, was purified to homogeneity by ammonium sulfate precipitation followed by diethylaminoethyl (DEAE)-cellulose chromatography and Sephacryl S-300 gel filtration. The protease migrated as a single band of 63 kDa upon electrophoresis in both denaturing and non-denaturing gel co-polymerised with gelatin. The enzyme was strongly active against Z-ARR-AFC peptide substrate, with a pH optimum of 7.0. The proteolytic activity was enhanced by dithiothreitol and inhibited by E-64, leupeptin, TPCK and antipain. The released proteolytic enzyme is putatively identified as a cathepsin B-like cysteine protease.

Subclass responses and their half-lives for antibodies against EBA175 and PfRh2 in naturally acquired immunity against Plasmodium falciparum malaria.

BackgroundPlasmodium falciparum EBA175 and PfRh2 belong to two main families involved in parasite invasion, and both are potential vaccine candidates. Current knowledge is limited regarding which target antigens and subclasses of antibodies are actually important for protection, and how naturally acquired immunity is achieved.MethodsRepeated blood samples were collected from individuals in Nigeria over a period of almost one year. ELISA was used to analyse subclasses of IgG responses.ResultsFor both EBA175 (region III-V) and (a fragment of) PfRh2, the dominant antibody responses consisted of IgG1 and IgG3 followed by IgG2, while for PfRh2 there was also a relatively prominent response for IgG4. High levels of IgG1, IgG2 and IgG3 for EBA175 and total IgG for PfRh2 correlated significantly with a lower parasitaemia during the study period. Children with HbAS had higher levels of some subclasses compared to children with HbAA, while in adults the pattern was the opposite. The half-lives of IgG2 and IgG4 against EBA175 were clearly shorter than those for IgG1 and IgG3.ConclusionEBA175 and PfRh2 are potential targets for protective antibodies since both correlated with lower parasitaemia. The shorter half-lives for IgG2 and IgG4 might explain why these subclasses are often considered less important in protection against malaria. Triggering the right subclass responses could be of critical importance in a successful vaccine. Further studies are needed to evaluate the role of haemoglobin polymorphisms and their malaria protective effects in this process.

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.

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.

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.

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.

Factors influencing the induction of high affinity antibodies to Plasmodium falciparum merozoite antigens and how affinity changes over time

Understanding the functional characteristics of naturally acquired antibodies against P. falciparum merozoite antigens is crucial for determining the protective functions of antibodies. Affinity (measured as kd) of naturally acquired antibodies against two key targets of acquired immunity, EBA175 and PfRh2, was determined using Surface Plasmon Resonance (SPR) in a longitudinal survey in Nigeria. A majority of the participants, 79% and 67%, maintained stable antibody affinities to EBA175 and PfRh2, respectively, over time. In about 10% of the individuals, there was a reciprocal interaction with a reduction over time in antibody affinity for PfRh2 and an increase for EBA175. In general, PfRh2 elicited antibodies with higher affinity compared to EBA175. Individuals with higher exposure to malaria produced antibodies with higher affinity to both antigens. Younger individuals (5–15 years) produced comparable or higher affinity antibodies than adults (>15 years) against EBA175, but not for PfRh2. Correlation between total IgG (ELISA) and affinity varied between individuals, but PfRh2 elicited antibodies with a higher correlation in a majority of the participants. There was also a correlation between antibody inhibition of erythrocyte invasion by merozoites and PfRh2 affinity. This work gives new insights into the generation and maintenance of antibody affinity over time.

Efficacy of Lophira alata Leaf Extract and its Combination with Artesunate in Mice Prior Exposed to Plasmodium berghei

Enhanced antimalarial activity of plant extracts used for treatment of malaria in endemic areas is attributed to partial immunity gained by prior infection. This suggests synergy between immunity and extract activity in treatment. Testing this hypothesis, rodent malaria was used to determine efficacy of Lophira alata leaf extracts in treating malaria in prior infected mice. One round of P. berghei infection and Pyrimethamine drug-cure was used to establish partial immunity in mice. Previously Exposed Mice (PEM) and Previously Unexposed Mice (PUM) mice challenged with P. berghei were used to determine influence of partial antimalarial immunity on efficacy of L. alata leaf extracts, administered alone or in combination with Artesunate (ART) in malaria treatment. There was a significant reduction in parasitemia in PEM when compared to PUM animals (P<0.001) irrespective of treatment regimen. Administration of L. alata combined with ART significantly reduced parasitemia (P<0.0032) and prolonged (P=0.0109) survival than when L. alata was administered alone in infected mice. These findings suggest that the action of L. alata in treating malaria infections in a murine model is enhanced by prior exposure to the malaria parasite. Thus the requirements of using plants in treating malaria in endemic populations may differ for those used in western systems, where trials are carried out with non-immune cohorts. Combining artemisinin derivatives and medicinal plants in malaria exposed populations may provide an alternative control measure in endemic regions and may justify the continued use of these plants by indigenous populations in treating malaria.