Hepatitis B virus-like particles expressing Plasmodium falciparum epitopes induce complement-fixing antibodies against the circumsporozoite protein

The repetitive structure of compact virus-like particles (VLPs) provides high density displays of antigenic sequences, which trigger key parts of the immune system. The hepatitis B virus (HBV) and human papilloma virus (HPV) vaccines exploit the assembly competence of structural proteins, which are the effective immunogenic components of the prophylactic HBV and HPV vaccines, respectively. To optimize vaccine designs and to promote immune responses against protective epitopes, the “Asp-Ala-Asp-Pro” (NANP)-repeat from the Plasmodium falciparum circumsporozoite protein (CSP) was expressed within the exposed, main antigenic site of the small HBV envelope protein (HBsAgS); this differs from the RTS,S vaccine, in which CSP epitopes are fused to the N-terminus of HBsAgS. The chimeric HBsAgS proteins are assembly competent, produce VLPs, and provide a high antigenic density of the NANP repeat sequence. Chimeric VLPs with four or nine NANP-repeats (NANP4 and NANP9, respectively) were expressed in mammalian cells, the HBsAgS- and CSP-specific antigenicity of the VLPs was determined, and the immunogenicity of the VLPs assessed in relation to the induction of anti-HBsAgS and anti-CSP antibody responses. The chimeric VLPs induced high anti-CSP titres in BALB/c mice independent of the number of the NANP repeats. However, the number of NANP repeats influenced the activity of vaccine-induced antibodies measured by complement fixation to CSP, one of the proposed effector mechanisms for Plasmodium neutralization in vivo. Sera from mice immunized with VLPs containing nine NANP repeats performed better in the complement fixation assay than the group with four NANP repeats. The effect of the epitope-specific density on the antibody quality may instruct VLP platform designs to optimize immunological outcomes and vaccine efficacy.     

Natural selection maintains a stable polymorphism at the circumsporozoite protein locus of Plasmodium falciparum in a low endemic area

Circumsporozoite protein gene sequences of Plasmodium falciparum were collected in 1996–1997 and in 2006–2007 from a single endemic area in Thailand. Repeat units were more similar within the same haplotype than between haplotypes, supporting the hypothesis that repeat arrays evolve by a process of concerted evolution. There was evidence that natural selection has favored amino acid changes in the Th2R and Th3R T-cell epitope regions. One haplotype in these epitopes, designated *5/*1, occurred in approximately 70% of sequences in both collection periods. The most common other haplotypes differed from *5/*1 by at least two amino acid replacements; and divergence in the epitopes was correlated with divergence in the repeats. These patterns are most consistent with balancing selection driven by interactions with the immune system of the vertebrate host, probably involving both T-cell recognition of the Th2R and Th3R epitopes and antibody responses to the repeats.     

Circumsporozoite antibodies and falciparum malaria incidence in children living in a malaria endemic area

In a case—control study we examined the association of Plasmodium falciparum circumsporozoite antibodies (anti-R32tet32) with subsequent P. falciparum infections. A study population of 140 children living in an endemic area was followed longitudinally for 25 weeks with weekly blood smears for malaria parasites and, once every two weeks, serum samples for circumsporozoite antibody determinations. From the malaria cases, antibody measurements occurring between two and six weeks prior to the onset of parasitaemia were utilized. For each case, two controls were selected. The results from 17 cases and 34 controls failed to show a statistically significant difference in antibody levels prior to the infection (P=0.07, one-tailed Student''s t-test). However, 8 of the 17 cases had antibody present, indicating a level that was not protective against patent infection.     

Recognition of dominant T cell-stimulating epitopes from the circumsporozoite protein of Plasmodium falciparum and relationship to malaria morbidity in Gambian children.

Cellular immune responses to the Plasmodium falciparum circumsporozoite (CS) protein were measured by proliferation and interferon-gamma production in a cohort of children aged 3 to 8 years, living in The Gambia. Anti-CS antibody titres, malariometric indices and sickle cell status were also determined. Malaria morbidity in the ensuing malaria transmission season was monitored by weekly health questionnaire, axillary temperature measurements and examination of blood films. Exposure to malaria was inferred from entomological data collected during the transmission season. Immunological and parasitological measurements were repeated at the end of the rainy season. Immunological findings were compared between children who experienced clinical malaria or asymptomatic infection and children who had no evidence of infection. No association was found between cellular immune responses to the CS protein at the beginning of the transmission season and subsequent susceptibility to infection except among children with high titres of antibody to (NANP)40. Seropositive children who did not become infected had a higher mean proliferative response to the Th3R epitope than seropositive children who did become infected. High titres of anti-(NANP)40 antibodies alone were not protective. Responses to the Th2R epitope were significantly higher at the end of the rainy season than at the beginning in children who experienced an asymptomatic infection. Responses to variant sequences of the 2 epitopes were highly correlated at an individual level but there was no correlation between proliferative and interferon responses to a particular epitope.     

Adenovirus particles that display the Plasmodium falciparum circumsporozoite protein NANP repeat induce sporozoite-neutralizing antibodies in mice

Adenovirus particles can be engineered to display exogenous peptides on their surfaces by modification of viral capsid proteins, and particles that display pathogen-derived peptides can induce protective immunity. We constructed viable recombinant adenoviruses that display B-cell epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) in the major adenovirus capsid protein, hexon. Recombinants induced high-titer antibodies against CSP when injected intraperitoneally into mice. Serum obtained from immunized mice recognized both recombinant PfCSP protein and P. falciparum sporozoites, and neutralized P. falciparum sporozoites in vitro. Replicating adenovirus vaccines have provided economical protection against adenovirus disease for over three decades. The recombinants described here may provide a path to an affordable malaria vaccine in the developing world.     

Development of multi-epitope driven subunit vaccine in secretory and membrane protein of Plasmodium falciparum to convey protection against malaria infection

Malaria infection is the severe health concern for a long time. As per the WHO reports, the malarial infection causes huge mortality all around the world and is incomparable with any other infectious diseases. The absence of effective treatment options and increasing drug resistance to the available therapeutics like artemisinin and other derivatives demand an efficient alternative to overcome this death burden. Here, we performed the literature survey and sorted the Plasmodium falciparum secretory and membrane proteins to design multi-epitope subunit vaccine using an adjuvant, B-cell- and T-cell epitopes. Every helper T-lymphocyte (HTL) epitope was IFN-γ positive and IL-4 non-inducer. The physicochemical properties, allergenicity, and antigenicity of designed vaccine were analyzed for the safety concern. Homology modeling and refinement were performed to obtain the functional tertiary structure of vaccine protein followed by its molecular docking with the toll-like receptor-4 (TLR-4) immune receptor. Molecular dynamics simulation was performed to check the interaction and stability of the receptor-ligand complex. Lastly, in silico cloning was performed to generate the restriction clone of designed vaccine for the futuristic expression in a microbial expression system. This way, we designed the multi-epitope subunit vaccine to serve the people living in the global endemic zone.     

Safety and immunogenicity in volunteers of a recombinant Plasmodium falciparum circumsporozoite protein malaria vaccine produced in Lepidopteran cells.

A recombinant Plasmodium falciparum circumsporozoite (CS) antigen (rPfCSA) was produced in insect cells using a baculovirus expression vector containing the entire CS gene. This near full-length CS antigen was adsorbed onto aluminium phosphate for use as a malaria vaccine. In a study of safety and immunogenicity, 20 volunteers were divided into four groups of five each and inoculated intramuscularly with 10, 100, 500 or 1000 micrograms of vaccine. Primary vaccinations were followed by two booster immunizations at 2 and 6 months. Three volunteers developed prominent local reactions manifested as tenderness, redness and swelling at the injection site following the second or third vaccination. All symptoms resolved spontaneously within 72 h. Postimmunization sera from six of 20 volunteers showed seroconversions as measured by Western blot, using rPfCSA as antigen. However, specific anti-CS protein antibody could not be detected by indirect immunoflourescence against intact sporozoites or by ELISA using rPfCSA or peptide to the repeat region. In addition, 18 of 20 volunteers developed antibody to baculovirus proteins as determined by ELISA and/or Western blot. Antigen-driven replication studies using peripheral blood mononuclear cells from vaccinees failed to detect proliferative responses specific to CS protein. This recombinant CS protein vaccine, as formulated, was minimally immunogenic in humans.     

Folate metabolism pathway and Plasmodium falciparum malaria infection in pregnancy

Malaria induced by Plasmodium falciparum is a major cause of mortality. P. falciparum has the ability to use host plasma folate as its primary folate source. Folate is a cofactor needed for both malaria parasite growth and host erythrocyte production. This review examines the possible impairment of the folate-mediated one-carbon metabolism pathway as a result of P. falciparum malaria infection during pregnancy. Folate deficiency during malaria infection is presented, with an emphasis on the controversy regarding the decrease of plasma or erythrocyte folate secondary to malaria. Maternal folate deficiency increases the risk of adverse pregnancy outcomes. Functional folate deficiency and/or increased plasma homocysteine levels during pregnancy of infected women in areas endemic for malaria is a probable scenario accentuating the impairment of placenta function leading to the occurrence of neural tube defects, low birth weights, and intrauterine growth retardations. Potential questions that may be answered in future investigations using an appropriate protocol to study pregnant women with malaria are also addressed.     

Detection of homologous and heterologous malaria antibodies by application of Plasmodium falciparum merozoite antigen to an ELISA

Sera of patients with anti-plasmodial antibodies determined by indirect fluorescence antibody test (IFAT) were used to evaluate an enzyme immunoassay (IgG ELISA) for determining antibodies to Plasmodium falciparum merozoite antigen. The two tests correlated well. Antibodies to species other than P. falciparum, however, were detected by P. falciparum merozoite antigen to a limited extent only.     

Clinical pattern of severe Plasmodium falciparum malaria in Sudan in an area characterized by seasonal and unstable malaria transmission

A hospital-based study was carried out in Gedarif town, eastern Sudan, an area of markedly unstable malaria transmission. Among the 2488 diagnosed malaria patients, 4.4% fulfilled the WHO criteria for severe malaria, and seven died of cerebral malaria. The predominant complication was severe malarial anemia (45.4%), followed by convulsions (21%), cerebral malaria (16. 4%) and hypotension (11.8%). Severe malaria was recognized in all age groups, but 44.5% of patients were aged 2 to 4 years. The mean ages of patients with severe anemia (5.6 years) and convulsions (5.9 years) were significantly lower than the mean ages of patients with cerebral malaria (14.1 years) or hypotension (35.2 years). Patients with convulsions and cerebral malaria had significantly higher mean parasite count (69972 and 56110 parasites/microL, respectively) than patients with severe anemia (24637 parasites/microL) or hypotension (13667 parasites/microL). The mean blood glucose level was higher in patients with cerebral malaria than in patients with anemia, and higher in patients who died than in patients who survived. In this setting, the clinico-epidemiological pattern of severe malaria varies considerably from that of hyperendemic regions in sub-Saharan Africa, and there is considerable variation between the individual complications of severe malaria.     

A longitudinal study of antibodies to the Plasmodium falciparum antigen Pf155/RESA and immunity to malaria infection in adult Liberians

118 adult Liberians from 2 villages were studied prospectively for one year with monthly blood examinations for malaria parasites. The crude parasite rate was 41.5% and the crude gametocyte rate was 6.1%. The inoculation rate varied between 0.075 in the dry season and almost 0.4 in the rainy season, which is in accordance with other data from holoendemic areas. 47.5% (56) had a titre to the Pf155/RESA antigen less than or equal to 1/50 ('low responders') and 52.5% (62) had a titre of greater than or equal to 1/250 ('high responders'). The response was not age-dependent in this adult population, which may suggest that genetic factors are determining whether the individual become a high or low responder. Antibodies against the Pf155/RESA antigen were measured in 2 surveys 8 months apart, and the mean antibody response to Pf155/RESA and its EENV sequence was constant without seasonal variation. Pf155/RESA high responders had lower parasite densities during all 3 seasons surveyed, and Pf155/RESA high responders, with high antibody reactivity against the (EENV)6 sequence from the 3' repeat region of Pf155/RESA, had significantly lower parasite densities in the rainy season of 1987. The data suggest that high titres of antibodies to the Pf155/RESA antigen, and especially to its EENV sequence, might play a role in protective immunity in adults.     

High levels of serum antibodies to merozoite surface protein 2 of Plasmodium falciparum are associated with reduced risk of clinical malaria in coastal Kenya

The merozoite surface protein (MSP) 2 is a vaccine candidate antigen of Plasmodium falciparum that is polymorphic in natural populations. In a prospective cohort study in two coastal populations of Kenya using recombinant proteins derived from the two major allelic types of MSP2, high serum levels of IgG to MSP2 were associated with protection from clinical malaria. This protection was independent of that associated with antibodies to another vaccine candidate antigen (AMA1) in these populations. However, low antibody levels to MSP2 appeared to be associated with increased susceptibility to malaria within people who were parasite negative at the time of serum collection. These data suggest that an MSP2 based vaccine should be designed to induce high level antibody responses against the different MSP2 types present globally in P. falciparum populations and that MSP2 could be combined with other P. falciparum antigens to form a multi-component malaria vaccine.     

Comparison of antibody responses to the circumsporozoite protein repeat region and to intact sporozoites during acute falciparum malaria

Most acute falciparum malaria patients mount an antibody response to the circumsporozoite (CS) protein which contains a dominant B-cell epitope. In order to investigate whether antibodies against other epitopes on the sporozoite surface may be important during a particular phase of infection or convalescence, we longitudinally studied the antibody responses of 13 Thai patients with acute falciparum malaria. Antibody comparisons were made using intact Plasmodium falciparum sporozoites in an indirect fluorescent antibody test and the recombinant peptide, R32tet32, as capture antigen in an enzyme-linked immunosorbent assay. Antibody response curves derived using the 2 methods were similar, and adsorption with R32tet32 greatly (greater than 95%) diminished anti-sporozoite activity in sera. Thus, peptide constructs containing the CS repeat region epitope, (NANP)n, can be used with confidence to assay anti-sporozoite antibodies, independent of both the time of infection and prior malaria history.     

Antibodies to blood stage antigens of Plasmodium falciparum in rural Gambians and their relation to protection against infection

Cross-sectional and longitudinal studies were performed in a rural population living in The Gambia to examine the relationship between several in vitro assays of the host immune response to asexual stages of Plasmodium falciparum and protection from malaria in vivo. Assays included an enzyme-linked immunosorbent assay for antibodies to schizont antigens; an indirect immunofluorescence assay for total antiblood-stage antibodies; an immunofluorescence assay on glutaraldehyde-fixed parasites to detect antibodies to antigen Pf 155; an assay for serum inhibition of red blood cell invasion; a micro-agglutination assay to detect antibodies to neo-antigens on the surface of infected red blood cells; and an assay using polymorphonuclear leucocytes to detect antibodies capable of opsonizing schizont infected red blood cells. There were marked differences in the age-related pattern of response for different assays performed on sera obtained at a cross-sectional survey of 280 individuals. Examination of the correlation between the various immune responses and malariometric indices at the population level and at the individual level provided no evidence that any of the in vitro assays were related to protective immunity. The relationship between in vitro measurements of the anti-malarial immune response and protection from clinical episodes of malaria was examined in a group of 134 children aged 11 years and under who were monitored weekly throughout an entire malaria transmission season. The only immune factor to show a consistent protective effect against clinical malaria was the titre of antibodies to neo-antigens on the infected erythrocyte surface (P = 0.01). The same longitudinal techniques were used to examine the effect of two non-immunological factors, sickle cell trait and mosquito net usage, both of which showed significant protection against clinical episodes and malaria.     

Bayesian analysis of an epidemiologic model of Plasmodium falciparum malaria infection in Ndiop, Senegal

Plasmodium falciparum has a complex transmission cycle. Public health planning and research would benefit from the ability of a calibrated model to predict the epidemiologic characteristics of populations living in areas of malaria endemicity. This paper describes the application of Bayesian calibration to a malaria transmission model using longitudinal data gathered from 176 subjects in Ndiop, Senegal, from July 1, 1993, to July 31, 1994. The model was able to adequately predict P. falciparum parasitemia prevalence in the study population. Further insight into the dynamics of malaria in Ndiop was provided. During the dry season, the estimated fraction of nonimmune subjects goes down to 20% and then increases up to 80%. The model-predicted time-weighted average incidences contributed by nonimmune and immune individuals are 0.52 cases per day and 0.47 cases per day, respectively. The median times needed to acquire infection (conversion delay) for nonimmune and immune individuals are estimated at 39 days and 285 days, respectively.     

Human antibodies to recombinant protein constructs of Plasmodium falciparum Apical Membrane Antigen 1 (AMA1) and their associations with protection from malaria

Serum antibodies from 1071 people in two Kenyan villages were assayed using eight different recombinant Apical Membrane Antigen 1 (AMA1) protein constructs to investigate their role in naturally acquired immunity. In both communities, antibodies against the full-length ectodomain (both FVO and 3D7 allele constructs) prior to a malaria transmission season were significantly associated with protection from malaria in the following 6 months, even after adjusting for age and antibody reactivity to whole parasite (schizont) extract. However, these protective associations of antibodies were only seen among subjects that were parasite slide positive at the time of pre-season serum sampling. Competition ELISAs with the FVO and 3D7 allele constructs showed that antibodies can recognise either conserved or allele-specific epitopes in AMA1. Results encourage the development of an AMA1 vaccine based on the full-length ectodomain, and indicate that the function of human antibodies to allele-specific and conserved epitopes in AMA1 should be studied further.     

Expression and immunogenicity of the Plasmodium falciparum circumsporozoite protein: the role of GPI signal sequence

Previous studies have shown that the immunogenicity of rodent malaria parasite-derived circumsporozoite protein (CS) can be improved by deleting the glycosyl-phosphatidyl-inositol (GPI) signal sequence. To study whether GPI signal sequence deletion would also improve immunogenicity of CS derived from the major plasmodium species causing mortality in humans (P. falciparum), we tested different variants of the P. falciparum CS protein in the context of a live vector-based vaccine carrier (rAd35). We demonstrate that deletion of the GPI signal sequence from CS did not result in altered expression or secretion. In contrast, cellular localization was clearly altered, which perhaps helps to explain the significant improvement of anti-CS antibody and T-cell responses observed in mice using deletion variants in the context of the rAd35 carrier. Our results show that rational design of antigens is warranted for further development of malaria vaccines.