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.     

The action of falciparum malaria on the human and chimpanzee genomes compared: Absence of evidence for a genomic signature of malaria at HBB and G6PD in three subspecies of chimpanzee

The historical association between Plasmodium and primates has meant that many Plasmodium species have coevolved with specific primate hosts. However, unlike humans that are infected by species such as P. falciparum that cause severe malaria, many non-human primates are infected by Plasmodium species that only cause mild disease. Here we investigate whether the genomic signatures of plasmodial infection found in humans are also present in chimpanzees. We find no evidence of the major deleterious mutations at HBB (β-globin) and G6PD in chimpanzees that confer resistance to malaria caused by P. falciparum nor evidence of long-term balancing selection at these loci. Our knowledge of malaria prevalence and pathogenesis in wild chimpanzees is severely limited, but it may be the case that β-globin and G6PD variation are not adaptive in chimpanzees because malaria is rare and/or less detrimental in this species. Alternatively, chimpanzees may utilise mechanisms that are different from those of humans to protect against malaria.     

Indirect ELISA test for malaria in blood donors

To determine the potential risk of transfusion malaria at the Hospital Militar Central in Bogota, Colombia, sera from 3114 blood donors were tested for malaria antibodies by the indirect ELISA technique. Positive results were found in 8·6 per thousand of the serum samples using P. falciparum antigen containing more than 60% mature forms as substrate. Three cases of transfusion-induced malaria were confirmed during the study. The first patient developed a P. vivax infection one week after the administration of one unit of infected blood. The other two patients received a red blood cell concentrate and a platelet preparation, respectively, derived from a single donor and developed a P. falciparum infection eight days after transfusion. The application of the ELISA technique would be of use in attempts to control transfusion-induced malaria.     

Antibody levels detected by the fluorescent antibody technique in mice infected with Plasmodium vinckei and P. chabaudi

The malaria parasites of mice are convenient models for immunological studies. Plasmodium vinckei and P. chabaudi are similar parasites which behave differently in mice, the former invariably being fatal whereas the latter seldom kills the host. The experiments described in this paper were performed in order to compare the antibody levels in the Ig, IgM and IgG serum fractions in mice cured of P. vinckei infections and naturally recovered from P. chabaudi. The technique involved using specific labelled antimouse-Ig, -IgM and -IgG sera and had not previously been applied to malaria infections. The results showed that the patterns of antibody production were similar in cured and naturally recovered mice. IgG antibodies were involved from an early stage of the infection and IgM antibodies persisted throughout the period of experiment and even after challenge. These results are significant in that they indicate a pattern of immune response different from the classically accepted one and draw attention to the fact that even in highly immune animals considerable amounts of IgM may be present.     

Pf332-C231-reactive antibodies affect growth and development of intra-erythrocytic Plasmodium falciparum parasites

The Plasmodium falciparum antigen 332 (Pf332), is a megadalton parasite protein expressed at the surface of infected red cells during later stages of the parasite's developmental cycle. Antibodies to different parts of this antigen have been shown to inhibit parasite growth and adherence to host cells with or without ancillary cells. However, the mechanisms involved in these inhibitions remain largely unknown. We further analysed the activities of specific antibodies with regard to their specific mechanisms of action. For these analyses, affinity purified human antibodies against epitopes in the C-terminal fragment of Pf332 (Pf332-C231) were employed. All purified antibodies recognized Pf332-C231 both by immunofluorescence and ELISA. IgG was the main antibody isotype detected, although all sera investigated had varying proportions of IgG and IgM content. All the antibodies showed a capacity to inhibit parasite growth in P. falciparum cultures to different extents, mainly by acting on the more mature parasite stages. Morphological analysis revealed the antibody effects to be characterized by the presence of a high proportion of abnormal schizonts (15-30%) and pyknotic parasites. There was also an apparent antibody effect on the red cell integrity, as many developing parasites (up to 10% of trophozoites and schizonts) were extracellular. In some cases, the infected red cells appeared to be disintegrating/fading, staining paler than surrounding infected and uninfected cells. Antigen reversal of inhibition confirmed that these inhibitions were antigen specific. Furthermore, the growth of parasites after 22-42 h exposure to antibodies was investigated. Following the removal of antibody pressure, a decreased growth rate of these parasites was seen compared to that of control parasites. The present study confirms the potential of Pf332 as a target antigen for parasite neutralizing antibodies, and further indicates that epitopes within the C231 region of Pf332 should constitute important tools in the dissection of the role of Pf332 in the biology of the malaria parasite, as well as in the design of a malaria vaccine.     

A multi-stage malaria vaccine candidate targeting both transmission and asexual parasite life-cycle stages

Effective control and eventual eradication of malaria drives the imperative need for clinical development of a malaria vaccine. Asexual parasite forms are responsible for clinical disease and death while apathogenic gametocytes are responsible for transmission from man to mosquito. Vaccines that combine antigens from both stages may provide direct protection and indirect benefit by reducing the force of infection. We constructed a chimeric antigen composed of a fragment of the Plasmodium falciparum (Pf) glutamate-rich protein fused in frame to a correctly folded fragment of Pfs48/45. The chimera was produced in Lactococcus lactis and induced robust antibody responses in rodents to the individual components. Specific antibodies showed strong transmission blocking activity against multiple Pf-strains in the standard membrane feeding assay and functional activity against asexual stages in the antibody dependent cellular inhibition assay. The combined data provide a strong rationale for entering the next phase of clinical grade production and testing.     

RTS,S/AS01E malaria vaccine induces IgA responses against CSP and vaccine-unrelated antigens in African children in the phase 3 trial

BackgroundThe evaluation of immune responses to RTS,S/AS01 has traditionally focused on immunoglobulin (Ig) G antibodies that are only moderately associated with protection. The role of other antibody isotypes that could also contribute to vaccine efficacy remains unclear. Here we investigated whether RTS,S/AS01_E elicits antigen-specific serum IgA antibodies to the vaccine and other malaria antigens, and we explored their association with protection.MethodsNinety-five children (age 5–17 months old at first vaccination) from the RTS,S/AS01_E phase 3 clinical trial who received 3 doses of RTS,S/AS01_E or a comparator vaccine were selected for IgA quantification 1 month post primary immunization. Two sites with different malaria transmission intensities (MTI) and clinical malaria cases and controls, were included. Measurements of IgA against different constructs of the circumsporozoite protein (CSP) vaccine antigen and 16 vaccine-unrelated Plasmodium falciparum antigens were performed using a quantitative suspension array assay.ResultsRTS,S vaccination induced a 1.2 to 2-fold increase in levels of serum/plasma IgA antibodies to all CSP constructs, which was not observed upon immunization with a comparator vaccine. The IgA response against 13 out of 16 vaccine-unrelated P. falciparum antigens also increased after vaccination, and levels were higher in recipients of RTS,S than in comparators. IgA levels to malaria antigens before vaccination were more elevated in the high MTI than the low MTI site. No statistically significant association of IgA with protection was found in exploratory analyses.ConclusionsRTS,S/AS01_E induces IgA responses in peripheral blood against CSP vaccine antigens and other P. falciparum vaccine-unrelated antigens, similar to what we previously showed for IgG responses. Collectively, data warrant further investigation of the potential contribution of vaccine-induced IgA responses to efficacy and any possible interplay, either synergistic or antagonistic, with protective IgG, as identifying mediators of protection by RTS,S/AS01_E immunization is necessary for the design of improved second-generation vaccines.Clinical trial registration: ClinicalTrials.gov: NCT008666191.     

Norovirus-VLPs expressing pre-erythrocytic malaria antigens induce functional immunity against sporozoite infection

Despite the development of prophylactic anti-malarial drugs and practices to prevent infection, malaria remains a health concern. Preclinical testing of novel malaria vaccine strategies achieved through rational antigen selection and novel particle-based delivery platforms is yielding encouraging results. One such platform, self-assembling virus-like particles (VLP) is safer than attenuated live viruses, and has been approved as a vaccination tool by the FDA. We explore the use of Norovirus sub-viral particles lacking the natural shell (S) domain forming the interior shell but that retain the protruding (P) structures of the native virus as a vaccine vector. Epitope selection and their surface display has the potential to focus antigen specific immune responses to crucial epitopes. Recombinant P-particles displaying epitopes from two malaria antigens, Plasmodium falciparum (Pf) CelTOS and Plasmodium falciparum (Pf) CSP, were evaluated for immunogenicity and their ability to confer protection in a murine challenge model. Immune responses induced in mice resulted either in sterile protection (displaying PfCelTOS epitopes) or in antibodies with functional activity against sporozoites (displaying PfCSP epitopes) in an in vitro liver-stage development assay (ILSDA). These results are encouraging and support further evaluation of this platform as a vaccine delivery system.     

Four year immunogenicity of the RTS,S/AS02(A) malaria vaccine in Mozambican children during a phase IIb trial

Previous studies with the malaria vaccine RTS,S/AS02_A in young children in a malaria endemic area of Mozambique have shown it to have a promising safety profile and to reduce the risk of Plasmodium falciparum infection and disease.In this study, we assessed the antibody responses to the P. falciparum and hepatitis B components of the RTS,S/AS02_A vaccine over a 45 months surveillance period in a large phase IIb trial which included 2022 children aged 1-4 years at recruitment.The RTS,S/AS02_A vaccine induced high anti-circumsporozoite antibody levels with at least 96% of children remaining seropositive during the entire follow-up period. IgG titers decayed over the first 6 months of follow-up to about 25% of the initial level, but still remained 30-fold higher until month 45 compared to controls. Children with higher levels of naturally acquired immunity at baseline, assessed by blood stage indirect fluorescent antibody test, had slightly higher anti-circumsporozoite levels, after adjusting for the effect of age.The RTS,S/AS02_A vaccine also induced high levels of anti-hepatitis B surface antigen antibodies (seroprotection >97%).RTS,S/AS02_A vaccine is immunogenic and induces long-lasting anti-circumsporozoite antibodies, persisting at least 42 months after immunization. These antibodies may play a role in protection against malaria.     

A quantitative approach to recommendations on malaria prophylaxis

In order to develop recommendations for malaria prophylaxis, a quantitative method is needed to balance the risk of Plasmodium falciparum malaria infections against the toxicity of antimalarial drugs. Using decision analysis, we estimated the expected mortality associated with three alternative regimens of prophylactic drugs for visitors to three areas with different risks of infection with chloroquine-resistant P. falciparum. The model used took into account the risks of malaria and of adverse reactions to antimalarial drugs. Estimates of the parameters used in the analysis were based on observations made on U.S. travellers. Reducing the risk of malaria infection was found to have a far greater impact on lowering the expected mortality than that of increasing the chemoprophylactic efficacy of the drugs used, thereby emphasizing the need for travellers to use anti-mosquito measures in malarious areas. The analytical method described can be used to define optimal malaria prevention strategies.     

Protective Antibodies against Placental Malaria and Poor Outcomes during Pregnancy,Benin

Placental malaria is caused by Plasmodium falciparum–infected erythrocytes that bind to placental tissue. Binding is mediated by VAR2CSA, a parasite antigen coded by the var gene, which interacts with chondroitin sulfate A (CSA). Consequences include maternal anemia and fetal growth retardation. Antibody-mediated immunity to placental malaria is acquired during successive pregnancies, but the target of VAR2CSA-specific protective antibodies is unclear. We assessed VAR2CSA-specific antibodies in pregnant women and analyzed their relationships with protection against placental infection, preterm birth, and low birthweight. Antibody responses to the N-terminal region of VAR2CSA during early pregnancy were associated with reduced risks for infections and low birthweight. Among women infected during pregnancy, an increase in CSA binding inhibition was associated with reduced risks for placental infection, preterm birth, and low birthweight. These data suggest that antibodies against VAR2CSA N-terminal region mediate immunity to placental malaria and associated outcomes. Our results validate current vaccine development efforts with VAR2CSA N-terminal constructs.     

Antibodies to Pf155, a major antigen of Plasmodium falciparum: seroepidemiological studies in Haiti

The presence of malaria parasites and the serological antibody responses against whole Plasmodium falciparum and the Pf155 antigen were studied in the population of a small rural locality in Haiti in December 1985. Only 7 (1.5%) of the individuals were found to be infected with P. falciparum, the only species observed. Antibodies to P. falciparum were detected in an ELISA in 38.2% of the sera, the positivity rates being age-related. Anti-Pf155 antibodies were detected in 12.5% and 13.6% of individuals by two different techniques used. The anti-Pf155 positivity rates increased only after 25 years of age. No trends were detected for a clear-cut protective value of Pf155 antibodies against clinical malaria and further longitudinally conducted field surveys are needed to satisfactorily assess the potential protective effect of Pf155 antibodies.     

Changing patterns in the humoral immune response to malaria before, during, and after the application of control measures: a longitudinal study in the West African savanna.

A longitudinal seroimmunological investigation of malaria was performed as part of the WHO research project conducted in the northern part of Nigeria from 1970 to 1975. The project included a preintervention phase, an intervention phase with application of malaria control measures (spraying of residual insecticide and mass drug administration), and a postintervention phase. Serological observations were made on the total population of eight villages consisting of approximately 3000 persons. Six immunological parameters were studied, namely, the serum levels of IgG and IgM, the number of bands of precipitation for Plasmodium falciparum in the double diffusion (Ouchterlony) test, the titres of antibodies for P. falciparum and P. malariae in the indirect fluorescent antibody (IFA) test, and the titres of agglutinating antibodies for P. falciparum by the indirect (passive) haemagglutination (IHA) test. The serological results were used to evaluate the impact on the humoral immune response of different levels of parasitaemia resulting, in the unprotected population, from natural factors such as seasons and ageing and in the protected population, from human intervention through the application of control measures and their interruption. The linkage by computer processing of the longitudinal data allowed analysis of the relationship between the results of a serological test in the same person at different surveys, and analysis of correlation between serological results and the concurrent parasitological findings. The correlation between parasitaemia and the results of the different serological tests at the same survey in the same person were also examined and analysed in terms of sensitivity and specificity of the tests.     

Serum immunoglobulin levels and malaria antibodies in Burkitt's lymphoma.

Data are presented to support a relationship between malaria infection and Burkitt's lymphoma in African children. IgG, IgM and IgA levels were measured in sera from Burkitt's lymphoma patients and from sex- and age-matched, nearest-neighbour controls. All three classes of immunoglobulins were present in significantly lower amounts in the sera from Burkitt's lymphoma patients than in the sera from controls. The mechanism of this apparent B-cell suppression is not yet clear. Malaria-specific IgG and IgM antibody titres were determined in the indirect immunofluorescence test. No significant difference in the IgG malaria-specific antibodies was detected between the two groups of sera. Malaria antibody levels measured using IgM specific conjugates were significantly lower in the sera from Burkitt's lymphoma patients in reactions with Plasmodium falciparum antigen. No significant difference was observed when P. malariae was used. Confirmation of this finding would serve as a positive link between Burkitt's lymphoma and P. falciparum infection.     

Congenital malaria due to Plasmodium vivax: a case report from Sri Lanka

A case of Plasmodium vivax malaria in an eight-week-old infant in Colombo is documented, with epidemiological and circumstantial evidence which strongly supports a transplacental route of infection. The malarial antibody levels detected by the indirect fluorescent antibody technique in both mother and child are discussed in terms of the present epidemiological pattern of malaria in the country. We also comment on the species incidence of congenital malaria, this case being the first caused by P. vivax in Sri Lanka, despite this species being more prevalent than P. falciparum which has been reported in six previous cases of congenital malaria in Sri Lanka.     

Identification of Plasmodium falciparum reticulocyte binding protein homologue 5-interacting protein,PfRipr, as a highly conserved blood-stage malaria vaccine candidate

Genetic variability in Plasmodium falciparum malaria parasites hampers current malaria vaccine development efforts. Here, we hypothesize that to address the impact of genetic variability on vaccine efficacy in clinical trials, conserved antigen targets should be selected to achieve robust host immunity across multiple falciparum strains. Therefore, suitable vaccine antigens should be assessed for levels of polymorphism and genetic diversity. Using a total of one hundred and two clinical isolates from a region of high malaria transmission in Uganda, we analyzed extent of polymorphism and genetic diversity in four recently reported novel blood-stage malaria vaccine candidate proteins: Rh5 interacting protein (PfRipr), GPI anchored micronemal antigen (PfGAMA), rhoptry-associated leucine zipper-like protein 1 (PfRALP1) and Duffy binding-like merozoite surface protein 1 (PfMSPDBL1). In addition, utilizing the wheat germ cell-free system, we expressed recombinant proteins for the four candidates based on P. falciparum laboratory strain 3D7 sequences, immunized rabbits to obtain specific antibodies (Abs) and performed functional growth inhibition assay (GIA). The GIA activity of the raised Abs was demonstrated using both homologous 3D7 and heterologous FVO strains in vitro. Both pfripr and pfralp1 are less polymorphic but the latter is comparatively more diverse, with varied number of regions having insertions and deletions, asparagine and 6-mer repeats in the coding sequences. Pfgama and pfmspdbl1 are polymorphic and genetically diverse among the isolates with antibodies against the 3D7-based recombinant PfGAMA and PfMSPDBL1 inhibiting merozoite invasion only in the 3D7 but not FVO strain. Moreover, although Abs against the 3D7-based recombinant PfRipr and PfRALP1 proteins potently inhibited merozoite invasion of both 3D7 and FVO, the GIA activity of anti-PfRipr was much higher than that of anti-PfRALP1. Thus, PfRipr is regarded as a promising blood-stage vaccine candidate for next-generation vaccines against P. falciparum.     

Preliminary studies on the immunogenicity of a prime-and-trap malaria vaccine in nonhuman primates

Development of next-generation vaccines against Plasmodium falciparum (Pf) is a priority. Many malaria vaccines target the pre-erythrocytic sporozoite (SPZ) and liver stages. These include subunit vaccines based on the Pf circumsporozoite protein (CSP) and attenuated PfSPZ vaccines. However, these strategies require 3–4 doses and have not achieved optimal efficacy against field-transmitted malaria. Prime-and-trap is a recently developed two-step heterologous vaccine strategy that combines priming with DNA encoding CSP followed by a single dose of attenuated SPZ. This strategy aims to induce CD8⁺ T cells that can eliminate parasites in the liver. Prior data has demonstrated that prime-and-trap with P. yoelii CSP and PySPZ was immunogenic and protective in mice. Here we report preliminary data on the immunogenicity of PfCSP prime and PfSPZ trap vaccine in rhesus macaques. This vaccine induced PfCSP-specific antibodies and T cell responses in all animals. However, response magnitude differed between individuals, suggesting further study is required.     

The influence of malaria and gestation on the immune response to one and two doses of adsorbed tetanus toxoid in pregnancy

The effect of Plasmodium falciparum infection on the response to immunization with tetanus toxoid in pregnancy is of importance because malaria is more frequent and severe in pregnant women. This paper presents the results of a study in west Kenya of the antibody response to an adsorbed tetanus toxoid in primigravidae and multigravidae living under holoendemic conditions. There was no apparent influence of either P. falciparum infection or gestational age on the immune response to one and two doses of adsorbed toxoid. The antibody response in pregnant women with and without malaria was comparable to that in non-pregnant healthy adults. Previous studies of responses to primary immunization schedules in pregnant and non-pregnant women are reviewed.     

Differences in automated depolarization patterns of Plasmodium faiciparum and P. vivax malaria infections defined by the Cell-Dyn® CD4000 haematology analyser

Of 1014 samples submitted for full blood count analysis and malaria screening, 854 were designated malaria-negative by blood film microscopy, 79 were unequivocally identified as Plasmodium vivax and 81 as P. falciparum. All samples were additionally analysed with the Abbott Cell-Dyn® CD4000 haematology instrument, and leucocyte differential plots of 90° polarized vs. 90° depolarized (NEU-EOS plot) and 90° depolarized vs. 0° light (EOS I plot) scatter were specifically examined for abnormal depolarization patterns. Depolarization pattern types were correlated with microscopy (species) results, and these correlations were consolidated by polymerase chain reaction analysis. All 854 microscopically-designated malaria-negative samples showed a type 1 (normal) CD4000 depolarization pattern. Abnormal pattern types 2, 3a and 3b were entirely restricted to one of the two malaria categories. Plasmodium falciparum malaria showed two CD4000 pattern types only; a ‘normal’ type 1 pattern was seen in 36/75 (48%) cases and the remaining 39 cases were all abnormal pattern type 3a. In contrast, most (79/85) P. vivax malaria cases showed a distinctive clustered EOS I population (types 2 and 3b patterns) that was not seen with P. falciparum. Automated depolarization analysis provides an effective means of detecting malaria-associated haemozoin, and the patterns of intracellular haemozoin further appear to provide species differentiation between P. falciparum and P. vivax.