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Immune correlates of protection against clinical malaria are difficult to ascertain in low-transmission areas because of the limited number of malaria cases. We collected blood samples from 5,753 individuals in a Kenyan highland area, ascertained malaria incidence in this population over the next 6 years, and then compared antibody responses to 11 Plasmodium falciparum vaccine candidate antigens in individuals who did versus did not develop clinical malaria in a nested case-control study (154 cases and 462 controls). Individuals were matched by age and village. Antigens tested included circumsporozoite protein (CSP), liver-stage antigen (LSA)-1, apical membrane antigen-1 FVO and 3D7 strains, erythrocyte-binding antigen-175, erythrocyte-binding protein-2, merozoite surface protein (MSP)-1 FVO and 3D7 strains, MSP-3, and glutamate-rich protein (GLURP) N-terminal non-repetitive (R0) and C-terminal repetitive (R2) regions. After adjustment for potential confounding factors, the presence of antibodies to LSA-1, GLURP-R2, or GLURP-R0 was associated with decreased odds of developing clinical malaria (odds ratio [OR], [95% CI] 0.56 [0.36-0.89], 0.56 [0.36-0.87], and 0.77 [0.43-1.02], respectively). Levels of antibodies to LSA-1, GLURP-R2, and CSP were associated with decreased odds of developing clinical malaria (OR [95% CI]; 0.61 [0.41-0.89], 0.60 [0.43-0.84], and 0.49 [0.24-0.99], for every 10-fold increase in antibody levels, respectively). The presence of antibodies to CSP, GLURP-R0, GLURP-R2, and LSA-1 combined best-predicted protection from clinical malaria. Antibodies to CSP, GLURP-R0, GLURP-R2, and LSA-1 are associated with protection against clinical malaria in a low-transmission setting. Vaccines containing these antigens should be evaluated in low malaria transmission areas.

Prevalence and levels of antibodies to multiple Plasmodium falciparum antigens show promise as tools for estimating malaria exposure. In a highland area of Kenya with unstable transmission, we assessed the presence and levels of antibodies to 12 pre-erythrocytic and blood-stage P. falciparum antigens by multiplex cytometric bead assay or ELISA in 604 individuals in August 2007, with follow-up testing in this cohort in April 2008, April 2009, and May 2010. Four hundred individuals were tested at all four time points. During this period, the only substantial malaria incidence occurred from April to August 2009. Antibody prevalence in adults was high at all time points (> 70%) for apical membrane antigen 1, erythrocyte-binding antigen 175, erythrocyte-binding protein-2, glutamate rich protein (GLURP)-R2, merozoite surface protein (MSP) 1 (19), MSP-1 (42), and liver-stage antigen-1; moderate (30–70%) for GLURP-R0, MSP-3, and thrombospondin-related adhesive protein; and low (< 30%) for SE and circumsporozoite protein (CSP). Changes in community-wide malaria exposure were best reflected in decreasing antibody levels overtime for highly immunogenic antigens, and in antibody seroprevalence overtime for the less-immunogenic antigens. Over the 3 years, antibody levels to all antigens except CSP and schizont extract (SE) decreased in an age-dependent manner. Prevalence and levels of antibodies to all antigens except CSP and SE increased with age. Increases in antibody prevalence and levels to CSP and SE coincided with increases in community-wide malaria incidence. Antibody levels to multiple P. falciparum antigens decrease in the absence of consistent transmission. Multiplex assays that assess both the presence and level of antibodies to multiple pre-erythrocytic and blood-stage P. falciparum antigens may provide the most useful estimates of past and recent malaria transmission in areas of unstable transmission and could be useful tools in malaria control and elimination campaigns.

In 2016, there were more cases and deaths caused by malaria globally than in 2015. An effective vaccine would be an ideal additional tool for reducing malaria’s impact. Sanaria® PfSPZ Vaccine, composed of radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum (Pf) sporozoites (SPZ) has been well tolerated and safe in malaria-naïve and experienced adults in the United States and Mali and protective against controlled human malaria infection with Pf in the United States and field transmission of Pf in Mali, but had not been assessed in younger age groups. We, therefore, evaluated PfSPZ Vaccine in 93 Tanzanians aged 45 years to 6 months in a randomized, double-blind, normal saline placebo-controlled trial. There were no significant differences in adverse events between vaccinees and controls or between dosage regimens. Because all age groups received three doses of 9.0 × 105 PfSPZ of PfSPZ Vaccine, immune responses were compared at this dosage. Median antibody responses against Pf circumsporozoite protein and PfSPZ were highest in infants and lowest in adults. T-cell responses were highest in 6–10-year olds after one dose and 1–5-year olds after three doses; infants had no significant positive T-cell responses. The safety data were used to support initiation of trials in > 300 infants in Kenya and Equatorial Guinea. Because PfSPZ Vaccine–induced protection is thought to be mediated by T cells, the T-cell data suggest PfSPZ Vaccine may be more protective in children than in adults, whereas infants may not be immunologically mature enough to respond to the PfSPZ Vaccine immunization regimen assessed.

Equatorial Guinea (EG) has implemented a successful malaria control program on Bioko Island. A highly effective vaccine would be an ideal complement to this effort and could lead to halting transmission and eliminating malaria. Sanaria® PfSPZ Vaccine (Plasmodium falciparum sporozoite Vaccine) is being developed for this purpose. To begin the process of establishing the efficacy of and implementing a PfSPZ Vaccine mass vaccination program in EG, we decided to conduct a series of clinical trials of PfSPZ Vaccine on Bioko Island. Because no clinical trial had ever been conducted in EG, we first successfully established the ethical, regulatory, quality, and clinical foundation for conducting trials. We now report the safety, tolerability, and immunogenicity results of the first clinical trial in the history of the country. Thirty adult males were randomized in the ratio 2:1 to receive three doses of 2.7 × 105 PfSPZ of PfSPZ Vaccine (N = 20) or normal saline placebo (N = 10) by direct venous inoculation at 8-week intervals. The vaccine was safe and well tolerated. Seventy percent, 65%, and 45% of vaccinees developed antibodies to Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) by enzyme-linked immunosorbent assay, PfSPZ by automated immunofluorescence assay, and PfSPZ by inhibition of sporozoite invasion assay, respectively. Antibody responses were significantly lower than responses in U.S. adults who received the same dosage regimen, but not significantly different than responses in young adult Malians. Based on these results, a clinical trial enrolling 135 subjects aged 6 months to 65 years has been initiated in EG; it includes PfSPZ Vaccine and first assessment in Africa of PfSPZ-CVac. ClinicalTrials.gov identifier: NCT02418962.

Vaccine development against Plasmodium vivax malaria lags behind that for Plasmodium falciparum. To narrow this gap, we administered recombinant antigens based on P. vivax circumsporozoite protein (CSP) to mice.

Molecular tools to distinguish strains of Plasmodium vivax are important for studying the epidemiology of malaria transmission. Two sets of markers-tandem repeat (TR) polymorphisms and MSP3α-were used to study Plasmodium vivax in patients in the Peruvian Amazon region of Iquitos. Of 110 patients, 90 distinct haplotypes were distinguished using 9 TR markers. An MSP3α polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using HhaI and AluI revealed 8 and 9 profiles, respectively, and 36 profiles when analyzed in combination. Combining TR and PCR-RFLP markers, 101 distinct molecular profiles were distinguished among these 110 patients. Nine TR markers arrayed along a 100 kB stretch of a P. vivax chromosome containing the gene for circumsporozoite protein showed non-linear linkage disequilibrium (I(SA) = 0.03, P = 0.001). These findings demonstrate the potential use of TR markers for molecular epidemiology studies.

Circumsporozoite (CS) protein is a malaria antigen involved in sporozoite invasion of hepatocytes, and thus considered to have good vaccine potential. We evaluated the polymorphism of the Plasmodium vivax CS gene in 24 parasite isolates collected from malaria-endemic areas of Colombia. We sequenced 27 alleles, most of which (25/27) corresponded to the VK247 genotype and the remainder to the VK210 type. All VK247 alleles presented a mutation (Gly → Asn) at position 28 in the N-terminal region, whereas the C-terminal presented three insertions: the ANKKAGDAG, which is common in all VK247 isolates; 12 alleles presented the insertion GAGGQAAGGNAANKKAGDAG; and 5 alleles presented the insertion GGNAGGNA. Both repeat regions were polymorphic in gene sequence and size. Sequences coding for B-, T-CD4(+), and T-CD8(+) cell epitopes were found to be conserved. This study confirms the high polymorphism of the repeat domain and the highly conserved nature of the flanking regions.

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate previously assessed in animals and humans. Here, combinations of three synthetic polypeptides corresponding to amino (N), central repeat (R), and carboxyl (C) regions of the CS protein formulated in Montanide ISA 720 or Montanide ISA 51 adjuvants were assessed for immunogenicity in rodents and primates. BALB/c mice and Aotus monkeys were divided into test and control groups and were immunized three times with doses of 50 and 100 μg of vaccine or placebo. Antigen-specific antimalarial antibodies were determined by enzyme-linked immunosorbent assay, immunofluorescent antibody test, and IFN-γ responses by enzyme-linked immunosorbent spot (ELIspot). Both vaccine formulations were highly immunogenic in both species. Mice developed better antibody responses against C and R polypeptides, whereas the N polypeptide was more immunogenic in monkeys. Anti-peptide antibodies remained detectable for several months and recognized native proteins on sporozoites. Differences between Montanide ISA 720 and Montanide ISA 51 formulations were not significant.

We assessed the safety, tolerability, and immunogenicity of a mixture of three synthetic peptides derived from the Plasmodium vivax circumsporozoite protein formulated in Montanide ISA 720 or Montanide ISA 51. Forty healthy malaria-naive volunteers were allocated to five experimental groups (A–E): four groups (A–D) were immunized intramuscularly with 50 and 100 μg/dose injections of a mixture of N, R, and C peptides formulated in the two different adjuvants at 0, 2, and 4 months and one group was administered placebo. Vaccines were immunogenic, safe, well tolerated, and no serious adverse events related to the vaccine occurred. Seroconversion occurred in > 90% of the vaccines and antibodies recognized the sporozoite protein on immunofluorescent antibody test. Vaccines in Montanide ISA 51 showed a higher sporozoite protein recognition and interferon production. Results encourage further testing of the vaccine protective efficacy.

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate. We describe the characterization of specific immune responses induced in 21 malaria-naive volunteers vaccinated with long synthetic peptides derived from the CS protein formulated in Montanide ISA 720. Both antibody- and cell-mediated immune responses were analyzed. Antibodies were predominantly of IgG1 and IgG3 isotypes, recognized parasite proteins on the immunofluorescent antibody test, and partially blocked sporozoite invasion of hepatoma cell lines in vitro. Peripheral blood mononuclear cells from most volunteers (94%) showed IFN-γ production in vitro upon stimulation with both long signal peptide and short peptides containing CD8+ T-cell epitopes. The relatively limited sample size did not allow conclusions about HLA associations with the immune responses observed. In summary, the inherent safety and tolerability together with strong antibody responses, invasion blocking activity, and the IFN-γ production induced by these vaccine candidates warrants further testing in a phase II clinical trial.

Mosquitoes of the Anopheles gambiae complex (N = 1,336) were sampled (2001-2005) across Angola to identify taxa, study inversion polymorphisms, and detect the circumsporozoite protein of Plasmodium falciparum. Anopheles gambiae s.s. was found in all sites; it was characterized as M-form in localities of the tropical dry and semi-desertic belts, whereas the S-form was predominant in comparatively more humid and less anthropized sites. Both forms were characterized by low degrees of chromosomal polymorphism based solely on the 2La inversion, a pattern usually associated with An. gambiae populations from forested, humid, and derived savanna areas. Unexpectedly, this pattern was also observed in M-form populations collected in dry/pre-desertic areas, where this form largely predominates over An. arabiensis, which was also detected in central/inland sites. Anopheles melas was found in northern coastal sites. Three of 534 An. gambiae s.s. were positive for P. falciparum CS-protein, whereas none of the 105 An. melas were positive.

Parasite genotyping studies have indicated that the Plasmodium falciparum populations circulating in Iran are genetically diverse and that multiple genotype infections are observed regularly. We wished to extend the analysis to the Pfcsp gene, coding for the dominant sporozoite surface antigen on which the leading malaria vaccine candidate RTS,S is based. Infected blood samples were collected mainly from Iranian, as well as Afghani and Pakistani, patients on admission with falciparum malaria. DNA was purified from 90 isolates, and from these, 21 fragments corresponding to Pfcsp and 69 fragments corresponding to the 3'-end conserved domain were amplified and sequenced. Overall diversity was low. Six patterns were noted for the repeat region, but mixed genotypes were not observed in any of the isolates. T cell epitopes also displayed limited diversity, with only five haplotypes (combined Th2R/Th3R epitopes) noted, and of these, three were dominant, accounting for 94% of the 90 sequences. These observations are akin to those observed in Thai P. falciparum isolates, where a particular Pfscp Th2R/Th3R haplotype seems to be maintained in an otherwise genetically diverse parasite population. The data imply that the selective pressure that maintains a restricted T cell epitope is caused by factors outside the mammalian host immune responses. Furthermore, they sustain the notion that protective responses induced by RTS,S vaccination are not strain-specific.

Genetic strategies for controlling malaria transmission based on engineering pathogen resistance in Anopheles mosquitoes are being tested in a number of animal models. A key component is the effector molecule and the efficiency with which it reduces parasite transmission. Single-chain antibodies (scFvs) that bind the circumsporozoite protein of the avian parasite, Plasmodium gallinaceum, can reduce mean intensities of sporozoite infection of salivary glands by two to four orders of magnitude in transgenic Aedes aegypti. Significantly, mosquitoes with as few as 20 sporozoites in their salivary glands are infectious for a vertebrate host, Gallus gallus. Although scFvs hold promise as effector molecules, they will have to reduce mean intensities of infection to zero to prevent parasite transmission and disease. We conclude that similar endpoints must be reached with human pathogens if we are to expect an effect on disease transmission.

Vector incrimination studies were conducted from April 2003 to February 2005 at three riverine villages 1.5 km to 7.0 km apart, along the Matapi River, Amapa State, Brazil. A total of 113,117 mosquitoes were collected and placed in pools of

Approximately 55% of malaria infections in the Guyana Amazon region are attributed to Plasmodium falciparum while the other 45% are attributed to non-falciparum, mostly Plasmodium vivax. However, little is known about the P. vivax strain types circulating in the region. Using PCR for Plasmodium detection and two genetic markers specific to P. vivax to detect the polymorphic circumsporozoite protein (CSP) and the conserved 19-kDa region of the merozoite surface protein-1 (MSP-1), we investigated the overall Plasmodium strain distribution and population diversity within P. vivax in isolates collected from the blood of infected individuals in the interior Amazon region of Guyana, South America. Out of a total of 250 samples positive for Plasmodium, P. vivax was detected in 30% (76/250) and P. falciparum was detected in 76% (189/250). Mixed infections containing both P. falciparum and P. vivax constituted 6% (15/250) of the total positive samples. Further analysis of P. vivax strains showed that 92% (56/61) of the P. vivax samples hybridized with a probe specific to type VK210, 39% (24/61) hybridized with a probe specific for type VK247, and 25% (15/61) hybridized with a probe specific for the P. vivax-like CS genotype. DNA sequencing of the 19-kDa C-terminal domain in block 13 of MSP-1 amplified from 61 samples from patients infected with P. vivax demonstrated that this region is highly conserved, and all samples were identical at the nucleotide level to the Belem and Salvador-1 types. No synonymous or nonsynonymous mutations were observed in this region of the gene, indicating that current vaccine-development efforts based on the MSP-1(19) fragment would be applicable in Guyana.

We present evidence that a parasite with characteristics of Plasmodium vivax is being transmitted among Duffy blood group-negative inhabitants of Kenya. Thirty-two of 4,901 Anopheles gambiae and An. funestus (0.65%) collected in Nyanza Province were ELISA positive for the P. vivax circumsporozoite protein VK 247. All positives were found late in the rainy season, when An. funestus predominated, and disproportionately many were found at a single village. A P. vivax specific sequence of the SSU rRNA gene was amplified from three of six ELISA-positive mosquitoes. Erythrocytes from 31 children, including 9 microscopically diagnosed as infected with P. vivax, were negative by flow cytometry for the Fy3 or Fy6 epitopes, which indicate Duffy blood group expression. A DNA fragment specific for the C terminus of the gene for P. vivax merozoite surface protein 1 (MSP-1) was amplified from the blood of four of these children and subsequently sequenced from two.

We conducted a phase 1 trial of candidate malaria vaccine RTS,S/AS02A in western Kenya to determine its safety and immunogenicity in healthy adults in an area hyperendemic for malaria. Twenty adults were enrolled and received RTS,S/AS02A (50 microg of RTS,S in 0.5 mL of AS02A) by intramuscular injection on a 0-, 28-, and 178-day schedule. All 60 scheduled immunizations were given, and 18 of 20 volunteers completed the last study visit on day 210. The vaccine was safe and well-tolerated. There were no vaccine-related severe adverse events. The most common solicited adverse events associated with immunization were injection site pain and headache. The geometric mean concentration of antibodies to circumsporozoite protein was 1.9 microg/mL at baseline and it increased 2-4 weeks after each dose to 16, 17.8, and 36.6 microg/mL, respectively. These safety and immunogenicity data from adults in hyperendemic Kenya are comparable to data reported earlier from two trials in west African adults in hypo-endemic and meso-endemic areas of The Gambia. We conclude that in this small study, RTS,S/AS02A is safe and similarly immunogenic in malaria-exposed African adults of different ethnicity in different transmission settings.

High levels of antibodies to multiple antigens may be more strongly associated with protection from infection than antibodies to a single antigen. Antibody-associated protection against Plasmodium falciparum infection was assessed in a cohort of 68 adults living in an area of holoendemic malaria in Kenya. Antibodies to the pre- erythrocytic antigens circumsporozoite protein (CSP), liver-stage antigen-1 (LSA-1), thrombospondin-related adhesive protein (TRAP), and blood-stage antigens apical membrane antigen-1 (AMA-1), erythrocyte binding antigen-175 (EBA-175), and merozoite surface protein 1 (MSP-1) were tested. Peptides were used for CSP (NANP repeat) and LSA-1 (central repeat), and recombinant antigens were used for TRAP (aa D48-K394), AMA-1 (ectodomain, non- glycosylated), EBA-175 (non-glycosylated), and MSP-1 (MSP-119). Weekly microscopy testing for P. falciparum infec- tion was performed over a 12-week period after drug-mediated clearance of P. falciparum parasitemia. Individuals with high levels of IgG antibodies (> 2 arbitrary units) to CSP, LSA-1, and TRAP had a 57% decrease in the risk of infection (95% confidence interval 20-77%, P 0.016). This decreased risk remained significant after adjustment for age, prior parasitemia, bed net use, sickle cell trait, and village of residence. In contrast, protection against infection did not correlate with high levels of IgG antibodies to blood-stage antigens or IgM antibodies to pre-erythrocytic or blood-stage antigens. High levels of IgG antibodies to CSP, LSA-1, and TRAP may be useful immune correlates of protection against P. falciparum infection in malaria-endemic populations.

Transgenic mosquitoes resistant to malaria parasites are being developed to test the hypothesis that they may be used to control disease transmission. We have developed an effector portion of an antiparasite gene that can be used to test malaria resistance in transgenic mosquitoes. Mouse monoclonal antibodies that recognize the circumsporozoite protein of Plasmodium gallinaceum can block sporozoite invasion of Aedes aegypti salivary glands. An anti-circumsporozoite monoclonal antibody, N2H6D5, whose corresponding heavy- and light-chain gene variable regions were engineered as a single-chain antibody construct, binds to P. gallinaceum sporozoites and prevents infection of Ae. aegypti salivary glands when expressed from a Sindbis virus. Mean intensities of sporozoite infections of salivary glands in mosquitoes expressing N2scFv were reduced as much as 99.9% when compared to controls.

We analyzed the humoral immune response to the amino- (amino acids 22-125) and carboxy-terminal (amino acids 289-390) non-repetitive domains of the Plasmodium falciparum circumsporozoite protein (Pf CSP) in individuals belonging to three west African ethnic groups (the Fulani, Mossi, and Rimaibe ´) living in the same conditions of hyperendemic transmission in a Sudan savanna area of Burkina Faso. Previous surveys conducted in the same area showed obvious interethnic differences in the susceptibility and immune reactivity to malaria, with the Fulani showing lower infection and disease rates and higher humoral responses to various P. falciparum antigens than sympatric ethnic groups. A total of 764 subjects (311 Mossi, 273 Rimaibe ´, and 180 Fulani) of all age classes were tested. The total mean 6 SE anti-(CSPf-N-term) and anti-(CSPf-C-term) seroprevalences were 65.6 6 1.7% and 57.0 6 1.8%, respectively. These seroprevalences were lower than that recorded in the same sample for the central (NANP)40 repetitive domain (88.3 6 1.2%). As previously reported for other P. falciparum antigens (Pf CSP- (NANP)40, thrombospondin-related anonymous protein, merozoite surface protein-1, Pf 155-ring-infected eryhtrocyte surface antigen, and Pf 332), in spite of similar exposure to malaria, the Fulani showed higher immune reactivity than sympatric populations for both antigens tested. Our results confirm the presence of B cell epitopes in the non-repetitive regions of the Pf CSP; moreover a further evidence of interethnic differences in the capacity to mount humoral responses against P. falciparum malaria was obtained. The assessment of the biological basis of interethnic hetero- geneities in the susceptibility and in the humoral immune responses to malaria appears relevant in the development of anti-malaria vaccines. The circumsporozoite protein (CSP) is considered to be the major surface antigen of malaria sporozoites. The main structural and antigenic properties of CSP are identical in all Plasmodium species; the molecule contains a species-specif- ic repetitive domain encompassing about 40% of the primary structure of the protein, which corresponds to the B cell im- munodominant epitope. 1 In Plasmodium falciparum,the cen- tral domain contains about 40 repeats of the tetrapeptide NANP plus 3-4 NVDP repeats. The central domain is flanked on both sides by sequences containing conserved regions in different Plasmodium species. 2 The carboxy-ter- minal conserved region (region II) bears a striking homology to a cell adhesion domain of thrombospondin 3,4 and to re- gions of several other adhesive proteins. 5-8 The involvement of CSP in the invasion of sporozoites into hepatocytes has been shown by Cerami and others. 9 The flanking non-repeat

In Brazil, two types of activities have led to the worsening of malarial transmission in the Amazon region: prospecting/mining and agricultural settlements. In the present study, we analyze the cellular response of 52 of these individuals (14 gold-miners and 38 farmers) living within the same endemic area. Two Plasmodium falciparum major surface antigens (recombinant proteins) were used for cellular proliferative assays: circumsporozoite protein and merozoite surface protein-1. The frequency of these cellular responses were significantly higher among the miners (57-64%) than the farmers (10-20%) when either recombinant protein was used. Our data suggest that a higher exposure to malaria of the gold-miners contributed to their higher in vitro cellular response compared with the farmers. These findings point the way to further studies evaluating the influence of risk factors associated with the life styles of different social groups and the immune responses to these antigens.

Collections of human sera from malaria-endemic areas would be valuable for identifying and characterizing antigens as malaria vaccine candidates if the contributing serum donors' ability to resist infection were fully characterized. We prepared such a serum collection from 26 apparently immune Nigerian adults who failed to develop patent parasitemia for at least 20 weeks following a documented increase in antibodies to the circumsporozoite protein (CSP) from Plasmodium falciparum. Volunteers were evaluated five times per week for malaria symptoms and bimonthly for parasites by examining thick blood smears. The incidence rate over 13 months for the cohort was 42% (47 malaria-confirmed volunteers) and the risk of infection was 1.3 infections/year. Responses to CSP did not correlate with protection. Because antibody responses to antigens other than CSP may be associated with protection, the sera from these immune individuals may be useful for identifying and characterizing other potential malaria vaccine candidates.

The circumsporozoite antigen (CS) of the simian malarial parasite Plasmodium knowlesi consists of tandemly repeated immunodominant peptide units that are variable and may play a role in evading the immune system. To study the immunogenicity of this antigen in the absence of the immunodominant repeats, the entire nonrepetitive region of the antigen was expressed in Escherichia coli as two fusion proteins with glutathione-S-transferase (GST) representing the amino terminal (GST-CSN) and the carboxy terminal domains (GST-CSC) of the CS antigen. The immunogenicity of these fusion proteins was studied in rabbits and different strains of mice. Antibody raised against both the CSN and CSC domains in both rabbits and every strain of mice recognized the native protein, as detected by immunofluorescence assay (IFA) using P. knowlesi sporozoites. A positive IFA reaction was also obtained with P. vivax sporozoites using antisera raised against the CSC domain. High titer antisera were raised in rabbits against both the domains, whereas mice showed comparatively low titers. On Western blots, mice showed specific response against the CSC domain. In both rabbits and mice, significant titers of antibodies were raised against region II, which has been shown to be the putative sporozoite binding site for hepatocytes in the case of P. falciparum.

We have characterized the circumsporozoite (CS) gene sequences of Plasmodium malariae China-1 CDC, isolated recently from a person who was infected 50 years ago in China, and P. vivax Chesson, isolated 48 years ago from a patient who had returned from New Guinea. These protein sequences were compared with the CS protein sequences of recently isolated P. vivax and P. malariae parasites. In a similar manner, we compared the previously characterized CS protein gene of P. falciparum clone 7G8, derived from a Brazilian isolate collected in 1980, with the CS protein genes of recent P. falciparum field isolates. In the case of the P. malariae CS protein gene, with the exception of an additional copy of major (NAAG) and minor (NDAG) repeat sequences and the presence of one copy of NDEG sequence, the China-1 CDC P. malariae parasite is similar to the Uganda-1 CDC isolate of 1982. In the nonrepeat region, changes were noted in two amino acid residues, one of which is also seen in a closely related monkey malaria parasite, P. brasilianum. In the case of P. vivax CS proteins, the nonrepeat region of the protein in Chesson strain shares identity with nearly 71% of the CS clones characterized from field isolates. In the P. falciparum CS proteins, the 7G8 CS protein sequence is identical to 75% of the genes of recent field isolates in the Th1R-N1 region. In the Th2R and Th3R regions, 34% and 55% of the CS clones analyzed, respectively, had changes at two amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Reemerged Plasmodium vivax malaria in South Korea has not yet been eradicated despite continuous governmental efforts. It has rather become an endemic disease. Our study aimed to determine the genetic diversity in P. vivax merozoite surface protein-1 (PvMSP-1) and circumsporozoite protein (PvCSP) genes over an extended period after its reemergence to its current status. Sequence analysis of PvMSP-1 gene sequences from the 632 P. vivax isolates during 1996-2007 indicates that most isolates recently obtained were different from isolates obtained in the initial reemergence period. There was initially only one subtype (recombinant) present but its subtypes have varied since 2000; six MSP-1 subtypes were recently found. A similar variation was observed by CSP gene analysis; a new CSP subtype was found. Understanding genetic variation patterns of the parasite may help to analyze trends and assess extent of endemic malaria in South Korea.

To determine the nature and extent of variation in the T cell sites of the Plasmodium falciparum circumsporozoite (CS) protein, a candidate antigen in the development of a malaria vaccine, we cloned and sequenced 69 recombinant clones of the CS protein gene representing 18 and 17 P. falciparum isolates from infected individuals from Madang, Papua New Guinea (PNG), a holoendemic malaria region, and Paragaminos and Jacunda, Brazil, relatively low endemic regions, respectively. As previously known, the amino acid sequence polymorphism was restricted to the three immunodominant regions of the protein, Th1R-N1, Th2R, and Th3R. While some of the observed nonsilent mutations in the T cell determinants of the CS protein were similar to those previously identified, we have found new amino acid changes in each of the polymorphic sequences in parasites from PNG and Brazil. A comparison of the CS epitope sequences of parasites from PNG and Brazil with the previously known CS epitope sequences of parasites from Brazil and The Gambia showed the following: 1) polymorphism was found in the Th1R-N1, Th2R, and Th3R region; however, while amino acid substitutions in the Th1R-N1 and Th2R region tended to be conservative, the substitutions found in the Th3R region were not, suggesting that the Th3R epitope may be rapidly evolving to allow parasites to escape host antiparasite cytotoxic T cell-enforced immune responses, and 2) the CS proteins of P. falciparum from high malaria-transmission regions (PNG and The Gambia) appear more polymorphic than the CS proteins of parasites from relatively low malaria-endemic regions in Brazil, where P. falciparum infection has been recently established.

Antibody responses to the circumsporozoite (CS) protein of Plasmodium falciparum have previously been reported against the central repeating tetrapeptides of this protein. Segments of the protein flanking the repeat region also contain B-cell epitopes, but specific antibody responses have not been previously characterized. Longitudinal serum sets from 16 Thai adults who developed acute falciparum malaria were selected to represent a spectrum of antibody response to the repeat region (R32). These sera were assayed by enzyme-linked immunosorbent assay using as capture antigen a recombinant fusion protein, NS1(81)RLF, which contains both flanking regions, but lacks the NANP and NVDP repeats of the P. falciparum CS protein. Antibody responses to the repeatless flanking (RLF) regions were observed in all subjects, including five individuals who lacked detectable anti-R32 antibody responses. Anti-RLF antibody responses induced by natural infection appear to be short-lived and of low-to-moderate magnitude. Thus, if anti-RLF antibodies prove to be protective, derived vaccine candidates may require presentation of these epitopes with adjuvants or delivery systems that enhance immunogenicity.

In areas studied in the Rondonia State of Brazil, a high rate of malaria transmission and a low prevalence of anti-(NANP)4 antibodies are reported. The entomologic data are comparable to those observed in some malaria-endemic areas of Africa and Asia. However, the frequency of individuals with antibodies to the immunodominant epitope of the circumsporozoite protein of Plasmodium falciparum recorded in the four localities of Rondonia state was very low when compared with the frequencies recorded in other African and Asian endemic areas. Most of the studies performed in Africa and Asia concerned the native population of hyperendemic areas, whereas we studied a migrant population who were mostly from malaria-free areas of Brazil and living in Rondonia State for 2-4 years. In positive individuals the antibody production was influenced by previous malaria experience, suggesting that infective bites must occur in cumulative numbers before anti-(NANP)4 antibodies are detected. Therefore, it is possible that the individuals described in this report have not been exposed long enough to malaria infection to develop detectable levels of anti-(NANP)4 antibodies.

The antibody response to the prototype circumsporozoite (CS) protein of Plasmodium vivax (CSPV) was studied in Thai soldiers experiencing occupational malaria. Seventy-four (65%) of 114 men followed during assignment to a malaria transmission area developed blood-stage infection with P. vivax. IgG antibodies against the central repeat region of the CSPV protein were quantitated by ELISA using the recombinant protein, NS181V20, as the capture antigen. One quarter of the subjects had detectable anti-CSPV antibodies at the beginning of the study. CSPV antibody seroconversion was documented in 16 of 26 subjects assessed during their first observed episodes of vivax malaria. This antibody response was of moderate magnitude, fell off after the first week post-diagnosis and appeared, at the low levels observed, to be unassociated with protection. Continued assessment of anti-CSPV antibody after subjects left the transmission area found no increase associated with relapse of P. vivax. These findings indicate that CS antibody responses to P. vivax during occupational malaria share many characteristics with responses to P. falciparum.

We describe a membrane based immunodot assay for the detection of Plasmodium falciparum sporozoites mixed with mosquitoes. A crude sodium dodecyl sulfate extract of mosquitoes and sporozoites is passed through a bi-layered membrane system, the top layer being a polyvinyldiene difluoride hydrophilic pre-filter which screens out debris but allows the passage of antigen. Sporozoite, as well as mosquito, proteins are bound to the hydrophobic membrane below. This membrane was probed with a monoclonal antibody to the repeat region of the P. falciparum circumsporozoite protein, a peroxidase labeled second antibody and a tetramethyl-benzidine substrate. The method detects as few as 10 sporozoites/mosquito or 100 sporozoites in a pool of 10.

Passage of rat antibodies induced by Plasmodium falciparum circumsporozoite protein (anti-CS IgG) from the bloodmeal into the hemocoel of uninfected Anopheles stephensi mosquitoes was quantified using enzyme-linked immunosorbent assay (ELISA) techniques. Anti-CS IgG were present in hemolymph immediately upon cessation of mosquito feeding. Titers peaked at 3 hr post-ingestion then declined steadily, becoming negligible at 18 hr. Substantial titers were present in the bloodmeal at 24 hr post-ingestion. By 48 hr, anti-CS IgG in both mosquito hemolymph and bloodmeal were virtually absent. Estimated quantities of anti-CS IgG in the hemolymph at 3 hr post-ingestion were 905-958 ng/ml, representing approximately 0.5% of that present in the host serum. Rat IgG subclasses 1, 2a, and 2b passed into hemolymph more readily than did IgM and possibly IgG2c. Hemolymph volume of unengorged mosquitoes (0.53 microliters) increased after a bloodmeal (0.73 microliters at 3 hr post-ingestion), suggesting that anti-CS IgG may move into the hemocoel in an aqueous solution.

Protective devices against vectors are used by travelers in malaria-endemic areas but their efficacy for protection against mosquitoes has rarely been evaluated. The level of exposure to malaria transmission of 205 soldiers deployed in Africa and the efficacy of their anti-vector prophylaxis was evaluated by comparison of their IgM and IgG responses against five pre-erythrocytic Plasmodium falciparum antigens (circumsporozoite protein, sporozoite threonine- and asparagine-rich protein, sporozoite- and liver-stage antigen, liver stage antigen 1, and SR11.1) before and at the end of their deployment, and three months after returning to France for 106 of these soldiers. The immune responses increased significantly during the mission in 35% (95% confidence interval = 28-42%) of the individuals. The permanent use of insecticide-treated bed nets and long-sleeve battle dress at night were associated with protective efficacy. The analysis of these antibody responses was sensitive enough to evaluate exposure to malaria transmission and the efficacy of anti-vector devices in travelers using antimalarial chemoprophylaxis.

Using two polymorphic genetic markers, the merozoite surface protein-3alpha (MSP-3alpha) and the circumsporozoite protein (CSP), we investigated the population diversity of Plasmodium vivax in Mae Sod, Thailand from April 2000 through June 2001. Genotyping the parasites isolated from 90 malaria patients attending two local clinics for the dimorphic CSP gene revealed that the majority of the parasites (77%) were the VK210 type. Genotyping the MSP3-alpha gene indicated that P. vivax populations exhibited an equally high level of polymorphism as those from Papua New Guinea, a hyperendemic region. Based on the length of polymerase chain reaction products, three major types of the MSP-3alpha locus were distinguished, with frequencies of 74.8%, 18.7%, and 6.5%, respectively. The 13 alleles distinguished by restriction fragment length polymorphism analysis did not show a significant seasonal variation in frequency. Genotyping the MSP-3alpha and CSP genes showed that 19.3% and 25.6% of the patients had multiple infections, respectively, and the combined rate was 35.6%. Comparisons of MSP-3alpha sequences from nine clones further confirmed the high level of genetic diversity of the parasite and also suggested that geographic isolation may exist. These results strongly indicate that P. vivax populations are highly diverse and multiple clonal infections are common in this malaria-hypoendemic region of Thailand.

The present study was initiated to characterize antibody responses to repetitive epitopes of the circumsporozoite protein (CSP), liver stage antigen-1 (LSA-1), and merozoite surface protein-2 (MSP-2) of Plasmodium falciparum in infants residing in a P. falciparum-hyperendemic area of western Kenya. In this study, development and maintenance of these antibody responses in 28 infants were studied longitudinally by use of monthly serum samples collected from birth to age 1 year. Mother plasma and infant umbilical cord plasma were also tested to assess the transplacental transfer of maternal antibodies. Results showed that antibodies passively transferred from mothers were detectable for CSP, LSA-1, and MSP-2 repeat epitopes. Infants were able to mount and maintain a strong antibody response against LSA-1 in their first year of life. Infants often responded to CSP repeats, but with a much lower antibody titer. Antibody responses in infants against Fc27 and 3D7 repeats of MSP-2 were low throughout their first year. In addition, 51 infants whose first detected infection occurred at > 4 months of age were selected to determine antibody responses to the antigens tested upon their first and second detected infections. Antibody responses to LSA-1 and, to a lesser degree, CSP increased in positivity rates and titer upon second infection. Antibody responses to Fc27-type and 3D7-type repeats of MSP-2 were low upon both infections. There was no association between maternally transferred anti-LSA-1, anti-CSP, or anti-MSP-2 antibodies and an infant's first detected infection. No significant correlation was found between an infant's antibody responses to the 4 antigen repetitive epitopes and protection against malarial parasitemia during the first year of life.

The extent of genetically distinct parasite populations coinfecting individual human hosts (i.e., multiplicity) was studied by polymerase chain reaction amplification of 3 polymorphic genetic markers, circumsporozoite protein and merozoite surface antigens (MSA) 1 and 2, in symptomatic children and adults and analyzed in relation with age and initial parasitemia. Of the total of 177 DNA samples analyzed (of which 115 were paired pre- and posttreatment samples), 101 (57%) were composed of multiclonal infections, with up to 7 distinguishable parasite populations. Among the 3 polymorphic markers, msa-2 yielded the highest proportion of clinical isolates with multiclonal populations. Patients with multiclonal infections before treatment had, on average, 2.9 genetically distinct parasite populations. The extent of multiplicity decreased significantly (P < 0.05) in recrudescent parasites, but not with reinfections, as compared with the pretreatment samples. Neither age (5-60 years) nor initial parasitemia was correlated with multiplicity. Further studies in different epidemiological settings are required to understand the role of multiclonal Plasmodium falciparum infections in influencing malaria transmission.

We measured sporozoite- and total parasite antigen-specific IgG and IgM antibodies before and after treatment in matched groups of Gabonese children who presented with either mild or severe Plasmodium falciparum malaria. We investigated the influence of various parameters on these antibody responses, including clinical presentation, age, and post-treatment reinfection profiles. IgG but not IgM responses were strongly influenced by both clinical and parasitological status. IgG responses to the repeat region of the circumsporozoite protein, which were low at admission, particularly so in those with severe anemia, increased after treatment but showed no association with either age or reinfection profiles. Total parasite antigen-specific IgG responses were strongly influenced by parasitological status, and also differed significantly when segregated according to clinical status at admission, age, and reinfection histories. Most notably, anti-parasite IgG responses measured when children were parasite-free were higher and a good indicator of recent reinfections in those who presented with mild rather than with severe malaria. The profile of responses in the latter group suggests some immune system dysfunction, which may reflect the induction of tolerance to parasite antigens.

The present study was designed to investigate the genetic diversity of Plasmodium falciparum among field isolates from India. A total of 71 clinical isolates were analyzed by the polymerase chain reaction (PCR) for the amplification of repeat regions of malaria vaccine candidate antigen genes, i.e., merozoite surface antigen-1 (MSA-1), MSA-2, and circumsporozoite protein (CSP). All three genes showed variation; MSA-2 has the maximum number of 10 variant forms while MSA-1 and CSP had 8 and 6 variants, respectively. Some variant forms were more common than others among the clinical isolates. There were mixed alleles for each gene in several (27 of 71) cases. The MSA-2 gene showed the maximum number of cases with mixed alleles (22 of 65 [33.85%]) compared with MSA-1 (10 of 68 [14.7%]) and CSP (10 of 65 [15.38%]). Fifty-five (88.7%) of 62 clinical isolates of P. falciparum showed a different genotype. The malaria hyperendemic region (Orissa) not only showed the maximum number of variant forms of each gene but also the maximum number of cases with mixed alleles compared with the non-hyperendemic regions (Madhya Pradesh and Rajasthan). The presence of such large numbers of P. falciparum strains in India should be taken into account in future malaria vaccine programs.

The prevalence and severity of drug-resistant malaria is emerging rapidly in the Amazon basin of Brazil. In support of clinical trials using the new antimalarial drug combination of atovaquone and proguanil, we performed in vitro drug sensitivities, molecular characterization of parasite populations using the circumsporozoite protein, merozoite surface antigen-1 (MSA-1), and MSA-2 markers, and an analysis of the Plasmodium falciparum multidrug resistance (pfmdr1) gene sequence and copy number in 26 isolates of P. falciparum obtained in a gold-mining endemic area in Peixoto de Azevedo, Mato Grosso State. All 26 isolates were found to be resistant to chloroquine (50% inhibitory concentration [IC50] = 100-620 nM) and sensitive to mefloquine (IC50 < 23 nM) and halofantrine (IC50 < 6 nM). The isolates also show reduced susceptibility to quinine (IC50 = 48-280 nM). Sequence analysis of the pfmdr1 gene revealed Asn, Phe, Cys, Asp, and Tyr in positions 86, 184, 1034, 1042, and 1246, respectively. These point mutations were similar to that previously described in other Brazilian isolates. Southern blot analysis revealed no amplification of the pfmdr1 gene. These results suggest that three different mechanisms for drug resistance exist for chloroquine, mefloquine, and quinine.

Saimiri boliviensis monkeys were immunized with a multiple antigen construct [(PvCS)2]2(P2)2 directed against the circumsporozoite protein of Plasmodium vivax or a combination of the multiple antigen construct with nonionic copolymer P1005, with P1005 and lipopolysaccharide, with muramyl tripeptide Mf-75, or with alum. Following intravenous challenge with 10,000 sporozoites of the Salvador I strain of P. vivax, 11 of the 26 monkeys were protected against patent parasitemia. Ten additional animals were partially protected. Following rechallenge of the 26 monkeys with 30,000 sporozoites of the homologous strain of parasite, four monkeys were totally protected and nine animals were partially protected.

In the present work, we analyze the recognition of synthetic polypeptides encompassing the aminoterminal (amino acids 22-125) and the carboxy terminal (289-390) regions of the circumsporozoite (CS) protein of Plasmodium falciparum by sera from donors living in endemic area of South America and Africa. Two populations were studied: one on the Colombian Pacific coast, with low endemicity for malaria; and a western African village exposed to a very intense transmission of P. falciparum. Antibodies directed to the two polypeptides were found at high titers in both populations. Furthermore, this response was observed in individuals lacking antibodies to the highly repetitive central sequence of the CS protein (NANP). The epitopes responsible for this recognition were mapped to the region 81-125 and 316-346 of the N- and C-termini, respectively. When the two populations were compared, both showed high titers of antibodies to the two flanking peptides. However, while 95% of the sera from African adults showed antibodies against the repeat region of the CS protein, only 37% of the Colombian adults studied had these antibodies. Furthermore, African donors of various ages exhibited different patterns of recognition of the two polypeptides. In African children less than five years of age, antibodies were found in comparable levels to Colombian adults; however, in older African donors, the response to NANP became dominant. These findings may reflect the skewing effect of the humoral response towards the central repetitive epitope under conditions of frequent exposure to malaria infections. The production of such polypeptides encompassing regions that contain multiple epitopes for antibodies, T helper, and cytotoxic T lymphocyte epitopes would be advantageous in the generation of new and more efficient malaria vaccines.

Humoral response against sporozoites is not effective in protecting individuals from getting malaria. Reduction in the infectivity of sporozoites has not been quantified for most anti-sporozoite vaccines tested. Quantification requires animal models providing predictable prepatent periods, e.g., time elapsed between sporozoite inoculation and detection of parasitemia, to be used as an indicator of activity against sporozoites. A delay in prepatent period from vaccinated animals would therefore reflect a protective effect in reducing the number of parasites. We report the vaccination of rhesus monkeys with a synthetic peptide reproducing part of the repeated region of the circumsporozoite protein of Plasmodium cynomolgi. This peptide was conjugated to the carrier protein diphtheria toxoid and injected with four adjuvant formulations that differed only by the type of emulsion or immunomodulator. Because all five control animals had a synchronous prepatent period after challenge with live sporozoites, it was possible to quantify the protective efficacy for each vaccine formulation, even though all monkeys developed parasitemia. Sporozoite elimination correlated with the immunomodulator and the type of emulsion. Such elimination was related neither to antibody titer against the immunizing peptide or the whole sporozoite, nor to antibody isotype induced by the vaccine formulation.

Allelic variation in the Plasmodium falciparum circumsporozoite (CS) protein gene has been examined by sequencing the entire gene in 15 isolates from an endemic area of Thailand. The isolates contain a total of six new allelic forms of the tetrapeptide repeats and eight variants of the T cell epitope (TCE) region of the CS gene. All nucleotide substitutions in the TCE are nonsynonymous. There is no apparent association between the sequence patterns in the repeats and in the TCE. Comparison of the TCE with published sequences has shown that most variants of our isolates are not identical to those found in different geographic areas, suggesting geographic variation in genetic diversity of the CS protein. In a phylogenetic tree, the new Thai alleles did not cluster together, suggesting a considerable heterogeneity within some geographic areas. Furthermore, analyses of tetrapeptide repeats from a number of isolates and strains showed evidence of three genetic mechanisms for the generation of variation in the repeats of the CS gene: point mutation, duplication of one or more repeat units, and intragenic recombination., American Journal of Tropical Medicine and Hygiene, 51(5), pp.659-668; 1994

A Plasmodium falciparum circumsporozoite protein (PfCSP) recombinant fusion protein, R32NS1(81), formulated with monophosphoryl lipid A, cell wall skeleton of mycobacteria, and squalane (Detox) was administered to 12 volunteers. One volunteer had malaise and self-limited painful induration at the injection site after the second dose and declined further immunization. The other 11 volunteers tolerated the three doses of 1,230 micrograms of vaccine, but most complained of sore arms; in five cases the pain or malaise was severe enough to interfere with work or sleep. Two weeks after the third dose of vaccine, four of the 11 immunized volunteers hador = 14 micrograms/ml of antibodies to the repeat region of the PfCSP in their serum. Two of these four volunteers did not develop P. falciparum parasitemia when challenged by the bite of five mosquitoes carrying P. falciparum sporozoites. The seven volunteers with lower levels of antibodies and 11 of 11 controls developed parasitemia. These data are consistent with other studies, and indicate that vaccine-induced antibodies against the repeat region of PfCSP can prevent effective sporozoite infection of hepatocytes in humans. The challenge is to improve the immunogenicity of PfCSP-based vaccines, and to develop methods for including PfCSP peptides as components of multitarget malaria vaccines.

We have shown in a previous study that immunization of a rhesus monkey (Macaca mulatta) with inactivated liver stages of the simian malaria parasite Plasmodium cynomolgi (B strain) produced high antibody titers against sporozoites, liver stages, and blood stages of P. cynomolgi. In the present study, we demonstrate that these anti-P. cynomolgi immune sera recognized P. vivax (Salvador I) antigens. In an indirect immunofluorescence assay, both postimmunization and postchallenge sera reacted with antigens of sporozoite, liver-, and blood-stage parasites. In Western blot analysis, postimmunization sera recognized four bands of 97, 93, 70, and 65 kD in sporozoite antigens; postchallenge sera further recognized three doublet (set of two) bands of 86–90, 73–78, and 44–52 kD. When blood-stage extracts were used as antigens, five bands of 118, 105, 76, 68, and 47 kD reacted with postimmunization sera; two doublet bands of 81–87 and 49–52 kD further reacted with postchallenge sera. None of these sera reacted with asexual blood-stage extracts of P. falciparum and P. malariae, or with a recombinant circumsporozoite protein of P. vivax.

Enzyme-linked immunosorbent assays (ELISAs) highly specific for the characteristic repeat units of the circumsporozoite proteins of the VK 247 and VK 210 polymorphs of Plasmodium vivax were used to test sporozoites produced by feeding mosquitoes on 1,711 human volunteers presenting at four locations in Thailand over five years. There was no evidence for the existence of any polymorph other than the two already described. Based on the ELISAs, the overall prevalence of the VK 247 type was 29.5%, including those found mixed with VK 210. Relative proportions of VK 210 and VK 247 differed between collection sites. At all places, the ratio of VK 210 to VK 247 was significantly higher at the end of the nontransmission season than it was later during the annual monsoon, suggesting that there may be intrinsic biological differences between the polymorphs that affect their survival.

The immunogenicity of the cocktail vaccine SPf66 against Plasmodium falciparum was investigated in rabbits, monkeys, and human volunteers. This polymerized peptide vaccine incorporates a portion of each of the 35-kD, 55-kD, and 83-kD blood-stage proteins, linked by an amino acid sequence reproducing one repeat from the circumsporozoite protein of P. falciparum. Results from this study show that vaccination with this vaccine molecule elicited high titers of antibodies to SPf66 and its constituents, but these antibodies did not correlate with regular or sensitive indirect fluorescent antibody (IFA) titers to blood-stage malaria parasites. However, rabbits injected with individual peptides produced antibodies with low affinity to indefinite parasite structures by immunoelectron microscopy, and rabbits injected with SPf66 had high antibody titers against the peptide (NANP)40. Consequently, these anti-SPf66 serum samples recognized P. falciparum sporozoites in the IFA. Such reactivity was not observed in monkeys or human volunteers vaccinated with SPf66. In addition, SPf66 components were recognized by antibodies induced by natural infection in humans and by laboratory-monitored infections in Aotus monkeys. These results suggest that this vaccine candidate merits further developmental work to better define immune response elicited by the copolymer to the parasite.

The repeat region of the Plasmodium vivax circumsporozoite (CS) protein contains 20 copies of the nine-amino acid sequence DRA A/D GQPAG. A monoclonal antibody that passively protects monkeys against sporozoite challenge recognizes a four-amino acid linear sequence AGDR included within this nonamer, but when monkeys were immunized with a vaccine, NS181 V20, which contains 20 copies of the nonamer, they failed to produce antibodies to AGDR. To determine if natural exposure to sporozoites induces antibodies to AGDR, we tested sera from 176 individuals from a malaria-endemic area in Flores, Indonesia. Seventy-one percent of the adults had antibodies to the P. vivax repeat region; only 18% had detectable antibodies to AGDR. None of the subjects had antibodies to the P. vivax variant repeat ANGAGNQPG. We next tested sera from six human volunteers immunized with NS181 V20 and found that the vaccine, despite inducing antibodies against the nonamer, as it did in the monkeys, did not induce antibodies against AGDR. To further test our ability to raise anti-AGDR antibodies using synthetic peptides, we immunized Aotus monkeys and BALB/c mice with AGDR. Sera from the mice reacted strongly with both AGDR and a recombinant protein containing the 20 copies of the nonamer. Sera from the monkeys reacted only minimally with a protein (VIVAX-1) that contains monomeric AGDR within its sequence. Sera from the mice also bound air-dried P. vivax sporozoites, while sera from the monkeys did not. These data indicate that natural exposure induces very low levels of antibodies to the CS protein epitope AGDR, and that polyclonal antibodies that recognize native CS protein can be induced experimentally with a synthetic AGDR peptide.

Individuals living in a malaria-endemic area in northern Peru were found to have antibodies to the variant repeat sequence of the circumsporozoite (CS) protein of Plasmodium vivax. The presence of IgG antibody to the predominant repeat sequence GDRAA/DGQPA represented by the recombinant protein NS181 V20 (V20), and the variant repeat sequence ANGAGNQPG contained in the synthetic peptide Pvk247, was determined by enzyme-linked immunosorbent assay. IgG antibodies to the repeats were present in 78 (26%) of 298 serum samples; 56% of the positive serum samples had antibodies to V20 and 60% had antibodies to Pvk247. These findings stress the importance of considering the variant epitope in designing a vaccine based on the repeat region of the vivax CS protein. In a malaria-endemic area such as the one in this study, in which exposure to the variant repeat epitope may be as frequent as exposure to the predominant repeat, a vaccine based solely on the predominant repeat epitope may be ineffective against the variant form.

The circumsporozoite (CS) protein of Plasmodium vivax consists of a central repeat region flanked by highly conserved non-repeat regions. Serum samples from 33 individuals with naturally acquired infections of P. vivax were tested for antibodies to four antigens representing the vivax CS protein. Three recombinant proteins containing different overlapping sequences in the non-repeat regions and either the entire central repeat region (vivax-1 and vivax-2) or two of the repeat sequences (vivax-3) were used as antigens in an enzyme-linked immunosorbent assay (ELISA). Antibodies to two other proteins, one (NS1(81)V20) containing the entire predominant repeat region (GDRAA/DGQPA) and the other (Pvk247) containing the variant repeat sequence (ANGAGNQPG) that was recently reported from Thailand were also measured by ELISA. Immunoglobulin G antibodies to the antigen representing the predominant repeat were present in 15% of the patients on the first day of treatment (day 0) and in 24% of the patients two weeks later (post-treatment). Six and 12% of the patients had IgG antibodies to the antigen containing the variant repeat on day 0 and post-treatment, respectively. A larger proportion of the sera had antibodies to the three antigens containing the non-repeat sequences; on the first day of treatment and two weeks later, 79 and 97% of the patients, respectively, had antibodies to vivax-1, vivax-2, and vivax-3. In this sample of Peruvians naturally infected with P. vivax, the most prevalent antibody responses were targeted to epitopes in the non-repeat region of the CS protein rather than to epitopes in the repeat region.(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferative responses of peripheral blood lymphocytes to synthetic peptides representing major epitopes of two malaria antigens (the merozoite ring-infected erythrocyte surface antigen and the sporozoite circumsporozoite protein) were investigated in Madagascar during a clinical Plasmodium falciparum episode. Thirty-seven patients greater than 10 years of age were enrolled at the beginning of the malaria transmission season and followed for four weeks. At enrollment, when the subjects presented with an acute infection, lymphocytes recovered from approximately 30% of them proliferated after peptide stimulation. These proliferative responses decreased sharply one and two weeks after treatment, with less than 10% responding to each peptide. Four weeks after treatment, the responses were only partially restored. The amplitude of these variations was not related to the initial parasitemia. At the individual level, proliferative response to each peptide varied greatly during the followup period, and this variation was unrelated to the presence of parasites in the blood.