Circumsporozoite Research Articles

Monoclonal antibodies of three different immunoglobulin G isotypes produced by immunization with a synthetic peptide or native protein protect mice against challenge with Plasmodium yoelii sporozoites.

Passive transfer of monoclonal antibodies (MAbs) against malaria circumsporozoite (CS) proteins protects animals against malaria. Active immunization with synthetic or recombinant peptides induces a level of polyclonal antibodies to sporozoites comparable to those found after passive immunization but does not provide comparable protection. In the Plasmodium yoelii system, synthetic or recombinant peptide-induced antibodies have never been shown to protect. The current studies were designed to determine whether immunogen structure (native protein versus synthetic peptide) or immunoglobulin G (IgG) subclass of antibodies was responsible for the absolute differences between protective, passively transferred MAbs and nonprotective, actively induced polyclonal antibodies. In this study we produced two MAbs, QGP-S1 (IgG1) and QGP-S2 (IgG2b), by immunization with a synthetic peptide based on the P. yoelii CS major repeat, (QGPGAP)4, conjugated to keyhole limpet hemocyanin. These MAbs were compared tp NYS1 (IgG3), an anti-CS protein MAb previously produced by immunization with irradiated P. yoelii sporozoites, which recognizes (QGP GAP)2. QGP-S1 and QGP-S2 passively transferred protection. However, when compared with NYS1, there was a hierarchy of protection, NYS1 > QGP-S1 > QGP-S2. There was no correlation between antibody level at challenge as determined by immunofluorescent antibody test against sporozoites or enzyme-linked immunosorbent assay against (QGPGAP)2 or apparent antibody avidity for (QGPGAP)2 by sodium thiocyanate elution assay. The data demonstrate that a synthetic peptide can induce protective antibodies and that a specific antibody subclass is not required for protection. Work to determine whether antibody affinity or fine specificity can explain the hierarchy of protection among the MAbs is under way.

Plasmodium vivax VK247 and VK210 circumsporozoite proteins in Anopheles mosquitoes from Andoas, Peru.

Anopheles mosquitoes captured in Andoas, Peru, a Plasmodium vivax-endemic area in the Peruvian Amazon region, contained both VK210 and VK247 P. vivax circumsporozoite (CS) proteins. Approximately 0.9% of the 4,403 mosquitoes tested by enzyme-linked immunosorbent assay were positive; 28% and 72% of the positive mosquitoes contained VK210 and VK247 CS proteins, respectively. These findings correlate strongly with a recent report of the presence of antibodies in residents of this area that recognize the VK210 and VK247 repeats, clearly indicating that both P. vivax CS protein polymorphs are common in the region.

Humoral and Cellular Immune Responses to the Circumsporozoite Protein of Plasmodium vivax in Madagascar

Plasmodium vivax malaria is prevalent during the rainy season in the central highlands of Madagascar. In April 1991, we investigated the cellular and antibody immune responses of 53 inhabitants of Manarintsoa, a village in this area, to four antigens corresponding to B and T cell epitopes of the P. vivax circumsporozoite (CS) protein. Cellular responses were assessed by lymphocyte proliferation assay as well as by detection of interferon-gamma and interleukin-2 production in vitro. Cell culture was performed with two overlapping synthetic peptides (CSVTCGVGVRVRSRVNA [amino acids 311-326]) and VRVRSRVNAANKKPED [amino acids 319-334]) from the vicinity of the highly conserved region II of the CS protein. In at least one of the three assays, cells from seven subjects showed a positive response to CSVTCGVGVRVRSRVNA, while cells form 14 subjects responded to VRVRSRVNAANKKPED. Antibodies directed against the two recombinant antigens, NS1(81)V20 and rPvCS-2, both of which contain the entire central repeat region of the P. vivax CS protein, plus regions I and II in the case of rPvCS-2, were measured by the Falcon assay screening test-enzyme-linked immunosorbent assay. Eight and nine subjects had antibodies to NS1(81)V20 and rPvCS-2, respectively. The presence of antibody responses to both recombinant antigens was related (P = 0.02, by Fisher's exact test), but was not related to the presence of a cellular response to peptides from vicinity of region II (P > 0.1, by Fisher's exact test).(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of Plasmodium vivax-like human malaria parasite

There are four species of human malarial parasite and several monkey ones, and in evolutionary terms the human and non-human primate plasmodia may be related. The tools of molecular biology have lately pointed to the existence of two types of Plasmodium vivax. Using specific oligonucleotides we have identified a human malaria parasite resembling P vivax under the microscope but with circumsporozoite (CS) protein differing from those of P vivax types 1 and 2. The CS protein of this "P vivax-like" malaria parasite is identical to that of P simiovale, a monkey parasite resembling P ovale, a human one, morphologically. Polyclonal serum raised against a partial repeat sequence of the P vivax-like malaria parasite specifically reacted with P simiovalesporozoites but not with those of P vivax types 1 and 2, P ovale, or P simium. Sera collected from people living in malaria endemic regions of Papua New Guinea and Brazil contained antibodies that specifically reacted with the synthetic peptides representing the repeat sequences of CS protein of this P vivax-like parasite. A comparison of the CS protein gene sequences of P simiovale and the P vivax-like malaria parasites with those of other primate parasites, data on serological cross-reactivity, and 18 S ribosomal DNA analyses suggest that the new human malaria parasite described here is distinct from P falciparum, P malariae, P vivax, and P ovale, the four known species of human parasite.

Malaria vaccine: Immunization of mice with a synthetic T cell helper epitope alone leads to protective immunity

The immunogenicity of the non-repetitive sequences of the Plasmodium berghei circumsporozoite (CS) protein was studied using synthetic peptides. Two CS sequences (residues 20-39 and 57-70) exhibiting T cell helper activity were identified. Immunization of BALB/c mice with a branched peptide containing either the 20-39 or the 57-70 sequence and two repeats (B epitope) in a linear sequence induced high titers of anti-repeat and anti-sporozoite antibodies. Mice immunized with the T-B construct (high antibody titers) or with the 57-70 epitope alone (no serum anti-repeat or anti-peptide antibodies) were protected to a similar degree after challenge with infective sporozoites. No protection was obtained in mice immunized with the 20-39 epitope. These results indicate that BALB/c mice can be protected either by effector T cells or by high levels of anti-repeat antibodies. Thus, in the same strain, a double mechanism of protection can be obtained by a synthetic peptide vaccine.

The transcript encoding the circumsporozoite antigen of Plasmodium berghei utilizes heterogeneous polyadenylation sites.

We have employed polymerase chain reaction-based techniques to examine the transcript encoding the circumsporozoite (CS) antigen, the immunodominant coat protein of the infectious stage of the murine parasite Plasmodium berghei. Earlier studies suggested that the 3' terminus of the CS message might be determined by transcription termination rather than by cleavage and polyadenylation, as in most eukaryotes. Here we report that a subset of CS messages are polyadenylated. Moreover, the poly(A) tails are added at multiple sites clustered within a short region 300 bp downstream from the stop codon. Whether 3' end heterogeneity is peculiar to the CS message or a common feature of plasmodial transcripts remains to be determined.

Cytotoxic T lymphocyte (CTL) low-responsiveness to the Plasmodium falciparum circumsporozoite protein in naturally-exposed endemic populations: analysis of human CTL response to most known variants

Cytotoxic T lymphocytes (CTL) specific for epitope(s) within the circumsporozoite (CS) protein of malaria sporozoites have been shown to play an important role in protective immunity against malaria, at least in murine models. Their role in sporozoite immunity in the human host has, however, not yet been elucidated. Immunological non-responsiveness and antigenic diversity within T cell epitopes of the CS protein have been identified as potential problems in producing a sporozoite vaccine. These factors may contribute to the widespread lack of sporozoite immunity in endemic populations. In this study, 137 individuals with a history of natural endemic exposure to falciparum sporozoites (119 resident in north west Thailand and 18 resident in coastal Papua New Guinea) were tested for a CTL response to the Plasmodium falciparum CS protein. Fifty-four overlapping peptides, spanning the entire sequence of the CS protein of P. falciparum including most known variants, were studied. While most individuals had antibodies to the immunodominant B cell repeat, (NANP)n, and while CTL specific for an influenza virus matrix synthetic peptide could be generated from five of 23 Karen Thai individuals tested, no CS protein-specific CTL could be detected in these populations. Our data have important implications for vaccine programs.

MHC class I H-2Kd-restricted antigenic peptides: additional constraints for the binding motif.

The previously defined binding motif of MHC class I H-2Kd-restricted antigenic peptides consists of a Y residue in position P2 and a hydrophobic residue with a large aliphatic side chain (L, I, or V) in position P9/P10 of optimal 9- or 10-mer peptides. We show now that the presence of a charged or a F residue in position P5 reduces the Kd-restricted competitor activity of several cytotoxic T lymphocyte (CTL) epitopes and model peptides, at a degree comparable to A substitutions for the P2 or the P9/P10 anchor residues. Various charged, polar, aromatic, and aliphatic amino acids were substituted for S256 in the CTL epitope Plasmodium berghei circumsporozoite (CS) 253-260 8-mer and in its CS 252-260 9-mer form, whereas a more restricted panel of substitutions was tested in the CTL epitopes influenza nucleoprotein 147-155 9-mer and HLA-CW3 170-179 10-mer. Analysis of all the Kd-restricted epitopes so far defined also revealed an uncharged residue at this position. These additional structural constraints present in the Kd binding motif may thus improve the prediction of new epitopes recognized by T cells in the context of this MHC molecule.

Circumsporozoite protein gene of Plasmodium simium, a Plasmodium vivax-like monkey malaria parasite.

The circumsporozoite (CS) protein, a species-specific sporozoite surface protein and a vaccine candidate antigen, has been characterized from malaria parasites that naturally cause infections in humans, monkeys and chimpanzees [1-7]. Because of the related structural features of repetitive and nonrepetitive sequences the CS protein genes have also been used as markers for studies of evolutionary relatedness of malaria parasites [8]. The CS protein genes of the nonhuman primate malaria parasites Plasmodium brazilianurn and Plasmodium reichenowi, which are considered to be evolutionarily related to the human malaria parasites Plasrnodium malariae and Plasmodium falciparum, respectively, have been characterized and shown to bear structural and antigenic similarity [4,7]. Plasmodium simium is a nonhuman malaria parasite both morphologically and biologically similar to the human malaria parasite Plasmo-

The population dynamics in mosquitoes and humans of two Plasmodium vivax polymorphs distinguished by different circumsporozoite protein repeat regions.

The population dynamics of two Plasmodium vivax polymorphs were studied over a two-year period in a village in a hyperendemic area of Papua New Guinea in both the mosquito and human populations. Strains of P. vivax were distinguished by different circumsporozoite (CS) protein repeats, the VK210 (classic) and the VK247 (variant) polymorphs. In 1986, 34% of P. vivax CS protein-positive mosquitoes were of the VK247 type. Although the proportion of P. vivax sporozoite antigen-positive mosquitoes compared with all sporozoite-positive mosquitoes did not change from 1986 to 1987, the proportion of P. vivax-positive mosquitoes of the VK247 polymorph decreased significantly from 34% to 11% (5 of 45) in 1987. In 1986, 61% (47 of 77) of humans tested had IgGs that recognized the VK247 CS repeat, while only 26% (22 of 84) had IgGs that recognized the VK210 CS repeat. The observed fluctuation in the proportion of the two P. vivax CS protein polymorphs recorded in the mosquito population from 1986 to 1987 is consistent with a hypothesis of selection by humoral immune pressure on the VK247 strain.

Diversity in the immunodominant determinants of the circumsporozoite protein of Plasmodium falciparum parasites from malaria-endemic regions of Papua New Guinea and Brazil.

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.

Low immunogenicity of a Plasmodium vivax circumsporozoite protein epitope bound by a protective monoclonal antibody.

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, NS1(81)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 NS1(81)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.(ABSTRACT TRUNCATED AT 250 WORDS)

Malaria entomological inoculation rates in western Venezuela

Over 61 000 anophelines collected between January 1988 and October 1989 in three villages in western Venezuela were assayed by ELISA for Plasmodium vivax circumsporozoite (CS) protein. The six specimens confirmed positive belonged to three species: Anopheles ( Nyssorhynchus) nuneztovari Gabaldón, 1940, A. albitarsis Arribalzaga, 1878 sensu lato and A. oswaldoi (Peryassu, 1922). The estimated CS protein rate for all species combined was 0.01% (95% confidence limits 0.004-0.02%). This CS protein rate and the mean number of bites received by the collectors indicated a sporozoite inoculation rate of about 10.5 infective bites per person per year. From this value and the number of human malaria cases reported it was estimated that only 0.32% of bites by CS-positive mosquitoes led to a malaria infection. The CS protein rate is so low that this parameter would not be a practical indicator of the efficacy of control campaigns in this area.

Recognition of different domains of the Plasmodium falciparum CS protein by the sera of naturally infected individuals compared with those of sporozoite-immunized volunteers.

The fine specificities of antibodies to the circumsporozoite (CS) protein of Plasmodium falciparum, present in the sera of volunteers immunized with irradiated P. falciparum sporozoites, were defined and compared to those of sera from persons living in a malaria-endemic area in West Africa. The specificity of these anti-CS antibodies was determined by ELISA, using recombinant proteins and synthetic peptides containing repeat and nonrepeat sequences of this CS protein. All 10 serum samples of the five sporozoite-immunized volunteers displayed very high antibody titers to the immunodominant repeat (NANP)n of the CS protein. However, only three of the serum samples of these vaccinees reacted with a single nonrepeat region and only at low titers. In contrast, a high percentage of sera from adults living in the malaria-endemic area who had been exposed to sporozoites, as well as liver and blood stages of P. falciparum, had high antibody levels, not only to the repeats but also to several nonrepeat regions of the CS protein. Furthermore, a number of sera from children living in this endemic area displayed appreciable levels of antibodies to the nonrepeat regions, in the absence of any antirepeat reactivity. Sera of Saimiri monkeys, which had undergone multiple blood-induced P. falciparum infections, consistently contained high titers of antibodies to several nonrepeat sequences of the CS protein, whereas only a few of these sera had low titers of antirepeat antibodies. Antibody binding sites, in nonrepeat regions, were mapped using synthetic polymers containing multiple copies of selected C-terminal sequences of the P. falciparum CS protein. The binding to sporozoites of antibodies to nonrepeat regions of the CS protein was determined. The basis for the differences in antibody binding sites of sera from persons immunized with irradiated sporozoites, compared to those from an endemic area, is discussed.

Polymorphism in the circumsporozoite protein of the human malaria parasite Plasmodium vivax

The circumsporozoite (CS) protein that covers the surface of infectious sporozoites is a candidate antigen in malaria vaccine development. To determine the extent of B- and T-epitope polymorphism and to understand the mechanisms of antigenic variability, we have characterized the CS protein gene of Plasmodium vivax from field isolates representing geographically distant regions of Papua New Guinea (PNG) and Brazil. In the central repeat region of the CS protein, in addition to variation in the number of repeats, an array of mutations was observed which suggests that point mutations have led to the emergence of the variant CS repeat sequence ANGA(G/D)(N/D)QPG from GDRA(D/A)GQPA. Outside the repeat region of the protein, the nonsilent nucleotide substitutions of independent origin are localized in three domains of the protein that either harbor known T-cell determinants or are analogous to the Plasmodium falciparum immunodominant determinants, Th2R and Th3R. We have found that, with the exception of one CS clone sequence that was shared by one P. vivax isolate each from PNG and Brazil, the P. vivax CS protein types can be grouped into Papuan and Brazilian types. These results suggest that an in-depth study of parasite population dynamics is required before field trials for vaccine formulations based on polymorphic immunodominant determinants are conducted.

Characterization of naturally acquired antibodies to the non-repetitive flanking regions of the circumsporozoite protein of Plasmodium falciparum.

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.

T helper epitopes enhance the cytotoxic response of mice immunized with MHC class I-restricted malaria peptides

We have previously derived MHC class I (H-2K d) restricted cytotoxic T lymphocytes (CTL) from BALB/c mice immunized with irradiated sporozoites from Plasmodium (P.) berghei and P. yoelii. The CTL recognize synthetic peptides corresponding to a region of the circumsporozoite (CS) protein that is homologous in the two species. In the present study, we have attempted to induce CS-specific CTL by immunization with those peptides in incomplete Freund's adjuvant. Only a low level CTL response was detected in BALB/c mice immunized with synthetic peptides corresponding to the Pb or Py CTL epitopes. In contrast, CS-specific CTL responses could be readily detected in mice injected with mixtures of peptides that combined the P. berghei or P. yoelii CTL epitopes with previously defined T helper epitopes. Several different T helper epitopes were shown to enhance the response when injected as separate peptides in a mixture, or when covalently linked to a CTL epitope. These results may have general implications for the elicitation of CTL responses to defined CTL epitopes and for the design of peptide-based synthetic vaccines.

Monoclonal antibodies recognize a processing dependent epitope present in the mature CS protein of various plasmodial species.

In the present paper, we have characterized the specificity of a series of monoclonal antibodies (MoAbs) against Plasmodium berghei sporozoites, selected for their lack of reactivity with the repeat domain of the circumsporozoite (CS) protein. We found that these MoAbs recognize Pb44, the mature membrane form of the CS protein, but they do not react with Pb54, its precursor. Furthermore, these MoAbs do not react with any of the synthetic peptides representing the linear sequence of the P. berghei CS protein nor do they react with a recombinant CS (rCS) protein. These observations indicate that the epitope recognized by these antibodies is expressed only after the processing of the CS protein has occurred. This 'processing dependent epitope' is present in sporozoites of P. berghei, and also in those of P. falciparum, P. yoelii and P. brasilianum. It is not present in sporozoites of the P. cynomolgi-P. vivax complex and of P. gallinaceum. These anti-sporozoite MoAbs strongly inhibit sporozoite invasion of hepatoma cells, in vitro, however, they displayed no protective effect in an in vivo assay.

Plasmodium falciparum: Release of circumsporozoite protein by sporozoites in the mosquito vector

The release of Circumsporozoite (CS) protein by Plasmodium falciparum sporozoites was investigated to identify factors regulating this process within infected Anopheles gambiae mosquitoes. The potential for sporozoites to release CS protein in vitro was not dependent upon their site-specific developmental stage (i.e., mature oocysts, hemolymph, salivary glands), their duration in the vector, or their exposure to mosquito-derived components such as salivary glands or hemolymph. The capacity of sporozoites to release CS protein was depressed by mosquito blood feeding during periods of sporozoite migration to the salivary glands, but the effect was only temporary and those sporozoites already in the glands were not affected. Free CS protein in the salivary glands was present in 93.3% of 45 infective mosquitoes. Sporozoites from these same, individual mosquitoes were also tested in vitro for CS protein release. In both cases, the amount of soluble CS protein increased as a function of sporozoite density but the total amount of CS protein per sporozoite became progressively less with increasing numbers of sporozoites. Further experiments showed that sporozoite contact with increasing amounts of soluble CS protein caused a down-regulation of CS protein release. Thus, a primary factor regulating the production and release of CS protein by sporozoites is their contact with soluble CS protein within the mosquito.

Development and evaluation of an enzyme-linked immunosorbent assay for Plasmodium vivax-VK247 sporozoites.

An enzyme-linked immunosorbent assay (ELISA) for the Plasmodium vivax-VK247 (variant) circumsporozoite (CS) protein was developed and evaluated using sporozoites produced by feeding mosquitoes on Thai patients with parasitologically confirmed P. vivax infections. The ELISA had a detection threshold of fewer than 50 sporozoites. Using this assay in conjunction with an ELISA for the VK210 polymorph, nearly 16% of the 235 P. vivax cases produced sporozoites positive only for the variant; 69% produced sporozoites positive only in the VK210 assay; and 15% were positive in both assays, indicating mixed infections. Twelve cases (5%) produced sporozoites negative in one assay and with unexpectedly low activity in the other ELISA, indicating the possibility of other CS protein polymorphs.