Consecutive determinations of seroreactivities to Pf 155/RESA antigen and to its different repetitive sequences in adult men from a holoendemic area of Liberia

Sera from 32 adult men residing in a malaria holoendemic area of Liberia were investigated for seroreactivities to different asexual blood-stage malaria antigens on five consecutive occasions from 1984 to 1986. The seroreactivities to crude parasitic antigens and to Pf 155/RESA (EMIF) were determined by immunofluorescence and to repetitive sequences of Pf 155/RESA by enzyme-linked immunosorbent assay (ELISA). All sera were highly reactive against the crude parasitic antigens with reciprocal titres varying from 5000 to 100,000. The EMIF titres showed a wider variation from negative (less than 10) to 25,000, and when the same individuals were re-examined on subsequent surveys similar EMIF titres were found. The ELISA seroreactivities to three different repetitive sequences of Pf 155/RESA also showed different individual profiles which were rather consistent on consecutive surveys. High EMIF titres appeared to be correlated mainly to one of the peptide sequences, namely (EENV)2. The consistent individual profiles of the seroreactivities to Pf 155 and its repetitive sequences suggest genetic restriction of the humoral immune response. Although no significant correlation was found between EMIF titres and parasitic densities in the adult hyperimmune men the specific peptides, however, offer new possibilities of further investigating protective capacities of different immune responses to specific epitopes of the malaria parasite.     

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.     

Structural diversity in the Plasmodium falciparum merozoite surface antigen 2.

Antigens associated with the surface of merozoites of the malaria parasite Plasmodium falciparum are directly accessible to immune attack and therefore are prime vaccine candidates. We have previously shown that one of the two known merozoite surface antigens (merozoite surface antigen 2; MSA-2) exhibits considerable sequence and antigenic diversity in different isolates. The sequences of MSA-2 from three isolates revealed a central domain composed of repeats that vary in number, length, and sequence, flanked in turn by nonrepetitive variable sequences and by conserved N- and C-terminal domains. We report here the sequences of a further four MSA-2 alleles, containing repetitive sequences that are related but not identical to each other. The seven alleles of MSA-2 can be divided into two distinct allele families on the basis of nonrepetitive sequences. Hybridization studies with repeat probes indicated that all of the 44 P. falciparum isolates examined contained repeat regions similar to those defined in known MSA-2 sequences.     

Plasmodium falciparum merozoite surface protein 3 is a target of allele-specific immunity and alleles are maintained by natural selection

BACKGROUND: Plasmodium falciparum merozoite surface protein (MSP) 3 is an asexual blood-stage malaria vaccine candidate antigen. Sequence polymorphisms divide alleles into 2 major types, but the adaptive and immunological significance of the types has not been defined. METHODS: One hundred one msp3 allele sequences were sampled from 2 populations living in areas where malaria is endemic and were analyzed for evidence of natural selection. Recombinant antigens representing full-length sequences of different allelic types and a relatively conserved C-terminal region were produced, to evaluate immunization-induced antibody responses in mice and protective associations for naturally acquired antibodies in a cohort of 319 Gambian children under surveillance for malaria. RESULTS: Frequency-based statistical analyses indicated that polymorphisms are maintained by balancing selection in each of the 2 populations studied. Immunization of mice with full-length MSP3 antigens induced predominantly type-specific antibodies, and a large proportion of naturally acquired antibodies to MSP3 in humans also discriminated between the alleles. Among Gambian children, antibodies to allele-specific and conserved epitopes in MSP3 were associated prospectively with protection from clinical malaria, even after adjustment for age and for the presence of antibodies to other merozoite antigens. CONCLUSIONS: A vaccine incorporating both major allelic types of this promising candidate antigen could be particularly useful for induction of protective immunity in infants and young children.     

Circumsporozoite gene of plasmodium cynomolgi (Gombak):cDNA cloning and expression of the repetitive circumsporozoite epitope.

We report the identification, sequence, and expression in Escherichia coli of the immunodominant epitope of the circumsporozoite (CS) gene of Plasmodium cynomolgi (Gombak), a simian malaria parasite. This epitope is encoded by a DNA sequence that is tandemly repeated 10 times in the cDNA clone. Subclones that contain and express only repeats and in variable number have been constructed. We show that the binding of a specific anti-CS protein monoclonal antibody correlates positively with the number of repeats in each subclone. The CS gene of another strain of P. cynomolgi (NIH) encodes an immunodominant epitope that is immunologically distinct from that of the Gombak strain. We present evidence that these two CS genes share extensive overall homology, although the nucleotide sequences that encode the epitopes appear to be unrelated.     

Antibody responses to repetitive epitopes of the circumsporozoite protein,liver stage antigen-1, and merozoite surface protein-2 in infants residing in a Plasmodium falciparum-hyperendemic area of western Kenya. XIII. Asembo Bay Cohort Project

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.     

Sequence diversity of the merozoite surface protein 1 of Plasmodium falciparum in clinical isolates from the Kilombero District, Tanzania

Merozoite surface protein 1 of Plasmodium falciparum (PfMSP-1) is regarded as a key candidate antigen for malaria vaccine development. It exhibits significant antigenic polymorphism and has been divided into 17 building blocks based on the analysis of sequence diversity. Differences in the antigenic composition of PfMSP-1 in local P. falciparum populations may result in differences in the efficacy of vaccines, which contain sequences of particular allelic variant(s) of PfMSP-1. To contribute to the required knowledge of genetic diversity of malaria parasites in geographically diverse regions, we have used the polymerase chain reaction (PCR) to analyze the sequence diversity of blocks 1-4 of PfMSP-1 in disease isolates from the Kilombero District in Tanzania. In the semi-conserved block 1, in which dimorphic amino acid variances have been described at three positions, we found three of the five previously described combinations of these three pairs of amino acids. In addition one combination was found, which has not been reported before in parasite isolates from different locations worldwide. Of the two sequence variants, which were dominating, one (S44-Q47-V52) corresponded to the 83.1 sequence incorporated into the SPf66 malaria peptide vaccine, while the other one (G44-H47-I52) differed from the previous in all three dimorphic amino acids. The partial protection observed in a phase III SPf66 trial conducted in the Kilombero District in children aged 1-5, thus does not seem to be associated with a clear dominance of favourable variants of block 1 of PfMSP-1 in this area. All three different principle types of block 2, the major polymorphic region of PfMSP-1, were found in the Tanzanian isolates. Most of the sequences contained K1-type tripeptide repeats, but clones with MAD20-type repeats or no repetitive sequence (RO33-type block 2) were also present. K1- and MAD20-type tripeptide repeat motifs were never mixed within one parasite clone. In one sequence a hexapeptide repeat was found at the end of block 2, which has not been reported before. Dimorphism in 13 of the 17 previously described variable positions of the semi-conserved block 3 and three of four recombination types of block 4 (K/K, M/K and M/M) were found among the Tanzanian isolates. Apart from previously described dimorphic amino acid positions, polymorphism was rare in the non-repeated building blocks. Selection and spreading of parasite variants, which contain amino acid exchanges at other than the dimorphic positions thus, is not a common event. Parasite isolates frequently harboured more than one PfMSP-1 allele. Three of the four heterogeneous isolates analysed contained two different general types of sequences. One isolate contained at least four distinct clones, demonstrating the high endemicity of malaria in the Kilombero District, which is a well-established site for malaria vaccine field trials.     

Immunization of Aotus monkeys with Plasmodium falciparum blood-stage recombinant proteins.

The current spread of multidrug-resistant malaria demands rapid vaccine development against the major pathogen Plasmodium falciparum. The high quantities of protein required for a worldwide vaccination campaign select recombinant DNA technology as a practical approach for large-scale antigen production. We describe the vaccination of Aotus monkeys with two recombinant blood-stage antigens (recombinant p41 and 190N) that were considered as vaccine candidates because parasite-derived antigen preparations could protect susceptible monkeys from an otherwise lethal malaria infection. In contrast to the natural antigen, recombinant p41 protein (P. falciparum aldolase) could not protect monkeys, although all animals seroconverted. 190N antigen, a recombinant protein containing conserved sequences of the major merozoite surface antigen p190, protected two of five monkeys from critical levels of infection with the highly virulent FVO isolate of P. falciparum. However, the B- and T-cell responses to 190N antigen were similar in protected and unprotected animals so that other unknown factors may contribute to protection. Higher purity or lack of protective epitopes or different structure of protective epitopes in the recombinant proteins might explain the better performance of parasite-derived antigens in vaccination trials. The partial protection obtained with 190N antigen suggests that this molecule could contribute to a vaccine mixture against P. falciparum.     

约氏疟原虫与伯氏疟原虫侵入期抗原的初步研究

目的　用针对鼠约氏疟原虫(Plasmodiumyoelii)侵入期的8种单克隆抗体,对约氏疟原虫和伯氏疟原虫(P.berghei)侵入期即动合子、裂殖子和子孢子棒状体和表面抗原检测分析。　方法　间接免疫荧光实验(IFA)对各侵入期抗原进行亚细胞结构定位,SDSPAGE及Western印迹对两种鼠疟原虫的不同侵入期进行抗原组分分析。　结果　经上述两种方法检测发现,顶端复合体抗原成分复杂,约氏疟原虫和伯氏疟原虫的棒状体有共同的抗原表位,约氏疟原虫的动合子与其自身的裂殖子有类似成分,也有各自独特的抗原。两种鼠疟原虫动合子抗原有类似成分。约氏疟原虫的子孢子具有与裂殖子、动合子不同的抗原成分。　结论　疟原虫侵入期棒状体和表面抗原在同一虫种的不同侵入期和不同虫种中有共同的抗原表位,也有各自的独特组分。     

Further studies on the immunization of Saimiri sciureus boliviensis with recombinant vaccines based on the circumsporozoite protein of Plasmodium vivax.

Reported are the results of a trial in squirrel monkeys of 2 Plasmodium vivax malaria vaccine candidates based on the circumsporozoite (CS) protein, namely, rPvCs-2 and rPvCS-3. Compared with an earlier recombinant P. vivax CS construct, rPvCS-1, rPvCS-2 has an additional 24 amino acids at the C-terminal, which includes the thrombospondin region of homology and a putative T cell epitope. The rPvCS-3 was generated from a chemically synthesized gene that contained an additional 54 amino acids at the amino terminus and terminates at the same carboxy-terminal amino acid as rPvCS-2. In addition, rPvCS-3 contained only 1 each of the repeat sequences DRADGQPAG and DRAAGQPAG. Both antigens were administered with alum as adjuvant. Neither formulation caused toxic side effects and both recombinant molecules induced high antibody titers. Two monkeys were protected against sporozoite challenge by immunization with rPvCS-2 antigen, while none of the rPvCS-3 immunized animals displayed any degree of protection. While there was no correlation between protection and antibody titer or the in vitro proliferation of lymphocytes in response to the antigens, this is further evidence to support the role of the repeating epitopes in generating protective immunity.     

Novel cross-reactive epitopes on asexual blood stage antigens of Plasmodium falciparum

Summary An IgM monoclonal antibody that reacts with the merozoite membrane and internal merozoite antigens was shown to recognize several previously characterized asexual blood stage antigens of Plasmodium falciparum as well as new antigens. Among the reactive antigens identified were FIRA, GYMSSA, RESA and the S-antigen. Analysis of the cross-reactions between FIRA and GYMSSA by epitope scanning was performed. The most reactive peptides in GYMSSA had the common sequence STNS. The cross-reactive epitopes in FIRA could, in many cases, be explained by the results of a replacement net analysis performed on the STNS epitope. It is proposed that the cross-reactive epitopes, which in several cases have no obvious linear homology but possess high S, T and N content, may be present as loops or coils on the surface of the molecules.     

Meta-analysis of immune epitope data for all Plasmodia: overview and applications for malarial immunobiology and vaccine-related issues

We present a comprehensive meta-analysis of more than 500 references, describing nearly 5000 unique B cell and T cell epitopes derived from the Plasmodium genus, and detailing thousands of immunological assays. This is the first inventory of epitope data related to malaria-specific immunology, plasmodial pathogenesis, and vaccine performance. The survey included host and pathogen species distribution of epitopes, the number of antibody vs. CD4⁺ and CD8⁺ T cell epitopes, the genomic distribution of recognized epitopes, variance among epitopes from different parasite strains, and the characterization of protective epitopes and of epitopes associated with parasite evasion of the host immune response. The results identify knowledge gaps and areas for further investigation. This information has relevance to issues, such as the identification of epitopes and antigens associated with protective immunity, the design and development of candidate malaria vaccines, and characterization of immune response to strain polymorphisms.     

Novel cross-reactive epitopes on asexual blood stage antigens of Plasmodium falciparum.

An IgM monoclonal antibody that reacts with the merozoite membrane and internal merozoite antigens was shown to recognize several previously characterized asexual blood stage antigens of Plasmodium falciparum as well as new antigens. Among the reactive antigens identified were FIRA, GYMSSA, RESA and the S-antigen. Analysis of the cross-reactions between FIRA and GYMSSA by epitope scanning was performed. The most reactive peptides in GYMSSA had the common sequence STNS. The cross-reactive epitopes in FIRA could, in many cases, be explained by the results of a replacement net analysis performed on the STNS epitope. It is proposed that the cross-reactive epitopes, which in several cases have no obvious linear homology but possess high S, T and N content, may be present as loops or coils on the surface of the molecules.     

T-cell epitopes in Pf155/RESA, a major candidate for a Plasmodium falciparum malaria vaccine.

Immunogens included in a subunit vaccine should contain both B- and T-cell-activating sites to ensure anamnestic responses following reinfection after vaccination as well as antibody-independent cellular immunity. The Plasmodium falciparum antigen Pf155/RESA, a major candidate for a vaccine against the asexual blood stages of this malaria parasite, was investigated for T-cell epitopes in its C-terminal amino acid repeat region, a region known to be conserved in different P. falciparum strains. It was found to contain several related sequences that activated T cells from humans primed to P. falciparum by natural exposure, to proliferation, and/or interferon-gamma release in vitro. T cells from approximately half of the donor group investigated responded to the intact protein, and 65% of these responders also responded to short synthetic peptides, probably representing a small number of partly overlapping T-cell epitopes. Thus, sequences from the C terminus of Pf155 may be suitable constituents of a P. falciparum subunit vaccine and also provide a basis for epitope-specific epidemiological studies relating cellular immune responses in vitro to clinical immunity and P. falciparum endemicity.     

Single-nucleotide polymorphisms and genome diversity in Plasmodium vivax

The study of genetic variation in malaria parasites has practical significance for developing strategies to control the disease. Vaccines based on highly polymorphic antigens may be confounded by allelic restriction of the host immune response. In response to drug pressure, a highly plastic genome may generate resistant mutants more easily than a monomorphic one. Additionally, the study of the distribution of genomic polymorphisms may provide information leading to the identification of genes associated with traits such as parasite development and drug resistance. Indeed, the age and diversity of the human malaria parasite Plasmodium falciparum has been the subject of recent debate, because an ancient parasite with a complex genome is expected to present greater challenges for drug and vaccine development. The genome diversity of the important human pathogen Plasmodium vivax, however, remains essentially unknown. Here we analyze an approximately 100-kb contiguous chromosome segment from five isolates, revealing 191 single-nucleotide polymorphisms (SNPs) and 44 size polymorphisms. The SNPs are not evenly distributed across the segment with blocks of high and low diversity. Whereas the majority (approximately 63%) of the SNPs are in intergenic regions, introns contain significantly less SNPs than intergenic sequences. Polymorphic tandem repeats are abundant and are more uniformly distributed at a frequency of about one polymorphic tandem repeat per 3 kb. These data show that P. vivax has a highly diverse genome, and provide useful information for further understanding the genome diversity of the parasite.     

Natural antibody response to Plasmodium falciparum Exp-1, MSP-3 and GLURP long synthetic peptides and association with protection

A longitudinal study was undertaken in Burkina Faso among 293 children aged 6 months to 9 years in order to determine the correlation between an antibody response to several individual malarial antigens and malarial infection. It was found that the presence of a positive antibody response at the beginning of the rainy season to three long synthetic peptides corresponding to Plasmodium falciparum Exp-1 101-162, MSP-3 154-249 and GLURP 801-920 but not to CSP 274-375 correlated with a statistically significant decrease in malarial infection during the ongoing transmission season. The simultaneous presence of an antibody response to more than one antigen is indicative of a lower frequency of malarial infection. This gives scientific credibility to the notion that a successful malaria vaccine should contain multiple antigens.     

Specificity variants in monoclonal antibodies reactive with peptide epitopes of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum

It has been suggested that repeat sequence antigens of Plasmodium falciparum may serve the parasite in immune evasion by modifying the host antibody response and impairing the development of protective immunity. According to this proposal networks of cross-reactive, repeat sequence malarial antigens have the ability to stimulate a high proportion of all somatically mutated B cells with altered antibody specificity, and thus to hinder the normal process of antibody affinity maturation. To determine the rate at which immunoglobulin mutations produce new reactivities with repeat sequence antigens, hybridoma cell lines specific for the ring-infected erythrocyte surface antigen (RESA) were examined for the incidence of specificity variants that arose naturally or as a result of treatment with the chemical mutagen ethylmethane sulphonate (EMS). From one of the cell lines variants were readily isolated having reactivity towards a very closely related repeat sequence epitope within the same RESA antigen. However, the other hybridoma/antigen combinations revealed no variants. In general, mutations giving rise to antibodies with altered specificity for related repetitive antigens were not readily induced and only limited support of the hypothesis was obtained.     

An epidemiological study of humoral and cell-mediated immune response to the Plasmodium falciparum antigen PF155/RESA in adult Liberians

We investigated the seroreactivity and T cell reactivity against the Plasmodium falciparum antigen Pf155/RESA, different oligopeptides from the 3' and 5' repeat regions of the Pf155/RESA antigen, and crude Plasmodium falciparum antigens in 164 adult Liberians. We compared 2 long-term residential groups with high and low exposure to malaria. The seropositive rate to the peptides was significantly higher with increased exposure. There was no significant difference in response rates to the Pf155/RESA. This may indicate the level of persistent T cell memory in previously primed donors. The seropositive rates to 3 Pf155/RESA peptides and the rates measured by either 3H-thymidine incorporation or IFN-gamma release after stimulation with Pf155/RESA and the peptides were all lower in parasite positive individuals. Even low grade, asymptomatic parasitemia can impair the T cell response in vitro. The lower antibody response in parasite positive subjects may be explained by either antibody consumption or lower protection against malaria parasitemia in subjects with low concentrations of antibodies against the Pf155/RESA antigen.     

The antibody response to well-defined malaria antigens after acute malaria in individuals living under continuous malaria transmission.

The IgG and IgM antibody responses to the C-terminal 783 amino acids of the P. falciparum glutamate-rich protein, GLURP489-1271, expressed as an E. coli fusion protein, the IgG response to a 18-mer synthetic peptide EDKNEKGQHEIVEVEEIL (GLURP899-916) representing the C-terminal repeats of GLURP, and a synthetic peptide (EENV)6 representing the C-terminal repeats from Pf155/RESA, were investigated longitudinally in 13 children and 7 adults living under conditions of continuous, intense malaria transmission. Some subjects did not recognize the antigens after malaria infection, and in subjects recognizing the antigens, the responses were often short-lived. In adults, the antibody responses to the GLURP489-1271 fusion protein and the (EENV)6 peptide peaked after 2 weeks, and not all individuals responded to all antigens. The antibody response, even against large fragments of conserved antigens, is not uniformly elicited by natural malaria infection in previously primed donors.     

cDNA cloning of small nuclear and cytoplasmic RNA-associated proteins

cDNA cloning has been a highly successful tool in elucidating the structure of U- and Y-RNA associated proteins. Whereas sera from autoimmune patients have often been helpful in the procedure, the cloning has not yet elucidated why such patients recognize especially these nuclear antigens. The finding of a repetitive proline-rich epitope in A/B/B'/N/C is interesting with regard to the findings of Gleichmann et al. (48,49), who studied an SLE-like disease in Graft-versus-Host diseased mice. It was shown, that parental T-cells with which the mice were immunized, could give aspecific help to B-cells. These B-cells then started to produce autoantibodies exclusively against autoantigens which contained a repetitive determinant and which could cross-link the antigen receptors on B-cells. However, most epitopes on U- and Y-RNA associated proteins against which an antibody response has been found are not repetitive. With regard to immunoassays it may be stated that, the first ELISA's in which recombinant antigens are used have now been described. The use of recombinant antigens in these (quantitative) assays will probably increase in the near future. The clinical significance of quantitation of these autoantibodies has not yet been established.