Antigenic diversity amongst ten geographic isolates of Plasmodium falciparum defined by merozoite invasion inhibition assay.

The extent to which human antibodies involved in functional immunity react with antigenic determinants varying between different isolates or strains of human malaria parasite Plasmodium falciparum will influence the design of vaccine against malaria. In this study, in vitro inhibition of merozoite invasion in erythrocytes by an immune human serum was used to define the antigenic differences in 10 isolates of P. falciparum from three endemic areas, i.e. Africa, South America and Southeast Asia. The serum inhibited the invasion of merozoites of all the strains but the extent of inhibition varied from low to moderate to high degree indicating antigenic differences amongst isolates of P. falciparum. The antigenic differences could not be correlated to the geographic origin of the parasite isolate.     

间日疟原虫和恶性疟原虫可溶性抗原免疫反应性比较

目的分析和比较间日疟原虫(Plasmodiumvivax,P.v)和恶性疟原虫(P.falciparum,P.f)可溶性抗原与间日疟感染者混合血清和单克隆抗体(McAb)M26-32免疫反应性的差别,以期寻找潜在的疟疾诊断抗原。方法取P.v感染者血样,用Plasmodipur滤器分离去除白细胞,经60%Percoll浓集其中的感染红细胞(i RBC)。分别制备P.v、P.f和正常红细胞(nRBC)可溶性抗原,应用P.v感染者、正常对照混合血清和M26-32单抗与相应的抗原进行免疫印迹分析。结果免疫印迹分析表明,P.v感染者能特异性识别26k、33、49、115kDaP.v抗原;100、102、110、150、175kDaP.f抗原;能够交叉识别的条带有:31、59、63、70、120kDa。M26-32单抗能识别P.f、P.v抗原中31kDa等抗原条带。结论P.v感染者能特异识别间日疟抗原组份,并能交叉识别P.f抗原,其中31kDa抗原组分具有较强的免疫原性,同时能被M26-32单抗识别,其作为P.v特异性诊断抗原的价值有待于进一步研究。     

Generalized immunological recognition of the major merozoite surface antigen (gp195) of Plasmodium falciparum.

The antibody response to the Plasmodium falciparum major merozoite surface antigen (gp195) of congenic mouse strains differing in H-2 haplotype has been examined. All seven strains of mice were capable of producing gp195-specific antibodies. Generalized immune recognition of gp195 by mice of diverse H-2 haplotypes distinguished gp195 from the P. falciparum circumsporozoite protein and the 230-kDa and 48/45-kDa gamete surface antigens. However, the H-2 genetic locus appeared to influence the specificity of gp195-specific antibodies. Immunoblot patterns of mouse sera with parasite antigens revealed a complex pattern of reactivity with terminal and intermediate processing fragments of gp195. The majority of immunoblot bands observed were similar for all of the mouse strains; however, there were several strains that additionally recognized a few unique fragments or displayed more intense reactivities with specific processing fragments. These results suggest that while individuals of diverse major histocompatibility complex makeup are capable of recognizing the gp195 antigen, the recognition of specific gp195 B-cell and T-cell epitopes may be under control of the major histocompatibility complex.     

New haplotypes of the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene among chloroquine-resistant parasite isolates

Mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene were examined to assess their associations with chloroquine resistance in clinical samples from Armopa (Papua) and Papua New Guinea. In Papua, two of the five pfcrt haplotypes found were new: SVIET from Armopa and CVIKT from an isolate in Timika. There was also a strong association (P < 0.0001) between the pfcrt 76T allele and chloroquine resistance in 50 samples. In Papua New Guinea, mutations in the pfcrt gene were observed in 15 isolates with chloroquine minimum inhibitory concentrations (MICs) of 16-64 pmol, while the remaining six isolates, which had a wild-type pfcrt gene at codon 76, had MICs of 2-8 pmol. These observations confirm that mutations at codon 76 in the pfcrt gene are present in both in vivo and in vitro cases of chloroquine resistance, and that detection of the pfcrt 76T allele could predict potential chloroquine treatment failures.     

Characterization of an S antigen synthesized by several isolates of Plasmodium falciparum.

The S antigen of a Papua New Guinean isolate of Plasmodium falciparum was identified by immunoblotting as the dominant antigen in culture supernatants. An antigen identical in molecular weight (Mr 220,000), isoelectric point (pI 4.2), and immunoreactivity with sera from individuals exposed to malaria was expressed by four Papua New Guinean isolates and one isolate of unknown origin. The Mr 220,000 antigen was not detected in culture supernatants derived from two isolates from Thailand and one from Ghana. The Mr 220,000, pI 4.2 S antigen may characterize a subpopulation of parasites common to many isolates of P. falciparum, which is selected for by continuous culture in vitro. A variant S antigen, 30 kilodaltons larger but with similar immunoreactivity, was expressed by 1 of 26 clonal populations derived by limit-dilution culture from one of the Papua New Guinean isolates of P. falciparum. The characteristics of the S antigen, defined by immunoblotting, allowed it to be identified in two-dimensional separations of [35S]methionine-labeled parasite proteins, thus confirming the parasite origin of the antigen.     

Dot-immunobinding assay (DIBA) with integral Plasmodium falciparum as antigen in immuno-diagnosis of falciparum malaria

Among the available immuno-diagnostic methods of parasitoses, dot-immunobinding assay (DIBA) has been proved to be promising for its high sensitivity and specificity, easy performance, lack of need of special equipment, and consequently its practical usage in field work. In previously reported tests, soluble antigen was used, thus a sonicator and an ultracentrifuge were required to produce the antigen. This paper reports the application of integral P. falciparum as antigen in DIBA to detect antibodies in falciparum malaria cases. Of 52 sera from falciparum malaria patients tested, 49 (94.2%) showed positive reactions, which was similar to the result using soluble antigen in DIBA (96.2%) and was higher than that in IFA (86.5%) and ELISA (80.8%). No false positive was revealed in 48 control sera from healthy individuals and sera from visceral leishmaniasis, paragonimiasis, fasciolopsiasis and schistosomiasis patients.     

Antibodies to Pf155, a major antigen of Plasmodium falciparum: longitudinal studies in humans

Antibodies to Pf155, a major Plasmodium falciparum antigen detected in the membrane of glutaraldehyde-fixed and air-dried erythrocytes infected with P. falciparum, were studied in serum samples collected from patients treated for neurosyphilis by induced P. falciparum infection. In 3 patients with no previous documented exposure to malaria, the antibodies were detected late and reached low titers. In 5 patients with extensive previous malaria infections, the antibodies appeared rapidly and reached high titers. The immunofluorescence findings were confirmed by immunoblots. No correlation was observed between antibodies to Pf155 and antibodies detected by standard immunofluorescence with whole parasite antigen.     

Plasmodium falciparum circumsporozoite protein: epidemiological variations among field isolates prevalent in India

Objective  To investigate the extent of genetic variations in T-helper-cell epitopic regions of circumsporozoite (CS) protein in Plasmodium falciparum field isolates collected from different regions of India at different phases of malaria transmission.
Methods  Genomic DNA was isolated from 507 P. falciparum wild-parasite isolates obtained from six geographical locations of India at three time points coinciding with malaria transmissions. The T-helper-cell epitopic regions were polymerase chain reaction (PCR)-amplified and the products were purified and then sequenced.
Results  Based on sequences, nine variants were found among isolates and they were categorized into nine groups (V-1 to V-9), where V-1 and V-2 were observed in all three time points (TP). The variants V-1 to V-4 in TP-1; V-1, V-2, V-5 to V-8 in TP-2; and V-1, V-2, V-5 and V-9 in TP-3 were present and they showed restricted heterogeneity. During peak transmission (TP-2), parasite populations were more diverse and heterogeneous and the variants regionally unbiased and restricted. However, the alleles of V-6 and V-9 in both Th2R and Th3R showed identical sequence variation with those observed in other geographical regions of the world. The remaining seven groups did not show such similarity.
Conclusion  The Th2R and Th3R epitopes are implicated in host immune response to P. falciparum . The polymorphism in these epitopic regions indicates antigenic diversity, which may cause adverse outcome of a subunit vaccine including the CS prototype variant. Therefore, the formulation of a vaccine considering the restricted local repertoire parasite populations may be helpful.     

Immunochemical analyses of a major antigen of Mycobacterium szulgai.

Unheated culture filtrate of Mycobacterium szulgai and a homologous goat antiserum were prepared. Immunoelectrophoretic analysis demonstrated a dominant anodal antigen in the culture filtrate. By the use of diethylaminoethyl-cellulose chromatography, a fraction designated MSP, which was rich in this anodal antigen, was recovered. The major antigen of MSP was demonstrated to have partial identity with reference mycobacterial antigen 6, and evidence was obtained for separate shared and specific antigenic determinants. MSP was found to be a potent, delayed skin-test antigen of considerable specificity when used in sensitized guinea pigs. Arthus reactions were also observed and were not specific.     

In vitro antimalarial drug susceptibility and pfcrt mutation among fresh Plasmodium falciparum isolates from the Lao PDR (Laos)

Recent drug trials in Laos have shown high levels of Plasmodium falciparum resistance to chloroquine, but there are no published data on in vitro antimalarial drug susceptibility. We used the double-site enzyme-linked pLDH immunodetection (DELI) assay to estimate the in vitro antimalarial drug susceptibility of 108 fresh P. falciparum isolates from southern Laos. The geometric mean (95% confidence interval) 50% inhibitory concentration values (nmol/L) were 152.4 (123.8-187.6) for chloroquine, 679.8 (533.8-863.0) for quinine, 45.9 (37.9-55.7) for mefloquine, 5.0 (4.4-6.4) for artesunate, 6.3 (4.5-8.9) for dihydroartemisinin, and 59.1 (46.4-75.3) for lumefantrine. The proportion of isolates defined as resistant were 65%, 40%, and 8% for chloroquine, quinine, and mefloquine, respectively. Of 53 isolates genotyped for the pfcrt T76K chloroquine-resistance mutation, 48 (91%) were mutants. P. falciparum in Laos is multi-drug resistant; antimalarial immunity resulting from the use of ineffective chloroquine before 2005 probably contributes significantly to the therapeutic responses in clinical trials.     

Molecular monitoring of antimalarial drug resistance among Plasmodium falciparum field isolates from Odisha,India

In the absence of definite marker for artemisinin (ART) resistance, molecular monitoring of its partner drug sulfadoxine pyrimethamine (SP) in artemisinin based combination therapy (ACTs) together with chloroquine (CQ) for which ART is negatively correlated, may predict the effectiveness of ACT. We analyzed 201 Plasmodium falciparum field isolates for drug resistance markers for CQ (pfcrt and pfmdr1), pyrimethamine (pfdhfr) and sulfadoxine (pfdhps). Our study reveals high prevalence and non-random association of resistant mutants (K76T and N86Y) of CQ markers (pfcrt and pfmdr1). The predominance of highly resistant pfdhfr genotypes for SP with intragenic and intergenic pair-wise linkage disequilibrium between single nucleotide polymorphisms of resistant mutants of pfdhfr (C59R and S108N) and pfdhps (S436A, A437G, K540E) warn on further inclusion of SP in ACT. These findings suggest the replacement of SP in ACT with alternative partner drug for better efficacy.     

Plasmodium vivax apicoplast genome: a comparative analysis of major genes from Indian field isolates

The apicomplexan parasite Plasmodium vivax is responsible for causing more than 70% of human malaria cases in Central and South America, Southeastern Asia and the Indian subcontinent. The rising severity of the disease and the increasing incidences of resistance shown by this parasite towards usual therapeutic regimens have necessitated investigation of putative novel drug targets to combat this disease. The apicoplast, an organelle of procaryotic origin, and its circular genome carrying genes of possible functional importance, are being looked upon as potential drug targets. The genes on this circular genome are believed to be highly conserved among all Plasmodium species. Till date, the plastid genome of P. falciparum, P. berghei and P. chabaudi have been detailed while partial sequences of some genes from other parasites including P. vivax have been studied for identifying evolutionary positions of these parasites. The functional aspects and significance of most of these genes are still hypothetical. In one of our previous reports, we have detailed the complete sequence, as well as structural and functional characteristics of the Elongation factor encoding tufA gene from the plastid genome of P. vivax. We present here the sequences of large and small subunit rRNA (lsu and ssu rRNA) genes, sufB (ORF470) gene, RNA polymerase (rpo B, C) subunit genes and clpC (casienolytic protease) gene from the plastid genome of P. vivax. A comparative analysis of these genes between P. vivax and P. falciparum reveals approximately 5-16% differences. A codon usage analysis of major plastid genes has shown a high frequency of codons rich in A/T at any or all of the three positions in all the species. TTA, AAT, AAA, TAT, and ATA are the major preferred codons. The sequences, functional domains and structural analysis of respective proteins do not show any variations in the active sites. A comparative analysis of these Indian P. vivax plastid genome encoded genes has also been done to understand the evolutionary position of the Indian parasite in comparison to other Plasmodium species.     

Human leukocyte antigen class II control of the immune response to p126-derived amino terminal peptide from Plasmodium falciparum

We investigated the relationships between class II human leukocyte antigens (HLA) and the antibody response to Plasmodium falciparum p126 protein and to its amino-terminal portion (Nt47) in 2 malaria-endemic villages in Brazil, Colina and Ribeirinha. All people from the endemic areas had anti-p126 antibodies, and the frequencies of anti-Nt47 antibodies were similar in both communities (66% for Colina and 75% for Ribeirinha). Typing of HLA showed that Colina and Ribeirinha groups had no significant differences in HLA antigen frequencies. However, in both groups, significant associations between positive response to anti-Nt47 and presence of HLA-DR4, as well as between absence of response and presence of HLA-DR15, were observed. The predominance of positive responses to Nt47 among HLA-DR4 people was independent of the presence of any particular allele. There was no evidence for association between HLA-DQB1 alleles and antibody response to Nt47. Thus, naturally exposed people with different HLA class II antigens seem to respond differently to Nt47, indicating that the choice of relevant peptide sequences may have important consequences for subunit vaccine development.     

Population structure of recrudescent Plasmodium falciparum isolates from Western Uganda

It has been proposed that polymorphisms of the Merozoite Surface Protein 1 and 2 (MSP1 and MSP2) and the Glutamate Rich Protein (GLURP) genes can be considered as genetic markers for the genotyping of field populations of Plasmodium falciparum. During a field study on in vivo drug resistance against chloroquine, sulphadoxine/pyrimethamine (S/P) and cotrimoxazole in West Uganda, sensitive and resistant isolates were collected from patients by fingerprick for genotyping. 59 (72.8%) of the 81 P. falciparum samples isolated at day 0 showed multiclonal infection with 2-7 clones. Among the isolates we investigated, presence of the allelic family MAD20 of MSP1 at day 0 was significantly (P = 0.0041) associated with decreased resistance to antimalarials. Use of this method in a field study on in vivo drug resistance demonstrates another potential application of genotyping as a tool for epidemiological investigations.     

High prevalence of pfcrt K76t mutants among Plasmodium falciparum isolates from Sabah, Malaysia

Chloroquine (CQ) remains the first line drug for the prevention and treatment of malaria in Malaysia in spite of the fact that resistance to CQ has been observed in Malaysia since the 1960s. CQ-resistance is associated with various mutations in pfcrt, which encodes a putative transporter located in the digestive vacuolar membrane of P. falciparum. Substitution of lysine (K) to threonine (T) at amino acid 76 (K76T) in pfcrt is the primary genetic marker conferring resistance to CQ. To determine the presence of T76 mutation in pfcrt from selected areas of Kalabakan, Malaysia 619 blood samples were screened for P. falciparum, out of which 31 were positive. Blood samples were collected on 3 MM Whatman filter papers and DNA was extracted using QIAmp DNA mini kit. RFLP-PCR for the detection of the CQ-resistant T76 and sensitive K76 genotype was carried out. Twenty-five samples were shown to have the point mutation in pfcrt whereas the remaining samples were classified as CQ-sensitive (wild-type). In view of the fact that CQ is the first line anti-malarial drug in Malaysia, this finding could be an important indication that treatment with CQ may no longer be effective in the future.     

Sequence diversity of serine repeat antigen gene exon II of Plasmodium falciparum in worldwide collected wild isolates.

Field isolates of Plasmodium falciparum collected from endemic areas of Southeast Asia, Solomon Islands, tropical African countries and Brazil were analyzed for the genetic diversity of the exon II of serine repeat antigen gene (SERA) by sequencing of genomic DNA. Of sixty-nine isolates, as compared to the reported FCR3, K1 and Honduras-1 types of exon II sequences, 5, 9 and 20 new allelic forms were found in 23 isolates of the FCR3 type, 36 of the K1 type and 10 of the Honduras-1 type. A group of novel non-synonymous substitutions, 4 new insertions and 3 new deletions of octamer units were found in the octamer repeat region (OR) of the exon II, and most of them clustered within a 40-residues domain. An octamer "SNPVSSEP" revealed in the OR was confirmed as a new repeat unit. Based on the sequences of the serine repeat region (SR) of the exon II, the allelic forms of the Honduras-1 type were conjectured to be the recombinant forms between the K1 type and FCR3 type. The allelic forms of K1 type with less or more repeat serine residues in the serine stretch of the SR than the reported 21 serine residues had most of the variations in the OR. Moreover, a biased geographical distribution of allelic forms was observed. Isolates from African and Southeast Asian countries accounted for most of the new allelic forms (29/33). All of the three types were detected in Southeast Asia but none of the FCR3 type in Africa. One of two groups of FCR3 new allelic forms was found solely in Brazil while another was mainly in Solomon Islands.     

Genetic polymorphism of falciparum malaria vaccine candidate antigen genes among field isolates in India.

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.     

Antibodies to Plasmodium falciparum ring-infected erythrocyte surface antigen and P. falciparum and P. malariae circumsporozoite proteins: seasonal prevalence in Kenyan villages

Two cross-sectional surveys of 954 persons in Asembo Bay and Got Nyabondo, western Kenya, were performed in August-September 1986, after long rains, and in February-March 1987, after a comparatively dry season. Serologic testing was performed using an ELISA with synthetic peptides representing repeat amino acid sequences of the Plasmodium falciparum ring-infected erythrocyte surface antigen (RESA), (EENV)5, (EENVEHDA)4, and (DDEHVEEPTVA)2 and repeat sequences (PNAN)5 and (NAAG)5 of the P. falciparum and P. malariae circumsporozoite proteins. In 1986, 45%, 73%, 72%, 85%, and 59% of the persons in Asembo Bay had antibodies to the respective peptides. In Got Nyabondo, the rates were 44%, 67%, 56%, 36%, and 41%, respectively. All positivity rates increased with age. When next determined in 1987, the positivity rates and levels of reactivity were generally unchanged in Asembo Bay, but were decreased in Got Nyabondo.