Search

Background Crucial gaps in our understanding of Plasmodium vivax reticulocyte invasion and protective immunity have hampered development of vivax vaccines. P. vivax exclusively invades reticulocytes that is mediated by the P. vivax reticulocyte-binding proteins (PvRBPs) specifically PvRBP2c and PvRBP1a. Vivax infections in Duffy-null individuals have suggested the evolution of alternate invasion pathways that may be mediated by the PvRBPs. Thus, PvRBPs appear as potential targets for efficacious P. vivax neutralization. However, there are limited data validating their vaccine efficacy. In the absence of vivax invasion assays, binding-inhibitory activity of antibodies has been reported to be associated with protection and a measure of vaccine potential. Methods -based analysis was performed of the PvRBP reticulocyte-binding properties and binding-inhibitory activity of specific anti-PvRBP2c/PvRBP1a human antibodies. Results PvRBP2c and PvRBP1a displayed a distinct reticulocyte-binding specificity, and their specific reticulocyte-binding domains were mapped within their N-terminal regions. Importantly, naturally acquired antibodies against the reticulocyte-binding domains efficaciously blocked reticulocyte binding of native PvRBPs, suggesting that the human immune system produced functional binding-inhibitory antibodies through exposure to vivax malaria. Conclusions Reticulocyte-binding domains of PvRBP2c/PvRBP1a are targets of naturally acquired binding-inhibitory antibodies, substantiating their promise as candidate antigens against which vaccine-inducible immunity could potentially be boosted through natural infections.

Elucidating the molecular mechanisms of the host-parasite interaction during red cell invasion by Plasmodium is important for developing newer antimalarial therapeutics. Recently, we have characterized a Plasmodium vivax tryptophan-rich antigen PvTRAg38, which is expressed by its merozoites, binds to host erythrocytes, and interferes with parasite growth. Interaction of this parasite ligand with the host erythrocyte occurs through its two regions present at amino acid positions 167–178 (P2) and 197–208 (P4). Each region recognizes its own erythrocyte receptor. Previously, we identified band 3 as the chymotrypsin-sensitive erythrocyte receptor for the P4 region, but the other receptor, binding to P2 region, remained unknown. Here, we have identified basigin as the second erythrocyte receptor for PvTRAg38, which is resistant to chymotrypsin. The specificity of interaction between PvTRAg38 and basigin was confirmed by direct interaction where basigin was specifically recognized by P2 and not by the P4 region of this parasite ligand. Interaction between P2 and basigin is stabilized through multiple amino acid residues, but Gly-171 and Leu-175 of P2 were more critical. These two amino acids were also critical for parasite growth. Synthetic peptides P2 and P4 of PvTRAg38 interfered with the parasite growth independently but had an additive effect if combined together indicating involvement of both the receptors during red cell invasion. In conclusion, PvTRAg38 binds to two erythrocyte receptors basigin and band 3 through P2 and P4 regions, respectively, to facilitate parasite growth. This advancement in our knowledge on molecular mechanisms of host-parasite interaction can be exploited to develop therapeutics against P. vivax malaria.

Plasmodium tryptophan rich proteins play important role in host-parasite interaction. Earlier, we have described that one of the merozoite expressed Plasmodium vivax tryptophan-rich antigen PvTRAg35.2 binds to the host erythrocytes, have conserved sequences in parasite population, and generates humoral as well as cellular immune responses in humans during this parasitic infection. Here, we show that PvTRAg35.2 interferes with the parasite growth in a heterologous Plasmodium falciparum culture system. This probably suggests the recognition of the common erythrocyte receptor(s) by certain merozoite ligands of these two parasite species. We have mapped the erythrocyte binding activity of PvTRAg35.2 to its two different regions positioned at amino acid residues 155-190 and 263-283. Binding of these peptide domains to the erythrocytes was inhibited by anti-PvTRAg35.2 antibodies either raised in rabbit or produced by the P. vivax patients. The cross-competition between peptides of PvTRAg35.2 and PvTRAg33.5 or PvTRAg38 during erythrocyte binding assay suggested sharing of host cell receptors by these PvTRAgs. Further studies on these receptor-ligand interactions may lead to the development of therapeutic agents for P. vivax malaria.

Tryptophan-rich antigens of malarial parasites interact with host molecules and play an important role in parasite survival. Merozoite expressed Plasmodium vivax tryptophan-rich antigen PvTRAg38 binds to human erythrocytes and facilitates parasite growth in a heterlologous Plasmodium falciparum culture system. Recently, we identified band 3 in human erythrocytes as one of its receptors, although the receptor-ligand binding mechanisms remain unknown. In the present study, using synthetic mutated peptides of PvTRAg38, we show that multiple amino acid residues of its 12 amino acid domain (KWVQWKNDKIRS) at position 197-208 interact with three different ectodomains of band 3 receptor on human erythrocytes. Our findings may help in the design of new therapeutic approaches for malaria.

Clearance of non-infected red blood cells (nRBCs) is one of the main components of anemia associated with Plasmodium vivax malaria. Recently, we have shown that anemic patients with P. vivax infection had elevated levels of anti-RBCs antibodies, which could enhance in vitro phagocytosis of nRBCs and decrease their deformability. Using immunoproteomics, here we characterized erythrocytic antigens that are differentially recognized by autoantibodies from anemic and non-anemic patients with acute vivax malaria. Protein spots exclusively recognized by anemic P. vivax-infected patients were identified by mass spectrometry revealing band 3 and spectrin as the main targets. To confirm this finding, antibody responses against these specific proteins were assessed by ELISA. In addition, an inverse association between hemoglobin and anti-band 3 or anti-spectrin antibodies levels was found. Anemic patients had higher levels of IgG against both band 3 and spectrin than the non-anemic ones. To determine if these autoantibodies were elicited because of molecular mimicry, we used in silico analysis and identified P. vivax proteins that share homology with human RBC proteins such as spectrin, suggesting that infection drives autoimmune responses. These findings suggest that band 3 and spectrin are potential targets of autoantibodies that may be relevant for P. vivax malaria-associated anemia.

BACKGROUND Plasmodium vivax synthesizes the largest number of 36 tryptophan-rich proteins belonging to the Pv-fam-a family. These parasite proteins need to be characterized for their biological function because tryptophan-rich proteins from other Plasmodium species have been proposed as vaccine candidates. METHODS Recombinant P. vivax tryptophan-rich antigens (PvTRAgs) were used to determine their erythrocyte-binding activity by a cell-based enzyme-linked immunosorbent assay, flow cytometry, and a rosetting assay. RESULTS Only 4 (PvTRAg26.3, PvTRAg34, PvTRAg36, and PvTRAg36.6) of 21 PvTRAgs bind to host erythrocytes. The cross-competition data indicated that PvTRAg36 and PvTRAg34 share their erythrocyte receptors with previously described proteins PvTRAg38 and PvTRAg33.5, respectively. On the other hand, PvTRAg26.3 and PvTRAg36.6 cross-compete with each other and not with any other PvTRAg, indicating that these 2 proteins bind to the same but yet another set of erythrocyte receptor(s). Together, 10 of 36 PvTRAgs possess erythrocyte-binding activity in which each protein recognizes >1 erythrocyte receptor. Further, each erythrocyte receptor is shared by >1 PvTRAg. CONCLUSIONS This redundancy may be useful for the parasite to invade red blood cells and cause disease pathogenesis, and it can be exploited to develop therapeutics against P. vivax malaria.

Background Tryptophan-rich antigens of malarial parasites have been proposed to be the potential vaccine candidate antigens. Plasmodium vivax contains the largest number of such antigens, which need to be evaluated for their immune responses. Methods Recombinant proteins of 15 P. vivax tryptophan-rich antigens (PvTRAgs) were expressed, purified, and used for the human humoral and cellular immune responses. Genetic polymorphism of these 15 genes was also determined among clinical P. vivax isolates. Results The T lymphocytes of P. vivax exposed individuals expressed higher level of CD69 against all 15 PvTRAgs. These antigens also activated the large population of CD4(+) T cells and produced higher level of intracellular IL-2, INF-γ and IL-4. Although there was a mixed Th1 and Th2 response against these antigens, this response was biased toward Th2. The majority of P. vivax patients (75.7%-100%, n = 33) produced IgG antibodies against these antigens. Most of these antigens showed conserved T- and B-cell epitopes in the parasite population. Conclusions These results suggest the presence of memory T cells in humans against these antigens to generate faster and more specific immune responses to minimize the P. vivax infection. Further characterization of these PvTRAgs may lead to the identification of a potential therapeutic target.

Need for malaria vaccine necessitates the characterization of potential antigens of the Plasmodium parasite. Recently, we have identified several Plasmodium vivax tryptophan-rich antigens (PvTRAgs). Here, we describe the immunological characterization of hitherto undescribed two such antigens PvTRAg 35.2 and PvTRAg 80.6 which are respective homologue of Plasmodium falciparum merozoite associated tryptophan-rich antigen (PfMaTrA) and P. falciparum tryptophan and threonine rich antigen (PfTryThrA) involved in erythrocyte invasion. Each of the pvtrag genes is comprised of two exons where exon 2 encodes for major part of the protein. PvTRAg 35.2 and PvTRAg 80.6 showed 97.06% and 94.12% (n = 34) seropositivity rates, and 92.3% (n = 13) and 100% (n = 29) lymphoproliferative responses, respectively, among P. vivax exposed individuals. Geometric mean values of IL-12, IFN-γ, TNF-α, IL-4 and IL-10 in PBMC culture supernatants of P. vivax exposed individuals were 182.02, 60.3, 62.84, 196.01 and 177.17 pg/ml against PvTRAg 35.2 and 185.27, 58.15, 64.56, 142.01 and 157.2 pg/ml against PvTRAg 80.6 showing mixed immune response with distinct biased towards anti-inflammatory Th2 phenotype. The pvtrag 35.2 gene was highly conserved in the parasite population whereas pvtrag 80.6 showed minor variations in the N-terminal region but highly conserved in the C-terminal region containing tryptophan-rich domain.

Present study describes the characterization of apical membrane antigen 1 (PvAMA1) polymorphisms among Indian Plasmodium vivax isolates. The partial PvAMA1 gene (covering domain I and domain II regions) sequenced from sixty-one (n = 61) isolates in this study resulted into 49 haplotypes. Comparison with the previously available PvAMA1 sequences in the GenBank database revealed that 45 of these were new haplotypes that have never been reported till date. For further analyses, we also included 11 previously reported PvAMA1 sequences from India available in the database. Thus genetic diversity and effect of natural selection were analyzed both at domain I and domain II of this promising malaria vaccine candidate among 72 Indian P. vivax isolates. Non-synonymous mutations were found at 25 codons (16 at domain I and 9 at domain II) where 17 codons were dimorphic while rest of them (8 codons) were trimorphic. Thus codon polymorphisms were observed to be more at domain I as compared to domain II. Although the difference between the rate of non-synonymous (dN) and synonymous (dS) mutations was positive (dN–dS, 0.002 ± 0.004SE) at domain II, it was not significantly different from each other (P = 0.272), indicating tendency of stronger diversifying selection at this domain. The dN–dS difference for domain I (− 0.006 ± 0.009SE, P = 0.268) and for entire 900 bp region (− 0.002 ± 0.005E, P = 0.320) being negative and statistically insignificant suggests the role of both positive as well as purifying selection. Three-dimensional distributions of all polymorphic residues were mapped on a modeled PvAMA1 structure. Results suggested that almost all of the observed polymorphisms were located at one surface of the antigen. In conclusion, PvAMA1 antigen displays high diversity among Indian isolates with more diversifying selection at domain II. The result has significant value in malaria vaccine development using this antigen.

We describe here an ∼40-kDaPlasmodium vivaxtryptophan-rich antigen (PvTRAg40) which contains 321 amino acids and 11.4% tryptophan residues. This protein shows 65% homology (35% identity) with the previously described PvTRAg, besides sharing 23 of 27 positionally conserved tryptophan residues and similar genomic organization. The nucleotide sequence of the entire tryptophan-rich domain of PvTRAg40 was identical among 35P. vivaxclinical isolates. The protein is expressed by ring, trophozoite, and schizont stages of the parasite. The cDNA covering exon 2 of PvTRAg40 was cloned and expressed in the pPROEXHTa vector, and recombinant protein was purified. A high humoral immune response (90.7% seropositivity;n= 43) against this recombinant protein was detected in humans during the course of naturalP. vivaxinfection. Eighty percent of the total of 20P. vivax-exposed individuals exhibited lymphoproliferative responses against this antigen. The T cells of these individuals produced larger amounts of interleukin-12 (IL-12), IL-4, and IL-10 than gamma interferon and tumor necrosis factor alpha cytokines in response to the recombinant protein. Production of Th2-biased cytokines, conserved T- and B-cell epitopes, and an enhanced humoral immune response indicate that PvTRAg40 could possibly induce antibody-mediated immune protection against infection.

SUMMARY Plasmodium vivax, the most widespread species of human malaria parasite responsible for 70–80 million cases each year requires a vaccine. In recent years, many potential vaccine candidate antigens have been identified from P. vivax including PvTRAg. We describe here cellular immune response to recombinant PvTRAg expressed in Escherichia coli. The in vitro stimulation of PBMCs derived from P. vivax-exposed individuals (n = 16) showed strong proliferative response (SI > 2·2) to PvTRAg as compared to PBMCs from normal healthy controls (n = 8). Although both Th1 (IFN-γ, TNF-α and IL-12) and Th2 (IL-4 and IL-10) cytokines were secreted by the PBMCs of the P. vivax-exposed individuals in response to PvTRAg, the overall response was more inclined towards Th2. In conclusion, recombinant PvTRAg was found to elicit strong cellular immune response among the P. vivax-exposed individuals.

Plasmodium tryptophan-rich proteins are involved in host-parasite interaction and thus potential drug/vaccine targets. Recently, we have described several P. vivax tryptophan-rich antigens (PvTRAgs), including merozoite expressed PvTRAg38, from this noncultivable human malaria parasite. PvTRAg38 is highly immunogenic in humans and binds to host erythrocytes, and this binding is inhibited by the patient sera. This binding is also affected if host erythrocytes were pretreated with chymotrypsin. Here, Band 3 has been identified as the chymotrypsin-sensitive erythrocyte receptor for this parasite protein. Interaction of PvTRAg38 with Band 3 has been mapped to its three different ectodomains (loops 1, 3, and 6) exposed at the surface of the erythrocyte. The binding region of PvTRAg38 to Band3 has been mapped to its sequence, KWVQWKNDKIRSWLSSEW, present at amino acid positions 197-214. The recombinant PvTRAg38 was able to inhibit the parasite growth in in vitro Plasmodium falciparum culture probably by competing with the ligand(s) of this heterologous parasite for the erythrocyte Band 3 receptor. In conclusion, the host-parasite interaction at the molecular level is much more complicated than known so far and should be considered during the development of anti-malarial therapeutics.

Tryptophan-rich proteins play important biological functions for the Plasmodium parasite. Plasmodium vivax contains remarkably large numbers of such proteins belonging to the “Pv-fam-a” family that need to be characterized. Earlier, we reported the presence of memory T cells and naturally acquired antibodies against 15 of these proteins in P. vivax malaria-exposed individuals (M. Zeeshan, H. Bora, and Y. D. Sharma, J Infect Dis 207: 175–185, 2013, http://dx.doi.org/10.1093/infdis/jis650 ). Here, we sought to characterize and ascertain the cross talk between effector responses of T and B cells in malarial patients against all Pv-fam-a family proteins. Therefore, we expressed the remaining 21 of these proteins in Escherichia coli and studied the humoral and cellular immune responses based on the same parameters used in our previous study. Naturally acquired IgG antibodies were detected against all 21 antigens in P. vivax patient sera (37.7 to 94.4% seropositivity). These antigens were able to activate the lymphocytes of P. vivax -exposed individuals, and the activated CD4 + T lymphocytes produced higher levels of Th1 (interleukin-2 [IL-2] and gamma interferon [IFN-γ]) and Th2 (IL-4 and IL-10) cytokines than the healthy controls, but the response was Th2 biased. The combined results of present and previous studies seem to suggest a striking link between induction of the CD4 + T cell response and naturally acquired antibodies against all 36 proteins of the Pv-fam-a family, the majority of them having conserved sequences in the parasite population. Further work is required to utilize this information to develop immunotherapeutic treatments for this disease.

Our recent studies have focused on the identification and characterization of the tryptophan-rich proteins of the Plasmodium vivax parasite where their role in the elicitation of humoral and cellular responses and erythrocyte-binding activity was investigated. Here, we report the humoral responses of a 32.4-kDa P. vivax tryptophan-rich antigen (PvTRAg32.4) among the sera of P. vivax-infected patients. PvTRAg32.4 also contains an unusually high percentage of tryptophan residues (10.7 %) that are positionally conserved with its orthologues in Plasmodium yoelii (PypAg1 and PypAg2) and Plasmodium falciparum (PfTryThrA and PfMATRA). Thirty-four of the 40 (85.0 %) P. vivax isolates showed seropositivity to recombinant PvTRAg32.4 by ELISA. The mean ± SD values of optical density (OD) for P. vivax subjects and naive individuals were 1.02 ± 0.36 and 0.26 ± 0.11, respectively. In the Western blot analysis, majority of the subjects studied (n = 44) showed reactivity to the recombinant, purified PvTRAg32.4. This antigen does not show binding to the erythrocytes, but the immunofluorescence data reveals that it is expressed in the erythrocytic stages of the parasite. Sequence analysis of the clinical isolates from various parts of the country shows that PvTRAg32.4 is highly conserved. Functional in-depth characterization of more such type of novel proteins in the parasite is warranted for the development of successful malaria intervention methods.

Plasmodium vivax is a very common but non-cultivable malaria parasite affecting large human population in tropical world. To develop therapeutic reagents for this malaria, the parasite molecules involved in host-parasite interaction need to be investigated as they form effective vaccine or drug targets. We have investigated here the erythrocyte binding activity of a group of 15 different Plasmodium vivax tryptophan rich antigens (PvTRAgs). Only six of them, named PvTRAg, PvTRAg38, PvTRAg33.5, PvTRAg35.2 PvTRAg69.4 and PvATRAg74, showed binding to host erythrocytes. That the PvTRAgs binding to host erythrocytes was specific was evident from the competitive inhibition and saturation kinetics results. The erythrocyte receptors for these six PvTRAgs were resistant to trypsin and neuraminidase. These receptors were also chymotrypsin resistant except the receptors for PvTRAg38 and PvATRAg74 which were partially sensitive to this enzyme. The cross-competition studies showed that the chymotrypsin resistant RBC receptor for each of these two proteins was different. Altogether, there seems to be three RBC receptors for these six PvTRAgs and each PvTRAg has two RBC receptors. Both RBC receptors for PvTRAg, PvTRAg69.4, PvTRAg33.5, and PvTRAg35.2 were common to all these four proteins. These four PvTRAgs also shared one of their RBC receptors with PvTRAg38 as well as with PvATRAg74. The erythrocyte binding activity of these six PvTRAgs was inhibited by the respective rabbit polyclonal antibodies as well as by the natural antibodies produced by the P. vivax exposed individuals. It is concluded that only selective few PvTRAgs show erythrocyte binding activity involving different receptor molecules which can be blocked by the natural antibodies. Further studies on these receptor and ligands may lead to the development of therapeutic reagents for P. vivax malaria.

The immune status of the individuals living in the Delhi area of northern India where Plasmodium vivax is predominant is described. The majority of the individuals who were acutely infected with P. vivax had antibodies against crude blood-stage antigen and the recombinant antigen, PV9. In contrast, the peripheral blood mononuclear cells of the majority of these individuals did not show proliferation in the presence of either of these antigens. There was no correlation between antibody levels and T cell proliferation. Such an immune status of individuals living in endemic areas should be taken into account in the future in developing P. vivax malaria vaccines.

We have isolated a new recombinant antigen ‘PV12’ of Plasmodium vivax by immunological screening of the genomic library. The antigen shows a sequence homology with human apolipoprotein B-100. A large number of P. vivax infected individuals (79%) were seropositive against this antigen. The probable function of this antigen could be to evade the host defence system (i) by inactivating the perforin of natural killer cells and (ii) by engaging the host immune system to produce antibodies against this decoy antigen.

A total of 172 sera samples were collected from individuals who were living in Piyawli-Jaitwarpur village in Ghaziabad district (U.P.), India. They had suffered from falciparum malaria attack, and were cured with antimalarial drugs 1–2 weeks prior to sample collection. These samples were divided into nine groups according to their age. The pooled sera from each group were tested for the presence of anti-schizont and anti-heat shock protein (hsp) −70 antibodies, as well as for parasite growth inhibition in vitro. All sera samples showed significant levels of antibodies against schizont antigens and these levels increased with age. The sera also contained anti-hsp-70 antibodies but at lower levels and did not follow the same age-related pattern as seen with schizont antibodies. The sera from each group significantly inhibited merozoite invasion in vitro. However the same was not true for other blood stage parasites; the 2–15 years age group sera did not show significant growth inhibition of rings, trophozoites and schizonts. No correlation was observed between anti-hsp-70 antibody levels and inhibition of merozoite invasion. It is therefore concluded that the antibodies preventing the merozoite invasion could be other than anti-hsp-70 antibodies. The candidature of hsp-70 for P. falciparum malaria vaccine thus needs to be re-evaluated.

Pooled polyvalent sera from lepromatous leprosy patients were used to screen a lambda gt11 recombinant DNA expression library of Mycobacterium leprae in order to identify the relevant antigens recognized by the human immune response. Of the 300,000 phages screened, 4 clones were identified that coded for fusion proteins of the same molecular mass. The fusion protein from clone LSR2 was tested for immunoreactivity in assays using peripheral blood cells and sera from 11 laboratory personnel and 105 patients across the leprosy spectrum. LSR2 protein appears to be predominantly a T-cell antigen. It evokes similar lymphoproliferative responses as the native bacillus both at the individual level and in the leprosy spectrum as a whole. Though only 50% of patient sera with anti-M. leprae antibodies reacted with the fusion protein, the pattern of reactivity in the antibody responses was also similar for the various clinical types. The coding regions of clones LSR1 and LSR2 are identical. They show no homology with sequences stored in data banks and encode a protein of 89 amino acids with a calculated molecular mass of approximately 10 kDa.

1. 1. The sequestration of trophozoite and schizont infected erythrocytes (IRBC) in post-capillary venules of host internal organs causes most of the morbidity and mortality in falciparum malaria. It is a knob mediated cytoadherence phenomenon where knobs act as the focal junction between IRBC and host endothelial cell. Knobless (K − ) parasites, isolated from cultures (not yet isolated from in vivo ), do not cause virulent infections. Knobs thus play an important role in pathophysiology of falciparum malaria. 2. 2. The chemical composition of knobs is partly explored, several proteins (Known as knob proteins) have been identified. According to their function they can be classified as (a) knob-inducing protein, “KAHRP” (b) knob-associated cytoadherent proteins, e.g. PFEMP-1, modified band 3 and an antigen recognized by monoclonal 33G2 and (c) knob-associated structural protein, e.g. PFEMP2/MESA/PP-300. Most of them show size polymorphism among different isolates. Only KAHRP and MESA/PFEMP-2 have been studied at molecular level. Their chromosomal locations have been identified such as KAHRP on chromosome 2 and MESA/PFEMP-2 on chromosomes 5 and 6. 3. 3. The receptor molecules on endothelial cells for knob ligands have been identified and partially characterized. 4. 4. Knob ligands and their receptor molecules can play an important role in developing the immunotherapeutic reagents. 5. 5. Based on the available data a tentative hypothesis has been proposed about the loss of knobs in vitro . Nevertheless, this needs further support from other experimental evidence. 6. 6. Future work should be directed towards the structure and function of knob proteins and their interactions with each other as well as with host proteins. Regulation of expression of knobs and knob protein(s), evaluation of knob antigens for immunotherapy of severe faleiparum malaria and for a malaria vaccine also require further investigations.

Tryptophan-rich protein from P. yoelii called PypAg3 had shown protection in a murine model. We describe here its P. vivax orthologue named PvTRAg39.8 which is expressed during schizont stages of the parasite. The transcription initiation site was mapped to the −41 nucleotide position from the first AUG codon and thus established the ORF of PvTRAg39.8 which codes for a 322aa long protein. The sequence of this protein is highly conserved among the P. vivax isolates. Recombinant PvTRAg39.8 was expressed in E. coli , purified to electrophoretic homogeneity, and used for immunological studies. A majority of P. vivax -exposed individuals (90%, n = 50) contained antibodies against the recombinant PvTRAg39.8. There was a switching of immunoglobulin isotypes against this antigen from IgG1 to IgG4 among individuals from the acute phase of P. vivax infection to convalescence. The PBMCs of 80% of the total 20 P. vivax -exposed individuals showed proliferation when stimulated with this antigen. The cytokine pattern produced by the PBMCs of the P. vivax -exposed individuals, after stimulation with this antigen, indicated a mixed type of immune response with a distinct bias towards the Th2 anti-inflammatory profile. Because of its high natural immunogenicity, conserved nature, and the protective properties of its P. yoelii counterpart, this protein may be a potential candidate for developing the immunotherapeutic reagents.

Background The Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate antigen. The complete AMA1 protein is comprised of three domains where domain I exhibits high sequence polymorphism and is thus named as the hyper-variable region (HVR). The present study describes the extent of genetic polymorphism and natural selection at domain I of the ama1 gene among Indian P. falciparum isolates. Methods The part of the ama 1 gene covering domain I was PCR amplified and sequenced from 157 P. falciparum isolates collected from five different geographical regions of India. Statistical and phylogenetic analyses of the sequences were done using DnaSP ver. 4. 10. 9 and MEGA version 3.0 packages. Results A total of 57 AMA1 haplotypes were observed among 157 isolates sequenced. Forty-six of these 57 haplotypes are being reported here for the first time. The parasites collected from the high malaria transmission areas (Assam, Orissa, and Andaman and Nicobar Islands) showed more haplotypes (H) and nucleotide diversity π as compared to low malaria transmission areas (Uttar Pradesh and Goa). The comparison of all five Indian P. falciparum subpopulations indicated moderate level of genetic differentiation and limited gene flow (Fixation index ranging from 0.048 to 0.13) between populations. The difference between rates of non-synonymous and synonymous mutations, Tajima's D and McDonald-Kreitman test statistics suggested that the diversity at domain I of the AMA1 antigen is due to positive natural selection. The minimum recombination events were also high indicating the possible role of recombination in generating AMA1 allelic diversity. Conclusion The level of genetic diversity and diversifying selection were higher in Assam, Orissa, and Andaman and Nicobar Islands populations as compared to Uttar Pradesh and Goa. The amounts of gene flow among these populations were moderate. The data reported here will be valuable for the development of AMA1-based malaria vaccine.

Summary Despite the immense global efforts, the malaria vaccine is not yet available and requires the identification of newer target molecules. Since tryptophan-rich proteins of P. yoelii have been proposed as vaccine candidates, we describe here the expression, purification and immunological characterization of a 55 kDa Plasmodium vivax tryptophan- and alanine-rich antigen (PvTARAg55). This protein consists of 480 aa residues with a calculated molecular mass of 55.0 kDa. It shows 42% aa sequence identity (64% homology) with PyPAg1 of P. yoelii and shares positional conservation of tryptophan residues. Sequence analysis of PvTARAg55 from different P. vivax isolates revealed that typtophan-rich domain which contains most of the B-cell epitopes was highly conserved in the parasite population while the alanine-rich domain showed polymorphism. Exon-2 covering major part (420 aa) of the protein including both the domains was PCR amplified, cloned, expressed in Escherichia coli, and the recombinant protein purified to its homogeneity. Majority of P. vivax-infected individuals (82.5%, n = 40) produced antibodies against this antigen. Proliferative responses to the recombinant PvTARAg55 were observed in 60% (n = 20) of individuals who had recently been exposed to the P. vivax infection. Measurement of Th1- (IFN-γ, TNF-α, and IL-12) and Th2-type (IL-4 and IL-10) cytokine production in response to this recombinant antigen revealed a mixed type T-cell response with a Th2 response being more pronounced. These results demonstrate that PvTARAg55 elicits high humoral and cellular immune responses thus establishes its immunogenecity in humans.

Plasmodium vivax is most common but non-cultivable human malaria parasite which is poorly characterized at the molecular level. Here, we describe the identification and characterization of a P. vivax Tryptophan-Rich Antigen (PvTRAg) which contains unusually high (8.28%) tryptophan residues and is expressed by all blood stages of the parasite. The pvtrag gene comprises a 978bp open reading frame interrupted by two introns. The first intron is located in the 5'-untranslated region while the second one is positioned 174bp downstream to the ATG codon. The encoded approximately 40kDa protein contains a transmembrane domain near the N-terminus followed by a tryptophan-rich domain with significantly high surface probability and antigenic index. It is localized in the parasite cytoplasm as well as in the cytoplasm of the parasitized erythrocyte. The purified E. coli expressed recombinant PvTRAg protein showed a very high seropositivity rate for the presence of antibodies amongst the P. vivax patients, indicating that the antigen generates significant humoral immune response during the natural course of P. vivax infection. Analysis of various field isolates revealed that the tryptophan-rich domain is highly conserved except for three-point mutations. The PvTRAg could be a potential vaccine candidate since similar tryptophan-rich antigens of P. yoelii have shown protection against malaria in murine model.

The Plasmodium falciparum heat-shock protein 70 (PFHSP70) has been proposed as a potential candidate antigen for a malaria vaccine. In this study the human immune response to this antigen has been evaluated. Enzyme-linked immunosorbent assays (ELISA) were performed on serum samples collected from individuals during and after malarial infection. Only 70% of falciparum malaria patients responded to this antigen during the natural course of infection and 54% of the recovered cases who had no parasitaemia at the time of blood collection lacked detectable antibody. The non-responders to PFHSP70 exhibited an antibody response to total schizont antigens. There was no correlation between the response against total schizont antigens and PFHSP70 antigen. The non-responsiveness against PFHSP70 was not related to age, sex or regional variations. These qualities are undesirable for a vaccine candidate antigen.

The effect of various body temperatures, encountered during malaria fever, on the synthesis of Plasmodium falciparum heat-shock protein called PFHSP70-I and parasite growth rates among five different isolates are described. The results show that after the exposure of parasites at 39 degrees C for 30 min the amount of PFHSP70-I in all five isolates increased markedly and significantly, whereas parasite growth rates and the amount of total blood stage antigens remained almost unaffected. This indicates that the PFHSP70-I gene responds to heat-shock by producing higher amounts of PFHSP70-I protein, presumably to protect the parasite from being killed during malaria fever.

Plasmodium vivax is a very common human malaria parasite but it is poorly characterized at the molecular level. Here, we describe the isolation and characterization of an antigen coding gene of P. vivax which contains Alu elements. This gene, called Pv-Alu, is expressed during the erythrocytic phase of the parasite. The encoded 200 amino acid long polypeptide is highly hydrophobic, contains transmembrane domains, and is rich in leucine (19.4%), serine (15.9%), proline (15.4%) and phenylalanine (15.4%). The 5′-untranslated region and part of the 3′-end coding region of Pv-Alu show significant homology to different Alu families. The presence of Alu elements in the coding region of a parasite antigen gene is significant from a functional and evolutionary viewpoint.

A genomic library for Plasmodium vivax was constructed in lambda gt11 and immunologically screened with pooled serum samples from vivax patients. Six seroreactive clones were isolated, and one clone, denoted PV9, was studied further. This clone has an unusual base composition (65% G + C), does not share any homology with P. falciparum, and codes for an entirely new antigenic determinant. Antibodies (immunoglobulin G type) against the PV9-encoded polypeptide were produced in all vivax patients older than 15 years. This seroreactivity was lower among patients younger than 15 years (53%). The antigenic epitope(s) of the PV9-encoded polypeptide was recognized at a similar rate by serum samples from P. vivax patients who were living 350 to 973 km apart. Fifty percent of uninfected Indian adults were also seropositive, whereas all European and American (United States) sera tested were negative, suggesting that anti-PV9 antibodies persist after infection. The seroreactivity pattern of this antigen is similar to that of the immunity developed in malaria after repeated infections.

Background Plasmodium vivax uses duffy antigen to invade the human erythrocytes but reports of duffy negative infection by P. vivax indicate that this parasite may also be using some other alternative receptors for the invasion [1]. Studies are therefore required to identify these additional parasite molecules which are involved in host-parasite interaction. Plasmodium vivax, contains a family of tryptophan rich antigens containing positionally conserved tryptophan residues. These antigens can induced significant cellular and humoral responses in human individuals [2,3] and few showed interaction with erythrocytes. Materials and methods Recombinant Plasmodium vivax tryptophan-rich antigens were purified by Ni-NTA affinity chromatography. Heparinized blood was collected from healthy lab individuals to obtain human erythrocytes for binding assays, and from the microscopically confirmed P.vivax infected individuals for patients’ sera. Rabbit antibodies against PvTRAgs used here were raised in the lab. Erythrocyte binding assays were performed by cell ELISA and Flow cytometry [2,4]. Results PvTRAgs binds to the human erythrocytes in a concentration dependent manner and not to the human lymphocytes. The binding was specific as competition with untagged proteins inhibits the erythrocyte binding to 50% at equimolar concentration. Antibodies raised against rabbits and produced by infected patients inhibit the binding of respective PvTRAg at different dilutions. PvTRAg38 and PvATRAg74 was sensitive to chymotrypsin only and not to the trypsin and neuraminidase while all other PvTRAgs were resistant to all these proteases. Conclusions Six out of fifteen PvTRAgs bind specifically to the human erythrocytes and were inhibited by rabbit anti PvTRAgs and also by the antibodies produced during natural course of P. vivax infection. There may be more than one RBC receptor for these six PvTRAgs where only PvTRAg38 and PvATRAg74 are sensitive to chymorypsin while others using those molecules which are other than glycophorin and sialoglycoproteins. Studies are in progress to investigate the respective RBC receptors for these PvTRAgs and their role in erythrocyte invasion process.