Molecular Modeling and Optimization of Type II E.coli l-Asparginase Activity by in silico Design and in vitro Site-directed Mutagenesis.

INTRODUCTION: L-asparaginase (also known as L-ASNase) is a crucial therapeutic enzyme that is widely used in treatment of ALL (acute lymphoblastic leukemia) as a chemotherapeutic drug. Besides, this enzyme is used in the food industry as a food processing reagent to reduce the content of acrylamide in addition to the clinical industry. The improvement of activity and kinetic parameters of the L-ASNase enzyme may lead to higher efficiency resulting in practical achievement. In order to achieve this goal, we chosen glycine residue in position 88 as a potential mutation with advantageous outcomes. METHOD: In this study, firstly to find the appropriate mutation on glycine 88, various in silico analyses, such as MD simulation and molecular docking, were carried out. Then, the rational design was adopted as the best strategy for molecular modifications of the enzyme to improve its enzymatic properties. RESULT: Our in silico findings show that the four mutations G88Q, G88L, G88K, and G88A may be able to increase L-ASNase's asparaginase activity. The catalytic efficiency of each enzyme (kcat/Km) is the most important feature for comparing the catalytic activity of the mutants with the wild type form. The laboratory experiments showed that the kcat/Km for the G88Q mutant is 36.32% higher than the Escherichia coli K12 ASNase II (wild type), which suggests that L-ASNase activity is improved at lower concentration of L-ASN. Kinetic characterization of the mutants L-ASNase activity confirmed the high turnover rate (kcat) with ASN as substrate relative to the wild type enzyme. CONCLUSION: In silico analyses and laboratory experiments demonstrated that the G88Q mutation rather than other mutation (G88L, G88K, and G88A) could improve the kinetics of L-ASNase.

Root-preferential expression of Newcastle virus glycoproteins driven by NtREL1 promoter in tobacco hairy roots and evaluation of oral delivery in mice.

Newcastle disease virus (NDV) is a lethal virus in avian species with a disastrous effect on the poultry industry. NDV is enveloped by a host-derived membrane with two glycosylated haemagglutinin-neuraminidase (HN) and Fusion (F) proteins. NDV infection usually leads to death within 2-6 days, so the preexisting antibodies provide the most critical protection for this infection. The HN and F glycoproteins are considered the main targets of the immune system. In the present study, two constructs harboring the HN or F epitopes are sub-cloned separately under the control of a root-specific promoter NtREL1 or CaMV35S (35S Cauliflower Mosaic Virus promoter) as a constitutive promoter. The recombinant vectors were transformed into the Agrobacterium tumefaciens strain LBA4404 and then introduced to tobacco (Nicotiana tabacum L.) leaf disk explants. PCR with specific primers was performed to confirm the presence of the hn and f genes in the genome of the regenerated plants. Then, the positive lines were transformed via non-recombinant A. rhizogenes (strain ATCC15834) to develop hairy roots.HN and F were expressed at 0.37% and 0.33% of TSP using the CaMV35S promoter and at 0.75% and 0.54% of TSP using the NtREL1 promoter, respectively. Furthermore, the mice fed transgenic hairy roots showed a high level of antibody responses (IgG and IgA) against rHN and rF proteins.

The Immunogenicity of a Novel Chimeric Hemagglutinin-Neuraminidase-Fusion Antigen from Newcastle Disease Virus by Oral Delivery of Transgenic Canola Seeds to Chickens.

Newcastle disease (ND) is considered as one of the most devastating infectious diseases targeting domestic birds and has considerable threat to the commercial poultry production. Two surface glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F), act as antigens in the virus structure and also play important roles in infecting host cells. In the current study, the expression of the chimeric HN-F protein in canola seeds and its immunogenicity in chickens were investigated. The HN-F gene was cloned downstream of the fatty acid elongase 1 (FAE1) promoter in the binary expression vector, pBI1400-HN-F, and introduced into rapeseed (Brassica napus L.) using Agrobacterium-mediated transformation. The amount of the HN-F glycoprotein was estimated up to 0.18% and 0.11% of the total soluble protein (TSP) in transgenic seeds and leaves of canola, respectively. Confirmatory analyses of 36 transgenic lines revealed that the HN-F gene was integrated into the genome. Subsequently, HN-F protein could be expressed and accumulated in the seed tissue. Specific pathogen-free (SPF) chickens immunized orally with recombinant HN-F showed a significant rise in specific and hemagglutination inhibition (HI) antibodies 35 to 42 days post the first administration. The results implied the potential of transgenic canola seed-based expression for oral delivery of NDV immunogenic glycoproteins.

Immunogenicity of the Multi-Epitopic Recombinant Glycoproteins of Newcastle Disease Virus: Implications for the Serodiagnosis Applications.

BACKGROUND: Newcastle disease virus (NDV) is a dangerous viral disease, infecting a broad range of birds, and has a fatal effect on the poultry industries. The attachment and consequently fusion of the virus to the host cell membrane is directed by the two superficial glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion (F) which is considered as the important targets for the poultry immune response. OBJECTIVES: The principal goal of this investigation was to realize the potential efficacy of the E. coli expression system for the production of the multi-epitopic HN, and F proteins with respect to the ability for the stimulation of the immune system and production of the cross-reactive antibodies in mice. MATERIALS AND METHODS: The recombinant HN and F (rHN, rF) have accumulated almost 40% of the total bacterial proteins. The presence of rHN and rF proteins recognized by the Western blotting with specific anti-HN, anti-F, anti-Newcastle B1, and anti-poly 6x His-tag antibodies. Furthermore, both rHN and rF have shown the specific reactivity against the Newcastle B1 antiserum as a standard strain. RESULTS: The ELISA analysis showed that the higher dilutions of the antibody against Newcastle B1 could react with the as least quantity as 100 ng of the purified rHN, and rF. Cross-reactivity analysis of the sera from the mice immunized with Newcastle B1 in two time points indicated that the raise of anti-Newcastle B1, anti-HN and anti-F antibodies peaked at 28 days post immunization (dpi). Moreover, temporal variation in IgG titration between both time points was significant at 5% probability level. CONCLUSION: The results provided valuable information about the cross-reactivity patterns and biological activity of the multi-epitopic proteins compared to the NDV standard strain which was determined by the Western blotting and ELISA.

Functional Surface Display of Laccase in a Phenol-Inducible Bacterial Circuit for Bioremediation Purposes

Background: Phenolic compounds, which are produced routinely by industrial and urban activities, possess dangers to live organisms and environment. Laccases are oxidoreductase enzymes with the ability of remediating a wide variety of phenolic compounds to more benign molecules. The purpose of the present research is surface display of a laccase enzyme with adhesin involved in diffuse adhesion (AIDA-I) autotransporter system on the surface of Escherichia coli cells for bioremediation of phenolic compounds. Methods: The expression of laccase was regulated by a phenol-responsive promoter (a σ54 promoter). The constitutively-expressed CapR transcription activator was able to induce laccase expression in the presence of phenolic compounds. Results: Western blot analysis showed the expression and correct transfer of the enzyme to the outer membrane of E. coli cells in the presence of phenol. Activity assay confirmed the correct folding of the enzyme after translocation through the autotransporter system. HPLC analysis of residual phenol in culture medium showed a significant reduction of phenol concentration in the presence of cells displaying laccase on the surface. Conclusion: Our findings confirm that autodisplay enables functional surface display of laccase for direct substrate-enzyme availability by overcoming membrane hindrance.

Protection against Legionnaire's Disease: Recombinant Flagellin A of Legionella pneumophila Can Induce Protective Immunity against Bacteremia in a BALB/c Murine Model.

To investigate the immunoprotective effects of the recombinant type A flagellin (FLA), the flaA gene of Legionella pneumophila serogroup 1 strain Paris was cloned into pET28a(+). Recombinant FLA (rFLA) was overexpressed in E. coli BL21 (DE3) and purified by Ni2+ exchange chromatography. Female BALB/c aged 6-8 weeks were immunized with 20 µg of rFLA. Nonimmunized mice along with mice inoculated with a sublethal dose of live L. pneumophila intravenously were considered as negative and positive controls, respectively. A significant serum antibody response was observed in female BALB/c mice immunized with rFLA. Production of IFN-γ and IL-12, and TNF-α in the serum and the splenocyte cultures, and antigen-specific splenocyte proliferation suggested a strong innate and adaptive cell-mediated immunity response in rFLA-immunized mice. Intravenous lethal challenge with L. pneumophila serogroup 1 (strain Paris) showed that 60% of mice immunized with rFLA survived in a 10-day follow-up survey. These results show that rFLA from L. pneumophila can elicit strong innate and adaptive immune responses and suggest the possibility of a long-term immunity against lethal challenge with L. pneumophila.

In silico designing, cloning, and heterologous expression of novel chimeric human B lymphocyte CD20 extra loop.

Design and production of monoclonal antibody for the diagnosis and immunotherapy of non-Hodgkin lymphoma require a suitable CD20 antigen as an effective immunogen. In this study, a new chimeric human CD20 extra loop (hCD20EXL) protein was designed by bioinformatics tools and was expressed in Escherichia coli BL21 DE3. Amino acid sequences, protein structure, immunogenicity, and other physicochemical property of potential antigens were in silico analyzed. Antigenicity, codon optimization, and other predictions of designed protein were determined by bioinformatics tools. The designed protein was heterologously expressed in E. coli and verified by SDS-PAGE and Western blot. Immunogenicity of this antigen was tested in mice, and reactivity of the antibodies was evaluated using flow cytometry. Experimental analysis confirmed the in silico prediction of the designed chimeric hCD20 in this study. Therefore, based on these results, it is suggested that the new chimeric hCD20 antigen could be an appropriate immunogen for production of monoclonal antibody in immunotherapy purposes.

Immunogenic properties of trivalent recombinant protein composed of B-subunits of LT, STX-2, and CT toxins.

Infectious diarrhoea remains an emerging problem in the world health program. Among diarrheagenic agents, Vibrio cholerae and enterotoxigenic and enterohemorrhagic Escherichia coli are critical enteropathogens. AB5 toxin produced by these bacteria, heat-labile enterotoxin (LT), cholera enterotoxin (CT), and shiga-like cytotoxin (STX) can target the immune system and are subunit vaccine candidates. A chemically-synthesized chimeric construct composed of the binding subunits of these toxins (LTB, STXB, and CTXB) was developed based on bioinformatics studies. The whole chimeric protein (rLSC) and each of the segments (rLTB, rSTXB, and rCTXB) were expressed in a prokaryotic expression system (E. coli), purified, and analysed for their immunogenic properties. The results indicate that these recombinant proteins were effectively able to present appropriate epitopes to an animal model of the immune system which could result in and increase IgG in serum and immune responses that protect against the binding activity of these toxins. The immunological assays revealed that the sera of immunized mice prevented toxins from binding to their specific receptors and neutralized their toxic effects. The proposed construct should be considered as a potent immunogen to prevent toxicity and diarrhoea.

Co-expression of hepatitis C virus polytope-HBsAg and p19-silencing suppressor protein in tobacco leaves.

CONTEXT: Plants transformed by virus-based vectors have emerged as promising tools to rapidly express large amounts and inexpensive antigens in transient condition. OBJECTIVE: We studied the possibility of transient-expression of an HBsAg-fused polytopic construct (HCVpc) [containing H-2d and HLA-A2-restricted CD8+CTL-epitopic peptides of C (Core; aa 132-142), E6 (Envelope2; aa 614-622), N (NS3; aa 1406-1415), and E4 (Envelope2; aa 405-414) in tandem of CE6NE4] in tobacco (Nicotiana tabacum) leaves for the development of a plant-based HCV vaccine. MATERIALS AND METHODS: A codon-optimized gene encoding the Kozak sequence, hexahistidine (6×His)-tag peptide, and HCVpc in tandem was designed, chemically synthesized, fused to HBsAg gene, and inserted into Potato virus X (PVX-GW) vector under the control of duplicated PVX coat protein promoter (CPP). The resulted recombinant plasmids (after confirmation by restriction and sequencing analyses) were transferred into Agrobacterium tumefaciens strain GV3101 and vacuum infiltrated into tobacco leaves. The effect of gene-silencing suppressor, p19 protein from tomato bushy stunt virus, on the expression yield of HCVpc-HBsAg was also evaluated by co-infiltration of a p19 expression vector. RESULTS: Codon-optimized gene increased adaptation index (CAI) value (from 0.61 to 0.92) in tobacco. The expression of the HCVpc-HBsAg was confirmed by western blot and HBsAg-based detection ELISA on total extractable proteins of tobacco leaves. The expression level of the fusion protein was significantly higher in p19 co-agroinfiltrated plants. DISCUSSION AND CONCLUSION: The results indicated the possibility of expression of HCVpc-HBsAg constructs with proper protein conformations in tobacco for final application as a plant-derived HCV vaccine.

Production and Characterization of New Anti-Human CD20 Monoclonal Antibody.

The B-cell CD20 antigen is one of the most reliable surface targets in immunotherapy of B lymphoma. In this project, we studied the production and characterization of a new monoclonal antibody against chimeric human CD20 extra loops (hCD20 exl). The results showed that clone C12H, IgG2/k isotype reacted with the antigen in ELISA and immunoblot. The Kd value was found to be 2×10(-9)M and flow cytometry results showed that 99.9% and 99.7% of the Daudi and Raji cells respectively were stained with C12H monoclonal antibody (mab) but not with Jurkat cell lines. It also effectively competed with Rituximab, thus, the staining of the Daudi and Raji cell lines was reduced to 55.9% and 40.5% of cells respectively. Based on the high affinity reaction of C12H mab and appropriate reactivity of C12H mab with the native antigen on the surface of Raji and Daudi cells in flow cytometry, it was concluded that development and evaluation of C12H mab could be a beneficial candidate for further application in genetically engineered monoclonal antibody.

Substrate-dependent expression of laccase in genetically modified Escherichia coli: design and construction of an inducible phenol-degrading system.

Phenolic compounds that are produced by variety of industrial and urban activities pose dangers to live organisms and the environment. Here, an inducible phenol-degrading system was designed and constructed in Escherichia coli as the host. CapR as a transcription activator in Pseudomonas species shows sensitivity towards most common phenolic pollutants. Upon presence of inducible pollutants and conformational changes of CapR, an inducible promoter will trigger the expression of a bacterial laccase gene, which had been isolated previously from a local Bacillus species. Laccase as a multicopper oxidase has the ability to oxidize wide variety of mono and polyphenols. The sensitivity of the inducible system was verified in the presence of phenol with the concentration range of 1 nM-10 mM. A linear correlation was observed between laccase expression and phenol concentration up to 1 mM. Laccase was expressed even in the lowest concentration of phenol (1 nM) after 2 hr of exposure. 2,2-Azinobis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) as a mediator of laccase oxidative reactions could induce laccase expression through conformational changes of CapR. Recognition of ABTS by CapR not only results in expression of the remediating enzyme but also extends its substrate range to nonphenolic compounds.

Evolutionary characterization of hemagglutinin gene of H9N2 influenza viruses isolated from Asia.

The full length hemagglutinin (HA) genes of 287 H9N2 AI strains isolated from chickens in Asia during the period 1994-2009 were genetically analyzed. Phylogenetic analysis showed that G1-like viruses circulated in the Middle East and Indian sub-continent countries, whereas other sublineages existed in Far East countries. It also revealed G1-like viruses with an average 96.7% identity clustered into two subgroups largely based on their time of isolation. The Ka/Ks ratio was calculated 0.34 for subgroup 1 and 0.57 for subgroup 2 indicates purifying/stabilizing selection, but despite this there is evidence of localized positive selection when comparing the subgroups 1 and 2 protein sequences. Five sites in HA H9N2 viruses had a posterior probability >0.5 using the Bayesian method, indicating these sites were under positive selection. These sites were found to be associated with the globular head region of HA. To identify sites under positive selection; amino acid substitution classified depends on their radicalism and neutrality. The results indicate that, although most positions in HAs were under purifying selection and can be eliminated, a few positions located in the antigenic regions and receptor binding sites were subject to positive selection.

IgG subclass antibodies to three variants of Plasmodium falciparum merozoite surface protein-1 (PfMSP-1(19)) in an area with unstable malaria transmission in Iran.

Plasmodium falciparum remains globally an important cause of mortality and morbidity and despite decades of research, no effective vaccine is available against this deadly parasite. The 19-kDa C-terminal fragment of P. falciparum merozoite surface protein 1 (PfMSP-1(19)) is a target for protective immunity against malaria and the major concern in development of vaccine based on this antigen is the presence of polymorphisms. This investigation was designed to evaluate naturally acquired antibodies and antigen-binding avidity of IgG antibodies to three variant forms of PfMSP-1(19) antigen (E/TSG/L, E/KNG/F and Q/KNG/L) in malaria individuals who are living in hypoendemic areas in Iran (n=92, 4-75 years old). The three variant forms of PfMSP-1(19) were expressed in Escherichia coli and IgG isotype composition and avidity of naturally acquired antibodies to the 19-kDa antigen were measured by ELISA assay. Results showed that almost 72% of the studied individuals had positive antibody responses to three PfMSP-1(19) variants and the prevalence of responders did not differ significantly (P>0.05). High-avidity IgG (62.7%, 65.7% and 47.76%) and IgG1 (64.2%, 50.75%, and 50.75%) were found in positive sera for E/TSG/L, E/KNG/F and Q/KNG/L variants, respectively. Moreover, the prevalence and titers of IgG1 antibody responses to the three variants increased with age (P<0.05). In summary, individuals in low transmission areas in Iran can develop and maintain equal immune responses with high avidity to the PfMSP-1(19) variants (E/TSG/L, E/KNG/F and Q/KNG/L); however, the precise role of the total IgG and its isotypes in protection requires further investigation. These results could support the design of a universal PfMSP-1(19)-based vaccine.

Specific subtyping of influenza A virus using a recombinant hemagglutinin protein expressed in baculovirus.

Influenza A viruses are subtyped according to antigen characterization of hemagglutinin (HA) and neuraminidase surface glycoproteins. The hemagglutination inhibition (HI) assay using reference antiserum is currently applied to serologic screening of subtype-specific antibodies in sera. The reference antiserum is made by injecting chickens with live or inactivated whole virus preparations. Nonspecific inhibitors of antisera prepared by the conventional method may affect the specificity of HI assay. In this study, highly pure recombinant proteins generated using baculovirus expression vector system based on full-length of HA (HAF) and antigenic region of HA(1) genes of H9 subtype, and also inactivated whole virus were used to immunization of chickens. Measurable antibody titers were present for treated birds after 3 weeks and generally increased after each boost. The performance of the prepared antisera was evaluated by testing a panel of known standard strains of influenza virus representing five HA subtypes. Relative to the conventional method using whole virus immunization and recombinant HAF protein, the antiserum prepared by recombinant HA(1) had a specificity of 100% for all tested subtypes. The antiserum prepared by expression of HA1 protein in baculovirus has the potential for rapid and specific HA subtyping of influenza viruses without producing antibodies specific to other viral proteins.

Development of rHA1-ELISA for specific and sensitive detection of H9 subtype influenza virus.

A recombinant antigen-based single serum dilution ELISA was developed for simultaneous detection and subtyping of influenza viruses. Recombinant baculovirus encoding the hemagglutinin (HA(1) subunit) of H9N2 virus was generated. To evaluate the rHA1-ELISA, microplates were coated with purified HA1 protein and tested with reference control sera. Subsequently, 92 field sera collected from chickens suspected to be infected with H9N2 AIV were employed to test the efficacy of the rHA1-ELISA. The sera were tested simultaneously by HI and a commercial AIV ELISA kit. The rHA1-ELISA appeared to be highly specific and sensitive for direct detection of H9N2 antibodies in serum samples.

Leishmania major: disruption of signal peptidase type I and its consequences on survival, growth and infectivity.

Leishmania major (L. major) signal peptidase type I (SPase I) is an endopeptidase encoded by a single-copy gene. In all organisms, SPase I is responsible for removing the signal peptide from secretory pre-proteins and releasing mature proteins to cellular or extra-cellular space. In this study, the role of SPase I in L. major is investigated by gene deletion using homologous recombination (HR). The null mutant of SPase I was not possible to create, suggesting that SPase I is an essential gene for parasite survival. The obtained heterozygote mutant by disrupting one allele of SPase I in L. major showed significantly reduced level of infectivity in bone marrow-derived macrophages. In addition, the heterozygote mutants are unable to cause cutaneous lesion in susceptible BALB/c mice. This is the first report showing that SPase I may have an important role in Leishmania infectivity, e.g. in differentiation and survival of amastigotes. Apparently, the SPase I expression is not essential for in vitro growth of the parasite.

Ethnic and geographic differentiation of Helicobacter pylori within Iran.

The bacterium Helicobacter pylori colonizes the human stomach, with individual infections persisting for decades. The spread of the bacterium has been shown to reflect both ancient and recent human migrations. We have sequenced housekeeping genes from H. pylori isolated from 147 Iranians with well-characterized geographical and ethnic origins sampled throughout Iran and compared them with sequences from strains from other locations. H. pylori from Iran are similar to others isolated from Western Eurasia and can be placed in the previously described HpEurope population. Despite the location of Iran at the crossroads of Eurasia, we found no evidence that the region been a major source of ancestry for strains across the continent. On a smaller scale, we found genetic affinities between the H. pylori isolated from particular Iranian populations and strains from Turks, Uzbeks, Palestinians and Israelis, reflecting documented historical contacts over the past two thousand years.

IgG subclasses pattern and high-avidity antibody to the C-terminal region of merozoite surface protein 1 of Plasmodium vivax in an unstable hypoendemic region in Iran.

The C-terminal region of Plasmodium vivax merozoite surface protein 1 (PvMSP-1(19)) is a leading vaccine candidate for inclusion in a polyvalent malaria vaccine. In the present study, the IgG subclasses profile and the avidity of IgG to PvMSP-1(19) were evaluated in individuals (n=94) naturally exposed to P. vivax parasite in malaria endemic areas in Chabahar districts, Iran. In individuals with patent P. vivax malaria, 86.1% was sero-positive to PvMSP-1(19) and IgG1 (81.9%) was the predominant subclass. In addition, to determine the persistence of specific IgG, IgG1 and IgG3 antibodies to PvMSP-1(19), the frequency of antibodies was determined in the infected subjects (n=74) after treatment with standard chloroquine and it was detected that the frequency of responders was significantly reduced to 51.3%, 51% and 16.2%, respectively. The antigen-binding avidity of IgG antibodies to PvMSP-1(19) was measured in sero-positive sera and the high-avidity of IgG, IgG1 and IgG3 was found in 66.6%, 61% and 47% of the infected subjects with P. vivax, respectively. The present result shows that individuals who exposed to vivax malaria in the endemic region in Iran develop antibodies with high-avidity to PvMSP-1(19). These results could help to understand the interactions between the host and P. vivax parasite in development of MSP-1(19)-based vaccine.

Yeast of the oral cavity is the reservoir of Heliobacter pylori.

BACKGROUND: Frequent occurrence of Helicobacter pylori in the human gastrointestinal tract and its persistence due to unsuccessful antimicrobial therapy might be related to a stage in the life cycle of H. pylori in which the bacterium establishes itself as an intracellular symbiont in yeast. In this study, occurrence of non-culturable H. pylori in the oral yeast was assessed by targeting vacuolating cytotoxin A (vacA s1s2) and ureAB genes in the total DNAs of yeasts. METHODS: DNAs were extracted from 13 oral yeasts in which bacterium-like bodies, suspected to be H. pylori, were observed microscopically. Primers were recruited to amplify vacA s1s2 and ureAB genes. DNAs from H. pylori and E. coli were used as controls. The amplicons from one yeast and H. pylori were sequenced. Yeasts were identified as Candida albicans. RESULTS: Fragments of vacA s1s2 and ureAB genes were amplified from 13 yeasts. The size of PCR products was 286 bp for vacA s1s2 gene and 406 bp for ureAB gene. Similar bands were obtained from the control H. pylori, and the results for E. coli were negative. The data from sequencing of PCR products showed about 98% homology between the genes amplified from yeast and those from H. pylori. CONCLUSIONS: The results of this study showed the intracellular occurrence of H. pylori in yeast. This endosymbiotic relationship might explain the persistence of H. pylori in the oral cavity, the consequence of which could be reinoculation of the stomach by the bacterium and spread of infection among human populations.

Plasmodium falciparum: sequence analysis of the gene encoding the C-terminus region of the merozoite surface protein-1, a potential malaria vaccine antigen, in Iranian clinical isolates.

C-terminal region of merozoite surface protein-1 of Plasmodium falciparum (PfMSP-1) isolated from different parts of the world revealed sequence variability, however no data exist on sequence heterogeneity of this region from Iran. To address this question, DNA encoding the carboxyl (C)-terminal region of PfMSP-1 was amplified in 144 Iranian P. falciparum clinical isolates, using allele type-specific primers. In this study both MAD20 (88.2%) and K1 (7.6%) types were detected. Sequence analysis of 33 and 92 fragments corresponding to pfmsp-1(42) and pfmsp-1(19) revealed eight (15MAD1-15MAD7 and 15KCH) and five [A1 (E/TSR/L), A2 (Q/KNG/F), A3 (E/KNG/F), A4 (E/TSG/L), and A5 (Q/KNG/L)] distinct haplotypes, respectively. E/TSG/L variant type was the predominant haplotype, and reported only from Thailand and India, but E/KNG/L is widespread in Africa, Asia, and Latin America; but not found among Iranian isolates. In summary, result of this study indicates limited antigenic diversity, and thus support the potential utility of the C-terminal region of PfMSP-1 in designing polyvalent vaccine constructs.