Fragment Of Merozoite Surface Protein Research Articles

Conjugation strategies on functionalized iron oxide nanoparticles as a malaria vaccine delivery system

Vaccination has been used effectively to protect from infectious diseases and non-infectious diseases such as cancer and allergies. Different forms of particulate arrangements, including nanoparticles, virus-like particles (VLPs), and virosomes, have been built recently depending on the type of pathogen to be targeted. The ability to conjugate the recombinant Plasmodium yoelii, 19-kDa C-terminal fragment of merozoite surface protein 1 (PyMSP119) on the surface of superparamagnetic magnetite nanoparticles (SPIONs) was explored as a new technique of enhancing vaccination against malaria. Different conjugation strategies were performed to correlate the effects of nanoparticle chemistry surfaces to bind later with the malaria protein. (SPIONs) were prepared by chemical coprecipitation method and coated with 3-aminopropyltriethoxysilane (APTS) alone (as a surface coater), or with both APTS and polyethylene glycol (PEG) (as a shield to protect the malaria protein from proteolytic enzymes) by using a modified silanisation method. X-ray powder diffraction (XRD, Philips Model) patterns indicated that the SPIONs were of high purity with an inverse spinal structure. Fourier Transform Infrared Spectroscopy (FTIR) was collected using PerkinElmer Spectrum 100 Series; spectra of uncoated and coated magnetite nanoparticles confirmed that the silane layer had been coated on the surface Fe3O4. The SPIONs were superparamagnetic as investigated by Vibrating Sample Magnetometry (VSM, Princeton Applied Research, model ISS) and relatively stable in aqueous phase at room temperature and could also be quickly recovered from suspension using an external magnet. Introduce the carboxyl groups onto the SPIONs surfaces, resulting in a relatively high protein binding capacity onto the nanoparticle surfaces. The bare particles had a mean size of around 20 nm with a relatively narrow size distribution. 82% of African Green Monkey fibroblast (COS-7) were alive in nanoparticle suspension using the MTT assay method. The quantity of protein explicitly bound to particles was determined using Sodium Dodecyl Sulfate (SDS) - Polyacrylamide Gel Electrophoresis (PAGE). SDS–PAGE. When the conjugation blend was prepared in EDC, there was approximately 100% binding between PyMSP119 and the Fe3O4-COOH particles because no protein band was apparent at the expected molecular weight for PyMSP119 (45 kDa). The current study investigates the theory that the gradual, persistent release of the malaria antigen may stimulate and maintain an elevated level of immune response for an extended period in vivo, which will be the scope of future work.

Structural diversity, natural selection and intragenic recombination in the Plasmodium vivax merozoite surface protein 9 locus in Thailand

The merozoite surface protein 9 (MSP9) of malarial parasite forms co-ligand complex with the 19 kDa fragment of merozoite surface protein 1 (MSP1) prior to erythrocyte invasion. Interruption of this process could hamper subsequent asexual erythrocytic development of malaria parasites; therefore, these proteins are considered potential vaccine candidates. In Plasmodium vivax, MSP9 (PvMSP9) contains both conserved and polymorphic repetitive domains that were immunogenic upon natural malaria exposure and conferred protection in vaccination studies in animal models. To investigate the extent of sequence diversity at this locus, 104 P. vivax isolates from 4 major malaria endemic areas of Thailand were analyzed. Results revealed that pvmsp9 contained 3 repeat domains (R1–R3) flanked by conserved domains. Repeat domains exhibit extensive sequence and length variation, in which 14, 39 and 16 haplotypes for domains R1–R3, respectively, circulated in this country. Sequence diversity in pvmsp9 among P. vivax isolates from each endemic area displayed population structure. The extent of sequence diversity in pvmsp9 isolates from the provinces of Tak, Chanthaburi, Ubon Ratchathani and Prachuap Khiri Khan in northwestern, eastern, northeastern and southwestern areas, respectively, was almost comparable and was remarkably higher than that from Yala/Narathiwat population in southern Thailand. Evidence for intragenic recombination in this locus was observed within each P. vivax population except among isolates from Yala and Narathiwat. Synonymous nucleotide diversity significantly exceeded nonsynonymous nucleotide diversity in domains R2 and R3, indicating purifying selection. However, micro-scale signatures of positive and negative selections occurred in both conserved and repeat domains, implying two opposing forces, probably from functional or structural constraint and host immune pressure, could have influenced diversity at this locus. The immunodominant T and B cell epitopes so far identified were invariant or highly conserved across isolates. Further analysis of global isolates is warranted for vaccine design based on this protein.

Antibody responses within two leading Plasmodium vivax vaccine candidate antigens in three geographically diverse malaria-endemic regions of India

BackgroundIdentifying highly immunogenic blood stage antigens which can work as target for naturally acquired antibodies in different eco-epidemiological settings is an important step for designing malaria vaccine. Blood stage proteins of Plasmodium vivax, apical membrane antigen-1 (PvAMA-1) and 19 kDa fragment of merozoite surface protein (PvMSP-119) are such promising vaccine candidate antigens. This study determined the naturally-acquired antibody response to PvAMA-1 and PvMSP-119 antigens in individuals living in three geographically diverse malaria endemic regions of India.MethodsA total of 234 blood samples were collected from individuals living in three different eco-epidemiological settings, Chennai, Nadiad, and Rourkela of India. Indirect ELISA was performed to measure human IgG antibodies against recombinant PvAMA-1 and PvMSP-119 antigens. The difference in seroprevalence and factors associated with antibody responses at each site was statistically analysed.ResultsThe overall seroprevalence was 40.6% for PvAMA-1 and 62.4% for PvMSP-119. Seroprevalence to PvAMA-1 was higher in Chennai (47%) followed by Nadiad (46.7%) and Rourkela (27.6%). For PvMSP-119, seroprevalence was higher in Chennai (80.3%) as compared to Nadiad (53.3%) and Rourkela (57.9%). Seroprevalence for both the antigens were found to be higher in Chennai where P. vivax is the dominant malaria species. In addition, heterogeneous antibody response was observed for PvAMA-1 and PvMSP-119 antigens at each of the study sites. Two factors, age and malaria positivity were significantly associated with seropositivity for both the antigens PvAMA-1 and PvMSP-119.ConclusionThese data suggest that natural acquired antibody response is higher for PvMSP-119 antigen as compared to PvAMA-1 antigen in individuals living in three geographically diverse malaria endemic regions in India. PvMSP-119 appears to be highly immunogenic in Indian population and has great potential as a malaria vaccine candidate. The differences in immune response against vaccine candidate antigens in different endemic settings should be taken into account for development of asexual stage based P. vivax malaria vaccine, which in turn can enhance malaria control efforts.

Mosquito Bite-Induced Controlled Human Malaria Infection with Plasmodium vivax or P. falciparum Generates Immune Responses to Homologous and Heterologous Preerythrocytic and Erythrocytic Antigens.

Seroepidemiological studies on the prevalence of antibodies to malaria antigens are primarily conducted on individuals from regions of endemicity. It is therefore difficult to accurately correlate the antibody responses to the timing and number of prior malaria infections. This study was undertaken to assess the evolution of antibodies to the dominant surface antigens of Plasmodium vivax and P. falciparum following controlled human malaria infection (CHMI) in malaria-naive individuals. Serum samples from malaria-naive adults, collected before and after CHMI with either P. vivax (n = 18) or P. falciparum (n = 18), were tested for the presence of antibodies to the circumsporozoite protein (CSP) and the 42-kDa fragment of merozoite surface protein 1 (MSP-142) of P. vivax and P. falciparum using an enzyme-linked immunosorbent assay (ELISA). Approximately 1 month following CHMI with either P. vivax or P. falciparum, >60% of subjects seroconverted to homologous CSP and MSP-1. More than 50% of the subjects demonstrated reactivity to heterologous CSP and MSP-142, and a similar proportion of subjects remained seropositive to homologous MSP-142 >5 months after CHMI. Computational analysis provides insight into the presence of cross-reactive responses. The presence of long-lived and heterologous reactivity and its functional significance, if any, need to be taken into account while evaluating malaria exposure in field settings.

Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate.

Challenges with the production and suboptimal immunogenicity of malaria vaccine candidates have slowed the development of a Plasmodium falciparum multiantigen vaccine. Attempting to resolve these issues, we focused on the use of highly immunogenic merozoite surface protein 8 (MSP8) as a vaccine carrier protein. Previously, we showed that a genetic fusion of the C-terminal 19-kDa fragment of merozoite surface protein 1 (MSP119) to P. falciparum MSP8 (PfMSP8) facilitated antigen production and folding and the induction of neutralizing antibodies to conformational B cell epitopes of MSP119 Here, using the PfMSP1/8 construct, we further optimized the recombinant PfMSP8 (rPfMSP8) carrier by the introduction of two cysteine-to-serine substitutions (CΔS) to improve the yield of the monomeric product. We then sought to test the broad applicability of this approach using the transmission-blocking vaccine candidate Pfs25. The production of rPfs25-based vaccines has presented challenges. Antibodies directed against the four highly constrained epidermal growth factor (EGF)-like domains of Pfs25 block sexual-stage development in mosquitoes. The sequence encoding mature Pfs25 was codon harmonized for expression in Escherichia coli We produced a rPfs25-PfMSP8 fusion protein [rPfs25/8(CΔS)] as well as unfused, mature rPfs25. rPfs25 was purified with a modest yield but required the incorporation of refolding protocols to obtain a proper conformation. In comparison, chimeric rPfs25/8(CΔS) was expressed and easily purified, with the Pfs25 domain bearing the proper conformation without renaturation. Both antigens were immunogenic in rabbits, inducing IgG that bound native Pfs25 and exhibited potent transmission-reducing activity. These data further demonstrate the utility of PfMSP8 as a parasite-specific carrier protein to enhance the production of complex malaria vaccine targets.

A Plasmodium vivax Plasmid DNA- and Adenovirus-Vectored Malaria Vaccine Encoding Blood-Stage Antigens AMA1 and MSP142 in a Prime/Boost Heterologous Immunization Regimen Partially Protects Aotus Monkeys against Blood-Stage Challenge.

Malaria is caused by parasites of the genus Plasmodium, which are transmitted to humans by the bites of Anopheles mosquitoes. After the elimination of Plasmodium falciparum, it is predicted that Plasmodium vivax will remain an important cause of morbidity and mortality outside Africa, stressing the importance of developing a vaccine against P. vivax malaria. In this study, we assessed the immunogenicity and protective efficacy of two P. vivax antigens, apical membrane antigen 1 (AMA1) and the 42-kDa C-terminal fragment of merozoite surface protein 1 (MSP142) in a plasmid recombinant DNA prime/adenoviral (Ad) vector boost regimen in Aotus monkeys. Groups of 4 to 5 monkeys were immunized with plasmid DNA alone, Ad alone, prime/boost regimens with each antigen, prime/boost regimens with both antigens, and empty vector controls and then subjected to blood-stage challenge. The heterologous immunization regimen with the antigen pair was more protective than either antigen alone or both antigens delivered with a single vaccine platform, on the basis of their ability to induce the longest prepatent period and the longest time to the peak level of parasitemia, the lowest peak and mean levels of parasitemia, the smallest area under the parasitemia curve, and the highest self-cure rate. Overall, prechallenge MSP142 antibody titers strongly correlated with a decreased parasite burden. Nevertheless, a significant proportion of immunized animals developed anemia. In conclusion, the P. vivax plasmid DNA/Ad serotype 5 vaccine encoding blood-stage parasite antigens AMA1 and MSP142 in a heterologous prime/boost immunization regimen provided significant protection against blood-stage challenge in Aotus monkeys, indicating the suitability of these antigens and this regimen for further development.

Multiple Plasmodium falciparum Merozoite Surface Protein 1 Complexes Mediate Merozoite Binding to Human Erythrocytes

Successful invasion of human erythrocytes byPlasmodium falciparummerozoites is required for infection of the host and parasite survival. The early stages of invasion are mediated via merozoite surface proteins that interact with human erythrocytes. The nature of these interactions are currently not well understood, but it is known that merozoite surface protein 1 (MSP1) is critical for successful erythrocyte invasion. Here we show that the peripheral merozoite surface proteins MSP3, MSP6, MSPDBL1, MSPDBL2, and MSP7 bind directly to MSP1, but independently of each other, to form multiple forms of the MSP1 complex on the parasite surface. These complexes have overlapping functions that interact directly with human erythrocytes. We also show that targeting the p83 fragment of MSP1 using inhibitory antibodies inhibits all forms of MSP1 complexes and disrupts parasite growthin vitro.

Transdermal immunization of P. falciparum surface antigen (MSP-119) via elastic liposomes confers robust immunogenicity

abstractAs transdermal immunization results in poor immunogenicity, which is attributed to poor permeability of antigens through the skin, we believed ultradeformable lipid vesicles (elastic liposome) might address the challenges encountered during transdermal immunization. The elastic liposome, versatile carrier, proves better vehicle for transcutaneous delivery of protein, peptide and nucleic acid antigens. Our recently published article1 is suggestive of improved immunogenicity of carboxyl-terminal 19 kDa fragment of merozoite surface protein-1 (PfMSP-119) of Plasmodium falciparum when administered subcutaneously via elastic liposomes (Fig. 1).

Elastic liposome-mediated transdermal immunization enhanced the immunogenicity of P. falciparum surface antigen, MSP-119

Transdermal immunization results in poor immunogenicity, which can be attributed to poor permeability of antigens through the skin. Therefore, elastic liposome, ultradeformable lipid vesicles, may overcome the challenges faced during transdermal immunization. This versatile carrier proves better vehicle for transcutaneous delivery of protein, peptide and nucleic acid antigens. The present results are suggestive of improved immunogenicity of carboxyl-terminal 19kDa fragment of merozoite surface protein-1 (PfMSP-119) of Plasmodium falciparum when administered subcutaneously through elastic liposomes. The prepared elastic liposomes were characterized with respect to vesicles shape and surface morphology, size and size distribution, entrapment efficiency, elasticity, stability and in vitro release. Humoral and cell-mediated immune (CMI) response elicited by topically applied PfMSP-119-loaded elastic liposomes, intramuscularly administered alum-adsorbed PfMSP-119 solution, and topically applied PfMSP-119-loaded conventional liposomes were compared and normalized with vehicle control. Results suggest greater transcutaneous immunization via elastic liposomes, and induced robust and perdurable IgG-specific antibody and cytophilic isotype responses. We report to have achieved sizeable CMI activating factor (IFNγ), a crucial player in conferring resistance to asexual blood stage malaria, responses with elastic liposomes when compared with other formulations. The fluorescence microscopy and histopathology results are suggestive of prominent skin permeation and biodistribution, and demonstrate efficient delivery of malaria antigen via elastic liposomes to immunocompetent Langerhans cells (LC) and lymphatics. In conclusion, elastic liposomal formulation provided greater entrapment efficiency, enhanced penetration and heightened and long-lasting immune response. Moreover, effective immunoadjuvant property of this carrier justifies its potential for improved vaccine delivery, and opens new avenues to explore further on the development of malaria vaccine.

Malaria and HIV infection in Mozambican pregnant women are associated with reduced transfer of antimalarial antibodies to their newborns.

Malaria and human immunodeficiency virus (HIV) infection during pregnancy affect the transplacental transfer of antibodies against several pathogens from mother to fetus, although the effect of malaria and HIV infection on the transfer of antimalarial antibodies remains unclear. Levels of total immunoglobulin G (IgG), immunoglobulin M (IgM), and IgG subtypes against the following Plasmodium falciparum antigens were measured in 187 pairs of mother-cord plasma specimens from Mozambique: 19-kDa fragment of merozoite surface protein 1 (MSP119), erythrocyte binding antigen 175 (EBA175), apical membrane antigen 1 (AMA1), and parasite lysate. Placental antibody transfer was defined as the cord-to-mother ratio (CMR) of antibody levels. Maternal malaria was associated with reduced CMR of EBA175 IgG (P = .014) and IgG1 (P = .029), AMA1 IgG (P = .002), lysate IgG1 (P = .001), and MSP1 IgG3 (P = .01). Maternal HIV was associated with reduced CMR of MSP1 IgG1 (P = .022) and IgG3 (P = .023), lysate IgG1 (P = .027) and IgG3 (P = .025), AMA1 IgG1 (P = .001), and EBA175 IgG3 (P = .001). Decreased CMR was not associated with increased adverse pregnancy outcomes or augmented risk of malaria in the infant during the first year of life. Placental transfer of antimalarial antibodies is reduced in pregnant women with malaria and HIV infection. However, this decrease does not contribute to an increased risk of malaria-associated morbidity during infancy.

Cloning, overexpression and characterization of soluble 42 kDa fragment of merozoite surface protein-1 of Plasmodium vivax

Plasmodium vivax represents the second most prevalent malaria species of major public health importance and the global eradication of malaria requires the development of vaccines to prevent infection. The lack of in vitro culture and a suitable animal model for P. vivax malaria are the major problems for the delay in developing a functional vivax vaccine. A number of antigens have been identified for P. vivax as potential malaria vaccine candidates and among these 42kDa fragment of merozoite surface protein-1 (MSP-142) is one of most promising antigen of asexual blood stage. In most of the earlier studies, the MSP-142 of malaria parasites was expressed as insoluble protein in inclusion bodies and it is difficult to get purified protein in conformation form. In the present study, we have cloned, overexpressed and characterized the 42kDa fragment of P. vivax MSP-1 as soluble protein in Escherichiacoli. The 42kDa gene fragment of P. vivax MSP-1 was PCR amplified using specific primers, sequenced and subcloned into pTriEx-4 expression vector. The optimum expression of recombinant P. vivax protein was obtained in SOC growth medium by inducing with 0.2mM IPTG at 37°C for 4h. The SDS–PAGE analysis showed a fusion protein of 55kDa and about 80% was present in soluble form. The purified P. vivax MSP-142 was characterized and found to be correctly folded and in conformation form as evident by CD spectroscopy, presence of 1 free –SH group and the reactivity with reduction sensitive conformational monoclonals against P. vivax MSP-142.

The Merozoite Surface Protein 1 Complex Is a Platform for Binding to Human Erythrocytes by Plasmodium falciparum

Plasmodium falciparum is the causative agent of the most severe form of malaria in humans. The merozoite, an extracellular stage of the parasite lifecycle, invades erythrocytes in which they develop. The most abundant protein on the surface of merozoites is merozoite surface protein 1 (MSP1), which consists of four processed fragments. Studies indicate that MSP1 interacts with other peripheral merozoite surface proteins to form a large complex. Successful invasion of merozoites into host erythrocytes is dependent on this protein complex; however, the identity of all components and its function remain largely unknown. We have shown that the peripheral merozoite surface proteins MSPDBL1 and MSPDBL2 are part of the large MSP1 complex. Using surface plasmon resonance, we determined the binding affinities of MSPDBL1 and MSPDBL2 to MSP1 to be in the range of 2-4 × 10(-7) m. Both proteins bound to three of the four proteolytically cleaved fragments of MSP1 (p42, p38, and p83). In addition, MSPDBL1 and MSPDBL2, but not MSP1, bound directly to human erythrocytes. This demonstrates that the MSP1 complex acts as a platform for display of MSPDBL1 and MSPDBL2 on the merozoite surface for binding to receptors on the erythrocyte and invasion.

Cellular and humoral immune responses against the Plasmodium vivax MSP-119 malaria vaccine candidate in individuals living in an endemic area in north-eastern Amazon region of Brazil

BackgroundPlasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil.MethodsThe study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-119) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay.ResultsThe prevalence of activated CD4+ was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. A low proliferative response against PvMSP-119 and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-γ and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-119 stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient’s cells while low levels of IFN-γ and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-119 was evidenced, regardless class or IgG subclass.PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses.ConclusionsThe results presented here shows that PvMSP-119 was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-119 in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.

Production, characterisation and immunogenicity of a plant-made Plasmodium antigen—the 19 kDa C-terminal fragment of Plasmodium yoelii merozoite surface protein 1

Development of a safe, effective and affordable malaria vaccine is central to global disease control efforts. One of the most highly regarded proteins for inclusion in an asexual blood stage subunit vaccine is the 19-kDa C-terminal fragment of merozoite surface protein 1 (MSP1(19)). As production of vaccine antigens in plants can potentially overcome cost and delivery hurdles, we set out to produce MSP1(19) in plants, characterise the protein and test its immunogenicity using a mouse model. Plasmodium yoelii MSP1(19) (PyMSP1(19)) was produced in Nicotiana benthamiana using the MagnICON® deconstructed TMV-based viral vector. PyMSP1(19) yield of at least 23% total soluble protein (TSP;3-4 mg/g Fwt) were achieved using a codon-optimised construct that was targeted to the apoplast. Freeze-dried leaf powder contained at least 20 mg PyMSP1(19) per gram dry weight and the protein retained immunogenicity in this form for more than 2 years. Characterisation studies, including SDS-PAGE, mass spectrometry and circular dichroism, indicated that the plant-expressed PyMSP1(19) was similar to its Escherichia coli- and Saccharomyces cerevisiae-expressed counterparts. Purified plant-made PyMSP1(19) induced strong immune responses following intraperitoneal immunisation, although titres were lower than those induced by an equivalent dose of purified E. coli-expressed PyMSP1(19). The reason for this is uncertain but may be due to differences in the oligomerisation profile of the vaccines. The plant-made PyMSP1(19) vaccine was also found to be orally immunogenic when delivered alone or following immunisation with a PyMSP1(19) DNA vaccine. This study adds to an increasing body of research supporting the feasibility of plants as both a factory for the production of malaria antigens, and as a safe and affordable platform for oral delivery of a temperature-stable malaria vaccine.

Age-Dependent IgG Subclass Responses to Plasmodium falciparum EBA-175 Are Differentially Associated with Incidence of Malaria in Mozambican Children

Plasmodium falciparum blood-stage antigens such as merozoite surface protein 1 (MSP-1), apical membrane antigen 1 (AMA-1), and the 175-kDa erythrocyte binding antigen (EBA-175) are considered important targets of naturally acquired immunity to malaria. However, it is not clear whether antibodies to these antigens are effectors in protection against clinical disease or mere markers of exposure. In the context of a randomized, placebo-controlled trial of intermittent preventive treatment in infants conducted between 2002 and 2004, antibody responses to Plasmodium falciparum blood-stage antigens in a cohort of 302 Mozambican children were evaluated by immunofluorescence antibody test and enzyme-linked immunosorbent assay at 5, 9, 12, and 24 months of age. We found that IgG subclass responses to EBA-175 were differentially associated with the incidence of malaria in the follow-up period. A double amount of cytophilic IgG1 or IgG3 was associated with a significant decrease in the incidence of malaria (incidence rate ratio [IRR] = 0.49, 95% confidence interval [CI] = 0.25 to 0.97, and P = 0.026 and IRR = 0.44, CI = 0.19 to 0.98, and P = 0.037, respectively), while a double amount of noncytophilic IgG4 was significantly correlated with an increased incidence of malaria (IRR = 3.07, CI = 1.08 to 8.78, P = 0.020). No significant associations between antibodies to the 19-kDa fragment of MSP-1 (MSP-1(19)) or AMA-1 and incidence of malaria were found. Age, previous episodes of malaria, present infection, and neighborhood of residence were the main factors influencing levels of antibodies to all merozoite antigens. Deeper understanding of the acquisition of antibodies against vaccine target antigens in early infancy is crucial for the rational development and deployment of malaria control tools in this vulnerable population.

Age-Related Differences in Naturally Acquired T Cell Memory to Plasmodium falciparum Merozoite Surface Protein 1

Naturally acquired immunity to Plasmodium falciparum malaria in malaria holoendemic areas is characterized by the gradual, age-related development of protection against high-density parasitemia and clinical malaria. Animal studies, and less commonly, observations of humans with malaria, suggest that T-cell responses are important in the development and maintenance of this immunity, which is mediated primarily by antibodies that slow repeated cycles of merozoites through erythrocytes. To advance our rather limited knowledge on human T-cell immunity to blood stage malaria infection, we evaluated CD4 and CD8 T-cell effector memory subset responses to the 42 kDa C-terminal fragment of Merozoite Surface Protein 1 (MSP142), a malaria vaccine candidate, by 49 healthy 0.5 to ≥18 year old residents of a holoendemic area in western Kenya. The proportion of individuals with peripheral blood mononuclear cell MSP142 driven IFN-γ ELISPOT responses increased from 20% (2/20) among 0.5–1 year old children to 90% (9/10) of adults ≥18 years (P = 0.01); parallel increases in the magnitude of IFN-γ responses were observed across all age groups (0.5, 1, 2, 5 and ≥18 years, P = 0.001). Less than 1% of total CD4 and CD8 T-cells from both children and adults produced IFN-γ in response to MSP142. However, adults had higher proportions of MSP142 driven IFN-γ secreting CD4 and CD8 effector memory (CD45RA− CD62L−) T-cells than children (CD4: 50.9% vs. 28.8%, P = 0.036; CD8: 52.1% vs. 18.3%, respectively P = 0.009). In contrast, MSP142 driven IFN-γ secreting naïve-like, transitional (CD45RA+ CD62L+) CD4 and CD8 cells were the predominant T-cell subset among children with significantly fewer of these cells in adults (CD4: 34.9% vs. 5.1%, P = 0.002; CD8: 47.0% vs. 20.5%, respectively, P = 0.030). These data support the concept that meaningful age-related differences exist in the quality of T-cell immunity to malaria antigens such as MSP1.

Plasmodium falciparum Infection during Suppressive Prophylaxis with Mefloquine Does Not Induce an Antibody Response to Merozoite Surface Protein-1(42)

A sensitive biomarker of malaria infection would obviate the need for placebo control arms in clinical trials of malaria prophylactic drugs. Antibodies to the 42-kDa fragment of merozoite surface protein-1 (MSP1(42)) have been identified as a potential marker of malaria exposure in individuals receiving prophylaxis with mefloquine. We conducted an open-label trial to determine the sensitivity of seroconversion to MSP1(42), defined as a fourfold rise in enzyme-linked immunosorbant assay (ELISA) titer, among 23 malaria naïve volunteers receiving mefloquine prophylaxis and 6 controls after Plasmodium falciparum sporozoite challenge. All members of the control cohort but none of the mefloquine cohort developed patent parasitemia. Four of six controls but zero of the mefloquine cohort seroconverted to MSP1(42). We conclude that malaria infection during suppressive prophylaxis does not induce antibody response to the blood-stage antigen MSP1(42) in a malaria-naïve study population.

Superparamagnetic Nanoparticles for Effective Delivery of Malaria DNA Vaccine

Low efficiency is often observed in the delivery of DNA vaccines. The use of superparamagnetic nanoparticles (SPIONs) to deliver genes via magnetofection could improve transfection efficiency and target the vector to its desired locality. Here, magnetofection was used to enhance the delivery of a malaria DNA vaccine encoding Plasmodium yoelii merozoite surface protein MSP1(19) (VR1020-PyMSP1(19)) that plays a critical role in Plasmodium immunity. The plasmid DNA (pDNA) containing membrane associated 19-kDa carboxyl-terminal fragment of merozoite surface protein 1 (PyMSP1(19)) was conjugated with superparamagnetic nanoparticles coated with polyethyleneimine (PEI) polymer, with different molar ratio of PEI nitrogen to DNA phosphate. We reported the effects of SPIONs-PEI complexation pH values on the properties of the resulting particles, including their ability to condense DNA and the gene expression in vitro. By initially lowering the pH value of SPIONs-PEI complexes to 2.0, the size of the complexes decreased since PEI contained a large number of amino groups that became increasingly protonated under acidic condition, with the electrostatic repulsion inducing less aggregation. Further reaggregation was prevented when the pHs of the complexes were increased to 4.0 and 7.0, respectively, before DNA addition. SPIONs/PEI complexes at pH 4.0 showed better binding capability with PyMSP1(19) gene-containing pDNA than those at neutral pH, despite the negligible differences in the size and surface charge of the complexes. This study indicated that the ability to protect DNA molecules due to the structure of the polymer at acidic pH could help improve the transfection efficiency. The transfection efficiency of magnetic nanoparticle as carrier for malaria DNA vaccine in vitro into eukaryotic cells, as indicated via PyMSP1(19) expression, was significantly enhanced under the application of external magnetic field, while the cytotoxicity was comparable to the benchmark nonviral reagent (Lipofectamine 2000).

Polymorphism of the Pv200L Fragment of Merozoite Surface Protein-1 of Plasmodium vivax in Clinical Isolates from the Pacific Coast of Colombia

Merozoite surface protein 1 (MSP-1) is a polymorphic malaria protein with functional domains involved in parasite erythrocyte interaction. Plasmodium vivax MSP-1 has a fragment (Pv200L) that has been identified as a potential subunit vaccine because it is highly immunogenic and induces partial protection against infectious parasite challenge in vaccinated monkeys. To determine the extent of genetic polymorphism and its effect on the translated protein, we sequenced the Pv200L coding region from isolates of 26 P. vivax-infected patients in a malaria-endemic area of Colombia. The extent of nucleotide diversity (π) in these isolates (0.061 ± 0.004) was significantly lower (P ≤ 0.001) than that observed in Thai and Brazilian isolates; 0.083 ± 0.006 and 0.090 ± 0.006, respectively. We found two new alleles and several previously unidentified dimorphic substitutions and significant size polymorphism. The presence of highly conserved blocks in this fragment has important implications for the development of Pv200L as a subunit vaccine candidate.

Induction of humoral immune response against PfMSP-1 19 and PvMSP-1 19 using gold nanoparticles along with alum

The C-terminal 19 kDa fragment of merozoite surface protein 1(MSP-1 19) is an important vaccine candidate but a poor immunogen with available human compatible adjuvants like alum. We have investigated if Gold Nanoparticles (GNPs) based delivery system would enhance immune response to recombinant PfMSP-1 19 and PvMSP-1 19. We found that PfMSP-1 19/PvMSP-1 19 coated on GNPs or in alum was poorly immunogenic in mice, a robust antibody response was observed when PfMSP-1 19/PvMSP-1 19 coated GNPs were formulated with alum. PfMSP-1 19/PvMSP-1 19 coated GNPs strongly recognized conformation specific monoclonal antibodies showing that capping on to GNPs did not compromise with conformational integrity of these antigens, which is critical for the generation of functional antibody response. Anti-PfMSP-1 19 antibodies raised with GNP-alum formulation recognized the native protein on parasite surface and showed invasion inhibition of Plasmodium falciparum parasite in an in vitro assay. Antigen coated GNPs with alum may be useful in the development of an alternate adjuvant formulation.