Malaria Vaccine Candidate Antigen Research Articles

Antibodies against thrombospondin-related anonymous protein do not inhibit Plasmodium sporozoite infectivity in vivo.

Thrombospondin-related anonymous protein (TRAP), a candidate malaria vaccine antigen, is required for Plasmodium sporozoite gliding motility and cell invasion. For the first time, the ability of antibodies against TRAP to inhibit sporozoite infectivity in vivo is evaluated in detail. TRAP contains an A-domain, a well-characterized adhesive motif found in integrins. We modeled here a three-dimensional structure of the TRAP A-domain of Plasmodium yoelii and located regions surrounding the MIDAS (metal ion-dependent adhesion site), the presumed business end of the domain. Mice were immunized with constructs containing these A-domain regions but were not protected from sporozoite challenge. Furthermore, monoclonal and rabbit polyclonal antibodies against the A-domain, the conserved N terminus, and the repeat region of TRAP had no effect on the gliding motility or sporozoite infectivity to mice. TRAP is located in micronemes, secretory organelles of apicomplexan parasites. Accordingly, the antibodies tested here stained cytoplasmic TRAP brightly by immunofluorescence. However, very little TRAP could be detected on the surface of sporozoites. In contrast, a dramatic relocalization of TRAP onto the parasite surface occurred when sporozoites were treated with calcium ionophore. This likely mimics the release of TRAP from micronemes when a sporozoite contacts its target cell in vivo. Contact with hepatoma cells in culture also appeared to induce the release of TRAP onto the surface of sporozoites. If large amounts of TRAP are released in close proximity to its cellular receptor(s), effective competitive inhibition by antibodies may be difficult to achieve.

Assessing the binding of four Plasmodium falciparum T helper cell epitopes to HLA-DQ and induction of T-cell responses in HLA-DQ transgenic mice.

A subunit vaccine for Plasmodium falciparum malaria will need to contain well-defined T helper cell epitopes that induce protective immune responses to the parasite. One major barrier to the use of subunit vaccines is the requirement for T helper cell epitopes to be presented by the HLA class II molecules that are present in the population being vaccinated. Since the majority of malaria studies have focused on HLA-DR, little information on the role of HLA-DQ in the binding and immune response to malarial epitopes is available. This study used an in vitro peptide-binding assay to predict the extent of HLA-DQ binding of four conserved T helper cell epitopes identified from asexual-stage malaria vaccine candidate antigens. Epstein-Barr virus (EBV)-transformed human B-cell lines expressing 14 different DQ molecules (DQ2.1, -2.2, -4.1, -4.2, -5.1 to -5.3, -6.1, -6.2, -6.4, -7.1, -7.3, -8, and -9) representing all broad serological specificities, including common DQ molecules present in populations in areas where malaria is endemic, were used in the binding assay. Moreover, an HLA-DQ transgenic mouse model was employed to evaluate the correlation between the in vitro DQ binding of the peptides and the generation of in vivo immune responses following peptide immunization. This study identified two broad DQ-binding peptides, ABRA#14 and SERA#9. ABRA#14 also induced T-cell proliferation and Th1-associated cytokine production in DQ8(+) transgenic mice. The combination of peptide binding to EBV-transformed cell lines and DQ transgenic mice provides a method for identifying additional T-cell epitopes for inclusion in a vaccine.

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

The present study was designed to investigate the genetic diversity of Plasmodium falciparum among field isolates from India. A total of 71 clinical isolates were analyzed by the polymerase chain reaction (PCR) for the amplification of repeat regions of malaria vaccine candidate antigen genes, i.e., merozoite surface antigen-1 (MSA-1), MSA-2, and circumsporozoite protein (CSP). All three genes showed variation; MSA-2 has the maximum number of 10 variant forms while MSA-1 and CSP had 8 and 6 variants, respectively. Some variant forms were more common than others among the clinical isolates. There were mixed alleles for each gene in several (27 of 71) cases. The MSA-2 gene showed the maximum number of cases with mixed alleles (22 of 65 [33.85%]) compared with MSA-1 (10 of 68 [14.7%]) and CSP (10 of 65 [15.38%]). Fifty-five (88.7%) of 62 clinical isolates of P. falciparum showed a different genotype. The malaria hyperendemic region (Orissa) not only showed the maximum number of variant forms of each gene but also the maximum number of cases with mixed alleles compared with the non-hyperendemic regions (Madhya Pradesh and Rajasthan). The presence of such large numbers of P. falciparum strains in India should be taken into account in future malaria vaccine programs.

Levels of antibody to conserved parts of Plasmodium falciparum merozoite surface protein 1 in Ghanaian children are not associated with protection from clinical malaria.

The 19-kDa conserved C-terminal part of the Plasmodium falciparum merozoite surface protein 1 (PfMSP119) is a malaria vaccine candidate antigen, and human antibody responses to PfMSP119 have been associated with protection against clinical malaria. In this longitudinal study carried out in an area of stable but seasonal malaria transmission with an estimated parasite inoculation of about 20 infective bites/year, we monitored 266 3- to 15-year-old Ghanaian children clinically and parasitologically over a period of 18 months. Blood samples were collected at the beginning of the study before the major malaria season in April and after the season in November. Using enzyme-linked immunosorbent assay, we measured antibody responses to recombinant gluthathione S-transferase-PfMSP119 fusion proteins corresponding to the Wellcome and MAD20 allelic variants in these samples. Prevalence of antibodies recognizing the Wellcome 19 construct containing both epidermal growth factor (EGF)-like motifs in Wellcome type PfMSP119 was about 30%. Prevalence of antibodies to constructs containing only the first EGF domain from either Wellcome or MAD20 type PfMSP119 was about 15%, whereas antibodies recognizing a construct containing only the second EGF domain of MAD20 type PfMSP119 was found in only about 4% of the donors. Neither the prevalence nor the levels of any of the antibody specificities varied significantly with season, age, or sex. Significantly, and in contrast to previous reports from other parts of West Africa, we found no evidence of an association between antibody responses to PfMSP119 and clinical protection against malaria.

A longitudinal investigation of IgG and IgM antibody responses to the merozoite surface protein-1 19-kiloDalton domain of Plasmodium falciparum in pregnant women and infants: associations with febrile illness, parasitemia, and anemia.

This study was aimed at delineating characteristics of naturally acquired immunity against the merozoite surface antigen-1 (MSP-1) of Plasmodium falciparum, a candidate malaria vaccine antigen. A case/control study was performed on 75 case/control pairs of infants with febrile illness at the time of the first detected infection indicating a clinical case. The presence and level of antibodies at one month prior to the first infection and at the time of the first infection in the afebrile group was significantly higher than in the febrile group. Decreased parasite density and decreased infection-related loss of hemoglobin was seen in infants with anti-MSP-1(19kD) IgG antibodies. In addition, mothers who were positive for the presence of these antibodies conferred protection against placental infection and infection in their infants. In this study, development of anti-MSP-1(19kD) antibody responses in 24 infants were studied longitudinally using monthly serum samples collected from birth until approximately one year of age. In addition, umbilical cord blood sera and respective mothers' sera were analyzed. Longitudinal studies of antibody responses revealed several short-lived IgG and IgM peaks throughout an infant's first year that correlated with detection of parasitemia. The protection against parasitemia and febrile illness was observed in infants when anti-MSP-1(19kD) antibodies were present; when infants were negative for IgG, they had a 10-times greater risk of becoming parasitemic. These data from a longitudinal and prospective study of malaria suggest a protective role for anti-MSP-1(19kD) antibodies in infants and pregnant women.

Selection of an adjuvant for vaccination with the malaria antigen, MSA-2

Various formulations of the Plasmodium falciparum merozoite surface antigen, MSA-2, were made and tested in animals in order to select one for use in human vaccine trials. Recombinant constructs representing both major allelic forms of MSA-2 were formulated with a range of adjuvants and used to immunize rabbits, mice and sheep. After immunization, antibody responses obtained with the most potent adjuvants were at least tenfold greater than responses obtained with the least potent adjuvant Alhydrogel, which was used as the reference standard, although its lower potency indicated against its further use in clinical trials. Based on broadly similar results obtained with the three animal species, several adjuvants, including the water-in-oil adjuvant Montanide ISA 720, the oil-in-water adjuvant SAF-1, and liposomes containing lipid A formulated with Alhydrogel were demonstrated to be potent and potentially suitable for the clinical evaluation of MSA-2 as a candidate malaria vaccine antigen. Of these, ISA 720 was selected for further trial.

Re-investigation of the circumsporozoite protein-based induction of sterile immunity against Plasmodium berghei infection

Although the circumsporozoite protein (CSP) of the malaria parasite is the most immunologically characterized protein, the goal of using this protein in an effective vaccine has not yet been realized. Monoclonal antibody against the repetitive immunodominant B-epitope of the CSP can protect mice from malaria, but vaccines that induce antibody against this epitope do not consistently induce protection. Toward developing a rationale for a CSP-based effective vaccine, we have re-investigated the ability of anti-CSP repeat antibodies, as induced by different CSP vaccine formulations with several adjuvants, to confer sterile immunity against sporozoite challenge. Using Plasmodium berghei rodent malaria model and several CSP subunit vaccine constructs, we found that a formulation consisting of the P. berghei CSP repetitive epitope, (DPPPPNPN) 2 (CS), conjugated to BSA by carbodiimide, formulated in a block copolymer and detoxified lipopolysaccharide (RaLPS) adjuvant, was particularly promising. Mice were immunized and boosted with vaccines that contain varying malarial peptide-carrier ratios of 6:1 (CS 6-BSA), 55:1 (CS 55-BSA) and 170:1 (CS 170-BSA). Following immunization, the animals were challenged with live sporozoites. Two types of effects were observed in vaccinated mice. First, sterile immunity was induced in 100%, 50% and 29% of mice that were immunized with the CS 170-BSA, CS 55-BSA, and CS 6-BSA vaccine conjugates, respectively. The second effect of immunization was observed with the CS 170-BSA conjugate vaccine primed mice; a boost in IFA titers followed sporozoite challenge. In addition, we observed that IgG1 isotype titer against the surface of the sporozoite, as measured by IFA, and antibody avidity parallel sterile immunity. These findings reiterate the potential of the CSP as a malaria vaccine candidate antigen, and suggest that the induction of sterile immune responses depends on inducing antibody of the appropriate isotype, avidity and specificity.

Immunogenicity of recombinant Plasmodium falciparum rhoptry associated proteins 1 and 2.

Mice and rabbits immunized with recombinant forms of malaria vaccine candidate antigens rhoptry-associated proteins 1 and 2 (RAP-1, RAP-2 and rRAP-1, rRAP-2) produce antibodies at titres equivalent to monoclonal antibody ascites fluid raised against the native proteins. Sera from animals immunized with rRAP-1 contain antibodies which recognize the native protein by indirect immunofluorescence and immunoblotting, partially inhibit erythrocyte invasion in vitro and are long lasting. Epitope mapping shows these antibodies predominantly recognize epitopes in the N-terminal third of rRAP-1, some of which coincide with the targets of inhibitory monoclonal antibodies. By contrast, sera from animals immunized with rRAP-2 contain antibodies which recognize the recombinant but not the native protein.

Presentation of the Plasmodium falciparum antigen Pf155/RESA to human T cells. Variations in responsiveness induced by antigen presenting cells from different but MHC class II identical donors

The antibody response in humans naturally primed to a malaria vaccine candidate antigen (Pf155/RESA) is genetically regulated. Here, the impact of antigen presenting cells (APC) on the control of in vitro T-cell responses induced by Pf155/RESA or synthetic peptides corresponding to its major Pf155/RESA epitopes was studied. T cells and APC were from the peripheral blood of monozygotic or dizygotic twins and their age matched siblings, all living in the central highlands of Madagascar. When induced to proliferate (thymidine incorporation) in vitro by antigenic peptides, the T-cell responses varied less within the twin pairs than between them and their siblings or the entire group, implying that they were genetically regulated. Occasional MHC class II associations of some of the responses were weak and did not reflect underlying MHC class II restrictions. When T cells and APC from different but MHC class II identical donors were incubated in various combinations, antigen charged APC from homologous donors induced in vitro T-cell proliferation which differed from that induced by the T-cell donors' own APC. Pretreatment of the APC with either paraformaldehyde or anti-class II antibodies inhibited or abolished this antigen dependent T-cell proliferation. The results suggest that the observed differences in T-cell responses induced by APC from different donors reflect differences at the level of these cells. Whether they reflect differences in the proteases involved in antigen processing, in the costimulatory signals provided by the APC to the T cells or in the secretion of other regulatory factors remains to be elucidated.

Expression systems that best mimic native structure: which ones to try first and why.

Classic approaches to vaccine development based on killed or attenuated organisms cannot be used to develop a malaria vaccine due to the difficulty in producing sufficient parasites for large-scale vaccination and the biohazard risks associated with the use of organisms cultivated in human blood. Consequently, current malaria vaccine research has adopted biotechnological approaches to develop a vaccine based on specific malaria parasite antigens. Many different criteria have been applied to select candidate malaria vaccine antigens and several key observations have influenced this selection process. First, it is recognized that a degree of clinical immunity develops after repeated, natural infection with the malaria parasite. Second, it is possible to induce protective immunity against malaria infection under defined experimental circumstances. In the case of immunity to the sexual stages of the parasite, infection of animals and human volunteers with irradiated sporozoites confers a significant degree of immunity to sporozoite infection.

Use of novel adjuvants and delivery systems to improve the humoral and cellular immune response to malaria vaccine candidate antigens

The immune effector mechanisms responsible for the solid protection against malaria, as demonstrated by immunization with radiation-attenuated sporozoites, are poorly understood. An effective malaria vaccine must induce a well orchestrated combination of humoral and cellular immune responses directed against critical parasite antigens/epitopes expressed during different stages of the parasite's complicated life cycle. Currently licensed human vaccine adjuvants, such as alum, may improve antibody production but are poor stimulators of cellular effector mechanisms, while potent cellular stimulants such as Freund's adjuvant are too reactogenic for human use. Over the last 5 years we have systematically evaluated several methods of antigen presentation to include chemical conjugation to bacterial carrier proteins, emulsification in ‘Freund's-like’ preparations, and incorporation into liposomes. This work has resulted in the production of safe, potent vaccine delivery systems capable of targeting multiple antigenic determinants to the host's immune system. Further advances in malaria vaccine development now depend on the identification of appropriate parasite epitopes for inclusion in a multicomponent-multistage vaccine.

Association between immune recognition of the malaria vaccine candidate antigen Pf155/RESA and resistance to clinical disease: a prospective study in a malaria-endemic region of West Africa

We have measured cellular and humoral immune responses to short synthetic peptides representing epitopes of the malaria vaccine candidate antigen Pf155/RESA in a longitudinal, prospective study of clinical immunity to Plasmodium falciparum malaria in a cohort of 354 Gambian children aged 3–8 years. A significant association was observed between presence of antibodies to the 3′ repeat region peptide (EENV) 6 and resistance to clinical malaria. The prevalence of protective antipeptide antibodies varied significantly between different ethnic groups, suggesting that immune recognition of some Pf155/RESA epitopes may be genetically regulated. There was no obvious association between proliferative or interferon γ responses to T cell epitopes of Pf155/RESA and resistance to malaria infection or disease. At an individual level, the presence of peptide-binding antibodies was associated with the induction of interleukin 4 messenger ribonucleic acid expression in T cells activated with the overlapping T cell epitope EENVEHDA(EENV) 2. This suggests that measurement of interleukin 4 production by T cells may represent a functional assay for T helper activity.

Human response to a malaria vaccine candidate antigen

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.

Sequestrin, a CD36 recognition protein on Plasmodium falciparum malaria-infected erythrocytes identified by anti-idiotype antibodies.

The CD36 molecule expressed by human endothelial cells is a receptor for the adhesion of erythrocytes infected with the human malaria parasite Plasmodium falciparum. A CD36-specific monoclonal antibody, OKM8, inhibits the adhesion of malaria-infected erythrocytes (IRBC) to purified CD36 and cells expressing CD36. Monospecific polyclonal anti-idiotype (anti-Id) antibodies, raised against monoclonal antibody OKM8, expressed determinants molecularly mimicking the CD36 binding domain for the adhesion of IRBC. Purified rabbit anti-Id antibodies reacted with the surface of IRBC by immunofluorescence, directly supported the adhesion of wild-type P. falciparum malaria isolates, and inhibited IRBC cytoadherence to melanoma cells. An approximately 270-kDa protein was immunoprecipitated by the anti-Id antibodies from surface-labeled and metabolically labeled IRBC and was competitively inhibited by soluble CD36. These results support the hypothesis that CD36 is a receptor and the approximately 270-kDa protein, sequestrin, is a complementary ligand involved in the adhesion of IRBC to host-cell endothelium. Sequestrin is a candidate malaria vaccine antigen, and anti-Id antibodies that recognize this molecule may be useful for passive immunotherapy of cerebral and severe P. falciparum malaria.

Immunization of Owl Monkeys with a Combination of Plasmodium Falciparum Asexual Blood-Stage Synthetic Peptide Antigens

A mixture of 3 synthetic peptides (35.1, 55.1, and 83.1) corresponding to portions of the 35 kDa, 55 kDa, and 83 kDa proteins from the asexual blood stages of Plasmodium falciparum and a polymer of a syntheic peptide incorporating the 3 individual peptides (SPf66) were tested as candidate malaria vaccine antigens in Aotus nancymai. Monkeys were immunized with combinations of the 3 peptides from 2 separate sources (Centers for Disease Control [CDC], Atlanta, GA or Colombia) or with the synthetic polymer. Animals immunized with a combination of the 3 peptides from CDC had higher antibody titers to the 35.1 and 55.1 peptides than to the 83.1 peptide. Monkeys immunized with a combination of the 3 peptides produced in Colombia developed higher levels of antibody to the 55.1 than to the 83.1 and 35.1 peptides. Animals immunized with the polymer produced detectable antibodies to the 55.1 peptide alone. Following challenge with P. falciparum, no differences were observed between the 3 vaccine groups and 2 control groups with respect to the number of animals with parasitemias greater than or equal to 10%. The inconsistency of serologic response to all 3 peptides in these animals contrasted with previous trials performed in Colombia where the monkeys developed high antibody titers against the 3 peptides and were protected against the experimental infection.

Number of cells from Plasmodium falciparum-immune donors that produce gamma interferon in vitro in response to Pf155/RESA, a malaria vaccine candidate antigen

Secretion of gamma interferon (IFN-gamma) in response to stimulation of Plasmodium falciparum-primed T cells by specific antigens may be a useful indicator of cellular immunity to malaria. An enzyme-linked immunospot (ELISPOT) assay designed to detect IFN-gamma at the single-cell level was used to study IFN-gamma-producing cells from P. falciparum-primed donors from The Gambia after in vitro stimulation with various malarial antigens. IFN-gamma secreted into the culture supernatant was measured by conventional enzyme-linked immunosorbent assay (ELISA). There was a good correlation in individual donors between the level of IFN-gamma secreted into the culture supernatant and the number of IFN-gamma-secreting cells. However, the ELISPOT assay was apparently more sensitive in demonstrating low levels of IFN-gamma production than the ELISA was. Thus after stimulation with crude P. falciparum antigen from infected erythrocytes, 72% of the primed donors responded positively in the ELISPOT assay but only 55% responded positively in the ELISA. When stimulated with synthetic peptides representing immunodominant epitopes of the malarial antigen Pf155/RESA, a vaccine candidate, 31 to 55% responded in the ELISPOT assay and 21 to 36% responded in the ELISA. Unprimed Europeans did not respond positively to these antigens in either of the assays, and background in antigen-free controls was generally low. These results indicate that measurement of IFN-gamma by the ELISPOT assay or ELISA should have wide applications in large-scale epidemiological studies of malaria immunity. In addition, the ELISPOT assay makes it possible to analyze the T cells responding to malarial antigens in terms of both numbers and functional heterogeneity.

Characterisation of an inhibitory monoclonal antibody-defined epitope on a malaria vaccine candidate antigen

A monoclonal antibody that recognizes a recently characterised 45-kDa merozoite surface antigen of the human malaria parasite Plasmodium falciparum inhibits the growth of the asexual blood stages of the parasite in vitro. The corresponding epitope has been determined by testing the reactivity of the antibody with sequentially overlapping octapeptides. A synthetic peptide containing the epitope elicits antibodies that react with the native antigen. Epitope mapping in this manner is useful in the design of synthetic vaccines against malaria.

Differential non-Responsiveness in Humans of Candidate Plasmodium Falciparum Vaccine Antigens

Synthetic subunit vaccines to sporozoites, merozoites, and gametes are being developed for malaria. The vaccine strategy assumes that the population to be immunized will respond favorably to these vaccine antigens. Using sera of 35 adults and 50 children from the The Gambia, West Africa, where Plasmodium falciparum is highly endemic, we examined the humoral immune response to candidate malaria vaccine antigens from sporozoites, merozoites, and gametes. We observed widespread restricted immunogenicity to defined parasite antigens in children and adults. HLA typing of adult lymphocytes demonstrated a marked diversity in HLA haplotypes in this population. Our results and those from our studies in mice suggest that genetic factors may partly explain the immunological non-responsiveness. This may necessitate re-evaluation of the malaria vaccine strategy.

Limited immunological recognition of critical malaria vaccine candidate antigens.

Current vaccine development strategies for malaria depend on widespread immunological responsiveness to candidate antigens such as the zygote surface antigens and the sporozoite coat protein, the circumsporozoite (CS) protein. Since immunological responsiveness is controlled mainly by genes mapping within the major histocompatibility complex (MHC), the humoral immune response to the zygote surface antigens and the cytotoxic T lymphocyte (CTL) response to the CS protein were examined in MHC-disparate congenic mouse strains. Only two of six strains responded to the 230-kilodalton zygote surface antigen and another two strains responded to the 48/45-kilodalton surface antigen. From two mouse strains, expressing between them five different class I MHC molecules, there was recognition of only a single CTL epitope from the CS protein, which was from a polymorphic segment of the molecule. The restricted CTL response to this protein parallels the restricted antibody response to this protein observed in humans and mice. These findings suggest that subunit malaria vaccines now being developed may be ineffective.