Immunization of Aotus nancymai with recombinant C terminus of Plasmodium falciparum merozoite surface protein 1 in liposomes and alum adjuvant does not induce protection against a challenge infection.

Merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is an antimalarial vaccine candidate. The highly conserved 19-kDa C-terminal processing fragment of MSP-1 (MSP-1(19)) is of particular interest since it contains epitopes recognized by monoclonal antibodies which inhibit the invasion of erythrocytes in vitro. The presence of naturally acquired anti-MSP-1(19) antibodies in individuals exposed to malaria has been correlated with reduced morbidity, and immunization with an equivalent recombinant P. yoelii antigen induces substantial protection against this parasite in mice. We have expressed P. falciparum MSP-1(19) in Escherichia coli as a correctly folded protein and immunized Aotus nancymai monkeys by using the protein incorporated into liposomes and adsorbed to alum. After vaccination, the sera from these animals contained anti-MSP-1(19) antibodies, some of which competed for binding to MSP-1(19) with monoclonal antibodies that inhibit parasite invasion of erythrocytes in vitro. However, after challenge with either a homologous or a heterologous strain of parasite, all animals became parasitemic and required treatment. The immunization did not induce protection in this animal model.     

Immunogenicity and efficacy in aotus monkeys of four recombinant Plasmodium falciparum vaccines in multiple adjuvant formulations based on the 19-kilodalton C terminus of merozoite surface protein 1

The immunogenicity and protective efficacy of four versions of recombinant C-terminal 19-kDa epidermal growth factor-like region of the major surface protein 1 (rMSP1(19)) of Plasmodium falciparum was studied in Aotus monkeys. Vaccination with each of the four rMSP1(19) constructs elicited high levels of antibodies to MSP1(19) but only one construct, the 19-kDa fragment expressed as a secreted fusion protein from Saccharomyces cerevisiae (yP30P2MSP1(19)), induced a high degree of protective immunity in Aotus nancymai against lethal P. falciparum challenge. Protective formulation required Freund's adjuvant; vaccination with yP30P2MSP1(19) in six other adjuvants that are suitable for human use induced lower levels of antibody response and no protection. These results emphasize the need to continue the search for an adjuvant that is comparable to Freund's adjuvant in potency and is safe for use in humans.     

Immunological cross-reactivity of the C-terminal 42-kilodalton fragment of Plasmodium falciparum merozoite surface protein 1 expressed in baculovirus.

The roles of allelic and conserved epitopes in vaccine-induced immunity to the C-terminal 42-kDa fragment of the Plasmodium falciparum merozoite surface protein 1 (MSP1) were investigated. The C-terminal fragment of MSP1 was expressed as a baculovirus recombinant protein, BVp42. Rabbits were immunized with BVp42, and antibodies were tested for reactivity to MSP1s of the homologous and heterologous allelic forms, represented by the FUP, FVO, FC27, and Honduras parasite isolates, by enzyme-linked immunosorbent assay and indirect immunofluorescence antibody assay. Despite the fact that allelic sequences accounted for approximately 50% of the BVp42 molecule, anti-BVp42 antibodies cross-reacted extensively with parasites carrying heterologous MSP1 alleles. Enzyme-linked immunosorbent inhibition assays confirmed that an overwhelming majority of the anti-BVp42 antibodies were cross-reactive, suggesting that determinants within conserved block 17 are dominant B-cell epitopes in the anti-BVp42 response. Moreover, the BVp42 polypeptide could inhibit (> 90%) the cross-reactivity of anti-MSP1 antibodies in animals immunized with the complete native MSP1 protein. Anti-BVp42 antibodies were equally effective in inhibiting the in vitro growth of parasites carrying homologous or heterologous MSP1 alleles. Serotyping by monoclonal antibodies indicated that the immunological and biological cross-reactivities were not caused by identical variant-specific amino acid substitutions within conserved block 17. These results should provide the impetus to develop a vaccine based on the C-terminal conserved region(s) of MSP1 against parasites of diverse genetic makeup.     

Vaccine efficacy of recombinant Plasmodium falciparum merozoite surface protein 1 in malaria-naive, -exposed, and/or -rechallenged Aotus vociferans monkeys

Protection against a lethal challenge infection of Plasmodium falciparum was elicited in malaria-naive Aotus vociferans monkeys by vaccination with the C terminus 19-kDa protein of the major merozoite surface protein (MSP-1(19)) fused to tetanus toxoid universal T-cell epitopes P30 and P2. Three of four monkeys were protected against a 10(4)-parasite challenge. Four monkeys were challenged with 10(5) parasites; one self-cured the infection, two were protected against high parasitemia (<2%) but were treated for severe anemia (hematocrit of <25%), and the fourth was not protected. In this model system, anemia appears to be a manifestation of incomplete protection (prolonged low-level parasitemia). Enzyme-linked immunosorbent assay (ELISA) antibody titers correlated with protection. Antibodies from some protected monkeys inhibited secondary processing of MSP-1(42) to MSP-1(33) and MSP-1(19). To mimic the repeated reinfections seen in regions where malaria is endemic, a second malaria parasite challenge was administered 4 months later. All P30P2MSP-1(19)-vaccinated monkeys were protected; thus, a single challenge infection may underestimate vaccine efficacy. ELISA antibody titers correlated with protection against a second infection but had decreased compared to the first challenge. As most target populations for asexual blood-stage malaria vaccines will have been exposed to malaria parasites, a malaria parasite-exposed monkey was vaccinated with P30P2MSP-1(19). This monkey was completely protected, while a malaria parasite-naive P30P2MSP-1(19)-vaccinated monkey self-cured a low-grade parasitemia. Prior malaria parasite infection primed the production of anti-native MSP-1(19) antibodies, which were boosted by vaccination with recombinant P30P2MSP-1(19). Preliminary data suggest that immunogenicity studies of vaccines designed for malaria parasite-exposed populations should also be conducted in malaria parasite-exposed subjects.     

Protective immunity induced in Aotus monkeys by a recombinant SERA protein of Plasmodium falciparum: further studies using SERA 1 and MF75.2 adjuvant.

We describe the third of three vaccination trials of Panamanian Aotus monkeys with a recombinant blood-stage antigen derived from the malaria parasite Plasmodium falciparum. Immunization was performed with an N-terminal region of the SERA antigen (serine repeat antigen protein), SERA 1, that contains a 262-amino-acid fragment including amino acids 24 to 285 of the 989-amino-acid SERA protein. Vaccinations were carried out with the recombinant protein mixed with either Freund's, MF75.2, or MF59.2 adjuvant. A control group that did not receive SERA 1 but only MF75.2 adjuvant was included. Monkeys vaccinated with the antigen MF59.2 mixture produced low anti-SERA 1 titers and were not protected. Monkeys vaccinated with antigen and Freund's adjuvant had, in general, a higher average anti-SERA 1 titer (107,278) than did monkeys immunized with SERA 1 and MF75.2 (40, 143), yet monkeys in both groups were well protected. Monkeys that received only MF75.2 developed neither detectable anti-SERA 1 nor anti-P. falciparum antibodies prior to or 10 days after parasite challenge, yet were apparently protected against infection. Monkeys vaccinated with either SERA 1 and Freund's, SERA 1 and MF75.2, or MF75.2 alone and that had been challenged but did not develop a countable parasitemia were treated with a curative dose of mefloquine 100 days after parasite challenge and then rechallenged 40 days later. None of the five rechallenged monkeys that had originally received SERA 1 and Freund's developed a countable parasitemia. Only one of five rechallenged monkeys that originally received SERA 1 and MF75.2 developed a high countable parasitemia, while two animals developed a barely countable parasitemia. Four of the rechallenged monkeys that had originally received only MF75.2 developed a moderate to high countable parasitemia. The results indicate that vaccination with SERA 1 and either Freund's or MF75.2 adjuvant provides protection and vaccination with MF75.2 alone can provide a temporary protection unrelated to the induction of anti-SERA 1 or antimalarial antibodies.     

Protection against malaria in Aotus monkeys immunized with a recombinant blood-stage antigen fused to a universal T-cell epitope: correlation of serum gamma interferon levels with protection.

The major surface antigen p190 of the human malaria parasite Plasmodium falciparum contains nonpolymorphic, immunogenic stretches of amino acids which are attractive components for a subunit vaccine against malaria. One such polypeptide, termed 190L, is contained in the 80-kDa processing product of p190, which constitutes the major coat component of mature merozoites. We report here that immunization of Aotus monkeys with 190L gives only poor protection against P. falciparum challenge. However, addition by genetic engineering of a universal T-cell epitope (CS.T3) to 190L improved immunity, and as a result three of four monkeys were protected following challenge infection with blood-stage parasites. Neither antibody against the immunizing antigens or against blood-stage parasites nor the capacity of the monkeys' sera to inhibit in vitro parasite invasion correlated with protection. However, in contrast to sera from nonprotected monkeys, sera from protected animals contained elevated levels of gamma interferon. These results suggest that gamma interferon is directly or indirectly involved in the process of asexual parasite control in vivo.     

Analysis of antibodies directed against merozoite surface protein 1 of the human malaria parasite Plasmodium falciparum

The 190-kDa merozoite surface protein 1 (MSP-1) of Plasmodium falciparum, an essential component in the parasite's life cycle, is a primary candidate for a malaria vaccine. Rabbit antibodies elicited by the heterologously produced MSP-1 processing products p83, p30, p38, and p42, derived from strain 3D7, were analyzed for the potential to inhibit in vitro erythrocyte invasion by the parasite and parasite growth. Our data show that (i) epitopes recognized by antibodies, which inhibit parasite replication, are distributed throughout the entire MSP-1 molecule; (ii) when combined, antibodies specific for different regions of MSP-1 inhibit in a strictly additive manner; (iii) anti-MSP-1 antibodies interfere with erythrocyte invasion as well as with the intraerythrocytic growth of the parasite; and (iv) antibodies raised against MSP-1 of strain 3D7 strongly cross-inhibit replication of the heterologous strain FCB-1. Accordingly, anti-MSP-1 antibodies appear to be capable of interfering with parasite multiplication at more than one level. Since the overall immunogenicity profile of MSP-1 in rabbits closely resembles that found in sera of Aotus monkeys immunized with parasite-derived MSP-1 and of humans semi-immune to malaria from whom highly inhibiting antigen-specific antibodies were recovered, we consider the findings reported here to be relevant for the development of MSP-1-based vaccines against malaria.     

Biochemical and immunological characterization of bacterially expressed and refolded Plasmodium falciparum 42-kilodalton C-terminal merozoite surface protein 1

Protection against Plasmodium falciparum can be induced by vaccination in animal models with merozoite surface protein 1 (MSP1), which makes this protein an attractive vaccine candidate for malaria. In an attempt to produce a product that is easily scaleable and inexpensive, we expressed the C-terminal 42 kDa of MSP1 (MSP1(42)) in Escherichia coli, refolded the protein to its native form from insoluble inclusion bodies, and tested its ability to elicit antibodies with in vitro and in vivo activities. Biochemical, biophysical, and immunological characterization confirmed that refolded E. coli MSP1(42) was homogeneous and highly immunogenic. In a formulation suitable for human use, rabbit antibodies were raised against refolded E. coli MSP1(42) and tested in vitro in a P. falciparum growth invasion assay. The antibodies inhibited the growth of parasites expressing either homologous or heterologous forms of P. falciparum MSP1(42). However, the inhibitory activity was primarily a consequence of antibodies directed against the C- terminal 19 kDa of MSP1 (MSP1(19)). Vaccination of nonhuman primates with E. coli MSP1(42) in Freund's adjuvant protected six of seven Aotus monkeys from virulent infection with P. falciparum. The protection correlated with antibody-dependent mechanisms. Thus, this new construct, E. coli MSP1(42), is a viable candidate for human vaccine trials.     

Development and pre-clinical analysis of a Plasmodium falciparum Merozoite Surface Protein-1(42) malaria vaccine

Merozoite Surface Protein-1(42) (MSP-1(42)) is a leading vaccine candidate against erythrocytic malaria parasites. We cloned and expressed Plasmodium falciparum MSP-1(42) (3D7 clone) in Escherichia coli. The antigen was purified to greater than 95% homogeneity by using nickel-, Q- and carboxy-methyl (CM)-substituted resins. The final product, designated Falciparum Merozoite Protein-1 (FMP1), had endotoxin levels significantly lower than FDA standards. It was structurally correct based on binding conformation-dependent mAbs, and was stable. Functional antibodies from rabbits vaccinated with FMP1 in Freund's adjuvant inhibited parasite growth in vitro and also inhibited secondary processing of MSP-1(42). FMP1 formulated with GlaxoSmithKline Biologicals (GSK) adjuvant, AS02A or alum was safe and immunogenic in rhesus (Macaca mulatta) monkeys.     

Efficacy of two alternate vaccines based on Plasmodium falciparum merozoite surface protein 1 in an Aotus challenge trial

In an attempt to produce a more defined, clinical-grade version of a vaccine based on Plasmodium falciparum merozoite surface protein 1 (MSP1), we evaluated the efficacy of two recombinant forms of MSP1 in an Aotus nancymai challenge model system. One recombinant vaccine, bvMSP1(42), based on the 42-kDa C-terminal portion of MSP1, was expressed as a secreted protein in baculovirus-infected insect cells. A highly pure baculovirus product could be reproducibly expressed and purified at yields in excess of 8 mg of pure protein per liter of culture. This protein, when tested for efficacy in the Aotus challenge model, gave significant protection, with only one of seven monkeys requiring treatment for uncontrolled parasitemia after challenge with P. falciparum. The second recombinant protein, P30P2MSP1(19), has been used in previous studies and is based on the smaller, C-terminal 19-kDa portion of MSP1 expressed in Saccharomyces cerevisiae. Substantial changes were made in its production process to optimize expression. The optimum form of this vaccine antigen (as judged by in vitro and in vivo indicators) was then evaluated, along with bvMSP1(42), for efficacy in the A. nancymai system. The new formulation of P30P3MSP1(19) performed significantly worse than bvMSP1(42) and appeared to be less efficacious than we have found in the past, with four of seven monkeys in the vaccinated group requiring treatment for uncontrolled parasitemia. With both antigens, protection was seen only when high antibody levels were obtained by formulation of the vaccines in Freund's adjuvant. Vaccine formulation in an alternate adjuvant, MF59, resulted in significantly lower antibody titers and no protection.     

Protective immunity induced in Aotus monkeys by a recombinant SERA protein of Plasmodium falciparum: adjuvant effects on induction of protective immunity.

We report the results of vaccination trial 2 of Panamanian Aotus monkeys with a recombinant blood-stage antigen, SERA 1, of the malaria parasite Plasmodium falciparum. Monkeys were immunized with SERA 1, a 262-amino-acid fragment (amino acids 24 to 285) of the 989-amino-acid SERA protein produced by the Honduras 1 strain of the parasite. Immunization mixtures contained 100 micrograms of recombinant SERA 1 protein per dose mixed with one of five different adjuvants. The protein mixed with either Freund's adjuvant or MF75.2 adjuvant stimulated protective immunity. When other P. falciparum antigens were included in the SERA 1-Freund's adjuvant mixture, no protective immunity was observed, although high anti-SERA 1 antibody titers were produced. Three other adjuvants mixed with SERA 1 failed to induce a protective immune response. These results, their relationship to those reported previously in the first vaccination trial (trial 1), and their relationships to the quantitative measurement of anti-SERA 1 antibodies in enzyme-linked immunosorbent assays provided insights into the induction of a protective immune response in vaccinated monkeys.     

Enhanced protection against malaria by a chimeric merozoite surface protein vaccine

The 42-kDa processed fragment of Plasmodium falciparum merozoite surface protein 1 (MSP-1(42)) is a prime candidate for a blood-stage malaria vaccine. Merozoite surface protein 8 contains two C-terminal epidermal growth factor (EGF)-like domains that may function similarly to those of MSP-1(42). Immunization with either MSP-1 or MSP-8 induces protection that is mediated primarily by antibodies against conformation-dependent epitopes. In a series of comparative immunogenicity and efficacy studies using the Plasmodium yoelii rodent model, we tested the ability of recombinant P. yoelii MSP-8 (rPyMSP-8) to complement rPyMSP-1-based vaccines. Unlike MSP-1, PyMSP-8-dependent protection required immunization with the full-length protein and was not induced with recombinant antigens that contained only the C-terminal EGF-like domains. Unlike PyMSP-8, the immunogenicity of the PyMSP-1 EGF-like domains was low when present as part of the rPyMSP-1(42) antigen. Immunization with a mixture of rPyMSP-1(42) and rPyMSP-8 further inhibited the antibody response to protective epitopes of rPyMSP-1(42) and did not improve vaccine efficacy. To improve PyMSP-1 immunogenicity, we produced a chimeric antigen containing the EGF-like domains of PyMSP-1 fused to the N terminus of PyMSP-8. Immunization with the chimeric rPyMSP-1/8 antigen induced high and comparable antibody responses against the EGF-like domains of both PyMSP-1 and PyMSP-8. This enhanced MSP-1-specific antibody response and the concurrent targeting of MSP-1 and MSP-8 resulted in improved, nearly complete protection against lethal P. yoelii 17XL malaria. Unexpectedly, immunization with rPyMSP-1/8 failed to protect against challenge infection with reticulocyte-restricted P. yoelii 17X parasites. Overall, these data establish an effective strategy to improve the efficacy of P. falciparum MSP-based vaccines.     

Protection against Plasmodium chabaudi malaria induced by immunization with apical membrane antigen 1 and merozoite surface protein 1 in the absence of gamma interferon or interleukin-4

Strategies to optimize formulations of multisubunit malaria vaccines require a basic knowledge of underlying protective immune mechanisms induced by each vaccine component. In the present study, we evaluated the contribution of antibody-mediated and cell-mediated immune mechanisms to the protection induced by immunization with two blood-stage malaria vaccine candidate antigens, apical membrane antigen 1 (AMA-1) and merozoite surface protein 1 (MSP-1). Immunologically intact or selected immunologic knockout mice were immunized with purified recombinant Plasmodium chabaudi AMA-1 (PcAMA-1) and/or the 42-kDa C-terminal processing fragment of P. chabaudi MSP-1 (MSP-1(42)). The efficacy of immunization in each animal model was measured as protection against blood-stage P. chabaudi malaria. Immunization of B-cell-deficient JH(-/-) mice indicated that PcAMA-1 vaccine-induced immunity is largely antibody dependent. In contrast, JH(-/-) mice immunized with PcMSP-1(42) were partially protected against P. chabaudi malaria, indicating a role for protective antibody-dependent and antibody-independent mechanisms of immunity. The involvement of gammadelta T cells in vaccine-induced PcAMA-1 and/or PcMSP-1(42) protection was minor. Analysis of the isotypic profile of antigen-specific antibodies induced by immunization of immunologically intact mice revealed a dominant IgG1 response. However, neither interleukin-4 and the production of IgG1 antibodies nor gamma interferon and the production of IgG2a/c antibodies were essential for PcAMA-1 and/or PcMSP-1(42) vaccine-induced protection. Therefore, for protective antibody-mediated immunity, vaccine adjuvants and delivery systems for AMA-1- and MSP-1-based vaccines can be selected for their ability to maximize responses irrespective of IgG isotype or any Th1 versus Th2 bias in the CD4(+)-T-cell response.     

In vivo expression and immunological studies of the 42-kilodalton carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 in the baculovirus-silkworm system

The 42-kDa carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 (MSP-1(42)) is an anti-erythrocytic stage malaria vaccine candidate. In this study, MSP-1(42) was expressed by using the Bombyx mori nuclear polyhedrosis virus-silkworm expression system, and the antigenicity and immmunogenicity of the recombinant protein, Bmp42, were evaluated. The average yield of Bmp42, as determined by a sandwich enzyme-linked immunosorbent assay (ELISA), was 379 microg/ml of infected silkworm hemolymph, which was >100-fold higher than the level attainable in cell culture medium. N-terminal amino acid sequencing revealed that Bmp42 was correctly processed in silkworm cells. Data from immunoblotting, as well as from the inhibition ELISA, suggested that the conformational B-cell epitopes of MSP-1(42) were recreated in Bmp42. Immunization of rabbits with Bmp42 in complete Freund's adjuvant generated high-titer antibody responses against the immunogen. Specificity analyses of the anti-Bmp42 antibodies using several recombinant MSP-1(19) proteins expressing variant and conserved B-cell epitopes suggested that the anti-Bmp42 antibodies recognized primarily conserved epitopes on MSP-1(19). Furthermore, the anti-Bmp42 antibodies were highly effective in inhibiting the in vitro growth of parasites carrying homologous or heterologous MSP-1(42). Our results demonstrated that the baculovirus-silkworm expression system could be employed to express biologically and immunologically active recombinant MSP-1(42) at elevated levels; thus, it is an attractive alternative for producing a protective MSP-1(42) vaccine for human use.     

A recombinant 15-kilodalton carboxyl-terminal fragment of Plasmodium yoelii yoelii 17XL merozoite surface protein 1 induces a protective immune response in mice.

Since the developmental stages of malarial parasites which replicate within erythrocytes are responsible for the morbidity and mortality associated with this disease, antigens produced by these stages have been proposed as candidates for a vaccine. One surface protein of merozoites (MSP-1) has been shown to immunize both rodents and primates against virulent challenge infection in experimental systems. However, little is known of relevant epitopes on the molecule, and attempts to obtain recombinant MSP-1 polypeptides in a native configuration have proven difficult. We have found that the cysteine-rich, carboxyl-terminal region of the MSP-1 protein from the rodent malarial parasite Plasmodium yoelii yoelii can be expressed in a native configuration as a fusion protein in Escherichia coli. This recombinant polypeptide containing 15 kDa of the predicted 197-kDa protein elicits antibodies in mice which recognize the native parasite MSP-1. Most significantly, both inbred and outbred mice immunized with the fusion protein in Ribi adjuvant are partially and in some cases completely protected against challenge infection with an otherwise lethal parasite strain. This is the first observation of such significant protection obtained with a small portion of the MSP-1 produced in recombinant systems.     

The clinical-grade 42-kilodalton fragment of merozoite surface protein 1 of Plasmodium falciparum strain FVO expressed in Escherichia coli protects Aotus nancymai against challenge with homologous erythrocytic-stage parasites

A 42-kDa fragment from the C terminus of major merozoite surface protein 1 (MSP1) is among the leading malaria vaccine candidates that target infection by asexual erythrocytic-stage malaria parasites. The MSP1(42) gene fragment from the Vietnam-Oak Knoll (FVO) strain of Plasmodium falciparum was expressed as a soluble protein in Escherichia coli and purified according to good manufacturing practices. This clinical-grade recombinant protein retained some important elements of correct structure, as it was reactive with several functional, conformation-dependent monoclonal antibodies raised against P. falciparum malaria parasites, it induced antibodies (Abs) that were reactive to parasites in immunofluorescent Ab tests, and it induced strong growth and invasion inhibitory antisera in New Zealand White rabbits. The antigen quality was further evaluated by vaccinating Aotus nancymai monkeys and challenging them with homologous P. falciparum FVO erythrocytic-stage malaria parasites. The trial included two control groups, one vaccinated with the sexual-stage-specific antigen of Plasmodium vivax, Pvs25, as a negative control, and the other vaccinated with baculovirus-expressed MSP1(42) (FVO) as a positive control. Enzyme-linked immunosorbent assay (ELISA) Ab titers induced by E. coli MSP1(42) were significantly higher than those induced by the baculovirus-expressed antigen. None of the six monkeys that were vaccinated with the E. coli MSP1(42) antigen required treatment for uncontrolled parasitemia, but two required treatment for anemia. Protective immunity in these monkeys correlated with the ELISA Ab titer against the p19 fragment and the epidermal growth factor (EGF)-like domain 2 fragment of MSP1(42), but not the MSP1(42) protein itself or the EGF-like domain 1 fragment. Soluble MSP1(42) (FVO) expressed in E. coli offers excellent promise as a component of a vaccine against erythrocytic-stage falciparum malaria.     

Plasmodium falciparum: protein antigens identified by analysis of serum samples from vaccinated Aotus monkeys.

Serum samples from Aotus trivirgatus subsp. griseimembra monkeys obtained at different stages of a vaccination experiment were analyzed for total antibody titer to Plasmodium falciparum and were used for identifying protective antigens of the human malaria parasite. Total malarial antibody titers were higher in serum samples from protected monkeys (vaccinated with antigen in an adjuvant) than in those from unprotected monkeys (vaccinated with either antigen or adjuvant only). Parasite proteins were labeled with [3H]isoleucine, solubilized with nonionic detergent, and reacted with immune Aotus sera. Immunoprecipitates obtained were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Thirteen protein antigen bands in the molecular weight range 73,000 to 180,000 were resolved. Serum samples obtained from protected Aotus monkeys reacted more intensely with these proteins than samples from unprotected monkeys did. Evidence is presented that the protective antigen is not a single, normally nonimmunogenic, protein that is recognized only in protected monkeys. Rather, the present data indicate that a heightened immune response to multiple proteins correlated with in vivo protection to P. falciparum in Aotus monkeys. This finding may have a significant bearing on strategies for the development of a human P. falciparum vaccine.     

Comparison of protection induced by immunization with recombinant proteins from different regions of merozoite surface protein 1 of Plasmodium yoelii.

Vaccination with native full-length merozoite surface protein 1 (MSP1) or with recombinant C-terminal peptides protects mice against lethal challenge with virulent malaria parasites. To determine whether other regions of MSP1 can also induce protection, Plasmodium yoelii MSP1 was divided into four separate regions. Each was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The N-terminal fragment began after the cleavage site for the signal sequence and ended in the region comparable to the cleavage site for the C terminus of the 82-kDa peptide of Plasmodium falciparum. This expressed protein was 30 kDa smaller than the predicted peptide. One peptide from the middle region was produced, and the C terminus consisted of a 42-kDa fragment corresponding to the analogous peptide of P. falciparum and a 19-kDa fragment that extended 37 amino acids in the amino-terminal direction beyond the probable cleavage site. To test protection of mice against lethal P. yoelii challenge, three mouse strains (CAF1, BALB/c, and A/J) were vaccinated with each of the four recombinant proteins of MSP1. Mice vaccinated with the C-terminal 19-kDa protein were highly protected (described previously), as were those vaccinated with the 42-kDa protein that contained the 19-kDa fragment. The N-terminally expressed fragment of P. yoelii was not full length because of proteolytic cleavage in E. coli. The GST-82-kDa partial fragments induced some immunity, but the surviving mice still had high parasitemias. Vaccination with the peptide from the middle region of MSP1 gave minimal to no protection. Therefore, in addition to the C-terminal 19- and 42-kDa proteins, the only other fragment to give protection was the 82-kDa protein. The protection induced by the truncated 82-kDa protein was minimal compared with that of the C-terminal fragments.     

Plasmodium falciparum merozoite surface protein 8 is a ring-stage membrane protein that localizes to the parasitophorous vacuole of infected erythrocytes

To date, the following seven glycosylphosphatidylinositol (GPI)-anchored merozoite antigens have been described in Plasmodium falciparum: merozoite-associated surface protein 1 (MSP-1), MSP-2, MSP-4, MSP-5, MSP-8, MSP-10, and the rhoptry-associated membrane antigen. Of these, MSP-1, MSP-8, and MSP-10 possess a double epidermal growth factor (EGF)-like domain at the C terminus, and these modules are considered potential targets of protective immunity. In this study, we found that surprisingly, P. falciparum MSP-8 is transcribed and translated in the ring stage and is absent from the surface of merozoites. MSP-8 is the only GPI-anchored protein known to be expressed at this time. It is synthesized as a mature 80-kDa protein which is rapidly processed to a C-terminal 17-kDa species that contains the double EGF module. As determined by a combination of immunofluorescence and membrane purification approaches, it appears likely that MSP-8 initially localizes to the parasite plasma membrane in the ring stage. Although the C-terminal 17-kDa fragment is present in more mature stages, at these times it is found in the food vacuole. We successfully disrupted the MSP-8 gene in P. falciparum, a process that validated the specificity of the antibodies used in this study and also demonstrated that MSP-8 does not play a role essential to maintenance of the erythrocyte cycle. This finding, together with the observation that MSP-8 is exclusively intracellular, casts doubt over the viability of this antigen as a vaccine. However, it is still possible that MSP-8 is involved in an early parasitophorous vacuole function that is significant for pathogenesis in the human host.     

Passive Immunization with Antibodies against Three Distinct Epitopes on Plasmodium yoelii Merozoite Surface Protein 1 Suppresses Parasitemia

We have produced monoclonal antibodies against Plasmodium yoelii merozoite surface protein 1 (MSP-1) and have assessed their ability to suppress blood stage parasitemia by passive immunization. Six immunoglobulin G antibodies were characterized in detail: three (B6, D3, and F5) were effective in suppressing a lethal blood stage challenge infection, two (B10 and G3) were partially effective, and one (B4) was ineffective. MSP-1 is the precursor to a complex of polypeptides on the merozoite surface; all of the antibodies bound to this precursor and to an ~42-kDa fragment (MSP-1₄₂) that is derived from the C terminus of MSP-1. MSP-1₄₂ is further cleaved to an N-terminal ~33-kDa polypeptide (MSP-1₃₃) and a C-terminal ~19-kDa polypeptide (MSP-1₁₉) comprised of two epidermal growth factor (EGF)-like modules. D3 reacted with MSP-1₄₂ but not with either of the constituents MSP-1₃₃ and MSP-1₁₉, B4 recognized an epitope within the N terminus of MSP-1₃₃, and B6, B10, F5, and G3 bound to MSP-1₁₉. B10 and G3 bound to epitopes that required both C-terminal EGF-like modules for their formation, whereas B6 and F5 bound to epitopes in the first EGF-like module. These results indicate that at least three distinct epitopes on P. yoelii MSP-1 are recognized by antibodies that suppress parasitemia in vivo.