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Previous Article | Next Article 
Infection and Immunity, September 2003, p. 4961-4969, Vol. 71, No. 9
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.9.4961-4969.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Immunology, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910
Received 30 April 2003/ Returned for modification 30 May 2003/ Accepted 4 June 2003
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Evidence from field studies suggests that a reduction in parasite load is sufficient to reduce malaria-related morbidity and mortality. In areas where malaria is endemic, children and adults have been shown to acquire a certain level of clinical immunity following multiple infections, in that they do not exhibit symptoms of disease despite the presence of low levels of circulating parasites (2). There is strong evidence to suggest that antibodies are the primary mediators of this type of immunity, as demonstrated by a decrease in parasite burden in children following passive transfer of antibodies from immune adults (7, 27). Information from natural immunity can be utilized to develop a vaccination strategy to actively induce immunity to blood stage antigens in order to achieve protection from clinical disease. Of the several antigens expressed during the erythrocytic stages of the parasite, those involved in the process of merozoite invasion into erythrocytes can serve as potential vaccine candidates. The goal of such an intervention would be to reduce the rate of merozoite invasion, leading to a decrease in the overall parasite burden and thus causing a reduction in severe disease and death from malaria.
The 175-kDa erythrocyte binding antigen of Plasmodium falciparum, EBA-175, is one of the most extensively studied malarial antigens. EBA-175 has been shown to be a parasite ligand that binds to glycophorin A on the surface of erythrocytes. This process is thought to be one of the early events that lead to the entry of merozoites into erythrocyte. Studies conducted over the last several years show that EBA-175 belongs to the Ebl family of proteins (1), members of which are involved in invasion and share motifs with the Var family of proteins involved in cytoadherence (20, 30). The sequence (18) and domain structure of this molecule are conserved and bear similarity to those of other molecules involved in invasion, except for a duplication within the 5' cysteine-rich region II (RII) in EBA-175 of P. falciparum (1). Of the two domains in RII, designated F1 and F2, the red blood cell binding function is located primarily within the F2 domain (29). By definition, RII, as well as F2, is a highly cysteine-rich molecule. The number and position of the cysteines are conserved not only within the EBAs from various strains but also among the members of the Ebl/Var family, suggesting their potential involvement in receptor recognition.
There is evidence to show that EBA-175 is a target of a protective immune response. Antibodies generated against baculovirus-produced recombinant F2, fused to glutathione S-transferase, prevented the binding of EBA-RII to erythrocytes (24). In in vitro assays, antibodies generated against bacterially produced and refolded F2 domain of EBA-175 (26), and baculovirus-produced RII (23), inhibited merozoite invasion of P. falciparum into erythrocytes in a strain-independent manner. In an in vivo challenge study in Aotus monkeys, Jones et al. (13) observed partial protection with a DNA-protein prime boost immunization approach using EBA-RII but no protection with protein alone. Seroepidemiological studies indicate that antibodies to RII of EBA-175 are associated with protection against clinical disease (25). Together, these data indicate that EBA is a strong candidate for a vaccine against malaria and needs to be developed further to test its vaccine potential in monkeys and humans.
A major requirement for a successful recombinant protein-based vaccine is the ability to produce biologically active protein that can be easily scaled up for mass production. Research shows that production of a biologically active recombinant protein depends upon (i) the microenvironment within the host cell used for expression and (ii) the compatibility of codon usage between the native gene sequence and that of the expression host. Historically, Escherichia coli has been used for the expression of proteins in large amounts. However, due to the lack of a proper redox environment within the cytoplasmic compartment of E. coli, it is often difficult to produce correctly folded molecules that require the formation of disulfide bonds. In recent years Pichia pastoris, a eukaryotic expression host, has been developed as an alternate to the E. coli expression system, and it has the advantage of being able to produce complex molecules that are difficult to express and/or properly fold in bacteria. P. falciparum presents an additional challenge in that its genome is about 82% A+T rich, the highest A+T content of any known organism (9). This results in the use of different sets of codons for a given amino acid compared to other hosts (32), making it difficult to clone and express some of the malarial antigens in a heterologous system.
This study was undertaken to select an expression system to produce high levels of a conformationally folded, biologically active F2 domain of EBA-175 that required the least amount of postexpression manipulation to acquire native structure. In order to accomplish this, we determined the effects of codon modification on the expression of EBA-F2 in E. coli and P. pastoris. We also evaluated the effects of homologous and heterologous codon use in the two expression hosts on the conformation of recombinant F2, as assessed by red blood cell binding function, and their ability to induce antibodies that inhibit the binding of red blood cells to recombinant EBA. Our results show that compared to the native sequence, codon optimization significantly improved the expression of F2 in both the prokaryotic and eukaryotic systems. The protein produced in Pichia, however, was superior in terms of its expression levels, solubility, and biological activity.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Cloning and expression. (i) E. coli constructs. The native P. falciparum F2 gene was cloned into the pQE60 vector (Qiagen, Valencia, Calif.) by utilizing the bacteriophage T5 promoter transcription-translation machinery. The resultant plasmid was designated pQE60-Pf-F2. The E. coli codon-optimized gene was cloned into a modified version of pQE60. The vector was modified to introduce a kanamycin gene and a lacI gene that constitutively expresses the lac repressor protein to enable tight regulation of foreign protein expression. The modified plasmid, designated pQE60-AKI, contained the ColE1 origin of replication, expressed the foreign protein under control of the T5 promoter system, and was selectable under either ampicillin or kanamycin or under dual pressure. The plasmid carrying the E. coli codon-optimized gene was designated pQE60-AKI-eF2, or AKI-eF2 for short. The plasmids were transformed into E. coli XL-1 Blue (Stratagene, La Jolla, Calif.) cells, and after screening, recombinant clones were further transformed into the BL21(DE3) (Novagen, Madison, Wis.) host for expression.
(ii) F2 expression in E. coli. For bacterial expression of the native P. falciparum gene, MA21, a plasmid carrying RII of EBA-175 from the 3D7 strain of P. falciparum (24) was used as a template to amplify the F2 domain with KlenTaq polymerase (BD Biosciences, San Jose, Calif.). The gene was cloned via compatible restriction sites into the pQE60 vector and expressed in the BL21(DE3) host. The E. coli codon-optimized gene was synthesized with the required restriction sites and was cloned into the pQE60-AKI plasmid and also expressed in BL21(DE3) cells. Both clones were grown in Superbroth with phosphates and 0.8% glycerol in a BioFlow3000 10L fermentor (New Brunswick Scientific, Edison, N.J.). Cells were grown at 37°C and induced at an A600 of between 4 and 6 with 0.1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside). Based on the optimal expression condition for each clone, the cells were harvested at 2 h (pQE60-Pf-F2) or 4 h (AKI-eF2) postinduction. The cell paste was frozen at -70°C until processing.
(iii) P. pastoris constructs.
PicZ
(Invitrogen), a zeocin-selectable plasmid, was used for cloning and expression in P. pastoris. The plasmid contains an alcohol oxidase 1 promoter from P. pastoris fused to the -mating factor from Saccharomyces cerevisiae for directing the protein to the secretory pathway. Upon induction with methanol, the protein is expressed under control of the alcohol oxidase 1 promoter and secreted into the culture medium. E. coli XL-1 Blue (Stratagene) was used to maintain the plasmids. A histidine auxotroph, protease-deficient strain of the methylotrophic yeast P. pastoris, SMD1168 (Invitrogen), was used for expression.
(iv) F2 expression in P. pastoris.
PCR-blunt plasmids carrying the synthetic F2 constructs were amplified, and the inserts were digested and cloned into the appropriately digested PicZ vector. E. coli XL-1 blue cells were transformed with the constructs, and zeocin-resistant clones were screened for the insert by PCR and restriction digestion. Positive clones were digested with restriction enzyme SstI, and the linearized plasmid was used to transform P. pastoris SMD1168 with the Easy Comp kit (Invitrogen). The transformation mixture was plated on yeast-peptone-dextrose-sorbitol plates containing 125 µg of zeocin/ml. Mut+ clones were grown in YPD medium. For expression, the clones were grown in buffered glycerol medium (1% yeast extract, 2% peptone, 1.34% yeast nitrogen base, 4 x 10-5% biotin, 100 mM potassium phosphate [pH 6.0], 1% glycerol) for 
24 h. The cells were pelleted and induced with fresh medium containing 1% methanol for another 24 h. Supernatants were tested for expression by enzyme-linked immunosorbent assay (ELISA) or Western blotting with EBA-specific antibodies. All constructs used in the study are listed in Table 1.
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The Pichia-expressed protein yF2 was purified from culture supernatant. The supernatant was dialyzed extensively against phosphate-buffered saline (PBS) to remove all medium components. The sample was then loaded over Ni-NTA agarose and eluted with 200 mM imidazole in phosphate buffer.
Antibodies. Monoclonal antibodies (MAbs) generated against a baculovirus-produced RII of EBA-175 (A. Barbosa et al., unpublished) or polyclonal mouse sera against the recombinant F2 domain were used as the primary antibodies. The two MAbs used in the study were MAb RII.10, which recognizes a functional epitope on RII, and MAb RII.19, which recognizes a nonconformational epitope on RII. Polyclonal anti-F2 antibodies were generated by immunizing BALB/c mice four times with 10 µg (per dose) of nonreduced or reduced and alkylated E. coli-produced AKI-eF2 and nonreduced yF2 in Montanide IS270 (Sigma) adjuvant at 2-week intervals. Blood samples were collected 10 weeks following the primary immunization, and sera were stored at -20°C until use.
ELISA. Immulon 2HB plates (Dynatech, Alexandria, Va.) were coated overnight at 4°C with 50 µl (per well) of baculovirus-produced RII of EBA-175, at a concentration of 1 µg/ml, diluted with PBS (pH 7.4). After removal of the unbound antigen by washing the plates four times with PBS containing 0.1% Tween 20 (PBS-T), plates were blocked with PBS-casein (Pierce, Rockford, Ill.) to prevent nonspecific binding. Serum diluted in PBS-T was added and left for 2 h at 37°C. After washing, horseradish peroxidase-labeled anti-mouse immunoglobulin G (IgG) (Kirkegaard and Perry, Gaithersburg, Md.) was added to the plates and left for an hour at room temperature. Plates were washed, the reaction was developed with ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)], and the A405 was read after 30 min.
Western blotting. Native EBA-175 purified from culture supernatants (described below) or purified recombinant proteins were separated on Bis-Tris sodium dodecyl sulfate-4 to 20% polyacrylamide gels (Invitrogen) and run under nonreducing conditions with the MES (morpholineethanesulfonic acid) buffer system. Samples were transferred electrophoretically onto nitrocellulose membranes (Invitrogen). The membranes were blocked for up to 1 h with nonfat milk in PBS-T. After being washed with PBS-T, the blots were incubated overnight at 4°C with primary antibody diluted in PBS-T. Alkaline phosphatase-labeled anti-mouse IgG (Kirkegaard and Perry) was added after the primary antibody was washed off. The blots were developed with nitroblue tetrazolium-5-bromo-4 chloroindol-3-yl phosphate solution (Promega, Madison, Wis.).
Immunofluorescence. Sporozoites obtained from mosquitoes infected with P. falciparum or Percoll-purified schizonts were applied to slides, air dried, and fixed with methanol. The slides were blocked with bovine serum albumin (BSA) diluted to 1% in PBS (PBS-BSA) for 30 min. Primary antibodies diluted in PBS-BSA were added to the wells, and the slides were incubated in a humidified chamber for 1 h at room temperature. The slides were washed with PBS, and fluorescein isothiocyanate-labeled goat anti-mouse antibody (Promega) was added and left for 30 min at room temperature. Slides were washed and mounted in Vectashield (Vector Labs, Burlingame, Calif.) and viewed on an Olympus (Melville, N.Y.) microscope.
Preparation of native EBA. Normal human erythrocytes were used to maintain the 3D7 clone from the NF54 strain, the FVO strain, and the ItG strain of P. falciparum in culture in the presence of 10% human AB serum (12, 31). Native EBA-175 was purified according to the method described by Camus and Hadley (4) with slight modifications. Briefly, synchronized parasites were concentrated over Percoll-alanine to obtain over 95% pure schizonts. The enriched parasites were washed in RPMI 1640 and put back in culture, in complete medium (RPMI 1640 containing 10% normal human serum and 25 mM HEPES), at a concentration of 4 x 107 parasitized erythrocytes/ml. Culture supernatant, collected after overnight incubation at 37°C, was used to prepare EBA-175. The culture supernatant was diluted in RPMI 1640 and incubated with washed O-positive erythrocytes for an hour at room temperature with continuous mixing. The cells were washed in RPMI 1640, and bound EBA-175 was eluted by incubating with 1 M NaCl in 50 mM Tris (pH 7.4) with 2 mM phenylmethylsulfonyl fluoride for 10 min at room temperature.
Assays of erythrocyte binding and inhibition of erythrocyte binding. Two different erythrocyte binding assays were performed. For a direct binding assay, 50 µl of a packed suspension of erythrocytes was incubated with recombinant protein, diluted in RPMI, for 45 min at 37°C. The sample was spun down, and the supernatant (or unbound fraction) was incubated with fresh erythrocytes. The process was repeated for a total of five times. The results were analyzed by assessing the depletion of antigen in the supernatant as well as by eluting the bound protein from erythrocytes. For the former, supernatant was analyzed for depletion of protein (compared to the starting sample) by Coomassie blue staining. For further confirmation by Western blotting, the erythrocytes were washed with RPMI and the bound protein was eluted from the erythrocytes with 1 M NaCl. The eluted samples were run on a sodium dodecyl sulfate-polyacrylamide gel, transferred to nitrocellulose paper, and probed with polyclonal anti-yF2 antibodies.
An indirect plate binding assay was performed to determine the red blood cell binding of yF2 as well as to assess the inhibitory potential of anti-F2 sera. Anti-EBA MAb RII.19 was used as a primary capture antibody. Plastic petri dishes were coated with 20 µg of MAb RII.19/ml and incubated overnight at 4°C. After washing, the spots were blocked with 1% BSA diluted in PBS for 30 min. The BSA solution was aspirated and replaced with recombinant yF2 diluted in 0.1% BSA for 2 h at room temperature. To assess the ability of anti-yF2 or normal mouse serum to inhibit the binding of erythrocytes to yF2, the spots were washed and incubated with serum diluted in 0.1% BSA for an hour at room temperature. Control wells did not receive any serum. After washing, the spots were incubated with erythrocytes diluted to a final hematocrit of 2% in 0.1% BSA for 30 min at room temperature. The plates were washed gently with PBS and then fixed with 2% glutaraldehyde diluted in PBS. Results were scored qualitatively based on visual binding. Wells with no serum served as a positive control.
Assay of inhibition of receptor (glycophorin A) binding. Glycophorin A (Sigma) was biotinylated (Amersham Biosciences, Piscataway, N.J.) and gel purified on Sephadex G-25 to remove unincorporated biotin. Immulon 2HB plates were coated overnight with 3 µg of baculovirus RII diluted in PBS (pH 7.4)/ml. The unbound antigen was washed off with PBS-T, and the nonspecific sites were blocked with 1% BSA diluted in PBS (pH 7.4). The plates were incubated with different dilutions of normal mouse serum or sera from mice immunized with reduced or nonreduced AKI-eF2 or with yF2. Sera were diluted in PBS-T-0.1% BSA and incubated for 90 min at room temperature. The plates were washed and incubated for 1 h with biotinylated glycophorin A diluted to 0.25 µg/ml in PBS-T-BSA. After washing, streptavidin labeled with alkaline phosphatase (Amersham Biosciences) was added and left for 30 min at room temperature. Plates were developed with p-nitrophenylphosphate, and the A405 was read kinetically for 30 min.
Nucleotide sequence accession numbers. The sequences of the E. coli and Pichia codon-optimized constructs have been submitted to GenBank under accession numbers AY311508 and AY311509, respectively.
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RESULTS AND DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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For expression in E. coli, we compared the native F2 gene to a synthetic gene based on E. coli codons. Recombinant proteins expressed by both constructs were recognized by a conformational MAb, RII.10, in a Western blot (Fig. 1A). However, a comparison of the levels of expression, as estimated by a densitometric scan (Imagequant 5.0; Molecular Dynamics) of the Western blot, revealed that the E. coli codon-optimized construct expressed approximately fourfold more protein. The yield of purified protein from the wild-type construct (pQE60-Pf-F2) was very low (data not shown) and was not deemed suitable for further scale-up for a vaccine. The E. coli codon-optimized construct expressed significantly higher levels of recombinant protein. However, the protein was insoluble and required extraction with Sarkosyl, a strong anionic detergent. In contrast, the majority of the recombinant F2 encoded by the native codon-based gene was expressed as a nonionic-detergent-extractable protein (data not shown). While not all soluble proteins are correctly folded, it is known that incorrectly folded proteins tend to form insoluble aggregates in the form of inclusion bodies (5). We observed that a large percentage of the Ni-NTA-purified, E. coli codon-optimized protein tended to form reduction-sensitive aggregates (Fig. 1B), and only a portion of the protein was recognized by the conformational MAb in comparison to the polyclonal antibody (Fig. 1C). That only a portion of this protein was folded in the correct conformation was also evident from the fact that in a red blood cell binding assay, a critical test for determining the conformation of EBA, a population of the protein did not bind to red blood cells even on sequential incubation with fresh red blood cells (data not shown). The presence of a heterogeneous population of correctly and incorrectly folded protein also affects the quality of antibodies generated against it (discussed below). The difference in the solubilities of the proteins encoded by the P. falciparum codons versus the E. coli codons could be due to the fact that use of the most frequently used E. coli codons speeds up the translational machinery and thus influences the folding of the recombinant protein. Protein folding is known to be determined by several factors, including the availability of appropriate tRNAs and the amount of protein being expressed. The presence of low-abundance codons is a mechanism used by host cells to slow down protein translation to enable the protein to acquire the domain architecture needed for attaining its final conformation (19). High expression levels achieved by the codon-optimized construct in E. coli may have contributed to incorrect folding of the recombinant protein. Thus, while altering the codons can lead to an increase in levels of expression, it also compromises the quality of protein in terms of achieving its native conformation. Obtaining the correct conformation of a recombinant molecule in vitro involves refolding by using complex multistep processes that lead to a significant decrease in the final yield of protein (28). In order to avoid these postexpression manipulations, we explored the feasibility of using a eukaryotic expression system.
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All recombinant clones were tested for expression by ELISA and/or by Western blotting at 24-h intervals for up to 96 h. Expression data at 24 h for one clone of each of the three synthetic genes are shown in Fig. 2. The native F2 gene did not show any detectable expression by Western blotting or ELISA (data not shown). The eF2 and yF2 clones expressed protein that was detectable by Western blotting (Fig. 2) and ELISA (data not shown). However, densitometric analysis showed that the yF2 clone expressed protein at levels that were up to ninefold higher than those expressed by eF2. Representative E. coli and Pichia codon-based clones, designated eF2 and yF2, respectively, were grown in a shake flask to compare their expression levels. The yF2 clone produced a large amount of protein that was detectable by Coomassie blue staining of the culture supernatant run on a polyacrylamide gel (Fig. 3A). eF2, while detected by Western blotting, could be seen on a Coomassie blue-stained gel only following concentration over an Ni-NTA column (data not shown). Both the eF2 and yF2 proteins were conformational as determined by the fact that both were recognized by a conformational MAb (data not shown). Unlike the E. coli-produced protein (see above), the proteins made in P. pastoris showed very little aggregation. The protein showed a shift in mobility upon reduction with 2-mercaptoethanol, indicating the presence of disulfide bonds (Fig. 3B). Furthermore, polyclonal anti-yF2 and functional anti-EBA MAb RII.10 showed similar immunoblot profiles in a Western blot (Fig. 3C), suggesting that the majority of recombinant F2 expressed in P. pastoris is biologically functional. Reduced yF2 was recognized by polyclonal antiserum but was not recognized by MAb RII.10 (Fig. 3C).
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Functional characterization of yF2. EBA-175 has the unique advantage of having a defined functional role. The molecule has been shown to bind erythrocytes, and this binding is sensitive to removal of sialic acid residues from its receptor, glycophorin A, by treatment with neuraminidase. To determine if the recombinant protein produced in P. pastoris was biologically active, we tested its ability to bind to erythrocytes. yF2 bound to normal erythrocytes (Fig. 4). The Coomassie blue-stained gel showed depletion of yF2 following incubation with erythrocytes. Although there was some protein in the unbound supernatant after the first pass over red blood cells, we were able to deplete all protein after two passes (Fig. 4A), demonstrating that the residual protein in the first unbound fraction was due to a limitation of the number of red blood cells used in our assay and not to the presence of a population of protein that was unable to bind red blood cells. The specificity was confirmed by probing the washed and eluted fraction of erythrocytes with polyclonal anti-F2 antibodies (Fig. 4B). No protein was eluted from erythrocytes incubated with supernatant after three incubations. Similar to the case for native EBA-175, yF2 did not bind to neuraminidase-treated erythrocytes. The entire protein was recovered in the unbound fraction (Fig. 4A), and the eluted fraction showed no reactivity to anti-EBA antibodies (Fig. 4B). These results show that all of the soluble recombinant yeast-produced F2 binds to red blood cells in a specific manner. In contrast, only part of the E. coli-produced protein bound to red blood cells (data not shown). This suggests that P. pastoris-produced yF2 is correctly folded and biologically active. Our results further confirm earlier observations that F2 alone is sufficient to bind to red blood cells.
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In order to establish whether a protein that contains a mixed population of correctly and incorrectly folded conformers is still able to generate antibodies that are able to inhibit red blood cell binding, we immunized mice with the two recombinant F2 proteins. We also immunized mice with a reduced and alkylated protein to determine what kind of antibodies are generated following immunization with the deliberately altered antigen so as to ascertain the limits of the stringency of the conformational requirement of F2 to induce an inhibitory immune response. Thus, mice were immunized with E. coli-produced AKI-eF2 in either the reduced and alkylated (R-AKI-eF2) or the nonreduced (NR-AKI-eF2) form or with yeast-produced yF2 to assess the relative immunogenicities of F2 produced in E. coli and P. pastoris. Baculovirus-produced RII, which was shown previously to mimic native EBA-175 (24), was used as the plate antigen to ensure that the immune response measured was specific to EBA-175 and was not against contaminating host proteins present in the immunogen.
Mice immunized with either reduced or nonreduced AKI-eF2 had a higher reactivity to the reduced form of the plate antigen than the mice immunized with yF2 (Fig. 5A), suggesting that the E. coli-produced protein contains epitopes that are not present in the correctly folded molecule. Mice immunized with yF2 recognized the nonreduced plate antigen better than the nonreduced form (Fig. 5). Hence, it appears that the recombinant protein needs to closely mimic the native protein in order to produce antibodies that recognize a conformationally folded molecule.
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To determine if these antibodies recognized native EBA-175, we performed immunofluorescence assays (IFA) with asexual stages of the parasite. The antibodies recognized parasite-expressed EBA-175 antigen on the apical ends of parasitized erythrocytes by IFA (Fig. 7A). To test whether anti-yF2 antibodies were also capable of recognizing antigen on preerythrocytic stages, we performed an IFA with P. falciparum sporozoites. The antibodies showed a punctate recognition pattern, with a more intense staining towards the center of the sporozoite (Fig. 7B). Our data are consistent with recent reports by Gruner and colleagues (11), who demonstrated the presence of EBA-175 on the sporozoite and liver stages of the parasites. This dual recognition of antigen in the erythrocytic and preerythrocytic stages by anti-yF2 antibodies adds to the value of using this antigen as a potential vaccine that not only can target the blood stage infection but may also be able to control or limit the preerythrocytic stages as well.
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An alternative explanation could be that the production of antibodies against the F1 domain present in RII may either block the inhibitory antibodies against the F2 domain or prevent the formation of a sufficiently high titer of inhibitory antibodies against the F2 domain. We hypothesized that use of F2, the domain that is sufficient on its own to bind red blood cells, might maximize the chances of producing high-titer antibodies that prevent parasite interaction with red blood cells.
In summary, we report that codon optimization leads to enhanced expression of the F2 domain of EBA-175 in both E. coli and P. pastoris. However, protein produced in P. pastoris is superior in its biological function. Antibodies generated against yF2 are able to recognize native antigen from homologous and heterologous strains of parasite. These antibodies are functionally potent and are able to prevent receptor-ligand interaction. In addition, the ability of yF2 to generate antibodies that recognize antigen on the preerythrocytic and erythrocytic stages makes it a very strong candidate for preclinical development.
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ACKNOWLEDGMENTS |
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The views expressed here are those of the authors and do not necessarily represent those of the Department of the Army or Department of Defense.
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FOOTNOTES |
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) or nonreduced (•) AKI-eF2 had better reactivity to the reduced form of EBA-RII. Sera from mice immunized with yF2 (
) reacted poorly to the reduced plate antigen. (B) In contrast, sera from mice immunized with yF2 (
) did not react to either reduced or nonreduced plate antigen. O.D., optical density.
