Design and characterization of MAPs.
MAP-1 included multiple epitopes from PfCSP, a major protein on the malaria sporozoite surface. CS proteins from different Plasmodium
spp. have a few structural features in common, including a signal peptide, a variable central domain (composed of tandem repeats of amino acid sequences and two highly conserved regions), and a C-terminal hydrophobic sequence (30
). In P
, the central region is composed of major repeat units of NANP and a minor repeat unit of NVDP (NANP × 37 and NVDP × 2 for the 3D7 strain of parasite). CSP repeats from the human and murine Plasmodium
species are recognized as both B-cell and T-cell epitopes (18
). Antibodies against the repeat region can block sporozoites from entering cultured hepatocytes (2
). These antibodies can also passively protect mice from sporozoite challenge in murine malarias (14
). Although the earliest immunological and vaccination studies were predominantly focused on the NANP repeat units, several additional B-cell and T-cell epitopes have been identified on PfCSP (5
The peptide S-7 (amino acid sequence DPNNANPNVDPNANPNV) was chosen from the 5′ part of the minor repeat region that was identified as a CD4+
T-cell epitope from T-cell clones derived from a P
sporozoite-immunized volunteer (33
). The peptide S-8 (amino acid sequence PNANPNANPNANPNANP) was chosen from the region of tandem repeats of NANP that contain an immunodominant B-cell epitope (33
). Major constraints to the development of subunit-based malaria vaccines are the HLA-restricted immune responses and antigenic polymorphism prevalent in the major malaria vaccine candidates (13
). Likewise, some of the immunodominant T-cell epitopes recognized by individuals in areas where malaria is endemic are either HLA dependent and/or located in the highly polymorphic regions of the molecule (12
). To provide a wider immune responsiveness for the MAP-1 vaccine, epitopes S-9 and S-10 were included. Peptide S-9, located in the C-terminal hydrophobic polymorphic domain (amino acid sequence SVFNVVNSSIGLIMVLSFLFLN), has been identified as both a CD4+
T-cell and a CD8+
T-cell epitope, while peptide S-10, from the conserved N-terminal signal peptide region (amino acid sequence ILSVSSFLFV), was identified based on its binding with supertypes HLA-A*0202 and HLA-A*6802 and demonstrated cytotoxic T-lymphocyte (CTL) activity in peripheral blood mononuclear cells from an irradiated-sporozoite-immunized volunteer (12
). Figure shows the locations and amino acid sequences of MAP-1 peptides on PfCSP.
Amino acid sequences of the MAP peptides and their physical locations on P. falciparum antigens. (A) MAP-1 vaccine. (B) MAP-2 vaccine. (C) MAP-3 vaccine.
MAP-2 includes two pre-erythrocytic (PfCSP and LSA-1) and two blood stage (MSP-1 and MSP-3) antigens. We designed peptide S-11 as a fusion of sequences from two previously identified B-cell epitopes from PfCSP that are outside the repeat region, sequences KPKHKKLKQPGDGNP (S-11a) and ENANANNAV (S-11b). Epitope S-11a was recognized by sera from individuals living in Gabon, where malaria is endemic (9
). Epitope S-11b was recognized more frequently by sera from Brazil and Papua New Guinea than by those from Kenya (47
). Epitope S-12 is a combination of two strong T-cell epitopes from LSA-1. The LSA-J peptide is located at the junction of the last repeat unit and the next seven residues from the nonrepetitive region (amino acid sequence ERRAKEKLQEQQRDLEQRKADTKK), while Ls6 is from the C-terminal region of LSA-1 (amino acid sequence KPIVQYDNF). Both of these peptides have proven T-cell-stimulatory activity (15
). Peptide S-13 (amino acid sequence GISYYEKVLAKYKDDLE) was chosen from block 15-16, the dimorphic region of P
. In a study in western Kenya, 55% of the individuals tested had proliferative responses to peptide S-13 (57
). Finally, the fourth peptide, S-14 (amino acid sequence AKEASSYDYILGWEFGGGVPEHKKEEN), for MAP-2 was selected from MSP-3b. This 27-amino-acid peptide from MSP-3b was identified as the target for antibody-dependent cellular inhibition (ADCI) in the sera from African adults who had clinical immunity to malaria. Affinity-purified antibodies against MSP-3b peptides are effective in killing P
parasites in vitro
through an ADCI mechanism that required the presence of monocytes (35
). Figure shows the locations and amino acid sequences of the MAP-2 peptides.
MAP-3 was based on asexual blood stage antigens of P
that are known to play important roles in the invasion of erythrocytes by merozoites. The first epitope (peptide S-15 [amino acid sequence LVAQKEEFEYDENMEKAKQDKKKAL]) is from RAP-1 and was recognized as a potent B-cell epitope. An anti-RAP-1 MAb that recognizes a sequence within this epitope has been shown to inhibit parasite growth in vitro
). Peptide S-16 (amino acid sequence SSPSSTKSSPSNVKSASETEFSKLY) contains a T-cell epitope (SSPSSTKSSPSNVKSAS) from RAP-1 (45
) and a B-cell epitope (ETEFSKLY) from RAP-2 (52
). The third epitope of MAP-3 is based on a red-cell-binding domain from the serine repeat antigen (amino acid sequence YDNILVKMFKTNENNDKSELI) (41
). The fourth epitope is again a red-cell-binding domain from the 33-kDa processing fragment of MSP-1 (amino acid sequence DVIYLKPLAGVYRSLKKQLE) and has been shown to be involved in a receptor ligand-type interaction between merozoites and red blood cells (58
). Figure shows the locations and amino acid sequences of MAP-3 peptides.
Characterization of MAPs was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and amino acid analysis (data not shown). In addition to MAPs, individual peptides were also synthesized and characterized for use in immunological assays.
Immunogenicity of MAPs. (i) Antibody responses measured by ELISA.
The pooled sera from mice that were collected 2 weeks after the third immunization were used to measure the total IgG responses by ELISA. The responses against MAP-1 vaccine were evaluated in HLA-A2, C57BL/6, BALB/c, A/J, and CD1 strains of mice. The responses against MAP-2 and MAP-3 vaccines were evaluated in C57BL/6, BALB/c, A/J, and CD1 strains of mice.
(a) Responses to MAP-1.
MAP-1 is a combination of four epitopes from PfCSP. ELISA anti-MAP-1 IgG titers were measured using either MAP-1 or individual MAP-1 peptides or rPfCSP as a coating antigen. The highest mean anti-MAP-1 IgG titer was observed in congenic HLA-A2 (50,659 ± 5,431), followed by C57BL/6 mice (39,030 ± 7,454) when MAP-1 was used as a coating antigen. In comparison, MAP-1 was significantly less immunogenic in BALB/c (1,137 ± 222) and A/J (114 ± 19) mice. The response in CD1 mice was similar to that seen in BALB/c mice, with an IgG titer of 1,306 ± 320 (Fig. ).
FIG. 2. ELISA IgG responses in HLA-A2, C57BL/6, A/J, BALB/c, and CD1 strains of mice immunized by three subcutaneous injections of MAP vaccines in Montanide ISA 51 or injected with Montanide ISA 51 alone. ELISA IgG titers were determined in pooled sera (n = (more ...)
When rPfCSP was used as a coating antigen, the responses were again highest in HLA-A2 mice (84,302 ± 6,191), followed by C57BL/6 mice (76,304 ± 2,182) and BALB/c mice (204 ± 28), and negligible in A/J mice (Fig. ). The response in CD1 mice was 60-fold lower than in HLA-A2 mice (84,302 ± 6,191 versus 1,405 ± 133). The IgG titers in mice receiving the Montanide ISA 51 control were almost negligible, ranging from 14 to 40. No ELISA IgG reactivities were observed in the sera from MAP-1 immunized mice when individual peptides were used as a coating antigens (data not shown), suggesting that these synthetic peptides, while serving as potent B-cell immunizing epitopes, are not optimal ELISA coating antigens.
(b) Responses to MAP-2.
MAP-2 includes B- and T-cell epitopes from two pre-erythrocytic-stage (CSP and LSA-1) and two erythrocytic-stage (MSP-1 and MSP-3) antigens. This vaccine induced high-level antibody responses in all four strains of immunized mice when MAP-2 was used as a coating antigen. The highest mean IgG titers were in BALB/c mice (98,621 ± 2,990), followed by A/J mice (70,640 ± 6,307) and CD1 mice (65,969 ± 17,399), while lower titers were observed in C57BL/6 mice (22,688 ± 3,869). These results show that the combination epitopes contained within MAP-2 were able to induce relatively high-level, wide-ranging immune responses in diverse major histocompatibility complex (MHC) types (Fig. ).
Although MAP-2 contained a strong B-cell epitope from PfCSP (peptide S-11), sera from all four strains of mice failed to react when rPfCSP was used as a coating antigen (data not shown). We further tested the reactivity of sera from mice immunized with MAP-2 with individual peptides. The sera from the four strains of mice also failed to recognize the S-11 peptide. S-12 is a T-cell epitope from LSA-1. When S-12 was used as a coating antigen, there was a moderate but consistent reactivity with sera from A/J (372 ± 155), BALB/c (401 ± 90), and CD1 mice (447 ± 133). However, sera from C57BL/6 mice failed to recognize the S-12 peptide. These results strongly indicate that peptide sequence S-12 also contains a previously unrecognized, novel B-cell epitope. Peptide S-13, a known T-cell epitope, also showed a low level of recognition in A/J mice (154 ± 63) which was ~12-fold higher than the sera from Montanide ISA 51 control mice (13 ± 1). Peptide S-14 had both a T- and a B-cell epitope from MSP-3. Among the four epitopes from MAP-2, S-14 was maximally recognized and sera from all four strains of mice reacted with the S-14 peptide. The ELISA IgG titers were 21,222 ± 2,525 (A/J mice), 18,759 ± 4,933 (BALB/c mice), 10,177 ± 3,270 (C57BL/6 mice), and 13,887 ± 9,445 (CD1 mice) (Fig. ). There was no detectable reactivity with the sera of mice immunized with Montanide ISA 51 only (data not shown).
(c) Responses to MAP-3.
MAP-3 contains sequences only from blood stage antigens that include epitopes from PfRAP-1 and PfRAP-2 and two epitopes from red-cell-binding domains (SERA and MSP-1). This vaccine induced extremely high-level antibody responses in all of the mouse strains tested when MAP-3 was used as a coating antigen. The highest titers were found in A/J (309,801 ± 18,765) and BALB/c (245,734 ± 30,307) mice, followed by CD1 mice (73,433 ± 33,807); the lowest (but still substantial) responses were observed in C57BL/6 mice (37,995 ± 5,018) (Fig. ).
IgG responses to individual peptides were also evaluated. Although peptide S-15 is a potent B-cell epitope from RAP-1, sera from mice immunized with MAP-3 failed to react with the S-15 peptide. The sera from all four strains of mice reacted strongly with the S-16 peptide, which possesses sequences representing both T- and B-cell epitopes from RAP-1 and RAP-2. When S-16 was used as a coating antigen, the highest titers were observed in BALB/c mice (112,172 ± 65,755). The A/J and CD1 mice had comparable IgG titers of 51,400 ± 16,322 and 49,912 ± 5,668, respectively, while the responses were lower in C57BL/6 mice (529 ± 177). Peptide S-17, which contains sequences from the red-cell-binding antigen (SERA), was immunogenic in all of the mouse strains tested. With the S-17 peptide as a coating antigen, BALB/c mice responded maximally, with an IgG titer of 82,888 ± 23,910, followed by A/J mice (50,049 ± 10,114). The responses were comparable in the CD1 and C57BL/6 strains of mice (with IgG titers of 21,551 ± 2,792 in CD1 mice and 13,880 ± 2,567 in C57BL/6 mice). The S-18 peptide, a red blood cell-binding epitope from MSP-1, was nonimmunogenic in all groups of immunized mice (Fig. ).
(ii) Antibody responses measured by IIF.
We next wanted to know if antibodies against MAP vaccines react with native antigens expressed on the parasite during their respective stages of the life cycle. To accomplish this, we evaluated the reactivity of pooled sera from mice immunized with MAP vaccines with sporozoite stage, liver stage, or asexual blood stage parasites by IIF. Sera from MAP-1 (four epitopes from CSP)-immunized mice were tested against P. falciparum sporozoites. MAP-1 vaccine induced extremely high titers of antibodies against CSP present on the sporozoite surface. The sera from HLA-A2 mice reacted with the sporozoites with an endpoint titer of 409,600. This was followed by the reactivity of sera from BALB/c (102, 400) and C57BL/6 (25, 600) mice. The sera from CD1 and A/J mice also recognized the sporozoites, but their endpoint titers were lower (800 for CD1 mice and 100 for A/J mice) (Table ).
IIF IgG titers in sera from HLA-A2, C57BL/6, A/J, BALB/c, and CD1 strains of mice immunized with MAP vaccines delivered in Montanide ISA 51
Since the MAP-2 vaccine construct contains a peptide based on the LSA-1 sequence, we determined the reactivity of sera from the mice immunized with MAP-2 with the liver stage malaria parasites. P. falciparum sporozoites were allowed to develop to the liver stage form in human hepatocyte cell line HC-04 for 3 days. When tested at a 1:50 dilution, sera from immunized BALB/c, C57BL/6, and CD1 mice recognized the liver stage parasite (Fig. ); the intensity of fluorescence of these sera was equivalent to the reactivity seen with a polyclonal mouse antibody generated by immunizing with a DNA plasmid containing the LSA-1 gene (A). The recognition of liver stage P. falciparum parasites by MAP-2-immunized mouse sera indicates that the LSA-1 epitope (peptide S-12) in the MAP-2 vaccine was able to generate antibodies that may be effective against liver stage parasites.
FIG. 3. IIF IgG reactivity against P. falciparum liver stage parasites in C57BL/6, A/J, BALB/c, and CD1 strains of mice that received three subcutaneous injections of MAP vaccines in Montanide ISA 51. The reactivity for liver stage parasites was tested against (more ...)
MAP-2 and MAP-3 contained epitopes from the asexual blood stages of the life cycle, and consequently, sera raised against the MAP-2 and MAP-3 vaccines were tested against the asexual blood stage P. falciparum parasites by IIF. MAP-2 vaccine induced various levels of antiparasite antibodies in the four strains of mice tested. The highest reciprocal endpoint titer was observed in A/J mice (1:1,600). The titer in BALB/c and C57BL/6 mice was 1:400, followed by 1:200 in CD1 mice (Table ). The sera from MAP-3-immunized mice also recognized the blood stage schizonts. The titer was highest in CD1 mice (1:3,200), followed by BALB/c (1:1,600), A/J (1:800), and C57BL/6 (1:400) mice (Table ). The antibody responses measured by ELISA and IIF showed that MAPs were generally highly immunogenic and they induced antibodies that specifically recognized the epitopes on the P. falciparum sporozoite, liver, or asexual blood stage of the life cycle.
(iii) Induction of T-cell responses.
We also investigated whether the T-cell epitopes contained in the MAP vaccines were able to generate cell-mediated immunity by assessing the ability of the spleen cells to generate IFN-γ and IL-4 responses following immunization with MAP-1 or MAP-2 vaccine. Spleen cells were harvested from MAP-1-immunized HLA-A2, C57BL/6, and BALB/c mice and MAP-2-immunized C57BL/6 and BALB/c mice on day 14 after the third immunization and cultured for 36 h in the presence of MAP constructs or individual peptides.
The spleen cells from the three strains of MAP-1-immunized mice were able to produce IFN-γ and IL-4 after in vitro stimulation with MAP-1 (Fig. ). The IFN-γ responses were comparable in the spleen cells from HLA-A2 (469 SFC/106 splenocytes) and C57BL/6 (463 SFC/106 splenocytes) mice, while 235 SFC/106 splenocytes were observed in BALB/c mice. While investigating the contributions of individual peptides, we found that peptide S-8, a NANP repeat and a known T- and B-cell epitope, was responsible for the induction of a large portion of the IFN-γ-secreting cells by MAP-1 in HLA-A2 (341 SFC/106 splenocytes) and C57BL/6 (275 SFC/106 splenocytes) mice. The IFN-γ responses induced following stimulation with the S-8 peptide in spleen cells from BALB/c mice were lower (80 spots/106 cells), suggesting that the cellular response induced by the S-8 peptide in the MAP-1 vaccine was under the regulation of MHC genes. The spleen cells also responded to the other three MAP-1 peptides, but the responses were significantly lower (Fig. ). The IL-4 production by spleen cells followed the IFN-γ production pattern, except at a lower level, when the cells were cultured in the presence of the MAP-1 construct. The responses were equivalent in HLA-A2 (195 SFC/106 splenocytes) and C57BL/6 (178 SFC/106 splenocytes) mice, followed by that of BALB/c (94 SFC/106 splenocytes) mice.
FIG. 4. (A) ELISPOT IFN-γ and IL-4 responses in HLA-A2, C57BL/6, and BALB/c mice immunized by three subcutaneous injections of MAP-1 vaccine in Montanide ISA 51. Freshly isolated spleen cells were pooled from five mice per group and cultured in the presence (more ...)
We then evaluated IFN-γ and IL-4 production in response to MAP-2 immunization in C57BL/6 and BALB/c mice. The spleen cells from both strains of mice induced IFN-γ and IL-4 production when cultured in the presence of either the MAP-2 construct or individual peptides (Fig. ). The responses of C57BL/6 mice were always stronger than those of BALB/c mice. The frequency of IFN-γ-secreting spleen cells in C57BL/6 mice (436 SFC/106
splenocytes) was highest in response to the S-14 peptide, which is an ADCI epitope from MSP-3. This epitope has been previously described as a T-cell epitope recognized by the majority of individuals in areas where malaria is endemic (36
). This was followed by response to the MAP-2 construct (290 SFC/106
splenocytes) and the S-11 peptide (150 SFC/106
splenocytes), which is a B-cell epitope from PfCSP. The responses to the S-12 (77 SFC/106
splenocytes) and S-13 (76 SFC/106
splenocytes) constructs, though lower, were also significant. In BALB/c mice, the highest IFN-γ responses were observed in response to the S-13 peptide (205 SFC/106
splenocytes), which is a known T-cell epitope from MSP-1. This was again followed by 112 SFC/106
splenocytes in response to the MAP-2 construct and 70 SFC/106
splenocytes in response to the S-14 peptide. The responses to the S-11 and S-12 peptides were weaker (Fig. ).
We also investigated IL-4 responses in MAP-2-immunized mice. The IL-4 responses also followed the pattern of IFN-γ responses, but at a lower level. In C57BL/6 mice, the responses to the S-14 peptide were the strongest (186 SFC/106 splenocytes). The IL-4 production was equivalent in response to MAP-2 or the individual peptide S-11, S-12, or S-13. In BALB/c mice, the IL-4 (130 SFC/106 splenocytes) responses to the S-13 peptide, which is a T-cell epitope from MSP-1, were the strongest. The responses to the MAP-2 construct and individual peptides S-11, S-12, and S-14 were comparable. Overall, while MAP vaccines and their component peptides were able to generally induce high levels of cellular responses in mice of disparate MHC types, in some instances, these responses were under the regulation of the host genetic background.
Biological activity of anti-MAP antibodies. (i) In vitro ISI in HepG2 cells.
Having established that immunizations with MAP-1 vaccine that incorporated CSP molecule sequences induced high levels of antibodies in mice of diverse MHC types, we wanted to determine if these antibodies have any effect on the growth of liver stage parasites in vitro. To this end, we measured the biological activity in sera by testing the ability to inhibit sporozoite invasion of the hepatoma cell line HepG2. The ISI assay revealed that antibodies against the MAP-1 vaccine have the capacity to inhibit the invasion of HepG2 cells by P. falciparum sporozoites in vitro in mouse strains of diverse MHC types (Table ). Pooled sera from immunized HLA-A2 mice showed the highest level of inhibition (95.16%) of sporozoite invasion, followed by sera from C57BL/6 (88.72%) and CD1 (66.08%) mice. The ISI effect observed in sera from immunized HLA-A2 mice was almost equivalent to that of an anti-CSP MAb (NFS1) used as a positive control. The ability of the antibodies generated against the MAP-1 construct to inhibit sporozoite invasion in vitro shows that the antibodies are biologically active and effective in reducing the invasion of liver cells by sporozoites.
In vitro ISI of HepG2 cells by sera from mice immunized with MAP-1 vaccine
(ii) Merozoite GIA.
Since both MAP-2 and MAP-3 contained epitopes from the asexual blood stage of P. falciparum, antibodies generated against these MAPs were tested to determine if they could affect the growth of blood stage parasites in erythrocytes. GIA was performed with the P. falciparum 3D7, FVO, and Camp strains in suspension cultures. MAP-2 contained epitopes from two blood stage antigens, PfMSP-142 and PfMSP-3b (Fig. ). Immunizations with MAP-2 had induced high ELISA IgG antibody levels but moderate levels of IIF IgG antibodies in all four strains of mice tested. Various levels of P. falciparum strain-dependent GIA activity were seen in sera from MAP-2-immunized BALB/c mice (3D7, 4%; FVO, 16%; Camp, 19%) and CD1 mice (3D7, 7%; FVO, 25%; Camp, 35%); sera from C57BL/6 and A/J mice were not effective in the GIA. In MAP-3, all four epitopes were from antigens present in the blood stage of the life cycle (Fig. ). Sera from immunized C57BL/6 mice demonstrated the highest level of GIA activity against all three P. falciparum strains tested (3D7, 54%; FVO, 73%; Camp, 62%), while the lowest GIA activity was seen in CD1 mice (3D7, 11%; FVO, 24%; Camp, 29%) (Fig. ). Thus, the generation of growth-inhibitory antibodies by MAP vaccines appeared to be dependent on the mouse MHC background. We also noted some variations in the level of GIA activity in immunized sera when they were tested against different P. falciparum strains, suggesting that sequence diversity in antigenic epitopes incorporated in the MAP constructs may influence the protective efficacy of MAP vaccine.
FIG. 5. Merozoite growth/invasion-inhibitory activity of anti-MAP-3 sera against the P. falciparum 3D7, FVO, and Camp strains of asexual blood stage parasites. Synchronized ring stage parasites at 0.2% parasitemia were mixed with anti-MAP-3 sera (1:10 (more ...)