Analysis of immune responses against T- and B-cell epitopes from Plasmodium falciparum liver-stage antigen 1 in rodent malaria models and malaria-exposed human subjects in India

Abstract

Liver-stage antigen 1 (LSA-1) is a potential vaccine candidate against preerythrocytic stages of malaria. We report here the immunogenicity of linear synthetic constructs delineated as T(H)-cell determinants from the nonrepeat regions of Plasmodium falciparum LSA-1 in murine models and human subjects from areas where malaria is endemic in Rajasthan State, India. Seven peptide constructs (LS1.1 to LS1.7) corresponding to predicted T-cell sites from both the N- and C-terminal regions and peptide LS1R from a repeat region of PfLSA-1 were synthesized to analyze the cellular immune responses. These linear peptides were also tested for humoral responses in order to determine if there were any overlapping B-cell epitopes in the predicted T-cell sites. Most peptides induced cellular responses in peptide-immunized BALB/c and C57BL/6 mice as measured by proliferation and cytokine analysis. Cross-reactive T-cell recognition of P. falciparum-based peptides in Plasmodium berghei-immune animals was evaluated, but only one peptide, LS1.2 (amino acids 1742 to 1760) triggered T-cell proliferation and interleukin-2 and gamma interferon secretion in P. berghei-immune splenocytes of BALB/c and C57BL/6 mice as well as in Thamnomys gazellae (natural host of P. berghei ANKA). In an enzyme-linked immunosorbent assay with the peptides, only one peptide, LS1.1, was recognized by anti-P. berghei liver-stage serum. Three peptides (LS1. 1, LS1.2, and LS1.3) of the eight peptides tested in this study were recognized by a relatively large percentage of P. falciparum-exposed human subjects; the reactivities ranged from approximately 45% for LS1.3 to approximately 60% for LS1.1 and LS1.2. Interestingly, all of the eight putative T-cell determinants were also recognized by the sera collected from malaria patients, although the response was variable in nature. These T(H)- and B-cell epitopes may be of potential value for preerythrocytic antigen-based malaria subunit vaccine formulations.

FIG. 1

(a) Schematic representation of P. falciparum LSA-1; (b) sequences of peptides used in this study. All the peptides represent amino acid sequences deduced from the genetic sequence of the P. falciparum NF54 isolate.

FIG. 2

Lymph node T-cell proliferative responses in BALB/c and C57BL/6 mice immunized with PfLSA-1-based synthetic peptides. All mice were immunized with 60 to 70 μg of peptide in CFA, and 10 to 12 days later cells from the draining lymph nodes were cultured for 100 h with various concentrations of homologous peptides. Concanavalin A (5 μg/ml) was used as a positive control. All values are means of quadruplicate determinations with standard deviations not more than 20% of the means. The results are expressed as SIs, defined as counts per minute in stimulated cultures divided by counts per minute in unstimulated cultures. An SI value of more than 2.0 was scored as positive result. Background counts per minute ranged from 235 to 5,560 for BALB/c mice and from 160 to 6,446 for C57BL/6 mice in all cases. The results shown are from three experiments performed separately, and results are expressed as maximum SI obtained at a given peptide concentration.

FIG. 3

Proliferative responses in P. berghei-immune splenocytes of BALB/c and C57BL/6 mice. Immune mice were obtained after immunization with irradiated P. berghei sporozoites. Immune splenocytes were stimulated with PfLSA-1-based peptides ex vivo at a concentration of 20 μg/ml. The results were expressed as SIs, and a value of more than 2.0 was scored as a positive result. Background counts per minute were 5,336 and 3,136 for BALB/c and C57BL/6 mice, respectively. The results shown are from a representative experiment performed more than three times.

FIG. 4

Reactivity of anti-P. berghei liver-stage serum antibodies to PfLSA-1-based peptides in an ELISA. Anti-P. berghei liver-stage sera were generated by injecting 1 million infected hepatocytes intravenously into a group of 10 naive BALB/c mice. Two weeks later, sera were collected from all immunized mice and pooled together. Negative-control values were obtained by incubating peptides with normal mouse serum antibodies and did not exceed an OD of 0.02.

FIG. 5

PBMC proliferation and IFN-γ responses in P. falciparum-exposed human subjects and nonexposed control donors. Blood was collected during the convalescence period, since the in vitro cellular response to parasite antigens is diminished during acute infections (30, 33, 38). Results are expressed as SIs, and only the maximum SI obtained at any of the peptide concentrations used is taken into consideration. Proliferative responses were considered positive if the SI was more than 2. □, negative response (SI < 2); ▤, positive response with 2 < SI < 5; ■, positive response with SI > 5. For IFN-γ: □, negative response; ▩, positive response (IFN-γ concentration in culture supernatant > 400 pg/ml). Mean IFN-γ production in the culture supernatants of control cultures (without antigen) of all donors was 180 ± 20 pg/ml. For antibody response: □, negative; ■, positive. PBMC from control individuals with no known history of malaria were tested in parallel (CON1 to CON7). As negative and positive controls, an unrelated T_H epitope from HEL and PHA, respectively, were included in the study at single concentrations of 20 and 5 μg/ml, respectively. Background count-per-minute ranges: for malaria exposed donors, 69 to 3,480 (geometric mean = 378 cpm); for control donors, 190 to 911 (geometric mean = 480 cpm). We summarized the results only from malaria-exposed individuals. n.d., not determined; Sum., summary; % + Ve, percent positive.

FIG. 6

Antibody responses in human subjects exposed to P. falciparum malaria. Human serum samples were diluted to 1:200 and used in ELISA to detect antibody levels against PfLSA-1-derived peptides. The positive responders were defined as those who had ODs above the mean plus 2 SD of 10 normal control sera. The cutoff OD for each peptide is indicated in Materials and Methods.