Induction of CD4(+) T cell-dependent CD8(+) type 1 responses in humans by a malaria DNA vaccine

Abstract

We assessed immunogenicity of a malaria DNA vaccine administered by needle i.m. or needleless jet injection [i.m. or i.m./intradermally (i.d.)] in 14 volunteers. Antigen-specific IFN-gamma responses were detected by enzyme-linked immunospot (ELISPOT) assays in all subjects to multiple 9- to 23-aa peptides containing class I and/or class II restricted epitopes, and were dependent on both CD8(+) and CD4(+) T cells. Overall, frequency of response was significantly greater after i.m. jet injection. CD8(+)-dependent cytotoxic T lymphocytes (CTL) were detected in 8/14 volunteers. Demonstration in humans of elicitation of the class I restricted IFN-gamma responses we believe necessary for protection against the liver stage of malaria parasites brings us closer to an effective malaria vaccine.

Figure 1

Antigen-specific IFN-γ responses induced by DNA vaccination. IFN-γ responses against: (a) HLA-A2.1-restricted positive control peptide from influenza matrix protein (Flu. A2.1); (b) HLA-A2.1-restricted peptide from PfCSP (A2.386); or (c) HLA-DR-restricted peptide from PfCSP (DR.375) with coded and double-blinded frozen PBMCs collected from eight volunteers pre- and postimmunization with PfCSP DNA. BJ2, -3, and -4 from Biojector i.m./i.d.; BJ6, -9, and -10 from Biojector i.m.; and BJ11 and -12 from needle i.m.

Figure 2

Breadth of DNA-induced IFN-γ responses. Fresh PBMCs from volunteer BJ10 (Biojector i.m.), who expressed the alleles HLA-A*0101, -A*0201, -B*0701, -DRB1*1501, and -DRB5*0101, were cultured with (a) six class I-restricted PfCSP peptides, an HLA-A2.1-restricted positive control peptide (Flu. A2.1), or an HLA-A2.1-restricted negative control peptide (HIV gag A2.1); or (b) six class II-restricted PfCSP peptides or an HLA-DR restricted negative control peptide (P. falciparum Exp-1 DR).

Figure 3

Comparison of frequency of IFN-γ responses that were induced by the three different routes of immunization: needle i.m., Biojector i.m., and Biojector i.m./i.d. IFN-γ responses were assessed by using fresh PBMCs from all of the volunteers at each of the three time points against the four HLA-A2.1-restricted epitopes and five HLA-DR-restricted epitopes, which were tested in all individuals in all assays. Data are presented as the number of responding volunteers (y axis) at each of the time points (x axis) for each of the peptides (z axis). P values were calculated by using Student's t test (two-tailed), and only the significant differences (P = 0.05) between groups are indicated.

Figure 4

CD8⁺ and CD4⁺ T cell dependency of IFN-γ responses. Frozen PBMCs from (a) volunteer BJ11 (needle i.m.) or (b) volunteer BJ7 (Biojector i.m.) collected 2 weeks after the second immunization or 6 weeks after the third immunization, respectively, were either untreated or selectively depleted of CD4⁺, CD8⁺, both CD4⁺ and CD8⁺ T cells, or CD45RO⁺ cells immediately before culture with peptide A2.386 or peptide DR.375. Alternatively, frozen PBMCs from the same volunteer were either untreated (whole PBMCs), or selectively enriched for CD8⁺ T cells, CD4⁺ T cells, or CD8⁺ plus APCs or CD4⁺ plus APCs immediately before culture with peptides A2.386 or DR.375. Two distinct patterns are presented. Pattern 1: (a) IFN-γ induced against both A2.386 and DR.375 peptides depended on CD4⁺, CD8⁺, and CD45RO⁺ T cells. (c) Either positively selected CD8⁺ or CD4⁺ T cells, with or without APCs, failed to reconstitute the effector response. Pattern 2: (b) IFN-γ induced against peptides A2.386 and DR.375 depended on CD8⁺ and CD45RO⁺ T cells, but not CD4⁺ T cells. (d) Positively selected CD4⁺ cells with or without APCs, or of CD8⁺ cells without APCs, failed to reconstitute the effector response. Positively selected CD8⁺ T cells with APCs completely restored the effector response to the class I peptide A2.386 and partially restored the response to the class II peptide, which contains a nested class I epitope.