Intranasal administration of a synthetic lipopeptide without adjuvant induces systemic immune responses

Abstract

Parenteral injection of a lipopeptide containing a human leucocyte antigen (HLA)-A*0201-restricted cytotoxic T-lymphocyte (CTL) epitope from the human cytomegalovirus (HCMV) immunodominant matrix protein pp65 efficiently induces systemic CTL responses in HLA-A*0201 transgenic mice. In this study, we demonstrate that intranasal (i.n.) administration of this lipopeptide, covalently linked to a universal T helper (Th) epitope (PADRE), also induces potent systemic CTL responses. Immune responses were substantially reduced when the unlipidated peptide analogue was used (P<0.01). The induced CTL were CD8+, major histocompatibility complex (MHC) class I-restricted and CMV specific. Moreover, i.n. administration of this lipidated peptide elicited both systemic and local mucosal CD4+ T-cell proliferative responses, as well as antigen-specific delayed type hypersensitivity (DTH) immune responses. In contrast, mice receiving the unlipidated peptide analogue developed substantially reduced Th or DTH responses (P<0.05). These results highlight the usefulness and potential of lipopeptides delivered via mucosal routes as painless, safe, and non-invasive vaccines.

Figure 1

A schematic representation of PAM₂-K25V dipalmitoylated peptide containing the amino acid sequence 495–503 from the human cytomegalovirus (HCMV) matrix protein pp65 (pp65_495–503) and the T helper (Th) epitope PADRE. The pp65_495–503 cytotoxic T-lymphocyte (CTL) epitope was covalently linked to the C-terminal end of the PADRE Th epitope, and the peptide was then modified with two palmitic acids moieties at the N-terminus by automated synthesis on ABI 432. X, cyclohexylalanine.

Figure 5

Induction of an antigen-specific delayed-type hypersensitivity (DTH) response following intranasal administration of the PAM₂-K25V peptide. (a) PAM₂-K25V was administered to groups of human leucocyte antigen (HLA)-A*0201 transgenic (Tg) mice (n=3), and DTH immune responses were recalled 2 weeks later by injecting PADRE, pp65_495–503 or the control TT_615–629 peptide into the footpad. Footpad swelling was measured 48 hr after the booster immunization. The standard deviation (SD) for each group is indicated by a bar. (b) Histological analysis of footpads from HLA-A*0201 Tg mice primed intranasally (i.n.) (A and C) or subcutaneously (s.c.) (B and D) with PAM₂-K25V and boosted with PADRE (A and B) or control peptide (C and D). Mice were killed 48 hr after the second immunization, and tissues from the distal footpad were stained with haematoxylin and eosin. Original magnification: ×400 for all sections. The data are similar and representative of two independent experiments.

Figure 2

Intranasal immunization with a lipopeptide induces systemic T helper (Th) cell responses. (a) Human leucocyte antigen (HLA)-A*0201 transgenic (Tg) mice (n=4) were immunized intranasally (i.n.) with either PAM₂-K25V lipopeptide or an equimolar amount of the unlipidated version. Th responses were stimulated in vitro utilizing either PADRE (filled squares) or TT_615–629 (filled triangles) for PAM₂-K25V, and equivalently for the unlipidated version (open squares, triangles). Comparison between vaccine groups was significant at P<0·01. (b) Same nomenclature as (a), except that peptides were introduced using the subcutaneous (s.c.) route. Spleen cells were isolated and PADRE-specific Th-cell responses were determined as described in the Materials and methods. The TT_615–629 peptide was used as a control for measurement of Th responses. The results are reported as average of triplicate cultures and proliferation is indicated as stimulation indices (SI) [mean counts per minute (c.p.m.) in experimental wells ÷ mean c.p.m. in control wells without antigen). The standard deviation for each group is indicated by a bar. The c.p.m. of cells without antigen was 1566–1921. [³H]TdR, [³H]thymidine.

Figure 3

Intranasal administration with a lipopeptide results in local mucosal T helper (Th) cell responses. Groups of human leucocyte antigen (HLA)-A*0201 transgenic (Tg) mice (n=4) were immunized intranasally with either PAM₂ K25V (filled squares and triangles) or with equimolar amounts of the unlipidated version (open squares and triangles). PADRE-specific (filled and open squares) Th cell responses were assessed (a) in local cervical lymph node (LN) cells and (b) in remote inguinal LN cells. The TT_615–629 peptide was used as a control for measurement of Th responses (open triangles and squares). Data represent an average of triplicate cultures of a representative experiment and proliferation is indicated as stimulation indices (SI). The standard deviation for each group is indicated by a bar. Counts per minute (c.p.m.) of cells without antigen was 2743–2987.

Figure 4

Systemic CD8⁺ cytotoxic T lymphocyte (CTL) responses induced following intranasal (i.n.) administration of a lipopeptide. (a) Systemic pp65_495–503-specific (open and filled squares) CTL responses were assessed in the spleens of human leucocyte antigen (HLA)-A*0201 transgenic (Tg) mice (n=3) after i.n. administration of PAM₂-K25V (filled squares and triangles) or the unlipidated peptide version (open squares and triangles) using a chromium release assay (CRA), as described in the Materials and methods. The p53_149–157 peptide was used as a control (open and filled triangles). (b) At an effector : target (E : T) ratio of 30, monoclonal antibody (mAb) blocking of CTL activity was performed by adding serial dilutions of anti-CD8 or anti-CD4 during the lysis part of the CRA assay. (c) Cytomegalovirus (CMV)-specific and HLA-restricted CTL responses determined against either HLA matched or mismatched CMV-infected fibroblasts in a CRA assay. The standard deviation for each group is indicated by a bar.