Article Figures & Data
Figures
- FIG. 1.
Schematic representation of the synthetic peptide immunogens used in this study. The CTL peptide represents the CD8 T-cell determinant NP (147-155) present in the NP of influenza A viruses of different strains and subtypes. The TH peptide represents the CD4 determinant HA2 (166-180) present in the light chain of the HA molecule of A/Memphis/1/71 virus and highly conserved within the H3 influenza virus subtype. The amino acid sequence of these determinants is shown in single-letter code above the monomer peptides. Pal, palmitic acid; K, lysine. The α- and ε-amino groups of certain lysine residues are indicated for clarity, as well as the C and N termini of the CTL and TH peptides. Within the text, peptide immunogens are referred to by the abbreviated term shown next to each schematic diagram.
- FIG. 2.
Cytotoxic T-cell response induced by the synthetic immunogens based on the CTL peptide alone (A) or the CTL-TH construct (B). A 51Cr release assay was performed with 104 uninfected or 104 Mem 71 virus-infected 51Cr-labeled P815 target cells and various numbers of effector cells. Secondary effectors were generated from lymph node cells of groups of five BALB/c mice that had been primed 7 days previously with 45 nmol of a peptide immunogen emulsified in CFA. The lymph node cell suspensions were cultured for 5 days with Mem 71 virus-infected autologous spleen cells and then tested in the 51Cr release assay. Each point on every curve represents the mean of triplicate cultures. For all effector populations, background lysis measured on uninfected P815 targets was <20% at an effector/target ratio of 100:1.
- FIG. 3.
Clearance of pulmonary viral infection by primary effector responses induced by synthetic immunogens. Groups of five mice were immunized s.c. with 9 nmol of the specified immunogens emulsified in CFA. Seven days later mice were challenged i.n. with 104.5 PFU of Mem 71 influenza virus and, 5 days postchallenge, mice were sacrificed, the lungs were removed, and lung homogenates were prepared. Homogenates were assayed for infectious virus by plaque formation on MDCK cell monolayers. Closed circles represent the lung virus titers of individual mice, and the bar represents the geometric mean titer for the group of mice.
- FIG. 4.
Recall of pulmonary viral clearing responses in mice inoculated with synthetic immunogens. Groups of five mice were immunized s.c. with 9 nmol of the specified peptide immunogens emulsified in CFA. Mice receiving one dose of the immunogens (x1) were challenged 28 days after priming; mice receiving two doses (x2) were primed on day 0, boosted in an identical manner on day 21, and then challenged i.n. with 104.5 PFU of Mem 71 influenza virus on day 42. Titers of infectious virus in lung homogenates sampled 5 days after challenge were determined by plaque formation. Closed circles represent the lung virus titers of individual mice, and the bar represents the geometric mean titer of the group of mice.
- FIG. 5.
Comparison of the lytic activity of cytotoxic T cells from CTL peptide-primed mice versus lipopeptide-primed mice at 7 days and at several months postpriming. A 51Cr release assay was performed by using uninfected P815 targets (open symbols) or Mem 71 virus-infected P815 targets (closed symbols) and various numbers of effector cells. Lymph node effectors (A) or spleen cell effectors (B, C, and D) were generated from mice that had been primed either 7 days (A) or at least 2 months previously (B, C, and D) with 9 nmol of CTL peptide (squares), Pal2-CTL-TH (circles), or Pal4-CTL-TH (triangles) emulsified in CFA. Lymph node or spleen cells were then cultured for 5 days with either virus-infected or peptide-pulsed autologous spleen cells, as indicated above each panel, and then tested in a 51Cr release assay.
- FIG. 6.
Comparison of the number of CTL determinant-specific IFN-γ-secreting cells in lipopeptide- and CTL peptide-primed mice. ELISPOT assays were performed on cells from the inguinal and popliteal lymph nodes of mice primed 7 days previously (left panel) or from the spleens of mice primed 28 days previously (right panel) with the indicated immunogens, emulsified in CFA. The data represent the number of ELISPOTs per total cells recovered, with backgrounds in cultures lacking antigen subtracted. The results are expressed as the mean value obtained from three individual mice, and the error bars represent one standard deviation of the mean.
- FIG. 7.
Lipopeptides elicit potent CD8 T-cell memory responses. (A) Cytolytic effector cells were generated in vitro from spleen cells of mice that had been primed 15 months previously with 9 nmol of the indicated constructs emulsified in CFA. The cells were cultured for 5 days with Mem 71 virus-infected autologous spleen cells and then tested in the 51Cr release assay. For each immunogen, the background lysis measured on uninfected P815 targets was <15% at an effector/target ratio of 100:1. (B) CTL determinant-specific IFN-γ-secreting cells were enumerated in the spleens of mice inoculated 3 months previously with 9 nmol of the indicated immunogens emulsified in CFA. The data are presented as the number of ELISPOTS per 106 spleen cells, with backgrounds in cultures lacking antigen subtracted. The results are expressed as the mean value obtained from three individual mice, and the error bars represent one standard deviation of the mean.
- FIG. 8.
CD4 T-cell depletion of mice after lipopeptide immunization but prior to viral challenge. Two groups of eight BALB/c mice were immunized s.c. with either Pal2-CTL-TH emulsified in CFA or adjuvant alone. At 28 days postpriming, four mice from each group were depleted of CD4 T cells by treatment with 400 μg of MAb GK1.5 and the other four mice received 400 μg of normal rat IgG. Successful depletion of CD4 T cells was verified by flow cytometric analysis, and all mice were subsequently challenged with Mem 71 virus 8 days after treatment. Five days later, the mice were sacrificed, and the lung viral titers were determined by plaque assay. Closed circles represent the lung virus titers of individual mice, and the bar represents the geometric mean titer of the group of mice.
- FIG. 9.
Cytotoxic T-cell activity and viral clearance elicited by immunogens administered in the absence of adjuvant. (A) Spleen effectors were generated from mice that had been primed s.c. 28 days previously with 9 nmol of the indicated immunogens in PBS. Spleen cells were cultured for 5 days with Mem 71 virus-infected spleen cells and then tested in the 51Cr release assay. For all immunogens, background lysis measured on uninfected P815 targets was <15% at an effector/target ratio of 100:1. (B) Groups of five mice were immunized s.c. with 9 nmol of the peptide constructs in PBS and then challenged i.n. 29 days later with Mem 71 virus. At 5 days after challenge, mice were sacrificed and the lung viral titers determined. Closed circles represent the lung virus titers of individual mice, and the bar represents the geometric mean titer of the group of mice.
