Fig 1.
PfTrx-based vaccine constructs induce antigen-specific CD8+ T-cell responses.
(A) Design of the constructs ‘PADRE-Trx-OVA’, ‘PADRE-Trx-E7’ and ‘PADRE-Trx-flank E7’. PADRE: universal CD4+ T-cell epitope, pan HLA DR-restricted in humans and I-Ab-restricted in C57BL/6 mice [32]. PfTrx: thioredoxin scaffold from Pyrococcus furiosus. OVA257–264: ovalbumin-derived CTL epitope (SIINFEKL, H2-Kb restricted). HPV16 E749–57: HPV16 E7-derived CTL epitope (RAHYNIVTF, H2-Db restricted) [24]. Flank E749-57: extended E749-57 epitope, flanked on both sides by the five amino acids that in the HPV16 E7 protein are located upstream (QAEPD) and downstream (CCKCD) to the sequence of the E749-57 epitope (RAHYNIVTF). (B) Numbers of IFN-γ spots per 106 splenocytes (shown as SFU (spot-forming unit)/million cells) are compared among groups of mice immunized with OVA257-264 and PADRE peptide mix or the PADRE-Trx-OVA protein, respectively. Both groups were adjuvanted with IFA (Incomplete Freund’s adjuvant). Splenocytes were stimulated in vitro with either the OVA257-264 or the PADRE peptide. (C) Comparison of different adjuvants. Numbers of IFN-γ spots per 106 splenocytes are compared in three groups of mice vaccinated with PADRE-Trx-OVA formulated with different adjuvants (IFA, AddaVax or Alum/MPLA). Splenocytes were stimulated with the OVA257-264 peptide. (D) Influence of amino acids flanking the E749-57 epitope. Mice were immunized with HPV16 E749-57 and PADRE peptides mix formulated with IFA, or PADRE-Trx-E7 or PADRE-Trx-flank E7 formulated with AddaVax. Splenocytes were stimulated with the HPV16 E749-57 peptide. Shown are the mean and SD (standard deviation) of triplicate values on each mouse. P-value ≤ 0.05 was considered as significant and are labeled as follows: *, P-value < 0.05; **, P-value < 0.01; ***, P-value < 0.001; ****, P-value < 0.0001.
Fig 2.
OVA-specific T-cell epitopes and HPV16 L2-specific B-cell epitopes can be combined in a single construct without compromising each other’s immunogenicity.
(A) Antigen structure. Design a: Construct PADRE-Trx-OVA only containing the T-cell epitope OVA257-264. Design b: Construct PADRE-Trx-L2-OVA bearing both the T-cell epitope OVA257-264 and the B-cell epitope HPV16 L220-38. Design c: Construct PADRE-Trx-L2 only containing the HPV16 L220-38 B-cell epitope. (B) Influence of L2 B-cell epitopes on the induction of OVA-specific CTL responses. Numbers of IFN-γ spots per 106 splenocytes are compared in three groups of mice immunized with PADRE-Trx-OVA, PADRE-Trx-L2-OVA or PADRE-Trx-L2 (negative control). Splenocytes were stimulated with the OVA257-264 peptide. Shown are the mean and SD of triplicate values on each mouse. (C) Influence of OVA epitopes on the induction of anti-HPV16 and anti-HPV18 neutralizing antibodies. Sera were collected one month after the last immunization with PADRE-Trx-L2 or PADRE-Trx-L2-OVA (10 mice/group) and analyzed against HPV16 and HPV18 pseudovirions using the L1-PBNA. Each symbol represents the neutralization titer (EC50) of an individual animal; geometric means of the titers for each group are indicated by horizontal lines. P-value ≤ 0.05 was considered as significant (*).
Fig 3.
Addition of the OVX313 domain leads to multimerization of the antigens.
(A) Antigenic constructs used for immunization. OVX313: Chimeric version of the avian C4-binding protein heptamerization domain (OligoDOM technology, OSIVAX). L220-38 8mer: polytope comprising the L220-38 epitopes from eight different HPV types (16-18-31-33-35-6-51-59), selected on the basis of sequence homology to the major cross-neutralization HPV16 L220-38 epitope. (B) SDS-PAGE analysis of the purified antigens under reducing (left) and non-reducing conditions (right). Note: the molecular weight shift of the monomer Trx-8mer-OVA results from intensive L2 epitope-related formation of disulfide bonds in the non-reducing conditions.
Fig 4.
Heptamerization and addition of the L2 polytope boosts T-cell immunogenicity.
(A) IFN-γ ELISpot analysis of the T-cell responses induced by the heptameric Trx-8mer-OVA-OVX313 (left) and Trx-8mer-flank E7-OVX313 (right) antigens compared to the responses elicited by the corresponding monomeric PADRE-Trx-L2-OVA and PADRE-Trx-L2-flank E7 antigens; the Trx-8mer-OVX313 construct served as a negative control. Splenocytes were stimulated with either the OVA257-264 or the E749-57 peptide, as indicated. (B) Recognition of OVA-expressing EG7 cells (left) or E7-expressing RMA/E7 transfectants and TC-1 cells (right) by splenocytes from C57BL/6 mice immunized with the Trx-8mer-OVA-OVX313 (left) or the Trx-8mer-flank E7-OVX313 (right). Parental EL4 cells (left) and RMA cells (right) served as negative controls; the bar colors refer to the different animals utilized as 'biological replicates' in these experiments, as indicated. (C) Presence of the 8mer polytope induces stronger OVA-specific T-cell responses. During the assay, splenocytes were stimulated with the OVA257-264 peptide. Shown are the mean and SD of triplicate values on each mouse. (D) Absolute number of OVA-specific CD8+ T cells among splenocytes from immunized mice determined by flow cytometry using H2-Kb/OVA257-264 (SIINFEKL) tetramers. Representative examples from three different mice are shown on the left; the percentages of IFN-γ-producing CD8+ T cells are reported in the upper right quadrant. The diagram on the right shows the cumulative data obtained from the experiments performed with the tetramers; each symbol represents one mouse; the mean percentage (OVA257-264–specific CD8+ T cells/total CD8+ T cells) is indicated by horizontal bars. (E) Influence of the OVX313 domain on OVA- and E7-specific CTL responses. In the left graph, the numbers of IFN-γ spots per 106 splenocytes, measured upon stimulation with the OVA257-264 peptide, are compared in two groups of mice immunized with Trx-8mer-OVA-OVX313 or Trx-8mer-OVA, as indicated. The results of the same assay, performed with splenocytes from Trx-8mer-flank E7-OVX313 or Trx-8mer-flank E7 immunized mice, stimulated with the E749-57 peptide, are shown on the right. Shown are the mean and SD of triplicate values on each mouse. P-value ≤ 0.05 was considered as significant and are labeled as follows:*, P-value < 0.05; **, P-value < 0.01; ***, P-value < 0.001; ****, P-value < 0.0001.
Fig 5.
OVX313 domain enhanced generation of L2-specific antibodies.
(A) Sera collected from vaccinated mice (10 animals per group) one month after the last immunization with Trx-8mer-flank E7 or Trx-8mer-flank E7-OVX313, were analyzed against HPV16 (left) and HPV18 (right) pseudovirions using the L1-PBNA. Each symbol represents the neutralization titer (EC50) of an individual mouse; geometric means of the titers for each group are indicated by horizontal lines. EC50 values of three Trx-8mer-flank E7 immunized mice are lower than 50 (the EC50 value set as threshold for neutralization positivity); the corresponding data are thus not shown in the graph. P-value ≤ 0.05 was considered as significant. *, P-value < 0.05; **, P-value < 0.01. (B) Analysis of isotype distribution of L2-specific antibodies measured by antibody isotype ELISA; the values are the mean ±SD of data obtained from duplicate assays performed on the pooled sera from each immunization group. (C) A T-helper response is induced by the ‘OVX313-I5’ peptide. Mice were immunized twice at 5 days intervals with the Trx-8mer-flank E7-OVX313 antigen. The splenocytes were stimulated with the OVX313 peptide panel (OVX313-I1 to OVX313-I9; see ‘Materials and methods‘ for details). Data, representing the numbers of IFN-γ spots per 106 splenocytes, are the mean ±SD of three mice for each peptide stimulation.
Fig 6.
The antigen Trx-8mer-flank E7-OVX313 induces robust humoral immune responses.
(A) The presence of the OVA- or flank E7-specific epitopes does not compromise the induction of L2-specific B-cell responses. Sera collected from mice (10 animals/ group) one month after the last immunization with Trx-8mer-OVX313, Trx-8mer-OVA-OVX313 or Trx-8mer-flank E7-OVX313, were analyzed against HPV16 and HPV18 pseudovirions using the L1-PBNA. Each symbol represents the neutralization titer (EC50) of an individual mouse; geometric means of the titers for each group are indicated by the horizontal lines. (B) In vivo protection afforded by anti-Trx-8mer-flank E7-OVX313 sera. Sera from nonimmunized animals were used as control. Sera from 10 mice immunized with the Trx-8mer-flank E7-OVX313 antigen were pooled and passively transferred into naive mice. Recipient animals were then challenged with HPV11 and HPV39 PsVs. Images of in vivo challenge show the magnitude of vaginal infection by HPV11 and 39 PsVs. The color scales indicate the intensity of luciferase expression; a region-of-interest (ROI) analysis for average radiance (photons per second per square centimeter per steradian) was performed using the Living Image 2.50.1 software. Note that due to the different in vivo transduction activities of the various HPV PsV types, different scales were used. The diagrams on the right show the analytical data obtained from the experiments. Each symbol represents the average radiance of an individual mouse with the horizontal lines indicating the geometric means. P-value ≤ 0.05 was considered as significant. *, P-value < 0.05; **, P-value < 0.01.
Fig 7.
Vaccination with Trx-8mer-flank E7-OVX313 suppresses the outgrowth of grafted TC-1 tumors in mice.
(A) E7-specific CD8+ T-cell responses were evaluated using H2-Db/E749-57 streptamer staining. Splenocytes from immunized mice were double-stained with an anti-CD8-PE antibody and an APC-conjugated H2-Db/E749-57 streptamer and visualized by flow cytometry. Representative raw data for two mice are shown in the left panels; the percentages of IFN-γ-producing CD8+ T cells are shown in the upper right quadrant. The diagram on the right shows the cumulative data obtained from the experiments performed with the streptamer; each symbol represents one mouse; the mean percentage (E749-57–specific CD8+ T cells/total CD8+ T cells) is indicated by horizontal bars. (B) Experimental scheme of tumor challenge and therapeutic vaccination. (C) Tumor growth curves of vaccinated and unvaccinated mice (12 mice per group). (D) Animal survival rate shown by Kaplan-Meier curves; the Log-rank test indicates a significant difference in survival (p = 0.0005). (E) Tumor growth kinetics after re-challenge. Sixty-eight days after inoculation of tumor cells, five vaccinated mice (indicated by green arrows in Fig 7C) were re-challenged with 0.3×106 TC-1 cells. As a reference and internal control, five naïve mice were implanted in parallel with the same amount of TC-1 cells. Red crosses indicate mice that had to be sacrificed ahead of time due to an excessive tumor burden (tumor diameter over 1.5 cm). (F) A potent E7-specific T-cell response is apparent in vaccinated, tumor-regressed mice. Numbers of IFN-γ spots per 106 splenocytes are determined. Splenocytes were stimulated with TC-1 cells, E749-57 or E648-57 [37] peptide. Shown are the mean and SD of triplicate values on each mouse under the specific stimulation agent.
Fig 8.
Vaccination with Trx-8mer-flank E7-OVX313 induces regression of advanced TC-1 tumors.
(A) Experimental scheme of tumor challenge and therapeutic vaccination. Specifically, mice were inoculated with 0.3×106 TC-1 cells suspended in 100 μl of PBS on their right flank. When tumor size reached a diameter of 4-6mm, half of the tumor-bearing animals were immunized subcutaneously at the base of the tail with 20 μg of the Trx-8mer-flank E7-OVX313 antigen, whereas the remaining tumor-bearing mice received 20 μg of the control Trx-8mer-OVX313 antigen. Both antigens were adjuvanted with AddaVax, and two vaccine doses were administered 9 and 14 days after the challenge. Mice were excluded from the experiment when tumor diameter exceeded 1.5 cm. (B) Tumor growth curves of Trx-8mer-flank E7-OVX313 and Trx-8mer-OVX313 immunized mice (15 mice per group). (C) Animal survival rate displayed as Kaplan-Meier curves; the Log-rank test indicates a significant difference in survival (p<0.0001). Red crosses indicate mice that had to be sacrificed ahead of time due to an excessive tumor burden (tumor diameter over 1.5 cm). The mouse indicated by the green cross was sacrificed owing to skin lesions irrelevant to the tumor.