PB1 as a potential target for increasing the breadth of T-cell mediated immunity to Influenza A

Abstract

Recently, we showed that combined intranasal and subcutaneous immunization with a non-replicating adenoviral vector expressing NP of influenza A, strain PR8, induced long-standing protection against a range of influenza A viruses. However, H-2^b mice challenged with an influenza A strain mutated in the dominant NP₃₆₆ epitope were not efficiently protected. To address this problem, we envision the use of a cocktail of adenovectors targeting different internal proteins of influenza A virus. Consequently, we investigated the possibility of using PB1 as a target for an adenovector-based vaccine against influenza A. Our results showed that PB1 is not as immunogenic as the NP protein. However, by tethering PB1 to the murine invariant chain we were able to circumvent this problem and raise quite high numbers of PB1-specific CD8⁺ T cells in the circulation. Nevertheless, mice immunized against PB1 were not as efficiently protected against influenza A challenge as similarly NP-vaccinated animals. The reason for this is not a difference in the quality of the primed cells, nor in functional avidity. However, under similar conditions of immunization fewer PB1-specific cells were recruited to the airways, and surface expression of the dominant PB1 peptide, PB1₇₀₃, was less stable than in the case of NP₃₆₆.

Figure 1. Systemic vaccination with AdIiPB1 induces a potent PB1-specific CD8⁺ T-cell response.

(A) C57BL/6 mice were vaccinated subcutaneously (s.c.) in the foot pad with 2 × 10⁷ of PFU AdIiPB1 or AdPB1 and splenocytes were isolated 14 days later. Antigen-specific CD8⁺ T cells were enumerated using ICS and flow cytometry after peptide stimulation. Each dot represents one animal and bars represent medians. Dotted line represents background. Splenocytes from mice vaccinated with an adenovector expressing the irrelevant glycoprotein of lymphocytic choriomeningitis virus (AdIiGP) were included to document the antigen-specificity of CD8⁺ T cells induced by Ii-tethered antigen. (B) Representative dot plots of gated CD8⁺ T cells from each group. In both cases results in the upper row represent ex vivo stimulation with PB1₇₀₃, and in the lower row with GP₃₃.

Figure 2. Despite the induction of a potent CD8⁺ T-cell response, AdIiPB1 does not protect against influenza challenge.

(A) C57BL/6 mice were vaccinated s.c. in the foot pad with 2 × 10⁷ of PFU AdIiPB1 or AdPB1 and splenocytes was isolated 11, 14, 17, 20 and 30 days later. Antigen-specific CD8⁺ T cells were enumerated using flow analysis after peptide stimulation. Each dot represents one animal and bars represent medians. (B) C57BL/6 mice were vaccinated s.c. with 2 × 10⁷ of PFU AdIiPB1 or AdPB1 and challenged intranasal (i.n.) 30 days later with PR8 virus. Lungs were isolated 3 and 5 days post challenge and viral titer was measured using MDCK plaque assay. Each dot represents one animal and lines represent medians. Dotted line represents the detection limit of the MDCK plaque assay.

Figure 3. Clinical consequences of local and systemic vaccination.

C57BL/6 mice were vaccinated with 2 × 10⁷ of PFU AdIiPB1 s.c., i.n. or both s.c. and i.n. 30 days after vaccination, mice were challenged with PR8 virus. (A) 3 and 5 days post challenge, lungs were isolated and viral titer determined using MDCK plaque assay. Each dot represents one animal and lines represent medians. The dotted line represents the detection limit of the assay. (B) Percentage weight loss of initial weight post challenge. Dots represent mean and bars SEM. Dotted line represents end point for experiment, 75%. *p < 0.05.

Figure 4. A higher number of antigen specific cells are present in the lungs when mice are vaccinated both systemically and locally.

C57BL/6 mice were vaccinated with 2 × 10⁷ of PFU AdIiPB1 s.c., i.n. or both s.c. and i.n. 17 and 30 days after vaccination spleen and MLN were isolated from half of the mice and the number of IFN-γ⁺ CD8⁺ T cells in each organ site was determined through flow analysis after peptide stimulation. The remaining mice were challenged 17 and 30 days post vaccination with PR8 virus and 5 days post challenge spleen, MLN and BAL were isolated. The number of IFN-γ⁺ CD8⁺ T cells in each organ site was determined through flow analysis after peptide stimulation. Each dot represents one animal and bars represent medians (spleen and MLN). In BAL bars represent average number of IFN-γ⁺ CD8⁺ T cells per animal based on a pool from 5 animals.

Figure 5. Systemic and local AdIiPB1 vaccination combined induces long lasting protection.

(A) C57BL/6 mice were vaccinated with 2 × 10⁷ of PFU AdIiPB1 s.c., i.n. or both s.c. and i.n. 60 days post vaccination, spleen and MLN were isolated from half of the mice and the number of IFNγ⁺ CD8⁺ T cells in each organ site was determined though flow analysis after peptide stimulation. The remaining mice were challenged with PR8 virus and 5 days post challenge spleen, MLN and BAL were isolated. The number of IFN-γ⁺ CD8⁺ T cells in each organ site was determined though flow analysis after peptide stimulation. Each dot represents one animal and bars represent medians (spleen and MLN). In BAL bars represent the average number of IFN-γ⁺ CD8⁺ T cells per animal based on a pool from 5 animals. (B) C57BL/6 mice were vaccinated with 2 × 10⁷ of PFU AdIiPB1 s.c., i.n. or both s.c. and i.n. 60 days post vaccination mice were challenged with PR8, and 5 and 7 days post challenge lungs were isolated and viral titer determined using MDCK plaque assay. Each dot represents one animal and lines represent medians. The dotted line represents the detection limit of the assay. (C) Percentage weight loss of the initial weight in C57BL/6 mice vaccinated and challenged as described in B up until 5 days post challenge. *p < 0.05.

Figure 6. Systemic and local AdIiPB1 vaccination combined decreases morbidity and mortality.

C57BL/6 mice were vaccinated with 2 × 10⁷ of PFU AdIiPB1 s.c., i.n. or both s.c. and i.n. 60 days post vaccination mice were challenged with PR8 virus. Percentage weight loss of initial weight (A) and survival rate (B) was recorded up to 21 days post challenge.

Figure 7. AdIiPB1 vaccination does not match AdNP vaccination with regard to antiviral protection.

C57BL/6 mice were vaccinated i.n. plus s.c. with one of the following adenovector constructs: AdIiPB1, AdNP or AdIiGP for control. Thirty days later these mice and a group of naïve controls were challenged i.n. with PR8. Five days later lungs were isolated and viral titer determined using MDCK plaque assay. Each dot represents one animal and lines represent medians. The dotted line represents the detection limit of the assay.

Figure 8. AdNP and AdIiPB1 induce similar number of CD8 T cells.

(A) Groups of C57BL/6 mice were vaccinated s.c. with one of the doses, 10⁶, 10⁷ and 10⁸ PFU, of one of the following adenovectors: AdPB1, AdIiPB1, AdNP and AdIiNP. Fourteen days later numbers of antigen-specific CD8 T cells in the spleen were enumerated though flow analysis after stimulation with matching peptide. Each dot represents one animal and bars represent medians. (B) C57BL/6 mice were vaccinated with 2 × 10⁷ PFU AdIiPB1 or AdNP s.c. and i.n. and 30 days post vaccination spleen, MLN and BAL were isolated from animals. The number of IFN-γ⁺ CD8⁺ T cells in each organ site were determined though flow analysis after stimulation with matching peptide. Each dot represents one animal and bars represent medians (spleen and MLN). In BAL bars represent average number of IFN-γ⁺ CD8⁺ T cells per animal based on a pool from 5 animals.

Figure 9. Comparison of T cells targeting PB1₇₀₃ and NP₃₆₆.

(A) C57BL/6 mice were vaccinated with 2 × 10⁷ of PFU AdIiPB1 or AdNP s.c. and i.n. and 14 days post vaccination spleenocytes were isolated. The percentages of CD107a⁺, IL-2⁺ or TNFα⁺ cells out of the IFN-γ⁺ CD8⁺ T cells were determined though ICS and flow analysis after stimulation with matching peptide. (B) C57BL/6 mice were vaccinated with 2 × 10⁷ of PFU AdIiPB1 or AdNP s.c. and i.n. 30 days later mice were intravenously injected with a 1:1 mixed population of spleen cells loaded with PB1₇₀₃ or NP₃₆₆ under similar conditions. 16 hours after cell transfer, the fraction of injected cells recovered in the host spleens was determined by flow cytometry. (C) C57BL/6 mice were vaccinated with 2 × 10⁷ of PFU AdIiPB1 or AdNP s.c. and i.n. Fourteen days post vaccination splenocytes were isolated from 3 mice and pooled. The fraction of CD8⁺ T cells producing IFN-γ as a function of the peptide concentration used for loading of stimulator cells (functional avidity) was determined though flow analysis after 5 hours of co-incubation with peptide-loaded splenocytes (left hand side). To study the temporal stability of surface MHC I-peptide complexes effector cells were also added 6 hours later, and again the fraction of CD8⁺ T cells producing IFN-γ as a function of the peptide concentration used to load the stimulators cells were determined though flow analysis after 5 hours of co-incubation (right hand side).