Prime-boost immunization with adenoviral and modified vaccinia virus Ankara vectors enhances the durability and polyfunctionality of protective malaria CD8+ T-cell responses

Abstract

Protection against liver-stage malaria relies on the induction of high frequencies of antigen-specific CD8+ T cells. We have previously reported high protective levels against mouse malaria, albeit short-lived, by a single vaccination with adenoviral vectors coding for a liver-stage antigen (ME.TRAP). Here, we report that prime-boost regimens using modified vaccinia virus Ankara (MVA) and adenoviral vectors encoding ME.TRAP can enhance both short- and long-term sterile protection against malaria. Protection persisted for at least 6 months when simian adenoviruses AdCh63 and AdC9 were used as priming vectors. Kinetic analysis showed that the MVA boost made the adenoviral-primed T cells markedly more polyfunctional, with the number of gamma interferon (INF-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-2 (IL-2) triple-positive and INF-gamma and TNF-alpha double-positive cells increasing over time, while INF-gamma single-positive cells declined with time. However, IFN-gamma production prevailed as the main immune correlate of protection, while neither an increase of polyfunctionality nor a high integrated mean fluorescence intensity (iMFI) correlated with protection. These data highlight the ability of optimized viral vector prime-boost regimens to generate more protective and sustained CD8+ T-cell responses, and our results encourage a more nuanced assessment of the importance of inducing polyfunctional CD8(+) T cells by vaccination.

FIG. 1.

Kinetics of the CD8 responses induced in Ad/MVA prime-boost regimens and stimulation of antibodies by prime-boost regimens. (A) Groups of BALB/c mice (n = 3) were immunized with 1 × 10¹⁰ vp of human (AdH) and chimpanzee (AdC) adenovirus expressing ME.TRAP and boosted 8 weeks later with 1 × 10⁷ PFU of MVA ME.TRAP. (B) Total number of CD8⁺ T cells per spleen expressing IFN-γ upon stimulation with Pb9 peptide after Ad or FP9 prime and MVA boost. The number represents the average of 3 mice per group. (C) Antibody responses against TRAP in four different heterologous prime-boost regimens. Responses were assessed on week 4 after a boost with MVA. The graph displays only three Ad regimens (high, intermediate, and low) out of five tested and one poxviral regimen (FP9-MVA). O.D., optical density. Data are represented as means ± standard errors of the means (SEM). Adenoviral vectors used for immunizations are from the human serotype 5 (AdH5) and simian serotypes (AdC6, AdC7, AdC9, and AdCh63). The poxviral vectors are fowlpox 9 (FP9) and modified Vaccinia virus ankara (MVA).

FIG. 2.

Kinetic responses of the generation of polyfunctional CD8⁺ T-cell responses upon a prime or a prime-boost regimen. (A) Groups of 5 BALB/c mice were immunized with 5 × 10⁹ vp of adenovirus and 1 × 10⁶ PFU of the poxvirus MVA. Intracellular cytokine staining (ICS) to quantify production of IFN-γ, TNF-α, and IL-2 from CD8⁺ T-cells was performed in blood at different time points upon stimulation with the immunodominant Pb9 peptide. Graphs were generated after performing a Boolean analysis in FlowJo (Graphpad) and data analysis in SPICE software (Mario Roederer, VRC, NIH). (B) Kinetics of the CD8⁺ responses after a single prime with AdC9 ME.TRAP or a prime-boost with AdC9-MVA. Pie charts display relative percentages of CD8 cells producing one (1+), two (2+), or three (3+) cytokines at various time points. (C) Kinetics of the polyfunctional responses in absolute numbers showing 1+, 2+, and 3+ cells. (D) CD8⁺ responses in liver induced by various regimens 2 weeks after immunization. Mice were vaccinated as mentioned above, and T cells were isolated from perfused livers. Plots represent the % of IFN-γ in the CD8⁺ compartment upon stimulation with Pb9. Pie charts show relative percentages of CD8 cells producing one, two, or three cytokines. Plots shown are from representative mice, and multifunctionality data in pie charts were calculated from 3 mice per group.

FIG. 3.

Generation of multifunctional CD8⁺ T-cell responses after single-, double-, or triple-vaccination regimens. Groups of 6 BALB/c mice were immunized as described (A). Intervals between prime and boost were 8 weeks, and immune responses were assessed 2 and 8 weeks after the last vaccination. CD8⁺ T cells from blood were stimulated, stained, and analyzed as described in the legend to Fig. 2. (B) Percentage of CD8⁺ T cells producing IFN-γ in different prime-boost regimens, without taking into consideration coproduction of other cytokines. (C) Percentage of CD8⁺ T cells coproducing the three cytokines (3+) IFN-γ, TNF-α, and IL-2. (D) Frequencies of CD8⁺ T cells coproducing the two cytokines (2+) IFN-γ and TNF-α. (E) Percentage of CD8⁺ T-cells producing only IFN-γ (1+) and none of the other cytokines. Data are represented as means ± SEM.

FIG. 4.

Immunogenicity and protective efficacy of prime-boost regimens using heterologous Ad-Ad or Ad-MVA regimens. Groups of 3 to 5 BALB/c mice were immunized as described in the legend to Fig. 2 with AdC7 ME.TRAP (5 × 10⁹ vp) and subsequently boosted after 8 weeks with MVA (1 × 10⁷ PFU) or AdC9 (5 × 10⁹ vp), both coding for the same ME.TRAP transgene. CD8⁺ T-cell responses and protective efficacy were assessed at different time points after the boost, at 2 weeks (A), 8 weeks (B), and 26 weeks (C). (D) Comparison of the antibody responses in the A-A versus A-M regimens. Sera were taken on week 4 after a boost, and IgG responses were assessed as described in Materials and Methods. The graph shows the optical density at 405 nm in serial dilutions of the sera. SFC, spot-forming cells. Data are represented as means ± SEM.