Highly persistent and effective prime/boost regimens against tuberculosis that use a multivalent modified vaccine virus Ankara-based tuberculosis vaccine with interleukin-15 as a molecular adjuvant

Abstract

Novel immunization strategies are needed to enhance the global control of tuberculosis (TB). In this study, we assessed the immunizing activity of a recombinant modified vaccinia Ankara (MVA) construct (MVA/IL-15/5Mtb) which overexpresses five Mycobacterium tuberculosis antigens (antigen 85A, antigen 85B, ESAT6, HSP60, and Mtb39), as well as the molecular adjuvant interleukin-15 (IL-15). Homologous prime/boost studies showed that the MVA/IL-15/5Mtb vaccine induced moderate but highly persistent protective immune responses for at least 16 months after the initial vaccination and that the interval between the prime and boost did not significantly alter vaccine-induced antituberculosis protective immunity. At 16 months, when the Mycobacterium bovis BCG and MVA/IL-15/5Mtb vaccine-induced protection was essentially equivalent, the protective responses after a tuberculous challenge were associated with elevated levels of gamma interferon (IFN-gamma), IL-17F, Cxcl9, and Cxcl10. To amplify the immunizing potential of the MVA/IL-15/5Mtb vaccine, a heterologous prime/boost regimen was tested using an ESAT6-antigen 85B (E6-85) fusion protein formulated in dimethyldiotacylammonium bromide/monophosphoryl lipid A (DDA/MPL) adjuvant as the priming vaccine and the MVA/IL-15/5Mtb recombinant virus as the boosting agent. When MVA/IL-15/5Mtb vaccine boosting was done at 2 or 6 months following the final fusion protein injections, the prime/boost regimen evoked protective responses against an aerogenic M. tuberculosis challenge which was equivalent to that induced by BCG immunization. Long-term memory after immunization with the E6-85-MVA/IL-15/5Mtb combination regimen was associated with the induction of monofunctional CD4 and CD8 IFN-gamma-producing T cells and multifunctional CD4 and CD8 T cells expressing IFN-gamma/tumor necrosis factor alpha (TNF-alpha), TNF-alpha/IL-2, and IFN-gamma/TNF-alpha/IL-2. In contrast, BCG-induced protection was characterized by fewer CD4 and CD8 monofunctional T cells expressing IFN-gamma and only IFN-gamma/TNF-alpha and IFN-gamma/TNF-alpha/IL-2 expressing multifunctional T (MFT) cells. Taken together, these results suggest that a heterologous prime/boost protocol using an MVA-based tuberculosis vaccines to boost after priming with TB protein/adjuvant preparations should be considered when designing long-lived TB immunization strategies.

FIG. 1.

Immunization schedules for three MVA/IL-15/5Mtb vaccine experiments. For study I, mice immunized with the MVA/IL-15/5Mtb construct were challenged with virulent M. tuberculosis at either 2 (A) or 12 (B) months after the priming vaccination (see Table 1). In study II, the impact of different prime/boost intervals was evaluated (Table 2). Mice in group C were boosted 1 month after the priming vaccination and then were challenged at 4 months postprime. For group D, mice were boosted at 7 months and challenged at 10 months after the prime. In group E, mice were boosted at 7 months and challenged at 16 months postprime. In study III, the heterologous boost immunizations were administered at either 3 and 4 months (group F) or 7 and 8 months following the initial E6-85 protein/adjuvant vaccination (Table 4). For study III, mice were challenged 1 month after the final booster immunization. For all of these studies, BCG vaccine was given at day 0.

FIG. 2.

Persistence of the MVA/IL-15/5Mtb vaccine-induced protective immune responses in the lung after an aerosol infection with M. tuberculosis. Mice were vaccinated subcutaneously (SC) with MVA/IL-15/5Mtb recombinant virus or wild-type MVA virus and intranasally (IN) with MVA/IL-15/5Mtb recombinant virus. As controls, other groups of mice remained nonvaccinated (naive) or were vaccinated subcutaneously with BCG. Two months after the initial vaccination, the mice were challenged with a low dose of M. tuberculosis and then sacrificed at 1 or 4 months postchallenge. Lung homogenates were plated on Middlebrook 7H11 plates, and mycobacterial CFU were counted 2 to 3 weeks later.

FIG. 3.

Vaccine-induced multifunctional T cells. After recovery of splenocytes from mice that had been vaccinated with BCG, the E6-85 adjuvant preparation, the E6-85/MVA/IL-15/5Mtb prime-boost regimen, or the adjuvant/MVA/IL-15/5Mtb (controls), the spleen cells were incubated overnight with BCG, stained the next day for CD4 and CD8 T-cell markers and cytokine expression, and then analyzed by multiparameter flow cytometry. Panels A (CD4) and B (CD8) show the frequency of T cells expressing either single or multiple cytokines. These data represent the mean responses + SEM for 5 individual mice.