Immunization with a DNA vaccine cocktail protects mice lacking CD4 cells against an aerogenic infection with Mycobacterium tuberculosis

Tuberculosis (TB) is the most common opportunistic disease and a potentially fatal complication among immunocompromised individuals infected with human immunodeficiency virus (HIV). Effective vaccination against TB in persons with HIV has been considered unlikely because of the central role that CD4 cells play in controlling tuberculous infections. Here we show that the vaccination of CD8^−/− mice with a TB DNA vaccine cocktail did not significantly enhance protective responses to a Mycobacterium tuberculosis infection. In contrast, immunization with a DNA vaccine cocktail or with the current TB vaccine, Mycobacterium bovis BCG, induced considerable antituberculosis protective immunity in immune-deficient mice lacking CD4 cells. In vaccinated CD4^−/− animals, substantially reduced bacterial burdens in organs and much improved lung pathology were seen 1 month after an aerogenic M. tuberculosis challenge. Importantly, the postchallenge mean times to death of vaccinated CD4^−/− mice were significantly extended (mean with DNA cocktail, 172 ± 7 days; mean with BCG, 156 ± 22 days) compared to that of naïve CD4^−/− mice (33 ± 6 days). Furthermore, the treatment of DNA-vaccinated CD4^−/− mice with an anti-CD8 or anti-gamma interferon (IFN-γ) antibody significantly reduced the effect of immunization, and neither IFN-γ^−/− nor tumor necrosis factor receptor-deficient mice were protected by DNA immunization; therefore, the primary vaccine-induced protective mechanism in these immune-deficient mice likely involves the secretion of cytokines from activated CD8 cells. The substantial CD8-mediated protective immunity that was generated in the absence of CD4 cells suggests that it may be possible to develop effective TB vaccines for use in HIV-infected populations.Tuberculosis (TB) remains a significant global threat to public health, with two million people dying from Mycobacterium tuberculosis infections each year and eight million cases of TB developing annually (6). The increasing linkage of TB with the human immunodeficiency virus (HIV) pandemic has magnified this tragedy in the past decade. The World Health Organization estimates that at least six million people worldwide are coinfected with M. tuberculosis and HIV (18). The HIV-M. tuberculosis coinfection rates exceed 5% in eight African countries, and in South Africa alone two million adults are coinfected (9). In these developing countries, TB is the most prevalent cause of morbidity and mortality for HIV-positive adults. In contrast to immunocompetent individuals, who have a 10% lifetime risk of disease following TB infection, persons coinfected with M. tuberculosis and HIV have a nearly 10% annual risk of developing disease.A primary reason for the continued failure to curb the global TB epidemic is the absence of a highly effective vaccine. Although the current TB vaccine, Mycobacterium bovis BCG, has been widely used for decades, its efficacy in controlled clinical trials has been extremely variable and its value in protecting against the most prevalent form of the disease, adult pulmonary TB, is doubtful (7). Moreover, the effectiveness of BCG in preventing TB in HIV-infected individuals is uncertain. Since BCG is a live vaccine (attenuated but not avirulent) and since clinical cases of reactivated BCG have been reported for HIV-infected persons, BCG vaccination has not been indicated for immunocompromised individuals (33).The development of a new vaccine against TB for use in HIV-positive persons has been considered unlikely because of the presumed essential roles that CD4 cells play in controlling TB infections. Mice that lack CD4 cells or that are aberrant in major histocompatibility complex class II presentation are extremely sensitive to a TB challenge and cannot effectively control acute TB infections (3, 13). The greatly enhanced susceptibility of HIV patients to both primary and reactivated disease argues that CD4 cells also play a prominent role in protective immune responses against human TB. The primary antituberculosis effector function of CD4⁺ T cells involves the production and secretion of cytokines which activate macrophages to control or eliminate the intracellular bacilli (13). In addition to this more direct role of CD4 cells in limiting tuberculous infections, CD4 cells also assist in the development of primary CD8 T-cell responses (23). CD4 cells stimulate professional antigen-presenting cells (APCs) primarily via CD40-CD40 ligand interactions; these activated APCs efficiently costimulate antigen-specific naïve CD8 T cells. Additionally, cytokine production from CD4 cells enhances the proliferation of primed CD8 cells.Despite the critical immune functions of CD4 cells, recent studies have demonstrated that protective immunity to pathogens can be generated in the absence of CD4 cells. For instance, substantial CD8 T-cell responses to the influenza virus, lymphocytic choriomeningitis virus (LCMV), and pathogenic fungi can occur in mice lacking CD4 cells (17, 26, 34, 35). In a Listeria monocytogenes model, similar levels of activated CD8 T cells were detected in CD4^−/− and wild-type (WT) C57BL/6 mice after an infection. Importantly, the epitope-specific CD8 T cells that were generated established long-term memory in CD4^−/− mice and were capable of producing an effective recall response (21). Under circumstances in which CD4 help is not required for the generation of effective CD8 T-cell responses, alternate pathways for the activation of APCs exist. For influenza virus, a direct infection of dendritic cells results in the upregulation of costimulatory molecules. For bacteria, dendritic cells can be activated through recognition by Toll-like receptors (TLRs) of bacterial products such as peptidoglycan, glycolipids, and lipoproteins (2, 29, 30).Based on these findings in other immune-deficient disease models, we evaluated whether cell-mediated antituberculosis protective immunity could be induced in mice lacking CD4 cells by immunization with a DNA vaccine cocktail or with BCG. Previously, members of our laboratory generated a DNA cocktail that expressed mycobacterial proteins fused at the N terminus to ubiquitin (UB), a eukaryotic intracellular targeting sequence (10, 11). These UB-conjugated proteins were designed to enhance major histocompatibility complex class I presentation. In studies using WT C57BL/6 mice, it was shown that vaccination with this combination was more effective than immunization with the individual single components and that the sustained protective immunity induced by the plasmid mixture was equivalent to the level of protection elicited by the BCG vaccine (10, 11). We report here that substantial antituberculosis protective immunity can be induced in the absence of CD4 cells. Immunization of CD4^−/− mice with either the DNA vaccine cocktail or BCG leads to significantly decreased lung and spleen bacterial burdens and much improved lung pathology, relative to naïve controls, after an aerogenic M. tuberculosis infection. Most importantly, the survival of the vaccinated animals was substantially extended compared to nonimmunized CD4^−/− controls.