Heterogeneity in lung macrophage control of Mycobacterium tuberculosis is modulated by T cells.

Following Mycobacterium tuberculosis infection, alveolar macrophages are initially infected but ineffectively restrict bacterial replication. The distribution of M. tuberculosis among different cell types in the lung changes with the onset of T cell immunity when the dominant infected cellular niche shifts from alveolar to monocyte-derived macrophages (MDM). We hypothesize that changes in bacterial distribution among different cell types is driven by differences in T cell recognition of infected cells and their subsequent activation of antimicrobial effector mechanisms. We show that CD4 and CD8 T cells efficiently eliminate M. tuberculosis infection in alveolar macrophages, but they have less impact on suppressing infection in MDM, which may be a bacterial niche. Importantly, CD4 T cell responses enhance MDM recruitment to the lung. Thus, the outcome of infection depends on the interaction between the T cell subset and the infected cell; both contribute to the resolution and persistence of the infection.

Intranasal multivalent adenoviral-vectored vaccine protects against replicating and dormant M.tb in conventional and humanized mice.

Viral-vectored vaccines are highly amenable for respiratory mucosal delivery as a means of inducing much-needed mucosal immunity at the point of pathogen entry. Unfortunately, current monovalent viral-vectored tuberculosis (TB) vaccine candidates have failed to demonstrate satisfactory clinical protective efficacy. As such, there is a need to develop next-generation viral-vectored TB vaccine strategies which incorporate both vaccine antigen design and delivery route. In this study, we have developed a trivalent chimpanzee adenoviral-vectored vaccine to provide protective immunity against pulmonary TB through targeting antigens linked to the three different growth phases (acute/chronic/dormancy) of Mycobacterium tuberculosis (M.tb) by expressing an acute replication-associated antigen, Ag85A, a chronically expressed virulence-associated antigen, TB10.4, and a dormancy/resuscitation-associated antigen, RpfB. Single-dose respiratory mucosal immunization with our trivalent vaccine induced robust, sustained tissue-resident multifunctional CD4+ and CD8+ T-cell responses within the lung tissues and airways, which were further quantitatively and qualitatively improved following boosting of subcutaneously BCG-primed hosts. Prophylactic and therapeutic immunization with this multivalent trivalent vaccine in conventional BALB/c mice provided significant protection against not only actively replicating M.tb bacilli but also dormant, non-replicating persisters. Importantly, when used as a booster, it also provided marked protection in the highly susceptible C3HeB/FeJ mice, and a single respiratory mucosal inoculation was capable of significant protection in a humanized mouse model. Our findings indicate the great potential of this next-generation TB vaccine strategy and support its further clinical development for both prophylactic and therapeutic applications.

Limited recognition of Mycobacterium tuberculosis-infected macrophages by polyclonal CD4 and CD8 T cells from the lungs of infected mice.

Immune responses following Mycobacterium tuberculosis (Mtb) infection or vaccination are frequently assessed by measuring T-cell recognition of crude Mtb antigens, recombinant proteins, or peptide epitopes. We previously showed that not all Mtb-specific T cells recognize Mtb-infected macrophages. Thus, an important question is what proportion of T cells elicited by Mtb infection recognize Mtb-infected macrophages. We address this question by developing a modified elispot assay using viable Mtb-infected macrophages, a low multiplicity of infection and purified T cells. In C57BL/6 mice, CD4 and CD8 T cells were classically MHC restricted. Comparable frequencies of T cells that recognize Mtb-infected macrophages were determined using interferon-γ elispot and intracellular cytokine staining, and lung CD4 T cells more sensitively recognized Mtb-infected macrophages than lung CD8 T cells. Compared to the relatively high frequencies of T cells specific for antigens such as ESAT-6 and TB10.4, low frequencies of total pulmonary T cells elicited by aerosolized Mtb infection recognize Mtb-infected macrophages. Finally, we demonstrate that BCG vaccination elicits T cells that recognize Mtb-infected macrophages. We propose that the frequency of T cells that recognize infected macrophages could correlate with protective immunity and may be an alternative approach to measuring T-cell responses to Mtb antigens.

Single-Dose Mucosal Immunotherapy With Chimpanzee Adenovirus-Based Vaccine Accelerates Tuberculosis Disease Control and Limits Its Rebound After Antibiotic Cessation.

BACKGROUND: The development of strategies to accelerate disease resolution and shorten antibiotic therapy is imperative in curbing the global tuberculosis epidemic. Therapeutic application of novel vaccines adjunct to antibiotics represents such a strategy. METHODS: By using a murine model of pulmonary tuberculosis (TB), we have investigated whether a single respiratory mucosal therapeutic delivery of a novel chimpanzee adenovirus-vectored vaccine expressing Ag85A (AdCh68Ag85A) accelerates TB disease control in conjunction with antibiotics and restricts pulmonary disease rebound after premature (nonsterilizing) antibiotic cessation. RESULTS: We find that immunotherapy via the respiratory mucosal, but not parenteral, route significantly accelerates pulmonary mycobacterial clearance, limits lung pathology, and restricts disease rebound after premature antibiotic cessation. We further show that vaccine-activated antigen-specific T cells, particularly CD8 T cells, in the lung play an important role in immunotherapeutic effects. CONCLUSIONS: Our results indicate that a single-dose respiratory mucosal immunotherapy with AdCh68Ag85A adjunct to antibiotic therapy has the potential to significantly accelerate disease control and shorten the duration of conventional treatment. Our study provides the proof of principle to support therapeutic applications of viral-vectored vaccines via the respiratory route.

Spray dried human and chimpanzee adenoviral-vectored vaccines are thermally stable and immunogenic in vivo.

Cold chain-free vaccine technologies are needed to ensure effective vaccine delivery and coverage, particularly in resource-poor countries. However, the immunogenicity and thermostability of spray dried live viral vector-based vaccines such as recombinant adenoviral-vectored vaccines remain to be investigated. To address this issue, we have spray dried human adenoviral (AdHu5)- and chimpanzee adenoviral (AdCh68)-vectored tuberculosis vaccines in a mannitol and dextran matrix. Spray dried powders containing these two vaccines display the morphologic and chemical properties desired for long-term thermostability and vaccination. Upon reconstitution, they effectively transfected the cells in vitro with relatively small losses in viral infectivity related to the spray drying process. Following in vivo vaccination, AdHu5- and AdCh68-vectored vaccines were as immunogenic as the conventional fresh, cryopreserved liquid vaccine samples. Of importance, even after cold chain-free storage, at ambient temperatures and relatively low humidity for 30 and 90days, the vaccines retained their in vivo immunogenicity, while the liquid vaccine samples stored under the same conditions lost their immune-activating capability almost entirely. Our results support further development of our spray drying technologies for generating thermally stable adenoviral-vectored and other viral-vectored vaccines.

Age at Mycobacterium bovis BCG Priming Has Limited Impact on Anti-Tuberculosis Immunity Boosted by Respiratory Mucosal AdHu5Ag85A Immunization in a Murine Model.

Tuberculosis (TB) remains a global pandemic despite the use of Bacillus Calmette-Guérin (BCG) vaccine, partly because BCG fails to effectively control adult pulmonary TB. The introduction of novel boost vaccines such as the human Adenovirus 5-vectored AdHu5Ag85A could improve and prolong the protective immunity of BCG immunization. Age at which BCG immunization is implemented varies greatly worldwide, and research is ongoing to discover the optimal stage during childhood to administer the vaccine, as well as when to boost the immune response with potential novel vaccines. Using a murine model of subcutaneous BCG immunization followed by intranasal AdHu5Ag85A boosting, we investigated the impact of age at BCG immunization on protective efficacy of BCG prime and AdHu5Ag85A boost immunization-mediated protection. Our results showed that age at parenteral BCG priming has limited impact on the efficacy of BCG prime-AdHu5Ag85A respiratory mucosal boost immunization-enhanced protection. However, when BCG immunization was delayed until the maturity of the immune system, longer sustained memory T cells were generated and resulted in enhanced boosting effect on T cells of AdHu5Ag85A respiratory mucosal immunization. Our findings hold implications for the design of new TB immunization protocols for humans.