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Aside from centrally induced trained immunity in the bone marrow (BM) and peripheral blood by parenteral vaccination or infection, evidence indicates that mucosal-resident innate immune memory can develop via a local inflammatory pathway following mucosal exposure. However, whether mucosal-resident innate memory results from integrating distally generated immunological signals following parenteral vaccination/infection is unclear. Here we show that subcutaneous Bacillus Calmette–Guérin (BCG) vaccination can induce memory alveolar macrophages (AMs) and trained immunity in the lung. Although parenteral BCG vaccination trains BM progenitors and circulating monocytes, induction of memory AMs is independent of circulating monocytes. Rather, parenteral BCG vaccination, via mycobacterial dissemination, causes a time-dependent alteration in the intestinal microbiome, barrier function and microbial metabolites, and subsequent changes in circulating and lung metabolites, leading to the induction of memory macrophages and trained immunity in the lung. These data identify an intestinal microbiota-mediated pathway for innate immune memory development at distal mucosal tissues and have implications for the development of next-generation vaccine strategies against respiratory pathogens.

Purpose: Intratracheal delivery and consistent dosing of dry powder vaccines is especially challenging in mice. To address this issue, device design of positive pressure dosators and actuation parameters were assessed for their impacts on powder flowability and in vivo dry powder delivery. Methods: A chamber-loading dosator assembled with stainless-steel, polypropylene or polytetrafluoroethylene needle-tips was used to determine optimal actuation parameters. Powder loading methods including tamp-loading, chamber-loading and pipette tip-loading were compared to assess performance of the dosator delivery device in mice. Results: Available dose was highest (45%) with a stainless-steel tip loaded with an optimal mass and syringe air volume, primarily due to the ability of this configuration to dissipate static charge. However, this tip encouraged more agglomeration along its flow path in the presence of humidity and was too rigid for intubation of mice compared to a more flexible polypropylene tip. Using optimized actuation parameters, the polypropylene pipette tip-loading dosator achieved an acceptable in vivo emitted dose of 50% in mice. After administering two doses of a spray dried adenovirus encapsulated in mannitol-dextran, high bioactivity was observed in excised mouse lung tissue three days post-infection. Conclusions: This proof-of-concept study demonstrates for the first time that intratracheal delivery of a thermally stable, viral-vectored dry powder can achieve equivalent bioactivity to the same powder, reconstituted and delivered intratracheally. This work may guide the design and device selection process for murine intratracheal delivery of dry powder vaccines to help progress this promising area of inhalable therapeutics.

Bacillus Calmette-Guérin (BCG) still remains the only licensed vaccine for TB and has been shown to provide nonspecific protection against unrelated pathogens. This has been attributed to the ability of BCG to modulate the innate immune system, known as trained innate immunity (TII). TII is associated with innate immune cells being in a hyper-responsive state leading to enhanced host defense against heterologous infections. Both epidemiological evidence and prospective studies demonstrate cutaneous BCG vaccine-induced TII provides enhanced innate protection against heterologous pathogens. Regardless of the extensive amounts of progress made thus far, the effect of cutaneous BCG vaccination against heterologous respiratory bacterial infections and the underlying mechanisms remain unknown. Here we show for the first time that s.c BCG vaccine-induced TII provides enhanced heterologous innate protection against pulmonary S. pneumoniae infection. We further demonstrate that this enhanced innate protection is mediated by accelerated neutrophilia in the lung and is independent of centrally trained circulating monocytes. New insight from this study will help design novel effective vaccination strategies against unrelated respiratory bacterial pathogens.

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis (TB), is responsible for millions of infections and deaths annually. Decades of TB vaccine development have focused on adaptive T cell immunity, whereas the importance of innate immune contributions toward vaccine efficacy has only recently been recognized. Airway macrophages (AwM) are the predominant host cell during early pulmonary M. tuberculosis infection and, therefore, represent attractive targets for vaccine-mediated immunity. We have demonstrated that respiratory mucosal immunization with a viral-vectored vaccine imprints AwM, conferring enhanced protection against heterologous bacterial challenge. However, it is unknown if innate immune memory also protects against M. tuberculosis. In this study, by using a murine model, we detail whether respiratory mucosal TB vaccination profoundly alters the airway innate immune landscape associated with AwM prior to M. tuberculosis exposure and whether such AwM play a critical role in host defense against M. tuberculosis infection. Our study reveals an important role of AwM in innate immune protection in early stages of M. tuberculosis infection in the lung.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the most formidable challenge to humanity in a century. It is widely believed that prepandemic normalcy will never return until a safe and effective vaccine strategy becomes available and a global vaccination programme is implemented successfully. Here, we discuss the immunological principles that need to be taken into consideration in the development of COVID-19 vaccine strategies. On the basis of these principles, we examine the current COVID-19 vaccine candidates, their strengths and potential shortfalls, and make inferences about their chances of success. Finally, we discuss the scientific and practical challenges that will be faced in the process of developing a successful vaccine and the ways in which COVID-19 vaccine strategies may evolve over the next few years., This Review outlines the guiding immunological principles for the design of coronavirus disease 2019 (COVID-19) vaccine strategies and analyses the current COVID-19 vaccine landscape and the challenges ahead.

In the past few years, our understanding of immunological memory has evolved remarkably due to a growing body of new knowledge in innate immune memory and immunity. Immunological memory now encompasses both innate and adaptive immune memory. The hypo-reactive and hyper-reactive types of innate immune memory lead to a suppressed and enhanced innate immune protective outcome, respectively. The latter is also named trained innate immunity (TII). The emerging information on innate immune memory has not only shed new light on the mechanisms of host defense but is also revolutionizing our long-held view of vaccination and vaccine strategies. Our current review will examine recent progress and knowledge gaps in innate immune memory with a focus on tissue-resident Mϕs, particularly lung Mϕs, and their relationship to local antimicrobial innate immunity. We will also discuss the impact of innate immune memory and TII on our understanding of vaccine concept and strategies and the significance of respiratory mucosal route of vaccination against respiratory pathogens.

SummaryBackgroundAdenoviral (Ad)-vectored vaccines are typically administered via intramuscular injection to humans, but this route of delivery is unable to induce respiratory mucosal immunity which requires respiratory mucosal route of vaccination. However, inhaled aerosol delivery of Ad-vectored vaccines has remained poorly characterized and its ability to induce respiratory mucosal immunity in humans is still unknown. The goal of our study was to evaluate and compare the safety and immunogenicity of a human serotype 5 Ad-based tuberculosis (TB) vaccine (AdHu5Ag85A) delivered to healthy humans via inhaled aerosol or intramuscular injection.MethodsIn this open-labeled phase 1b trial, 31 healthy adults between 18 and 55 years of age with a history of BCG vaccination were enrolled at McMaster University Medical Centre, Hamilton, Ontario, Canada. AdHu5Ag85A was administered by a single-dose aerosol using the Aeroneb® Solo Vibrating Mesh Nebulizer or by intramuscular (IM) injection; 11 in the low dose (LD, 1×106 PFU) aerosol group, 11 in the high dose (HD, 2×106 PFU) aerosol group and 9 in the IM (1×108 PFU) group. The primary outcome was safety of a single administration of vaccine delivered to the respiratory tract by aerosol or by IM injection. The vaccine-related local and systemic adverse events were collected from participants from a self-completed diary for 14 days after vaccination and at scheduled follow-up visits. Routine laboratory biochemical and haematological tests were measured at 2, 4 and 12 weeks after vaccination and lung function was measured at 2, 4, 8 and 12 weeks after vaccination. The secondary outcome was comparison of immunogenicity among the different routes and aerosol dose groups. Immunogenicity to aerosol or IM vaccination was measured both in the peripheral blood and bronchoalveolar lavage samples by Luminex, and cell surface and intracellular cytokine immunostaining. Anti-AdHu5 antibodies and neutralization titers were determined before and after vaccination using ELISA and bioassay, respectively. This trial is registered with ClinicalTrial.gov, NCT02337270.ResultsThe aerosol droplets generated by Aeroneb® Solo Nebulizer were mostly InterpretationInhaled aerosol delivery of Ad-vectored vaccine is a safe, economical and superior way to elicit respiratory mucosal immunity. The results of this study encourage further development of aerosol vaccine strategies against not only TB but also other respiratory pathogens including COVID-19.FundingThe Canadian Institutes for Health Research and the Natural Sciences and Engineering Research Council of Canada.Research in contextEvidence before this studyWe searched PubMed for published research articles without language or date restrictions by using search terms “adenovirus”, “aerosol” and “clinical trial” or “virus”, “vaccine”, “aerosol”, and “clinical trial”. Our search results indicate that adenoviral-vectored vaccine has rarely been delivered via inhaled aerosol into the respiratory tract of healthy humans. Adenoviral-vectored TB or HIV vaccine was delivered via inhaled aerosol to non-human primates, providing support for its further development for human application. An aerosolized adenoviral vector expressing CFTR was tested as a gene replacement therapeutic in cystic fibrosis patients. Recently, although an adenoviral-vectored COVID-19 vaccine was delivered via aerosol to humans, there were no evidence presented that it induced local mucosal immunity. Given the increased recognition of its value, inhaled aerosol has been explored in humans with non-adenoviral, viral vaccines against respiratory infections of global importance including measles and TB. However, since different aerosol technologies were used in these studies and aerosol characteristics including delivery efficiency vary according to the viral platform, aerosol delivery technology and its efficiency in inducing respiratory mucosal immunity remain to be established for administering adenoviral-vectored vaccine to healthy humans.Added value of this studyThis represents the first study to demonstrate the characteristics of a single-dose aerosolized human serotype 5 adenoviral-vectored vaccine, and its safety and immunogenicity in BCG-vaccinated healthy humans. It is also the first to show the superiority of inhaled aerosol immunization with this type of vaccine platform, over intramuscular (IM) route of immunization, in inducing respiratory mucosal immunity. Robust adaptive immune memory responses were induced in the respiratory tract with an aerosol vaccine dose up to 100 times smaller than the dose for IM immunization. Besides local mucosal immunity induced by aerosol immunization, it also induces levels of systemic immunity comparable to those by IM immunization. Also for the first time, we show that contrast to IM immunization, aerosol immunization does not increase either local or systemic anti-adenoviral neutralizing antibodies.Implications of all the available evidenceCollectively our findings show the technical feasibility, safety and potency of needle/pain-free delivery of a recombinant adenoviral-vectored vaccine to the respiratory tract of healthy adults. This vaccine strategy differs from parenteral route of immunization in its potency to elicit much desired respiratory mucosal immunity consisting of trained macrophages and tissue-resident memory T cells. The study provides the proof of concept to endorse inhaled aerosol vaccine strategies against pulmonary TB. Of further importance, as a number of currently authorized COVID-19 vaccines are also adenoviral-vectored, our study offers important technological details and scientific rationale for the development of inhalable next-generation COVID-19 vaccines aiming to induce all-around protective respiratory mucosal immunity in humans.

SummaryThe emerging SARS-CoV-2 variants of concern (VOC) increasingly threaten the effectiveness of current first-generation COVID-19 vaccines that are administered intramuscularly and are designed to only target the spike protein. There is thus a pressing need to develop next-generation vaccine strategies to provide more broad and long-lasting protection. By using adenoviral vectors (Ad) of human and chimpanzee origin, we developed Ad-vectored trivalent COVID-19 vaccines expressing Spike-1, Nucleocapsid and RdRp antigens and evaluated them following single-dose intramuscular or intranasal immunization in murine models. We show that respiratory mucosal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the three-arm immunity, consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells, and mucosal trained innate immunity. We further show that single-dose intranasal immunization provides robust protection against not only the ancestral strain of SARS-CoV-2, but also two emerging VOC, B.1.1.7 and B.1.351. Our findings indicate that single-dose respiratory mucosal delivery of an Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy against current and future VOC. This strategy has great potential to be used not only to boost first-generation vaccine-induced immunity but also to expand the breadth of protective T cell immunity at the respiratory mucosa.

The emerging SARS-CoV-2 variants of concern (VOCs) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing spike-1, nucleocapsid, and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC.

The COVID‐19 pandemic has unfolded to be the most challenging global health crisis in a century. In 11 months since its first emergence, according to WHO, the causative infectious agent SARS‐CoV‐2 has infected more than 100 million people and claimed more than 2.15 million lives worldwide. Moreover, the world has raced to understand the virus and natural immunity and to develop vaccines. Thus, within a short 11 months a number of highly promising COVID‐19 vaccines were developed at an unprecedented speed and are now being deployed via emergency use authorization for immunization. Although a considerable number of review contributions are being published, all of them attempt to capture only a specific aspect of COVID‐19 or its therapeutic approaches based on ever‐expanding information. Here, we provide a comprehensive overview to conceptually thread together the latest information on global epidemiology and mitigation strategies, clinical features, viral pathogenesis and immune responses, and the current state of vaccine development.

Considering the substantive potential benefits of thermally stable dry powder vaccines to public health, causes for inactivation of their sensitive viral vectors during preparation require intensive study. The focus of this work was atomization of suspensions containing encapsulating excipients and a human type 5 adenovirus, involving a detailed investigation of shear stresses in the nozzle of a spray dryer. Samples were sprayed at 25 °C into falcon tubes and immediately evaluated for viral activity by in vitro testing, minimizing the confounding of thermal effects on the deactivation of the virus, although interfacial stresses could not be decoupled from shear stresses. Despite the expectations of only virus deactivation with ever-increasing shear stresses in the spray nozzle, some conditions were found to show better activity than the positive control, leading to investigations of viral aggregation. It was found that the adenovirus experienced minor aggregation when mixed with the excipient solutions, which was reversed by subjecting samples to moderate shear conditions in the spray nozzle. At very high shear rates, the activity diminished again because of damage to the viral capsid fibers, which also led to the production of new aggregates after atomization. Despite these findings, activity losses caused by shear were small compared to the overall spray drying process loss. However, formulation composition, solution viscosity, and process conditions should be considered carefully for optimization because of their impact on aggregation. This is the first known report comparing shear, aggregation, and biological activity loss during the atomization step of spray drying viral vaccines.

Tuberculosis (TB) is one of the major global health concerns. There has been a lack of an effective vaccine strategy. The Bacillus Calmette-Guerin (BCG), the only licensed vaccine against TB, is not effective against adult pulmonary TB, the highly contagious form of TB. In the past two decades or so, many novel TB vaccines have been developed, and some of them were evaluated in clinical trials. However, the lack of validated immune correlates to assess the clinical relevance of novel TB vaccines before their entry into costly efficacy trials is a huge challenge to the field of TB vaccine development. Here we describe a general protocol for the procedure of a systematic immunological approach that can be utilized to better assess the clinical relevance of TB vaccine-activated T cells in early phases of clinical studies.

Tuberculosis (TB) is one of the major global health concerns. There has been a lack of an effective vaccine strategy. The Bacillus Calmette-Guerin (BCG), the only licensed vaccine against TB, is not effective against adult pulmonary TB, the highly contagious form of TB. In the past two decades or so, many novel TB vaccines have been developed, and some of them were evaluated in clinical trials. However, the lack of validated immune correlates to assess the clinical relevance of novel TB vaccines before their entry into costly efficacy trials is a huge challenge to the field of TB vaccine development. Here we describe a general protocol for the procedure of a systematic immunological approach that can be utilized to better assess the clinical relevance of TB vaccine-activated T cells in early phases of clinical studies.

Despite some major progress made in developing tuberculosis (TB) vaccine strategies, with a dozen novel vaccines currently in the clinical pipeline, the world is still missing an effective TB vaccine. This questions whether any major breakthroughs can be achieved without making a drastic departure from the current strategy, which creates a state of ‘near-natural immunity', imitating the natural immunity developed after Mycobacterium tuberculosis ( Mtb ) infection. Here, we argue instead that mounting evidence suggests an effective strategy ought to induce a state of all-around ‘un-natural' immunity comprising trained innate immunity (TII), tissue-resident memory T cells (T RM ), and anti- Mtb surface antibodies in the lung. Thus, here we summarize the latest information, thinking, and development in the field of TB and vaccines.

Although most novel tuberculosis (TB) vaccines are designed for delivery via the muscle or skin for enhanced protection in the lung, it has remained poorly understood whether systemic vaccine-induced memory T cells can readily home to the lung mucosa prior to and shortly after pathogen exposure. We have investigated this issue by using a model of parenteral TB immunization and intravascular immunostaining. We find that systemically induced memory T cells are restricted to the blood vessels in the lung, unable to populate either the lung parenchymal tissue or the airway under homeostatic conditions. We further find that after pulmonary TB infection, it still takes many days before such T cells can enter the lung parenchymal tissue and airway. We have identified the acquisition of CXCR3 expression by circulating T cells to be critical for their entry to these lung mucosal compartments. Our findings offer new insights into mucosal T cell biology and have important implications in vaccine strategies against pulmonary TB and other intracellular infections in the lung.

Background The translation of preclinically promising novel tuberculosis vaccines to ultimate human applications has been challenged by the lack of animal models with an immune system equivalent to the human immune system in its genetic diversity and level of susceptibility to tuberculosis. Methods We have developed a humanized mice (Hu-mice) tuberculosis model system to investigate the clinical relevance of a novel virus-vectored (VV) tuberculosis vaccine administered via respiratory mucosal or parenteral route. Results We find that VV vaccine activates T cells in Hu-mice as it does in human vaccinees. The respiratory mucosal route for delivery of VV vaccine in Hu-mice, but not the parenteral route, significantly reduces the humanlike lung tuberculosis outcomes in a human T-cell-dependent manner. Conclusions Our results suggest that the Hu-mouse can be used to predict the protective efficacy of novel tuberculosis vaccines/strategies before they proceed to large, expensive human trials. This new vaccine testing system will facilitate the global pace of clinical tuberculosis vaccine development.

Tissue-resident memory T cells (TRM) are critical to host defense at mucosal tissue sites. However, the parenteral route of immunization as the most commonly used immunization route in practice is not effective in inducing mucosal TRM cells particularly in the lung. While various respiratory mucosal (RM)-pull strategies are exploited to mobilize parenteral vaccine-primed T cells into the lung, whether such RM-pull strategies can all or differentially induce Ag-specific TRM cells in the lung remains unclear. Here, we have addressed this issue by using a parenteral TB vaccine-primed and RM-pull model. We show that both Ag-independent and Ag-dependent RM-pull strategies are able to mobilize Ag-specific CD8 T cells into the lung. However, only the RM-pull strategy with cognate antigens can induce robust Ag-specific CD8 TRM cells in the lung. We also show that the cognate Ag-based RM-pull strategy is the most effective in inducing TRM cells when carried out during the memory phase, as opposed to the effector phase, of T cell responses to parenteral prime vaccination. We further find that cognate Ag-induced CD4 T cells play an important role in the development of CD8 TRM cells in the lung. Our study holds implications in developing effective vaccine strategies against respiratory pathogens.

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.

BACKGROUND Whether a candidate tuberculosis vaccine induces clinically relevant protective T-cell repertoires in humans will not be known until the completion of costly efficacy clinical trials. METHODS We have developed an integrated immunologic approach to investigate the clinical relevance of T cells induced by a novel tuberculosis vaccine in a phase 1 trial. This approach consists of screening for likely dominant T-cell epitopes, establishing antigen-specific memory T-cell lines for identifying CD8+ and CD4+ T-cell epitopes, determining the ability of vaccine-induced T cells to inhibit mycobacterial growth in infected cells, and examining the genetic diversity of HLA recognition and the clinical relevance of identified T-cell epitopes. RESULTS A single-dose immunization in BCG-primed adults with an adenovirus-based tuberculosis vaccine elicits a repertoire of memory T cells capable of recognizing multiple Ag85A epitopes. These T cells are polyfunctional and cytotoxic and can inhibit mycobacterial growth in infected target cells. Some identified T-cell epitopes are promiscuous and recognizable by the common HLA alleles. These epitopes are clinically relevant to the epitopes identified in people with latent Mycobacterium tuberculosis infection and treated patients with tuberculosis. CONCLUSIONS These data support further clinical development of this candidate vaccine. Our approach helps fill the gap in clinical tuberculosis vaccine development.

Pulmonary tuberculosis (TB) remains to be a major global health problem despite many decades of parenteral use of Bacillus Calmette-Guérin (BCG) vaccine. Developing safe and effective respiratory mucosal TB vaccines represents a unique challenge. Over the past decade or so, the human serotype 5 adenovirus (AdHu5)-based TB vaccine has emerged as one of the most promising candidates based on a plethora of preclinical and early clinical studies. However, anti-AdHu5 immunity widely present in the lung of humans poses a serious gap and limitation to its real-world applications. In this study we have developed a novel chimpanzee adenovirus 68 (AdCh68)-vectored TB vaccine amenable to the respiratory route of vaccination. We have evaluated AdCh68-based TB vaccine for its safety, T-cell immunogenicity, and protective efficacy in relevant animal models of human pulmonary TB with or without parenteral BCG priming. We have also compared AdCh68-based TB vaccine with its AdHu5 counterpart in both naive animals and those with preexisting anti-AdHu5 immunity in the lung. We provide compelling evidence that AdCh68-based TB vaccine is not only safe when delivered to the respiratory tract but, importantly, is also superior to its AdHu5 counterpart in induction of T-cell responses and immune protection, and limiting lung immunopathology in the presence of preexisting anti-AdHu5 immunity in the lung. Our findings thus suggest AdCh68-based TB vaccine to be an ideal candidate for respiratory mucosal immunization, endorsing its further clinical development in humans.

Innate immune memory is an emerging area of research. However, innate immune memory at major mucosal sites remains poorly understood. Here, we show that respiratory viral infection induces long-lasting memory alveolar macrophages (AMs). Memory AMs are programed to express high MHC II, a defense-ready gene signature, and increased glycolytic metabolism, and produce, upon re-stimulation, neutrophil chemokines. Using a multitude of approaches, we reveal that the priming, but not maintenance, of memory AMs requires the help from effector CD8 T cells. T cells jump-start this process via IFN-γ production. We further find that formation and maintenance of memory AMs are independent of monocytes or bone marrow progenitors. Finally, we demonstrate that memory AMs are poised for robust trained immunity against bacterial infection in the lung via rapid induction of chemokines and neutrophilia. Our study thus establishes a new paradigm of immunological memory formation whereby adaptive T-lymphocytes render innate memory of mucosal-associated macrophages.

Emerging evidence suggests a role of B cells in host defense against primary pulmonary tuberculosis (TB). However, the role of B cells in TB vaccine–induced protective T cell immunity still remains unknown. Using a viral-vectored model TB vaccine and a number of experimental approaches, we have investigated the role of B cells in respiratory mucosal vaccine–induced T cell responses and protection against pulmonary TB. We found that respiratory mucosal vaccination activated Ag-specific B cell responses. Whereas respiratory mucosal vaccination elicited Ag-specific T cell responses in the airway and lung interstitium of genetic B cell–deficient (Jh−/− knockout [KO]) mice, the levels of airway T cell responses were lower than in wild-type hosts, which were associated with suboptimal protection against pulmonary Mycobacterium tuberculosis challenge. However, mucosal vaccination induced T cell responses in the airway and lung interstitium and protection in B cell–depleted wild-type mice to a similar extent as in B cell–competent hosts. Furthermore, by using an adoptive cell transfer approach, reconstitution of B cells in vaccinated Jh−/− KO mice did not enhance anti-TB protection. Moreover, respiratory mucosal vaccine–activated T cells alone were able to enhance anti-TB protection in SCID mice, and the transfer of vaccine-primed B cells alongside T cells did not further enhance such protection. Alternatively, adoptively transferring vaccine-primed T cells from Jh−/− KO mice into SCID mice only provided suboptimal protection. These data together suggest that B cells play a minimal role, and highlight a central role by T cells, in respiratory mucosal vaccine–induced protective immunity against M. tuberculosis.

Pulmonary TB remains a leading global health issue, but the current Bacille Calmette-Guérin (BCG) vaccine fails to control it effectively. Much effort has gone into developing safe and effective boost vaccine candidates for use after the BCG prime vaccination. To date, almost all the lead candidates are being evaluated clinically via a parenteral route. Abundant experimental evidence suggests that parenteral boosting with a virus-based vaccine is much less effective than respiratory mucosal boosting, because the former fails to activate a type of T cell capable of rapidly transmigrating into the airway luminal space in the early phase of the Mycobacterium tuberculosis infection. The next few years will determine whether parenteral boosting with some of the lead vaccine candidates, particularly the protein-based vaccines, improves protection in humans over that by BCG. Much effort is needed to develop respiratory mucosal boost vaccines and to identify the reliable immune protective correlates in humans.

Interaction of mycobacteria with the host leads to retarded expression of T helper cell type 1 (Th1) immunity in the lung. However, the immune mechanisms remain poorly understood. Using in vivo and in vitro models of Mycobacterium tuberculosis (M. tb) infection, we find the immunoadaptor DAP12 (DNAX-activating protein of 12 kDa) in antigen-presenting cells (APCs) to be critically involved in this process. Upon infection of APCs, DAP12 is required for IRAK-M (interleukin-1 receptor-associated kinase M) expression, which in turn induces interleukin-10 (IL-10) and an immune-suppressed phenotype of APCs, thus leading to suppressed Th1 cell activation. Lack of DAP12 reduces APC IL-10 production and increases their Th1 cell-activating capability, resulting in expedited Th1 responses and enhanced protection. On the other hand, adoptively transferred DAP12-competent APCs suppress Th1 cell activation within DAP12-deficient hosts, and blockade of IL-10 aborts the ability of DAP12-competent APCs to suppress Th1 activation. Our study identifies the DAP12/IRAK-M/IL-10 to be a novel molecular pathway in APCs exploited by mycobacterial pathogens, allowing infection a foothold in the lung.

The immune mechanisms underlying delayed induction of Th1-type immunity in the lungs following pulmonary mycobacterial infection remain poorly understood. We have herein investigated the underlying immune mechanisms for such delayed responses and whether a selected innate immune-modulating strategy can accelerate Th1-type responses. We have found that, in the early stage of pulmonary infection with attenuated Mycobacterium tuberculosis (M.tb H37Ra), the levels of infection in the lung continue to increase logarithmically until days 14 and 21 postinfection in C57BL/6 mice. The activation of innate immune responses, particularly DCs, in the lung is delayed. This results in a delay in the subsequent downstream immune responses including the migration of antigen-bearing DCs to the draining lymph node (dLN), the Th1-cell priming in dLN, and the recruitment of Th1 cells to the lung. However, single lung mucosal exposure to the TLR agonist FimH postinfection is able to accelerate protective Th1-type immunity via facilitating DC migration to the lung and draining lymph nodes, enhancing DC antigen presentation and Th1-cell priming. These findings hold implications for the development of immunotherapeutic and vaccination strategies and suggest that enhancement of early innate immune activation is a viable option for improving Th1-type immunity against pulmonary mycobacterial diseases.

Background. One-third of the world’s population has latent infection with Mycobacterium tuberculosis, and 10%–15% of cases of reactivation occur at extrapulmonary sites without active pulmonary tuberculosis. Methods. To establish the frequency and location of mycobacterial DNA, organ specimens from 49 individuals who died from causes other than tuberculosis were studied by means of polymerase chain reaction (PCR), PCR plus DNA hybridization, in situ PCR, real-time PCR, and spoligotyping. Results. Lung specimens from most subjects (36) were positive for M. tuberculosis, as were specimens from the spleen (from 35 subjects), kidney (from 34), and liver (from 33). By in situ PCR, mycobacterial DNA was found in endothelium, pneumocytes, and macrophages from the lung and in Bowman’s parietal cells and convoluted proximal tubules from the kidney. In spleen, macrophages and sinusoidal endothelial cells were positive, whereas in liver, Kupffer cells and sinusoidal endothelium were commonly positive. Spoligotyping of 54 pulmonary and extrapulmonary positive tissues from 30 subjects showed 43 different genotypes, including 36 orphan types. To confirm the viability of mycobacteria, 10 positive tissue samples were selected for isolation of mycobacterial RNA. All samples showed 16S ribosomal RNA expression, while 8 and 4 samples showed expression of the latent infection genes encoding isocitrate lyase and α-crystallin, respectively. Conclusions. M. tuberculosis persists in several sites and cell types that might constitute reservoirs that can reactivate infection, producing extrapulmonary tuberculosis without lung involvement.

The granuloma, a hallmark of host defense against pulmonary mycobacterial infection, has long been believed to be an active type 1 immune environment. However, the mechanisms regarding why granuloma fails to eliminate mycobacteria even in immune-competent hosts, have remained largely unclear. By using a model of pulmonary Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection, we have addressed this issue by comparing the immune responses within the airway luminal and granuloma compartments. We found that despite having a similar immune cellular profile to that in the airway lumen, the granuloma displayed severely suppressed type 1 immune cytokine but enhanced chemokine responses. Both antigen-presenting cells (APCs) and T cells in granuloma produced fewer type 1 immune molecules including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and nitric oxide. As a result, the granuloma APCs developed a reduced capacity to phagocytose mycobacteria and to induce T-cell proliferation. To examine the molecular mechanisms, we compared the levels of immune suppressive cytokine IL-10 in the airway lumen and granuloma and found that both granuloma APCs and T cells produced much more IL-10. Thus, IL-10 deficiency restored type 1 immune activation within the granuloma while having a minimal effect within the airway lumen. Hence, our study provides the first experimental evidence that, contrary to the conventional belief, the BCG-induced lung granuloma represents a symbiotic host-microbe microenvironment characterized by suppressed type 1 immune activation.

Protection against pulmonary tuberculosis (TB) by vaccination is often ascribed to the presence of TB-reactive T cells in the lung before infection. Challenging this view, new studies analyzing vaccine-induced T cells in various tissue compartments after parenteral immunization suggest a poor correlation between the presence of anti-TB T cells in the lung interstitium and spleen before Mycobacterium tuberculosis exposure and protection. In contrast, respiratory mucosal immunization leads to distribution of T cells not only in the lung interstitium and spleen, but also in the airway lumen, and the presence of these cells correlates well with protection. Furthermore, airway luminal recruitment of parenteral vaccine-induced T cells in peripheral tissues prior to M. tuberculosis challenge restores protection. We propose that understanding the biology of airway luminal T cells holds important implications for developing effective TB vaccination strategies.

The airway luminal memory CD8 T cells induced by respiratory mucosal immunization in a murine model have been found to be critical to antituberculosis immunity. However, the mechanisms of their maintenance on airway mucosal surface still remain poorly understood.Using a model of adenovirus-based intranasal immunization we investigated the immune property and the mechanisms of maintenance of airway luminal CD8 T cells.Immune properties of airway luminal Mycobacterium tuberculosis antigen-specific CD8 T cells were examined. Proliferation of airway luminal CD8 T cells was determined by in vivo T cell-labeling techniques. The role of peripheral T cell recruitment in maintaining airway luminal CD8 T cells was investigated by blocking lymphocyte trafficking from lymphoid and peripheral tissues. The requirement of M. tuberculosis antigens for in situ T cell proliferation was evaluated using a T cell transfer approach. An airway M. tuberculosis challenge model was used to study the relationship between CD8 T cell-mediated protection and peripheral T cell recruitment.Intranasal immunization leads to elicitation of persisting M. tuberculosis antigen-specific CD8 T cells in the airway lumen, which display an activated effector memory phenotype different from those in peripheral tissues. Airway luminal T cells continuously proliferate in an antigen-dependent manner, and can be maintained even in the absence of peripheral T cell recruitment. The lungs equipped with such CD8 T cells are protected from airway M. tuberculosis challenge independent of both peripheral T cell supply and CD4 T cells.Vaccine-inducible airway luminal antituberculosis memory CD8 T cells are self-renewable in an antigen-dependent manner, and can be maintained independent of peripheral T cell supply.

Recombinant virus-vectored vaccines hold great promise for tuberculosis (TB) vaccination strategies. However, there is a lack of side-by-side comparative investigations to dissect the functional differences and support the advantage of multivalent virus-vectored vaccine over its monovalent counterpart. We previously successfully developed a monovalent adenovirus (Ad)-vectored vaccine expressing Ag85a (AdAg85a) and demonstrated its superior protective efficacy in models of pulmonary TB. In this study, we have developed a bivalent Ad TB vaccine expressing Ag85a and TB10.4 antigens as a fusion protein (AdAg85a:TB10.4) and compared its T-cell-activating and immune protective efficacy with that by monovalent AdAg85a. A single intranasal (i.n.) administration of AdAg85a:TB10.4 induced robust T-cell responses toward the respective antigens within the airway lumen and spleen, although the level of Ag85a-specific T-cell responses in the airway lumen triggered by bivalent AdAg85a:TB10.4 was lower than that by its monovalent counterpart at earlier time points. Thus, a single i.n. delivery of AdAg85a:TB10.4 conferred a markedly improved and sustained level of protection in the lung against Mycobacterium tuberculosis (M.tb) challenge over that by AdAg85a or by conventional BCG immunization with similarly induced levels of protection in the spleen. Our results indicate a unique advantage of multivalent viral-vectored TB vaccines for immunization against pulmonary TB.

Protection by parenteral immunization with plasmid DNA vaccines against pulmonary tuberculosis (TB) is very modest. In this study, we have investigated the underlying mechanisms for the poor mucosal protective efficacy and the avenues and mechanisms to improve the efficacy of a single i.m. immunization with a monogenic plasmid DNA TB vaccine in a murine model. We show that i.m. DNA immunization fails to elicit accumulation of Ag-specific T cells in the airway lumen despite robust T cell responses in the spleen. Such systemically activated T cells cannot be rapidly mobilized into the airway lumen upon Mycobacterium tuberculosis exposure. However, airway deposition of low doses of soluble mycobacterial Ags in previously immunized mice effectively mobilizes the systemically activated T cells into the airway lumen. A fraction of such airway luminal T cells can persist in the airway lumen, undergo quick, robust expansion and activation and provide marked immune protection upon airway M. tuberculosis exposure. Airway mucosal deposition of soluble mycobacterial Ags was found to create a tissue microenvironment rich in proinflammatory molecules including chemokines and hence conducive to T cell recruitment. Thus, in vivo neutralization of MIP-1α or IFN-inducible protein-10 markedly inhibited the accumulation of Ag-specific T cells in the airway lumen. Our data suggest that immunoprotective efficacy on the mucosal surface by i.m. plasmid DNA immunization could be substantially improved by simple mucosal soluble Ag inoculation and restoration of mucosal luminal T cells. Our study holds implication for the future design of DNA vaccination strategies against intracellular infections.

In vitro manipulated dendritic cells (DC) have increasingly been used as a promising vaccine formulation against cancer and infectious disease. However, improved understanding of the immune mechanisms is needed for the development of safe and efficacious mucosal DC immunization. We have developed a murine model of respiratory mucosal immunization by using a genetically manipulated DC vaccine. Within 24 h of intranasal delivery, the majority of vaccine DCs migrated to the lung mucosa and draining lymph nodes and elicited a significant level of T cells capable of IFN-γ secretion and CTL in the airway lumen as well as substantial T cell responses in the spleen. And such T cell responses were associated with enhanced protection against respiratory mucosal intracellular bacterial challenge. In comparison, parenteral i.m. DC immunization did not elicit marked airway luminal T cell responses and immune protection regardless of strong systemic T cell activation. Although repeated mucosal DC delivery boosted Ag-specific T cells in the airway lumen, added benefits to CD8 T cell activation and immune protection were not observed. By using MHC-deficient vaccine DCs, we further demonstrated that mucosal DC immunization-mediated CD8 and CD4 T cell activation does not require endogenous DCs. By using IL-12-deficient vaccine DCs, we also observed that IL-12−/− DCs failed to migrate to the lymph nodes but remained capable of T cell activation. Our observations indicate that mucosal delivery of vaccine DCs represents an effective approach to enhance mucosal T cell immunity, which may operate independent of vaccine IL-12 and endogenous DCs.

Studies seeking Mycobacterium avium subsp. paratuberculosis in Crohn's disease by PCR have generated inconsistent findings. As an alternative, microscopy offers a number of advantages, including direct visualization of organisms in tissue. Experimental infections have demonstrated that M. avium organisms can be seen by both acid-fast staining and species-specific in situ hybridization, but because they are smaller than M. tuberculosis, oil-immersion microscopy (x 1,000 magnification) is needed. We performed a blinded search for M. avium in paraffin-embedded surgical resections from Crohn's and control subjects at two centres. Specimens were coded and subjected to acid-fast staining and ribosomal RNA in situ hybridization for M. avium rRNA. Agreement between these two methods was good (42/52 patients, kappa=0.60) and similar results were observed for patients from two centers. Together, both methods provided positive results in 10 of 17 Crohn's subjects (59%, 95% CI: 36-78), contrasting with only 5 of 35 control subjects (Odds ratio for Crohn's vs. controls=8.6, p=0.002). M. avium organisms had an intracellular localization within inflammatory lesions, but were often observed as lone organisms outside of granulomas. Using two assays in two settings, presence of M. avium organisms was strongly associated with Crohn's disease.