Xizhong Zhang

Intranasal boosting with an adenovirus-vectored vaccine markedly enhances protection by parenteral Mycobacterium bovis BCG immunization against pulmonary tuberculosis.

ABSTRACT Parenterally administered Mycobacterium bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans. There is a need for developing effective boosting vaccination strategies. We examined a heterologous prime-boost regimen utilizing BCG as a prime vaccine and our recently described adenoviral vector expressing Ag85A (AdAg85A) as a boost vaccine. Since we recently demonstrated that a single intranasal but not intramuscular immunization with AdAg85A was able to induce potent protection from pulmonary Mycobacterium tuberculosis challenge in a mouse model, we compared the protective effects of parenteral and mucosal booster immunizations following subcutaneous BCG priming. Protection by BCG prime immunization was not effectively boosted by subcutaneous BCG or intramuscular AdAg85A. In contrast, protection by BCG priming was remarkably boosted by intranasal AdAg85A. Such enhanced protection by intranasal AdAg85A was correlated to the numbers of gamma interferon-positive CD4 and CD8 T cells residing in the airway lumen of the lung. Our study demonstrates that intranasal administration of AdAg85A represents an effective way to boost immune protection by parenteral BCG vaccination.

Mechanisms of Mucosal and Parenteral Tuberculosis Vaccinations: Adenoviral-Based Mucosal Immunization Preferentially Elicits Sustained Accumulation of Immune Protective CD4 and CD8 T Cells within the Airway Lumen

The mechanisms underlying better immune protection by mucosal vaccination have remained poorly understood. In our current study we have investigated the mechanisms by which respiratory virus-mediated mucosal vaccination provides remarkably better immune protection against pulmonary tuberculosis than parenteral vaccination. A recombinant adenovirus-based tuberculosis (TB) vaccine expressing Mycobacterium tuberculosis Ag85A (AdAg85A) was administered either intranasally (i.n.) or i.m. to mice, and Ag-specific CD4 and CD8 T cell responses, including frequency, IFN-γ production, and CTL, were examined in the spleen, lung interstitium, and airway lumen. Although i.m. immunization with AdAg85A led to activation of T cells, particularly CD8 T cells, in the spleen and, to a lesser extent, in the lung interstitium, it failed to elicit any T cell response in the airway lumen. In contrast, although i.n. immunization failed to effectively activate T cells in the spleen, it uniquely elicited higher numbers of Ag-specific CD4 and CD8 T cells in the airway lumen that were capable of IFN-γ production and cytolytic activities, as assessed by an intratracheal in vivo CTL assay. These airway luminal T cells of i.n. immunized mice or splenic T cells of i.m. immunized mice, upon transfer locally to the lungs of naive SCID mice, conferred immune protection against M. tuberculosis challenge. Our study has demonstrated that the airway luminal T cell population plays an important role in immune protection against pulmonary TB, thus providing mechanistic insights into the superior immune protection conferred by respiratory mucosal TB vaccination.

Mucosal Luminal Manipulation of T Cell Geography Switches on Protective Efficacy by Otherwise Ineffective Parenteral Genetic Immunization

Genetic immunization holds great promise for future vaccination against mucosal infectious diseases. However, parenteral genetic immunization is ineffective in control of mucosal intracellular infections, and the underlying mechanisms have remained unclear. By using a model of parenteral i.m. genetic immunization and pulmonary tuberculosis (TB), we have investigated the mechanisms that determine the failure and success of parenteral genetic immunization. We found that lack of protection from pulmonary Mycobacterium tuberculosis (M.tb) challenge by i.m. immunization with a recombinant adenovirus-vectored tuberculosis vaccine was linked to the absence of M.tb Ag-specific T cells within the airway lumen before M.tb challenge despite potent T cell activation in the systemic compartments. Furthermore, pulmonary mycobacterial challenge failed to recruit CD8 T cells into the airway lumen of i.m. immunized mice. Such defect in T cell recruitment, intra-airway CTL, and immune protection was restored by creating acute inflammation in the airway with inflammatory agonists such as virus. However, the Ag-specific T cells recruited as such were not retained in the airway lumen, resulting in a loss of protection. In comparison, airway exposure to low doses of soluble M.tb Ags not only recruited but retained Ag-specific CD8 T cells in the airway lumen over time that provided robust protection against M.tb challenge. Thus, our study reveals that mucosal protection by parenteral immunization is critically determined by T cell geography, i.e., whether Ag-specific T cells are within or outside of the mucosal lumen and presents a feasible solution to empower parenteral immunization strategies against mucosal infectious diseases.

Gamma Interferon Responses of CD4 and CD8 T-Cell Subsets Are Quantitatively Different and Independent of Each Other during Pulmonary Mycobacterium bovis BCG Infection

ABSTRACT Gamma interferon (IFN-γ) is a key cytokine in host defense against intracellular mycobacterial infection. It has been believed that both CD4 and CD8 T cells are the primary sources of IFN-γ. However, the relative contributions of CD4 and CD8 T-cell subsets to IFN-γ production and the relationship between CD4 and CD8 T-cell activation have not been examined. By using a model of pulmonary mycobacterial infection and various immunodetection assays, we found that CD4 T cells mounted a much stronger IFN-γ response than CD8 T cells at various times after mycobacterial infection, and this pronounced IFN-γ production by CD4 T cells was attributed to both greater numbers of antigen-specific CD4 T cells and a greater IFN-γ secretion capacity of these cells. By using major histocompatibility complex class II-deficient or CD4-deficient mice, we found that the lack of CD4 T cells did not negatively affect primary or secondary CD8 T-cell IFN-γ responses. The CD8 T cells activated in the absence of CD4 T cells were capable of immune protection against secondary mycobacterial challenge. Our results suggest that, whereas both CD4 and CD8 T cells are capable of IFN-γ production, the former represent a much greater cellular source of IFN-γ. Moreover, during mycobacterial infection, CD8 T-cell IFN-γ responses and activation are independent of CD4 T-cell activation.

Airway Delivery of Soluble Mycobacterial Antigens Restores Protective Mucosal Immunity by Single Intramuscular Plasmid DNA Tuberculosis Vaccination: Role of Proinflammatory Signals in the Lung

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.

Organ distribution of transgene expression following intranasal mucosal delivery of recombinant replication-defective adenovirus gene transfer vector

It is believed that respiratory mucosal immunization triggers more effective immune protection than parenteral immunization against respiratory infection caused by viruses and intracellular bacteria. Such understanding has led to the successful implementation of intranasal immunization in humans with a live cold-adapted flu virus vaccine. Furthermore there has been an interest in developing effective mucosal-deliverable genetic vaccines against other infectious diseases. However, there is a concern that intranasally delivered recombinant viral-based vaccines may disseminate to the CNS via the olfactory tissue. Initial experimental evidence suggests that intranasally delivered recombinant adenoviral gene transfer vector may transport to the olfactory bulb. However, there is a lack of quantitative studies to compare the relative amounts of transgene products in the respiratory tract, lung, olfactory bulb and brain after intranasal mucosal delivery of viral gene transfer vector. To address this issue, we have used fluorescence macroscopic imaging, luciferase quantification and PCR approaches to compare the relative distribution of transgene products or adenoviral gene sequences in the respiratory tract, lung, draining lymph nodes, olfactory bulb, brain and spleen. Intranasal mucosal delivery of replication-defective recombinant adenoviral vector results in gene transfer predominantly in the respiratory system including the lung while it does lead to a moderate level of gene transfer in the olfactory bulb. However, intranasal inoculation of adenoviral vector leads to little or no viral dissemination to the major region of the CNS, the brain. These experimental findings support the efficaciousness of intranasal adenoviral-mediated gene transfer for the purpose of mucosal immunization and suggest that it may not be of significant safety concern.

Immunization With a Bivalent Adenovirus-vectored Tuberculosis Vaccine Provides Markedly Improved Protection Over Its Monovalent Counterpart Against Pulmonary Tuberculosis

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.

Heterologous Boosting of Recombinant Adenoviral Prime Immunization With a Novel Vesicular Stomatitis Virus-vectored Tuberculosis Vaccine

Pulmonary tuberculosis (TB) remains a serious health problem worldwide. Effective vaccination strategies are needed. We report the development of a novel TB vaccine using vesicular stomatitis virus (VSV) as a viral vector system to express Ag85A. VSVAg85A was shown to be immunogenic when given to mice by either an intranasal or an intramuscular (IM) route. Although distinct T-cell profiles resulted from both routes of immunization, only intranasal delivery generated a mucosal T-cell response that was protective upon pulmonary Mycobacterium tuberculosis (M.tb) challenge. While this protection manifested at an early time-point after immunization, it was not sustained. The potential of VSVAg85A to be used as a mucosal booster for parenteral priming by an adenoviral TB vaccine expressing Ag85A (AdAg85A) was investigated. VSVAg85A immunization markedly boosted antigen-specific T-cell responses in the airway lumen while also augmenting immune activation in the systemic compartment, after AdAg85A priming. This translated into significantly better protective efficacy against pulmonary challenge with M.tb than either vaccine used alone. Our study therefore suggests that VSV as a vector system is a promising candidate to be used in a heterologous viral prime-boost immunization regimen against intracellular bacterial infection.

Mucosally Delivered Dendritic Cells Activate T Cells Independently of IL-12 and Endogenous APCs

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.

Manipulation of dendritic cells for host defence against intracellular infections

Dendritic cells (DCs) are an important innate immune cell type which is the bridge between innate and adaptive immunity. Mounting experimental evidence suggests that manipulating DCs represents a powerful means to enhance host defence against intracellular infectious diseases. We have developed several strategies to manipulate DCs either in vivo or in vitro for the purpose of enhancing the effect of vaccination or immunotherapeutics. In vivo delivery of transgene encoding GM-CSF (granulocyte/macrophage colony-stimulating factor), a DC-activating cytokine, increases the number and activation status of DCs at various tissue sites and enhances antimicrobial immune responses in murine models. Co-expression or co-delivery of GM-CSF gene transfer vector with an antimicrobial vaccine enhances microbial antigen-specific T-cell responses and immune protection. Murine bone marrow-derived DCs are being manipulated in vitro and exploited as a vaccine delivery system. Transduction of DCs with a virus-vectored tuberculosis vaccine is a powerful way to activate T-cells in vivo. Such genetically modified DC vaccines can be administered either parenterally or mucosally via the respiratory tract.