To examine the association between fragile X mental retardation protein (FMRP) expression and astrocytoma characteristics.

We have investigated the role of CD4(+) T cells in the development of the CD8(+) T-cell response after immunization with recombinant adenovirus (rAd). In the absence of CD4(+) T cells, the "unhelped" CD8(+) T-cell population exhibited a reduction in primary expansion and long-term survival that appeared to be due to inadequate priming of naïve T cells. There were few functional or phenotypic differences between the helped and unhelped CD8(+) T-cell populations with the exception of O-glycosylated CD43, a marker of effector cells, which was augmented on the unhelped CD8(+) T-cell population. In some cases, the unhelped CD8(+) T-cell population exhibited reduced ability to control virus infection; however, this appeared to be a function of the reduced frequency of antigen-specific CD8(+) T cells. Most notably, the unhelped CD8(+) T-cell population exhibited no defect in secondary expansion. These results provide insight into the role of CD4(+) T cells during the primary CD8(+) T-cell response generated by rAd vaccines and identify potential benefits and issues that must be considered when using adenovirus vaccines under conditions where CD4(+) T-cell function may be limiting, such as vaccination of human immunodeficiency virus patients.

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.

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.

Chemotherapeutic intervention remains the primary strategy in treating and controlling tuberculosis (TB). However, a complex interplay between therapeutic and patient-related factors leads to poor treatment adherence. This in turn continues to give rise to unacceptably high rates of disease relapse and the growing emergence of drug-resistant forms of TB. As such, there is considerable interest in strategies that simultaneously improve treatment outcome and shorten chemotherapy duration. Therapeutic vaccines represent one such approach which aims to accomplish this through boosting and/or priming novel anti-TB immune responses to accelerate disease resolution, shorten treatment duration, and enhance treatment success rates. Numerous therapeutic vaccine candidates are currently undergoing pre-clinical and clinical assessment, showing varying degrees of efficacy. By dissecting the underlying mechanisms/correlates of their successes and/or shortcomings, strategies can be identified to improve existing and future vaccine candidates. This mini-review will discuss the current understanding of therapeutic TB vaccine candidates, and discuss major strategies that can be implemented in advancing their development.

Increasing viral dosage within dry powder vaccines reduces the powder mass required to elicit an immune response through pulmonary delivery. This work analyzes how cryoprotective agents affect viral activity, particle properties and thermal stability of a spray dried, inhalable vaccine vector under high viral loading. Stock suspensions of a human serotype 5 adenovirus (AdHu5) vector in either neat phosphate buffered saline (PBS), 10% glycerol in PBS, or 5% trehalose in PBS were added to a mannitol-dextran formulation prior to spray drying. At high viral loading, spray dried powder containing glycerol had a viral titre log loss of 2.8 compared to 0.7 log loss using neat PBS. Powders containing glycerol had a lower glass transition temperature (Tg) compared to all other formulations, permitting greater viral mobility and exposure to heat damage. Inclusion of glycerol also promoted particle cohesion during spray drying and lower yields. Using 5% trehalose as a cryogenic alternative, viral powders had a viral log loss of 1.5 and the highest displayed thermal stability over time. Additionally, trehalose-containing powders had smaller particles with lower water moisture content and higher powder yield compared to glycerol-containing powders. These findings demonstrate the importance of cryoprotective agent selection when developing thermostable vaccine powders.

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.

Thermally stable, spray dried vaccines targeting respiratory diseases are promising candidates for pulmonary delivery, requiring careful excipient formulation to effectively encapsulate and protect labile biologics. This study investigates the impact of dextran mass ratio and molecular weight on activity retention, thermal stability and aerosol behaviour of a labile adenoviral vector (AdHu5) encapsulated within a spray dried mannitol-dextran blend.

Clinical and experimental studies have shown that estradiol (E2) confers protection against HIV and other sexually transmitted infections. Here, we investigated the underlying mechanism. Better protection in E2-treated mice, immunized against genital HSV-2, coincided with earlier recruitment and higher proportions of Th1 and Th17 effector cells in the vagina post-challenge, compared to placebo-treated controls. Vaginal APCs isolated from E2-treated mice induced 10-fold higher Th17 and Th1 responses, compared to APCs from progesterone-treated, placebo-treated, and estradiol-receptor knockout mice in APC-T cell co-cultures. CD11c+ DCs in the vagina were the predominant APC population responsible for priming these Th17 responses, and a potent source of IL-6 and IL-1β, important factors for Th17 differentiation. Th17 responses were abrogated in APC-T cell co-cultures containing IL-1β KO, but not IL-6 KO vaginal DCs, showing that IL-1β is a critical factor for Th17 induction in the genital tract. E2 treatment in vivo directly induced high expression of IL-1β in vaginal DCs, and addition of IL-1β restored Th17 induction by IL-1β KO APCs in co-cultures. Finally, we examined the role of IL-17 in anti-HSV-2 memory T cell responses. IL-17 KO mice were more susceptible to intravaginal HSV-2 challenge, compared to WT controls, and vaginal DCs from these mice were defective at priming efficient Th1 responses in vitro, indicating that IL-17 is important for the generation of efficient anti-viral memory responses. We conclude that the genital mucosa has a unique microenvironment whereby E2 enhances CD4+ T cell anti-viral immunity by priming vaginal DCs to induce Th17 responses through an IL-1-dependent pathway.

Monocyte phenotype and output changes with age, but why this occurs and how it impacts anti-bacterial immunity are not clear. We found that, in both humans and mice, circulating monocyte phenotype and function was altered with age due to increasing levels of TNF in the circulation that occur as part of the aging process. Ly6C+ monocytes from old (18-22 mo) mice and CD14+CD16+ intermediate/inflammatory monocytes from older adults also contributed to this "age-associated inflammation" as they produced more of the inflammatory cytokines IL6 and TNF in the steady state and when stimulated with bacterial products. Using an aged mouse model of pneumococcal colonization we found that chronic exposure to TNF with age altered the maturity of circulating monocytes, as measured by F4/80 expression, and this decrease in monocyte maturation was directly linked to susceptibility to infection. Ly6C+ monocytes from old mice had higher levels of CCR2 expression, which promoted premature egress from the bone marrow when challenged with Streptococcus pneumoniae. Although Ly6C+ monocyte recruitment and TNF levels in the blood and nasopharnyx were higher in old mice during S. pneumoniae colonization, bacterial clearance was impaired. Counterintuitively, elevated TNF and excessive monocyte recruitment in old mice contributed to impaired anti-pneumococcal immunity since bacterial clearance was improved upon pharmacological reduction of TNF or Ly6C+ monocytes, which were the major producers of TNF. Thus, with age TNF impairs inflammatory monocyte development, function and promotes premature egress, which contribute to systemic inflammation and is ultimately detrimental to anti-pneumococcal immunity.

In people living with HIV, Mycobacterium tuberculosis (Mtb) is the major cause of death. Due to the increased morbidity/mortality in co-infection, further research is urgently required. A limiting factor to research in HIV and HIV/Mtb co-infection is the lack of accessible in vivo models. Next-generation humanized mice expressing HLA transgenes report improved human immune reconstitution and functionality, which may better recapitulate human disease. This study compares well-established huNRG mice and next-generation HLA I/II-transgenic (huDRAG-A2) mice for immune reconstitution, disease course, and pathology in HIV and TB. HuDRAG-A2 mice have improved engraftment of key immune cell types involved in HIV and TB disease. Upon intravaginal HIV-1 infection, both models developed significant HIV target cell depletion in the blood and tissues. Upon intranasal Mtb infection, both models sustained high bacterial load within the lungs and tissue dissemination. Some huDRAG-A2 granulomas appeared more classically organized, characterized by focal central necrosis, multinucleated giant cells, and foamy macrophages surrounded by a halo of CD4+ T cells. HIV/Mtb co-infection in huNRG mice trended towards worsened TB pathology and showed potential for modeling co-infection. Both huNRG and huDRAG-A2 mice are viable options for investigating HIV and TB, but the huDRAG-A2 model may offer advantages.

Vaccination route dictates the quality and localization of immune responses within tissues. Intranasal vaccination seeds tissue-resident adaptive immunity, alongside trained innate responses within the lung/airways, critical for superior protection against SARS-CoV-2. This protocol encompasses intranasal vaccination in mice, step-by-step bronchoalveolar lavage for both cellular and acellular airway components, lung mononuclear cell isolation, and detailed flow cytometric characterization of lung tissue-resident memory T cell responses, and airway macrophage-trained innate immunity. For complete details on the use and execution of this protocol, please refer to Afkhami et al. (2022).

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.

Pneumococcal infections are the leading cause of community-acquired pneumonia. Although the type 1 interferon-α (IFN-α) is a well-known antiviral cytokine, the role of IFN-α in antipneumococcal host defense and its therapeutic potential remain poorly understood. We have investigated these issues by using a murine transgene expression model. We found that in control animals, Streptococcus pneumoniae infection caused severe weight loss and excessive lung inflammation, associated with rapid bacterial outgrowth. In contrast, the animals that received a single dose of an adenoviral vector expressing IFN-α prior to pneumococcal infection demonstrated rapid and effective control of bacterial replication and lung inflammation and improved clinical outcome. Enhanced protection by IFN-α was due to increased activation of neutrophils and macrophages with increased release of reactive oxygen and nitrogen species and bacterial killing. Furthermore, we found that raised levels of IFN-α in the lung remained immune protective even when the gene transfer vector was given at a time postpneumococcal infection. Our study thus shows that the classically antiviral type 1 IFN can be exploited for enhancing immunity against pneumococcal infection via its activating effects on innate immune cells. Our findings hold implications for the therapeutic use of IFN-α gene transfer strategies to combat pneumococcal infections.

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.

The incidence of bovine tuberculosis (bTB) in the GB has been increasing since the 1980s. Immunisation, alongside current control measures, has been proposed as a sustainable measure to control bTB. Immunisation with Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been shown to protect against bTB. Furthermore, much experimental data indicates that pulmonary local immunity is important for protection against respiratory infections including Mycobacterium tuberculosis and that pulmonary immunisation is highly effective. Here, we evaluated protection against M. bovis, the main causative agent of bTB, conferred by BCG delivered subcutaneously, endobronchially or by the new strategy of simultaneous immunisation by both routes. We also tested simultaneous subcutaneous immunisation with BCG and endobronchial delivery of a recombinant type 5 adenovirus expressing mycobacterial antigen 85A. There was significantly reduced visible pathology in animals receiving the simultaneous BCG/BCG or BCG/Ad85 treatment compared to naïve controls. Furthermore, there were significantly fewer advanced microscopic granulomata in animals receiving BCG/Ad85A compared to naive controls. Thus, combining local and systemic immunisation limits the development of pathology, which in turn could decrease bTB transmission.

Tuberculosis (TB) vaccine-induced airway luminal T cells (ALT) have recently been shown to be critical to host defense against pulmonary TB. However, the mechanisms that maintain memory ALT remain poorly understood. In particular, whether respiratory mucosal exposure to environmental agents such as endotoxin may regulate the size of vaccine-induced ALT population is still unclear. Using a murine model of respiratory genetic TB vaccination and respiratory LPS exposure, we have addressed this issue in the current study. We have found that single or repeated LPS exposure increases the number of antigen-specific ALT which are capable of robust secondary responses to pulmonary mycobacterial challenge. To investigate the potential mechanisms by which LPS exposure modulates the ALT population, we have examined the role of ALT proliferation and peripheral T cell recruitment. We have found that LPS exposure-increased ALT is not dependent on increased ALT proliferation as respiratory LPS exposure does not significantly increase the rate of proliferation of ALT. But rather, we find it to be dependent upon the recruitment of peripheral T cells into the airway lumen as blockade of peripheral T cell supplies markedly reduces the initially increased ALT. Thus, our data suggest that environmental exposure to airborne agents such as endotoxin has a profound modulatory effect on TB vaccine-elicited T cells within the respiratory tract. Our study provides a new, M.tb antigen-independent mechanism by which the respiratory mucosal anti-TB memory T cells may be maintained.

Pulmonary tuberculosis (TB), caused by Mycobacterium tuberculosis, is the leading cause of death due to a bacterial pathogen. Emerging epidemiologic evidence suggests that the leading risk factor associated with TB mortality is cigarette smoke exposure. Despite this, it remains poorly understood what is the effect of cigarette smoke exposure on anti-TB immunity and whether its potential detrimental effect can be reversed by cigarette smoking cessation. In our current study, we have investigated the impact of both continuous and discontinuous cigarette smoke exposure on the development of anti-mycobacterial type 1 immunity in murine models. We find that while continuous cigarette smoke exposure severely impairs type 1 immunity in the lung, a short-term smoking cessation allows rapid restoration of anti-mycobacterial immunity. The ability of continuous cigarette smoke exposure to dampen type 1 protective immunity is attributed locally to its affects on innate immune cells in the lung. Continuous cigarette smoke exposure locally, by not systemically, impairs APC accumulation and their production of TNF, IL-12, and RANTES, blunts the recruitment of CD4+IFN-γ+ T cells to the lung, and weakens the formation of granuloma. On the other hand, smoking cessation was found to help restore type 1 immunity by rapidly improving the functionality of lung APCs, enhancing the recruitment of CD4+IFN-γ+ T cells to the lung, and promoting the formation of granuloma. Our study for the first time demonstrates that continuous, but not discontinuous, cigarette smoke exposure severely impedes the lung expression of anti-TB Th1 immunity via inhibiting innate immune activation and lung T cell recruitment. Our findings thus suggest cigarette smoking cessation to be beneficial to the control of pulmonary TB.

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 (i.m.) 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.

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.