Aerosol delivery, but not intramuscular injection, of adenovirus-vectored tuberculosis vaccine induces respiratory-mucosal immunity in humans.

BackgroundAdenovirus-vectored (Ad-vectored) vaccines are typically administered via i.m. injection to humans and are incapable of inducing respiratory mucosal immunity. However, aerosol delivery of Ad-vectored vaccines remains poorly characterized, and its ability to induce mucosal immunity in humans is unknown. This phase Ib trial evaluated the safety and immunogenicity of human serotype-5 Ad-vectored tuberculosis (TB) vaccine (AdHu5Ag85A) delivered to humans via inhaled aerosol or i.m. injection.MethodsThirty-one healthy, previously BCG-vaccinated adults were enrolled. AdHu5Ag85A was administered by single-dose aerosol using Aeroneb Solo Nebulizer or by i.m. injection. The study consisted of the low-dose (LD) aerosol, high-dose (HD) aerosol, and i.m. groups. The adverse events were assessed at various times after vaccination. Immunogenicity data were collected from the peripheral blood and bronchoalveolar lavage samples at baseline, as well as at select time points after vaccination.ResultsThe nebulized aerosol droplets were < 5.39 µm in size. Both LD and HD of AdHu5Ag85A administered by aerosol inhalation and i.m. injection were safe and well tolerated. Both aerosol doses, particularly LD, but not i.m., vaccination markedly induced airway tissue-resident memory CD4+ and CD8+ T cells of polyfunctionality. While as expected, i.m. vaccination induced Ag85A-specific T cell responses in the blood, the LD aerosol vaccination also elicited such T cells in the blood. Furthermore, the LD aerosol vaccination induced persisting transcriptional changes in alveolar macrophages.ConclusionInhaled aerosol delivery of Ad-vectored vaccine is a safe and superior way to elicit respiratory mucosal immunity. This study warrants further development of aerosol vaccine strategies against respiratory pathogens, including TB and COVID-19.Trial registrationClinicalTrial.gov, NCT02337270.FundingThe Canadian Institutes for Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada funded this work.

BCG vaccination induces enhanced frequencies of memory T cells and altered plasma levels of common γc cytokines in elderly individuals.

BCG vaccination is known to induce innate immune memory, which confers protection against heterologous infections. However, the effect of BCG vaccination on the conventional adaptive immune cells subsets is not well characterized. We investigated the impact of BCG vaccination on the frequencies of T cell subsets and common gamma c (γc) cytokines in a group of healthy elderly individuals (age 60-80 years) at one month post vaccination as part of our clinical study to examine the effect of BCG on COVID-19. Our results demonstrate that BCG vaccination induced enhanced frequencies of central (p<0.0001) and effector memory (p<0.0001) CD4+ T cells and diminished frequencies of naïve (p<0.0001), transitional memory (p<0.0001), stem cell memory (p = 0.0001) CD4+ T cells and regulatory T cells. In addition, BCG vaccination induced enhanced frequencies of central (p = 0.0008), effector (p<0.0001) and terminal effector memory (p<0.0001) CD8+ T cells and diminished frequencies of naïve (p<0.0001), transitional memory (p<0.0001) and stem cell memory (p = 0.0034) CD8+T cells. BCG vaccination also induced enhanced plasma levels of IL-7 (p<0.0001) and IL-15 (p = 0.0020) but diminished levels of IL-2 (p = 0.0033) and IL-21 (p = 0.0020). Thus, BCG vaccination was associated with enhanced memory T cell subsets as well as memory enhancing γc cytokines in elderly individuals, suggesting its ability to induce non-specific adaptive immune responses.

Trained immunity contributes to the prevention of Mycobacterium tuberculosis infection, a novel role of autophagy.

Mycobacterium tuberculosis (M. tuberculosis) is the pathogen which causes tuberculosis (TB), a significant human public health threat. Co-infection of M. tuberculosis and the human immunodeficiency virus (HIV), emergence of drug resistant M. tuberculosis, and failure to develop highly effective TB vaccines have limited control of the TB epidemic. Trained immunity is an enhanced innate immune response which functions independently of the adaptive/acquired immune system and responds non-specifically to reinfection with invading agents. Recently, several studies have found trained immunity has the capability to control and eliminate M. tuberculosis infection. Over the past decades, however, the consensus was adaptive immunity is the only protective mechanism by which hosts inhibit M. tuberculosis growth. Furthermore, autophagy plays an essential role in the development of trained immunity. Further investigation of trained immunity, M. tuberculosis infection, and the role of autophagy in this process provide new possibilities for vaccine development. In this review, we present the general characteristics of trained immunity and autophagy. We additionally summarize several examples where initiation of trained immunity contributes to the prevention of M. tuberculosis infection and propose future directions for research in this area.

Detection and quantification of Mycobacterium tuberculosis antigen CFP10 in serum and urine for the rapid diagnosis of active tuberculosis disease.

Outside of the ongoing COVID-19 pandemic, tuberculosis is the leading cause of infectious disease mortality globally. Currently, there is no commercially available point-of-care diagnostic that is rapid, inexpensive, and highly sensitive for the diagnosis of active tuberculosis disease. Here we describe the development and optimization of a novel, highly sensitive prototype bioelectronic tuberculosis antigen (BETA) assay to detect tuberculosis-specific antigen, CFP10, in small-volume serum and urine samples. In this proof-of-concept study we evaluated the performance of the BETA assay using clinical specimens collected from presumptive tuberculosis patients from three independent cohorts. Circulating CFP10 antigen was detected in ALL serum (n = 19) and urine (n = 3) samples from bacteriologically confirmed tuberculosis patients who were untreated or had less than one week of treatment at time of serum collection, successfully identifying all culture positive tuberculosis patients. No CFP10 antigen was detected in serum (n = 7) or urine (n = 6) samples from individuals who were determined to be negative for tuberculosis disease. Additionally, antigen quantification using the BETA assay of paired serum samples collected from tuberculosis patients (n = 8) both before and after treatment initiation, indicate consistently declining within-person levels of CFP10 antigen during treatment. This novel, low-cost assay demonstrates potential as a rapid, non-sputum-based, point-of-care tool for the diagnosis of tuberculosis disease.

Synthetic protein conjugate vaccines provide protection against Mycobacterium tuberculosis in mice.

The global incidence of tuberculosis remains unacceptably high, with new preventative strategies needed to reduce the burden of disease. We describe here a method for the generation of synthetic self-adjuvanted protein vaccines and demonstrate application in vaccination against Mycobacterium tuberculosis Two vaccine constructs were designed, consisting of full-length ESAT6 protein fused to the TLR2-targeting adjuvants Pam2Cys-SK4 or Pam3Cys-SK4 These were produced by chemical synthesis using a peptide ligation strategy. The synthetic self-adjuvanting vaccines generated powerful local CD4+ T cell responses against ESAT6 and provided significant protection in the lungs from virulent M. tuberculosis aerosol challenge when administered to the pulmonary mucosa of mice. The flexible synthetic platform we describe, which allows incorporation of adjuvants to multiantigenic vaccines, represents a general approach that can be applied to rapidly assess vaccination strategies in preclinical models for a range of diseases, including against novel pandemic pathogens such as SARS-CoV-2.

Association of the past epidemic of Mycobacterium tuberculosis with mortality and incidence of COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has created a remarkable and varying impact in every country, inciting calls for broad attention. Recently, the Bacillus Calmette-Guérin (BCG) vaccination has been regarded as a potential candidate to explain this difference. Herein, we hypothesised that the past epidemic of Mycobacterium tuberculosis (M. tuberculosis) may act as a latent explanatory factor for the worldwide differences seen in COVID-19 impact on mortality and incidence. We compared two indicators of past epidemic of M. tuberculosis, specifically, incidence (90 countries in 1990) and mortality (28 countries in 1950), with the mortality and incidence of COVID-19. We determined that an inverse relationship existed between the past epidemic indicators of M. tuberculosis and current COVID-19 impact. The rate ratio of the cumulative COVID-19 mortality per 1 million was 2.70 (95% confidence interval [CI]: 1.09-6.68) per 1 unit decrease in the incidence rate of tuberculosis (per 100,000 people). The rate ratio of the cumulative COVID-19 incidence per 1 million was 2.07 (95% CI: 1.30-3.30). This association existed even after adjusting for potential confounders (rate of people aged 65 over, diabetes prevalence, the mortality rate from cardiovascular disease, and gross domestic product per capita), leading to an adjusted rate ratio of COVID-19 mortality of 2.44, (95% CI: 1.32-4.52) and a COVID-19 incidence of 1.31 (95% CI: 0.97-1.78). After latent infection, Mycobacterium survives in the human body and may continue to stimulate trained immunity. This study suggests a possible mechanism underlying the region-based variation in the COVID-19 impact.

Relationship of SARS-CoV-2-specific CD4 response to COVID-19 severity and impact of HIV-1 and tuberculosis coinfection.

T cells are involved in control of coronavirus disease 2019 (COVID-19), but limited knowledge is available on the relationship between antigen-specific T cell response and disease severity. Here, we used flow cytometry to assess the magnitude, function, and phenotype of SARS coronavirus 2-specific (SARS-CoV-2-specific) CD4+ T cells in 95 hospitalized COVID-19 patients, 38 of them being HIV-1 and/or tuberculosis (TB) coinfected, and 38 non-COVID-19 patients. We showed that SARS-CoV-2-specific CD4+ T cell attributes, rather than magnitude, were associated with disease severity, with severe disease being characterized by poor polyfunctional potential, reduced proliferation capacity, and enhanced HLA-DR expression. Moreover, HIV-1 and TB coinfection skewed the SARS-CoV-2 T cell response. HIV-1-mediated CD4+ T cell depletion associated with suboptimal T cell and humoral immune responses to SARS-CoV-2, and a decrease in the polyfunctional capacity of SARS-CoV-2-specific CD4+ T cells was observed in COVID-19 patients with active TB. Our results also revealed that COVID-19 patients displayed reduced frequency of Mycobacterium tuberculosis-specific CD4+ T cells, with possible implications for TB disease progression. These results corroborate the important role of SARS-CoV-2-specific T cells in COVID-19 pathogenesis and support the concept of altered T cell functions in patients with severe disease.

A century of BCG: Impact on tuberculosis control and beyond.

BCG turns 100 this year and while it might not be the perfect vaccine, it has certainly contributed significantly towards eradication and prevention of spread of tuberculosis (TB). The search for newer and better vaccines for TB is an ongoing endeavor and latest results from trials of candidate TB vaccines such as M72AS01 look promising. However, recent encouraging data from BCG revaccination trials in adults combined with studies on mucosal and intravenous routes of BCG vaccination in non-human primate models have renewed interest in BCG for TB prevention. In addition, several well-demonstrated non-specific effects of BCG, for example, prevention of viral and respiratory infections, give BCG an added advantage. Also, BCG vaccination is currently being widely tested in human clinical trials to determine whether it protects against SARS-CoV-2 infection and/or death with detailed analyses and outcomes from several ongoing trials across the world awaited. Through this review, we attempt to bring together information on various aspects of the BCG-induced immune response, its efficacy in TB control, comparison with other candidate TB vaccines and strategies to improve its efficiency including revaccination and alternate routes of administration. Finally, we discuss the future relevance of BCG use especially in light of its several heterologous benefits.

In Vivo Antigen Expression Regulates CD4 T Cell Differentiation and Vaccine Efficacy against Mycobacterium tuberculosis Infection.

New vaccines are urgently needed against Mycobacterium tuberculosis (Mtb), which kills more than 1.4 million people each year. CD4 T cell differentiation is a key determinant of protective immunity against Mtb, but it is not fully understood how host-pathogen interactions shape individual antigen-specific T cell populations and their protective capacity. Here, we investigated the immunodominant Mtb antigen, MPT70, which is upregulated in response to gamma interferon (IFN-γ) or nutrient/oxygen deprivation of in vitro-infected macrophages. Using a murine aerosol infection model, we compared the in vivo expression kinetics of MPT70 to a constitutively expressed antigen, ESAT-6, and analyzed their corresponding CD4 T cell phenotype and vaccine protection. For wild-type Mtb, we found that in vivo expression of MPT70 was delayed compared to ESAT-6. This delayed expression was associated with induction of less differentiated MPT70-specific CD4 T cells but, compared to ESAT-6, also reduced protection after vaccination. In contrast, infection with an MPT70-overexpressing Mtb strain promoted highly differentiated KLRG1+CX3CR1+ CD4 T cells with limited lung-homing capacity. Importantly, this differentiated phenotype could be prevented by vaccination, and against the overexpressing strain, vaccination with MPT70 conferred protection similar to vaccination with ESAT-6. Together, our data indicate that high in vivo antigen expression drives T cells toward terminal differentiation and that targeted vaccination with adjuvanted protein can counteract this phenomenon by maintaining T cells in a protective less differentiated state. These observations shed new light on host-pathogen interactions and provide guidance on how future Mtb vaccines can be designed to tip the immune balance in favor of the host.IMPORTANCE Tuberculosis, caused by Mtb, constitutes a global health crisis of massive proportions and the impact of the current coronavirus disease 2019 (COVID-19) pandemic is expected to cause a rise in tuberculosis-related deaths. Improved vaccines are therefore needed more than ever, but a lack of knowledge on protective immunity hampers their development. The present study shows that constitutively expressed antigens with high availability drive highly differentiated CD4 T cells with diminished protective capacity, which could be a survival strategy by Mtb to evade T cell immunity against key antigens. We demonstrate that immunization with such antigens can counteract this phenomenon by maintaining antigen-specific T cells in a state of low differentiation. Future vaccine strategies should therefore explore combinations of multiple highly expressed antigens and we suggest that T cell differentiation could be used as a readily measurable parameter to identify these in both preclinical and clinical studies.

COVID-19 and Tuberculosis Coinfection in a 51-Year-Old Taxi Driver in Mexico City.

BACKGROUND Coinfection with severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) and Mycobacterium tuberculosis (MBT) has been reported, albeit rarely, in various parts of the world and has received attention from health systems because up to one-third of the world's population has been infected with SARS-CoV-2. Mexico was not included in the first-ever report on a global cohort of patients with this coinfection. We report on a case of SARS-CoV-2/MBT coinfection in a 51-year-old taxi driver from Mexico City that underscores the importance of rapid and accurate laboratory testing, diagnosis, and treatment. CASE REPORT We present the case of a man in the sixth decade of life who was admitted to the National Institute of Respiratory Diseases (INER) with a diagnosis of COVID-19 pneumonia, which was confirmed by nasopharyngeal exudate using real-time polymerase chain reaction (RT-PCR) for the identification of SARS-CoV-2. Findings from imaging studies suggested that the patient might be coinfected with MBT. That suspicion was confirmed with light microscopy of a sputum sample after Ziehl-Neelsen staining and when a Cepheid Xpert MTB/RIF assay, an automated semi-quantitative RT-PCR assay, failed to detect rifampicin resistance. The patient was discharged from the hospital 10 days later. CONCLUSIONS The present report underscores the importance of using validated molecular diagnostic tests to identify coinfections in areas where there is a high prevalence of other causes of pneumonia, such as MBT, as a way to improve clinical outcomes in patients during the COVID-19 pandemic. While it is imperative to control the COVID-19 pandemic, the medical community must not forget about the other pandemics to which populations are still prey, and tuberculosis is one of them. We must remain alert to any clinical subtleties so as to ensure timely and accurate diagnosis and stay one step ahead of COVID-19.

Pathology of TB/COVID-19 Co-Infection: The phantom menace.

Tuberculosis (TB) and coronavirus disease 2019 (COVID-19) are currently the two main causes of death among infectious diseases. There is an increasing number of studies trying to elucidate the interactions between Mycobacterium tuberculosis and SARS-CoV-2. Some of the first case reports point to a worsening of respiratory symptoms in co-infected TB/COVID-19 individuals. However, data from the cohort studies has shown some conflicting results. This study proposes to conduct a systematic review on the current literature on TB/COVID-19 co-infection cohorts, evaluating clinical and epidemiological data, focusing on its implications to the immune system. From an immunological perspective, the TB/COVID-19 co-infection has the potential to converge in a "perfect storm". The disorders induced by each pathogen to the immunomodulation tend to induce an unbalanced inflammatory response, which can promote the progression and worsening of both diseases. Understanding the nature of the interactions between M. tuberculosis and SARS-CoV-2 will be crucial for the development of therapeutic strategies against co-infection.

BCG epidemiology supports its protection against COVID-19? A word of caution.

The COVID-19 pandemic, caused by type 2 Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), puts all of us to the test. Epidemiologic observations could critically aid the development of protective measures to combat this devastating viral outbreak. Recent observations, linked nation based universal Bacillus Calmette-Guerin (BCG) vaccination to potential protection against morbidity and mortality from SARS-CoV-2, and received much attention in public media. We wished to validate the findings by examining the country based association between COVID-19 mortality per million population, or daily rates of COVID-19 case fatality (i.e. Death Per Case/Days of the endemic [dpc/d]) and the presence of universal BCG vaccination before 1980, or the year of the establishment of universal BCG vaccination. These associations were examined in multiple regression modeling based on publicly available databases on both April 3rd and May 15th of 2020. COVID-19 deaths per million negatively associated with universal BCG vaccination in a country before 1980 based on May 15th data, but this was not true for COVID-19 dpc/d on either of days of inquiry. We also demonstrate possible arbitrary selection bias in such analyses. Consequently, caution should be exercised amidst the publication surge on COVID-19, due to political/economical-, arbitrary selection-, and fear/anxiety related biases, which may obscure scientific rigor. We argue that global COVID-19 epidemiologic data is unreliable and therefore should be critically scrutinized before using it as a nidus for subsequent hypothesis driven scientific discovery.

Upregulation of CD47 Is a Host Checkpoint Response to Pathogen Recognition.

It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.