Recombinant BCG Vaccines Reduce Pneumovirus-Caused Airway Pathology by Inducing Protective Humoral Immunity

The Human Respiratory Syncytial Virus (hRSV) and the Human Metapneumovirus (hMPV) are two pneumoviruses that are leading agents causing acute lower respiratory tract infections (ALRTIs) affecting young infants, the elderly, and immunocompromised patients worldwide. Since these pathogens were first discovered, many approaches for the licensing of safe and effective vaccines have been explored being unsuccessful to date. We have previously described that immunization with recombinant strains of Mycobacterium bovis Bacillus Calmette-Guérin (rBCG) expressing the hRSV nucleoprotein (rBCG-N) or the hMPV phosphoprotein (rBCG-P) induced immune protection against each respective virus. These vaccines efficiently promoted viral clearance without significant lung damage, mainly through the induction of a T helper 1 cellular immunity. Here we show that upon viral challenge, rBCG-immunized mice developed a protective humoral immunity, characterized by production of antibodies specific for most hRSV and hMPV proteins. Further, isotype switching from IgG1 to IgG2a was observed in mice immunized with rBCG vaccines and correlated with an increased viral clearance, as compared to unimmunized animals. Finally, sera obtained from animals immunized with rBCG vaccines and infected with their respective viruses exhibited virus neutralizing capacity and protected naïve mice from viral replication and pulmonary disease. These results support the notion that the use of rBCG strains could be considered as an effective vaccination approach against other respiratory viruses with similar biology as hRSV and hMPV

Autoantibodies Against Proteins Previously Associated With Autoimmunity in Adult and Pediatric Patients With COVID-19 and Children With MIS-C

The antibody profile against autoantigens previously associated with autoimmune diseases and other human proteins in patients with COVID-19 or multisystem inflammatory syndrome in children (MIS-C) remains poorly defined. Here we show that 30% of adults with COVID-19 had autoantibodies against the lung antigen KCNRG, and 34% had antibodies to the SLE-associated Smith-D3 protein. Children with COVID-19 rarely had autoantibodies; one of 59 children had GAD65 autoantibodies associated with acute onset of insulin-dependent diabetes. While autoantibodies associated with SLE/Sjögren’s syndrome (Ro52, Ro60, and La) and/or autoimmune gastritis (gastric ATPase) were detected in 74% (40/54) of MIS-C patients, further analysis of these patients and of children with Kawasaki disease (KD), showed that the administration of intravenous immunoglobulin (IVIG) was largely responsible for detection of these autoantibodies in both groups of patients. Monitoring in vivo decay of the autoantibodies in MIS-C children showed that the IVIG-derived Ro52, Ro60, and La autoantibodies declined to undetectable levels by 45-60 days, but gastric ATPase autoantibodies declined more slowly requiring >100 days until undetectable. Further testing of IgG and/or IgA antibodies against a subset of potential targets identified by published autoantigen array studies of MIS-C failed to detect autoantibodies against most (16/18) of these proteins in patients with MIS-C who had not received IVIG. However, Troponin C2 and KLHL12 autoantibodies were detected in 2 of 20 and 1 of 20 patients with MIS-C, respectively. Overall, these results suggest that IVIG therapy may be a confounding factor in autoantibody measurements in MIS-C and that antibodies against antigens associated with autoimmune diseases or other human proteins are uncommon in MIS-C

Immunopathological signatures in multisystem inflammatory syndrome in children and pediatric COVID-19

: Pediatric Coronavirus Disease 2019 (pCOVID-19) is rarely severe; however, a minority of children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might develop multisystem inflammatory syndrome in children (MIS-C), with substantial morbidity. In this longitudinal multi-institutional study, we applied multi-omics (analysis of soluble biomarkers, proteomics, single-cell gene expression and immune repertoire analysis) to profile children with COVID-19 (n = 110) and MIS-C (n = 76), along with pediatric healthy controls (pHCs; n = 76). pCOVID-19 was characterized by robust type I interferon (IFN) responses, whereas prominent type II IFN-dependent and NF-κB-dependent signatures, matrisome activation and increased levels of circulating spike protein were detected in MIS-C, with no correlation with SARS-CoV-2 PCR status around the time of admission. Transient expansion of TRBV11-2 T cell clonotypes in MIS-C was associated with signatures of inflammation and T cell activation. The association of MIS-C with the combination of HLA A*02, B*35 and C*04 alleles suggests genetic susceptibility. MIS-C B cells showed higher mutation load than pCOVID-19 and pHC. These results identify distinct immunopathological signatures in pCOVID-19 and MIS-C that might help better define the pathophysiology of these disorders and guide therapy

Virulence of drug-resistant Mycobacterium tuberculosis and activity of drug combinations against drug-resistant and drug-susceptible isolates in ex-vivo and in vitro models

[eng] Tuberculosis (TB) remains a global threat worldwide with nearly nine million incident cases of TB. The emergence of M. tuberculosis resistance to antituberculous drugs has led to the need for careful TB surveillance and control. Further knowledge of drug-resistant M. tuberculosis plays an important role in avoiding TB transmission as well as designing more efficient schedules of treatment. The objectives were: 1) to analyse of clinical isolates of Mycobacterium tuberculosis resistant to antituberculous drugs to penetrate and grow within murine macrophages compared with drug-susceptible isolates; 2) to determine the in vitro synergistic activity of the following combinations against clinical isolates of M. tuberculosis resistant to isoniazid (INH) compared with drug-susceptible isolates: a) INH-rifampicin (RIF)-ethambutol (EMB), b) ofloxacin (OFL)-RIF-EMB; 3) to determine the in vitro synergistic activity of the following combinations against clinical isolates of multi-drug resistant M. tuberculosis compared with drug-susceptible isolates: a) levofloxacin (LEV)-linezolid(LNZ)-amikacin(AMK), b) LEV-LNZ-EMB, c) LEV-AMK-EMB; 4) to determine the antimicrobial and the synergistic activity of drug combinations of objective 3 against multidrug-resistant (MDR) and drug-susceptible clinical isolates of M. tuberculosis in a cell culture model of human macrophages from the THP-1 cell line. In the study of murine macrophages, we found that INH-resistant and MDR isolates with mutations in the katG gene showed decreased multiplication inside murine macrophages, suggesting a lower fitness of M. tuberculosis with these resistance patterns. With the results of this thesis, the reliability of the method of the three-drug chequerboard assay has been showed. The combination including INH, RIF and EMB could be efficient to treat TB cases with low level INH resistance (MICs ≤ 0.8µg/ml) due to the synergistic effect of the combination showed. The combination including OFL, RIF and EMB shows better efficacy than that of INH, RIF and EMB, being of potential use in drug-susceptible and in INH-resistant isolates. The combinations including second-choice drugs (LEV-AMK-EMB; LEV-AMK-LNZ; LEV-EMB-LNZ) are equally effective to the combination of INH, RIF and EMB with the checkerboard assay. On the other hand, these drug combinations including second-choice drugs tested against M. tuberculosis-infected macrophages show antimicrobial activity, with the combinations including LNZ and LEV displaying an antagonistic effect.[cat] La Tuberculosi (TB) continua sent una de les malalties més important en salut pública arreu del món. La preocupació sobre el control de la TB ha augmentat a conseqüència de l’aparició de soques resistents als fàrmacs disponibles actualment. Un millor coneixement de les soques de M. tuberculosis resistents a fàrmacs és clau per evitar la transmissió així com per dissenyar pautes de tractament més efectives contra la TB. Els objectius d’aquesta tesi van ser: 1) analitzar l’habilitat de soques clíniques de M. tuberculosis resistents a fàrmacs de penetrar i créixer dintre dels macròfags murins comparat amb soques sensibles; 2) determinar l’activitat sinèrgica in vitro de les següents combinacions davant de soques clíniques de M. tuberculosis resistents a isoniazida (INH) comparant amb soques sensibles: a) INH-rifampicina(RIF)–etambutol (EMB) i b) ofloxacin (OFL)–RIF-EMB; 3) determinar l’activitat sinèrgica in vitro de les següents combinacions davant de soques clíniques de M. tuberculosis multiresistents comparant amb soques sensibles; a) levofloxacin(LEV)-linezolid(LNZ)-amikacin(AMK), b)LEV-LNZ-EMB, c)LEV-AMK-EMB; 4) determinar l’activitat microbiana i sinèrgica de les combinacions descrites al objectiu 3 davant de soques clíniques de M. tuberculosis multiresistents i sensibles en un model de macròfags humans de la línia cel•lular THP-1. En l’estudi en macròfags murins, es va trobar que les soques resistents a INH i les multiresistents amb mutació en el gen katG van mostrar una multiplicació en l’interior dels macròfags disminuïda, suggerint una fitness disminuïda de les soques amb aquests patrons de resistència de M. tuberculosis. En aquesta tesi s’ha vist com l’adaptació del mètode de tauler d’escacs, és una tècnica fiable per l’estudi in vitro de combinacions de tres fàrmacs. La combinació incloent INH, RIF i EMB podria ser eficaç per tractar casos de TB amb una baixa resistència a INH (CIMs ≤ 0.8µg/ml) degut al efecte sinergístic de la combinació mostrat. Les combinacions de INH-RIF-EMB i la de OFL-EMB-RIF poden ser útils pel tractament tot i que la combinació que conté OFL presenta una eficàcia major, sent d’us potencial per tractar casos de soques resistents com de sensibles. In vitro, les combinacions que inclouen fàrmacs de segona línia (LEV-AMK-EMB; LEV-AMK-LNZ; LEV-EMB-LNZ) són igual de eficaces que la combinació de INH-RIF-EMB utilitzant el mètode de tauler d’escacs. En canvi, aquestes combinacions que inclouen fàrmacs de segona línia davant de macròfags humans THP-1 infectats amb M. tuberculosis mostren una activitat antimicrobiana, i un efecte antagonista de les combinacions que inclouen LEV i LNZ

Immunological Features of Respiratory Syncytial Virus-Caused Pneumonia—Implications for Vaccine Design

The human respiratory syncytial virus (hRSV) is the causative agent for high rates of hospitalizations due to viral bronchiolitis and pneumonia worldwide. Such a disease is characterized by an infection of epithelial cells of the distal airways that leads to inflammation and subsequently to respiratory failure. Upon infection, different pattern recognition receptors recognize the virus and trigger the innate immune response against the hRSV. Further, T cell immunity plays an important role for virus clearance. Based on animal studies, it is thought that the host immune response to hRSV is based on a biased T helper (Th)-2 and Th17 T cell responses with the recruitment of T cells, neutrophils and eosinophils to the lung, causing inflammation and tissue damage. In contrast, human immunity against RSV has been shown to be more complex with no definitive T cell polarization profile. Nowadays, only a humanized monoclonal antibody, known as palivizumab, is available to protect against hRSV infection in high-risk infants. However, such treatment involves several injections at a significantly high cost. For these reasons, intense research has been focused on finding novel vaccines or therapies to prevent hRSV infection in the population. Here, we comprehensively review the recent literature relative to the immunological features during hRSV infection, as well as the new insights into preventing the disease caused by this virus

A safe and efficient BCG vectored vaccine to prevent the disease caused by the human Respiratory Syncytial Virus

The human Respiratory Syncytial Virus (hRSV) causes lower respiratory tract infections including pneumonia and bronchiolitis. Such infections also cause a large number of hospitalizations and affects mainly newborns, young children and the elderly worldwide. Symptoms associated with hRSV infection are due to an exacerbated immune response characterized by low levels of IFN-γ, recruitment of neutrophils and eosinophils to the site of infection and lung damage. Although hRSV is a major health problem, no vaccines are currently available. Different immunization approaches have been developed to achieve a vaccine that activates the immune system, without triggering an unbalanced inflammation. These approaches include live attenuated vaccine, DNA or proteins technologies, and the use of vectors to express proteins of the virus. In this review, we discuss the host immune response to hRSV and the immunological mechanisms underlying an effective and safe BCG vectored vaccine against hRSV

TCR Repertoire Characterization for T Cells Expanded in Response to hRSV Infection in Mice Immunized with a Recombinant BCG Vaccine

T cells play an essential role in the immune response against the human respiratory syncytial virus (hRSV). It has been described that both CD4+ and CD8+ T cells can contribute to the clearance of the virus during an infection. However, for some individuals, such an immune response can lead to an exacerbated and detrimental inflammatory response with high recruitment of neutrophils to the lungs. The receptor of most T cells is a heterodimer consisting of α and β chains (αβTCR) that upon antigen engagement induces the activation of these cells. The αβTCR molecule displays a broad sequence diversity that defines the T cell repertoire of an individual. In our laboratory, a recombinant Bacille Calmette–Guérin (BCG) vaccine expressing the nucleoprotein (N) of hRSV (rBCG-N-hRSV) was developed. Such a vaccine induces T cells with a Th1 polarized phenotype that promote the clearance of hRSV infection without causing inflammatory lung damage. Importantly, as part of this work, the T cell receptor (TCR) repertoire of T cells expanded after hRSV infection in naïve and rBCG-N-hRSV-immunized mice was characterized. A more diverse TCR repertoire was observed in the lungs from rBCG-N-hRSV-immunized as compared to unimmunized hRSV-infected mice, suggesting that vaccination with the recombinant rBCG-N-hRSV vaccine triggers the expansion of T cell populations that recognize more viral epitopes. Furthermore, differential expansion of certain TCRVβ chains was found for hRSV infection (TCRVβ+8.3 and TCRVβ+5.1,5.2) as compared to rBCG-N-hRSV vaccination (TCRVβ+11 and TCRVβ+12). Our findings contribute to better understanding the T cell response during hRSV infection, as well as the functioning of a vaccine that induces a protective T cell immunity against this virus

Contribution of autophagy to antiviral immunity

AbstractAlthough identified in the 1960’s, interest in autophagy has significantly increased in the past decade with notable research efforts oriented at understanding as to how this multi-protein complex operates and is regulated. Autophagy is commonly defined as a “self-eating” process evolved by eukaryotic cells to recycle senescent organelles and expired proteins, which is significantly increased during cellular stress responses. In addition, autophagy can also play important roles during human diseases, such as cancer, neurodegenerative and autoimmune disorders. Furthermore, novel findings suggest that autophagy contributes to the host defense against microbial infections. In this article, we review the role of macroautophagy in antiviral immune responses and discuss molecular mechanisms evolved by viral pathogens to evade this process. A role for autophagy as an effector mechanism used both, by innate and adaptive immunity is also discussed

A single, low dose of a cGMP recombinant BCG vaccine elicits protective T cell immunity against the human respiratory syncytial virus infection and prevents lung pathology in mice

International audienceHuman respiratory syncytial virus (hRSV) is a major health burden worldwide, causing the majority of hospitalizations in children under two years old due to bronchiolitis and pneumonia. HRSV causes year-to-year outbreaks of disease, which also affects the elderly and immunocompromised adults. Furthermore, both hRSV morbidity and epidemics are explained by a consistently high rate of re-infections that take place throughout the patient life. Although significant efforts have been invested worldwide, currently there are no licensed vaccines to prevent hRSV infection. Here, we describe that a recombinant Bacillus Calmette-Guerin (BCG) vaccine expressing the nucleoprotein (N) of hRSV formulated under current good manufacture practices (cGMP rBCG-N-hRSV) confers protective immunity to the virus in mice. Our results show that a single dose of the GMP rBCG-N-hRSV vaccine retains its capacity to protect mice against a challenge with a disease-causing infection of 1×107 plaque-forming units (PFUs) of the hRSV A2 clinical strain 13018-8. Compared to unimmunized infected controls, vaccinated mice displayed reduced weight loss and less infiltration of neutrophils within the airways, as well as reduced viral loads in bronchoalveolar lavages, parameters that are characteristic of hRSV infection in mice. Also, ex vivo re-stimulation of splenic T cells at 28days post-immunization activated a repertoire of T cells secreting IFN-γ and IL-17, which further suggest that the rBCG-N-hRSV vaccine induced a mixed, CD8+ and CD4+ T cell response capable of both restraining viral spread and preventing damage of the lungs. All these features support the notion that rBCG-N-hRSV is a promising candidate vaccine to be used in humans to prevent the disease caused by hRSV in the susceptible population