protective-efficacy-pulmonary-immune-response-following-subcutaneous

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

University of Zaragoza

Substantial efforts have been made over the past decade to develop vaccines against tuberculosis. We review recent developments in tuberculosis vaccines in the global portfolio, including those designed for use in a prophylactic setting, either alone or as boosts to Bacille Calmette-Guérin, and therapeutic vaccines designed to improve chemotherapy. While there is no doubt that progress is still being made, there are limitations to our animal model screening processes, which are further amplified by the lack of understanding of the immunological responses involved and the precise type of long-lived immunity that new vaccines need to induce. The challenge ahead is to optimize the planning for advanced clinical trials in poor endemic settings, which could be greatly facilitated by identifying correlates of protection.

Recent developments in tuberculosis vaccines.

The major Mycobacterium tuberculosis virulence factor ESAT-6 exported by the ESX-1 secretion system has been described as a pro-apoptotic factor by several independent groups in recent years, sustaining a role for apoptosis in M. tuberculosis pathogenesis. This role has been supported by independent studies in which apoptosis has been shown as a hallmark feature in human and mouse lungs infected with virulent strains. Nevertheless, the role of apoptosis during mycobacterial infection is subject to an intense debate. Several works maintain that apoptosis is more evident with attenuated strains, whereas virulent mycobacteria tend to inhibit this process, suggesting that apoptosis induction may be a host mechanism to control infection. In this review, we summarize the evidences that support the involvement of ESX-1-induced apoptosis in virulence, intending to provide a rational treatise for the role of programmed cell death during M. tuberculosis infection.

ESX-1-induced apoptosis during mycobacterial infection: to be or not to be, that is the question.

Worldwide, the Mycobacterium bovis BCG vaccine is one of the most widely used vaccines. However, it appears to be ineffective in preventing pulmonary tuberculosis. Here, we show that pulmonary BCG vaccination of mice with a broad dose range provides superior protection against Mycobacterium tuberculosis challenge compared to that of subcutaneous vaccination.

Pulmonary Mycobacterium bovis BCG vaccination confers dose-dependent superior protection compared to that of subcutaneous vaccination.

Among the tuberculosis (TB) vaccine candidates, SO2 is the prototype of the first live-attenuated vaccine that recently entered into clinical trials. To investigate the capacity of SO2 to stimulate an appropriate immune response in vitro within a human immunological context, a comparative analysis of the effects promoted by SO2, the current Bacille Calmette-Guerin (BCG) vaccine and Mycobacterium tuberculosis (Mtb) was conducted in human primary dendritic cells (DC), which are critical modulators of vaccine-induced immunity. In particular, we found that SO2 promotes the expression of maturation markers similarly to BCG but at a lower extent than Mtb. Moreover, SO2-infected DC released higher levels of interleukin (IL)-23 than BCG-infected cells, which account for the expansion of interferon (IFN)-γ-producing T cells in an IL-12-independent manner. In the autologous mixed leukocyte reaction setting, the expansion of IL-17-producing T cells was also observed in response to SO2 infection. Interestingly, apoptosis and autophagic flux, events required for the antigen presentation within MHC class II complex, were not affected in DC infected with SO2, conversely to what observed upon Mtb stimulation. Collectively, our results indicate that SO2 represents a promising TB vaccine candidate, which displays an attenuated phenotype and promotes in DC a stronger capacity to stimulate the Th response than BCG vaccine. Interestingly, the data obtained by using the human DC-based experimental setting mirrored the results derived from studies in animal models, suggesting that this system could be used for an efficient and rapid down-selection of new TB vaccine candidates, contributing to achieve the "3Rs" objective.

A human dendritic cell-based in vitro model to assess Mycobacterium tuberculosis SO2 vaccine immunogenicity.

The ESX-1 secreted virulence factor ESAT-6 is one of the major and most well-studied virulence factors of Mycobacterium tuberculosis, given that its inactivation severely attenuates virulent mycobacteria. In this work, we show that clinical isolates of M. tuberculosis produce and secrete larger amounts of ESAT-6 than the widely used M. tuberculosis H37Rv laboratory strain. A search for the genetic polymorphisms underlying this observation showed that whiB6 (rv3862c), a gene upstream of the ESX-1 genetic locus that has not previously been found to be implicated in the regulation of the ESX-1 secretory apparatus, presents a unique single nucleotide insertion in its promoter region in strains H37Rv and H37Ra. This polymorphism is not present in any of the other publicly available M. tuberculosis complex genomes or in any of the 76 clinical M. tuberculosis isolates analyzed in our laboratory. We demonstrate that in consequence, the virulence master regulator PhoP downregulates whiB6 expression in H37Rv, while it upregulates its expression in clinical strains. Importantly, reintroduction of the wild-type (WT) copy of whiB6 in H37Rv restored ESAT-6 production and secretion to the level of clinical strains. Hence, we provide clear evidence that in M. tuberculosis--with the exception of the H37Rv strain--ESX-1 expression is regulated by WhiB6 as part of the PhoP regulon, which adds another level of complexity to the regulation of ESAT-6 secretion with a potential role in virulence adaptation.

A specific polymorphism in Mycobacterium tuberculosis H37Rv causes differential ESAT-6 expression and identifies WhiB6 as a novel ESX-1 component.

During bacterial sepsis, proinflammatory cytokines contribute to multiorgan failure and death in a process regulated in part by cytolytic cell granzymes. When challenged with a sublethal dose of the identified mouse pathogen Brucella microti, wild-type (WT) and granzyme A (gzmA)(-/-) mice eliminate the organism from liver and spleen in 2 or 3 weeks, whereas the bacteria persist in mice lacking perforin or granzyme B as well as in mice depleted of Tc cells. In comparison, after a fatal challenge, only gzmA(-/-) mice exhibit increased survival, which correlated with reduced proinflammatory cytokines. Depletion of natural killer (NK) cells protects WT mice from sepsis without influencing bacterial clearance and the transfer of WT, but not gzmA(-/-) NK, cells into gzmA(-/-) recipients restores the susceptibility to sepsis. Therefore, infection-related pathology, but not bacterial clearance, appears to require gzmA, suggesting the protease may be a therapeutic target for the prevention of bacterial sepsis without affecting immune control of the pathogen.

Elucidating sources and roles of granzymes A and B during bacterial infection and sepsis.

Safety of individuals at risk of immune suppression is an important concern for live vaccines. The new-generation tuberculosis vaccine candidate MTBVAC, a genetically engineered doubly attenuated Mycobacterium tuberculosis mutant with deletions in phoP and fadD26 virulence genes has demonstrated comparable safety in different relevant animal models and superior protection in mice as compared to the only currently licensed tuberculosis vaccine Mycobacterium bovis BCG. Here we describe the construction of a highly attenuated MTBVAC-based live vaccine by an additional gene inactivation generated in erp of MTBVAC. The gene product of erp is an exported repeated protein (Erp), a virulence factor described to be involved in intracellular replication of M. tuberculosis. The resultant strain, MTBVAC erp(-), was tested in severe combined immunodeficiency (SCID) mouse model showing to be severely attenuated when compared to BCG and MTBVAC. Experiments conducted in immunocompetent mice revealed that the hyper-attenuated profile observed with MTBVAC erp(-) strain did not compromise its protective efficacy profile in comparison with BCG. These results postulate MTBVAC erp(-) as a potential tuberculosis vaccine candidate for use in high-risk populations of immune suppression (e.g., due to HIV infection), where the use of BCG is not recommended.

Hyper-attenuated MTBVAC erp mutant protects against tuberculosis in mice.

Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB(+)Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB(+)Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-XL, the anti-apoptotic counterparts of Bim. In contrast, gzmB(+)Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB(+)Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB(+)Tc-induced death pathways.

Mouse cytotoxic T cell-derived granzyme B activates the mitochondrial cell death pathway in a Bim-dependent fashion.

Natural killer (NK) cells recognize and eliminate transformed or infected cells that have downregulated MHC class-I and express specific activating ligands. Recent evidence indicates that allogeneic NK cells are useful to eliminate haematological cancer cells independently of MHC-I expression. However, it is unclear if transformed cells expressing mutations that confer anti-apoptotic properties and chemoresistance will be susceptible to NK cells. Allogeneic primary human NK cells were activated using different protocols and prospectively tested for their ability to eliminate diverse mutant haematological and apoptotic-resistant cancer cell lines as well as patient-derived B-cell chronic lymphocytic leukemia cells with chemotherapy multiresistance. Here, we show that human NK cells from healthy donors activated in vitro with Epstein Barr virus positive (EBV(+))-lymphoblastoid cells display an enhanced cytotoxic and proliferative potential in comparison to other protocols of activation such a K562 cells plus interleukin (IL)2. This enhancement enables them to kill more efficiently a variety of haematological cancer cell lines, including a panel of transfectants that mimic natural mutations leading to oncogenic transformation and chemoresistance (e.g., overexpression of Bcl-2, Bcl-XL and Mcl-1 or downregulation of p53, Bak/Bax or caspase activity). The effect was also observed against blasts from B-cell chronic lymphocytic leukemia patients showing multi-resistance to chemotherapy. Our findings demonstrate that particular in vitro activated NK cells may overcome anti-apoptotic mechanisms and oncogenic alterations frequently occurring in transformed cells, pointing toward the use of EBV(+)-lymphoblastoid cells as a desirable strategy to activate NK cells in vitro for the purpose of treating haematological neoplasia with poor prognosis.

Human NK cells activated by EBV(+) lymphoblastoid cells overcome anti-apoptotic mechanisms of drug resistance in haematological cancer cells.

Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K. pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of phosphoinositide 3-kinase (PI3K). Microscopy studies revealed that K. pneumoniae resides within a vacuolar compartment, the Klebsiella-containing vacuole (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV-killed bacteria, the majority of live bacteria did not co-localize with markers of the lysosomal compartment. Our data suggest that K. pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K. pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K-Akt-Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the down-regulation of the expression of cps. Altogether, this study proves evidence that K. pneumoniae survives killing by macrophages by manipulating phagosome maturation that may contribute to Klebsiella pathogenesis.

Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes.

Some of the most promising novel tuberculosis vaccine strategies currently under development are based on respiratory vaccination, mimicking the natural route of infection. In this work, we have compared pulmonary and subcutaneous delivery of BCG vaccine in the tuberculosis-susceptible DBA/2 mouse strain, a model in which parenterally administered BCG vaccine does not protect against tuberculosis. Our data show that intranasally but not subcutaneously administered BCG confers robust protection against pulmonary tuberculosis challenge. In addition, our results indicate that pulmonary vaccination triggers a Mycobacterium tuberculosis-specific mucosal immune response orchestrated by interleukin 17A (IL-17A). Thus, IL-17A neutralization in vivo reduces protection and abrogates M. tuberculosis-specific immunoglobulin A (IgA) secretion to respiratory airways and lung expression of polymeric immunoglobulin receptor induced following intranasal vaccination. Together, our results demonstrate that pulmonary delivery of BCG can overcome the lack of protection observed when BCG is given parenterally, suggesting that respiratory tuberculosis vaccines could have an advantage in tuberculosis-endemic countries, where intradermally administered BCG has inefficient effectiveness against pulmonary tuberculosis.

Pulmonary but Not Subcutaneous Delivery of BCG Vaccine Confers Protection to Tuberculosis-Susceptible Mice by an Interleukin 17-Dependent Mechanism.

Tuberculosis remains one of the world's deadliest transmissible diseases despite widespread use of the BCG vaccine. MTBVAC is a new live tuberculosis vaccine based on genetically attenuated Mycobacterium tuberculosis that expresses most antigens present in human isolates of M tuberculosis. We aimed to compare the safety of MTBVAC with BCG in healthy adult volunteers.

Safety of human immunisation with a live-attenuated Mycobacterium tuberculosis vaccine: a randomised, double-blind, controlled phase I trial.

The immunogenicity and diagnostic interference caused by M. tuberculosis SO2, a prototype vaccine first time tested in goats was evaluated. Tuberculosis-free goats were distributed in four groups: [1], non-vaccinated; [2], subcutaneously (SC) BCG vaccinated; [3], intranasally (IN) SO2 vaccinated and [4], SC SO2 vaccinated. Intradermal tuberculin and IFN-γ tests using PPDs and alternative antigenic cocktails containing mainly ESAT-6 and CFP-10 (E/C) were applied at different times post-vaccination. Results showed a significant (p<0.05) increase in the number of reactors detected using both PPD-based intradermal and IFN-γ tests at different times in all the vaccinated groups. No intradermal reactivity was detected in the vaccinated goats using a cocktail containing E/C, Rv3615c and Rv3020c. A higher overall reactivity was observed in the group [4] in comparison with the other vaccinated groups. Results showed that antigens used to differentiate BCG vaccinated animals could be potentially used to differentiate SO2 vaccinated ones.

Evaluation of the immunogenicity and diagnostic interference caused by M. tuberculosis SO2 vaccination against tuberculosis in goats.

Development of novel more efficient preventive vaccines against tuberculosis (TB) is crucial to achieve TB eradication by 2050, one of the Millennium Development Goals (MDG) for the current century. MTBVAC is the first and only live attenuated vaccine based on a human isolate of Mycobacterium tuberculosis developed as BCG-replacement strategy in newborns that has entered first-in-human adult clinical trials. In this work, we characterize the safety, immunogenicity and protective efficacy of MTBVAC in a model of newborn C57/BL6 mice. Our data clearly indicate that MTBVAC is safe for newborn mice, and does not affect animal growth or organ development. In addition, MTBVAC-vaccinated mice at birth showed enhanced immunogenicity and better protection against M. tuberculosis challenge in comparison with BCG.

MTBVAC vaccine is safe, immunogenic and confers protective efficacy against Mycobacterium tuberculosis in newborn mice.

Granzyme A, a serine protease expressed in the granules of cytotoxic T and Natural Killer cells, is involved in the generation of pro-inflammatory cytokines by macrophages. Granzyme A has been described to induce in macrophages in vitro the activation of pro-inflammatory pathways that impair intracellular mycobacterial replication. In the present study, we explored the physiological relevance of Granzyme A in the control of pulmonary Mycobacterium tuberculosis infection in vivo. Our results show that, even though Granzyme A is expressed by cytotoxic cells from mouse lungs during pulmonary infection, its deficiency in knockout mice does not have an effect in the control of M. tuberculosis infection. In addition our findings indicate that absence of Granzyme A does not affect the protection conferred by the live-attenuated M. tuberculosis vaccine MTBVAC. Altogether, our findings are in apparent contradiction with previously published in vitro results and suggest that Granzyme A does not have a crucial role in vivo in the protective response to tuberculosis.

Article	Year
Recent developments in tuberculosis vaccines. Expert review of vaccines\| PubMed ID: 24195481	2013
ESX-1-induced apoptosis during mycobacterial infection: to be or not to be, that is the question. Frontiers in cellular and infection microbiology\| PubMed ID: 24364000	2013
Pulmonary Mycobacterium bovis BCG vaccination confers dose-dependent superior protection compared to that of subcutaneous vaccination. Clinical and vaccine immunology : CVI\| PubMed ID: 24501340	2014
A human dendritic cell-based in vitro model to assess Mycobacterium tuberculosis SO2 vaccine immunogenicity. ALTEX\| PubMed ID: 24840067	2014
A specific polymorphism in Mycobacterium tuberculosis H37Rv causes differential ESAT-6 expression and identifies WhiB6 as a novel ESX-1 component. Infection and immunity\| PubMed ID: 24891105	2014
Elucidating sources and roles of granzymes A and B during bacterial infection and sepsis. Cell reports\| PubMed ID: 25017060	2014
Hyper-attenuated MTBVAC erp mutant protects against tuberculosis in mice. Vaccine\| PubMed ID: 25066740	2014
Mouse cytotoxic T cell-derived granzyme B activates the mitochondrial cell death pathway in a Bim-dependent fashion. The Journal of biological chemistry\| PubMed ID: 25605735	2015
Human NK cells activated by EBV(+) lymphoblastoid cells overcome anti-apoptotic mechanisms of drug resistance in haematological cancer cells. Oncoimmunology\| PubMed ID: 25949911	2015
Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes. Cellular microbiology\| PubMed ID: 26045209	2015
Pulmonary but Not Subcutaneous Delivery of BCG Vaccine Confers Protection to Tuberculosis-Susceptible Mice by an Interleukin 17-Dependent Mechanism. The Journal of infectious diseases\| PubMed ID: 26494773	2016
Safety of human immunisation with a live-attenuated Mycobacterium tuberculosis vaccine: a randomised, double-blind, controlled phase I trial. The Lancet. Respiratory medicine\| PubMed ID: 26598141	2015
Evaluation of the immunogenicity and diagnostic interference caused by M. tuberculosis SO2 vaccination against tuberculosis in goats. Research in veterinary science\| PubMed ID: 26679799	2015
MTBVAC vaccine is safe, immunogenic and confers protective efficacy against Mycobacterium tuberculosis in newborn mice. Tuberculosis (Edinburgh, Scotland)\| PubMed ID: 26786657	2016
Granzyme A Is Expressed in Mouse Lungs during Mycobacterium tuberculosis Infection but Does Not Contribute to Protection In Vivo. PloS one\| PubMed ID: 27055232	2016

Nacho Aguilo

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