Clinical and Experimental Determination of Protection Afforded by BCG Vaccination against Infection with Non-Tuberculous Mycobacteria: A Role in Cystic Fibrosis?

Mycobacterium abscessus is a nontuberculous mycobacterium (NTM) of particular concern in individuals with obstructive lung diseases such as cystic fibrosis (CF). Treatment requires multiple drugs and is characterised by high rates of relapse; thus, new strategies to limit infection are urgently required. This study sought to determine how Bacille Calmette-Guérin (BCG) vaccination may impact NTM infection, using a murine model of Mycobacterium abscessus infection and observational data from a non-BCG vaccinated CF cohort in Sydney, Australia and a BCG-vaccinated CF cohort in Cape Town, South Africa. In mice, BCG vaccination induced multifunctional antigen-specific CD up sup T cells circulating in the blood and was protective against dissemination of bacteria to the spleen. Prior infection with M. abscessus afforded the highest level of protection against M. abscessus challenge in the lung, and immunity was characterised by a greater frequency of pulmonary cytokine-secreting CD4 T cells compared to BCG vaccination. In the clinical CF cohorts, the overall rates of NTM sampling during a three-year period were equivalent; however, rates of NTM colonisation were significantly lower in the BCG-vaccinated (Cape Town) cohort, which was most apparent for M. abscessus. This study provides evidence that routine BCG vaccination may reduce M. abscessus colonisation in individuals with CF, which correlates with the ability of BCG to induce multifunctional CD4T cells recognising M. abscessus in a murine model. Further research is needed to determine the optimal strategies for limiting NTM infections in individuals with CF

Delta inulin-based adjuvants promote the generation of polyfunctional CD4+ T cell responses and protection against Mycobacterium tuberculosis infection

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.There is an urgent need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as Mycobacterium tuberculosis. Advax™ is a novel adjuvant based on delta inulin microparticles that enhances immunity with a minimal inflammatory profile and has entered human trials to protect against viral pathogens. In this report we determined if Advax displays broad applicability against important human pathogens by assessing protective immunity against infection with M. tuberculosis. The fusion protein CysVac2, comprising the M. tuberculosis antigens Ag85B (Rv1886c) and CysD (Rv1285) formulated with Advax provided significant protection in the lungs of M. tuberculosis-infected mice. Protection was associated with the generation of CysVac2-specific multifunctional CD4+ T cells (IFN-γ+TNF+IL-2+). Addition to Advax of the TLR9 agonist, CpG oligonucleotide (AdvaxCpG), improved both the immunogenicity and protective efficacy of CysVac2. Immunisation with CysVac2/AdvaxCpG resulted in heightened release of the chemoattractants, CXCL1, CCL3, and TNF, and rapid influx of monocytes and neutrophils to the site of vaccination, with pronounced early priming of CysVac2-specific CD4+ T cells. As delta inulin adjuvants have shown an excellent safety and tolerability profile in humans, CysVac2/AdvaxCpG is a strong candidate for further preclinical evaluation for progression to human trials

TBVAC2020 : advancing tuberculosis vaccines from discovery to clinical development

TBVAC2020 is a research project supported by the Horizon 2020 program of the European Commission (EC). It aims at the discovery and development of novel tuberculosis (TB) vaccines from preclinical research projects to early clinical assessment. The project builds on previous collaborations from 1998 onwards funded through the EC framework programs FP5, FP6, and FP7. It has succeeded in attracting new partners from outstanding laboratories from all over the world, now totaling 40 institutions. Next to the development of novel vaccines, TB biomarker development is also considered an important asset to facilitate rational vaccine selection and development. In addition, TBVAC2020 offers portfolio management that provides selection criteria for entry, gating, and priority settings of novel vaccines at an early developmental stage. The TBVAC2020 consortium coordinated by TBVI facilitates collaboration and early data sharing between partners with the common aim of working toward the development of an effective TB vaccine. Close links with funders and other consortia with shared interests further contribute to this goal

TBVAC2020: Advancing tuberculosis vaccines from discovery to clinical development

TBVAC2020 is a research project supported by the Horizon 2020 program of the European Commission (EC). It aims at the discovery and development of novel tuberculosis (TB) vaccines from preclinical research projects to early clinical assessment. The project builds on previous collaborations from 1998 onwards funded through the EC framework programs FP5, FP6, and FP7. It has succeeded in attracting new partners from outstanding laboratories from all over the world, now totaling 40 institutions. Next to the development of novel vaccines, TB biomarker development is also considered an important asset to facilitate rational vaccine selection and development. In addition, TBVAC2020 offers portfolio management that provides selection criteria for entry, gating, and priority settings of novel vaccines at an early developmental stage. The TBVAC2020 consortium coordinated by TBVI facilitates collaboration and early data sharing between partners with the common aim of working toward the development of an effective TB vaccine. Close links with funders and other consortia with shared interests further contribute to this goal

Preclinical evaluation of novel candidate vaccines in a murine model of tuberculosis

Tuberculosis (TB) remains a major cause of mortality and morbidity worldwide, yet the currently vaccine available, Mycobacterium bovis Bacille Calmette et Guérin (BCG), is only partially effective against infection with M. tuberculosis. Therefore the development of a new effective TB vaccine is urgently required. A novel fusion protein vaccine, termed CysVac2, was developed by fusing CysD, a major component of the sulphate activation pathway of M. tuberculosis, to the immunodominant-secreted antigen Ag85B. Vaccination of C57BL/6 mice with CysVac2, combined with the adjuvant MPL/DDA, induced the generation of multifunctional CD4+ T cells and conferred protection against challenge with aerosol M. tuberculosis to an extent comparable to BCG. Furthermore, the CysVac2 vaccine could boost the protective effect of BCG. Vaccination with BCG overexpressing CysVac2 (rBCG:CysVac2) resulted in enhanced CD4+ T cell priming and immunogenicity compared to parental BCG, however rBCG:CysVac2 did not demonstrate improve protective efficacy compared to BCG alone. Combination of CysVac2 with the novel polysaccharide adjuvant Advax reduced the bacterial burden in the lungs of M. tuberculosis-challenged mice. This improved protection correlated with the rapid influx of neutrophils and macrophages to the site of vaccination as well as pronounced priming of M. tuberculosis-specific CD4+ T cells with a multifunctional phenotype. In conclusion, this thesis has defined the vaccine potential of CysD-based TB vaccines and identified candidates that merit further preclinical evaluation for progression to human trials

Interazione diretta tra Mycobacterium bovis bacillo di Calmette e Guérin e cellule natural killer umane: studio dei possibili recettori coinvolti

Le cellule natural killer (NK) rappresentano circa il 10% dei linfociti del sangue periferico dell’uomo e sono caratterizzate dalla mancanza del marcatore CD3 e dall'espressione del marcatore CD16 e/o CD56. Sebbene inizialmente descritte per la loro spiccata attività citotossica nei confronti di cellule infettate da virus o di cellule tumorali, è oggi noto che tali cellule svolgono funzioni immunitarie importanti anche nei meccanismi di difesa innati contro agenti patogeni batterici. Una delle più importanti acquisizioni della biologia delle cellule NK negli ultimi anni è stata quella che ha evidenziato che l'attività di tali cellule è il risultato di un delicato equilibrio tra stimoli attivatori e stimoli inibitori mediati da altrettanti recettori cellulari. I recettori inibitori includono i Killer immunoglobin-like receptors (KIR) che, riconoscendo molecole MHC di classe I sulle cellule target, inibiscono l’attivazione delle funzioni citotossiche. Tra i recettori attivatori hanno recentemente acquisito particolare importanza quelli appartenenti alla famiglia dei natural citotoxicity receptors (NCR) che includono tre molecole, NKp30, NKp44, NKp46, espresse esclusivamente da cellule NK e coinvolte nel riconoscimento di ligandi microbici e/o cellulari ancora in gran parte da caratterizzare. Recentemente si sono, inoltre, accumulati dati sperimentali che dimostrano che le cellule NK possano esprimere anche alcuni membri della famiglia dei Toll-like receptors (TLR), suggerendo che esse possano, al pari di altre cellule dell’immunità innata, riconoscere ed interagire direttamente con gli agenti patogeni. Studi precedenti, effettuati nel laboratorio in cui è stato svolto il lavoro di tesi, hanno dimostrato che Mycobacterium bovis bacillo di Calmette e Guérin (BCG), un ceppo attenuato di M. bovis, usato correntemente come vaccino antitubercolare, è in grado di interagire direttamente con cellule NK umane, inducendo la produzione di IFN-gamma, la proliferazione e l’attività citotossica di tali cellule. Scopo del presente lavoro di tesi è stato quello di studiare i possibili recettori cellulari coinvolti nel riconoscimento di BCG da parte di cellule NK umane e, probabilmente, responsabili dell’attivazione delle loro funzioni effettrici in risposta al microrganismo. A tale scopo, popolazioni cellulari altamente purificate di cellule NK sono state ottenute da sangue periferico di soggetti sani per selezione negativa mediante l’uso di biglie magnetiche commerciali. In seguito a stimolazione in vitro per vari intervalli di tempo con BCG vivo, è stata valutata l’espressione di superficie di TLR e NCR, mediante colorazione con anticorpi monoclonali ed analisi in citofluorimetria a flusso. I risultati ottenuti non hanno evidenziato alcuna espressione di superficie di TLR-2 e TLR-4 (due recettori toll-like per i quali è stata dimostrata l’esistenza di ligandi micobatterici) a nessuno dei tempi analizzati né su cellule NK non stimolate né dopo stimolazione con BCG. Al contrario, BCG è risultato in grado di indurre l’espressione di superficie del recettore NKp44, ma non degli altri due recettori appartenenti alla stessa famiglia, NKp30 e NKp46. Tale induzione, valutata come intensità di fluorescenza media (MFI), era evidente a partire da 3-4 giorni di stimolazione con BCG quando i valori di MFI per tale recettore risultavano statisticamente più elevati rispetto alle cellule non stimolate. L’induzione di NKp44, inoltre, era principalmente ascrivibile ad una particolare sottopopolazione di cellule CD56+ che esprime tale marcatore ad alta densità, le cellule CD56bright. Allo scopo di valutare se l’induzione da parte di BCG di NKp44 su cellule NK isolate potesse essere dovuta ad un’interazione diretta del batterio con tale recettore, forme solubili di tutti e tre i recettori NCR, chimera per il frammento Fc delle immunoglobuline G umane (IgG), disponibili commercialmente (NKp30Fc, NKp44Fc, NKp46Fc), sono stati incubati con un ceppo di BCG vivo, ricombinante per il gene codificante per la green fluorescent protein (gfp), a disposizione del laboratorio. L’eventuale legame recettore-batterio è stato rilevato mediante colorazione con un anticorpo secondario di capra diretto contro il frammento Fc delle IgG umane marcato con ficoeritrina (PE) ed analisi in citofluorimetria a flusso a due colori dei complessi. I risultati ottenuti hanno dimostrato che, rispetto al controllo negativo, rappresentato dai batteri incubati in presenza di IgG umane e successivamente con l’anticorpo secondario, i batteri trattati con NKp44Fc solubile mostravano un netto incremento della fluorescenza corrispondente al fluorocromo PE, al contrario di quanto osservato con i batteri incubati con la forma solubile di NKp30Fc e di NKp46Fc. In particolare, la fluorescenza media dei batteri trattati con NKp44Fc era di almeno 50 volte superiore rispetto a quella del controllo negativo. Analoghi saggi di binding hanno dimostrato che NKp44Fc era in grado di legarsi anche ad altre specie di micobatteri, quali M. smegmatis, M. avium e M. tuberculosis, ma non a Streptococcus pyogenes (es di batterio Gram-positivo) o a Salmonella enteritidis (es di batterio Gram-negativo). Allo scopo di verificare se il legame tra NKp44Fc e BCG, evidenziato tramite citofluorimetria, potesse essere confermato con un approccio metodologico diverso, BCG incubato con tutti e tre i recettori NCR solubili è stato trattato con anticorpi, marcati con particelle d’oro colloidale, diretti verso il frammento Fc delle immunoglobuline umane. Le varie sospensioni batteriche sono state quindi osservate mediante microscopia elettronica a trasmissione che ha rivelato una netta positività, costituita da microparticelle d’oro distribuite uniformemente su tutta la superficie del batterio, sui batteri incubati con NKp44Fc, ma non con gli altri due NCR. Nel loro insieme, i risultati ottenuti dimostrano che: i) l’interazione diretta di BCG vivo con cellule NK umane altamente purificate induce su quest’ultime l’espressione di superficie di NKp44, ma non di NKp30, NKp46, TLR-2 e TLR-4; ii) NKp44Fc, ma non NKp30Fc e NKp46Fc, in forma solubile formano complessi con BCG vivo, suggerendo l’esistenza di ligandi micobatterici per tali recettori cellulari; iii) il putativo ligando per NKp44 sembra conservato nell’ambito del genere Mycobacterium, ma non in almeno due specie di batteri Gram-positivi o Gram-negativi. Il legame diretto del germe a tale recettore cellulare potrebbe indurne l’espressione ed, eventualmente, da solo od in combinazione con altri recettori cellulari, attivare le funzioni cellulari delle cellule NK

Preclinical evaluation of novel candidate vaccines in a murine model of tuberculosis

Tuberculosis (TB) remains a major cause of mortality and morbidity worldwide, yet the currently vaccine available, Mycobacterium bovis Bacille Calmette et Guérin (BCG), is only partially effective against infection with M. tuberculosis. Therefore the development of a new effective TB vaccine is urgently required. A novel fusion protein vaccine, termed CysVac2, was developed by fusing CysD, a major component of the sulphate activation pathway of M. tuberculosis, to the immunodominant-secreted antigen Ag85B. Vaccination of C57BL/6 mice with CysVac2, combined with the adjuvant MPL/DDA, induced the generation of multifunctional CD4+ T cells and conferred protection against challenge with aerosol M. tuberculosis to an extent comparable to BCG. Furthermore, the CysVac2 vaccine could boost the protective effect of BCG. Vaccination with BCG overexpressing CysVac2 (rBCG:CysVac2) resulted in enhanced CD4+ T cell priming and immunogenicity compared to parental BCG, however rBCG:CysVac2 did not demonstrate improve protective efficacy compared to BCG alone. Combination of CysVac2 with the novel polysaccharide adjuvant Advax reduced the bacterial burden in the lungs of M. tuberculosis-challenged mice. This improved protection correlated with the rapid influx of neutrophils and macrophages to the site of vaccination as well as pronounced priming of M. tuberculosis-specific CD4+ T cells with a multifunctional phenotype. In conclusion, this thesis has defined the vaccine potential of CysD-based TB vaccines and identified candidates that merit further preclinical evaluation for progression to human trials

Deciphering protective immunity against tuberculosis: implications for vaccine development

Introduction: The development of more effective tuberculosis (TB) vaccines is essential for the global control of TB. Recently, there have been major advances in the field, but an important hindrance remains the lack of correlates of protection against TB. This requires each vaccine candidate to undergo clinical efficacy trials based on data from animal protection studies, but the results from animal models do not necessarily predict efficacy in humans. Area covered: In this review we summarize our current knowledge of immune mechanisms that may contribute to protective immunity against TB following vaccination and relate these to protective efficacy in animal models and recent clinical trials. Although some initial trials did not reproduce protection against TB in humans, recent trials have demonstrated promising efficacy for three vaccine approaches. Expert opinion: Although CD4+ T lymphocytes are essential for protection against TB, there is no clear correlation between conventional CD4+ or CD8+ T cell responses and protective efficacy of TB vaccines. Recent attention has focused on other immune responses, including donor unrestricted T cells, B lymphocytes, and antibodies. Prospective studies on samples from vaccinated individuals protected in recent trials will allow evaluation of these alternative immune mechanisms as potential correlates of protection

Characterization of the Protective Immune Responses Conferred by Recombinant BCG Overexpressing Components of <i>Mycobacterium tuberculosis</i> Sec Protein Export System

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is the only approved vaccine against tuberculosis (TB). However, its efficacy in preventing pulmonary TB in adults is limited. Despite its variable efficacy, BCG offers a number of unique and beneficial characteristics, which make it suitable as a vaccine vehicle to express recombinant molecules. In Mycobacterium tuberculosis, the general Sec pathway is an essential cellular process, and it is responsible for exporting the majority of proteins across the cytoplasmic membrane, including potent immune-protective antigens, such as members of the antigen 85 (Ag85) complex. We engineered BCG to overexpress the M. tuberculosis SecDFG proteins in order to improve the efficiency of the Sec-dependent export system and, thus, enhance the secretion of immunogenic proteins. BCGSecDFG displayed increased intracellular survival within macrophages in vitro and greater persistence in the lymphoid organs of vaccinated mice than parental BCG. In addition, vaccination with BCGSecDFG generated higher numbers of IFN-γ-secreting T cells in response to secreted mycobacterial antigens compared to BCG, particularly members of the Ag85 complex. Furthermore, vaccination with BCGSecDFG significantly reduced the bacterial load in the lungs and spleens of M. tuberculosis-infected mice, which was comparable to the protection afforded by parental BCG. Therefore, the modification of protein secretion in BCG can improve antigen-specific immunogenicity