Genetic Modification of the BCG Vaccine to Overcome Its Limited Efficacy in Adults: A Specialized Review

Genetic Modification of the BCG Vaccine to Overcome Its Limited Efficacy in Adults: A Specialized Review | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Systematic Review Genetic Modification of the BCG Vaccine to Overcome Its Limited Efficacy in Adults: A Specialized Review Ssembuusi Allan Francis, Eloghosa Nosa-Ihaza This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8714621/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract While the Bacillus Calmette-Guérin (BCG) vaccine protects against severe childhood tuberculosis, it does not consistently protect against adult pulmonary tuberculosis, the main driver of global transmission and mortality. This review summarizes the scientific rationale and evolution of genetic engineering approaches to address BCG's immunological limitations, from initial strategies such as overexpressing Mycobacterium tuberculosis antigens and immunomodulatory cytokines to second-generation methods using subtractive and integrative genomic editing. Key candidates, such as VPM1002—engineered for phagosomal escape and improved CD8 + T-cell activation via MHC-I cross-presentation—and MTBVAC, a live-attenuated M. tuberculosis strain with a broad antigenic repertoire, are now in advanced clinical trials within a diverse pipeline monitored by WHO. These advances constitute a paradigm shift in vaccine design. However, adoption remains challenged by major barriers: the lack of immune correlates of protection, complex regulatory pathways for GMOs, and significant economic and manufacturing hurdles. Genetic modification offers the most feasible route to an effective adult TB vaccine, but its promise depends on overcoming obstacles in the translational landscape and aligning with the WHO's End TB Strategy. Recombinant BCG vaccines Genetic modification of BCG Adult pulmonary tuberculosis VPM1002 MTBVAC TB vaccine development Figures Figure 1 Figure 2 Figure 3 1 Introduction Tuberculosis (TB) is a serious infectious disease that remains a leading cause of death worldwide, especially among adults. For over 100 years, the Bacillus Calmette-Guérin (BCG) vaccine has been the only vaccine available to fight it. ( 1 ) While BCG works very well to protect babies and young children from the most severe forms of TB, it does not work consistently to protect adolescents and adults from lung TB, which is the most common and infectious form of the disease. ( 2 ) To address these limitations, researchers have employed genetic modification to enhance BCG's immunogenicity, a research priority underscored by the WHO's End TB Strategy. Specifically, this process involves the deliberate alteration of the BCG genome to confer novel immunological properties, primarily to elicit more robust and durable immune responses in vaccines. These improved responses are critical for preventing the establishment of latent infection and reactivation disease in adults. ( 3 , 4 ) Early strategies, initiated several decades ago, focused on the overexpression of antigens and cytokines. For example, recombinant BCG (rBCG) strains were engineered to express additional Mycobacterium tuberculosis antigens, such as Ag85B, a major secreted protein that is a dominant target of T-cell immunity, to broaden antigenic recognition. ( 5 – 7 ) Concurrently, cytokine-armed rBCG variants were developed to secrete immunomodulators like interleukin-2 (IL-2), which promotes T-cell proliferation; interleukin-12 (IL-12), a key inducer of Th1 responses; or interferon-gamma (IFN-γ), the central macrophage-activating cytokine. ( 8 , 9 ) These modifications aimed to create a localized Th1-polarizing microenvironment and enhance macrophage activation and T-cell recruitment, as evidenced by studies in murine models showing improved bacterial clearance. ( 10 – 12 ) Building on these findings, this foundational work established the principle that genetic modification could significantly enhance BCG's immunogenicity, paving the way for more sophisticated second-generation constructs. As research progressed, subsequent strategies evolved from additive approaches to more refined subtractive and integrated genomic engineering, moving beyond simple overexpression to more nuanced immune modulation. ( 13 ) A pivotal advancement within this evolution was the development of VPM1002, created through the deletion of the urease C (ureC) gene and insertion of listeriolysin O (hly) from Listeria monocytogen. This design facilitates phagosomal escape by perforating the phagosomal membrane in an acid-dependent manner, thereby promoting antigen escape into the cytosol and subsequent cross-presentation on MHC class I molecules.les. This, in turn, significantly enhances CD8 + T-cell activation—a response critical for adult protection that is poorly induced by the parent BCG strain. ( 14 – 17 ) Demonstrating promising immunogenicity and safety in Phase II and III clinical trials, VPM1002 received regulatory approval in India in 2021, marking a milestone as the first genetically modified BCG vaccine to be licensed. ( 18 , 19 ) Alongside VPM1002, other next-generation constructs, such as MTBVAC—a live-attenuated M. tuberculosis double-deletion mutant—and those incorporating multi-gene edits, such as disruption of the immune-suppressive phoP regulator or the nuoG gene, continue to undergo evaluation in global clinical trials. ( 20 ) Together, these efforts form a core part of the current TB vaccine pipeline as tracked by the WHO. The primary aim is to develop a vaccine effective against pulmonary disease across all age groups, an essential step toward meeting the WHO’s target of a 90% reduction in TB incidence by 2035. ( 21 , 22 ) 2 Methodology 2.1 Literature Search and Selection Criteria This review systematically searched PubMed, Scopus, and Web of Science for studies (2000–2024) on genetically modified BCG vaccines. Search terms included "BCG," "genetic modification," "recombinant BCG," "VPM1002," "MTBVAC," "adult pulmonary tuberculosis," and "vaccine efficacy." Of the 587 initial publications, 142 studies met the inclusion criteria: peer-reviewed articles, clinical trial data, and WHO reports on novel vaccine candidates and immunological outcomes ( 23 , 24 ). Priority was given to studies with specific genetic changes, quantifiable immunogenicity (≥ 2-fold CD8 + T-cell activation or significant antigen cross-presentation), and Phase II/III trial results from the WHO TB vaccine pipeline (2022–2024) ( 25 ). 2.2 Data Analysis Framework Extracted data were synthesized to evaluate three aspects: ( 1 ) scientific rationale for genetic engineering strategies, ( 2 ) immunogenic profile and efficacy of pipeline candidates, and ( 3 ) translational challenge ( 26 , 27 ). A comparative analysis traced the shift from animal proof-of-concept studies to recent human trials, highlighting candidates such as VPM1002, which demonstrated 45% efficacy in a South African adult trial. ( 28 ) 3 Genetic Engineering Strategies The genetic modification of Bacillus Calmette-Guérin (BCG) represents a sophisticated and targeted approach to overcome its limitations in protecting adults against pulmonary tuberculosis. ( 29 ) These engineering strategies have evolved systematically from initial enhancement approaches to complex multi-gene modifications, each designed to address specific immunological deficiencies of the parental BCG strain. ( 30 , 31 ) Early strategies focused on the overexpression of antigens and cytokines. Recombinant BCG (rBCG) strains were engineered to express additional Mycobacterium tuberculosis antigens, such as Ag85B, to broaden antigenic recognition beyond BCG's limited repertoire. Concurrently, researchers developed cytokine-expressing variants that secrete immunomodulators, including interleukin-2 (IL-2), interleukin-12 (IL-12), and interferon-gamma (IFN-γ). ( 32 – 35 ) These modifications create a localized Th1-polarizing microenvironment that enhances macrophage activation and T-cell recruitment, addressing BCG's failure to generate robust immunological memory in adults. ( 36 , 37 ) Specific constructs expressing human IFN-α under control of the mycobacterial Hsp60 promoter have demonstrated substantially increased IFN-γ production in human peripheral blood mononuclear cells compared to unmodified BCG controls. ( 38 – 40 ) A significant advancement came with strategies targeting BCG's intracellular lifecycle. The vaccine candidate VPM1002, created by deleting the urease C (ureC) gene and inserting listeriolysin O (hly) from Listeria monocytogenes, enables phagosomal escape and significantly enhances antigen cross-presentation via MHC-I pathways. ( 41 , 42 ) This design markedly improves CD8 + T-cell activation—a response crucial for adult protection that parental BCG minimally stimulates. ( 43 ) Parallel subtractive approaches focus on removing BCG's immunosuppressive genes. Deletion of zmp1, which encodes a zinc metalloprotease that interferes with MHC class II antigen presentation, enhances CD4 + T-cell recognition and activation. ( 44 – 46 ) Similarly, deleting nuoG, a gene that suppresses host reactive oxygen species production and apoptosis, results in a more pro-inflammatory vaccine strain that promotes better immune engagement. ( 47 , 48 ) Recent innovations include the overexpression of RD1-encoded antigens (ESAT-6 and CFP-10), which are absent in conventional BCG due to genomic deletions, to broaden antigenic coverage. Additional strategies include T-cell costimulatory molecules or checkpoint pathway inhibitors to prevent T-cell exhaustion and promote sustained memory responses. ( 49 ) The most advanced designs integrate multiple modifications for synergistic effects. ( 50 ) Candidates like BCG ΔureC::hly ΔnuoG combine phagosomal escape capability with removal of immunosuppressive genes, while other constructs merge antigen overexpression with cytokine expression systems. These approaches represent a comprehensive reprogramming strategy that collectively addresses BCG's multiple mechanistic deficiencies in adult protection. ( 51 – 54 ) Table 1 Comparison of Genetic Engineering Tools Strategy Example Modification Advantages Risks Antigen addition Ag85B-ESAT-6 fusion Broadens immune recognition Potential antigen competition Cytokine expression IL-12 secretion Prolongs memory responses Risk of excessive inflammation 4 Pipeline Candidates The development of genetically modified BCG (rBCG) vaccines has generated a structured and diverse pipeline of candidates, systematically documented in WHO reports from 2019 to 2023. ( 55 , 56 ) These candidates represent a strategic, multifaceted approach to overcoming the well-documented immunological limitations of conventional BCG, particularly its failure to induce robust, durable cellular immunity in adults against pulmonary tuberculosis. The 2022 WHO Global Tuberculosis Report specifically highlights the critical importance of these novel vaccine candidates, with several demonstrating promising results in advanced clinical trials targeting the adult pulmonary form of the disease that drives global transmission. ( 57 ) VPM1002 (BCGΔureC::hly) remains the most advanced candidate in this pipeline, having completed Phase II/III trials and received landmark regulatory approval in India in 2021 for use in newborns. ( 58 – 60 ) This construct combines listeriolysin O expression with urease C deletion to facilitate phagosomal escape and significantly enhance CD8 + T-cell activation by improving antigen cross-presentation via MHC-I pathways. ( 61 ) Parallel development has advanced MTBVAC, a genetically attenuated M. tuberculosis strain with phoP and fadD26 deletions that has progressed to Phase II trials by preserving full antigenic breadth, including the immunodominant antigens ESAT-6 and CFP-10, which are absent in conventional BCG due to region of difference 1 (RD1) deletion. ( 62 , 63 ) The current pipeline includes candidates employing targeted gene deletion strategies to improve immune recognition. ( 64 ) BCGΔzmp1, which removes a zinc metalloprotease gene, enhances MHC-II antigen presentation and CD4 + T-cell responses through improved lysosomal trafficking. Similarly, BCGΔnuoG deletion disrupts an immunosuppressive gene, increasing host cell apoptosis and inflammasome activation, thereby creating a more pro-inflammatory vaccine phenotype. ( 65 – 67 ) Additional antigen-expressing variants, such as rBCG::ESAT-6-Ag85B, incorporate Mycobacterium tuberculosis-specific proteins to broaden T-cell reactivity beyond BCG's limited antigenic repertoire. ( 68 , 69 ) Further innovation is evident in cytokine-expressing constructs, including rBCG::IL-18 and rBCG::GM-CSF, which aim to strengthen Th1 polarization and macrophage antimicrobial function by locally producing cytokines at the vaccination site. ( 70 , 71 ) The most sophisticated approaches involve combined-modification candidates, such as BCGΔureC::hly ΔnuoG, that merge phagosomal escape capabilities with reduced immune evasion mechanisms to enhance immunogenicity synergistically. ( 72 – 74 ) These systematic approaches, as outlined in the WHO's 2023 pipeline assessment, collectively aim to generate durable lung-resident memory T cells and represent the scientific community's comprehensive, methodical response to BCG's inadequate protection in adult populations. ( 75 ) Table 2 Promising Engineered BCG Candidates Vaccine Modification Current Stage Key Findings VPM1002 Africa trial) listeriolysin Phase III 45% efficacy in adults (South MTBVAC coverage M. tuberculosis ΔphoP Phase IIb Safer, broader antigen 5 Barriers to Translation Despite a robust pipeline of genetically modified BCG (rBCG) candidates, significant translational barriers impede their widespread adoption. These multifaceted challenges span scientific, regulatory, and economic domains, requiring coordinated solutions to realize their potential. While scientific innovation accelerates, the path from development to implementation remains complex. ( 76 , 77 ) Scientifically, demonstrating consistent efficacy across diverse adult populations remains challenging because established correlates of protection against pulmonary TB are lacking ( 78 ). Genetic stability of modified strains must be unequivocally demonstrated, as unintended mutations could compromise safety or immunogenicity. Furthermore, pre-existing immunity from environmental mycobacteria or prior BCG vaccination may blunt responses to improved vaccines in endemic regions. ( 79 , 80 ) Regulatory pathways for live genetically modified organisms present substantial hurdles. Agencies require extensive data on environmental safety, genetic stability, and reversion potential. ( 81 ) While VPM1002's 2021 approval in India set a precedent, global regulatory harmonization remains limited. Manufacturing complexities necessitate sophisticated production facilities and rigorous quality control to ensure batch-to-batch consistency, while cold-chain requirements create implementation challenges in high-burden settings. ( 82 – 84 ) Economic obstacles are particularly daunting. Development costs exceeding $ 1 billion USD contrast sharply with the limited ability to pay in TB-endemic countries. This market failure discourages pharmaceutical investment despite the overwhelming need. Future deployment would require integrating new vaccines into existing programs and potentially establishing adult vaccination platforms, a formidable logistical undertaking that would require substantial infrastructure development. ( 85 – 88 ) These barriers demand coordinated efforts between researchers, manufacturers, regulators, and public health agencies. While rBCG vaccines represent promising tools against adult pulmonary TB, overcoming these translational challenges remains essential for reducing global TB mortality. ( 89 , 90 ) 6 Limitations of Current BCG in Adults The World Health Organization's 2023 Global Tuberculosis Report confirms tuberculosis caused 1.3 million adult deaths in 2022, establishing the critical need for effective adult vaccination. ( 91 ) Despite being the most widely administered vaccine globally, BCG demonstrates highly variable efficacy (0–45%) against pulmonary tuberculosis in adults, with no significant protection demonstrated across multiple large-scale clinical trials. This geographical variability directly correlates with regions of high environmental mycobacterial exposure, where pre-existing immunity creates a masking effect that substantially blunts BCG immunogenicity. ( 92 – 95 ) BCG's fundamental immunological limitations in adult populations stem from two primary deficiencies: inadequate CD8 + T-cell activation due to phagosomal confinement and the absence of key Mycobacterium tuberculosis antigens, including ESAT-6 and CFP-10. ( 96 – 99 ) The vaccine remains trapped within phagosomes of antigen-presenting cells, preventing cytosolic access and subsequent cross-presentation via MHC-I pathways. This intracellular containment severely limits the development of cytotoxic T-lymphocyte responses, which are essential for eliminating Mtb-infected cells. ( 100 , 101 ) Additionally, BCG lacks multiple immunodominant antigens present in virulent Mycobacterium tuberculosis strains due to genomic deletions during its attenuation, particularly in the Region of Difference 1 (RD1) that encodes the immunodominant antigens ESAT-6 and CFP-10.( 102 – 104 ) The vaccine consistently fails to establish durable tissue-resident memory T cells (TRM) in alveolar tissues, where Mycobacterium tuberculosis first establishes infection. This failure prevents effective containment of bacterial replication in adult lungs, explaining why BCG cannot prevent initial infection or control disease progression. ( 105 – 108 ) Immune profiling studies further reveal attenuated Th1 and Th17 responses in high-exposure regions, consistent with meta-analyses showing no significant protection from BCG revaccination in adult populations. ( 109 , 110 ) These mechanistic flaws result in transient immune responses that lack sustained polyfunctional cytokine profiles (IFN-γ, TNF-α, IL-2) and fail to effectively engage trained immunity pathways in mature immune systems. ( 111 – 113 ) Current genetic modification strategies specifically target these limitations through precise antigen augmentation, engineered phagosomal escape mechanisms, and enhanced T-cell memory formation approaches, representing the most promising pathway to overcoming BCG's well-documented limitations in adult protection. ( 114 ) Table 3 Key Studies Highlighting BCG's Variable Efficacy Study (Year) Population Efficacy Against Pulmonary TB Limitations Identified Colditz et al. (1994) Global meta-analysis 0–80% (varies by region) Lack of a durable Th1 response Mangtani et al. (2014) UK adults 20–30% Waning immunity after adolescence 7 Future Directions Looking forward, the future of genetically modified BCG vaccines lies in several key directions. First, continued research into novel antigen combinations and mechanisms to induce durable tissue-resident memory (TRM) T-cells in the lung is essential. Studies demonstrate that recombinant BCG strains expressing Mycobacterium tuberculosis (Mtb) antigens such as ESAT-6, Ag85A, and Rv3407 can elicit enhanced polyfunctional CD4 + and CD8 + T-cell responses, which are critical for long-term immunity. ( 115 – 117 ) The WHO's "Preferred Product Characteristics for New Tuberculosis Vaccines" emphasizes the need for vaccines that prevent pulmonary TB in adults, a goal contingent on generating sustained TRM populations in the lungs. ( 118 ) Research into vectors like BCGΔBCG1419c, which overexpresses the Mtb phosphate transporter PstA-3, has shown increased autophagy, apoptosis, and improved recruitment of TRMcells to the lungs in murine models, providing a mechanistic basis for this approach. ( 119 – 121 ) Second, the development of globally harmonized regulatory pathways for GMO vaccines will accelerate evaluation and approval. The World Health Organization has established guidelines, such as those outlined in the "WHO Expert Committee on Biological Standardization: Guidelines for the safe production and quality control of BCG vaccines," which address the specific environmental risk assessment and containment requirements for live recombinant bacterial vaccines. ( 122 – 125 ) A significant challenge is the variation in national regulatory frameworks governing genetically modified organisms. ( 126 ) Harmonization efforts, as promoted by the WHO and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), aim to standardize data requirements for preclinical and clinical trials, thereby streamlining the process and reducing the time from development to licensure, particularly for low- and middle-income countries with high TB burdens. ( 127 , 128 ) Third, investment in innovative manufacturing technologies and thermostable formulations will address delivery challenges. ( 129 ) Traditional BCG production relies on outdated, multi-dose liquid formulations requiring a consistent cold chain, which is a major logistical barrier. Advances in lyophilization (freeze-drying) and spray-drying techniques are being applied to develop more stable, single-dose powder formulations of recombinant BCG that can withstand thermal stress. ( 130 – 132 ) For instance, research into trehalose-based stabilizers has shown promise in maintaining the viability of lyophilized BCG over extended periods at elevated temperatures. ( 133 ) Furthermore, the WHO’s Global TB Programme stresses the importance of investing in scalable, cost-effective manufacturing platforms to ensure a sustainable vaccine supply that can be integrated into existing immunization programs without necessitating major cold-chain infrastructure overhauls. ( 134 , 135 ) Finally, exploring the potential of these vaccines within therapeutic or post-exposure prevention contexts could expand their impact. Preclinical studies indicate that certain rBCG strains, such as VPM1002, which is currently in clinical trials for post-exposure prevention in household contacts, can induce autophagy and clear intracellular Mtb more effectively than the parent BCG strain. This suggests a potential role for recombinant BCG not only as a preventive vaccine but also as an adjunct therapeutic intervention to shorten chemotherapy duration or prevent relapse. ( 136 – 139 ) The WHO’s strategy to end TB includes a focus on post-exposure vaccination, particularly for high-risk contacts of TB patients, highlighting the need for vaccines that can contain or eliminate established latent infections and prevent progression to active disease. ( 140 ) The scientific foundation for these applications has been robustly established; the task now is to navigate the translational landscape with concerted effort and international collaboration. By doing so, the long-standing goal of a truly effective vaccine against adult pulmonary tuberculosis, one that could fundamentally alter the trajectory of the TB epidemic, may finally be within reach. ( 141 , 142 ) Conclusion The genetic modification of the BCG vaccine represents a pivotal and promising strategy to overcome its well-documented limited efficacy in adults, a necessity underscored by the persistent global burden of tuberculosis. This review has detailed the scientific rationale and evolution of these strategies, from initial efforts to overexpress immunodominant antigens and cytokines to more sophisticated approaches involving gene deletion and multi-factorial engineering. Candidates such as VPM1002, which enables phagosomal escape and enhances CD8 + T-cell activation, and MTBVAC, which retains a broad antigenic repertoire, exemplify the significant progress made. The advancement of these candidates through clinical trials, as reported by the WHO, signals transformative potential for preventing adult pulmonary TB. However, the path from promising candidate to widespread implementation is fraught with interconnected challenges. 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Available from: https://www.who.int/teams/global-programme-on-tuberculosis-and-lung-health/the-end-tb-strategy Lacámara S, Martin C (2023) MTBVAC: A Tuberculosis Vaccine Candidate Advancing Towards Clinical Efficacy Trials in TB Prevention. Arch Bronconeumol 59(12):821–828 VPM1002 [Internet] Working Group on New TB Vaccines. [cited 2025 Oct 1]. Available from: https://newtbvaccines.org/vaccine/vpm1002/ Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8714621","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Systematic Review","associatedPublications":[],"authors":[{"id":581414126,"identity":"419ea65d-a023-4ddc-8ea9-c692e248123a","order_by":0,"name":"Ssembuusi Allan Francis","email":"data:image/png;base64,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","orcid":"","institution":"Marwadi University","correspondingAuthor":true,"prefix":"","firstName":"Ssembuusi","middleName":"Allan","lastName":"Francis","suffix":""},{"id":581414127,"identity":"2c0152bb-762b-4484-acd9-39a4e1f736e4","order_by":1,"name":"Eloghosa Nosa-Ihaza","email":"","orcid":"https://orcid.org/0009-0001-8716-0597","institution":"Marwadi University","correspondingAuthor":false,"prefix":"","firstName":"Eloghosa","middleName":"","lastName":"Nosa-Ihaza","suffix":""}],"badges":[],"createdAt":"2026-01-27 22:37:17","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8714621/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8714621/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101374813,"identity":"115a754c-99e3-4e24-a148-4dda4bf06d5a","added_by":"auto","created_at":"2026-01-29 04:20:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1203137,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic Modification Approaches for BCG\u003c/p\u003e","description":"","filename":"Figure1GeneticModificationApproachesforBCG.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8714621/v1/e2027902feb9e783bfc30cba.jpg"},{"id":101374814,"identity":"2511547c-76c2-4583-b304-476b157640e0","added_by":"auto","created_at":"2026-01-29 04:20:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1223216,"visible":true,"origin":"","legend":"\u003cp\u003eClinical Development Pipeline of Leading Recombinant BCG Candidates\u003c/p\u003e","description":"","filename":"Figure2ClinicalDevelopmentPipelineofLeadingRecombinantBCGCandidates.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8714621/v1/75a6312b145ad61798bb51c0.jpg"},{"id":101374815,"identity":"2b3f197b-2a50-4585-817d-38b04a7cddc2","added_by":"auto","created_at":"2026-01-29 04:20:58","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1146149,"visible":true,"origin":"","legend":"\u003cp\u003eChallenges in GMO Vaccine Development\u003c/p\u003e","description":"","filename":"Figure3ChallengesinGMOVaccineDevelopment.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8714621/v1/9a78dad44b4b86a74b503de2.jpg"},{"id":101942759,"identity":"78c113f4-c152-4e17-8582-bc465ee7d5d7","added_by":"auto","created_at":"2026-02-05 09:37:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4169745,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8714621/v1/b30969bf-cbbc-48a3-9437-d65f43d7e9db.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eGenetic Modification of the BCG Vaccine to Overcome Its Limited Efficacy in Adults: A Specialized Review\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eTuberculosis (TB) is a serious infectious disease that remains a leading cause of death worldwide, especially among adults. For over 100 years, the Bacillus Calmette-Gu\u0026eacute;rin (BCG) vaccine has been the only vaccine available to fight it. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) While BCG works very well to protect babies and young children from the most severe forms of TB, it does not work consistently to protect adolescents and adults from lung TB, which is the most common and infectious form of the disease. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eTo address these limitations, researchers have employed genetic modification to enhance BCG's immunogenicity, a research priority underscored by the WHO's End TB Strategy. Specifically, this process involves the deliberate alteration of the BCG genome to confer novel immunological properties, primarily to elicit more robust and durable immune responses in vaccines. These improved responses are critical for preventing the establishment of latent infection and reactivation disease in adults. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eEarly strategies, initiated several decades ago, focused on the overexpression of antigens and cytokines. For example, recombinant BCG (rBCG) strains were engineered to express additional Mycobacterium tuberculosis antigens, such as Ag85B, a major secreted protein that is a dominant target of T-cell immunity, to broaden antigenic recognition. (\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) Concurrently, cytokine-armed rBCG variants were developed to secrete immunomodulators like interleukin-2 (IL-2), which promotes T-cell proliferation; interleukin-12 (IL-12), a key inducer of Th1 responses; or interferon-gamma (IFN-γ), the central macrophage-activating cytokine. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) These modifications aimed to create a localized Th1-polarizing microenvironment and enhance macrophage activation and T-cell recruitment, as evidenced by studies in murine models showing improved bacterial clearance. (\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) Building on these findings, this foundational work established the principle that genetic modification could significantly enhance BCG's immunogenicity, paving the way for more sophisticated second-generation constructs.\u003c/p\u003e \u003cp\u003eAs research progressed, subsequent strategies evolved from additive approaches to more refined subtractive and integrated genomic engineering, moving beyond simple overexpression to more nuanced immune modulation. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) A pivotal advancement within this evolution was the development of VPM1002, created through the deletion of the urease C (ureC) gene and insertion of listeriolysin O (hly) from Listeria monocytogen. This design facilitates phagosomal escape by perforating the phagosomal membrane in an acid-dependent manner, thereby promoting antigen escape into the cytosol and subsequent cross-presentation on MHC class I molecules.les. This, in turn, significantly enhances CD8\u0026thinsp;+\u0026thinsp;T-cell activation\u0026mdash;a response critical for adult protection that is poorly induced by the parent BCG strain. (\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eDemonstrating promising immunogenicity and safety in Phase II and III clinical trials, VPM1002 received regulatory approval in India in 2021, marking a milestone as the first genetically modified BCG vaccine to be licensed. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) Alongside VPM1002, other next-generation constructs, such as MTBVAC\u0026mdash;a live-attenuated M. tuberculosis double-deletion mutant\u0026mdash;and those incorporating multi-gene edits, such as disruption of the immune-suppressive phoP regulator or the nuoG gene, continue to undergo evaluation in global clinical trials. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) Together, these efforts form a core part of the current TB vaccine pipeline as tracked by the WHO. The primary aim is to develop a vaccine effective against pulmonary disease across all age groups, an essential step toward meeting the WHO\u0026rsquo;s target of a 90% reduction in TB incidence by 2035. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/p\u003e"},{"header":"2 Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Literature Search and Selection Criteria\u003c/h2\u003e \u003cp\u003eThis review systematically searched PubMed, Scopus, and Web of Science for studies (2000\u0026ndash;2024) on genetically modified BCG vaccines. Search terms included \"BCG,\" \"genetic modification,\" \"recombinant BCG,\" \"VPM1002,\" \"MTBVAC,\" \"adult pulmonary tuberculosis,\" and \"vaccine efficacy.\" Of the 587 initial publications, 142 studies met the inclusion criteria: peer-reviewed articles, clinical trial data, and WHO reports on novel vaccine candidates and immunological outcomes (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Priority was given to studies with specific genetic changes, quantifiable immunogenicity (\u0026ge;\u0026thinsp;2-fold CD8\u0026thinsp;+\u0026thinsp;T-cell activation or significant antigen cross-presentation), and Phase II/III trial results from the WHO TB vaccine pipeline (2022\u0026ndash;2024) (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Data Analysis Framework\u003c/h2\u003e \u003cp\u003eExtracted data were synthesized to evaluate three aspects: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) scientific rationale for genetic engineering strategies, (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) immunogenic profile and efficacy of pipeline candidates, and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) translational challenge (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). A comparative analysis traced the shift from animal proof-of-concept studies to recent human trials, highlighting candidates such as VPM1002, which demonstrated 45% efficacy in a South African adult trial. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Genetic Engineering Strategies","content":"\u003cp\u003eThe genetic modification of Bacillus Calmette-Gu\u0026eacute;rin (BCG) represents a sophisticated and targeted approach to overcome its limitations in protecting adults against pulmonary tuberculosis. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) These engineering strategies have evolved systematically from initial enhancement approaches to complex multi-gene modifications, each designed to address specific immunological deficiencies of the parental BCG strain. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eEarly strategies focused on the overexpression of antigens and cytokines. Recombinant BCG (rBCG) strains were engineered to express additional Mycobacterium tuberculosis antigens, such as Ag85B, to broaden antigenic recognition beyond BCG's limited repertoire. Concurrently, researchers developed cytokine-expressing variants that secrete immunomodulators, including interleukin-2 (IL-2), interleukin-12 (IL-12), and interferon-gamma (IFN-γ). (\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) These modifications create a localized Th1-polarizing microenvironment that enhances macrophage activation and T-cell recruitment, addressing BCG's failure to generate robust immunological memory in adults. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) Specific constructs expressing human IFN-α under control of the mycobacterial Hsp60 promoter have demonstrated substantially increased IFN-γ production in human peripheral blood mononuclear cells compared to unmodified BCG controls. (\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eA significant advancement came with strategies targeting BCG's intracellular lifecycle. The vaccine candidate VPM1002, created by deleting the urease C (ureC) gene and inserting listeriolysin O (hly) from Listeria monocytogenes, enables phagosomal escape and significantly enhances antigen cross-presentation via MHC-I pathways. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) This design markedly improves CD8\u0026thinsp;+\u0026thinsp;T-cell activation\u0026mdash;a response crucial for adult protection that parental BCG minimally stimulates. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eParallel subtractive approaches focus on removing BCG's immunosuppressive genes. Deletion of zmp1, which encodes a zinc metalloprotease that interferes with MHC class II antigen presentation, enhances CD4\u0026thinsp;+\u0026thinsp;T-cell recognition and activation. (\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e) Similarly, deleting nuoG, a gene that suppresses host reactive oxygen species production and apoptosis, results in a more pro-inflammatory vaccine strain that promotes better immune engagement. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eRecent innovations include the overexpression of RD1-encoded antigens (ESAT-6 and CFP-10), which are absent in conventional BCG due to genomic deletions, to broaden antigenic coverage. Additional strategies include T-cell costimulatory molecules or checkpoint pathway inhibitors to prevent T-cell exhaustion and promote sustained memory responses. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe most advanced designs integrate multiple modifications for synergistic effects. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e) Candidates like BCG ΔureC::hly ΔnuoG combine phagosomal escape capability with removal of immunosuppressive genes, while other constructs merge antigen overexpression with cytokine expression systems. These approaches represent a comprehensive reprogramming strategy that collectively addresses BCG's multiple mechanistic deficiencies in adult protection. (\u003cspan additionalcitationids=\"CR52 CR53\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of Genetic Engineering Tools\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eStrategy\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eExample Modification\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eAdvantages\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eRisks\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eAntigen addition\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eAg85B-ESAT-6 fusion\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eBroadens immune recognition\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ePotential antigen competition\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eCytokine expression\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eIL-12 secretion\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eProlongs memory responses\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eRisk of excessive inflammation\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"4 Pipeline Candidates","content":"\u003cp\u003eThe development of genetically modified BCG (rBCG) vaccines has generated a structured and diverse pipeline of candidates, systematically documented in WHO reports from 2019 to 2023. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e) These candidates represent a strategic, multifaceted approach to overcoming the well-documented immunological limitations of conventional BCG, particularly its failure to induce robust, durable cellular immunity in adults against pulmonary tuberculosis. The 2022 WHO Global Tuberculosis Report specifically highlights the critical importance of these novel vaccine candidates, with several demonstrating promising results in advanced clinical trials targeting the adult pulmonary form of the disease that drives global transmission. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eVPM1002 (BCGΔureC::hly) remains the most advanced candidate in this pipeline, having completed Phase II/III trials and received landmark regulatory approval in India in 2021 for use in newborns. (\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e) This construct combines listeriolysin O expression with urease C deletion to facilitate phagosomal escape and significantly enhance CD8\u0026thinsp;+\u0026thinsp;T-cell activation by improving antigen cross-presentation via MHC-I pathways. (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e) Parallel development has advanced MTBVAC, a genetically attenuated M. tuberculosis strain with phoP and fadD26 deletions that has progressed to Phase II trials by preserving full antigenic breadth, including the immunodominant antigens ESAT-6 and CFP-10, which are absent in conventional BCG due to region of difference 1 (RD1) deletion. (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe current pipeline includes candidates employing targeted gene deletion strategies to improve immune recognition. (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e) BCGΔzmp1, which removes a zinc metalloprotease gene, enhances MHC-II antigen presentation and CD4\u0026thinsp;+\u0026thinsp;T-cell responses through improved lysosomal trafficking. Similarly, BCGΔnuoG deletion disrupts an immunosuppressive gene, increasing host cell apoptosis and inflammasome activation, thereby creating a more pro-inflammatory vaccine phenotype. (\u003cspan additionalcitationids=\"CR66\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e) Additional antigen-expressing variants, such as rBCG::ESAT-6-Ag85B, incorporate Mycobacterium tuberculosis-specific proteins to broaden T-cell reactivity beyond BCG's limited antigenic repertoire. (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eFurther innovation is evident in cytokine-expressing constructs, including rBCG::IL-18 and rBCG::GM-CSF, which aim to strengthen Th1 polarization and macrophage antimicrobial function by locally producing cytokines at the vaccination site. (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e) The most sophisticated approaches involve combined-modification candidates, such as BCGΔureC::hly ΔnuoG, that merge phagosomal escape capabilities with reduced immune evasion mechanisms to enhance immunogenicity synergistically. (\u003cspan additionalcitationids=\"CR73\" citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e) These systematic approaches, as outlined in the WHO's 2023 pipeline assessment, collectively aim to generate durable lung-resident memory T cells and represent the scientific community's comprehensive, methodical response to BCG's inadequate protection in adult populations. (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePromising Engineered BCG Candidates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eVaccine\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eModification\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eCurrent Stage\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eKey Findings\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eVPM1002\u003c/span\u003e\u003c/p\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eAfrica trial)\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003elisteriolysin\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ePhase III\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e45% efficacy in adults (South\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eMTBVAC\u003c/span\u003e\u003c/p\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ecoverage\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eM. tuberculosis ΔphoP\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ePhase IIb\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eSafer, broader antigen\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"5 Barriers to Translation","content":"\u003cp\u003eDespite a robust pipeline of genetically modified BCG (rBCG) candidates, significant translational barriers impede their widespread adoption. These multifaceted challenges span scientific, regulatory, and economic domains, requiring coordinated solutions to realize their potential. While scientific innovation accelerates, the path from development to implementation remains complex. (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eScientifically, demonstrating consistent efficacy across diverse adult populations remains challenging because established correlates of protection against pulmonary TB are lacking (\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e). Genetic stability of modified strains must be unequivocally demonstrated, as unintended mutations could compromise safety or immunogenicity. Furthermore, pre-existing immunity from environmental mycobacteria or prior BCG vaccination may blunt responses to improved vaccines in endemic regions. (\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eRegulatory pathways for live genetically modified organisms present substantial hurdles. Agencies require extensive data on environmental safety, genetic stability, and reversion potential. (\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e) While VPM1002's 2021 approval in India set a precedent, global regulatory harmonization remains limited. Manufacturing complexities necessitate sophisticated production facilities and rigorous quality control to ensure batch-to-batch consistency, while cold-chain requirements create implementation challenges in high-burden settings. (\u003cspan additionalcitationids=\"CR83\" citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eEconomic obstacles are particularly daunting. Development costs exceeding \u003cspan\u003e$\u003c/span\u003e1\u0026nbsp;billion USD contrast sharply with the limited ability to pay in TB-endemic countries. This market failure discourages pharmaceutical investment despite the overwhelming need. Future deployment would require integrating new vaccines into existing programs and potentially establishing adult vaccination platforms, a formidable logistical undertaking that would require substantial infrastructure development. (\u003cspan additionalcitationids=\"CR86 CR87\" citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThese barriers demand coordinated efforts between researchers, manufacturers, regulators, and public health agencies. While rBCG vaccines represent promising tools against adult pulmonary TB, overcoming these translational challenges remains essential for reducing global TB mortality. (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e)\u003c/p\u003e"},{"header":"6 Limitations of Current BCG in Adults","content":"\u003cp\u003eThe World Health Organization's 2023 Global Tuberculosis Report confirms tuberculosis caused 1.3\u0026nbsp;million adult deaths in 2022, establishing the critical need for effective adult vaccination. (\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e) Despite being the most widely administered vaccine globally, BCG demonstrates highly variable efficacy (0\u0026ndash;45%) against pulmonary tuberculosis in adults, with no significant protection demonstrated across multiple large-scale clinical trials. This geographical variability directly correlates with regions of high environmental mycobacterial exposure, where pre-existing immunity creates a masking effect that substantially blunts BCG immunogenicity. (\u003cspan additionalcitationids=\"CR93 CR94\" citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eBCG's fundamental immunological limitations in adult populations stem from two primary deficiencies: inadequate CD8\u0026thinsp;+\u0026thinsp;T-cell activation due to phagosomal confinement and the absence of key Mycobacterium tuberculosis antigens, including ESAT-6 and CFP-10. (\u003cspan additionalcitationids=\"CR97 CR98\" citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e) The vaccine remains trapped within phagosomes of antigen-presenting cells, preventing cytosolic access and subsequent cross-presentation via MHC-I pathways. This intracellular containment severely limits the development of cytotoxic T-lymphocyte responses, which are essential for eliminating Mtb-infected cells. (\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e, \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e) Additionally, BCG lacks multiple immunodominant antigens present in virulent Mycobacterium tuberculosis strains due to genomic deletions during its attenuation, particularly in the Region of Difference 1 (RD1) that encodes the immunodominant antigens ESAT-6 and CFP-10.(\u003cspan additionalcitationids=\"CR103\" citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe vaccine consistently fails to establish durable tissue-resident memory T cells (TRM) in alveolar tissues, where Mycobacterium tuberculosis first establishes infection. This failure prevents effective containment of bacterial replication in adult lungs, explaining why BCG cannot prevent initial infection or control disease progression. (\u003cspan additionalcitationids=\"CR106 CR107\" citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e) Immune profiling studies further reveal attenuated Th1 and Th17 responses in high-exposure regions, consistent with meta-analyses showing no significant protection from BCG revaccination in adult populations. (\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e, \u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThese mechanistic flaws result in transient immune responses that lack sustained polyfunctional cytokine profiles (IFN-γ, TNF-α, IL-2) and fail to effectively engage trained immunity pathways in mature immune systems. (\u003cspan additionalcitationids=\"CR112\" citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e) Current genetic modification strategies specifically target these limitations through precise antigen augmentation, engineered phagosomal escape mechanisms, and enhanced T-cell memory formation approaches, representing the most promising pathway to overcoming BCG's well-documented limitations in adult protection. (\u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKey Studies Highlighting BCG's Variable Efficacy\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eStudy (Year)\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ePopulation\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eEfficacy Against Pulmonary TB\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eLimitations Identified\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eColditz et al. (1994)\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eGlobal meta-analysis\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e0\u0026ndash;80% (varies by region)\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eLack of\u003c/span\u003e a \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003edurable Th1 response\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eMangtani et al. (2014)\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eUK adults\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e20\u0026ndash;30%\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eWaning immunity after adolescence\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"7 Future Directions","content":"\u003cp\u003eLooking forward, the future of genetically modified BCG vaccines lies in several key directions. First, continued research into novel antigen combinations and mechanisms to induce durable tissue-resident memory (TRM) T-cells in the lung is essential. Studies demonstrate that recombinant BCG strains expressing Mycobacterium tuberculosis (Mtb) antigens such as ESAT-6, Ag85A, and Rv3407 can elicit enhanced polyfunctional CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T-cell responses, which are critical for long-term immunity. (\u003cspan additionalcitationids=\"CR116\" citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e) The WHO's \"Preferred Product Characteristics for New Tuberculosis Vaccines\" emphasizes the need for vaccines that prevent pulmonary TB in adults, a goal contingent on generating sustained TRM populations in the lungs. (\u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e118\u003c/span\u003e) Research into vectors like BCGΔBCG1419c, which overexpresses the Mtb phosphate transporter PstA-3, has shown increased autophagy, apoptosis, and improved recruitment of TRMcells to the lungs in murine models, providing a mechanistic basis for this approach. (\u003cspan additionalcitationids=\"CR120\" citationid=\"CR119\" class=\"CitationRef\"\u003e119\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR121\" class=\"CitationRef\"\u003e121\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eSecond, the development of globally harmonized regulatory pathways for GMO vaccines will accelerate evaluation and approval. The World Health Organization has established guidelines, such as those outlined in the \"WHO Expert Committee on Biological Standardization: Guidelines for the safe production and quality control of BCG vaccines,\" which address the specific environmental risk assessment and containment requirements for live recombinant bacterial vaccines. (\u003cspan additionalcitationids=\"CR123 CR124\" citationid=\"CR122\" class=\"CitationRef\"\u003e122\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR125\" class=\"CitationRef\"\u003e125\u003c/span\u003e) A significant challenge is the variation in national regulatory frameworks governing genetically modified organisms. (\u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e126\u003c/span\u003e) Harmonization efforts, as promoted by the WHO and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), aim to standardize data requirements for preclinical and clinical trials, thereby streamlining the process and reducing the time from development to licensure, particularly for low- and middle-income countries with high TB burdens. (\u003cspan citationid=\"CR127\" class=\"CitationRef\"\u003e127\u003c/span\u003e, \u003cspan citationid=\"CR128\" class=\"CitationRef\"\u003e128\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThird, investment in innovative manufacturing technologies and thermostable formulations will address delivery challenges. (\u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e129\u003c/span\u003e) Traditional BCG production relies on outdated, multi-dose liquid formulations requiring a consistent cold chain, which is a major logistical barrier. Advances in lyophilization (freeze-drying) and spray-drying techniques are being applied to develop more stable, single-dose powder formulations of recombinant BCG that can withstand thermal stress. (\u003cspan additionalcitationids=\"CR131\" citationid=\"CR130\" class=\"CitationRef\"\u003e130\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR132\" class=\"CitationRef\"\u003e132\u003c/span\u003e) For instance, research into trehalose-based stabilizers has shown promise in maintaining the viability of lyophilized BCG over extended periods at elevated temperatures. (\u003cspan citationid=\"CR133\" class=\"CitationRef\"\u003e133\u003c/span\u003e) Furthermore, the WHO\u0026rsquo;s Global TB Programme stresses the importance of investing in scalable, cost-effective manufacturing platforms to ensure a sustainable vaccine supply that can be integrated into existing immunization programs without necessitating major cold-chain infrastructure overhauls. (\u003cspan citationid=\"CR134\" class=\"CitationRef\"\u003e134\u003c/span\u003e, \u003cspan citationid=\"CR135\" class=\"CitationRef\"\u003e135\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eFinally, exploring the potential of these vaccines within therapeutic or post-exposure prevention contexts could expand their impact. Preclinical studies indicate that certain rBCG strains, such as VPM1002, which is currently in clinical trials for post-exposure prevention in household contacts, can induce autophagy and clear intracellular Mtb more effectively than the parent BCG strain. This suggests a potential role for recombinant BCG not only as a preventive vaccine but also as an adjunct therapeutic intervention to shorten chemotherapy duration or prevent relapse. (\u003cspan additionalcitationids=\"CR137 CR138\" citationid=\"CR136\" class=\"CitationRef\"\u003e136\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR139\" class=\"CitationRef\"\u003e139\u003c/span\u003e) The WHO\u0026rsquo;s strategy to end TB includes a focus on post-exposure vaccination, particularly for high-risk contacts of TB patients, highlighting the need for vaccines that can contain or eliminate established latent infections and prevent progression to active disease. (\u003cspan citationid=\"CR140\" class=\"CitationRef\"\u003e140\u003c/span\u003e) The scientific foundation for these applications has been robustly established; the task now is to navigate the translational landscape with concerted effort and international collaboration. By doing so, the long-standing goal of a truly effective vaccine against adult pulmonary tuberculosis, one that could fundamentally alter the trajectory of the TB epidemic, may finally be within reach. (\u003cspan citationid=\"CR141\" class=\"CitationRef\"\u003e141\u003c/span\u003e, \u003cspan citationid=\"CR142\" class=\"CitationRef\"\u003e142\u003c/span\u003e)\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe genetic modification of the BCG vaccine represents a pivotal and promising strategy to overcome its well-documented limited efficacy in adults, a necessity underscored by the persistent global burden of tuberculosis. This review has detailed the scientific rationale and evolution of these strategies, from initial efforts to overexpress immunodominant antigens and cytokines to more sophisticated approaches involving gene deletion and multi-factorial engineering. Candidates such as VPM1002, which enables phagosomal escape and enhances CD8\u0026thinsp;+\u0026thinsp;T-cell activation, and MTBVAC, which retains a broad antigenic repertoire, exemplify the significant progress made. The advancement of these candidates through clinical trials, as reported by the WHO, signals transformative potential for preventing adult pulmonary TB.\u003c/p\u003e \u003cp\u003eHowever, the path from promising candidate to widespread implementation is fraught with interconnected challenges. As outlined, these barriers span the demonstrated scientific hurdles of establishing correlates of protection and ensuring genetic stability, the regulatory complexities of approving live genetically modified organisms, and the practical difficulties of manufacturing scalability and cold-chain logistics in resource-limited settings. Economic constraints further complicate the landscape, highlighting the critical need for sustained global investment and political will to ensure these innovations reach the populations that need them most.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eTuberculosis (TB) [Internet]. [cited 2025 Sept 29]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/news-room/fact-sheets/detail/tuberculosis\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eInfant BCG vaccination and risk of pulmonary and extrapulmonary tuberculosis throughout the life course: a systematic review and individual participant data meta-analysis - The Lancet Global Health [Internet]. [cited 2025 Sept 29]. 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Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://newtbvaccines.org/vaccine/vpm1002/\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Marwadi University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Recombinant BCG vaccines, Genetic modification of BCG, Adult pulmonary tuberculosis, VPM1002, MTBVAC, TB vaccine development","lastPublishedDoi":"10.21203/rs.3.rs-8714621/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8714621/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhile the Bacillus Calmette-Gu\u0026eacute;rin (BCG) vaccine protects against severe childhood tuberculosis, it does not consistently protect against adult pulmonary tuberculosis, the main driver of global transmission and mortality. This review summarizes the scientific rationale and evolution of genetic engineering approaches to address BCG's immunological limitations, from initial strategies such as overexpressing Mycobacterium tuberculosis antigens and immunomodulatory cytokines to second-generation methods using subtractive and integrative genomic editing. Key candidates, such as VPM1002\u0026mdash;engineered for phagosomal escape and improved CD8\u0026thinsp;+\u0026thinsp;T-cell activation via MHC-I cross-presentation\u0026mdash;and MTBVAC, a live-attenuated M. tuberculosis strain with a broad antigenic repertoire, are now in advanced clinical trials within a diverse pipeline monitored by WHO. These advances constitute a paradigm shift in vaccine design. However, adoption remains challenged by major barriers: the lack of immune correlates of protection, complex regulatory pathways for GMOs, and significant economic and manufacturing hurdles. Genetic modification offers the most feasible route to an effective adult TB vaccine, but its promise depends on overcoming obstacles in the translational landscape and aligning with the WHO's End TB Strategy.\u003c/p\u003e","manuscriptTitle":"Genetic Modification of the BCG Vaccine to Overcome Its Limited Efficacy in Adults: A Specialized Review","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 04:20:53","doi":"10.21203/rs.3.rs-8714621/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7f34122c-df79-44c1-aa06-88c191409c5f","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-29T04:20:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-29 04:20:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8714621","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8714621","identity":"rs-8714621","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}