Bacillus Calmette-Guérin (BCG) immunotherapy has been a cornerstone treatment for non-muscle-invasive bladder cancer for decades and still faces challenges, such as severe immune adverse reactions, which reduce its use as a first-line treatment. This review examines BCG therapy\'s history, mechanisms, and current status, highlighting how nanotechnology and bioengineering are revitalizing its application. We discuss novel nanocarrier systems aimed at enhancing BCG\'s efficacy while mitigating specific side effects. These approaches promise improved tumor targeting, better drug loading, and an enhanced stimulation of anti-tumor immune responses. Key strategies involve using materials such as liposomes, polymers, and magnetic particles to encapsulate BCG or functional BCG cell wall components. Additionally, co-delivering BCG with chemotherapeutics enhances drug targeting and tumor-killing effects while reducing drug toxicity, with some studies even achieving synergistic effects. While most studies remain experimental, this research direction offers hope for overcoming BCG\'s limitations and advancing bladder cancer immunotherapy. Further elucidation of BCG\'s mechanisms and rigorous safety evaluations of new delivery systems will be crucial for translating these innovations into clinical practice.

UNASSIGNED: cGMP-dependent protein kinase 1 (PRKG1) has shown to be associated with some tumorigenesis, while the role of PRKG1 in bladder cancer is unclear.
UNASSIGNED: To investigate the biological and clinical significance of PRKG1 in bladder cancer, we detected the expression of PRKG1 and explored the function of PRKG1 in bladder cancer cells. The PRKG1 transcripts data was downloaded from The Cancer Genome Atlas (TCGA) database, and immunohistochemistry staining was conducted on formalin-fixed paraffin-embedded (FFPE) sample tissues. Relationship between clinical characteristics of patients and expression of PRKG1 was analyzed in FFPE samples, TCGA database, and GSE19423 dataset. PRKG1 was over-expressed, and cell proliferation, migration, invasion, apoptosis, and spheroidizing ability were then detected. Chemosensitivity to cisplatin was detected with cell viability, and half-maximal drug inhibitory concentration (IC50) was calculated. In addition, the relation between PRKG1 expression and the infiltration level of tumor immune cells in tumor microenvironment were analyzed.
UNASSIGNED: The results showed expression of PRKG1 was lower in bladder cancer, compared with normal tissues both at protein and transcript levels. Lower PRKG1 expression was related to higher tumor grade, T stage, and muscle invasion, also predicted worse overall survival and recurrence free survival in patients treated with Bacillus Calmette-Guerin (BCG) intravesical immunotherapy. Analysis of tumor immune cells infiltration showed lower PRKG1 was associated with non-inflamed tumor microenvironment.
UNASSIGNED: The present study firstly identified the anti-tumor role and tumor immune regulatory role of PRKG1, also found loss of PRKG1 could be used as a prognosis factor. The present study provided a potential biomarker and therapy target to bladder cancer.

UNASSIGNED: The current prophylactic tuberculosis vaccine Bacille Calmette-Guérin (BCG), was derived in the 1920s, but the humoral immune responses induced by BCG vaccination have not been fully elucidated to date. In this study, our aim was to reveal the profiles of antibody responses induced by BCG vaccination in adults and identify the potential biomarkers for evaluating the BCG vaccination response.
UNASSIGNED: Proteome microarrays were performed to reveal the serum profiles of antibody responses induced by BCG vaccination in adults. ELISA was used to validate the potential biomarkers in validation cohort (79 healthy controls and 58 BCG-vaccinated subjects). Then combined panel was established by logistic regression analysis based on OD values of potential biomarkers.
UNASSIGNED: Multiple antigens elicited stronger serum IgG or IgM antibody responses in BCG vaccinated subjects than healthy subjects at 12 weeks post BCG vaccination; among the antigens, Rv0060, Rv2026c and Rv3379c were further verified using 137 serum samples and presented the moderate performance in assessment of the BCG vaccination response by receiver operating characteristic analysis. Furthermore, a combined panel exhibited an improved AUC of 0.923, and the sensitivity and specificity were 77.59 % and 91.14 %, respectively. In addition, the antibody response against Rv0060, Rv2026c and Rv3379c was related to the clinical background to a certain extent.
UNASSIGNED: The novel antigens identified in our study could offer better knowledge towards developing a more efficacious vaccine based on humoral immune responses, and they could be potential biomarkers in assessments of BCG vaccination responses.

UNASSIGNED: Tuberculosis, caused by Mycobacterium tuberculosis complex (MTBC), remains a global health concern in both human and animals. However, the absence of rapid, accurate, and highly sensitive detection methods to differentiate the major pathogens of MTBC, including M. tuberculosis, M. bovis, and BCG, poses a potential challenge.
UNASSIGNED: In this study, we have established a triplex droplet digital polymerase chain reaction (ddPCR) method employing three types of probe fluorophores, with targets M. tuberculosis (targeting CFP-10-ESAT-6 gene of RD1 and Rv0222 genes of RD4), M. bovis (targeting CFP-10-ESATs-6 gene of RD1), and BCG (targeting Rv3871 and Rv3879c genes of ΔRD1), respectively.
UNASSIGNED: Based on optimization of annealing temperature, sensitivity and repeatability, this method demonstrates a lower limit of detection (LOD) as 3.08 copies/reaction for M. tuberculosis, 4.47 copies/reaction for M. bovis and 3.59 copies/reaction for BCG, without cross-reaction to Mannheimia haemolytica, Mycoplasma bovis, Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Ochrobactrum anthropi, Salmonella choleraesuis, Brucella melitensis, and Staphylococcus aureus, and showed repeatability with coefficients of variation (CV) lower than 10%. The method exhibits strong milk sample tolerance, the LOD of detecting in spike milk was 5 × 103 CFU/mL, which sensitivity is ten times higher than the triplex qPCR. 60 clinical DNA samples, including 20 milk, 20 tissue and 20 swab samples, were kept in China Animal Health and Epidemiology Center were tested by the triplex ddPCR and triplex qPCR. The triplex ddPCR presented a higher sensitivity (11.67%, 7/60) than that of the triplex qPCR method (8.33%, 5/60). The positive rates of M. tuberculosis, M. bovis, and BCG were 1.67, 10, and 0% by triplex ddPCR, and 1.67, 6.67, and 0% by triplex qPCR, with coincidence rates of 100, 96.7, and 100%, respectively.
UNASSIGNED: Our data demonstrate that the established triplex ddPCR method is a sensitive, specific and rapid method for differentiation and identification of M. tuberculosis, M. bovis, and BCG.

Tuberculosis (TB) remains a major infectious disease partly due to the lack of an effective vaccine. Therefore, developing new and more effective TB vaccines is crucial for controlling TB. Mycobacterium tuberculosis (M. tuberculosis) usually parasitizes in macrophages; therefore, cell-mediated immunity plays an important role. The maintenance of memory T cells following M. tuberculosis infection or vaccination is a hallmark of immune protection. This review analyzes the development of memory T cells during M. tuberculosis infection and vaccine immunization, especially on immune memory induced by BCG and subunit vaccines. Furthermore, the factors affecting the development of memory T cells are discussed in detail. The understanding of the development of memory T cells should contribute to designing more effective TB vaccines and optimizing vaccination strategies.

BCG vaccination is increasingly reconsidered in the effective prevention of bovine tuberculosis (bTB). However, the primary challenge in BCG vaccination for cattle is the lack of a technique for differentiating between infected and vaccinated animals (DIVA). This study aimed to establish a novel DIVA diagnostic test based on an interferon-gamma in vitro release assay (IGRA). The plasmid encoding three differential antigens (Rv3872, CFP-10, and ESAT-6) absent in BCG genes but present in virulent M. bovis was previously constructed. Thus, a recombinant protein called RCE (Rv3872, CFP-10, and ESAT-6) was expressed, and an RCE-based DIVA IGRA (RCE-IGRA) was established. The RCE concentration was optimized at 4 μg/mL by evaluating 97 cattle (74 of which were bTB-positive, and 23 were negative) using a commercial IGRA bTB diagnostic kit. Further, 84 cattle were tested in parallel with the RCE-IGRA and commercial PPD-based IGRA (PPD-IGRA), and the results showed a high correlation with a kappa value of 0.83. The study included BCG-vaccinated calves (n = 6), bTB-positive cattle (n = 6), and bTB-negative non-vaccinated calves (n = 6). After 3 months post-vaccination, PPD-IGRA generated positive results in both vaccinated and infected calves. However, RCE-IGRA developed positive results in infected calves but negative results in vaccinated calves. In conclusion, this DIVA method has broad prospects in differentiating BCG vaccination from natural infection to prevent bTB.

BACKGROUND: Bacillus Calmette-Guérin (BCG) vaccination has off-target protective effects against infections unrelated to tuberculosis. Among these, murine and human studies suggest that BCG vaccination may protect against malaria. We investigated whether BCG vaccination influences neonatal in vitro cytokine responses to Plasmodium falciparum. Blood samples were collected from 108 participants in the Melbourne Infant Study BCG for Allergy and Infection Reduction (MIS BAIR) randomised controlled trial (Clinical trials registration NCT01906853, registered July 2013), seven days after randomisation to neonatal BCG (n = 66) or no BCG vaccination (BCG-naïve, n = 42). In vitro cytokine responses were measured following stimulation with P. falciparum-infected erythrocytes (PfIE) or E. coli.
RESULTS: No difference in the measured cytokines were observed between BCG-vaccinated and BCG-naïve neonates following stimulation with PfIE or E. coli. However, age at which blood was sampled was independently associated with altered cytokine responses to PfIE. Being male was also independently associated with increased TNF-a responses to both PfIE and E. coli.
CONCLUSIONS: These findings do not support a role for BCG vaccination in influencing in vitro neonatal cytokine responses to P. falciparum. Older neonates are more likely to develop P. falciparum-induced IFN-γ and IFN-γ-inducible chemokine responses implicated in early protection against malaria and malaria pathogenesis.

To preliminarily assess the immunogenicity of Mtb-HAg in mice and the synergistic effect provided by HAg when co-immunised with BCG.
Mice were randomly grouped for different immunisations and then spleens were aseptically removed and lymphocytes were extracted for immediate detection of cytokines transcript levels and stimulation index(SI), cytokine secretion and multifunctional antigen-specific T cells were detected after incubation for different times.
HAg extracted from active Mtb is a group of mixed polypeptides with molecular weights of (10-14) kDa. It can significantly stimulate lymphocytes proliferation and increase SI. Injection of HAg alone and in combination with BCG induced significantly higher numbers of multifunctional antigen-specific T cells including CD4+ IFN-γ+, CD4+ IL-2+, CD8+ IFN-γ+, and CD8+ IL-2+ cells than that in BCG-treated mice. Co-immunisation induced the secretion of higher levels of IFN-γ, TNF-α, IL-2 and IL-4 and increased their mRNA expression levels. Significant increases in the transcription levels of IL-10, IL-12 and IL-17 were observed in the co-immunised group with the assistance of HAg.
We demonstrated that HAg has favourable immunogenicity, triggers a stronger Th1-type immune response and proposed the hypothesis that HAg can be used as a BCG booster to further enhance the benefits of BCG.

As one of the scheduled immunization vaccines worldwide, virtually all individuals have been vaccinated with BCG vaccine. In order to verify the hypothesis that delivering BCG high-affinity peptides to tumor areas could activate the existing BCG memory T cells to attack tumor, we firstly predicted the HLA-A*0201 high-affinity peptides of BCG Ag85A protein (KLIANNTRV, GLPVEYLQV), and then, A375 melanoma cells and HLA-A*0201 PBMCs (from PPD-positive adults) were added to co-incubated with the predicted peptides in vitro. We found that the predicted BCG high-affinity peptides could be directly loaded onto the surface of tumor cells, enhancing the tumor-killing efficacy of PBMCs from PPD-positive volunteer. Then, we constructed PPD-positive mice model bearing B16F10 subcutaneous tumors and found that intratumor injection of BCG Ag85A high-affinity peptides (SGGANSPAL, YHPQQFVYAGAMSGLLD) enhanced the anti-tumor efficacy in PPD-positive melanoma mice. Along with the better anti-tumor efficacy, the expression of PDL1 on tumor cell surface was also increased, and stronger antitumor effects occurred when further combined with anti-PD1 antibody. For microenvironment analysis, the proportion of effector memory T cells was increased and the better treatment efficacy may be attributed to the elevated effector memory CD4 + T cells within the tumor. In conclusion, using the existing immune response of BCG vaccine by delivering high-affinity peptides of BCG to tumor area is a safe and promising therapy for cancer.

OBJECTIVE: To evaluate the impact of adjuvant therapy on oncological outcomes in patients with intermediate-risk non-muscle-invasive bladder cancer (NMIBC), as due to the poorly-defined and overlapping diagnostic criteria optimal decision-making remains challenging in these patients.
METHODS: In this multicentre study, patients treated with transurethral resection of bladder tumour for Ta disease were retrospectively analysed. All patients with low- or high-risk NMIBC were excluded from the analysis. Associations between adjuvant therapy administration with recurrence-free survival (RFS) and progression-free survival (PFS) rates were assessed in Cox regression models.
RESULTS: A total of 2206 patients with intermediate-risk NMIBC were included in the analysis. Among them, 1427 patients underwent adjuvant therapy, such as bacille Calmette-Guérin (n = 168), or chemotherapeutic agents, such as mitomycin C or epirubicin (n = 1259), in different regimens up to 1 year. The median (interquartile range) follow-up was 73.3 (38.4-106.9) months. The RFS at 1 and 5 years in patients treated with adjuvant therapy and those without were 72.6% vs 69.5% and 50.8% vs 41.3%, respectively. Adjuvant therapy was associated with better RFS (hazard ratio [HR] 0.79, 95% confidence interval [CI] 0.70-0.89, P < 0.001), but not with PFS (P = 0.09). In the subgroup of patients aged ≤70 years with primary, single Ta Grade 2 <3 cm tumours (n = 328), adjuvant therapy was not associated with RFS (HR 0.71, 95% CI 0.50-1.02, P = 0.06). While in the subgroup of patients with at least one risk factor including patient age >70 years, tumour multiplicity, recurrent tumour and tumour size ≥3 cm (n = 1878), adjuvant intravesical therapy was associated with improved RFS (HR 0.78, 95% CI 0.68-0.88, P < 0.001).
CONCLUSIONS: In our study, patients with intermediate-risk NMIBC benefit from adjuvant intravesical therapy in terms of RFS. However, in patients without risk factors, adjuvant intravesical therapy did not result in a clear reduction in the recurrence rate.