Genetic engineering employing MPB70 and its promoter enables efficient secretion and expression of foreign antigen in bacillus Calmette Guérin (BCG) Tokyo.

Vaccination is an important factor in public health. The recombinant bacillus Calmette Guérin (rBCG) vaccine, which expresses foreign antigens, is expected to be a superior vaccine against infectious diseases. Here, we report a new recombination platform in which the BCG Tokyo strain is transformed with nucleotide sequences encoding foreign protein fused with the MPB70 immunogenic protein precursor. By RNA-sequencing, mpb70 was found to be the most transcribed among all known genes of BCG Tokyo. Small oligopeptide, namely, polyhistidine tag, was able to be expressed in and secreted from rBCG through a process in which polyhistidine tag fused with intact MPB70 were transcribed by an mpb70 promoter. This methodology was applied to develop an rBCG expressing the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2. Immunoblotting images and mass spectrometry data showed that RBD was also secreted from rBCG. Sera from mice vaccinated with the rBCG showed a tendency of weak neutralizing capacity. The secretion was retained even after a freeze-drying process. The freeze-dried rBCG was administered to and recovered from mice. Recovered rBCG kept secreting RBD. Collectively, our recombination platform offers stable secretion of foreign antigens and can be applied to the development of practical rBCGs.

Characterization of cellular immune response in hamsters immunized with recombinant vaccines against leptospirosis based on LipL32:LemA:LigAni chimeric protein.

In the last 20 years, various research groups have endeavored to develop recombinant vaccines against leptospirosis to overcome the limitations of commercially available bacterins. Numerous antigens and vaccine formulations have been tested thus far. However, the analysis of cellular response in these vaccine formulations is not commonly conducted, primarily due to the scarcity of supplies and kits for the hamster animal model. Our research group has already tested the Q1 antigen, a chimeric protein combining the immunogenic regions of LipL32, LemA, and LigANI, in recombinant subunit and BCG-vectored vaccines. In both strategies, 100 % of the hamsters were protected against clinical signs of leptospirosis. However, only the recombinant BCG-vectored vaccine provided protection against renal colonization. Thus, the objective of this study is to characterize the cellular immune response in hamsters immunized with different vaccine formulations based on the Q1 antigen through transcriptional analysis of cytokines. The hamsters were allocated into groups and vaccinated as follows: recombinant subunit (rQ1), recombinant BCG (rBCG:Q1), and saline and BCG Pasteur control vaccines. To assess the cellular response induced by the vaccines, we cultured and stimulated splenocytes, followed by RNA extraction from the cells and analysis of cytokines using real-time PCR. The results revealed that the recombinant subunit vaccine elicited a Th2-type response, characterized by the expression of cytokines IL-10, IL-1α, and TNF-α. This pattern closely resembles the cytokines expressed in severe cases of leptospirosis. On the other hand, the rBCG-vectored vaccine induced a Th1-type response with significant up-regulation of IFN-γ. These findings suggest the involvement of the cellular response and the IFN-γ mediated inflammatory response in the sterilizing immunity mediated by rBCG. Therefore, this study may assist future investigations in characterizing the cellular response in hamsters, aiming to elucidate the mechanisms of efficacy and establish potential correlates of protection.

The Induction of Antigen 85B-Specific CD8+ T Cells by Recombinant BCG Protects against Mycobacterial Infection in Mice.

Mycobacterium tuberculosis (Mtb) infection remains a major health problem worldwide. Although the Bacillus Calmette-Guérin (BCG) vaccine is the most widely used vaccination for preventing tuberculosis (TB), its efficacy is limited. We previously developed a new recombinant BCG (rBCG)-based vaccine encoding the Ag85B protein of M. kansasii (Mkan85B), termed rBCG-Mkan85B, and its administration is followed by boosting with plasmid DNA expressing the Ag85B gene (DNA-Mkan85B). Previously, we identified MHC-I (H2-Kd)-restricted epitopes that highly cross-react with those of Mtb in BALB/c (H2d) and CB6F1 (H2b/d) mice. We also reported that the rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination protocol protected CB6F1 mice against M. kansasii infection. In this study, to investigate the protective effect of our novel rBCG against Mtb infection, CB6F1 mice were either left unimmunized or immunized with the BCG, rBCG-Mkan85B, or rBCG-Mkan85B/DNA-Mkan85B vaccine for 10 weeks prior to inhalation exposure to the virulent Mtb Erdman strain for another 6 weeks. Compared with the BCG and rBCG-Mkan85B vaccinations, the rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination protocol significantly reduced the numbers of pulmonary colony-forming units (CFUs). Moreover, the rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination induced antigen-specific polyfunctional CD4+ and CD8+ T cells. These results suggest that CD8+ T-cell immunity to immunodominant epitopes of Mtb is enhanced by rBCG vector-based immunization. Thus, rBCG vector-based vaccinations may overcome the limited ability of the current BCG vaccine to elicit TB immunity.

CD8 T Cells Show Protection against Highly Pathogenic Simian Immunodeficiency Virus (SIV) after Vaccination with SIV Gene-Expressing BCG Prime and Vaccinia Virus/Sendai Virus Vector Boosts.

Toward development of a dual vaccine for human immunodeficiency virus type 1 (HIV-1) and tuberculosis infections, we developed a urease-deficient bacillus Calmette-Guérin (BCG) strain Tokyo172 (BCGΔurease) to enhance its immunogenicity. BCGΔurease expressing a simian immunodeficiency virus (SIV) Gag induced BCG antigen-specific CD4+ and CD8+ T cells more efficiently and more Gag-specific CD8+ T cells. We evaluated its protective efficacy against SIV infection in cynomolgus monkeys of Asian origin, shown to be as susceptible to infection with SIVmac251 as Indian rhesus macaques. Priming with recombinant BCG (rBCG) expressing SIV genes was followed by a boost with SIV gene-expressing LC16m8Δ vaccinia virus and a second boost with SIV Env-expressing Sendai virus. Eight weeks after the second boost, monkeys were repeatedly challenged with a low dose of SIVmac251 intrarectally. Two animals out of 6 vaccinees were protected, whereas all 7 control animals were infected without any early viral controls. In one vaccinated animal, which had the most potent CD8+ T cells in an in vitro suppression activity (ISA) assay of SIVmac239 replication, plasma viremia was undetectable throughout the follow-up period. Protection was confirmed by the lack of anamnestic antibody responses and detectable cell-associated provirus in various organs. Another monkey with a high ISA acquired a small amount of SIV, but it later became suppressed below the detection limit. Moreover, the ISA score correlated with SIV acquisition. On the other hand, any parameter relating anti-Env antibody was not correlated with the protection.IMPORTANCE Because both AIDS and tuberculosis are serious health threats in middle/low-income countries, development of a dual vaccine against them would be highly beneficial. To approach the goal, here we first assessed a urease-deficient bacillus Calmette-Guérin (BCG) for improvement of immunogenicity against both Mycobacterium tuberculosis and SIV. Second, we demonstrated the usefulness of Asian-origin cynomolgus monkeys for development of a preclinical AIDS vaccine by direct comparison with Indian rhesus macaques as the only validated hosts that identically mirror the outcomes of clinical trials, since the availability of Indian rhesus macaques is limited in countries other than the United States. Finally, we report the protective effect of a vaccination regimen comprising BCG, the highly attenuated vaccinia virus LC16m8Δ strain, and nontransmissible Sendai virus as safe vectors expressing SIV genes using repeated mucosal challenge with highly pathogenic SIVmac251. Identification of CD8+ T cells as a protective immunity suggests a future direction of AIDS vaccine development.

Deletion of BCG_2432c from the Bacillus Calmette-Guérin vaccine enhances autophagy-mediated immunity against tuberculosis.

BACKGROUND: Mycobacterium bovis bacillus Calmette-Guérin (BCG) is an attenuated live vaccine that provides insufficient protection against tuberculosis (TB), the underlying mechanisms for which remain unknown. Assuming that the BCG vaccine inherits immune evasive strategies from virulent parent M. bovis strains, we aimed to identify the associated genes and assess their effects on the vaccine efficacy. METHODS: Three genes, BCG_3174, BCG_1782, and BCG_2432c, associated with immune evasion were first identified via bioinformatics analysis and then confirmed in the genome of M. bovis and 12 commercial BCG vaccine substrains using Polymerase Chain Reaction (PCR) and DNA sequencing. These genes were disrupted to develop mutant strains, and their effects on autophagy and their protective efficacy were further compared with the BCG vaccine in vitro and in vivo. RESULTS: Of the three identified genes, only the disruption of BCG_2432c, namely ΔBCG_2432c, conferred stronger protection against intranasal TB in vaccinated mice, when compared with the BCG vaccine. ΔBCG_2432c showed a stronger ability to trigger intracellular ROS-mediated complete autophagic flux in infected THP-1 cells that resulted in higher antigen presentation. The improved protection could be attributed to early and increased IFN-γ+ CD4+ TEM and IL-2+ CD4+ TCM cells in the spleens and lungs of ΔBCG_2432c-vaccinated mice. CONCLUSIONS: The insufficient efficacy of the BCG vaccine is attributable to the important autophagy-inhibition gene BCG_2432c that blocks the autophagosome-lysosome pathway of antigen presentation. ΔBCG_2432c provides a promising platform to either replace the current BCG vaccine or develop vaccines that are more effective against TB.

The inhibitory effect of rBCG on EB virus-positive tumours using an EB virus fusion gene.

At present, studies have found that latent Epstein-Barr virus (EBV) infection is associated with a variety of human tumours, and a vaccine is not available in this field. In this research, RT-PCR was used to obtain BZLF1 (immediately expressed early antigen Z) and LMP2 (latent membrane protein 2) cDNA from EBV. A ZLP2 fusion gene containing a linker sequence that encoded the polypeptide (Gly4Ser)3 was obtained using the sequence splicing overlap extension method. Then, ZLP2 was inserted into pMV261 cells, and the recombinant plasmid pMV-ZLP2 was transformed into BCG competent cells. After EB virus-positive tumour cell (NPRC18) cancer models were established with C57BL/6 J mice, tumour weight, tumour formation time and mouse survival conditions were analyzed, and flow cytometry was used to analyze the quantities of CD8 + and CD4 + T cells. HE staining was used to detect and analyze lymphocyte infiltration, and statistical analysis was used to analyze the immunological effect of recombinant BCG (rBCG). Compared with the control group, rBCG could significantly prolong the survival time of mice, slow tumour growth and delay tumour formation time. Recombinant BCG exhibits an obvious immune effect in mice and an inhibitory effect on EBV-positive cancer.Key points• AZLP2 fusion gene with BZLF1 and LMP2 of EB virus was constructed.• ZLP2 fusion gene was expressed with rBCG.• rBCG with ZLP2 has an obvious effect on EBV-positive cancer.

Vaccine-Induced Subcutaneous Granulomas in Goats Reflect Differences in Host-Mycobacterium Interactions between BCG- and Recombinant BCG-Derivative Vaccines.

Tuberculous granulomas are highly dynamic structures reflecting the complex host-mycobacterium interactions. The objective of this study was to compare granuloma development at the site of vaccination with BCG and its recombinant derivatives in goats. To characterize the host response, epithelioid cells, multinucleated giant cells (MNGC), T cell subsets, B cells, plasma cells, dendritic cells and mycobacterial antigen were labelled by immunohistochemistry, and lipids and acid-fast bacteria (AFB) were labelled by specific staining. Granulomas with central caseous necrosis developed at the injection site of most goats though lesion size and extent of necrosis differed between vaccine strains. CD4+ T and B cells were more scarce and CD8+ cells were more numerous in granulomas induced by recombinant derivatives compared to their parental BCG strain. Further, the numbers of MNGCs and cells with lipid bodies were markedly lower in groups administered with recombinant BCG strains. Microscopic detection of AFB and mycobacterial antigen was rather frequent in the area of central necrosis, however, the isolation of bacteria in culture was rarely successful. In summary, BCG and its recombinant derivatives induced reproducibly subcutaneous caseous granulomas in goats that can be easily monitored and surgically removed for further studies. The granulomas reflected the genetic modifications of the recombinant BCG-derivatives and are therefore suitable models to compare reactions to different mycobacteria or TB vaccines.

A New Mouse Model of Chronic Myocarditis Induced by Recombinant Bacille Calmette-Guèrin Expressing a T-Cell Epitope of Cardiac Myosin Heavy Chain-α.

Dilated cardiomyopathy (DCM) is a potentially lethal disorder characterized by progressive impairment of cardiac function. Chronic myocarditis has long been hypothesized to be one of the causes of DCM. However, owing to the lack of suitable animal models of chronic myocarditis, its pathophysiology remains unclear. Here, we report a novel mouse model of chronic myocarditis induced by recombinant bacille Calmette-Guérin (rBCG) expressing a CD4+ T-cell epitope of cardiac myosin heavy chain-α (rBCG-MyHCα). Mice immunized with rBCG-MyHCα developed chronic myocarditis, and echocardiography revealed dilation and impaired contraction of ventricles, similar to those observed in human DCM. In the heart, CD62L-CD4+ T cells were increased and produced significant amounts of IFN-γ and IL-17 in response to cardiac myosin. Adoptive transfer of CD62L-CD4+ T cells induced myocarditis in the recipient mice, which indicated that CD62L-CD4+ T cells were the effector cells in this model. rBCG-MyHCα-infected dendritic cells produced proinflammatory cytokines and induced MyHCα-specific T-cell proliferation and Th1 and Th17 polarization. This novel chronic myocarditis mouse model may allow the identification of the central pathophysiological and immunological processes involved in the progression to DCM.

MHC-restricted Ag85B-specific CD8+ T cells are enhanced by recombinant BCG prime and DNA boost immunization in mice.

Despite efforts to develop effective treatments and vaccines, Mycobacterium tuberculosis (Mtb), particularly pulmonary Mtb, continues to provide major health challenges worldwide. To improve immunization against the persistent health challenge of Mtb infection, we have studied the CD8+ T cell response to Bacillus Calmette-Guérin (BCG) and recombinant BCG (rBCG) in mice. Here, we generated CD8+ T cells with an rBCG-based vaccine encoding the Ag85B protein of M. kansasii, termed rBCG-Mkan85B, followed by boosting with plasmid DNA expressing the Ag85B gene (DNA-Mkan85B). We identified two MHC-I (H2-Kd )-restricted epitopes that induce cross-reactive responses to Mtb and other related mycobacteria in both BALB/c (H2d ) and CB6F1 (H2b/d ) mice. The H2-Kd -restricted peptide epitopes elicited polyfunctional CD8+ T cell responses that were also highly cross-reactive with those of other proteins of the Ag85 complex. Tetramer staining indicated that the two H2-Kd -restricted epitopes elicit distinct CD8+ T cell populations, a result explained by the X-ray structure of the two peptide/H2-Kd complexes. These results suggest that rBCG-Mkan85B vector-based immunization and DNA-Mkan85B boost may enhance CD8+ T cell response to Mtb, and might help to overcome the limited effectiveness of the current BCG in eliciting tuberculosis immunity.

Mycobacterium bovis BCG in metastatic melanoma therapy.

Melanoma is the most aggressive form of skin cancer, with a high mortality rate and with 96,480 new cases expected in 2019 in the USS. BRAFV600E, the most common driver mutation, is found in around 50% of melanomas, contributing to tumor growth, angiogenesis, and metastatic progression. Dacarbazine (DTIC), an alkylate agent, was the first chemotherapeutic agent approved by the US Food and Drug Administration (FDA) used as a standard treatment. Since then, immunotherapies have been approved for metastatic melanoma (MM) including ipilimumab and pembrolizumab checkpoint inhibitors that help decrease the risk of progression. Moreover, Mycobacterium bovis Bacillus Calmette-Guerin (BCG) serves as an adjuvant therapy that induces the recruitment of natural killer NK, CD4+, and CD8+ T cells and contributes to antitumor immunity. BCG can be administered in combination with chemotherapeutic and immunotherapeutic agents and can be genetically manipulated to produce recombinant BCG (rBCG) strains that express heterologous proteins or overexpress immunogenic proteins, increasing the immune response and improving patient survival. In this review, we highlight several studies utilizing rBCG immunotherapy for MM in combination with other therapeutic agents.