Nanocages engineered from Bacillus Calmette-Guerin facilitate protective Vγ2Vδ2 T cell immunity against Mycobacterium tuberculosis infection.

Tuberculosis (TB), induced by Mycobacterium tuberculosis (Mtb) infection, remains a top killer among infectious diseases. While Bacillus Calmette-Guerin (BCG) is the sole TB vaccine, the clumped-clustered features of BCG in intradermal immunization appear to limit both the BCG protection efficacy and the BCG vaccination safety. We hypothesize that engineering of clumped-clustered BCG into nanoscale particles would improve safety and also facilitate the antigen-presenting-cell (APC)'s uptake and the following processing/presentation for better anti-TB protective immunity. Here, we engineered BCG protoplasts into nanoscale membraned BCG particles, termed as "BCG-Nanocage" to enhance the anti-TB vaccination efficiency and safety. BCG-Nanocage could readily be ingested/taken by APC macrophages selectively; BCG-Nanocage-ingested macrophages exhibited better viability and developed similar antimicrobial responses with BCG-infected macrophages. BCG-Nanocage, like live BCG bacilli, exhibited the robust capability to activate and expand innate-like T effector cell populations of Vγ2+ T, CD4+ T and CD8+ T cells of rhesus macaques in the ex vivo PBMC culture. BCG-Nanocage immunization of rhesus macaques elicited similar or stronger memory-like immune responses of Vγ2Vδ2 T cells, as well as Vγ2Vδ2 T and CD4+/CD8+ T effectors compared to live BCG vaccination. BCG-Nanocage- immunized macaques developed rapidly-sustained pulmonary responses of Vγ2Vδ2 T cells upon Mtb challenge. Furthermore, BCG- and BCG-Nanocage- immunized macaques, but not saline controls, exhibited undetectable Mtb infection loads or TB lesions in the Mtb-challenged lung lobe and hilar lymph node at endpoint after challenge. Thus, the current study well justifies a large pre-clinical investigation to assess BCG-Nanocage for safe and efficacious anti-TB vaccination, which is expected to further develop novel vaccines or adjuvants.

Immunostimulatory Response of RWFV Peptide-Targeted Lipid Nanoparticles on Bladder Tumor Associated Cells.

Bladder carcinoma is the most expensive tumor type to treat on a cost-per-patient basis from diagnosis to death. Treatment with Bacillus Calmette Guerin (BCG) instillation is the only approved immunotherapy in the clinic for the remission of superficial bladder carcinoma. Unfortunately, frequent relapses, high local morbidity, risk of systemic mycobacterial infection, and occasional supply chain interruptions limit the utility of BCG for bladder cancer treatment. It is well known that BCG utilizes an adhesin protein known as fibronectin attachment protein that possesses a crucial RWFV peptide sequence for binding to the bladder tumor microenvironment prior to the initiation of the immunotherapeutic response. We report a RWFV-targeted, pH-responsive stabilized lipid nucleic acid nanoparticle (LNP) vehicle for the effective delivery of an immunotherapeutic oligonucleotide, CpG, that is assembled using a glass microfluidic Chemtrix 3221 reactor. Our small-angle X-ray scattering studies revealed a layer-by-layer assembly of the oligonucleotides with a repeat distance of 6.04 nm within the LNP. Using flow cytometry to evaluate the different cell types found in the bladder tumor microenvironment, RWFV-targeted LNPs were found to attach specifically to fibronectin-secreting cells in culture during a 2 h incubation period. The trafficking and cellular fate of these targeted LNPs were revealed by confocal microscopy of RAW264.7 macrophages to enter the endocytotic pathway within 4 h post treatment. Importantly, control studies reveal that only the pH-sensitive LNP formulation is capable of efficiently releasing the payload within 12 h. As a result, the targeted pH-sensitive LNP resulted in higher expression levels of costimulatory molecules CD83, CD 86, and MHC II, while also inducing higher levels of TNF-α secretion from macrophages. These results demonstrate that RWFV-targeted, pH-sensitive LNP formulations are capable of maximum immunotherapeutic response, potentially making them a highly efficient, lower risk, and readily manufactured alternative to BCG immunotherapy.

Biological Rationale for the Repurposing of BCG Vaccine against SARS-CoV-2.

The Bacillus Calmette-Guerin vaccine is still widely used in the developing world. The vaccination prevents infant death not only from tuberculosis but also from unrelated infectious agents, especially respiratory tract infections and neonatal sepsis. It is proposed that these off-target protective effects of the BCG vaccine are mediated by the general long-term boosting of innate immune mechanisms, also termed "trained innate immunity". Recent studies indicate that both COVID-19 incidence and total deaths are strongly associated with the presence or absence of national mandatory BCG vaccination programs and encourage the initiation of several clinical studies with the expectation that revaccination with BCG could reduce the incidence and severity of COVID-19. Here, presented results from the bioinformatics analysis of the Mycobacterium bovis (strain BCG/Pasteur 1173P2) proteome suggests four immunodominant antigens that could induce an immune response against SARS-CoV-2.

The Apoptosis Mechanism of Epirubicin Combined with BCG on Human Bladder Cancer Cells.

AIMS: The purpose of our study was to explore the combination effect of epirubicin and Bacillus Calmette Guerin (BCG) and its mechanism. BACKGROUND: Bladder cancer is a threat to human health worldwide. Commonly used chemotherapy drugs and biotherapy have significant therapeutic effects on bladder cancer, but the mechanism and combined effects are still unclear. OBJECTIVE: To evaluate the anti-cancer effect of epirubicin combined with BCG on human bladder cancer cells, our studies were carried out. METHODS: The viability of human bladder cancer cells with epirubicin and/or BCG treatments was examined by Cell Counting Kit-8 (CCK-8) assay. Apoptosis and cell cycle phase were determined by flow cytometry analysis. Pre-apoptosis factors of caspase-3, p53, B-cell lymphoma 2 associated X protein (Bax) and anti-apoptosis factor of B-cell lymphoma 2 (Bcl-2) were detected by western blot. RESULTS: The viability of human bladder cancer with epirubicin or BCG treatment was decreased and the viability with epirubicin combined with BCG treatment was decreased more, which were determined by CCK-8 assay. Both epirubicin and BCG increased the apoptosis rate of human bladder cancer and arrested more cells into G0/G1 phase, which were tested by flow cytometry. The expression of caspase-3, p53 and Bax was increased and the expression of Bcl-2 was decreased with epirubicin treatment on human bladder cells, which were analyzed by western blot. The expression of caspase-3 and p53 was increased with BCG treatment, which was examined by western blot. CONCLUSION: Epirubicin induced apoptosis in human bladder cancer cells by up-regulating the expression of proapoptotic factors (caspase-3, p53 and Bax) and down-regulating the expression of anti-apoptotic factor (Bcl-2). BCG promoted apoptosis of human bladder cancer cells by up-regulating the expression of caspase-3 and p53. BCG plays a potential role at the time of the combination of epirubicin and BCG on bladder cancer cells in early stage. Both epirubicin and BCG affected cell cycle distribution via arresting more bladder cancer cells at G0/G1 phase, which ultimately led bladder cancer proliferation in vitro and promoted apoptosis.

Bacillus Calmette-Guérin Overexpressing an Endogenous Stimulator of Interferon Genes Agonist Provides Enhanced Protection Against Pulmonary Tuberculosis.

BACKGROUND: Stimulator of interferon genes (STING) is a key cytosolic receptor for small nucleotides and plays a key role in anticancer and antiviral immunity. Cyclic dinucleotide STING agonists may comprise a novel class of vaccine adjuvants capable of inducing cellular immune responses and protective efficacy against intracellular pathogens. METHODS: We generated a recombinant Bacillus Calmette-Guérin ([BCG] BCG-disA-OE) that overexpresses the endogenous mycobacterial diadenylate cyclase gene and releases high levels of the STING agonist bis-(3'-5')-cyclic dimeric adenosine monophosphate (c-di-AMP). We used a 24-week guinea pig vaccination-Mycobacterium tuberculosis (M.tb.) challenge model to test the protective efficacy of BCG-disA-OE versus wild-type BCG and measured lung weights, pathology scores, and M.tb. organ colony-forming unit (CFU) counts. RESULTS: BCG-disA-OE elicited significantly stronger tumor necrosis factor-α, interleukin (IL)-6, IL-1ß, interferon (IFN) regulatory factor 3, and IFN-ß levels than BCG-wild type (WT) in vitro in murine macrophages. In vivo in guinea pigs, we found that BCG-disA-OE reduced lung weights, pathology scores, and M.tb. CFU counts in lungs by 28% (P < .05), 34%, and 2.0 log10 CFU units (P < .05) compared with BCG-WT, respectively. CONCLUSIONS: We report a strategy of delivering a STING agonist from within live BCG. Overproduction of the STING agonist c-di-AMP significantly enhanced the protective efficacy of BCG against pulmonary and extrapulmonary tuberculosis. Our findings support the development of BCG-vectored STING agonists as a tuberculosis vaccine strategy.

Engineering and characterisation of BCG-loaded polymeric microneedles.

The aim of this study was to fabricate Bacillus Calmette-Guérin (BCG)-loaded microneedle patches using micromould casting technique and compare their efficacy with the injectable counterparts. The microneedle patches were formulated using sodium alginate (10% w/v) and trehalose (20% of polymer). The patches were characterised using optical microscopy, scanning electron microscopy and folding endurance. Serum IgG, TLC, granulocyte count, lymphocyte count and CRP were assessed and results were compared to that of intradermal injections alongside controls. The results showed that polymeric patches had a thickness of 0.8 mm, microneedle projections of 272 ± 12 µm and folding endurance of more than 300. Based on haematological and IgG ELISA assays, microneedle-based BCG administration significantly activated the immune cells and induced production of lymphocytes, granulocytes and peptide-specific IgG in immunised rats that were comparable to injectable counterparts. There was an increase in IgG antibodies from 3 g/L to 5.98 g/L and an increase in leucocytes from 2.6 × 109/L to 18.45 × 109/L. There was also an increase in granulocytes from 14.4% to 29.15% and lymphocyte count from 58.75% to 85.3%. It was concluded that BCG-coated polymeric microneedle patches are suitable for the transdermal delivery of vaccine without inducing discomfort usually observed with injections.

Design and Optimization of a Temperature-Stable Dry Powder BCG Vaccine.

PURPOSE: Loss of vaccine potency due to extreme temperature exposure during storage and transport remains a significant obstacle to the success of many vaccines, including the Bacille Calmette-Guérin (BCG) vaccine, the only vaccine available against Mycobacterium tuberculosis. BCG is a live, attenuated vaccine requiring refrigerated storage for viability. In this study, we formulated a temperature-stable BCG dry powder using the spray drying technique. METHODS: We employed a factorial design to optimize our formulation of stabilizing excipients that included L-leucine, bovine serum albumin, polyvinylpyrrolidone, mannitol, and trehalose. Powders were characterized for their particle size, yield, water retention and uptake, glass transition temperature, and aerosol performance. Three optimal powder carrier mixtures were selected from the factorial design for BCG incorporation based on their stability-promoting and powder flow characteristics. Vaccine powders were also assessed for BCG viability and in vivo immunogenicity after long-term storage. RESULTS: Live BCG was successfully spray-dried using the optimized carriers. Dry powder BCG showed no loss in viability (25°C, up to 60% relative humidity; RH) and ~2-log loss in viability (40°C, 75% RH) after one year of storage. The aerodynamic size of the powders was in the respirable range. Further, when healthy mice were immunized intradermally with reconstituted BCG powders (storage for 2 years), the vaccine retained its immunogenicity. CONCLUSION: We developed a spray-dried BCG vaccine that was viable and antigenic after long-term storage. To our knowledge, this is a first study to show room temperature stability of live BCG vaccine without any loss in viability for 12 months.

Tuberculosis vaccine candidates based on mycobacterial cell envelope components.

Even after decades searching for a new and more effective vaccine against tuberculosis, the scientific community is still pursuing this goal due to the complexity of its causative agent, Mycobacterium tuberculosis (Mtb). Mtb is a microorganism with a robust variety of survival mechanisms that allow it to remain in the host for years. The structure and nature of the Mtb envelope play a leading role in its resistance and survival. Mtb has a perfect machinery that allows it to modulate the immune response in its favor and to adapt to the host's environmental conditions in order to remain alive until the moment to reactivate its normal growing state. Mtb cell envelope protein, carbohydrate and lipid components have been the subject of interest for developing new vaccines because most of them are responsible for the pathogenicity and virulence of the bacteria. Many indirect evidences, mainly derived from the use of monoclonal antibodies, support the potential protective role of Mtb envelope components. Subunit and DNA vaccines, lipid extracts, liposomes and membrane vesicle formulations are some examples of technologies used, with encouraging results, to evaluate the potential of these antigens in the protective response against Mtb.

Selective delipidation of Mycobacterium bovis BCG enables direct pulmonary vaccination and enhances protection against Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), is the leading killer due to an infectious organism. Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine approved against TB, however, its efficacy against pulmonary TB is poor. While BCG is currently inoculated intradermally, the natural route of M.tb infection is through the lung. Excessive lung pathology caused by pulmonary inoculation of BCG has prevented the use of this immunization route. Here, we show that selective chemical treatment of BCG with petroleum ether removes inflammatory lipids from the bacterial surface while keeping BCG viable. Pulmonary vaccination using this modified BCG attenuated inflammatory responses, prevented immunopathology of the lung, and significantly increased protection against M.tb infection in mice. We further directly linked IL-17A as the responsible contributor of improved immunity against M.tb infection. These results provide evidence that selective removal of cytotoxic lipids from the BCG surface attenuates inflammation and offers a safer and superior vaccine against TB causing less damage post-infectious challenge with M.tb.

Enhancement of the effect of BCG vaccine against tuberculosis using DDA/TDB liposomes containing a fusion protein of HspX, PPE44, and EsxV.

Tuberculosis has been a major health problem worldwide for years; therefore, it is important to develop and produce an effective vaccine against this disease. In this study, the immunogenicity of Mycobacterium tuberculosis fusion protein (FP) encapsulated in liposomes containing DDA/TDB was evaluated. The FP was expressed in E. coli BL21 and encapsulated in liposomal formulations. Three weeks after the last subcutaneous immunization, IFN-γ, IL-4, IL-17, and IL-12 in spleen cell culture supernatants, and IgG2a, IgG1, and IgG2b titres in sera were measured. The greatest IFN-γ and IL-12 interleukin concentrations were observed in the DDA/TDB/CHOL liposomes containing the FP. Initial injection with BCG improved the efficacy of the DDA/TDB/CHOL/FP vaccine. The IgG2a/IgG1 ratio was also high in the DDA/TDB/CHOL/FP group; furthermore, the IgG2a/IgG1 ratio was increased in the BCG-primed, DDA/TDB/CHOL/FP-boosted group, indicating induction of a cellular immune response. Our study showed that the FP-containing DDA/TDB/CHOL liposomes induced a Th1 response. However, the groups that first received BCG and then DDA/TDB/CHOL/FP had the greatest Th1 response in terms of IFN-γ and IL-12 production of all the groups. This suggests that these formulations enhance the BCG vaccine's effectiveness.

Cationized liposomal keto-mycolic acids isolated from Mycobacterium bovis bacillus Calmette-Guérin induce antitumor immunity in a syngeneic murine bladder cancer model.

Intravesical therapy using Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the most established cancer immunotherapy for bladder cancer. However, its underlying mechanisms are unknown. Mycolic acid (MA), the most abundant lipid of the BCG cell wall, is suspected to be one of the essential active components of this immunogenicity. Here, we developed cationic liposomes incorporating three subclasses (α, keto, and methoxy) of MA purified separately from BCG, using the dendron-bearing lipid D22. The cationic liposomes using D22 were efficiently taken up by the murine bladder cancer cell line MB49 in vitro, but the non-cationic liposomes were not. Lip-kMA, a cationic liposome containing keto-MA, presented strong antitumor activity in two murine syngeneic graft models using the murine bladder cancer cell lines MB49 and MBT-2 in comparison to both Lip-aMA and Lip-mMA, which contained α-MA and methoxy-MA, respectively. Interestingly, Lip-kMA(D12), which was made of D12 instead of D22, did not exhibit antitumor activity in the murine syngeneic graft model using MB49 cells, although it was successfully taken up by MB49 cells in vitro. Histologically, compared to the number of infiltrating CD4 lymphocytes, the number of CD8 lymphocytes was higher in the tumors treated with Lip-kMA. Antitumor effects of Lip-kMA were not observed in nude mice, whereas weak but significant effects were observed in beige mice with natural killer activity deficiency. Thus, a cationized liposome containing keto-MA derived from BCG induced in vivo antitumor immunity. These findings will provide new insights into lipid immunogenicity and the underlying mechanisms of BCG immunotherapy.

Extended Freeze-Dried BCG Instructed pDCs Induce Suppressive Tregs and Dampen EAE.

Several clinical observations have shown that Bacillus Calmette-Guérin (BCG) vaccine has beneficial impact on patients suffering from different chronic inflammatory diseases. Here we evaluated whether BCG inactivated by Extended Freeze-Drying (EFD) which circumvents all the side effects linked to the live bacteria, could influence the development of experimental autoimmune encephalomyelitis (EAE), a mouse model for Multiple Sclerosis. EFD BCG strongly attenuates inflammation, both systemically and at the central nervous system (CNS) level, alleviating EAE. Mechanistically, EFD BCG directly impacts the phenotype of plasmacytoid dendritic cells (pDCs), and promotes their ability to induce suppressive IL-10 secreting regulatory T cells (Tregs) that inhibit encephalitogenic CD4+ T cells. When co-cultured with human allogenic naive CD4+ T cells, EFD BCG exposed human pDCs similarly induce the differentiation of IL-10 producing Tregs. Our study provides evidence that EFD BCG could be used as an immunomodulator of encephalitogenic T cells in multiple sclerosis patients.

Can colony-forming unit testing be used to extend the shelf life of BCG vaccines?

A recent shortage in supply of Bacille Calmette-Guérin (BCG), the live attenuated vaccine given to protect against tuberculosis (TB) caused major disruption to global vaccination programs. In this study, we assessed whether quantification of viable bacteria, could be used to inform the use of the BCG vaccine beyond its manufacturer-assigned expiration date. The viability of a single batch of BCG-Denmark was tested in three independent laboratories. There was high inter-vial and inter-laboratory variability in viability counts, however all three laboratories detected a decrease in BCG viability over time. Despite this, there was no difference in the rate of BCG scar formation in infants who were vaccinated with this batch of BCG before and after its manufacturer-assigned expiration date. This study demonstrates the potential for using BCG viability counts to guide the use of BCG vaccine beyond the manufacturer-assigned expiration date.

In silico design of Mycobacterium tuberculosis epitope ensemble vaccines.

Effective control of Mycobacterium tuberculosis is a global necessity. In 2015, tuberculosis (TB) caused more deaths than HIV. Considering the increasing prevalence of multi-drug resistant forms of M. tuberculosis, the need for effective TB vaccines becomes imperative. Currently, the only licensed TB vaccine is Bacillus Calmette-Guérin (BCG). Yet, BCG has many drawbacks limiting its efficacy and applicability. We applied advanced computational procedures to derive a universal TB vaccine and one targeting East Africa. Our approach selects an optimal set of highly conserved, experimentally validated epitopes, with high projected population coverage (PPC). Through rigorous data analysis, five different potential vaccine combinations were selected each with PPC above 80% for East Africa and above 90% for the World. Two potential vaccines only contained CD8+ epitopes, while the others included both CD4+ and CD8+ epitopes. Our prime vaccine candidate was a putative seven-epitope ensemble comprising: SRGWSLIKSVRLGNA, KPRIITLTMNPALDI, AAHKGLMNIALAISA, FPAGGSTGSL, MLLAVTVSL, QSSFYSDW and KMRCGAPRY, with a 97.4% global PPC and a 92.7% East African PPC.

Evolution and Strain Variation in BCG.

BCG vaccines were derived by in vitro passage, during the years 1908-1921, at the Pasteur Institute of Lille. Following the distribution of stocks of BCG to vaccine production laboratories around the world, it was only a few decades before different BCG producers recognized that there were variants of BCG, likely due to different passaging conditions in the different laboratories. This ultimately led to the lyophilization of stable BCG products in the 1950s and 1960s, but not before considerable evolution of the different BCG strains had taken place. The application of contemporary research methodologies has now revealed genomic, transcriptomic and proteomic differences between BCG strains. These molecular differences in part account for phenotypic differences in vitro between BCG strains, such as their variable secretion of antigenic proteins. Yet, the relevance of BCG variability for immunization policy remains elusive. In this chapter we present an overview of what is known about BCG evolution and its resulting strain variability, and provide some speculation as to the potential relevance for a vaccine given to over 100 million newborns each year.

Raw starch microparticles have immunostimulant activity in mice vaccinated with BCG and challenged with Mycobacterium tuberculosis.

The main challenge for vaccine development or improvement is the lack of safe adjuvants or immunostimulants that induce protective immune responses and can be used for mucosal immunization, which is a highly desirable strategy for vaccination against infectious diseases acquired by oral or intranasal routes. One promising alternative is the use of biodegradable and biocompatible polymeric microparticles. Recently, we developed an immobilization and delivery system with starch microparticles (SMPs) and a starch-binding domain (SBDtag) suitable for the mucosal administration of antigens and the induction of antigen-specific immune responses. Here, we explore the immunostimulant and reinforcing potential of the system using BALB/c mice with progressive pulmonary tuberculosis (PPT). The heat shock protein alpha-crystallin from Mycobacterium tuberculosis immobilized on SMPs (µAcr-SBDtag) or SMPs alone were administered nasally as boosters to BCG-vaccinated mice without any extra adjuvant. The mice were challenged intratracheally with either moderately virulent or highly virulent M. tuberculosis strains. Our results showed that the administration of either the immobilized antigen or SMPs asa booster for the BCG vaccination induced a significant reduction of bacterial loads in the lungs of mice, even more than in mice that received the BCG vaccination alone. Since no difference was observed in pulmonary bacillary burdens between the two reinforced groups, the obtained effect was most likely primarily caused by the starch. As determined by histological study, the administration of boosters did not contribute to the progress of pneumonia, which diminishes the safety concerns related to the administration of SMPs intranasally. Taken together, our findings suggest that this system may be considered asa new carbohydrate-based adjuvant suitable for mucosal vaccines against tuberculosis and other infectious diseases, and more generally, they highlight the potential of particulate α-glucans as immune response modifiers.

BCG-loaded chitosan microparticles: interaction with macrophages and preliminary in vivo studies.

The aim of this study was to develop a novel BCG-loaded chitosan vaccine with high association efficiency which can afford efficient interaction with APC and elicit local and Th1-type-specific immune response after intranasal administration. Chitosan-suspended BCG and BCG-loaded chitosan-alginate microparticles were prepared by ionotropic gelation. Interaction with APC was evaluated by fluorescence microscopy using rBCG-GFP. Specific immune responses were evaluated following intranasal immunisation of mice. Cellular uptake was approximately two-fold higher for chitosan-suspended BCG. A single dose of BCG-loaded microparticles or chitosan-suspended BCG by intranasal route improved Th1-type response compared with subcutaneous BCG. Chitosan-suspended BCG originated the highest mucosal response in the lungs by intranasal route. These positive results indicate that the proposed approach of whole live BCG microencapsulation in chitosan-alginate for intranasal immunisation was successful in allowing efficient interaction with APC, while improving the cellular immune response, which is of interest for local immunisation against tuberculosis.

A simplified ATP method for the rapid control of cell viability in a freeze-dried BCG vaccine.

We propose a simple and cost-effective ATP method for controlling the specific activity of a freeze-dried BCG vaccine. A freeze-dried BCG vaccine is reconstituted with 1ml saline and incubated for 15min at room temperature and then for 1h at 37°C. The vaccine is then treated with apyrase to remove extracellular ATP. After that, the cells are lysed with DMSO and the ATP content in the lysate is measured by the bioluminescence method. To implement the method, we developed a kit that requires no time-consuming preparation before the analysis. We demonstrated the linear relationship between the experimental values of the specific activity (106CFU/mg) and intracellular ATP content (ATP, pmol/mg) for different batches of the studied BCG vaccines; the proportionality coefficient was К=0.36±0.02. We proposed a formula for calculating the specific activity from the measured content of intracellular ATP (ATP, pmol/mg). The comparison of the measured and calculated values of the specific activity (106CFU/mg) shows that these values are similar; their differences fall within the allowable range of deviations for the specific activity values of the BCG vaccine.

Mixture of polysaccharide and nucleic acid extracted from Bacillus Calmette-Guerin (BCG) enhances immune response of infectious bursal disease virus vaccine in chickens.

In this study, the immune response induced by a mixture of polysaccharide and nucleic acid extracted from Bacillus Calmette-Guerin (BCG) was evaluated in chickens inoculated with infectious bursal disease virus (IBDV) vaccine. After the mixture was injected intramuscularly at a dose of 0.075, 0.15 or 0.3 mg·kg(-1)·day(-1) for 3 days, the 14-day-old chickens were inoculated with the attenuated IBDV vaccine via intranasal and ocular routes. The relative weight of bursa of Fabricius (BF) and thymus, the serum IBD antibody titer, the CD4+/CD8+ ratio, and the concentrations of IFN-γ, IL-2 and IL-6 in peripheral blood were investigated on days 5, 15 and 25. The IBD antibody titer in BCG-treated groups was higher than in the negative control and only IBD-vaccinated chickens, indicating that the mixture of BCG can significantly enhance chicken humoral response. CD4+/CD8+ and the secretions of IFN-γ, IL-2 and IL-6 were also clearly increased compared with that in the negative control and IBD-vaccinated chickens, indicating that the mixture can also enhance the cell-mediated immune response. The results also showed that the relative weights of BF and thymus increased after chickens were inoculated with BCG, indicating that the BCG mixture can clearly enhance the immunity of IBD-vaccine and can be expected to be viewed as a candidate for a new type of immune adjuvant.

Effect of Experimental Parameters on Alginate/Chitosan Microparticles for BCG Encapsulation.

The aim of the present study was to develop novel Mycobacterium bovis bacille Calmette-Guérin (BCG)-loaded polymeric microparticles with optimized particle surface characteristics and biocompatibility, so that whole live attenuated bacteria could be further used for pre-exposure vaccination against Mycobacterium tuberculosis by the intranasal route. BCG was encapsulated in chitosan and alginate microparticles through three different polyionic complexation methods by high speed stirring. For comparison purposes, similar formulations were prepared with high shear homogenization and sonication. Additional optimization studies were conducted with polymers of different quality specifications in a wide range of pH values, and with three different cryoprotectors. Particle morphology, size distribution, encapsulation efficiency, surface charge, physicochemical properties and biocompatibility were assessed. Particles exhibited a micrometer size and a spherical morphology. Chitosan addition to BCG shifted the bacilli surface charge from negative zeta potential values to strongly positive ones. Chitosan of low molecular weight produced particle suspensions of lower size distribution and higher stability, allowing efficient BCG encapsulation and biocompatibility. Particle formulation consistency was improved when the availability of functional groups from alginate and chitosan was close to stoichiometric proportion. Thus, the herein described microparticulate system constitutes a promising strategy to deliver BCG vaccine by the intranasal route.