Immunogenicity and vaccine potential of clinical isolate Mycobacterium kansasii strain against Mycobacterium tuberculosis infection.

Mycobacterium kansasii is a bacterium included in non-tuberculous mycobacteria (NTM) that can cause lung disease. It shares a significant number of antigens with Mycobacterium tuberculosis (Mtb), suggesting that it has the potential to be used as a tuberculosis (TB) vaccine. Therefore, we subcutaneously vaccinated mice with reference strain, M. kansasii-ATCC12478 [M. kansasii-American Type Culture Collection (ATCC)], and clinically isolated strain, M. kansasii-SM-1 to evaluate potential as a TB vaccine by comparing with bacille Calmette-Guerin (BCG) vaccine. Ten weeks after vaccination, we evaluated immunogenicity of M. kansasii-ATCC and M. kansasii-SM-1, and M. kansasii-SM-1 immunization induces potent Mtb antigen-specific IFN-γ-producing CD4+ T cells than M. kansasii-ATCC. Upon Mtb infection, M. kansasii-SM-1 provided better protection than M. kansasii-ATCC, which was comparable to the efficacy of BCG. These results showed that the clinical strain M. kansasii-SM-1, which exhibits an enhanced Mtb antigen-specific Th1 response, shows greater vaccine efficacy compared to M. kansasii-ATCC. In this study, we demonstrated that vaccine efficacy can vary depending on the strain of M. kansasii and that its efficacy can be comparable to BCG. This suggests that M. kansasii has the potential to be a live TB vaccine candidate.IMPORTANCEMycobacterium kansasii, a non-tuberculous mycobacteria (NTM) species causing lung disease, shares key antigens with Mycobacterium tuberculosis (Mtb), indicating its potential for TB vaccine development. Subcutaneous vaccination of mice with M. kansasii strains reference strain M. kansasii-ATCC12478 [(M. kansasii-American Type Culture Collection (ATCC)] and clinically isolated strain M. kansasii-SM-1 revealed differences in immunogenicity. M. kansasii-SM-1 induced a robust Mtb antigen-specific IFN-γ-producing CD4+ T cell response compared to M. kansasii-ATCC. Additionally, M. kansasii-SM-1 conferred better protection against Mtb infection than M. kansasii-ATCC, which is comparable to bacille Calmette-Guerin (BCG). These findings underscore the variable vaccine efficacy among M. kansasii strains, with M. kansasii-SM-1 exhibiting promising potential as a live TB vaccine candidate, suggesting its comparative effectiveness to BCG.

Adjuvant system AS01: from mode of action to effective vaccines.

INTRODUCTION: The use of novel adjuvants in human vaccines continues to expand as their contribution to preventing disease in challenging populations and caused by complex pathogens is increasingly understood. AS01 is a family of liposome-based vaccine Adjuvant Systems containing 2 immunostimulants: 3-O-desacyl-4'-monophosphoryl lipid A and the saponin QS-21. AS01-containing vaccines have been approved and administered to millions of individuals worldwide. AREAS COVERED: Here we report advances in our understanding of the mode of action of AS01 that contributed to the development of efficacious vaccines preventing disease due to malaria, herpes zoster, and respiratory syncytial virus. AS01 induces early innate immune activation that induces T cell-mediated and antibody-mediated responses with optimized functional characteristics and induction of immune memory. AS01-containing vaccines appear relatively impervious to baseline immune status translating into high efficacy across populations. Currently licensed AS01-containing vaccines have shown acceptable safety profiles in clinical trials and post-marketing settings. EXPERT OPINION: Initial expectations that adjuvantation with AS01 could support effective vaccine responses and contribute to disease control have been realized. Investigation of the utility of AS01 in vaccines to prevent other challenging diseases, such as tuberculosis, is ongoing, together with efforts to fully define its mechanisms of action in different vaccine settings.

Adjuvants are added to vaccines to increase the immune response produced after vaccination. Adjuvant Systems contain 2 or more molecules that stimulate the immune system. AS01 is an Adjuvant System that contains 2 components, MPL and QS-21, that stimulate the immune system. AS01 is included in 3 approved vaccines: a malaria vaccine for children, a herpes zoster vaccine for older adults, and a respiratory syncytial virus vaccine also for older adults. Vaccines containing AS01 have been extensively evaluated in clinical trials and administered to millions of individuals during market use. These vaccines are effective in preventing disease and have acceptable safety in different age groups. Experiments have been done to investigate how AS01 works in vaccines to produce an efficient immune response that helps to protect against the disease being targeted. A key effect of AS01 is to encourage specific immune cells to produce chemicals that stimulate the immune system. We now know that this effect is due to co-operation between MPL and QS-21. Experiments have shown that AS01 induces a sophisticated immune 'gene signature' in blood within 24 hours after vaccination, and people who developed this 'gene signature' had a stronger response to vaccination. AS01 seems to be able to stimulate the immune system of most people ­ even if they are older or have a weakened immune system. This means that AS01 could be included in other vaccines against other challenging diseases, such as tuberculosis, or could be used in the treatment of some disease, such as chronic hepatitis B.

Lessons from a decade of adult vaccine rollout in low- and middle-income countries: a scoping review.

INTRODUCTION: The historical focus of vaccines on child health coupled with the advent of novel vaccines targeting adult populations necessitates exploring strategies for adult vaccine implementation. AREAS COVERED: This scoping review extracts insights from the past decade's experiences introducing adult vaccines in low- and middle-income countries. Among 25 papers reviewed, 19 focused on oral cholera vaccine, 2 on Meningococcal A vaccines, 2 on tetanus toxoid vaccine, 1 on typhoid vaccine, and 1 on Ebola vaccine. Aligned with WHO's Global Framework for New TB Vaccines for Adults and Adolescents, our findings center on vaccine availability, accessibility, and acceptance. EXPERT OPINION: Availability findings underscore the importance of understanding disease burden for prioritization, multi-sectoral collaboration during planning, and strategic resource allocation and coordination. Accessibility results highlight the benefits of leveraging existing health infrastructure and adequately training healthcare workers, and contextually tailoring vaccine delivery approaches to reach challenging sub-groups like working male adults. Central to fostering acceptance, resonant sensitization, and communication campaigns engaging the communities and utilizing trusted local leaders countered rumors and increased awareness and uptake. As we approach the introduction of a new adult TB vaccine, insights from this review equips decision-makers with key evidence-based recommendations to support successful and equitable vaccinations targeting adults.

Promotion of Th17 Polarized Immunity via Co-Delivery of Mincle Agonist and Tuberculosis Antigen Using Silica Nanoparticles.

Adjuvants and immunomodulators that effectively drive a Th17-skewed immune response are not part of the standard vaccine toolkit. Vaccine adjuvants and delivery technologies that can induce Th17 or Th1/17 immunity and protection against bacterial pathogens, such as tuberculosis (TB), are urgently needed. Th17-polarized immune response can be induced using agonists that bind and activate C-type lectin receptors (CLRs) such as macrophage inducible C-type lectin (Mincle). A simple but effective strategy was developed for codelivering Mincle agonists with the recombinant Mycobacterium tuberculosis fusion antigen, M72, using tunable silica nanoparticles (SNP). Anionic bare SNP, hydrophobic phenyl-functionalized SNP (P-SNP), and cationic amine-functionalized SNP (A-SNP) of different sizes were coated with three synthetic Mincle agonists, UM-1024, UM-1052, and UM-1098, and evaluated for adjuvant activity in vitro and in vivo. The antigen and adjuvant were coadsorbed onto SNP via electrostatic and hydrophobic interactions, facilitating multivalent display and delivery to antigen presenting cells. The cationic A-SNP showed the highest coloading efficiency for the antigen and adjuvant. In addition, the UM-1098-adsorbed A-SNP formulation demonstrated slow-release kinetics in vitro, excellent stability over 12 months of storage, and strong IL-6 induction from human peripheral blood mononuclear cells. Co-adsorption of UM-1098 and M72 on A-SNP significantly improved antigen-specific humoral and Th17-polarized immune responses in vivo in BALB/c mice relative to the controls. Taken together, A-SNP is a promising platform for codelivery and proper presentation of adjuvants and antigens and provides the basis for their further development as a vaccine delivery platform for immunization against TB or other diseases for which Th17 immunity contributes to protection.

The Immunogenicity and Safety of Mycobacterium tuberculosis-mosR-Based Double Deletion Strain in Mice.

Mycobacterium tuberculosis (M. tuberculosis) remains a significant global health threat, accounting for ~1.7 million deaths annually. The efficacy of the current vaccine, M. bovis BCG, ranges from 0 to 80% in children and does not prevent adulthood tuberculosis. We explored the immune profile and safety of a live-attenuated M. tuberculosis construct with double deletions of the mosR and echA7 genes, where previously, single mutations were protective against an M. tuberculosis aerosol challenge. Over 32 weeks post-vaccination (WPV), immunized mice with M. tuberculosisΔmosRΔechA7 (double mutant) were sacrificed to evaluate the vaccine persistence, histopathology, and immune responses. Interestingly, despite similar tissue colonization between the vaccine double mutant and wild-type M. tuberculosis, the vaccine construct showed a greater reaction to the ESAT-6, TB.10, and Ag85B antigens with peptide stimulation. Additionally, there was a greater number of antigen-specific CD4 T cells in the vaccine group, accompanied by significant polyfunctional T-cell responses not observed in the other groups. Histologically, mild but widely distributed inflammatory responses were recorded in the livers and lungs of the immunized animals at early timepoints, which turned into organized inflammatory foci via 32WPV, a pathology not observed in BCG-immunized mice. A lower double-mutant dose resulted in significantly less tissue colonization and less tissue inflammation. Overall, the double-mutant vaccine elicited robust immune responses dominated by antigen-specific CD4 T cells, but also triggered tissue damage and vaccine persistence. The findings highlight key features associated with the immunogenicity and safety of the examined vaccine construct that can benefit the future evaluation of other live vaccines.

Airway T cells are a correlate of i.v. Bacille Calmette-Guerin-mediated protection against tuberculosis in rhesus macaques.

Bacille Calmette-Guerin (BCG), the only approved Mycobacterium tuberculosis (Mtb) vaccine, provides limited durable protection when administered intradermally. However, recent work revealed that intravenous (i.v.) BCG administration yielded greater protection in macaques. Here, we perform a dose-ranging study of i.v. BCG vaccination in macaques to generate a range of immune responses and define correlates of protection. Seventeen of 34 macaques had no detectable infection after Mtb challenge. Multivariate analysis incorporating longitudinal cellular and humoral immune parameters uncovered an extensive and highly coordinated immune response from the bronchoalveolar lavage (BAL). A minimal signature predicting protection contained four BAL immune features, of which three remained significant after dose correction: frequency of CD4 T cells producing TNF with interferon γ (IFNγ), frequency of those producing TNF with IL-17, and the number of NK cells. Blood immune features were less predictive of protection. We conclude that CD4 T cell immunity and NK cells in the airway correlate with protection following i.v. BCG.

Preclinical Evaluation of TB/FLU-04L-An Intranasal Influenza Vector-Based Boost Vaccine against Tuberculosis.

Tuberculosis is a major global threat to human health. Since the widely used BCG vaccine is poorly effective in adults, there is a demand for the development of a new type of boost tuberculosis vaccine. We designed a novel intranasal tuberculosis vaccine candidate, TB/FLU-04L, which is based on an attenuated influenza A virus vector encoding two mycobacterium antigens, Ag85A and ESAT-6. As tuberculosis is an airborne disease, the ability to induce mucosal immunity is one of the potential advantages of influenza vectors. Sequences of ESAT-6 and Ag85A antigens were inserted into the NS1 open reading frame of the influenza A virus to replace the deleted carboxyl part of the NS1 protein. The vector expressing chimeric NS1 protein appeared to be genetically stable and replication-deficient in mice and non-human primates. Intranasal immunization of C57BL/6 mice or cynomolgus macaques with the TB/FLU-04L vaccine candidate induced Mtb-specific Th1 immune response. Single TB/FLU-04L immunization in mice showed commensurate levels of protection in comparison to BCG and significantly increased the protective effect of BCG when applied in a "prime-boost" scheme. Our findings show that intranasal immunization with the TB/FLU-04L vaccine, which carries two mycobacterium antigens, is safe, and induces a protective immune response against virulent M. tuberculosis.

Safety and Immunogenicity of Recombinant Bacille Calmette-Guérin Strain VPM1002 and Its Derivatives in a Goat Model.

A more effective vaccine against tuberculosis than Bacille Calmette-Guérin (BCG) is urgently needed. BCG derived recombinant VPM1002 has been found to be more efficacious and safer than the parental strain in mice models. Newer candidates, such as VPM1002 Δpdx1 (PDX) and VPM1002 ΔnuoG (NUOG), were generated to further improve the safety profile or efficacy of the vaccine. Herein, we assessed the safety and immunogenicity of VPM1002 and its derivatives, PDX and NUOG, in juvenile goats. Vaccination did not affect the goats' health in regards to clinical/hematological features. However, all three tested vaccine candidates and BCG induced granulomas at the site of injection, with some of the nodules developing ulcerations approximately one month post-vaccination. Viable vaccine strains were cultured from the injection site wounds in a few NUOG- and PDX- vaccinated animals. At necropsy (127 days post-vaccination), BCG, VPM1002, and NUOG, but not PDX, still persisted at the injection granulomas. All strains, apart from NUOG, induced granuloma formation only in the lymph nodes draining the injection site. In one animal, the administered BCG strain was recovered from the mediastinal lymph nodes. Interferon gamma (IFN-γ) release assay showed that VPM1002 and NUOG induced a strong antigen-specific response comparable to that elicited by BCG, while the response to PDX was delayed. Flow cytometry analysis of IFN-γ production by CD4+, CD8+, and γδ T cells showed that CD4+ T cells of VPM1002- and NUOG-vaccinated goats produced more IFN-γ compared to BCG-vaccinated and mock-treated animals. In summary, the subcutaneous application of VPM1002 and NUOG induced anti-tuberculous immunity, while exhibiting a comparable safety profile to BCG in goats.

Recombinant Pichinde viral vector expressing tuberculosis antigens elicits strong T cell responses and protection in mice.

Introduction: Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remains a major global health threat. The only available vaccine Bacille Calmette-Guérin (BCG) does not prevent adult pulmonary TB. New effective TB vaccines should aim to stimulate robust T cell responses in the lung mucosa to achieve high protective efficacy. We have previously developed a novel viral vaccine vector based on recombinant Pichinde virus (PICV), a non-pathogenic arenavirus with low seroprevalence in humans, and have demonstrated its efficacy to induce strong vaccine immunity with undetectable anti-vector neutralization activity. Methods: Using this tri-segmented PICV vector (rP18tri), we have generated viral vectored TB vaccines (TBvac-1, TBvac-2, and TBvac-10) encoding several known TB immunogens (Ag85B, EsxH, and ESAT-6/EsxA). A P2A linker sequence was used to allow for the expression of two proteins from one open-reading-frame (ORF) on the viral RNA segments. The immunogenicity of TBvac-2 and TBvac-10 and the protective efficacy of TBvac-1 and TBvac-2 were evaluated in mice. Results: Both viral vectored vaccines elicited strong antigen-specific CD4 and CD8 T cells through intramuscular (IM) and intranasal (IN) routes as evaluated by MHC-I and MHC-II tetramer analyses, respectively. The IN inoculation route helped to elicit strong lung T cell responses. The vaccine-induced antigen-specific CD4 T cells are functional, expressing multiple cytokines as detected by intracellular cytokine staining. Finally, immunization with TBvac-1 or TBvac-2, both expressing the same trivalent antigens (Ag85B, EsxH, ESAT6/EsxA), reduced Mtb lung tissue burden and dissemination in an aerosol challenge mouse model. Conclusions: The novel PICV vector-based TB vaccine candidates can express more than two antigens via the use of P2A linker sequence and elicit strong systemic and lung T cell immunity with protective efficacy. Our study suggests the PICV vector as an attractive vaccine platform for the development of new and effective TB vaccine candidates.

Modeling of MT. P495, an mRNA-based vaccine against the phosphate-binding protein PstS1 of Mycobacterium tuberculosis.

Tuberculosis (TB) is a contagious disease that predominantly affects the lungs, but can also spread to other organs via the bloodstream. TB affects about one-fourth population of the world. With age, the effectiveness of Bacillus Calmette-Guérin (BCG), the only authorized TB vaccine, decreases. In the quest for a prophylactic and immunotherapeutic vaccine, in this study, a hypothetical mRNA vaccine is delineated, named MT. P495, implementing in silico and immunoinformatics approaches to evaluate key aspects and immunogenic epitopes across the PstS1, a highly conserved periplasmic protein of Mycobacterium tuberculosis (Mtb). PstS1 elicited the potential to generate 99.9% population coverage worldwide. The presence of T- and B-cell epitopes across the PstS1 protein were validated using several computational prediction tools. Molecular docking and dynamics simulation confirmed stable epitope-allele interaction. Immune cell response to the antigen clearance rate was verified by the in silico analysis of immune simulation. Codon optimization confirmed the efficient translation of the mRNA in the host cell. With Toll-like receptors, the vaccine exhibited stable and strong interactions. Findings suggest that the MT. P495 vaccine probably will elicit specific immune responses against Mtb. This mRNA vaccine model is a ready source for further wet-lab validation to confirm the efficacy of this proposed vaccine candidate.

Non-clinical evaluation of local and systemic immunity induced by different vaccination strategies of the candidate tuberculosis vaccine M72/AS01.

A new efficacious tuberculosis vaccine targeting adolescents/adults represents an urgent medical need. The M72/AS01E vaccine candidate protected half of the latently-infected adults against progression to pulmonary tuberculosis in a Phase IIb trial (NCT01755598). We report that three immunizations of mice, two weeks apart, with AS01-adjuvanted M72 induced polyfunctional, Th1-cytokine-expressing M72-specific CD4+/CD8+ T cells in blood and lungs, with the highest frequencies in lungs. Antigen-dose reductions across the three vaccinations skewed pulmonary CD4+ T-cell profiles towards IL-17 expression. In blood, reducing antigen and adjuvant doses of only the third injection (to 1/5th or 1/25th of those of the first injections) did not significantly alter CD4+ T-cell/antibody responses; applying a 10-week delay for the fractional third dose enhanced antibody titers. Delaying a full-dose booster enhanced systemic CD4+ T-cell and antibody responses. Cross-reactivity with PPE and non-PPE proteins was assessed, as Mycobacterium tuberculosis (Mtb) virulence factors and evasion mechanisms are often associated with PE/PPE proteins, to which Mtb39a (contained in M72) belongs. In silico/in vivo analyses revealed that M72/AS01 induced cross-reactive systemic CD4+ T-cell responses to epitopes in a non-vaccine antigen (putative latency-associated Mtb protein PPE24/Rv1753c). These preclinical data describing novel mechanisms of M72/AS01-induced immunity could guide future clinical development of the vaccine.

Pam2CSK4-adjuvanted SARS-CoV-2 RBD nanoparticle vaccine induces robust humoral and cellular immune responses.

As the global COVID-19 pandemic continues and new SARS-CoV-2 variants of concern emerge, vaccines remain an important tool for preventing the pandemic. The inactivated or subunit vaccines themselves generally exhibit low immunogenicity, which needs adjuvants to improve the immune response. We previously developed a receptor binding domain (RBD)-targeted and self-assembled nanoparticle to elicit a potent immune response in both mice and rhesus macaques. Herein, we further improved the RBD production in the eukaryote system by in situ Crispr/Cas9-engineered CHO cells. By comparing the immune effects of various Toll-like receptor-targeted adjuvants to enhance nanoparticle vaccine immunization, we found that Pam2CSK4, a TLR2/6 agonist, could mostly increase the titers of antigen-specific neutralizing antibodies and durability in humoral immunity. Remarkably, together with Pam2CSK4, the RBD-based nanoparticle vaccine induced a significant Th1-biased immune response and enhanced the differentiation of both memory T cells and follicular helper T cells. We further found that Pam2CSK4 upregulated migration genes and many genes involved in the activation and proliferation of leukocytes. Our data indicate that Pam2CSK4 targeting TLR2, which has been shown to be effective in tuberculosis vaccines, is the optimal adjuvant for the SARS-CoV-2 nanoparticle vaccine, paving the way for an immediate clinical trial.

Histopathologic differences in granulomas of Mycobacterium bovis bacille Calmette Guérin (BCG) vaccinated and non-vaccinated cattle with bovine tuberculosis.

Mycobacterium bovis (M. bovis) is the zoonotic bacterium responsible for bovine tuberculosis. An attenuated form of M. bovis, Bacillus Calmette-Guerin (BCG), is a modified live vaccine known to provide variable protection in cattle and other species. Protection for this vaccine is defined as a reduction in disease severity rather than prevention of infection and is determined by evaluation of the characteristic lesion of tuberculosis: the granuloma. Despite its recognized ability to decrease disease severity, the mechanism by which BCG imparts protection remains poorly understood. Understanding the histopathologic differences between granulomas which form in BCG vaccinates compared to non-vaccinates may help identify how BCG imparts protection and lead to an improved vaccine. Utilizing special stains and image analysis software, we examined 88 lymph nodes obtained from BGC-vaccinated and non-vaccinated animals experimentally infected with M. bovis. We evaluated the number of granulomas, their size, severity (grade), density of multinucleated giant cells (MNGC), and the amounts of necrosis, mineralization, and fibrosis. BCG vaccinates had fewer granulomas overall and smaller high-grade granulomas with less necrosis than non-vaccinates. The relative numbers of high- and low- grade lesions were similar as were the amounts of mineralization and the density of MNGC. The amount of fibrosis was higher in low-grade granulomas from vaccinates compared to non-vaccinates. Collectively, these findings suggest that BCG vaccination reduces bacterial establishment, resulting in the formation of fewer granulomas. In granulomas that form, BCG has a protective effect by containing their size, reducing the relative amount of necrosis, and increasing fibrosis in low-grade lesions. Vaccination did not affect the amount of mineralization or density of MNGC.

CRISPR/Cas9 Approach to Generate an Auxotrophic BCG Strain for Unmarked Expression of LTAK63 Adjuvant: A Tuberculosis Vaccine Candidate.

Tuberculosis is one of the deadliest infectious diseases and a huge healthcare burden in many countries. New vaccines, including recombinant BCG-based candidates, are currently under evaluation in clinical trials. Our group previously showed that a recombinant BCG expressing LTAK63 (rBCG-LTAK63), a genetically detoxified subunit A of heat-labile toxin (LT) from Escherichia coli, induces improved protection against Mycobacterium tuberculosis (Mtb) in mouse models. This construct uses a traditional antibiotic resistance marker to enable heterologous expression. In order to avoid the use of these markers, not appropriate for human vaccines, we used CRISPR/Cas9 to generate unmarked mutations in the lysA gene, thus obtaining a lysine auxotrophic BCG strain. A mycobacterial vector carrying lysA and ltak63 gene was used to complement the auxotrophic BCG which co-expressed the LTAK63 antigen (rBCGΔ-LTAK63) at comparable levels to the original construct. The intranasal challenge with Mtb confirmed the superior protection induced by rBCGΔ-LTAK63 compared to wild-type BCG. Furthermore, mice immunized with rBCGΔ-LTAK63 showed improved lung function. In this work we showed the practical application of CRISPR/Cas9 in the tuberculosis vaccine development field.

Considering human challenge trials for tuberculosis vaccine development.

Human challenge trials provide a promising route to cut down on the high, sometimes prohibitive, costs of vaccine development. By requiring fewer participants than a conventional clinical trial and reducing the time required for a trial, human challenge trials improve economic feasibility and reduce the risk of vaccine development. Tuberculosis is one disease where challenge trials could be very impactful, given that it is a widespread and dangerous disease that is nonetheless difficult to track in a standard clinical vaccine trial. Various attenuated strains of Mycobacterium tuberculosis are in development and can be used in a challenge trial in order to reduce the risk to challenge trial participants and their surrounding communities.

Lyophilization Process Engineering and Thermostability of ID93 + GLA-SE, a Single-Vial Adjuvanted Subunit Tuberculosis Vaccine Candidate for Use in Clinical Studies.

Promising clinical efficacy results have generated considerable enthusiasm for the potential impact of adjuvant-containing subunit tuberculosis vaccines. The development of a thermostable tuberculosis vaccine formulation could have significant benefits on both the cost and feasibility of global vaccine distribution. The tuberculosis vaccine candidate ID93 + GLA-SE has reached Phase 2 clinical testing, demonstrating safety and immunogenicity as a two-vial point-of-care mixture. Earlier publications have detailed efforts to develop a lead candidate single-vial lyophilized thermostable ID93 + GLA-SE vaccine formulation. The present report describes the lyophilization process development and scale-up of the lead candidate thermostable ID93 + GLA-SE composition. The manufacture of three full-scale engineering batches was followed by one batch made and released under current Good Manufacturing Practices (cGMP). Up to 4.5 years of stability data were collected. The cGMP lyophilized ID93 + GLA-SE passed all manufacturing release test criteria and maintained stability for at least 3 months when stored at 37°C and up to 24 months when stored at 5°C. This work represents the first advancement of a thermostable adjuvant-containing subunit tuberculosis vaccine to clinical testing readiness.

Enhancement of the Local CD8+ T-Cellular Immune Response to Mycobacterium tuberculosis in BCG-Primed Mice after Intranasal Administration of Influenza Vector Vaccine Carrying TB10.4 and HspX Antigens.

BCG is the only licensed vaccine against Mycobacterium tuberculosis (M.tb) infection. Due to its intramuscular administration route, BCG is unable to induce a local protective immune response in the respiratory system. Moreover, BCG has a diminished ability to induce long-lived memory T-cells which are indispensable for antituberculosis protection. Recently we described the protective efficacy of new mucosal TB vaccine candidate based on recombinant attenuated influenza vector (Flu/THSP) co-expressing TB10.4 and HspX proteins of M.tb within an NS1 influenza protein open reading frame. In the present work, the innate and adaptive immune response to immunization with the Flu/THSP and the immunological properties of vaccine candidate in the BCG-prime → Flu/THSP vector boost vaccination scheme are studied in mice. It was shown that the mucosal administration of Flu/THSP induces the incoming of interstitial macrophages in the lung tissue and stimulates the expression of co-stimulatory CD86 and CD83 molecules on antigen-presenting cells. The T-cellular immune response to Flu/THSP vector was mediated predominantly by the IFNγ-producing CD8+ lymphocytes. BCG-prime → Flu/THSP vector boost immunization scheme was shown to protect mice from severe lung injury caused by M.tb infection due to the enhanced T-cellular immune response, mediated by antigen-specific effector and central memory CD4+ and CD8+ T-lymphocytes.

Expression and purification of Rv2654 recombinant protein from Mycobacterium tuberculosis and its characteristics of immune response. / ç»“æ ¸åˆ†æžæ†èŒæŠ—åŽŸRv2654é‡ç»„è›‹ç™½çš„è¡¨è¾¾å’Œçº¯åŒ–åŠå ¶è¯±å¯¼çš„å ç–«åº”ç­”ç‰¹å¾.

OBJECTIVES: With the decreased protective effect of Bacille Calmette-Guérin (BCG) vaccine, widespread drug-resistant strains of tuberculosis as well as the lack of effective vaccines, global morbidity and mortality of tuberculosis remains high. The purpose of this study is to evaluate the potential of Mycobacterium tuberculosis antigen Rv2654 as a candidate vaccine against tuberculosis, and to verify the characteristics of cellular and humoral immune responses in mice induced by this protein. METHODS: Isopropyl-beta-D-thiogalactoside (IPTG) was added to induce the expression of Rv2654 recombinant protein in the logarithmic growth stage of bacteria. The recombinant protein was purified by affinity chromatography and identified by Western blotting. The immunoreactivity of Rv2654 recombinant protein with human sera was detected by ELISA. After immunization with Rv2654 recombinant protein, the levels of Th1/Th2 cytokines in peripheral blood of mice was measured using cytokine magnetic bead arrays, and the T and B lymphocyte subsets in spleen of mice was analyzed by flow cytometry. RESULTS: The recombinant protein Rv2654 was successfully induced and expressed. The ELISA data showed that the recombinant protein Rv2654 was responsive to the serum of BCG-inoculated patients or active pulmonary tuberculosis patients. In immunized mice with recombinant protein Rv2654, the expression of IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10 and other cytokines in peripheral blood was elevated (all P<0.05). Flow cytometry analysis showed that the recombinant protein significantly stimulated the differentiation of CD4+T cells and CD8+T cells into effector T cells, and this effect was more obvious when combined with BCG. The recombinant protein Rv2654 also stimulated the activation and proliferation of B cells and their differentiation into memory cells. However, less plasma cells were produced. CONCLUSIONS: Rv2654 protein could induce the cell immune responses and it has good binding ability with serum of BCG-inoculated patients and active pulmonary tuberculosis patients, suggesting that it may serve as a good candidate antigen for tuberculosis immunological prophylaxis and diagnostic vaccines.

Engineering a new vaccine platform for heterologous antigen delivery in live-attenuated Mycobacterium tuberculosis.

Live vaccines are attractive vehicles for antigen delivery as a strategy to immunize against heterologous pathogens. The live vaccine MTBVAC is based on rational attenuation of Mycobacterium tuberculosis with the objective of improving BCG protection against pulmonary tuberculosis. However, the development of recombinant mycobacteria as antigen-presenting microorganisms has been hindered due to their fastidious genetic manipulation. In this study, we used MTBVAC as a genetic platform to deliver diphtheria, tetanus, or pertussis toxoids, which are the immunogenic constituents of the DTP vaccine. When using nonoptimal genetic conditions, the expression of these immunogens was barely detectable. Accordingly, we pursued a rational, step-by-step optimization of the genetic components to achieve the expression and secretion of these toxoids. We explored variants of the L5 mycobacteriophage promoter to ensure balanced antigen expression and plasmid stability. Optimal signal sequences were identified by comparative proteomics of MTBVAC and its parental strain. It was determined that proteins secreted by the Twin Arginine Translocation pathway displayed higher secretion in MTBVAC, and the Ag85A secretion sequence was selected as the best candidate. Because the coding regions of diphtheria, tetanus, and pertussis toxoids significantly differ in G + C content relative to mycobacterial genes, their codon usage was optimized. We also placed a 3xFLAG epitope in frame with the C-terminus of these toxoids to facilitate protein detection. Altogether, these optimizations resulted in the secretion of DTP antigens by MTBVAC, as demonstrated by western blot and MRM-MS. Finally, we examined specific antibody responses in mice vaccinated with recombinant MTBVAC expressing DTP antigens.

Evaluation of the stability of a spray-dried tuberculosis vaccine candidate designed for dry powder respiratory delivery.

Particle engineering via spray drying was used to develop a dry powder presentation of an adjuvanted tuberculosis vaccine candidate. This presentation utilizing a trileucine-trehalose excipient system was designed to be both thermostable and suitable for respiratory delivery. The stability of the spray-dried vaccine powder was assessed over one year at various storage temperatures (-20, 5, 25, 40, 50 °C) in terms of powder stability, adjuvant stability, and antigen stability. A formulation without trileucine was included as a control. The results showed that the interior particle structure and exterior particle morphology of the powder was maintained for one year at 40 °C, while the control case exhibited a small extent of particle fusing under the same storage conditions. Moisture content was maintained, and powder solid state remained amorphous for all storage temperatures. Aerosol performance was assessed with a commercial dry powder inhaler in combination with a human mouth-throat model. The emitted dose and lung dose were maintained for all samples after one year at temperatures up to 40 °C. Nanoemulsion size and oil content of the adjuvant system were maintained after one year at temperatures up to 40 °C, and the agonist content was maintained after one year at temperatures up to 25 °C. The antigen was completely degraded in the control formulation at seven months of storage at 40 °C; by contrast, 45% of the antigen was still present in the trehalose-trileucine formulation after one year of storage at 50 °C. Comparatively, the antigen was completely degraded in a liquid sample of the vaccine candidate after only one month of storage at 37 °C. The spray-dried trehalose-trileucine vaccine powder clearly maintained its inhalable properties after one year's storage at high temperatures and improved overall thermostability of the vaccine.