Interferon γ and Tumor Necrosis Factor Are Not Essential Parameters of CD4+ T-Cell Responses for Vaccine Control of Tuberculosis.

BACKGROUND: Mycobacterium tuberculosis infects one third of the world's population and causes >8 million cases of tuberculosis annually. New vaccines are necessary to control the spread of tuberculosis. T cells, interferon γ (IFN-γ), and tumor necrosis factor (TNF) are necessary to control M. tuberculosis infection in both humans and unvaccinated experimental animal models. However, the immune responses necessary for vaccine efficacy against M. tuberculosis have not been defined. The multifunctional activity of T-helper type 1 (TH1) cells that simultaneously produce IFN-γ and TNF has been proposed as a candidate mechanism of vaccine efficacy. METHODS: We used a mouse model of T-cell transfer and aerosolized M. tuberculosis infection to assess the contributions of TNF, IFN-γ, and inducible nitric oxide synthase (iNOS) to vaccine efficacy. RESULTS: CD4(+) T cells were necessary and sufficient to transfer protection against aerosolized M. tuberculosis, but neither CD4(+) T cell-produced TNF nor host cell responsiveness to IFN-γ were necessary. Transfer of Tnf(-/-) CD4(+) T cells from vaccinated donors to Ifngr(-/-) recipients was also sufficient to confer protection. Activation of iNOS to produce reactive nitrogen species was not necessary for vaccine efficacy. CONCLUSIONS: Induction of TH1 cells that coexpress IFN-γ and TNF is not a requirement for vaccine efficacy against M. tuberculosis, despite these cytokines being essential for control of M. tuberculosis in nonvaccinated animals.

Stressed stability and protective efficacy of lead lyophilized formulations of ID93+GLA-SE tuberculosis vaccine.

With the recent exception of coronavirus disease 2019 (COVID-19), tuberculosis (TB) causes more deaths globally than any other infectious disease, and approximately 1/3 of the world's population is infected with Mycobacterium tuberculosis (Mtb). However, encouraging progress in TB vaccine development has been reported, with approximately 50% efficacy achieved in Phase 2b clinical testing of an adjuvanted subunit TB vaccine candidate. Nevertheless, current lead vaccine candidates require cold-chain transportation and storage. In addition to temperature stress, vaccines may be subject to several other stresses during storage and transport, including mechanical, photochemical, and oxidative stresses. Optimal formulations should enable vaccine configurations with enhanced stability and decreased sensitivity to physical and chemical stresses, thus reducing reliance on the cold chain and facilitating easier worldwide distribution. In this report, we describe the physicochemical stability performance of three lead thermostable formulations of the ID93 + GLA-SE TB vaccine candidate under various stress conditions. Moreover, we evaluate the impact of thermal stress on the protective efficacy of the vaccine formulations. We find that formulation composition impacts stressed stability performance, and our comprehensive evaluation enables selection of a lead single-vial lyophilized candidate containing the excipient trehalose and Tris buffer for advanced development.

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.

Development and Testing of a Spray-Dried Tuberculosis Vaccine Candidate in a Mouse Model.

Converting a vaccine into a thermostable dry powder is advantageous as it reduces the resource burden linked with the cold chain and provides flexibility in dosage and administration through different routes. Such a dry powder presentation may be especially useful in the development of a vaccine towards the respiratory infectious disease tuberculosis (TB). This study assesses the immunogenicity and protective efficacy of spray-dried ID93+GLA-SE, a promising TB vaccine candidate, against Mycobacterium tuberculosis (Mtb) in a murine model when administered via different routes. Four administration routes for the spray-dried ID93+GLA-SE were evaluated along with relevant controls-1) reconstitution and intramuscular injection, 2) reconstitution and intranasal delivery, 3) nasal dry powder delivery via inhalation, and 4) pulmonary dry powder delivery via inhalation. Dry powder intranasal and pulmonary delivery was achieved using a custom nose-only inhalation device, and optimization using representative vaccine-free powder demonstrated that approximately 10 and 44% of the maximum possible delivered dose would be delivered for intranasal delivery and pulmonary delivery, respectively. Spray-dried powder was engineered according to the different administration routes including maintaining approximately equivalent delivered doses of ID93 and GLA. Vaccine properties of the different spray-dried lots were assessed for quality control in terms of nanoemulsion droplet diameter, polydispersity index, adjuvant content, and antigen content. Our results using the Mtb mouse challenge model show that both intranasal reconstituted vaccine delivery as well as pulmonary dry powder vaccine delivery resulted in Mtb control in infected mice comparable to traditional intramuscular delivery. Improved protection in these two vaccinated groups over their respective control groups coincided with the presence of cytokine-producing T cell responses. In summary, our results provide novel vaccine formulations and delivery routes that can be harnessed to provide protection against Mtb infection.

Enhanced Anti-Mycobacterium tuberculosis Immunity over Time with Combined Drug and Immunotherapy Treatment.

It is estimated that one third of the world's population is infected with Mycobacterium tuberculosis (Mtb). This astounding statistic, in combination with costly and lengthy treatment regimens make the development of therapeutic vaccines paramount for controlling the global burden of tuberculosis. Unlike prophylactic vaccination, therapeutic immunization relies on the natural pulmonary infection with Mtb as the mucosal prime that directs boost responses back to the lung. The purpose of this work was to determine the protection and safety profile over time following therapeutic administration of our lead Mtb vaccine candidate, ID93 with a synthetic TLR4 agonist (glucopyranosyl lipid adjuvant in a stable emulsion (GLA-SE)), in combination with rifampicin, isoniazid, and pyrazinamide (RHZ) drug treatment. We assessed the host inflammatory immune responses and lung pathology 7⁻22 weeks post infection, and determined the therapeutic efficacy of combined treatment by enumeration of the bacterial load and survival in the SWR/J mouse model. We show that drug treatment alone, or with immunotherapy, tempered the inflammatory responses measured in brochoalveolar lavage fluid and plasma compared to untreated cohorts. RHZ combined with therapeutic immunizations significantly enhanced TH1-type cytokine responses in the lung over time, corresponding to decreased pulmonary pathology evidenced by a significant decrease in the percentage of lung lesions and destructive lung inflammation. These data suggest that bacterial burden assessment alone may miss important correlates of lung architecture that directly contribute to therapeutic vaccine efficacy in the preclinical mouse model. We also confirmed our previous finding that in combination with antibiotics therapeutic immunizations provide an additive survival advantage. Moreover, therapeutic immunizations with ID93/GLA-SE induced differential T cell immune responses over the course of infection that correlated with periods of enhanced bacterial control over that of drug treatment alone. Here we advance the immunotherapy model and investigate reliable correlates of protection and Mtb control.

Pulmonary immunity and durable protection induced by the ID93/GLA-SE vaccine candidate against the hyper-virulent Korean Beijing Mycobacterium tuberculosis strain K.

The majority of tuberculosis (TB) vaccine candidates advanced to clinical trials have been evaluated preclinically using laboratory-adapted strains. However, it has been proposed that challenge with clinical isolates in preclinical vaccine testing could provide further and more practical validation. Here, we tested the ID93/GLA-SE TB vaccine candidate against the clinical Mycobacterium tuberculosis (Mtb) strain K (Mtb K) belonging to the Beijing family, the most prevalent Mtb strain in South Korea. Mice immunized with ID93/GLA-SE exhibited a significant reduction in bacteria and reduced lung inflammation against Mtb K when compared to non-immunized controls. In addition, we analyzed the immune responses in the lungs of ID93/GLA-SE-immunized mice, and showed that ID93/GLA-SE was able to elicit sustained Th1-biased immune responses including antigen-specific multifunctional CD4(+) T cell co-producing IFN-γ, TNF-α, and IL-2 as well as a high magnitude of IFN-γ response for up to 10 weeks post-challenge. Notably, further investigation of T cell subsets in the lung following challenge showed remarkable generation of CD8(+) central memory T cells by ID93/GLA-SE-immunization. Our findings showed that ID93/GLA-SE vaccine confers a high level of robust protection against the hypervirulent Mtb Beijing infection which was characterized by pulmonary Th1-polarized T-cell immune responses. These findings may also provide relevant information for potential utility of this vaccine candidate in East-Asian countries where the Beijing genotype is highly prevalent.

Mucosal delivery switches the response to an adjuvanted tuberculosis vaccine from systemic TH1 to tissue-resident TH17 responses without impacting the protective efficacy.

Pulmonary tuberculosis (TB) remains one of the leading causes of infectious disease death despite widespread usage of the BCG vaccine. A number of new TB vaccines have moved into clinical evaluation to replace or boost the BCG vaccine including ID93+GLA-SE, an adjuvanted subunit vaccine. The vast majority of new TB vaccines in trials are delivered parenterally even though intranasal delivery can augment lung-resident immunity and protective efficacy in small animal models. Parenteral immunization with the adjuvanted subunit vaccine ID93+GLA-SE elicits robust TH1 immunity and protection against aerosolized Mycobacterium tuberculosis in mice and guinea pigs. Here we describe the immunogenicity and efficacy of this vaccine when delivered intranasally. Intranasal delivery switches the CD4 T cell response from a TH1 to a TH17 dominated tissue-resident response with increased frequencies of ID93-specific cells in both the lung tissue and at the lung surface. Surprisingly these changes do not affect the protective efficacy of ID93+GLA-SE. Unlike intramuscular immunization, ID93+GLA does not require the squalene-based oil-in-water emulsion SE to elicit protective CD4 T cells when delivered intranasally. Finally we demonstrate that TNF and the IL-17 receptor are dispensable for the efficacy of the intranasal vaccine suggesting an alternative mechanism of protection.