10 research outputs found
The TLR4 Agonist Vaccine Adjuvant, GLA-SE, Requires Canonical and Atypical Mechanisms of Action for TH1 Induction.
The Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant formulated in a stable emulsion (GLA-SE) promotes strong TH1 and balanced IgG1/IgG2 responses to protein vaccine antigens. This enhanced immunity is sufficient to provide protection against many diseases including tuberculosis and leishmaniasis. To better characterize the adjuvant action it is important to understand how the different cytokines and transcription factors contribute to the initiation of immunity. In the present study using T-bet-/- and IL-12-/- mice and a blocking anti-IFNαR1 monoclonal antibody, we define mechanisms of adjuvant activity of GLA-SE. In accordance with previous studies of TLR4 agonist based adjuvants, we found that TH1 induction via GLA-SE was completely dependent upon T-bet, a key transcription factor for IFNγ production and TH1 differentiation. Consistent with this, deficiency of IL-12, a cytokine canonical to TH1 induction, ablated TH1 induction via GLA-SE. Finally we demonstrate that the innate immune response to GLA-SE, including rapid IFNγ production by memory CD8+ T cells and NK cells, was contingent on type I interferon, a cytokine group whose association with TH1 induction is contextual, and that they contributed to the adjuvant activity of GLA-SE
Development of a thermostable nanoemulsion adjuvanted vaccine against tuberculosis using a design-of-experiments approach
Ryan M Kramer, Michelle C Archer, Mark T Orr, Natasha Dubois Cauwelaert, Elyse A Beebe, Po-wei D Huang, Quinton M Dowling, Alicia M Schwartz, Dawn M Fedor, Thomas S Vedvick, Christopher B Fox Infectious Disease Research Institute, Seattle, WA, USA Background: Adjuvants have the potential to increase the efficacy of protein-based vaccines but need to be maintained within specific temperature and storage conditions. Lyophilization can be used to increase the thermostability of protein pharmaceuticals; however, no marketed vaccine that contains an adjuvant is currently lyophilized, and lyophilization of oil-in-water nanoemulsion adjuvants presents a specific challenge. We have previously demonstrated the feasibility of lyophilizing a candidate adjuvanted protein vaccine against Mycobacterium tuberculosis (Mtb), ID93 + GLA-SE, and the subsequent improvement of thermostability; however, further development is required to prevent physicochemical changes and degradation of the TLR4 agonist glucopyranosyl lipid adjuvant formulated in an oil-in-water nanoemulsion (SE). Materials and methods: In this study, we took a systematic approach to the development of a thermostable product by first identifying compatible solution conditions and stabilizing excipients for both antigen and adjuvant. Next, we applied a design-of-experiments approach to identify stable lyophilized drug product formulations. Results: We identified specific formulations that contain disaccharide or a combination of disaccharide and mannitol that can achieve substantially improved thermostability and maintain immunogenicity in a mouse model when tested in accelerated and real-time stability studies. Conclusion: These efforts will aid in the development of a platform formulation for use with other similar vaccines. Keywords: adjuvant, lyophilization, tuberculosis, formulation development, design of experiments, controlled temperature chain, GRA
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
IFNα is produced early after GLA-SE immunization.
<p>B6 mice were immunized with saline or ID93+GLA-SE. Sera and draining inguinal LNs were collected0, 6, 24, or 48 hours later and analyzed for IFNα protein expression by ELISA. Data are shown as mean ± SEM and are the combined results of two independent experiments with similar results with 3 or 4 mice/group. Statistics by unpaired t test; *p≤0.05 compared to Saline group.</p
IFNαR1 signaling contributes to T<sub>H</sub>1 skewing.
<p>B6 mice were treated with IFNαR1 antibody or its isotype and immunized with ID93+GLA-SE and responses were analyzed one week after prime. (A) ID93 stimulated splenocytes were analyzed for the production of CD154, IFNγ, TNF and IL-5 by CD4 T cells. (B) One week after prime CD4 T cells were isolated and stained with an I-A<sup>b</sup> tetramer presenting the dominant epitope for Rv3619 and analyzed for T-bet induction. (C) Sera were collected one week after prime and serially diluted to assess levels of anti-ID93 IgG1 and IgG2c. Data are shown as mean ± SEM of N = 5 animal/group and are from one experiment representative of two experiments performed except for IL-5 and T-bet levels measurement which were only done once. Statistics by simple or multiple t test corrected for multiple comparisons using the Holm-Sidak method between IgG or cytokine groups; *p≤0.05</p
IFNαR1 signaling is essential for lymphocyte activation and IFNγ production upon immunization with ID93+GLA-SE.
<p>B6 mice were treated with IFNαR1 antibody or its isotype and immunized with ID93+GLA-SE. Innate responses were assessed in the ipsilateral draining LN or in the contralateral LN by flow cytometry on LN cells or ELISA on LN supernatant The results are shown for the ipsilateral LN when not stated otherwise. (A) MFI and representative histograms of CD69 staining (B) IFNγ production assessed by ELISA, (C) IFNγR occupancy as indicated by decreased MFI of IFNγR staining with the monoclonal antibody GR-20 at 12 hrs after immunization. (D) IFNγ staining on CD8+ T cells. (E) IFNγ staining on NK cells. (F) IL-12 production assessed by ELISA. Data are shown as mean ± SEM of N = 4 animal/group and are from one experiment. Statistics by one-way ANOVA, *p≤0.05 compare to anti-IFNαR1 treated animals, #p≤0.05 compare to all the other groups.</p