Lyophilized and stabilized formulated live attenuated vaccine against tularemia

Abstract

A lyophilized, stabilized preparation containing a live attenuated vaccine strain against tularemia.

Description

[Technical Field]

[0001] government support

[0001] This invention was made with United States government support under Agreement No. W15QKN-16-0-1002 awarded by ACC-NJ to MCDC. The government has certain rights in this invention.

[0002] Field The present invention relates to a freeze-dried, stabilized formulation containing a live attenuated vaccine strain against tularemia. [Background technology]

[0003] background Tularemia is caused by the Gram-negative intracellular pathogen Francisella tularensis. The bacterium was first identified in 1912. Francisella tularensis is currently listed as a Category A designated biological agent. Since the pathogen's discovery, at least four subspecies have been identified, each exhibiting unique virulence and biochemical profiles. Human disease is primarily associated with two F. tularensis subspecies: the highly virulent F. tularensis subsp. tularensis (Ftt), found in North America, and the moderately virulent F. tularensis subsp. holarctica, endemic throughout the Northern Hemisphere. See, for example, "Francisella tularensis in the United States," Farlow et al., Emerging Infectious, Vol. 11, No. 12 (Dec. 2005).

[0004]

[0004] Currently, the only vaccine used to treat tularemia, caused by Francisella tularensis, is the live vaccine strain (LVS), an attenuated type B (F. tularensis subsp. holarctica) strain. Although it does not appear to cause disease in humans, its genetic stability and safety are unknown, making it a poor candidate for FDA approval. F. tularensis itself is classified as a Category A scheduled biological agent, posing a threat to civilians as well as military personnel.

[0005] Another relatively recent attempt was to develop Fn-igID OAg as a candidate tularemia vaccine. FTT The use of genetically engineered forms of F. novicida (Fn) in the form of FTT See expression in F. novicida, X. Zogay, A. Adelani, J. Nguyen, P. Chu, K. Klose, 9th International Conference on Tuleremia, October 2018. Summary of the Invention [Problem to be solved by the invention]

[0006]

[0006] There remains a need to produce an FDA-approved formulated vaccine against F. tularensis that allows for intradermal delivery and provides relatively high levels of protective immunity while being shelf stable. [Means for solving the problem]

[0007] BRIEF DESCRIPTION OF THE DRAWINGS

[0007] A lyophilized, stabilized formulation containing a live attenuated vaccine strain against tularemia is further described herein with reference to the accompanying drawings identified below. [Brief explanation of the drawings]

[0008] [Figure 1]

[0008] Survival rates in F344 rats are illustrated following intradermal administration of a 10% TMC-stabilized lyophilized vaccine and booster challenge with F. tularensis Schu S4 mutant by the intratracheal route. [Figure 2A]

[0009] 10A-10C show plots of polymorphonuclear leukocyte population levels in the lungs of 10% TMC lyophilized KKF768 and sham-vaccinated rats at days 3, 5, and 7 following intratracheal challenge with a potentially lethal dose of Ft SCHU S4. [Figure 2B]

[0010] Levels of natural killer cell populations in the lungs of 10% TMC lyophilized KKF768 and sham-vaccinated rats at days 3, 5, and 7 following intratracheal challenge with a potentially lethal dose of Ft SCHU S4 are plotted. [Figure 2C]

[0011] Levels of CD43hi monocytes in the lungs of 10% TMC lyophilized KKF768 and sham vaccinated rats at days 3, 5, and 7 following intratracheal challenge with a potentially lethal dose of Ft SCHU S4 are plotted. [Figure 2D]

[0012] Levels of His48hi monocytes in the lungs of 10% TMC lyophilized KKF768 and sham vaccinated rats at days 3, 5, and 7 following intratracheal challenge with a potentially lethal dose of Ft SCHU S4 are plotted. [Figure 3A]

[0013] CD4+ Th cell levels are plotted on days 3, 5, and 7 in the lungs of 10% TMC lyophilized KK768 and sham-vaccinated rats following intratracheal challenge with a potentially lethal dose of Ft SCHU S4. [Figure 3B]

[0014] CD8+ Tc cell levels in the lungs of 10% TMC lyophilized KK768 and sham vaccinated rats at days 3, 5, and 7 following intratracheal challenge with a potentially lethal dose of Ft SCHU S4 are plotted. [Figure 3C]

[0015] B cell levels in the lungs of 10% TMC lyophilized KK768 and sham vaccinated rats at days 3, 5, and 7 following intratracheal challenge with a potentially lethal dose of Ft SCHU S4 are plotted. [Figure 4A]

[0016] Levels of Th1 cells in the lungs of 10% TMC lyophilized KK768 and sham vaccinated rats at days 3, 5, and 7 following intratracheal challenge with a potentially lethal dose of Ft SCHU S4 are plotted. [Figure 4B]

[0017] Levels of Th2 cells in the lungs of 10% TMC lyophilized KK768 and sham vaccinated rats at days 3, 5, and 7 following intratracheal challenge with a potentially lethal dose of Ft SCHU S4 are plotted. [Figure 4C]

[0018] Levels of T171 cells in the lungs of 10% TMC lyophilized KK768 and sham-vaccinated rats at days 3, 5, and 7 following intratracheal challenge with a potentially lethal dose of Ft SCHU S4 are plotted. [Figure 4D]

[0019] The levels of Tc1 cells in the lungs of 10% TMC lyophilized KK768 and sham vaccinated rats at days 3, 5, and 7 following intratracheal challenge with a potentially lethal dose of Ft SCHU S4 are plotted. [Figure 4E]

[0020] The levels of Tc2 cells in the lungs of 10% TMC lyophilized KK768 and sham vaccinated rats at days 3, 5, and 7 following intratracheal challenge with a potentially lethal dose of Ft SCHU S4 are plotted. [Figure 4F]

[0021] The levels of Tc17 cells in the lungs of 10% TMC lyophilized KK768 and sham vaccinated rats at days 3, 5, and 7 following intratracheal challenge with a potentially lethal dose of Ft SCHU S4 are plotted. DETAILED DESCRIPTION OF THE INVENTION

[0009] Detailed Description

[0022] The present invention begins with the consideration of F. novicida (Fn), a species closely related to Francisella tularensis subsp. tularensis (Ftt), which is avirulent in humans. Specifically, Fn (Fn-iglD), which harbors a mutation in iglD, a T6SS gene required for escape from the phagosome and replication within macrophages, inhibits FTT-derived lipopolysaccharide (LPS) O antigen (Ftt(OAg)). FTT ), which is preferably achieved by removing the entire 12-gene OAg cluster from Fn-iglD, followed by OAg FTT This was achieved by stepwise replacement of the 15-gene OAg cluster from the Ft subsp. holarctica LVS strain, which expresses Fn-iglD. The resulting strain, KKF768 (Fn-iglD OAg FTT ) is the OAg bound to that LPS. FTT is expressed.

[0010]

[0023] For lyophilization of KKF768, a preferred stabilizing formulation was used containing a stabilizer mixture of 10.0% trehalose (w / v), 5.0% mannitol (w / v), and 0.2% cysteine ​​(w / v) as cryoprotectants, which mixture is identified below as "10% TMC." 10 An aqueous solution of this stabilized mixture, preferably containing a suspension of 1000 cfu / mL, was prepared, followed by lyophilization.

[0011]

[0024] The preferred procedure for freeze-drying is as follows: FTT was suspended in 10% TM solution, divided into aliquots (e.g., 0.5 ml) and placed into 5.0 ml (20 mm x 40 mm) vials with fluoropolymer stoppers (e.g., FluroTec stoppers), then transferred to a Labconco Freezone 12 L equipped with a stopper ring tray dryer attachment. Table 1 below shows the preferred ranges of lyophilization parameters used to provide the 10% TMC lyophilized formulation.

[0012] [Table 1]

[0013]

[0025] From the above, it can be appreciated that freezing is preferably carried out at a temperature range of -45.0 to -35.0°C for a period ranging from 2.5 to 3.5 hours, the first drying is carried out at a temperature of -35.0 to -25.0°C at 0.45 to 0.55 mbar for a period ranging from 40 to 55 hours, and the second drying is carried out at 0 to 10°C, then the chamber is purged with nitrogen and the samples are stoppered in vials and can be removed.

[0014]

[0026] The viability of lyophilized KK768 is first summarized in Table 2 below.

[0015] [Table 2]

[0016]

[0027] FIG. 1 illustrates the survival rate of F344 rats after intradermal administration of 10% TMC-stabilized, lyophilized vaccine and boosting by exposure to F. tularensis Schu S4 mutant strain via the intratracheal route.

[0017]

[0028] Next, correlates of immunity were evaluated for 10% TMC lyophilized KKF768, and as illustrated by the data below, immune cells were upregulated with vaccination, resulting in protective immunity after vaccination and boosting.

[0018]

[0029] Figure 2A plots the levels of polymorphonuclear leukocyte populations in the lungs of 10% TMC lyophilized KKF768- and sham-vaccinated rats on days 3, 5, and 7 following intratracheal exposure to a potentially lethal dose of Ft SCHU S4. Figure 2B plots the levels of natural killer cell populations in the lungs of 10% TMC lyophilized KKF768- and sham-vaccinated rats on days 3, 5, and 7 following intratracheal exposure to a potentially lethal dose of Ft SCHU S4. Figure 2C plots the levels of CD43hi monocytes in the lungs of 10% TMC lyophilized KKF768- and sham-vaccinated rats on days 3, 5, and 7 following intratracheal exposure to a potentially lethal dose of Ft SCHU S4. FIG. 2D plots the levels of His48hi monocytes in the lungs of 10% TMC lyophilized KKF768 and sham-vaccinated rats on days 3, 5, and 7 following intratracheal challenge with a potentially lethal dose of Ft SCHU S4.

[0019]

[0030] Figure 3A plots the levels of CD4+ Th cells in the lungs of 10% TMC lyophilized KK768 and sham-vaccinated rats on days 3, 5, and 7 following intratracheal exposure to a potentially lethal dose of Ft SCHU S4. Figure 3B plots the levels of CD8+ Tc cells in the lungs of 10% TMC lyophilized KK768 and sham-vaccinated rats on days 3, 5, and 7 following intratracheal exposure to a potentially lethal dose of Ft SCHU S4. Figure 3C plots the levels of B cells in the lungs of 10% TMC lyophilized KK768 and sham-vaccinated rats on days 3, 5, and 7 following intratracheal exposure to a potentially lethal dose of Ft SCHU S4.

[0020]

[0031] Figure 4A plots the levels of Th1 cells in the lungs of 10% TMC lyophilized KK768- and sham-vaccinated rats on days 3, 5, and 7 following intratracheal exposure to a potentially lethal dose of Ft SCHU S4. Figure 4B plots the levels of Th2 cells in the lungs of 10% TMC lyophilized KK768- and sham-vaccinated rats on days 3, 5, and 7 following intratracheal exposure to a potentially lethal dose of Ft SCHU S4. Figure 4C plots the levels of T171 cells in the lungs of 10% TMC lyophilized KK768- and sham-vaccinated rats on days 3, 5, and 7 following intratracheal exposure to a potentially lethal dose of Ft SCHU S4. Figure 4D plots the levels of Tc1 cells in the lungs of 10% TMC lyophilized KK768- and sham-vaccinated rats on days 3, 5, and 7 following intratracheal exposure to a potentially lethal dose of Ft SCHU S4. Figure 4E plots the levels of Tc2 cells in the lungs of 10% TMC lyophilized KK768 and sham-vaccinated rats on days 3, 5, and 7 following intratracheal exposure to a potentially lethal dose of Ft SCHU S4. Figure 4F plots the levels of Tc17 cells in the lungs of 10% TMC lyophilized KK768 and sham-vaccinated rats on days 3, 5, and 7 following intratracheal exposure to a potentially lethal dose of Ft SCHU S4.

[0021]

[0032] As can be appreciated from the above, the present disclosure provides an immunogenic agent for use in the prevention or treatment of infection with Francisella tularensis in an animal, the immunogenic agent comprising Fn-iglD OAg FTT The present invention relates to an immunogenic agent comprising a lyophilized and stabilized formulation of Fn-iglD OAg. The animal may be a human. FTT In process form, the present disclosure provides a method for producing an immunogenic agent for use in the prevention or treatment of infection with Francisella tularensis in an animal, comprising administering to an animal a lyophilized, stabilized formulation of Fn-iglD OAg. FTTThe present disclosure provides a method for treating an animal in need of treatment for prevention of or for infection with Francisella tularensis, comprising administering to an animal an immunogenic agent comprising Fn-iglD OAg FTT The present invention also relates to methods of administering an immunogenic agent comprising the lyophilized, stabilized formulation of the present invention.

Claims

1. An immunogenic agent for use in the prevention or treatment of infection with Francisella tularensis in an animal, comprising Fn-iglD OAg ＦＴＴ 1. An immunogenic agent comprising a lyophilized and stabilized formulation of

2. The immunogenic agent of claim 1, wherein the stabilized formulation contains 10.0% trehalose (w / v), 5.0% mannitol (w / v) and 0.2% cysteine ​​(w / v) as cryoprotectants.

3. The immunogenic agent of claim 1 , wherein the animal is a human.

4. 1. A pharmaceutical composition comprising an immunogenic agent for use in the prevention or treatment of infection with Francisella tularensis in an animal, wherein the immunogenic agent is Fn-iglD OAg ＦＴＴ 1. A pharmaceutical composition comprising a lyophilized, stabilized formulation of

5. 1. A method for producing an immunogenic agent for use in the prevention or treatment of infection with Francisella tularensis in an animal, comprising: Fn-iglD OAg ＦＴＴ providing said immunogenic agent comprising: subjecting the immunogenic agent to lyophilization and recovering a lyophilized and stabilized form of the immunogenic agent; A method comprising:

6. 6. The method of claim 5, wherein the freeze-drying comprises freezing the immunogenic agent at a temperature of −45.0 to −35.0° C. for a period ranging from 2.5 to 3.5 hours, followed by drying at a temperature of −35.0 to −25.0° C. at 0.45 to 0.55 mbar for a period ranging from 40 to 55 hours.

7. 1. A method of treating an animal in need thereof for the prevention of or for infection with Francisella tularensis, comprising administering to said animal a therapeutically effective amount of Fn-iglD OAg ＦＴＴ administering an immunogenic agent comprising a lyophilized, stabilized formulation of

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