Compositions and methods for treating viral infections

Abstract

The present disclosure provides compositions and methods useful for treating viral infections. As described herein, the compositions and methods are based on the development of immunogenic compositions that include an inactivated virus in combination with a non-ionic surfactant vesicle (NISV). In certain embodiments at least a portion of the antigen present in the composition is physically associated with the NISV. In certain embodiments the compositions are lyophilized and subsequently rehydrated after a period of storage. In certain embodiments the rehydrated compositions exhibit greater potency as compared to otherwise equivalent compositions that lack the NISV. In certain embodiments the lyophilized compositions are stored at temperatures in excess of 8° C. prior to rehydration. In certain embodiments, the rehydrated compositions exhibit greater potency as compared to otherwise equivalent compositions that lack the NISV and that were also stored at temperatures in excess of 8° C. prior to rehydration. In certain embodiments the antigen is taken from a licensed vaccine and the administered dose of antigen is less than the standard human dose for the licensed vaccine.

Claims

1. A method of preparing a thermostable lyophilized composition comprising (i) an inactivated viral antigen, said antigen comprising an inactivated polio virus, an inactivated rabies virus, an inactivated hepatitis A virus, or a combination thereof and (ii) lipid vesicles, wherein the lipid vesicles comprise a non-ionic surfactant comprising 1-monopalmitoyl glycerol, the method comprising: melting lipids comprising the non-ionic surfactant to produce molten lipids; combining the molten lipids with an aqueous solution comprising the inactivated viral antigen; homogenizing the resulting product, wherein the molten lipids and aqueous solution are combined in relative amounts and volumes that achieve a lipid concentration of about 6.25 mg/ml to about 25 mg/ml in the resulting product; and lyophilizing the homogenized mixture to produce the lyophilized composition, wherein the lyophilized composition is thermostable when stored for a period of up to nine months at a temperature of 8° C. to 40° C.

2. The method of claim 1, wherein the molten lipids are added to the aqueous solution.

3. The method of claim 1, wherein the aqueous solution is added to the molten lipids.

4. The method of claim 1, wherein the lyophilized composition is thermostable when stored for a period of up to nine months at a temperature of 8° C. to 25° C.

5. The method of claim 1, wherein the thermostable lyophilized composition, when stored for a period of up to nine months at a temperature of 8° C. to 40° C., exhibits a lower level of antigen loss relative to a corresponding composition comprising the antigen and lacking lipid vesicles.

6. The method of claim 4, wherein the thermostable lyophilized composition, when stored for a period of up to nine months at a temperature of 8° C. to 25° C., exhibits a lower level of antigen loss relative to a corresponding composition comprising the antigen and lacking lipid vesicles.

7. A method of treating an individual suffering from, or at risk for, infection from a polio virus, a rabies virus, a hepatitis A virus or a combination thereof, the method comprising: a) melting lipids comprising a non-ionic surfactant comprising 1-monopalmitoyl glycerol to produce molten lipids; combining the molten lipids with an aqueous solution comprising an inactivated polio virus, an inactivated rabies virus, an inactivated hepatitis A virus, or a combination thereof; homogenizing the resulting product to produce a homogenized mixture, wherein the molten lipids and aqueous solution are combined in relative amounts and volumes that achieve a lipid concentration of about 6.25 mg/ml to about 25 mg/ml in the homogenized mixture; b) lyophilizing the homogenized mixture to produce a lyophilized composition, wherein the lyophilized composition is thermostable when stored for a period of time at a temperature in excess of 8° C.; c) rehydrating the lyophilized composition with an aqueous solution to form a rehydrated composition comprising a vesicle comprising 1-monopalmitoyl glycerol and the inactivated polio virus, the inactivated rabies virus, the inactivated hepatitis A virus, or the combination thereof; and d) administering to the individual a therapeutically effective amount of the rehydrated composition.

8. The method of claim 7, wherein the lyophilized composition is thermostable when stored for a period of time at a temperature in excess of 25° C.

9. The method of claim 7, wherein the lyophilized composition is thermostable when stored for a period of time at a temperature in excess of 30° C.

10. The method of claim 7, wherein the lyophilized composition is thermostable when stored for a period of time at a temperature in excess of 35° C.

11. The method of claim 7, wherein the composition is administered by intramuscular injection.

12. The method of claim 7, wherein the composition is administered by subcutaneous injection.

13. The method of claim 7, wherein the composition elicits an immune response in the individual at a first level that is higher than a second level of an immune response elicited by a second composition comprising the inactivated polio virus, the inactivated rabies virus, the inactivated hepatitis A virus, or the combination thereof and lacking the vesicle.

Description

EXAMPLES

(1) The following examples describe some exemplary modes of making and practicing certain compositions that are described herein. It should be understood that these examples are for illustrative purposes only and are not meant to limit the scope of the compositions and methods described herein.

Example 1: Inverted Melt Formulation Method for Preparing Antigen-Containing Vesicles

(2) This example describes an inverted melt formulation method for preparing antigen-containing non-ionic surfactant vesicles (NISV). In Step 1, a 5:4:1 molar ratio of the following lipids: 1-monopalmitoyl glycerol (MPG), cholesterol (CHO) and dicetyl phosphate (DCP) was placed in a flat bottom 50 ml glass beaker, ensuring none of the powder stuck to the side of the glass beaker. The lipids were melted in a heated oil bath at about 120-125° C. for 10 minutes, with occasional swirling in the glass beaker covered with aluminum foil.

(3) At this stage, a stock solution of inactivated antigen vaccine (Imovax® Rabies vaccine reconstituted as per manufacturer Sanofi Pasteur's instructions) was pre-incubated for 5-10 minutes at about 30-35° C. in a heated water bath. In Step 2, the resulting vaccine stock solution was homogenized at 8,000 rpm at 30-35° C., and the molten lipid mixture was added into the homogenizing vaccine stock solution (to give either a 6.25 mg/ml—test article 1 (TA 1), 12.5 mg/ml—test article 2 (TA 2) or 25 mg/ml—test article 3 (TA 3) total lipid concentration homogenate) and homogenization was continued for a further 30 seconds at about 30° C. The resulting liposomal suspension homogenate was transferred into a closed bottle and shaken for 30 minutes at 220±10 rpm and about 30-35° C. An equivalent volume of a 400 mM sucrose solution in WFI water was added to the shaken homogenate and the homogenate was further shaken for 5 minutes at 220±10 rpm at about 30-35° C. This mixture was aliquoted (0.5 ml aseptically transferred into sterile 2 cc vials sealed with a rubber stopper) and frozen at −78 to −82° C., then lyophilized and reconstituted with sterile water for injection (WFI) prior to use in thermostability studies or in vivo immunogenicity studies in animals.

Example 2: Thermostability Studies of Inverted Melt Method Formulated Antigen-Containing Vesicles

(4) To assess thermostability, NISVs were prepared as described in Example 1, and lyophilized aliquots were stored (prior to reconstitution) at two different thermal storage temperatures (5±3° C. and 40±2° C.). The freeze-dried Imovax® Rabies vaccine, used in this Study, is stable if stored in the refrigerator at 2° C. to 8° C.; while reconstituted vaccine is not stable and should be used immediately. The Imovax® Rabies vaccine is also not stable at elevated temperatures in either lyophilized or reconstituted forms. At specified times, stability samples were removed from the temperature chambers, reconstituted in WFI and analyzed by appearance, pH, microscopy, Zeta Potential, nanosizing and ELISA (antigen content). Vaccine controls (Test article 7 (TA 7)—unformulated lyophilized Imovax® Rabies vaccine) were stored as above but without addition of NISVs and were also tested.

(5) Rabies antigen content in NISV formulations was determined by performing a sandwich ELISA assay. Prior to the ELISA analysis, samples and standards were extracted by adding an equal volume of 100 mM carbonate-bicarbonate buffer (pH 9.5) with 0.5% Triton X-100 and pipetting up and down 10 times. Briefly, each well of 96 well ELISA plates was coated overnight at 4° C. with rabies virus monoclonal antibody (Ms Mab to Rabies virus (4.2 mg/ml) ab1002, Abcam) diluted 1/2000 in 25 mM bicarbonate buffer pH 9.7. The next day the coating solution was removed and the plates were blocked (1-3 hours at 37° C.) with 5% FBS in 0.05% Tween 20 in PBS. After the incubation time, plates were washed six times in wash buffer (0.05% Tween 20 in PBS). Then four to eight 2-fold serial dilutions of each extracted sample and standard were prepared using 5% FBS in 0.05% Tween 20 in PBS. The extracted and diluted samples and standards were added to the 96 well ELISA plates and were incubated for 1.5 h at 37° C. The plates were washed six times in wash buffer and incubated for 1 h at 37° C. with primary antibody ( 1/500 dilution of ferret sera in blocking solution). The plates were washed six times in wash buffer and incubated for 1 h at 37° C. with a 1/10,000 dilution of a goat anti-ferret IgG-Fc HRP conjugated secondary antibody (Bethyl). The plates were washed six times and developed using TMB substrate for 10 min at room temperature. Stop solution was added to each well and absorbance was read at 450 nm within 1 hour using an ELISA plate reader (Bio-Rad).

(6) In Table 2 in vitro antigen content results are shown for TA 1 (Imovax® Rabies vaccine formulated in 6.25 mg/ml total lipid concentration NISVs), TA 2 (Imovax® Rabies vaccinc in 12.5 mg/ml total lipid concentration NISVs), TA 3 (Imovax® Rabies vaccine in 25 mg/ml total lipid concentration NISVs) and TA 7 (unformulated lyophilized Imovax® Rabies vaccine) stability samples stored at either 4° C. or 40° C. for 0, 5 or 9 months. (Percent antigen content reflects the ratio of antigen detected in extracts from NISVs relative to the initial amount of inactivated antigen vaccine used in the preparation of NISVs).

(7) TABLE-US-00002 TABLE 2 Test Article 0 months 5 months 9 months TA 1-4° C. 71% 78% 79% TA 1-40° C. NA 80% 78% TA 2-4° C. 69% 64% 63% TA 2-40° C. NA 65% 65% TA 3-4° C. 65% 39% 43% TA 3-40° C. NA 43% 47% TA 7-4° C. 88% 81% 73% TA 7-40° C. NA 75% 60%

(8) As can be seen in Table 2 there is no difference in thermostability between 4° C. and 40° C. stored samples of the same test articles for up to 9 months but overall the higher lipid concentration NISVs formulations stored at both temperatures were found to have a lower in vitro antigen content.

(9) In Table 3 is shown the in vitro antigen content loss between 4° C. and 40° C. stored samples for TA 1 (Imovax® Rabies vaccine formulated in 6.25 mg/ml total lipid concentration NISVs) and TA 7 (unformulated lyophilized Imovax® Rabies vaccine) stability samples stored at either 4° C. or 40° C. for 0, 5, 9 or 18 months.

(10) TABLE-US-00003 TABLE 3 Test Article 0 months 5 months 9 months 18 months TA 1 0%   0%  1.3% 13.8% TA 7 0% 7.4% 17.8% 63.6%

(11) As can be seen in Table 3 no appreciable loss in antigen content occurred between the 4° C. and 40° C. stored NISVs formulated Rabies Imovax® vaccine (TA 1-6.25 mg/ml total lipid concentration NISVs) for up to 18 months indicative of thermostability; while TA 7 (unformulated Rabies vaccine) loses significant antigen content between the 4° C. and 40° C. stored samples at the same time points which indicates lack of thermostability.

(12) In Table 4 is presented the physical-chemical data derived on testing NISVs formulated Imovax® Rabies vaccine (TA 1-6.25 mg/ml total lipid concentration NISVs stored for 18 months at 4° C. and 40° C.) versus unformulated Imovax® Rabies vaccine (TA 7 stored for 18 months at 4° C. and 40° C.).

(13) TABLE-US-00004 TABLE 4 Z-Ave Zeta Potential Osmolality Test Article (d, nm) PDI (mV) (mmol/kg) pH TA 1-4° C. 1111 0.530 −76.2 692 9.18 TA 1-40° C. 2126 0.790 −60.0 690 9.33 TA 7-4° C. 18.83 0.741 −20.6 288 9.24 TA 7-40° C. 17.42 0.508 −16.6 299 9.33

(14) As expected the Z-average and zeta potential were different between the two test articles as TA 1 was formulated to have lipid-based antigen-containing vesicles and TA 7 was the unformulated vaccine control that did not contain any vesicles. Also as expected the Osmolality between TA 1 and TA 7 was different due to TA 1 containing sucrose whereas TA 7 did not contain any sucrose. Test Articles stored at the two different temperatures did not show any significant differences in physical-chemical parameters when compared to the other similarly formulated test articles.

Example 3: In Vivo Animal Testing of Inverted Melt Method Formulated Antigen-Containing Vesicles

(15) Female Balb/C mice (6-8 weeks old; body weight 18 to 28 grams, Charles River Canada Inc.) were immunized (n=8) intramuscularly once on day 0 (with 0.1 ml of indicated vaccine samples). Pre-immunization and post-1st immunization blood samples were collected to assess humoral immune responses to formulated and unformulated Imovax® Rabies Vaccine. Humoral immune responses were determined by performing an IgG ELISA Serological Assay. An indirect ELISA was performed to assess anti-rabies specific IgG titres in immune serum. Briefly, each well of 96 well ELISA plates was coated overnight at 4° C. with rabies antigen (Imovax® Vaccine, Sanofi Pasteur) diluted 1/25 in 25 mM bicarbonate buffer pH 9.7. The next day the plates were washed with PBS containing 0.05% Tween 20 and then blocked (1 h at 37° C.) with 10% goat sera in PBS. After the incubation time, plates were washed six times in wash buffer (0.05% Tween 20 in PBS). Then four to eight 2-fold serial dilutions of each serum sample were prepared using 10% goat sera in PBS. The sample and the controls were added to the 96 well ELISA plates and were incubated for 1.5 h at 37° C. The plates were washed six times in wash buffer and incubated for 1 h at 37° C. with a 1/5000 dilution of a goat anti-mouse IgG-Fc HRP conjugated secondary antibody (Bethyl). The plates were washed six times and developed using TMB substrate for 3 min at room temperature. Absorbance was read at 450 nm with an ELISA plate reader (Bio-Rad). The inverted end point titre is considered the highest sera dilution for which the OD450 reading is higher or equal with 0.1. Results on Geometric Mean (GM) of OD450 reading of 1/800 dilution of serum samples are presented in Table 5 for Imovax® Rabies Vaccine formulated with lipids as described previously versus unformulated Imovax® Rabies Vaccine.

(16) TABLE-US-00005 TABLE 5 GM of OD450 of 1/800 Test Article Storage Antigen Dose Formulation Total Serum Group (n = 8) Temp (IU/volume) Method Lipid Homogenization Dilution) TA 1 4° C. Imovax ® Inverted Melt 6.25 mg 30 sec at 0.77 Rabies (0.25 with Sucrose 8,000 rpm IU/100 μL) TA 7 4° C. Imovax ® Commercial — — 0.43 Rabies (0.25 Formulation IU/100 μL)

(17) The GM of OD450 reading for a 1/800 serum dilution of TA 1 (Imovax® Rabies Vaccine formulated with 6.25 mg/ml total lipid concentration NISVs stored at 4° C. for 18 months) was significantly higher than the GM of OD450 reading for a 1/800 serum dilution of TA 7 (unformulated Imovax® Rabies Vaccine stored at 4° C. for 18 months) indicating that the inverted melt non-ionic surfactant (NISVs) lipid based formulation appeared to have an adjuvant effect on the Rabies Vaccine.

Other Embodiments

(18) Other embodiments of the disclosure will be apparent to those skilled in the art from a consideration of the specification or practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope of the disclosure being indicated by the following claims. The contents of any reference that is referred to herein are hereby incorporated by reference in their entirety.