ADJUVANTED NANOPARTICULATE INFLUENZA VACCINE
20220088176 · 2022-03-24
Inventors
Cpc classification
C12N2760/16134
CHEMISTRY; METALLURGY
A61K39/39
HUMAN NECESSITIES
A61K2039/58
HUMAN NECESSITIES
A61K2039/55555
HUMAN NECESSITIES
C12N2760/16234
CHEMISTRY; METALLURGY
International classification
Abstract
Vaccine compositions comprising influenza antigens formulated as nanoparticulate water in oil miniemulsions. The vaccines may be formulated at the point of use and are useful in emergency response conditions.
Claims
1-18. (canceled)
19. A kit for the point-of-use administration of a nanoparticulate water-in-oil emulsion vaccine against an infectious agent comprising: a vial of an adjuvant oil comprising mannide monooleate, squalene and squalane, a vial of aqueous PBS for combining with an antigen, at least one syringe, a lipid resistant three-way stopcock, at least one needle, and a vial for storing the formulated water-in-oil emulsion vaccine.
20. The kit of claim 19 wherein metabolizable oils comprise from about 85% to about 90% by weight of the oil.
21. The kit of claim 19 wherein a first emulsifier comprises from about 9% to about 12% by weight of the oil.
22. The kit of claim 19, wherein a second emulsifier comprises from about 0.5% to about 0.7% by weight of the oil.
23. The kit of claim 19, wherein said squalene comprises a range from about 40% to about 60% by weight.
24. The kit of claim 19, wherein said squalane comprises a range from about 40% to about 60% by weight.
25. The kit of claim 24 wherein said squalene and said squalane comprise a ratio from about 50% squalene to about 50% squalane by weight.
26. The kit of claim 19, wherein said nanoparticulate water-in-oil emulsion comprises an adjuvant oil vehicle from about 65% to about 75% by weight.
27. The kit of claim 19, wherein said nanoparticulate water-in-oil emulsion vaccine comprises an adjuvant oil vehicle.
28. The kit of claim 19, wherein said nanoparticulate water-in-oil emulsion vaccine comprises an adjuvant oil vehicle from about 65% to about 75% by weight.
29. The kit of claim 19, wherein said nanoparticulate water-in-oil emulsion vaccine comprises an aqueous phase further comprising a protein antigen.
30. The kit of claim 19, wherein said nanoparticulate water-in-oil emulsion vaccine comprises an aqueous phase from about 25% to about 35% by weight.
31. The kit of claim 19, wherein said nanoparticulate water in oil emulsion vaccine comprises an aqueous phase from about 27% to about 33% by weight.
32. The kit of claim 19, wherein said water in oil emulsion vaccine is used in conjunction with a protein solubilizer.
33. The kit of claim 19, wherein said protein solubilizer is Urea or DMSO.
34. A method for a point of use formulation of an adjuvanted nanoparticulate water-in-oil emulsion vaccine comprising: preparing an aqueous solution of a proteinaceous antigen, transferring a suitable amount of the aqueous solution to a sterile container containing a predetermined amount of an adjuvant oil comprising mannide monooleate, squalene and squalane, shaking the container so as to mix the contents to form a milky pre-emulsion, transferring an aliquot of the milky pre-emulsion to a first sterile syringe, connecting the first sterile syringe to a sterile three-way stopcock, attaching a second sterile syringe to the three-way stopcock at a 90 degree angle to the first syringe, passing the emulsion from one syringe to the other by manually depressing the plunger of each respective syringe in a serial fashion for a sufficient number of cycles so as to create an emulsion of the aqueous solution in the adjuvant oil wherein the median globule size is from about 0.3 μm to about 1 μm.
35. A method of producing an influenza vaccine composition comprising: (i) preparing an aqueous phase comprising proteinaceous antigens for each of a plurality of influenza vaccine strains; (ii) preparing a water-in-oil emulsion of the aqueous phase with an oil phase wherein: the aqueous phase of the emulsion comprises from about 25% to about 35% by weight of the vaccine composition; and the oil phase comprises from about 65% to about 75% by weight of the emulsion, and comprises from about 85% to about 95% by weight of squalene and squalane, and from about 9% to about 12% by weight of mannide monooleate and from about 0.5% to about 0.7% of polyoxyl-40-hydrogenated castor oil; and (iii) processing the emulsion to form a nanoparticulate water-in-oil emulsion comprising globules of the aqueous phase of a median diameter from about 0.3 μm to about 1 μm; wherein: (i) said vaccine has enhanced cross-reactivity to influenza antigens not contained in the vaccine as compared to an unadjuvanted vaccine consisting essentially of the proteinaceous antigens; (ii) said vaccine has increased response rate in elderly and immunocompromised subjects as compared to an unadjuvanted vaccine consisting essentially of the proteinaceous antigens; and (iii) said vaccine provides an extended period of protective antibody titers as compared to an unadjuvanted vaccine consisting essentially of the proteinaceous antigens.
36. The method of claim 35, wherein said squalene and said squalane are in a ratio of about 1:1 by weight.
37. The method of claim 35, wherein the median globule size is about 300 nm.
38. The method of claim 35, wherein said water in oil emulsion vaccine further comprises a protein solubilizer that is urea or DMSO.
39. The method of claim 35, wherein the vaccine has enhanced immunopotency.
40. A method of producing an influenza vaccine composition comprising: (i) preparing an aqueous phase comprising proteinaceous antigens for each of a plurality of influenza vaccine strains; (ii) preparing a water-in-oil emulsion of the aqueous phase with an oil phase wherein: the aqueous phase of the emulsion comprises from about 25% to about 35% by weight of the vaccine composition; and the oil phase comprises from about 65% to about 75% by weight of the emulsion, and comprises from about 85% to about 95% by weight of squalene and squalane, and from about 9% to about 12% by weight of mannide monooleate and from about 0.5% to about 0.7% of polyoxyl-40-hydrogenated castor oil; and (iii) processing the emulsion to form a nanoparticulate water-in-oil emulsion comprising globules of the aqueous phase of a median diameter from about 0.3 μm to about 1 μm; wherein said vaccine has enhanced immunopotency.
41. The method of claim 40, wherein said squalene and said squalane are in a ratio of about 1:1 by weight.
42. The method of claim 40, wherein the median globule size is about 300 nm.
43. The method of claim 40, wherein said water in oil emulsion vaccine further comprises a protein solubilizer.
44. The method of claim 43, wherein said protein solubilizer is urea or DMSO.
45. The method of claim 40, wherein the vaccine has enhanced immunopotency relative to unadjuvanted trivalent inactivated vaccine (TIV).
46. The method of claim 40, wherein the vaccine provides a greater than 1.3-fold increase in antibody titers compared to an unadjuvanted influenza vaccine.
47. The method of claim 40, wherein the vaccine provides a greater than 1.3-fold increase in antibody titers compared to an unadjuvanted trivalent inactivated vaccine.
48. A method of providing protection against influenza to a human subject comprising administering by intramuscular injection to the subject an effective amount of the vaccine produced by the method of any one of claims 35 to 47.
49. A method of producing an immune response to influenza, comprising administering to a patient a nanoparticle water-in-oil adjuvant influenza vaccine, said vaccine comprising: a nanoparticulate water-in-oil emulsion having a viscosity of 100 cP or less; an aqueous phase which comprises about 25% to about 35% by weight of the emulsion, which aqueous phase is in the form of globules of a median diameter from about 0.3 μm to about 1 and contains said one or more influenza antigens; and an oil phase which comprises from about 65% to about 75% by weight of the emulsion, which oil phase contains from about 85% to about 90% of squalene and squalane, from about 9% to about 12% of mannide monooleate, and from about 0.5% to about 0.7% of polyoxyl-40-hydrogenated castor oil, each by weight of the oil phase; wherein the nanoparticle water-in-oil adjuvant vaccine has: enhanced immunopotency to the influenza antigens compared to an unadjuvanted vaccine comprising the same or a higher concentration of the same influenza antigens; enhanced cross-reactivity to influenza antigens not contained in the vaccine; increased efficacy in elderly and immunocompromised subjects; and enhanced durability.
Description
DESCRIPTION OF THE FIGURES
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[0030]
DETAILED DESCRIPTION
[0031] In one example, the vaccines of the invention can be produced either “point-of use” or as a “bulk filled” final drug product.
[0032] As a “point-of-use” product, the vaccine is formulated as a nanoparticulate emulsion made by a simple, but robust and reproducible hand mixing procedure. This is illustrated by the globule size diameter 50% distribution results (D(v,0.5) for emulsions determined by laser light diffraction between multiple operators and their stability at room temperature. At T zero, globule size distribution for “point-of-use” emulsions prepared by 4 operators at the lower and upper mixing limits of the method were N=29; Mean D(v,0.5) SEM=0.0.44±0.03 μm and N=26; Mean D(v,0.5)±SEM=0.66±0.03 μm, respectively. Globule size measurements at 2, 18-24 and 48 hours after preparation indicated that there was no significant change in this parameter over the two-day storage period.
[0033] “Bulk-filled” vaccine emulsion is manufactured aseptically and filled into single use containers such as vials or ampules. The mechanized, bulk fill process should typically produce emulsions with a mean globule size diameter of approximately 0.30 Protein antigen up to at least 9.25 mg/mL in the aqueous phase has no effect on globule size distribution for emulsions made by either the point-of-use or the bulk fill method.
[0034] The vaccine emulsions have a low viscosity affording syringeability so that as little as 0.1 mL doses can be administered to patients with high precision and reproducibility.
[0035] The components of the oil adjuvant vehicle suitable for use in the invention, comprise a first sugar ester emulsifier such as mannide monooleate (MMO) or sorbitan monooleate, a second emulsifier such as a hydrogenated castor oil, for example, polyoxyl-40-hydrogenated castor oil (POCO), and naturally occurring and metabolizable oils, preferably squalene and squalane. The metabolizable oils typically comprise from about 85% to about 90% by weight of the oil, the first sugar ester emulsifier from about 6% to 15%, i.e., about 9% to about 12%, or about 10% or 11% by weight of the oil, and the second emulsifier from about 0.1%-1.1%, i.e., 0.2% to about 1%, 0.4 to about 0.8%, 0.5% to about 0.7%, or about 0.6% by weight of the oil. The metabolizable oil component may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% squalene, and 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% squalane by weight, but the concentration of these components may vary plus or minus 10% within this component. A suitable adjuvant vehicle for use in the invention is MAS-1, which is comprised of naturally occurring and metabolizable components derived from vegetable sources, and is commercially available from Mercia Pharma, Inc, Scarsdale, N.Y. (www.merciapharma.com). Point-of-use (POU) vaccines may be produced with these adjuvant oils using a robust and reproducible hand-mixing method described herein.
[0036] The components of the oil vehicle, including their starting materials, which may be derived from either animal or vegetable sources, or combinations thereof, are all commercially available from multiple sources. Suitable sugar esters as the first emulsifier in addition to MMO include polysorbates, particularly sorbitan monooleate. In addition to POCO as the second emulsifier sorbitan esters, such as sorbitan monopalmitate, polysorbates, such as the Tweens family of emulsifiers, and Hypermers B239 and B246 may be useful.
[0037] The nanoparticulate vaccine emulsions of the invention typically contain from about 65% by weight to about 75% by weight of the adjuvant oil vehicle, but may also contain about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% by weight of the adjuvant oil vehicle. The nanoparticulate vaccine emulsions of the invention typically contain from about 25% to about 35% by weight of an aqueous phase containing the protein antigen, but may also contain from about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight of an aqueous phase containing the protein antigen. In certain embodiments of the invention the aqueous phase comprises from about 27% to about 33% by weight of the vaccine emulsion.
[0038] The water-in-oil vaccine emulsions used in the invention should be formulated so that the aqueous globules in the emulsion carrying the antigen have median diameters less than 1 micron with median diameters in the range from about 100 nanometers to about 1 micron, and typically with an average diameter of about 300 nanometers. It is further contemplated that the aqueous globules may be 50 nanometers, 100 nanometers, 150 nanometers, 200 nanometers, 250 nanometers, 300 nanometers, 350 nanometers, 400 nanometers, 450 nanometers, 500 nanometers, 550 nanometers, 600 nanometers, 650 nanometers, 700 nanometers, 750 nanometers, 800 nanometers, 850 nanometers, 900 nanometers, or 950 nanometers. The oil components of the adjuvant are preferably naturally occurring biological oils that are metabolizable, unlike the mineral oil that comprises the oil phase of the well known Freund's adjuvants (both incomplete and complete formulations).
[0039] The vaccine emulsions of the invention should tolerate high concentrations of antigen, such as from 0.1 mg/mL to 20 mg/mL, i.e, 1 mg/mL, 5 mg/mL, 7 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 13 mg/mL, 15 mg/mL, 20 mg/mL, or up to at least 10 mg/mL and should be compatible with commonly used protein solubilizers (e.g., 4M urea, 30% DMSO). Unlike IFA emulsions, they should be compatible with aqueous phases having a wide range of pH, i.e., from about 2-9, 3-8, 4-6, i.e., about 5, and unaffected over a wide range salt concentrations. Unlike IFA emulsions (>1,500 cP), the vaccine emulsions of the invention should have a low viscosity (<100 cP) as free flowing emulsions permitting high precision low volume (0.1 mL) dosing. The physico-chemical characteristics of the vaccine emulsions of the invention should have a median distribution of globule size diameter of (D(v,0.5)) less than or equal to 1.0 μM, and be unaffected by high concentrations of protein in the aqueous phase (
[0040] At T zero, D(v,0.5) μm±SEM for POU vaccine emulsions prepared by four different operators at the lower and upper mixing limits of the method were; 0.44±0.30 (n=29) and 0.66±0.03 (n=26), respectively. D(v,0.5) after 2, 24 and 48 hours at ambient temperature indicated no significant change. At release, BMP vaccine emulsions should have a D,(v0.5) of 0.3 μm with an end of shelf life D,(v0.5) of ≤1 μm after 3 years at 2-8° C. without any loss in immunopotency in vivo (by contrast D(v,0.5) of IFA is 3-10 μm a T zero).
[0041] The invention is further described in the following examples. The examples are merely illustrative and do not in any way limit the scope of the invention as described and claimed.
Example 1
[0042] TIV Vaccines:
[0043] In one example of the invention the vaccines are comprised of standard TIV antigens derived from egg or cell cultures located in the aqueous phase, formulated as a 30:70 (w/w/) water-in-oil (w/o) nanoparticulate emulsion with MAS-1 having a median globule size diameter D(v,0.5) of 0.30 μm. A suitable adjuvant vehicle for use in the invention is MAS-1.
[0044] Formulation of TIV Influenza Vaccine:
[0045] Fluzone TIV, was obtained from Sanofi Pasteur, as the source of H antigen (Ag) for these studies. Fluzone contains 30 μg/mL of each H antigen, and yields 9 μg/mL of each H antigen formulated in a 30:70 emulsion with MAS-1 adjuvant. Fluzone TIV was concentrated up to 10-fold using Amicon Ultra 15 centrifugational concentrators (cut-off: 10,000 kd). During concentration of TIV approximately 50% of total protein determined by Lowry assay is recovered in the concentrate. Approx. 40% of total protein passes through the filters into the filtrate, and 12% is adsorbed to the ultrafilters (Table 1). TIV protein recovery was reproducible between batches. Total protein analyses of the aqueous phase by Lowry confirmed batch-to-batch consistency across the range of H Ag concentrations, with protein content of the aqueous phase varying ≤2.5% and 3.5% for Fluzone between batches (Table 2).
TABLE-US-00001 TABLE 1 Recovery and Mass Balance of TIV protein during centrifugal concentration Aqueous Phase Total Protein (μg) Total Protein (μg) Test Sample Batch 1 Batch 2 Fluzone 12,020 12,440 Concentrate 5,893 (49.0%) 5,928 (47.7%) filtrate 4,662 (38.8%) 5,130 (41.2%) Balance 12.2% 11.1%
[0046] Although total protein was lost, H antigen was not lost during the concentration process. Total H antigen by single radial immunodiffusion (SRID) test comprises 14-15% of the total protein measured by Lowry (against BSA standard) in Fluzone, and 30-31% of the total protein in the vaccine emulsion formulations (Table 2). The validated SRID assay is used for batch release of approved influenza vaccines. We established the SRID assay in our laboratory, using the WHO methods and reference antisera and antigens. By SRID, all three H antigens were retained in the concentrate with no loss into the filtrate. These results were confirmed by SDS-PAGE run on samples of concentrate and filtrate (
TABLE-US-00002 TABLE 2 Content and reproducibility of H antigen and total protein in MAS-1/TIV Protein % [H Ag]/ Protein % [H Ag]/ Total (μg/mL) by [SRID] (μg/mL) by [SRID] % Immunogen (μg/mL) Lowry.sup.2 for for Lowry.sup.2 for for (Batch-1/ For Group H Ag.sup.1 Batch 1 Batch 1 Batch 2 Batch 2 Batch-2) 1(Fluzone) 90.0 601 15% 622 14% −3.49% 2 150 496 30% 499 30% −0.60% 3 50 165 30% 166 30% −0.61% 4 50 159 31% 163 31% −2.52% 5 150 496 30% 499 30% −0.60% 6 16.7 55 30% 56 30% −1.82% 7 50 159 31% 163 31% −2.52% 8 5.7 18.3 31% 18.7 30% −2.19% 9 (Placebo) 0 0 NA 0 NA NA .sup.1Total H Ag concentration from Fluzone specification .sup.2Total protein (BSA equivalents) measured by Lowry against BSA standard; NA Not applicable
Example 2
[0047] POU Mixing Procedure Parameters:
[0048] The range of mixing rate and mixing cycles optimal for making water-in-oil miniemulsion nanoparticulate vaccine emulsions according to the invention were established using an aqueous phase comprising phosphate buffered saline (PBS), pH 7.2 and MAS-1 oily vehicle in a 30:70 (w/w) ratio made using the POU mixing procedure. A range of mixing rates and mixing cycles were evaluated at syringe sizes from 1.0 to 5.0 mL and emulsion volumes ranging from 1.0 to 3.0 mL. Samples were analyzed immediately after preparation (T-zero) and at select times after storage at ambient conditions for up to 1 week. The data are presented in Table 3.
TABLE-US-00003 TABLE 3 Physico-chemical characteristics of POU 30:70 (w/w) MAS-1 Vaccine emulsions Mixing Syringe Size/ Globule size diam, Replicates Mix Duration emulsion vol D(v, 0.5) Mean (SD) μm (N)* Cycles (sec) (mL) T-zero 4-6 hr 24 hr 48 hr 1 week 8 40 No Constraint 1/1 7.68 11.37 1.58 nr nr (10.2) (14.6) 3 50 No Constraint 1/1 1.46 2.04 nr nr nr (0.62) 3 60 No Constraint 1/1 12.00 nr 14.29 nr nr (17.76) (18.00) 6 75 No Constraint 1/1 1.16 1.40 nr nr nr (0.32) 1 100 No Constraint 1/1 0.62 nr 0.70 nr nr 11 125 No Constraint 1/1 0.64 nr 0.40 nr nr (0.20) 6 125 90 1/1 0.33 nr 0.36 0.44 0.40 (0.04) (0.11) 20 125 100 1/1 0.47 0.31 0.43 0.48 0.53 (0.20) (0.11) (0.01) (0.33) 6 125 110 1/1 0.74 nr nr nr nr (0.07) 17 125 120 1/1 0.60 0.57 0.70 0.83 0.82 (0.16) (0.13) (0.16) (0.07) (0.19) 2 125 150 1/1 nr nr 0.91 nr 0.99 (0.07) 3 125 90 3/2 0.31 0.30 0.31 0.30 nr (0.01) 1 125 180 3/2 0.75 nr nr nr nr 1 125 135 5/5 0.30 nr 0.31 nr nr Note: nr = not reported; *Replicates (N) refers to the number of emulsions produced under the stated condition
[0049] The combined (N=52) results for physico-chemical characteristics of 1 and 2 mL POU MAS-1 vaccine emulsions prepared with 1 and 3 mL syringes, respectively, made by 125 cycles in the range 90 to 120 seconds presented in Table 4.
TABLE-US-00004 TABLE 4 Physico-chemical characteristics of POU 30:70 (w/w) MAS-1 Vaccine emulsions Storage at Ambient Temperature Characteristics T zero 4-6 hr 24 hr 48 hr 1 wk Viscosity cP 86 nr nr nr nr Globule size D(v0.5) 0.52 0.44 0.47 0.56 0.61 Mean (SD) μm (0.2) (0.17 (0.17) (0.22) (0.27) Appearance score 0.52 0 0 0 nr (0)
Key to Appearance Scores
[0050] The samples, contained in clear glass vials, are visually examined under defined lighting for the presence of aqueous droplets collecting at the bottom of the emulsion. The method gives a macroscopic measure of the quality of the emulsion.
TABLE-US-00005 0 No aqueous droplets were observed 1-3 Increasing amounts (quantity and size) of aqueous droplets were observed 4 An aqueous pool was observed 5 An aqueous layer was observed 6 a complete phase separation was observed
[0051] The POU method produces reproducible, robust, and stable MAS-1 vaccine emulsions when mixed for 125 cycles within 90-120 seconds at the 1 to 2 mL. Scale and 90-150 seconds at the 5 mL scale.
Example 3
[0052] Immunopotency Enhancement in Mice of MAS-1/TIV:
[0053] The objective of this dose ranging study was to evaluate the immunopotency and dose sparing benefits of MAS-1 adjuvant on TN in mice. The study was designed to generate data on primary and secondary response parameters, including antibody titers, response kinetics, isotype and specificity following immunization with the adjuvanted vaccine of the invention compared to standard TIV vaccine. General well-being of the animals was monitored throughout the study period.
[0054] Test Formulations:
[0055] Prior to emulsification with MAS-1 vehicle, H antigen from TIV concentrate was diluted with PBS to yield aqueous phases with the correct concentrations of H antigen. H antigen concentrations were calculated from the H antigen content of the starting material (Fluzone) and the volume following concentration by centrifugal ultrafiltration. The aqueous phase protein contents were confirmed by Modified Lowry Assay (Pierce Chemical Co.). The TIV aqueous phase preparations were readily emulsified in MAS-1 oil phase vehicle using the POU mixing procedure.
[0056] The vaccine emulsions were broken to extract the aqueous phase and oil phases. SDS-PAGE analysis of antigen-bearing aqueous phase extracted from the emulsion demonstrated that the antigens are compatible with the MAS-1 adjuvant. Protein mobility and band numbers were the same between aqueous phase and extracted aqueous phase.
[0057] Study Outline:
[0058] The study was a seven-arm, placebo controlled study at doses/H antigen (A/H1N1; A/H3N2; and, B) of 4.0 μg as TIV (+control); MAS-1 placebo (−control), and 0.05 to 4.0 μg of TIV formulated as a nanoparticulate emulsion with MAS-1 according to the invention. BALB/c mice (female, 8-12 weeks old at start of study, 6 per group) were immunized with 0.1 mL test articles subcutaneously (dorsal, base of neck) on days 0 and 28. Blood samples were taken on days 0, 14, 28, 42, and 56. Sera were prepared and stored at −20° C. until assay. The formulations were prepared from clinical grade 2007/08 season TIV (Fluzone). Fluzone was concentrated 10-fold to 452 μg/mL total H antigen aqueous phase. The vaccine and placebo emulsions were prepared by the POU method. The aqueous phase protein content for the vaccine preparations were verified by Modified Lowry and SDS-PAGE.
[0059] General Safety:
[0060] None of the mice showed any signs of distress or ill health over the course of the study at any of the doses tested indicating that the formulations were safe and well tolerated.
[0061] Immunopotency and Enhancement:
[0062] The hemagglutination inhibition (HAI) titers against each of the three viral strains used in the 2007/08 TIV (A/Solomon Islands/3/2006 (H1N1); A/Wisconsin/67/2005 (H3N2); B/Malaysia/2506/200) were measured using the WHO assay protocol and reference standards. Testing was performed blinded to group assignment. Group geometric mean HAI titers at days 42 and 56 for the three influenza viral antigens and the dose adjusted enhancement of the immune response in MAS-1/TIV vaccine according to the invention for each antigen relative to TIV (Group 1) are presented in Table 5. MAS-1 significantly enhanced the immunopotency of TIV against all three component viral strains at both time points, particularly on day 56. At the matched 4 μg dose, MAS-1 enhanced the day 56 responses by 9 fold for A/H1N1 virus, 11.3 fold for A/H3N2 virus, and 6.3 fold for B virus. Higher HAI titers were observed at day 56 even at 0.15 μg/H antigen in the adjuvanted vaccine compared with 4 μg/H antigen in standard TIV group 1, with dose adjusted enhancement of 48-fold for A/H1N1, 85-fold for A/H3N2, and 23-fold for B/Malaysia. Overall, dose-adjusted enhancement of TIV in MAS-1/TIV at day 56 compared to standard TIV was 22 fold for A/H1N1, 26-fold for A/H3N2, and 9-fold for B/Malaysia.
TABLE-US-00006 TABLE 5 Geometric mean HAI titers of test groups and dose-adjusted enhancement of HAI titers relative to Fluzone positive control (group 1). Titer A/H1N1 A/H3N2 B Group Treatment Bleed day 42 56 42 56 42 56 1 TIV 4 μg Geo. Mean 1,280 905 1,611 905 285 226 (+) Control — — — — — — 2 MAS-1/TIV 4 μg Geo. Mean 5,747 8,127 25,803 10,240 905 1,437 Enhanced 4.5 9.0 16.0 11.3 3.2 6.3 3 MAS-1/TIV 1.3 μg Geo. Mean 2,874 4,561 905 2,032 137 359 Enhanced 6.8 15.2 1.7 6.8 1.4 4.8 4 MAS-1/TIV 0.4 μg Geo. Mean 1,016 1,613 118 149 9 16 Enhanced 7.2 16.2 0.7 1.5 0.3 0.6 5 MAS-1/TIV 0.15 μg Geo. Mean 806 1,613 1.016 2,874 20 194 Enhanced 16.8 47.5 16.8 84.7 1.9 22.9 6 MAS-1/TIV 0.05 μg Geo. Mean 5 7 3 3 1 1 Enhanced 0.03 0.6 0.1 0.3 0.3 0.4 7 MAS-1 placebo Geo. Mean 1 1 1 1 1 1 (−) Control — — — — — — Combo 0.15-4.0 Enhanced 8.8 22.0 8.8 26.1 1.7 8.7 Note: Enhancement = (test group mean/group 1 mean) × (H Ag dose of group 1/H Ag dose of test group)
[0063] Immunopharmacokinetics:
[0064] Antibody response kinetics were similar for each of the three component strains (
[0065] Dose Response:
[0066] The dose-related response of HAI titers for each of the three viral antigens, A/H1N1, A/H3N2, and B virus elicited by the adjuvanted formulations of the invention is further illustrated by the data for day 56 presented in
Example 4
[0067] Evaluation of Efficacy and Safety in Rabbits
[0068] Study Outline:
[0069] A dose ranging, placebo controlled 9 arm (N=5/group) study was performed in rabbits to evaluate enhancement of immunopotency, dose sparing potential, and duration of immunoprotective response versus standard TIV at doses and route of administration anticipated in humans. Additionally, the study incorporated both general safety and a formal evaluation of toxicology to evaluate injection site tolerance and systemic safety by necropsy and histopathology on a panel of organs. The study encompassed a range of variables, including: MAS-1 vs. no adjuvant (Fluzone), dose of H antigen in MAS-1 (0, 0.56, 1.67, 5.0, and 15.0 μg/H antigen), injection volume (0.1 and 0.3 mL), and formulation strategy (emulsified to strength or dilution of vaccine emulsion to H antigen strength with placebo emulsion). Animals were immunized intramuscularly on days 0 and 28. Blood samples were taken on day 0 and at 2 weekly intervals through day 84. Sera was prepared from the blood samples and stored at −20° C. until assay. Animals were sacrificed on day 84, necropsied and organs collected for gross evaluation and histopathology in compliance with GLP. Formulations were prepared from clinical grade 2007/08 season TIV (Fluzone), and vaccine and MAS-1 placebo emulsions were prepared by the POU method. The aqueous phase protein content for vaccine preparations was verified by Modified Lowry and SDS-PAGE.
[0070] Immunopotency and Enhancement:
[0071] HAI titers were measured against the three viral strains present in the 2007/08 TIV, and against the three antigenically dissimilar strains in the 2008/09 TIV to evaluate cross-immunoprotection. Testing was performed blinded to group assignment. Peak HAI titers for the three 2007/08 and 2008/09 season virus strains and dose adjusted enhancement of HAI titers by MAS-1 for each antigen are presented in Tables 6A and B, respectively. The immunopharmacokinetics of the immune response induced by TIV and MAS-1/TIV after IM immunization are shown in
TABLE-US-00007 TABLE 6A Peak Geometric mean HAI titers and dose adjusted enhancement of MAS-1/TIV Against 2007/08 Season Virus Strains 2007/08 A/H1N1 A/H3N2 B Season Dose Vol Solomon Islands/3/2006 Wisconsin/67/2005 Malaysia/2506/2004 Grp (μg) (mL) Titer Enhanc p value Titer Enhanc p value Titer Enhanc p value 1 15 0.5 2941 — — 3378 — — 2941 — — 2 .sup.a 15 0.3 8914 3.0 0.001 11763 3.5 0.015 5881 2.0 0.059 3 .sup.b 5 0.3 7760 7.9 0.005 7760 6.9 0.086 5881 6.0 0.089 4 .sup.a 5 0.3 11763 12.0 0.018 11763 10.4 0.004 8914 9.1 0.002 5 .sup.c 5 0.1 4457 4.5 0.207 6756 6.0 0.034 6756 6.9 0.031 6 .sup.a 1.67 0.3 3881 11.9 0.326 4457 11.9 0.242 2561 7.8 0.620 7 .sup.a 1.67 0.1 4458 13.6 0.167 8914 23.7 0.203 3379 10.3 0.658 8 .sup.b 0.56 0.3 5882 53.6 0.030 5881 46.6 0.066 3882 35.4 0.460 9 0 0.3 0 — — 0 — — 0 — — Overall — — — 15.21 — — 15.57 11.07 .sup.a Emulsified direct y to the indicated concentration of H antigen. .sup.b Emulsion prepared at strength for Group 2 was diluted 1:3 with placebo emulsion .sup.c Same emulsion as that used for Group 2. Enhancement over TIV = (test group mean/group 1 mean) × (H Ag dose of group 1/H Ag dose of test group)
Example 5
[0072] Dose Response:
[0073] The dose response for the adjuvanted vaccine seen in mice appeared to be in the linear range. By contrast, the dose response of HAI titers for each of the three viral antigens, A/H1N1, A/H3N2, and B virus elicited after IM immunization in rabbits indicate that the lowest dose tested, 0.56 μg/H antigen, in MAS-1/TIV is close to the plateau response above the dose titration range—lower doses were not evaluated.
[0074] Cross-Immunoprotection Against 2008/09 Season Strains:
[0075] Immunization with a vaccine according to the invention (MAS-1/TIV, using 2007/2008 TIV) was significantly more cross protective than standard TIV with HAI titers against 2008/09 TIV strains. At the 5 μg/H antigen dose all three MAS-1/TIV preparations were statistically equivalent by ANOVA, and enhanced on average 4.1-fold for A/H5N1, 3.4-fold for A/H1N1, and 2.4-fold for B viruses. The mean dose adjusted enhancement at 5 μg/H antigen was 12-fold for A/H1N1, 10-fold for A/H3N2, and 7-fold for B/Florida (Table 6B).
TABLE-US-00008 TABLE 6B Day 56 Geometric mean HAI titers and dose adjusted enhancement of MAS-1/TIV Against 2008/09 Season Virus Strains 2007/08 A/H1N1 A/H3N2 B Season Dose Vol Brisbane/59/2007 Brisbane/10/2007 Florida/4/2006 Grp (μg) (mL) Titer Enhanc p value Titer Enhanc p value Titer Enhanc p value 1 15 0.5 320 — — 320 — — 279 — — 2 15 0.3 1280 4.0 0.011 844 2.6 0.023 557 2.0 0.059 3 5 0.3 1470 13.8 0.010 1114 10.4 0.032 970 10.4 0.089 4 5 0.3 1470 13.8 0.002 1470 13.8 0.002 422 4.5 0.002 5 5 0.1 970 9.1 0.069 640 6.0 0.006 640 6.9 0.031 Overall 5-15 — — 10.2 — — 8.2 6.0 Enhancement over TIV = (test group mean/group 1 mean) × (H Ag dose of group 1/H Ag dose of test group) Student's T test in each case is compared to group 1 Fluzone positive control
[0076] Immunopharmacokinetcis:
[0077] Antibody response kinetics were similar for each of the three component strains, as shown in
Example 6
[0078] Safety/Toxicology Evaluations:
[0079] The scope of the rabbit immunopharmacology included: observations of animal general well-being during the in vivo phase, and formal evaluations of safety and toxicology, including animal weights, necropsy, visual inspection and organ weights under veterinary supervision, and histopathology assessments of the 20 selected organs in Table 7 from Groups 1 (Fluzone 15 μg), Group 2 (MAS-1/TIV 15 μg), and Group 9 (MAS-1 placebo), respectively; were performed in compliance with GLP. In addition, all injection sites were examined post mortem by visual and histological assessments on all rabbits.
TABLE-US-00009 TABLE 7 Tissues collected at necropsy for histopathology from each rabbit in Groups 1, 2 and 9. Tissue Collected Sample Type Heart, Spleen, Adrenal, Ovary, Popliteal Whole lymph node, Mandibular lymph node, Brain Kidney, Liver, Lung, Pancreas, Aorta, Section Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Esophagus, Trachea
[0080] General Safety:
[0081] Fluzone TIV and all MAS-1/TIV formulations appeared safe and well tolerated during the in vivo phase of the study assessed by independent observations made by animal welfare personnel. No animals at any adjuvanted vaccine dose or Fluzone showed signs of ill health at any point during the study. No statistically significant differences in body weights taken on day 72 were observed between the adjuvanted vaccine Groups 2-8 or MAS-1 placebo Group 9, or Fluzone Group 1.
[0082] Necropsy:
[0083] All rabbits in each of Groups 1, 2, and 9 were necropsied and the panel of 20 organs harvested in compliance with GLP. Three minor abnormalities were found, including two in the Fluzone group 1 (loose stool) and one in MAS-1 placebo group 9 (small lobe of extra-splenic tissue). None of these abnormalities were considered to be related to any of the test materials, nor were they believed to have any bearing on the study outcome. Eleven of the 20 organs collected from each rabbit in groups 1, 2 and 9 were weighed prior to fixation. The organ weights, the organ to body weight ratios, and the organ to brain weight ratios were compared between the three groups. No statistically significant differences between the groups were found in these comparisons, with three exceptions: First: the mandibular lymph nodes, which are distal to the injection sites in the rear legs, were heavier in group 1 than in groups 2 or 9 rabbits. Second: the right (but not left) popliteal lymph nodes were slightly larger in group 9 than in group 2 rabbits. Third: Brain/body weight ratio was statistically smaller in group 2 than group 9 rabbits. The actual brain weights between groups 2 and 9 were not different and apparent differences in brain/body weight ratio can be attributed to the differences in overall body weights between Groups 2 and 9. None of these observations were considered to be significant to the safety of any of the test articles.
[0084] Histopathology:
[0085] Histopathology on the 20 organs collected from groups 1, 2, and 9 rabbits was performed in compliance with GLP. Occasional instances of cellular infiltrates and/or congestion noted for some organs were concluded to be typical background findings. This study confirmed the expected lack of systemic toxicity and found no evidence of histomorphologic differences between rabbits treated with the adjuvanted vaccine of the invention at 15 μg/H antigen and rabbits treated with either Fluzone or MAS-1 placebo.
[0086] Injection Site Tolerance:
[0087] Immediately after sacrifice of the rabbits on study day 84, injection sites were scored visually (macroscopic) and biopsy specimens collected. The biopsies were subsequently studied for histopathology and graded by a Board Certified veterinary pathologist. Visual and histology evaluations are presented in Table 8.
TABLE-US-00010 TABLE 8 Visual and Histology Scores at sites 1 and 2 after IM injection in rabbit thigh muscle Injection site 1 (Day 0 Inj) Injection site 2 (Day 28 Inj) Vaccine Dose Vol Visual Histology Visual Histology Grp TIV μg/H mL Mean Range Mean Range Mean Range Mean Range 1 Fluzone 15.0 0.5 0 0 0 0 0 0 0 0 2 MAS-1/TIV 15.0 .sup.a 0.3 0 0 1.5 0-2.5 0.4 0-1 0.5 .sup. 0-1.5 3 MAS-1/TIV 5.0 .sup.b 0.3 0 0 0 0 0.3 0-1 1.5 .sup. 0-2.5 4 MAS-1/TIV 5.0 .sup.a 0.3 0.1 0-0.5 0 0 0.7 0-1 1.4 0-3 5 MAS-1/TIV 5.0 .sup.c 0.1 0 0 0 0 0.1 .sup. 0-0.5 1.0 0-3 6 MAS-1/TIV 1.67 .sup.a 0.3 0 0 0 0 0.5 0-1 1.5 0-3 7 MAS-1/TIV 1.67 .sup.a 0.1 0 0 0 0 0.4 0-1 1.1 0-3 8 MAS-1/TIV 0.56 .sup.b 0.3 0.2 0-0.5 0 0 0.3 0-1 0.7 0-2 9 Placebo 0 0.3 0 0 0.2 0-1 0.1 .sup. 0-0.5 0.7 0-1 .sup.a Emulsified directly to the indicated concentration of H antigen. .sup.b Emulsion prepared at strength for Group 2 was diluted 1:3 with placebo emulsion .sup.c Same emulsion as that used for Group 2 Key to visual pathology scores Normal tissue 0-0.5 Minimal pathology 1-1.5 Moderate pathology 2-2.5 3 Key to histolopathology scores Normal tissue or very mild inflammation 0-0.5 Mild active or residual chronic pathology 1-1.5 Moderate active chronic inflammation 2-2.5 Severe chronic inflammation/pathology 3
[0088] Visual injection site scores of ≤1, and histology scores of ≤2 are indicative of a tolerable formulation; some inflammation is anticipated and correlates with the immune response enhanced by the adjuvant. Fluzone was well tolerated at injection sites 1 and 2, but consistent with its lower immune response, only limited inflammation was seen histologically at both sites. MAS-1/TIV was well tolerated both visually and histologically at the site of the first injection. Of the 35 MAS-1/TIV injection sites examined, three (one in Group 4, two in Group 8) received visual scores of 0.5, indicating barely discernable difference between the injection site and surrounding muscle tissue. The remaining 32 MAS-1/TIV and all 5 Fluzone and 5 MAS-1 placebo first injection sites appeared normal. The second MAS-1/TIV injection was more reactogenic, with 7/35 sites at 0.5, 10/35 sites at 1.0 and 18/35 with 0 visual scores. The second MAS-1 placebo injection had 1/5 sites at 0.5 with 4/5 at a 0 visual score. All second injection sites with Fluzone had 0 visual score.
[0089] The macroscopic injection site scores were generally supported by histological examinations of biopsy specimens. Thus, histopathology was not observed at the first injection site in any rabbits except for those in group 2, where moderate microscopic reactions were noted in two rabbits and mild reactions seen in two others. Increased inflammation was found at the second injection site for all MAS-1/TIV formulations, with scores ranging from 0 to 3. Mild inflammation at site two was noted in two rabbits receiving MAS-1 placebo, while Fluzone did not elicit inflammation at the second injection sites.
[0090] Both visual and histopathology assessments support a single injection regimen anticipated for MAS-1 adjuvanted TIV to be administered IM as a prophylactic influenza vaccine in humans at doses of 15 μg/H antigen or less.
Example 7
[0091] Preparation of POU Vaccine Formulation from Commercial Seasonal Influenza Vaccine Supplies.
[0092] Preparation of the vaccine miniemulsions of the invention requires mixing and emulsification the aqueous phase containing the protein antigen with oily vehicle. Standard stock influenza vaccine contains 30 μg/mL (TIV 30) of each H antigen or TIV containing higher concentrations, (a 4-fold higher strength seasonal TIV vaccine for the elderly comprising 120 μg/mL (TIV 120) Sanofi Pasteur) may be used for each H antigen. The vaccines of the invention are preferably about a 30:70 (w/w) water-in-oil emulsion. To produce POU vaccine formulations of the invention for clinical purposes so as to provide doses at 1.0, 3.0, and 5.0±15% ng/H antigen, the TIV 30 and TIV 120 vaccines are combined with MAS-1 vehicle as indicated in the schematic shown in Table 9.
[0093] POU Process Outline:
[0094] Step 1: In each case, for 1, 3 and 5 μg/H antigen doses in MAS-1 formulation, 0.5 mL of Fluzone TIV 30 and TIV 120 are removed from the Fluzone vials according to the schema A and B shown in Table 9.
[0095] Step 2; The TIV aqueous phase solutions are then transferred by injection into single use, pre-filled sterile PBS vials and mixed by hand.
[0096] Step 3: In each case, 0.5 mL of each [diluted] aqueous phase is then transferred by injection into single use pre-filled, sterile vials containing 1.2 g of MAS-1 adjuvant. The vial contents are mixed by shaking vigorously for 30 seconds to produce a milky pre-emulsion.
[0097] Step 4: The aqueous and MAS-1 pre-emulsion mixture is transferred into a 2.0 ml syringe and emulsified using the double syringe method.
TABLE-US-00011 TABLE 9 POU formulations at 1, 3, and 5 ± 15% μg/H antigen for clinical purposes MAS-1/TIV Step Dose μg 1.0 μg/H 3.0 μg/H 5.0 μg/H A1 TIV 30 mL 0.5 0.5 0.5 A2 PBS for dilution mL 1.0 — — A3 Diluted Aq. Phase mL 0.5 0.5 0.5 MAS-1 g 1.2 1.2 1.2 A4 MAS-1/TIV yield mL 1.9 1.9 1.9 Dose Vol mL 0.3 0.3 0.5 B1 TIV 120 mL 0.5 0.5 0.5 B2 PBS for dilution mL 1.67 0.23 — B3 Diluted Aq. Phase mL 0.5 0.5 0.5 MAS-1 g 1.2 1.2 1.2 B4 MAS-1/TIV yield mL 1.9 1.9 1.9 Dose vol mL 0.1 0.1 0.12
[0098] Based on the results in rabbits (Table 6A) showing statistical equivalency between as low as 0.56 μg/H antigen in MAS-1/TIV and 15 μg/H antigen in standard TIV, we anticipate that from about 1 to 5 μg/H antigen dose in formulated in MAS-1 delivered in either 0.1 mL or 0.3 mL should be optimal for use in the elderly human patients.
[0099] POU Syringe Hand Mixing Process:
[0100] At 1 to 2 mL scale, the mixing procedure takes 90-120 seconds using a pre-set number of cycles. The geometry of the syringe method and flow characteristics are critical to successful emulsification. The emulsion pre-mix is drawn into a 2 mL Norm-Ject syringe and then attached to a 3-way stopcock. A second 2 mL syringe is then attached to the stopcock at 90°. The assembly is clasped firmly around the 3-way stopcock. The pre-emulsion is passed from one syringe to the second by carefully depressing the first syringe plunger with the palm of the other hand. This constitutes 1 pass or cycle. Full emulsification is then achieved by completing 125 cycles within 90-120 (or 150 seconds at 5 mL scale). (Syringes—2 to 5 mL Norm-Ject, sterile, single-use, all plastic Tuberculin, Air-tite Products Co. Inc, VA USA; Henke Sass Wolf GMBH, Tuttlingen, Germany; Kruuse UK Ltd, UK; Syringe needles—18 or 21 gauge, sterile, single use; 3-way stopcock, lipid resistant, Vygon Corp, catalog number 876.00)
[0101] Typically the emulsions are expelled into a clean sterile vial and can be transferred from the pharmacy to the patient area prior to removing the prescribed injection volume. The POU method is effective for producing from 1 to 5 mL of adjuvanted vaccine that should remain stable for at least 24 hours at ambient temperature (Tables 3 and 4). The low viscosity, free flowing emulsions enable accurate low volume dosing with as little as 0.1 to 0.3 mL injection volumes. This means that multiple (from 3 to 10 at 1 mL scale to 15 to 50 at 5 mL scale) POU doses of the adjuvanted influenza vaccine of the invention can be simply and quickly provided by this POU method, particularly useful in the event of a pandemic influenza outbreak.
[0102] The POU process is useful in epidemic emergency response situations where there is a need for a potent adjuvant system that can be formulated and administered with antigens that are in short supply. The oil component of the emulsion can be stockpiled with kits such as described above, comprising syringes, vials, stopcocks, etc. and distributed independently of the required vaccine antigen, which can subsequently be delivered as it becomes available. This type of POU system can be particularly useful for a rapid response in epidemic and biodefense situations where there is very short time period between the outbreak of the infectious agent and the identification of an effective target antigen and its production in sufficient quantity for vaccination of large populations.
[0103] A kit useful for the point-of-use administration of a water-in-oil emulsion vaccine against an infectious agent comprises a sterile vial of an adjuvant oil, a sterile vial of aqueous PBS for combining with an infectious agent antigen, two sterile syringes, a lipid resistant three-way stopcock, a 21 gauge sterile needle, a 25 gauge sterile needle and a sterile vial for storing the formulated water-in-oil emulsion vaccine. The adjuvant oil should be useful with a wide range of protein antigens. In one embodiment of the invention the adjuvant oil comprises mannide monooleate, squalene and squalane. Other oil adjuvants may be formulated as described above. The MAS-1 adjuvant available from Mercia Pharma, Inc. may be used as the adjuvant oil for the vaccines of the invention. Other oil adjuvants such as the Montanide adjuvants available from SEPPIC, SA, Paris, France, that are not mineral oil based but are comprised of animal or vegetable sourced oils may also be used to formulate vaccines of the invention according to the methods described herein. The kits may be used with a wide range of influenza antigens or antigens of other infectious agents or combinations of such antigens, including toxins derived from said pathogens.