Liquid vaccines of live enveloped viruses

Abstract

The present invention describes a liquid vaccine composition of a live enveloped virus and a pharmaceutically acceptable carrier, whereby the carrier is a natural deep-eutectic solvent (NADES), and the vaccine has a water activity of less than about 0.8. The NADES provides a stabilisation of the sensitive virus for prolonged time and at ambient temperature. In general, the liquid vaccine compositions according to the invention, in different compositions for the various enveloped viruses, show remarkable capabilities of stabilisation. This overcomes the need for lyophilisation, a great economic benefit. Also the liquid nature of the vaccines facilitates administration to human or animal targets.

Claims

1. A liquid vaccine composition comprising a live enveloped virus and a pharmaceutically acceptable carrier; wherein the carrier is a natural deep-eutectic solvent (NADES), the vaccine has a water activity between about 0.8 and about 0.1, and wherein the water content in said composition is less than about 50% w/v.

2. The liquid vaccine composition of claim 1, wherein the NADES comprises an organic salt and a polyol.

3. The liquid vaccine composition of claim 2, wherein the organic salt is selected from the group consisting of betaine, proline, carnitine, and choline.

4. The liquid vaccine composition of claim 2, wherein the polyol is a sugar or a sugar-alcohol.

5. The liquid vaccine composition of claim 2, wherein the polyol is selected from the group consisting of sucrose, glycerol, xylitol, and sorbitol.

6. The liquid vaccine composition of claim 1, wherein the enveloped virus has an RNA genome.

7. A method of preparing the liquid vaccine composition of claim 1, comprising the steps of: a. preparing the NADES, and b. admixing the NADES and a composition comprising the live enveloped viruses.

8. A method of vaccinating a human or animal target, comprising administering the liquid vaccine composition made by the method of claim 7.

9. A kit comprising at least two containers, wherein one container comprises the liquid vaccine composition of claim 1 and one container comprises a liquid diluent.

10. A method of vaccinating a human or animal target, comprising administering the liquid vaccine composition mixed with the diluent that both came in the kit of claim 9.

11. A method of vaccinating a human or animal target caused by a pathogenic form of the live enveloped virus, comprising administering the liquid vaccine composition of claim 1.

Description

EXAMPLES

Example 1: Methods and Materials

(1) 1.1 Preparing the NADES Formulations

(2) NADES can be prepared by mixing, which can be accelerated by heating and/or addition of a small amount of water. Some of the samples of NADES as further tested and described herein below were prepared by dissolving organic salts and polyols in an excess of water. Next, the excess of water was removed by rotational evaporation in a waterbath at 70° C. for several hours under reduced atmospheric pressure. The amount of water present in the NADES was calculated by taking into account the weight-loss due to the evaporation.

(3) Other samples of NADES were prepared by sonification method in a waterbath. The required quantities of powders were added to a flask. The flask was briefly shaken or vortexed, to mix the powders. Next the required amount of water was added. This mixture was placed in a sonication-waterbath, set at 45° C., with sonication up to several hours, until a NADES was formed. During sonication temperatures of up to 70° C. could be reached.

(4) 1.2 Measurements of Water Activity

(5) Samples

(6) For biological containment purposes the water activities were measured of samples of NADES preparations, so without live virus. However, when knowing the amount of aqueous virus composition that will be added to the NADES to form the liquid vaccine composition according to the invention, the resulting water activity of the combination of NADES and virus composition can be calculated according to Dai et al. (2015, Food Chem., vol. 187, p. 14-19).

(7) Sample Preparation

(8) An amount of NADES sample was transferred to a 10 ml glass vial in order to reach a fill height of approx. ¼ of the vial. For each sample coded vials were filled in duplicate. The vials were closed using an aluminium crimp cap with PTFE stopper, and were placed at room temperature for 12 hours to reach an equilibrium between sample and headspace.

(9) Equipment

(10) The moisture vapour pressure in the headspace of the samples was determined using the Lighthouse FMS-1400 Moisture/Pressure analyser. The samples are inserted into the sample chamber as such. Before sample measurement the system is calibrated using calibration headspace pressure standards in the range of 0-0.2 a.sub.w. Also, saturated salt standards were used to calibrate and recalculate the non-linear range from 0.2-1 a.sub.w.

(11) Calibration

(12) To calibrate the machine over the full range from a.sub.w 0-1, and to create a reference line of water activity values, a number of standard samples were measured: next to pure water 5 samples of specific saturated salt-solutions were used, and for each of the 6 standards 2 samples were measured in 3 fold. By setting the measured value for pure water to 1.000, the other standard values were determined at a.sub.w of: 0.8511, 0.7547, 0.5438, 0.3307, and 0.1131. Also a series of certified vapour pressure standards were measured.

(13) Results of a.sub.w measurements are included in Table 1.

(14) 1.3 Virus Compositions Used for Stability Assays

(15) The viruses used for stability incubation were: canine parainfluenza virus (CPiV); from the Paramyxoviridae family bovine respiratory syncytial virus (BRSV); also known as the species: bovine orthopneumovirus; from the Pneumoviridae family, Orthopneumovirus genus, and bovine parainfluenza 3 virus (PI3); from the Paramyxoviridae family.

(16) All these viruses are enveloped viruses, and have an RNA genome.

(17) 1.4 NADES Compositions Used for Stability Assays

(18) NADES compositions used for the stability assays were: NADES 1: betaine:sucrose:water, in 2:1:6,4 molar ratio NADES 2: carnitine:glycerol:water, in 2:1:4 molar ration, and NADES 3: proline:sorbitol:water, in 2:1:5,7 molar ratio.
1.5 Mixing NADES and Virus Compositions

(19) NADES preparations were blended 9:1 v/v with virus compositions in a laminar flow cabinet. To prevent osmotic shock, stabilizer was added gradually and portion-wise to the cooled virus solution. Samples were gently stirred using a sterile magnetic stirring. Care was taken not to entrap air bubbles into the mixture. The mixtures were stored at 2-8° C. until the start of filling into appropriate containers.

(20) Filling the NADES/virus mixture into 3 ml vials was performed in a laminar flow cabinet at 2 ml per vial using a pipetboy, samples were kept on ice. Duplicate samples were prepared for each stability time-point. Also NADES samples without virus were included as negative controls. After filling, vials were capped with an aluminium cap. Samples were then stored at their stability-incubation temperatures.

(21) 1.6 Stability Assays

(22) For a number of enveloped viruses, comprised in liquid vaccine compositions according to the invention, extended stability assays were performed. These were done at three temperatures: 4, 15, and 25° C., and during incubation for 1, 2, 4, or 8 weeks. Day zero samples were set apart.

(23) At the appropriate time points, NADES/virus mixtures were taken and used to determine the remaining virus titres, by titration on cells.

(24) Virus titrations were done on 96 well microtitration plates. Proper positive and negative controls were included. Titres were calculated from at least two readings of a sample, using the method of Spearman and Kärber via a computer program, and were expressed in Log 10 TCID50/ml.

(25) CPI virus was titrated on Vero cells, cultured in standard culture medium with 5% FCS and antibiotics. 2×10{circumflex over ( )}5 cells/ml were seeded, and 1:10 dilutions were made on the plates.

(26) Next, the microtiter plates were incubated for 7 days at 37° C. and 5% CO.sub.2. Each well was then inspected for cpe as evaluated by lightmicroscopy.

(27) To quantify the amount of infectious BRSV or PI3 in samples, serial tenfold dilutions of test samples were made in standard cell-culture medium and were pipetted into microtitration plates that were pre-seeded with Madin Darby Bovine Kidney (MDBK) cells. After an incubation period of 5-7 days for BRSV, or 4-6 days for PI3, at 37° C. in a humidified 5% CO.sub.2 atmosphere, the monolayers were examined for the presence or absence of virus. The plates were fixated and an immune-peroxidase monolayer assay was performed, using a peroxidase-labelled virus-specific monoclonal antibody, in combination with an enzyme-substrate. Detection was done by registering the presence of a coloured precipitate visually.

(28) Results of the stability assays are described in Example 3.

Example 2: Examples of NADES for Use in the Invention

(29) A large number of NADES formulations were prepared for use in the invention, with different compounds, with different molar ratios, and with different water contents.

(30) Table 1 below list a number of the most effective NADES formulations; these yielded clear liquid solutions, that were fluid at room temperature (20-25° C.). Details on their composition and water content are provided. For several of the samples prepared, the water activity was determined and the results are included. Not indicated: the molecular weight of water is 18 Da; trehalose was used as dihydrate.

(31) A number of observations could be made: choline, betaine, and proline were the most versatile and effective organic acids for use in the invention, and sorbitol, glycerol, and xylitol were the most effective polyols. NADES with water contents up to 37% w/v could readily be made, resulting in liquids of low viscosity water activities were generally found to be very low, often below 0.3, and even below 0.1 a.sub.w. although sugars and amines can react via the Maillard reaction, it was observed that the majority of the prepared NADES remained colourless and visually unchanged after several hours of heating up to 70° C. several samples were also fluid at refrigerated conditions of 2-8° C. or even at −20° C.

(32) TABLE-US-00001 TABLE 1 NADES liquid at room temperature NADES water water Organic salt Polyol Water molar ratio total content activity compound Mw grams compound Mw grams grams (organ. salt:polyol:water) (g) (% w/v) (a.sub.w) Proline 115 17.26 D-sorbitol 182 27.36 5.40 1 1 2 50.01 10.79% 0.299 11.27 35.27 3.51 1 2 2 50.12 7.00% 232.75 184.14 82.69 2 1 4.5 499.58 16.43% 0.307 307.03 242.86 311.96 2 1 13.7 861.85 37.44% 0.101 20.20 xylitol 152 26.64 3.15 1 1 1 49.99 6.29% 0.300 18.98 25.08 5.94 1 1 2 50.01 11.88% 18.14 glycerol 92 29.02 2.89 1 2 1 50.2 5.76% 0.119 Betaine 117 17.49 D-sorbitol 182 27.18 5.35 1 1 2 50.02 10.69% 0.238 18.47 28.72 2.90 1 1 1 50.09 5.80% 19.18 xylitol 152 24.91 5.91 1 1 2 50.01 11.82% 0.227 25.78 glycerol 92 20.26 3.95 1 1 1 49.99 7.90% 18.34 28.88 2.81 1 2 1 50.03 5.61% 0.089 5.86 trehalose 378 8.56 4.00 2 1 9.8 18.42 21.72% sucrose 342 4 1 13.6 696.11 1016.97 335.65 2 1 6.3 2048.73 16.45% 0.391 Choline-Cl 140 20.53 D-sorbitol 182 26.82 2.65 1 1 1 50 5.30% 0.097 19.54 25.47 5.10 1 1 2 50.11 10.17% 22.56 xylitol 152 24.56 2.90 1 1 1 50.03 5.80% 21.30 23.21 5.48 1 1 2 49.99 10.96% 1396.2 760.75 271.23 2 1 3 2428.18 11.13% 0.174 29.88 16.28 3.87 2 1 2 50.03 7.74% 0.109 20.63 glycerol 92 26.94 2.66 1 2 1 50.22 5.29% 27.94 18.45 3.60 1 1 1 50 7.20% 0.091 L-Carnitine 161.2 88.50 glycerol 92 25.28 19.78 2 1 4 133.56 14.81% 8.06 trehalose 378 8.56 6.00 2 1 14.7 22.62 26.53%

Example 3: Results of Viral Stability Assays

(33) For a number of enveloped viruses, comprised in liquid vaccine compositions according to the invention, extended stability assays were performed, as described above. These were done at three temperatures: 4, 14, and 25° C.

(34) The incubation at 25° C. for 8 weeks can serve as an expedited stability test, indicative of the effect of incubation for 1-2 years at 4° C. This is thus a very severe attack on the virus' stability, and not all samples were expected to survive that long under those conditions.

(35) TABLE-US-00002 TABLE 2 Stability results of BRSV and PI3 viruses Duration (weeks) Virus Temp. Stabilizer 0 1 2 4 BRSV  4° C. Medium Control 6.25 6.15 5.85 5.85 NADES 1 6.25 6.10 6.05 6.15 NADES 2 6.45 6.15 6.55 6.35 NADES 3 6.35 6.05 6.20 6.15 14° C. Medium Control 6.25 6.00 5.40 4.30 NADES 1 6.25 5.85 5.75 5.65 NADES 2 6.45 5.80 5.55 4.90 NADES 3 6.35 5.70 5.60 5.25 25° C. Medium Control 6.25 2.45 0.00 0.00 NADES 1 6.25 5.10 4.10 2.45 NADES 2 6.45 2.30 0.00 0.00 NADES 3 6.35 4.10 2.55 0.00 PI3  4° C. Medium Control 6.35 5.70 5.70 5.75 NADES 1 6.10 6.00 6.15 6.05 NADES 2 4.55 3.95 4.00 3.60 NADES 3 5.45 5.15 4.95 5.00 14° C. Medium Control 6.35 5.95 5.55 5.15 NADES 1 6.10 5.40 5.15 4.55 NADES 2 4.55 3.30 2.90 0.00 NADES 3 5.45 4.15 3.70 0.00 25° C. Medium Control 6.35 4.50 2.65 0.00 NADES 1 6.10 3.35 2.40 0.00 NADES 2 4.55 0.00 0.00 0.00 NADES 3 5.45 0.00 0.00 0.00

(36) TABLE-US-00003 TABLE 3 Stability results of CPiV Duration (weeks) CPiV Temp 0 1 2 4 8 PBS control  4° C. 8.85 8.20 8.20 7.91 8.15 14° C. 7.50 7.25 6.25 5.80 25° C. 6.10 3.90 2.50 — NADES 1  4° C. 8.55 8.45 8.40 8.25 7.90 betaine:sucrose:water 14° C. 8.25 7.85 7.30 6.40 2:1:6.4 25° C. 6.70 4.95 2.90 — NADES 2  4° C. 8.60 8.40 8.45 8.30 8.40 carnitine:glycerol:water 14° C. 8.25 7.95 7.45 6.85 2:1:4.0 25° C. 4.90 3.50 3.50 — NADES 3  4° C. 8.65 8.55 8.50 8.55 8.25 proline:sorbitol:water 14° C. 8.30 8.40 8.19 7.95 2:1:5.7 25° C. 8.15 7.75 7.35 6.10
Some Conclusions from the Stability Results

(37) Especially CPiV virus could be stabilised very well by NADES 3 (stab. 3) formulation; even after 8 weeks at 25° C., most of the virus remained infectious.

(38) Similar results were seen for BRSV and PI3 in NADES 1, up to 4 weeks at 25° C.

(39) In general, the liquid vaccine compositions according to the invention, in different compositions for the various enveloped viruses, show remarkable capabilities of stabilisation.

(40) This overcomes the need for lyophilisation, a great economic benefit. Also the liquid nature of the vaccines facilitates administration to human or animal targets.

Example 4: Long Term Stability Assay Results

(41) The experiments described in the Examples 1-3 above were continued over time, and regular samples were taken and measured.

(42) BRSV:

(43) The stability assays were continued with the Pneumovirus BRSV in liquid vaccine compositions based on NADES 1 (Betaine:Sucrose:Water for injection, at 2:1:6.4 molar ratio), NADES 3 (Proline:Sorbitol:WFI, at 2:1:5.7), or NADES 4 (Choline:Xylitol:WFI, at 2:1:3).

(44) Results are presented in FIG. 4, panels A-C, of continued storage at temperatures of 4, 14, or 28° C., and for 30, 8 or 8 weeks respectively. These are the extensions of the results as depicted in FIG. 1, panels A-C.

(45) It is clear is that liquid vaccine compositions based on all NADES tested (1, 2, and 4), provide a solid stabilising effect to the Pneumovirus BRSV, upon storage at 4 or at 14° C., see FIGS. 4A and 4B. Also, even at the very demanding temperature of 25° C., FIG. 4C, there is a significant stabilisation by NADES 4 and an even better stabilisation by NADES 1.

(46) Further results on BRSV are presented in Example 5

(47) PI3:

(48) For the Paramyxovirus PI3, all liquid vaccine compositions based on NADES 1, 2, or 3, provided some level of stabilisation, see FIG. 2, panels A-C, but the best stabilisation over time was obtained in NADES 1 (Betaine:Sucrose:Water for injection, at 2:1:6.4 molar ratio), as is depicted in FIG. 5: this composition was able to prevent the drop in live viral titre to little more than 1 Log 10, over a period of over 30 weeks at 4° C. In comparison, the control sample in standard culture medium showed a drop in titre of 4 Log 10 over the same period.

(49) CPiV:

(50) The Paramyxovirus CPiV was mixed into liquid vaccine compositions comprising two variants of the Proline:Sorbitol:WFI NADES 3 formulation: NADES 3A and NADES 3B; details are provided in Table 4 below.

(51) Amounts of live CPiV virus titres remaining after incubation at room temperature (about 20° C.) up to 12 weeks, are presented in FIG. 6. Both compositions were able to significantly reduce virus degradation at this challenging temperature: in NADES 3A titre loss was less than 2 Log 10, in 3B the loss was about 3 Log 10, while in PBS the loss of live CPiV titre was more than 5 Log 10.

Example 5: Variations on Previous Stability Results in NADES-Based Vaccines

(52) To further expand on the experiments in Examples 1-4 above, a number of variations were tested of NADES formulations for the invention.

(53) TABLE-US-00004 TABLE 4 Further variations of NADES formulations tested Ionic molar ratio wt % water Tg # species Polyol Ion. sp:Pol.:WFI water activity (° C.) 1A Betaine Sucrose 2.0 1.0 4.0 11.06 0.30 — 3A Proline Sorbitol 1.0 1.0 2.5 13.14 0.39 −41.9 3B Proline Sorbitol 1.0 1.0 10.0 37.74 0.76 −32.2 4 Choline Xylitol 2.0 1.0 2.5 9.49 0.18 −74.8 5 Betaine Sorbitol 1.0 1.0 2.5 13.07 0.32 −49.9 6 Choline Sorbitol 1.0 1.0 2.5 12.27 0.3 −77.9

(54) In this table, ‘Tg’ stands for: glass-transition temperature. This was determined using a standard set-up for differential scanning calorimetry. In these measurements, no crystalline phase-transition could be observed for the NADES samples tested in these experiments, only a glass-transition was observed of very small size. Together with the very low Tg values measured, this indicates that these samples do not contain a detectable amount of free water that could form ice-crystals.

(55) The NADES 3B formulation has a water activity of 0.76, which allows the addition of 1/10 volume part of virus in water, to 9/10 volume parts of this NADES, to obtain a liquid vaccine composition according to the invention with a water activity of 0.80.

(56) The various NADES formulations were tested in liquid vaccine compositions according to the invention, comprising BRSV. All virus samples were combined with 9 volume parts NADES, mixed, and incubated at 28° C. to perform a forced stability assay. Results are presented in FIG. 7. All NADES formulations listed in Table 4 were able to stabilise BRSV; best results were obtained with NADES using Proline or Betaine as the ionic species.

Example 6: Stability of a Further Paramyxovirus in NADES-Based Vaccines

(57) Canine distemper virus (CVD) is a further Paramyxovirus, like PI3 and CPiV. It is of high veterinary relevance, because of its capability to infect many species of canines and felines. Symptoms include intestinal-, respiratory-, and a variety of systemic signs of disease. Regular vaccinations of cats and dogs against CDV at young age is common. Typically the vaccine is a live attenuated virus, that is produced as a freeze-dried product.

(58) CDV was taken up into a liquid vaccine composition according to the invention, mixed with 9 volume parts NADES 3A (Proline:Sorbitol:WFI at 1:1:2.5 molar ratios). Stability was tested by incubation at 4° C. for up to 16 weeks.

(59) Results are presented in FIG. 8. This shows that a composition based on NADES 3A was able to effectively stabilise CDV at essentially the starting titre, during prolonged storage at refrigerated temperature. The control sample in standard culture medium, showed a persistent drop in titre over time. This difference of stabilisation effect between NADES and control for CDV, is more pronounced than that observed for PI3, see FIG. 2A.

Example 7: Stability of Coronavirus in NADES-Based Vaccines

(60) To assess the stability of a further viral family, a Coronavirus was taken up into liquid vaccine formulations according to the invention. Coronaviruses typically cause (severe) respiratory disease in humans or animals. The virus used was infectious bronchitis virus (IBV), which is an avian Coronavirus.

(61) Like other viruses of its family, IBV is an enveloped virus, and therewith is very sensitive to outside influences. Further, IBV has a positive-sense single-stranded RNA genome, which genome is one of the largest known for RNA viruses. This makes IBV particularly susceptible to degradation upon storage.

(62) IBV has a large impact on the health of poultry such as chickens, and a big negative potential impact on the economy of operation of poultry farming. Most chickens worldwide are vaccinated against IBV infection and disease, and much of those vaccines are live attenuated vaccines, requiring the storage and shipment of live IBV virus up to prolonged periods of time. For a long time, such live attenuated vaccines were therefore produced as freeze-dried product.

(63) To assess the stability of IBV in liquid vaccine formulations based on NADES, the virus was taken up into a variant of NADES 3, namely NADES 3A, composed of: Proline:Sorbitol:Water for injection, in a molar ratio of 1:1:2.5. More characteristics of this formulation are described in Table 4.

(64) The titration of IBV is extremely laborious, as this needs to be done in birds in vivo, or ex vivo on primary cells. A much used, but equally laborious, alternative is titration on embryonated chickens eggs. For this reason only one type of NADES and only one storage temperature was tested in these experiments with IBV.

(65) Materials and Methods:

(66) IBV strain 4/91 was obtained as a standard virus product harvested as the allantoic fluid from inoculated chicken eggs. Virus and NADES were combined 1+9 volume parts, mixed by vortexing, and incubated at 4° C. To be able to titrate all samples on the same batch of eggs, the various timed samples were each prepared fresh, then started incubation, with the longest incubations starting first.

(67) Results and Conclusions:

(68) The results of IBV stability in NADES 3A, up to 30 weeks at 4° C. are depicted in FIG. 9.

(69) From the data in FIG. 9 it is evident that there is a large positive difference in the stabilising effect of the liquid vaccine compositions based on NADES 3A, as compared to the control sample of IBV 4/91 in allantoic fluid: while all virus became degraded in the control samples after 29 weeks at 4° C., the amount of live IBV in NADES 3A remained unaffected, and was not significantly different from the starting titre even after 7 months.

(70) In conclusion, a liquid vaccine composition according to the invention, comprising a Coronavirus, IBV, and a NADES, showed an excellent and unprecedented stabilising effect for the live Coronavirus over a prolonged period of storage.

Example 8: Stability of Herpesvirus in NADES-Based Vaccines

(71) Similar to Example 7, this experiment tested the stabilising effect of a liquid vaccine composition according to the invention on yet a further enveloped virus family: Herpesvirus.

(72) Two representatives were tested: Feline Herpesvirus 1 (FHV1), and Bovine Herpesvirus 1 (BHV1). These viruses are both enveloped viruses with a large double stranded DNA genome. Consequently, from being an enveloped virus, as well as from being a large virus, most Herpesviruses are quite delicate and are known to be prone to rapid degradation under standard storage conditions. Inevitably, live Herpesvirus vaccines are therefore commonly produced as freeze-dried products.

(73) FHV1 causes a contagious and severe respiratory infection in felines, called Feline viral rhinotracheitis. BHV1 is also called Infectious bovine rhinotracheitis virus, and causes similar symptoms in bovines.

(74) Samples of FHV1 and of BHV 1 were obtained from standard cell-cultures.

(75) The FHV1 sample was mixed 1+9 v/v with NADES 3A and with NADES 3B formulations. Next the sample-tubes were mixed for 1 hour on a rotating wheel. Considering the significant in-mixing loss of about 1 Log 10, this method of mixing was probably too violent for this fragile virus.

(76) BHV1 was mixed also with NADES 3A and with NADES 3B, and in addition was also tested in NADES 5 (Betaine:Sorbitol:WFI, at 1:1:2.5 molar ratios). Details are given in Table 4.

(77) Results and Conclusions:

(78) The results of FHV1 and BHV1 stability in the different liquid vaccine compositions, are presented in FIGS. 10 and 11 respectively.

(79) From the data in FIG. 10 it is clear that FHV1 rapidly degrades in the control composition (standard culture medium). The initial drop in titre at time point zero, is more than compensated later on, as both in NADES 3A and in NADES 3B the live FHV1 was effectively stabilised at essentially the level of the starting titre of the experiment, for up to 4 weeks. Considering that the test temperature in this experiment was room temperature (about 20° C.), which are quite unfavourable conditions for virus stability, this is a remarkably positive result.

(80) Similarly, FIG. 11 indicates that the stability results for BHV1 in the different NADES formulations, at 4° C. are very comparable. All the three NADES tested provided good stabilisation for up to 6 weeks, while the control composition (PBS) showed a clear drop in titre.

(81) In conclusion: a liquid vaccine composition according to the invention, comprising a Herpesvirus, FHV1 or BHV1, and a NADES, showed an excellent stabilising effect for the live Herpesvirus upon storage at a demanding storage temperature, and over a significantly long period.

Example 9: Administration of Vaccine with NADES

(82) To assess the ease of administration, and the safety of a liquid vaccine composition according to the invention in target animals, examples of the vaccine were administered to experimental animals and to post mortem materials.

(83) For this purpose the two variants of the NADES 3 formulation, comprising Proline, Sorbitol, and water for injection, were made and tested: one with high (NADES 3A) and one with low (NADES 3B) viscosity. These served to compare the administration-properties of liquid vaccine compositions according to the invention at two extremes of the viscosity spectrum. The characteristics of these two compositions are described in Table 3. Further, their viscosities were measured using standard conditions: at about 20° C., using a Brookfield™ DV-I+ viscometer, utilising spindle type No. 62, for 30 seconds at 60 r.p.m., in a standard measuring cup of about 80 ml volume.

(84) Handling

(85) The high viscosity NADES, formulation number 3A, could be pushed through a hypodermic needle of size 21 G or less, i.e. a needle with an outer diameter of 0.8 mm or more.

(86) TABLE-US-00005 TABLE 5 NADES formulatons tested by administration Ionic molar ratio Viscosity # species Polyol Pro:Sor:WFI (mPa .Math. s) 3A Proline Sorbitol 1.0 1.0 2.5 5197 3B Proline Sorbitol 1.0 1.0 10.0 33.4 3C Proline Sorbitol 1.0 1.0 5.0 289
Subcutaneous Administration In Vivo

(87) The two NADES formulations tested were compared as placebo's administered by subcutaneous injection, in a safety study in dogs. Special attention was given to any adverse reactions that could occur after the administration of the stabilizer formulations, either locally or systemically.

(88) Both formulations were prepared as described before, by heating and sonification of proline, sorbitol and water for injection, in a closed vial, resulting in a clear and homogeneous liquid. Additionally they were filter-sterilised, and bioburden and endotoxins were determined. Both formulations were found to score below the lowest dilution standard of 1 unit/ml, meeting the general acceptance criteria for sterile formulations. The NADES 3C formulation was prepared by diluting 1 volume part NADES 3A with 9 volume parts WFI, resulting in a formulation with Proline:Sorbitol:WFI in 1:1:5 ratio, and a viscosity of 289 mPa.Math.s. NADES 3B has a viscosity of 33.4 mPa.Math.s.

(89) Six healthy beagle dogs of 7 months old, were divided into two groups of 3 dogs. Each group was assigned to a NADES formulation, and received two doses of 1 ml of the assigned formulation, using a 1 ml syringe and a 21 G 5/8 needle by subcutaneous inoculation, with a two week interval. Group 1 dogs were given the low viscosity NADES 3B, and the Group 2 dogs received the high viscosity NADES 3C. All dogs were clinically monitored daily throughout the study, including for local reactions at the site of the inoculations.

(90) The vaccinations were performed in the scruff of the neck; the first vaccination was done on the left side, and the second vaccination was done on the right side of the neck. The vaccination sites were shaved prior to each administration, and the injection site was circled with a marker pen. No discomfort was observed during any of the injections. At 4 hours post-injection, injection sites were palpated, and little or no material was palpable at the injection sites, and no swelling or redness was observed.

(91) Rectal temperatures were taken on days −3, −2, −1 before each of the vaccinations; at the day of vaccination: just before vaccination and 4 hours after vaccination; and next daily for seven days post vaccination.

(92) Throughout the whole study no significant increase in body temperature was observed in any of the dogs and no clinical adverse effects were observed.

(93) In conclusion: the inoculation of NADES formulations 3C or 3B by subcutaneous route did not induce any adverse inoculation reaction, neither locally, nor systemically.

(94) Intra-Nasal Administration

(95) Two NADES formulations: 3A and 3B were tested for their pattern of spread on the mucosal surface of the nose, when administered intranasally. Both NADES formulations were tested as such, without adding water, consequently, NADES 3A has a viscosity of 5197 mPa.Math.s, and NADES 3B of 33.4 mPa.Math.s. A spatula tip of ‘patent blue’ colourant was added to 30 ml of each of the NADES formulations, to obtain a deep blue colour. As a control PBS with colourant was used.

(96) Calf heads were obtained post-mortem from calves of about 2-4 weeks old. The administration was done holding the head in an upright position mimicking the position of a calf during i.n. vaccination in the field, with the nostrils tilted slightly upwards.

(97) The liquids were then administered according to a standard procedure for intranasal vaccination, whereby a 2.5 ml syringe without needle, containing 1.5 ml liquid, was placed into a nostril, and then the liquid was squirted out in one steady flow. Next the head was dissected into two halves and the septum was removed and inspected for presence of blue colour in the intranasal tract.

(98) For all samples it was observed that the blue liquids had moved easily through the nasal tract. The low viscosity of the PBS sample allowed for easy injection, but all liquid rushed through quickly. A blue colour was observed all through the whole of the nasal cavity including the beginning of the trachea.

(99) The injection of NADES 3A with the very high viscosity took a little force to inject out of the syringe. Also, as expected, it took longer for the blue liquid to emerge below the head. After dissection of the head, it was observed that also this viscous liquid had spread evenly through the intranasal tract, where it appeared to have formed a thick layer, leaving more material in the nasal tract. This indicates an improved retention and less progression to the upper respiratory tract would occur, when administered to a life calf during i.n. vaccination.

(100) The effect of the administration of the NADES 3B formulation, with the low viscosity, closely resembled that of the PBS sample, where the liquid rushed through the nasal cavity.

(101) In conclusion: it was possible to monitor the distribution of coloured preparations with different viscosity after intranasal injection into calve-heads. Distribution of the liquid as judged by the blue colour was comparable for the two NADES preparations and PBS, with some difference with regard to ease of administration and amount of residual material, whereby the NADES 3A formulation was a little more difficult to eject, but resulted in a thicker layer of residue within the nasal tract. This suggests a delayed release of antigen from such viscous NADES formulations may be obtained.

Example 10: Detailed Analysis of the Proline:Sorbitol:WFI NADES

(102) The NADES 3 type formulation, comprising proline, sorbitol, and water for injection, was subjected to detailed analysis. Many variations of ratio's of the three components were made and tested. This is depicted in FIG. 12, which represents a triangle plot of the compositions in weight ratios of the three components. The ratios that were able to generate NADES formulations are presented by the parallelogram inside the triangle. Ratios outside the parallelogram resulted in mixtures that were either solid, or aqueous, as is indicated in the triangle. The various NADES compositions that were tested and found to produce effective and stable variants of NADES 3 are indicated by balls. The specific formulations of NADES 3A and 3B as tested in more detail in the above Examples are also indicated.

LEGEND TO THE FIGURES

(103) FIG. 1:

(104) Stability results of BRSV in different NADES formulations

(105) FIG. 2:

(106) Stability results of bovine PI3 virus in different NADES formulations

(107) FIG. 3:

(108) Stability results of CPiV in different NADES formulations (NADES 1-3 are called stab. 1-3 in the legend; x-axis: time in weeks; y-axis: titre).

(109) FIGS. 4, 5, and 6:

(110) Long term stability results of BRSV, PI3 and CPiV in different NADES formulations, and at different storage temperatures.

(111) FIG. 7:

(112) Stability results for BRSV in variants of NADES formulations

(113) FIGS. 8, 9, 10 and 11:

(114) Stability results of further viruses and virus families: CDV, IBV, FHV1 and BHV1, respectively.

(115) FIG. 12:

(116) Triangle plot of the weight ratios of Proline, Sorbitol, and WFI, made and tested in a detailed analysis of variants of NADES 3. The grey parallelogram indicates effective NADES compositions, the balls indicate individual formulations made and tested.