阅读说明：本技术 热稳定的液体轮状病毒疫苗 (Thermostable liquid rotavirus vaccine ) 是由 S.V.卡普雷 I.A.奥拉夫 于 2018-02-14 设计创作，主要内容包括：本发明涉及一种由在非水性液体中的微粉化冻干轮状病毒颗粒乳剂和缓冲赋形剂组成的口服疫苗。这种IVT-06制剂通过在30℃和40℃的温度下保护病毒至少12个月而赋予热稳定性。从12个月的稳定性数据推断,表明保存期在30℃下超过2年,且在50℃下6个月。此外,为易于施用,配制的剂量的体积为0.5 Ml。(The present invention relates to an oral vaccine consisting of an emulsion of micronized lyophilized rotavirus particles in a non-aqueous liquid and a buffering excipient. This IVT-06 formulation confers thermal stability by protecting the virus at temperatures of 30 ℃ and 40 ℃ for at least 12 months. Inferred from the 12 month stability data, the shelf life was shown to be over 2 years at 30 ℃ and 6 months at 50 ℃. Furthermore, for ease of administration, the volume of the dose formulated is 0.5 Ml.)

1. A method of producing a vaccine in bulk comprising:

adding a buffering agent, a bulking agent, and an excipient comprising at least arginine to a virus-containing composition to form a mixture, wherein the mixture has a viral titer of log 8 or greater;

lyophilizing the mixture to form a lyophilized composition comprising less than or equal to about 0.8% moisture;

grinding the mixture to an approximately uniform particle size of about 5 μm or less; and

the particles are formulated as vaccines.

2. The method of claim 1 wherein the virus-containing composition comprises rotavirus.

3. The method of claim 2, wherein the rotavirus comprises multiple serotypes of rotavirus.

4. The method of claim 1, wherein the buffer comprises a mixture of citrate and calcium carbonate.

5. The method of claim 1, wherein the buffer maintains the pH of the mixture at about 7.0-8.0 without significant change after lyophilization.

6. The method of claim 1, wherein the compatibilizer comprises glucose, sucrose, or both glucose and sucrose.

7. The method of claim 1, wherein the compatibilizer is sucrose and the ratio of sucrose to arginine is about 1:1.1 to 1: 1.5.

8. The method of claim 1, wherein the mixture comprises about 2% arginine.

9. The method of claim 1, wherein the mixture comprises about 4% arginine.

10. The process of claim 1 wherein the mixture comprises Medium Chain Triglycerides (MCT).

11. The method of claim 1, wherein the vaccine is stored at about room temperature or higher for at least about 3 months without significant loss of efficacy.

12. The method of claim 1, wherein the bulk vaccine is stored at a temperature of about 30 ℃ or greater for at least 6 months without significant loss of efficacy.

13. The method of claim 1, wherein the bulk vaccine is stored at a temperature of about 30 ℃ or greater for at least 12 months without significant loss of efficacy.

14. The method of claim 1, wherein formulating comprises partitioning the mixture to contain about 5.9log₁₀FFU/mL virus and a volume of about 0.5mL of individual dose.

15. A vaccine produced according to claim 1.

16. A bulk vaccine formulation comprising a lyophilized rotavirus strain and an excipient which provides thermostability of at least about 2 years at about 30 ℃ and at least about 3 months at about 50 ℃.

17. The bulk vaccine formulation of claim 16, having a uniform particle size of less than or equal to about 5 μm.

18. The bulk vaccine formulation of claim 16, which is homogenized with Medium Chain Triglycerides (MCT).

19. The bulk vaccine formulation of claim 16, comprising about 5.9log of multiple doses in a volume of about 0.5mL₁₀FFU/mL virus.

20. The bulk vaccine formulation of claim 16, comprising citrate and calcium carbonate as buffering agents.

1. Field of the invention

The present invention provides compounds, compositions, vaccines and methods for the treatment and prevention of viral infections, particularly oral vaccines and formulations that are stable and effective over extended periods of time at room temperature and higher temperatures.

2. Description of the background

Rotavirus infection is a major cause of severe diarrhoeal disease in infants (Kotloff et al, 2013). Until 2008, diarrhea accounts for 37% of all deaths in infants and children under 5 years of age, and is estimated to cause nearly 50 million deaths each year, before introduction of the universal rotavirus immunization program (Tate et al, 2008; Kotloff et al, 2013;). Most of this death occurs in poor countries.

There are eight rotaviruses, named A, B, C, D, E, F, G and H. Humans are infected mainly with species A, B and C, most commonly with species a. Species a-E cause disease in other animals, species E and H cause disease in pigs, and D, F and G cause disease in birds. Within rotavirus a there are multiple serotypes (also called strains). The classification system used is based on two major surface proteins: VP7 (a glycoprotein that defines serotype G), VP4 (which defines serotype P). Genes that determine G-type and P-type are passed separately to progeny viruses, and many combinations have been identified.

Since 2006, three live oral attenuated rotaviruses-rotareq from Merck, ROTARIX from Glaxo SmithKline, and ROTAVAC from Bharat Biotech-were commercially available to help prevent severe rotavirus infection (Vesikari et al, 2006,&2007, Bhandari et al 2014a, b). The dosage is 1-2.8 x 10⁶CCID₅₀(ii)/dose.

Two of these vaccines are available as 1.5-2.0 mL liquid formulations and need to be stored at 2-8 ℃ (cold chain storage); while the third vaccine is a frozen formulation that requires constant refrigerator storage to maintain stability of the rotavirus in order to maintain its efficacy. According to the package insert, after removal of the vaccine from refrigeration, the vaccine must be administered as soon as possible (Matthias, 2007). Furthermore, the cold chain system is expensive to build, requires a lot of space, and large organizations and infrastructures, which are sometimes difficult to perform in poor countries where preventive help is most needed.

The refrigerated space for vaccines and their cold packaged materials also becomes irrelevant as the cold chain just becomes unnecessary from the manufacturer's premises. Furthermore, the reduction in dose volume will allow less space to be occupied in storing and shipping the vaccine, with the additional benefit that multiple dose forms of the vaccine can be prepared, similar to the forms currently used for oral polio vaccines. Furthermore, in this field, the dose volume of oral polio is also well established to be 0.5ml or less.

The product prepared upon lyophilization needs to be accompanied by a buffer for dissolution. When reconstituted, the volume is typically greater than 1.5 mL, which is difficult to administer to infants, especially those exhibiting infant regurgitation (which may lead to vaccine spitting). Of course, the greater the volume administered, the greater this probability. It is estimated that current vaccine manufacturers have the ability to produce 5000 ten thousand doses of rotavirus vaccine per year using a single dose model. If the demand for rotavirus vaccine is 1 hundred million doses or more, then the single dose model is not effective.

Thus, there is a need in the art for a new rotavirus vaccine with a lower dose volume so that the space occupied by multiple dose vaccines can be smaller, resulting in cost savings, while reducing packaging due to thermal stability to address the cold chain problem. Another important advantage of developing such thermostable rotavirus vaccines is to ensure proper vaccine efficacy during immune exercise.

Summary of The Invention

The present invention overcomes the problems and disadvantages associated with current strategies and designs and provides novel vaccines, compositions, and methods for treating infections.

One of the present inventionEmbodiments relate to a method for producing a bulk vaccine, the method comprising: combining a buffering agent, a bulking agent, and an excipient comprising at least arginine to a virus-containing composition to form a mixture, preferably wherein the mixture has a viral titer of log 8 or greater to reduce total solids, although thermal stability is good even at lower titers; lyophilizing the mixture to form a lyophilized composition comprising less than or equal to about 0.8% moisture; and grinding the mixture to an approximately uniform particle size of 5 μm or less. Preferably, the virus of the virus-containing composition comprises a rotavirus, and it is also preferred that the rotavirus comprises multiple serotypes. Preferably, the buffer comprises a mixture of citrate and calcium carbonate and maintains the pH of the mixture at about 7.0-8.0, which does not change significantly after lyophilization. Significant changes are about 1.0 unit, about 0.5 unit, or about 0.1 unit or less. Preferably, the compatibilizer comprises glucose, sucrose, or both glucose and sucrose. Wherein the compatibilizer is preferably sucrose and the ratio of sucrose to arginine is about 1:1.0 to 1.1.5. Preferably, the mixture comprises about 2% to 6% arginine, and the mixture contains Medium Chain Triglyceride (MCT) oil. Preferably, the bulk vaccine of the present invention can be stored at room temperature for 2 years or more, at 50 ℃ for at least 3 months without any loss of efficacy; storage at 30 ℃ or higher for at least 24 months without significant loss of efficacy; and/or at up to 40 ℃ for at least 12 months without significant loss of efficacy. A significant loss in efficacy should be a loss of about 10%, preferably a loss of about 5%, more preferably a loss of about 1% or less. Preferably, the method further comprises partitioning the mixture to contain about 5.9log₁₀An individual dose of FFU/mL virus and a volume of about 0.5mL or less.

Another embodiment of the invention relates to a bulk vaccine produced according to the method disclosed herein.

Another embodiment of the invention relates to a bulk vaccine formulation comprising a lyophilized rotavirus strain and an excipient which provides thermostability at 30 ℃ for at least 2 years and at 50 ℃ for at least 3 months. Preferably, the bulk vaccine formulation has a uniform particle size of at least 90% of less than or equal to 5 μm,the remainder does not exceed 10 microns, is homogenized with Medium Chain Triglyceride (MCT) oil, and/or contains citrate and calcium carbonate as buffering agents. It is also preferred that the bulk vaccine contains about 5.9log₁₀An individual dose of FFU/mL of virus in a volume of about 0.5mL or less.

Additional embodiments and advantages of the invention are set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Description of the drawings

FIG. 1. an example of a formulation screening method using 96-well plates. Each column (1-9) has one formulation type (A-I). Different excipient rations were tested in different columns.

FIG. 2 accelerated stability studies of IVT-00 and IVT-01 formulations at 40 ℃.

Fig. 3. a typical freeze-drying cycle for an IVT-05 formulation, which includes an annealing step prior to primary drying. The color of the lines is depicted in the figure. The left and right Y-axes depict the temperature and vacuum scales, respectively.

FIG. 4. Table of accelerated stability studies at 40 ℃ of different batches of IVT-05 formulations, showing that decreasing moisture increases thermal stability.

FIG. 5. Table of accelerated stability studies of 116E rotavirus in formulations IVT-05 and IVT-06 containing 2% or 4% arginine at 30 ℃ for 12 months.

FIG. 6. Table of accelerated stability studies of 116E rotavirus in formulations IVT-05 and IVT-06 containing 2% or 4% arginine at 40 ℃ for 12 months.

FIG. 7. Table of accelerated stability studies of 116E rotavirus in formulations IVT-05 and IVT-06 containing 2% or 4% arginine at 50 ℃ for 3 months.

Figure 8 is a graph showing stability data for vials containing lyophilized 116E rotavirus in IVT-06 formulation subjected to accelerated stability studies at 30 ℃ (blue diamond) for 12 months and 50 ℃ (red circle) for 3 months. The equation for the linear trend line for each temperature is shown.

FIG. 9. Table of accelerated stability studies of rotavirus in formulation IVT-06 with 2% or 4% arginine at 50 ℃ for 3 months.

FIG. 10 shows the linear trend line equation obtained based on the data given in FIG. 8, and assumes a starting viral titer of 5.6 log₁₀{FFU/mL]Table of expected rotavirus titers in IVT-06 formulations after incubation at 30 ℃ and 50 ℃ for different times.

FIG. 11. expected potency of IVT-06 with 2% or 4% arginine over time based on the linear curve of the trend line equation.

Figure 12 is a graph showing the average buffer capacity (n =3) of 0.2M citrate with different concentrations of calcium carbonate in a 0.5mL IVT-06 final formulation, analyzed by the Baby Rossett-Rice determination (BRR). The final formulation consisted of micronized IVT-06 formulation in MCT oil and buffering ingredients. The horizontal black line at pH4.0 indicates the lowest pH at which the rotavirus was stable. The buffered mixture containing 80 mg of calcium carbonate was able to maintain the pH above 4.0 for 19-20 minutes (black arrow).

Examples