阅读说明：本技术 单价疫苗制剂及其制备方法 (Monovalent vaccine formulations and methods of making the same ) 是由 古鲁斯瓦米·巴拉卡兰 玛尼坎姆·拉维汉德兰 古鲁纳森·阿鲁·辛尼亚 恩恩·陈 于 2018-12-26 设计创作，主要内容包括：本发明公开了一种根据说明性实施方案的疫苗制剂。所述制剂包含：活的减毒霍乱疫苗株VCUSM14P；疫苗培养基,所述疫苗培养基具有淀粉、纤维素、右旋糖和酵母提取物；以及磷酸盐缓冲盐水。(The present invention discloses a vaccine formulation according to an illustrative embodiment. The formulation comprises: live attenuated cholera vaccine strain VCUSM 14P; a vaccine medium having starch, cellulose, dextrose, and yeast extract; and phosphate buffered saline.)

1. A monovalent vaccine formulation against vibrio cholerae O139, comprising:

a live attenuated cholera vaccine strain;

a vaccine medium having starch, cellulose, dextrose, and yeast extract; and

phosphate buffered saline.

2. The formulation of claim 1, wherein the strain is VCUSM 14P.

3. The formulation of claim 1, wherein the vaccine medium comprises starch (3.25-6.75%), cellulose (2.50-4%), dextrose (18.25-24.50%), yeast extract (0.01-0.05%) and NaCl (0.05-0.15%).

4. The formulation of claim 1, wherein the formulation comprises at least 1000mL of phosphate buffered saline having a pH of 7.2 ± 0.2.

5. The formulation of claim 1, wherein the formulation is in an oral liquid form.

6. The use of claim 1, wherein the formulation does not require a cold chain.

7. The use of claim 1, wherein the formulation has a storage stability of 180 days at 25 ℃ ± 2 ℃.

8. The use of claim 1, wherein the formulation is stable for 180 days at 60% ± 5% humidity.

9. The use of claim 1, wherein the formulation is non-reactogenic.

10. The use of claim 1, wherein the agent elicits antibody and cytotoxic cell-mediated immunity.

11. Use of a monovalent vaccine formulation comprising a live attenuated cholera vaccine strain grown in a vaccine medium having starch, cellulose, dextrose, and yeast extract in phosphate buffered saline for the treatment of wild-type vibrio cholerae O139.

12. The use according to claim 12, wherein the strain is VCUSM 14P.

13. Use of a monovalent vaccine formulation comprising a live attenuated cholera vaccine grown in a vaccine medium having starch (3.25-6.75%), cellulose (2.50-4%), dextrose (18.25-24.50%), yeast extract (0.01-0.05%), and NaCl (0.05-0.15%) in 1000mL of phosphate buffered saline at pH 7.2 ± 0.2, for treating wild-type vibrio cholerae O139.

14. The use according to claim 14, wherein the strain is VCUSM 14P.

15. The use of claims 11 and 12, wherein the formulation does not require a cold chain.

16. The use according to claims 11 and 12, wherein the formulation has a storage stability of 180 days at 25 ℃ ± 2 ℃.

17. Use according to claims 11 and 12, wherein the formulation is stable for 180 days at a humidity of 60% ± 5%.

18. Use according to claims 11 and 12, wherein the preparation is non-reactogenic.

19. The use of claims 11 and 12, wherein the agent is antibody and cytotoxic cell mediated.

20. A method for preparing a monovalent cholera vaccine against O139, the method comprising:

culturing live attenuated bacterial cells of the strain in a vaccine growth medium;

recovering the bacterial cells from the culture; and

the recovered bacterial cells were suspended in phosphate buffered saline.

21. The method of claim 20, wherein the vaccine medium comprises starch, cellulose, dextrose, and yeast extract.

22. The method of claim 20, wherein the vaccine medium comprises starch (3.25-6.75%), cellulose (2.50-4%), dextrose (18.25-24.50%), yeast extract (0.01-0.05%) and NaCl (0.05-0.15%) in 1000mL of phosphate buffered saline at pH 7.2 ± 0.2.

Technical Field

The present invention generally relates to a vaccine formulation and a method of preparing the same. More specifically, the present invention relates to monovalent vaccine formulations for the treatment of vibrio cholerae (v.cholerae). The preparation does not need cold chain and is durable, and the invention also discloses a preparation method of the preparation.

Background

Vibrio cholerae (Vibrio cholerae) is a gram-negative, comma-shaped bacterium. The natural habitat of the bacteria is brackish or brackish water. Some strains of Vibrio cholerae cause cholera disease. Cholera may be endemic, epidemic or pandemic. Despite significant advances in research, this condition remains a challenge to the modern medical community. Although the disease may be asymptomatic or mild, severe cholera can cause dehydration and death within hours of onset. Cholera (diarrheal disease) causes a global epidemic with an estimated 430 million deaths and 129064 deaths per year in 47 countries.

The bacteria inhabit the aquatic ecosystem and the host intestinal tract. In an aquatic environment, virulent and non-virulent strains of Vibrio cholerae survive all year round. The toxigenic strains typically cause acute diarrheal diseases by ingesting contaminated water or food into a human host, colonizing the small intestine, multiplying and producing cholera toxin. In endemic regions, large-scale oral vaccine campaigns are organized annually throughout the world. However, existing oral cholera vaccines approved by WHO in developing countries, e.g.Andthere are operational challenges to performing a cholera vaccination campaign. Such vaccines are based on whole cell, inactivated vibrio cholerae O1 and O139 serogroups. Such vaccines are safe and induceAdequate short term protection. However, vaccines require a cold chain supply (2-8 ℃) to ensure the efficacy of the vaccine from production to the site of immunization, resulting in 14-20% increase in vaccination costs. In addition, such vaccines must be used repeatedly, resulting in high costs. Alternatively, cholera vaccines based on live attenuated strains mimic natural infection and are highly immunogenic without the need for repeated administrations. Furthermore, it does not require complex downstream processing and is relatively inexpensive to produce compared to highly purified subunit and whole cell inactivated vaccines. Various growth-inhibiting stresses in the environment, such as nutritional deprivation, fluctuations in temperature, salinity and oxygen content, cause a strict response in Vibrio cholerae involving the regulation of the expression of several genes to mediate its adaptation, persistence, spreading and dissemination of cholera.

For example, european patent publication EP1650315B1 and U.S. patent publication US7838016B2 disclose vibrio cholerae strains VCUSM1 and VCUSM4, methods of producing the same, and vaccine derivatives thereof. The method involves mutations in the hemA genes of strains VCUSM1 and YCUSM4, which render Vibrio cholerae incapable of de novo synthesis of aminolevulinic acid (ALA). Such vaccines show potential as new avirulent vaccine candidates that are toxic to vibrio cholerae O139 and are capable of eliciting high antibody titers and protective immune responses. However, similar to other cholera vaccines, formulations made with live attenuated strains VCUSM1 and VCUSM4 are also sensitive to heat, requiring cold chain supply.

In another example, U.S. patent publication US4169886A relates to a live vaccine against pasteurella multocida (pasteurella multocida) for administration to poultry by injection, orally or as an aerosol, containing an attenuated, avirulent, genetically stable strain of pasteurella multocida; a method for producing an attenuated, genetically stable, avirulent, hemorrhagic pasteurella strain comprising culturing a virulent field strain at 37 ℃ for 18 hours prior to selection. However, this vaccine is primarily for poultry and is cold chain dependent.

Therefore, it is inevitable to develop a thermostable live attenuated cholera vaccine that can be stored at room temperature to increase its coverage for the global immune program. Like other microorganisms, the Vibrio cholerae bacterial strain is also a major adopter because it is viable in harsh natural environments. Thus, by revealing its adaptation/survival mechanism under natural conditions, we can simulate similar conditions when formulating vaccine candidates to allow them to be stored without cold chain conditions to serve the bottom billion people in the last mile.

Therefore, there is a need to develop a vaccine formulation that can dispense with the cold chain, avoiding repeated administrations, resulting in lower cost and longer life.

Disclosure of Invention

The present invention has been made in view of the above problems, and discloses a monovalent vaccine formulation against vibrio cholerae O139 according to an illustrative embodiment. The formulation comprises: live attenuated cholera vaccine strain VCUSM 14P; a vaccine medium having starch, cellulose, dextrose, and yeast extract; and phosphate buffered saline.

In another embodiment, the invention discloses the use of a monovalent vaccine formulation. The formulation comprises live attenuated cholera vaccine strain VCUSM14P grown in a vaccine medium with starch, cellulose, dextrose, and yeast extract in phosphate buffered saline. The formulation is formulated for the treatment of Vibrio cholerae wild type O139.

In another embodiment, the invention discloses the use of a monovalent vaccine formulation. The formulation comprises live attenuated cholera vaccine strain VCUSM14P grown in a vaccine medium having starch in the range of 1% -5% in 1000mL phosphate buffered saline (pH 7.2), at least 0.3% cellulose, at least 20% dextrose, and at least 0.5% yeast extract. The formulation is formulated for the treatment of Vibrio cholerae wild type O139.

In another embodiment, the invention discloses a method for preparing a monovalent vaccine formulation against O139. The method comprises culturing live attenuated bacterial cells of strain VCUSM14P in a vaccine growth medium. The method further comprises recovering the bacterial cells from the culture. Finally, the recovered bacterial cells were suspended in phosphate buffered saline.

Drawings

Other objects, features and advantages of the present invention will become apparent from the following description when read in light of the accompanying drawings. In the drawings, wherein like reference numerals designate corresponding parts throughout the several views:

drawings

FIG. 1 illustrates a schematic diagram of the construction of live attenuated cholera vaccine strain VCUSM14P, according to an illustrative embodiment of the present invention;

FIG. 2A shows an image of an unvaccinated rabbit; figure 2B shows an image of a rabbit vaccinated with an unformulated oral monovalent vaccine formulation; and figure 2C shows an image of a rabbit vaccinated with a formulated oral monovalent vaccine formulation according to an exemplary embodiment of the present invention.

Table form

Table 01 represents a list of primers and their sequences, as well as annealing temperatures and functions used to develop monovalent vaccine formulations against O139 challenge according to illustrative embodiments of the invention.

Detailed Description

Reference will now be made in detail to the preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings.

In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be understood by those of ordinary skill in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and/or components have not been described in detail so as not to obscure the present invention.

The invention will be more clearly understood from the following description of the method of the invention, given by way of example only with reference to the accompanying drawings. In the description below, like numbers refer to like elements throughout. For example, in one figure, the number (2) is used to refer to a particular element, and the number (2) appearing in any other figure also refers to the same element.

Cholera is an acute, diarrheal disease caused by intestinal infection with vibrio cholerae. It is estimated that 3-5 million deaths occur annually worldwide, over 100,000 deaths. Infections are often mild or asymptomatic, but can sometimes be severe. One of approximately 10 infected persons (5-10%) may have severe disease characterized by massive watery diarrhea, vomiting, and leg cramps. In such humans, rapid loss of body fluids leads to dehydration and shock. Without treatment, death can occur within hours.

Currently, many vaccine formulations have been developed to get rid of diarrheal diseases. Such vaccines are based on whole inactivated vibrio cholerae or live attenuated vibrio cholerae. Both types of vaccines have potential as vaccine candidates against vibrio cholerae O139. Such vaccines have the ability to elicit high antibody titers and protective immune responses. However, vaccines present certain challenges. For example, vaccines are cold chain dependent and therefore require repeated administration. Such characteristics lead to higher costs and shorter durations.

Thus, the present invention discloses a live attenuated cholera vaccine formulation that does not require a cold chain, resulting in a single dose thereof, thus reducing the costs involved. In some embodiments, the formulation is stable at room temperature and therefore long lasting. In some embodiments, the present invention discloses a method of making a live attenuated cholera vaccine formulation. Various growth-inhibiting stresses in the environment, such as nutritional deprivation, fluctuations in temperature, salinity and oxygen content, cause a strict response in Vibrio cholerae involving the regulation of the expression of several genes to mediate its adaptation, persistence, spreading and dissemination of cholera. Thus, such an adoptive trait of vibrio cholerae can be exploited under in vitro conditions that best mimic environmental stress conditions, for long-term storage of viable attenuated cholera vaccine strains at ambient temperature.

In one embodiment, the invention discloses a monovalent vaccine formulation against O139. The formulation comprises a live attenuated cholera vaccine strain grown in a vaccine medium. In some embodiments, the vaccine strain is VCUSM 14P. VCUSM14P is a non-productive strain of aminolevulinic acid (ALA) prototroph constructed against the O139 serogroup of Vibrio cholerae. The scheme for constructing ALA prototype VCUSM14P is depicted in fig. 1. The wild-type hemA/M gene was excised from pARO-hemA/M by EcoRI restriction enzyme digestion as shown in FIG. 1. The 2.18kb fragment containing the 5' flanking hemA/M was then blunted with T4 DNA polymerase and cloned into the SalI restriction enzyme site in the linear suicide vector pCVD442 to yield pCVD-hemA/M. Successful cloning of pCVD-hemA/M was confirmed by PCR using VHF (5'-GAC CTG TGA TGT AAA GGA AC-3') and VHR (5'-CTT CAT AGC GCT CAA CAAGG-3') primers. pCVD-hemA/M containing the entire hemA/M gene was conjugately transferred from E.coli (E.coli) host BW 20767. lambda. -pir to strain VCUSM 14. The resulting merodiploid having suicide vector construct pCVD-hemA/M integrated into the chromosome of VCUSM14 as a result of the first crossing event was selected on LB agar containing ampicillin and polymyxin B. A modified LB medium containing 15% sucrose was used to select for prototrophic strains with the wild-type hemA/M gene. A strain sensitive to ampicillin, resistant to both polymyxin B and sucrose and showing the absence of ALA auxotrophy was selected and designated VCUSM 14P. The various primer sequences and the respective functions have been listed in table 01.

Experiment of

Secondary cultures of live attenuated cholera vaccine (VCUSM14P) were maintained on Sigma-Aldrich LB agar without any antibiotics and propagated routinely in LB broth (LB; 10g/l tryptone, 5g/l yeast extract and 5g/l sodium chloride) at 37 ℃. Wild Type (WT) Vibrio cholerae 0319 Bengal strain is maintained on Oxoid nutrient agar consisting of Bacto peptone 10g/l, beef extract powder (Lab lemco powder)10g/l, sodium chloride 5g/l and regular agar 15g/l supplemented with polymyxin 0.75 g/ml. In the intestinal colonization study, VCUSM14P was counted using thiosulfate citrate bile salt sucrose (TCBS) agar. Stock cultures of the strains were stored at-80 ℃ in 15% glycerol with brain heart infusion broth (BHIB, Difco) until use.

In some embodiments, strain VCUSM14P is grown in vaccine medium. The medium contained starch (3.25-6.75%), cellulose (2.50-4%), dextrose (18.25-24.50%), yeast extract (0.01-0.05%) and NaCl (0.05-0.15%) in 1000mL of phosphate buffered saline at pH 7.2 ± 0.2.

In the above embodiments, the agent mediates antibodies and cytotoxic T cells.

In some embodiments, the present invention discloses a method of making a cholera vaccine formulation. The order of the steps of the methods described below is exemplary in nature to understand techniques in the art. The method comprises culturing live attenuated bacterial cells of strain VCUSM14P in a vaccine growth medium. The method further comprises recovering the bacterial cells from the culture. Finally, the recovered bacterial cells were suspended in phosphate buffered saline.

Experiment of

A single colony of VCUSM14P from stock culture purity plates was inoculated into a 100ml Erlenmeyer flask (Erlenmeyer flash) containing 20ml LB broth and incubated at 37 ℃ for 24 hours in an orbital shaker set at 250 rpm. Bacterial growth was monitored using a spectrophotometer to make OD measurements at 600 nm. Such cultures were inoculated in 200ml fresh LB broth in 1000ml Erlenmeyer flasks and incubated for 4 hours under the same conditions. In addition, 4 hours culture inoculated 2L vaccine growth medium.

In a 5L bench-top fermenter (BIOSTAT a Plus, Sartorius, Germany), the vaccine strain (VCUSM14P) was incubated in a vaccine growth medium (LB broth supplemented with 1% starch and 0.3% cellulose) in a laboratory fermenter at 37 ℃, 0.6vvm aeration and 150rpm stirring for 48 hours. The cells were recovered by centrifugation at 10,000rpm for 20 minutes at 4 ℃. The recovered bacterial cells were suspended in 200ml of phosphate buffered saline (pH 7.2).

Sterile excipient solutions were prepared in 1000ml of phosphate buffered saline (pH 7.2) supplemented with 5% starch, 1.5% cellulose, 20% dextrose and 0.05% yeast extract. Live Attenuated Cholera Vaccine (LACV) formulation was accomplished by aseptically mixing 200ml of a bacterial cell saline suspension (6X 108CFU/ml) and 800ml of a sterile excipient solution together. The formulations were mixed homogeneously and incubated at 37 ℃ for 48 hours. After 48 hours, 5ml aliquots of the vaccine formulation were aseptically dispensed into 10ml glass vials, sealed with rubber stoppers and stored at room temperature (25 ℃ ± 2 ℃).

In addition, the purity, potency and viability of the formulations were evaluated for storage stability. Evaluation can be carried out by phenotypic and genotypic methods at 25 ℃. + -. 2 ℃ and 60%. + -. 5% humidity over an extended storage period of 180 days.

The viability of the strains in the formulation was reduced by 2log after 180 days of storage compared to storage at room temperature (25 ℃ ± 2 ℃); 6X 108CFU/ml, the colony reduction can be attributed to high humidity.

To identify the complete VCUSM14P culture in the formulation after 180 days of storage at 25 ℃ ± 2 ℃ and 60% + 5%, PCR can be performed using two different primer sets. PCR and gel electrophoresis determined the genetic purity of the VCUSM14P culture in formulations that did not require cold-chain.

The first PCR reaction was a mtx reaction using ctxA112MS-F and ctxBCDS-R primers to detect the presence of the mutated ctxA gene in the sample to verify that the mutated ctxA gene does not revert back to a toxigenic form. All culture-containing samples in the preparation as well as the positive control, which was the VCUSM14P strain (unformulated strain) from the glycerol stock, showed bands in the 700bps region, while no bands were observed in the negative control column. Such results indicate that the mutated ctxA gene is present in the culture and that it is free of contamination.

The second PCR reaction included the KanFse reaction. The KanFse reaction involved KanFse-2F and KanFse-R primers to detect the presence of the genetic marker inserted into VCUSM14P, which is a truncated aphA gene. The aphA gene is a kanamycin resistance gene. All samples containing cultures in the preparation and the positive control showed bands in the 500bps region. The presence of the aphA gene in the formulation culture indicated it to be a strain of VCUSM14P comparable to the positive control (VCUSM14P from the glycerol stock (unformulated) strain).

Bacterial colonies in LACV formulations were isolated periodically and streaked on TCBS agar during long storage periods at 25 ℃ ± 2 ℃ and 60% ± 5% humidity. Colonies of VCUSM14P in the formulation showed flat yellow colonies with diameters of 2-4mm with an opaque center on TCBS agar. Gram stain in optical microscopeThe presence of gram-negative "comma" (bent rod) shaped cells is shown below. In addition, HiVibrio is used^TMThe identification kit carries out a series of biochemical tests to verify the characteristics of the strain of Vibrio cholerae (VCUSM 14P). All the above experimental results confirmed the presence of a pure culture of strain VCUSM14P in the LACV formulation.

The potential for colonization in a young mouse model of a LACV formulation stored for 180 days at 25 ℃. + -. 2 ℃ and 60%. + -. 5% humidity was evaluated in this assay, which was recovered to 7 × 10 after inoculation with 5 × 106CFU/ml formulation⁷CFU/ml of VCUSM 14P. The colonization potential of formulated VCUSM14P has a one log higher recovery.

Experiment of

Reactogenicity studies can be performed in rabbits. In the ligation-ideal loop assay, 10 can be injected⁴、10⁵And 10⁶The rings of LACV formulation of CFU were recorded with significantly reduced fluid accumulation (0.2 fluid retention rate). However inject 10⁴、10⁵And 10⁶The loop of CFU wild-type vibrio cholerae O139 recorded a 4-fold increase in fluid accumulation (0.8 fluid accumulation rate) and indicated the presence of bleeding. In the ideal loop model of rabbits, the LACV formulation was at 10⁴-10⁶Has no reactogenicity under the dosage of (1).

The vaccine formulations were further evaluated for their protective ability in a rabbit model. Protective capacity can be measured using a reversible intestinal ligation rabbit diarrhea (RITARD) model. By using 10⁹The RITARD assay was performed on normal rabbits and rabbits immunized with the LACV preparation using a CFU/ml challenge with wild type Vibrio cholerae O139 as shown in FIGS. 2A, 2B and 2C, using 10ml of the LACV preparation (5 × 10)⁶CFU/ml) immunized rabbits did not show any signs of diarrhea or death by day 5, whereas in the RITARD model, 100% mortality was observed in normal rabbits after 18 hours 1 × 10⁹2 unvaccinated male rabbits, 3kg each, were challenged with CFU/ml of the wild type Vibrio cholerae O139 Bengal strain. After 18 hours, fluid accumulation was found in the small intestine, as shown in fig. 2A. 3 oral 10mL of unformulated VCUSMP14PVaccinated male rabbits (3.1 kg each) showed no signs of diarrhea after day 6 of vaccination. As shown in fig. 2B, there was no fluid accumulation in the small intestine. Similarly, when 3 male rabbits (3.2 kg each) were vaccinated with 10mL of formulated VCUSMP14P orally, the rabbits showed no signs of diarrhea after day 6 of vaccination. As shown in fig. 2C, there was no fluid accumulation in the small intestine.

The immune response of rabbits immunized with LACV preparation was assessed by measuring anti-CT IgG antibodies. anti-CT IgG was induced in rabbits vaccinated with the LACV formulation when compared to preimmune serum. In rabbits vaccinated with LACV preparations, the increase in IgG antibody response increased 6-fold starting at week 2, and the highest IgG response of 17-fold was recorded at week 4.

Thus, LACV formulations are stable for 180 days at 25 ℃ ± 2 ℃ and 60% + 5% humidity without the need for a cold chain, are non-reactogenic and immunogenic in vivo, and protect animals from lethal wild-type vibrio cholerae O139 challenge.

Although the preferred embodiments of the present invention and their advantages have been disclosed in the foregoing detailed description, the invention is not limited thereto but only by the scope of the appended claims.

It will be readily apparent to those skilled in the art that it is possible to embody the invention in other specific forms without departing from the essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.