MONOVALENT VACCINE FORMULATION AND A METHOD FOR PREPARATION THEREOF

Abstract

The present invention discloses a vaccine formulation in accordance with an illustrative embodiment. The formulation including a live attenuated cholera vaccine strain VCUSM14P; a vaccine medium having starch, cellulose, dextrose, and yeast extract; and a phosphate buffer saline.

Claims

1. A method for preparing a monovalent cholera vaccine against 0139, the method comprising: culturing of live attenuated bacterial cells of strain in a vaccine growth medium; recovering the bacterial cells from the culture; and suspending the recovered bacterial cells in phosphate buffer saline.

2. The method of claim 1, wherein the wherein the vaccine medium comprising starch, cellulose, dextrose, and yeast extract.

3. The method of claim 1, wherein the vaccine medium comprising 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 a 1000 mL of phosphate buffer saline at pH 7.2+0.2

Description

BRIEF DESCRIPTION OF DRAWINGS

[0012] Other objects, features, and advantages of the invention will be apparent from the following description when read with reference to the accompanying drawings. In the drawings, wherein like reference numerals denote corresponding parts throughout the several views:

[0013] Figures

[0014] FIG. 1 illustrates a schematic of construction of a live attenuated cholera vaccine strain VCUSM14P, in accordance with an illustrative embodiment of a present invention;

[0015] FIG. 2A shows an image of unvaccinated rabbit; FIG. 2B shows an image of a rabbit vaccinated with an oral unformulated monovalent vaccine formulation; and FIG. 2C shows an image of a rabbit vaccinated with an oral formulated monovalent vaccine formulation, in accordance with the illustrative embodiment of the present invention.

TABLE

[0016] Table 01 represents a list of primers along with sequences thereof, along with annealing temperature and functions used for developing a monovalent vaccine formulation against 0139 challenge, in accordance with an illustrative embodiment of a present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Reference will now be made in detail to the preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings.

[0018] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those or ordinary skill in the art that the 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 invention.

[0019] The invention will be more clearly understood from the following description of the methods thereof, given by way of example only with reference to the accompanying drawings. In the descriptions that follow, like numerals represent like elements in all figures. For example, where the numeral (2) is used to refer to a particular element in one figure, the numeral (2) appearing in any other figure refers to the same element.

[0020] Cholera is an acute, diarrheal illness caused by infection of the intestine with the bacterium Vibrio cholerae. An estimated 3-5 million cases and over 100,000 deaths occur each year around the world. The infection is often mild or without symptoms, but can sometimes be severe. Approximately one in 10 (5-10%) infected persons can have severe disease characterized by profuse watery diarrhea, vomiting, and leg cramps. In such people, rapid loss of body fluids leads to dehydration and shock. Without treatment, death can occur within hours.

[0021] Currently, there is a number of vaccine formulations developed to get rid of the diarrheal illness. Such vaccines are whole-killed or live attenuated Vibrio cholera based. Both types of the vaccines have potential as vaccine candidate against 0139 V. cholerae. Such vaccines have capability of eliciting high antibody titers and protective immune responses. However, there are certain challenges with the vaccines. For example, the vaccines are cold chain dependent, therefore requiring repetitive dosage thereof. Such characteristics thereof lead to higher cost and also short lasting.

[0022] Therefore, the present invention discloses a live attenuated cholera vaccine formulation which is cold chain free leading to a single dosage thereof and hence lowers the cost involved. In some embodiments, the formulation has stability at room temperature, therefore long lasting. In some embodiments, the present invention discloses a method for preparing the live attenuated cholera vaccine formulation. The various growth-inhibiting stresses in the environment such as deprivation of nutrients, fluctuations in temperature, salinity and oxygen level elicit stringent responses in V. cholerae involving modulation of expression of several genes to mediate their adaptation, persistence, dissemination and transmission of cholera. Therefore such adoptive traits of V. cholerae can be leveraged at an in-vitro condition that best mimic the environmental stress conditions for the extended storage period of live attenuated cholera vaccine strain at ambient temperature.

[0023] In an embodiment, the present invention discloses a monovalent vaccine formulation against 0139. The formulation includes a live attenuated cholera vaccine strain grown in a vaccine medium. In some embodiments, the vaccine strain is VCUSM14P. VCUSM14P is an aminoleuvulinic acid (ALA) prototroph and non-toxigenic strain constructed against 0139 serogroup of V. cholerae. A protocol for constructing the ALA prototroph, VCUSM14P is depicted in FIG. 1. As shown in FIG. 1, the wild type hemA/M genes are excised from pARO-hemA/M through EcoRI restriction enzyme digestion. The 2.18 kb fragment containing the 5′ flanking hemA/M are then blunted with T4 DNA polymerase and cloned into a linearized suicide vector, pCVD442, at the Sall restriction enzyme site to produce pCVD-hemA/M. The successful cloning of pCVD-hemA/M confirmed by PCR using VHF (5-GAC CTG TGA TGT AAA GGA AC-3′) and VHR (5′-CTT CAT AGC GCT CAA CAA GG-3′) primers. The pCVD-hemA/M containing intact hemA/M gene is conjugationally transferred from the E. coli host, BW 20767 1-pir, to YCUSM14 strain. The resulting merodiploids possessing the suicide vector construct, pCVD-hemA/M, integrated into VCUSM14 chromosome as a result of the first crossover event, are selected on LB agar containing ampicillin and polymyxin B. The prototrophic strains with wild type hemA/M gene are selected using modified LB medium containing 15% sucrose. A strain that is sensitive to ampicillin, resistant to both polymyxin B and sucrose and shows an absence of ALAauxotrophy is selected and designated as VCUSM14P. Various primer sequences along with respective functions have been enlisted in Table 01.

[0024] Experiment

[0025] The sub culture of live attenuated cholera vaccine (VCUSM14P) is maintained on Sigma-Aldrich LB agar without any antibiotics and routinely propagated at 37° C. in LB broth (LB; 10 g/l tryptone, 5 g/l yeast extract, and 5 g/l sodium chloride). Wild Type (WT) V. cholerae 0319 Bengal strain is maintained on Oxoid Nutrient Agar composed of Bacto peptone 10 g/l, Lab lemco powder 10 g/l, sodium chloride 5 g/l and plain agar 15 g/l supplemented with polymyxin 0.75 g/ml. Thiosulfate citrate bile salts sucrose (TCBS) agar enumerates the VCUSM14P in intestinal colonization studies. The stock culture of strains is stored in 15% glycerol with brain heart infusion broth (BHIB, Difco) at −80° C. until used.

[0026] In some embodiments, the strain VCUSM14P is grown in a vaccine medium. The medium further comprising 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 a 1000 mL of phosphate buffer saline at pH 7.2+0.2.

[0027] In the above embodiments, the formulations mediate antibody and cytotoxic T cells.

[0028] In some embodiments, the present invention discloses a method for preparing the cholera vaccine formulation. The sequence of the steps of the method described hereinafter is exemplary in nature to understand the skill in the art. The method includes culturing of live attenuated bacterial cells of strain VCUSM14P in a vaccine growth medium. The method further includes recovering the bacterial cells from the culture. Finally, the recovered bacterial cells are suspended in a phosphate buffer saline.

[0029] Experiment

[0030] A single colony of VCUSM14P from stock culture purity plates is inoculated in 100 ml Erlenmeyer flask containing 20 ml LB broth and incubated at 37° C. for 24 hours in an orbital shaker set at 250 rpm. Bacterial growth is monitored using a spectrophotometer for OD measurements at 600 nm. Such culture inoculates 200 ml of fresh LB broth in a 1000 ml Erlenmeyer flask and incubates at the same conditions for 4 hours. Further, 4 hours culture inoculates 2 L vaccine growth medium.

[0031] In a 5 L bench top fermenter (BIOSTAT A Plus, Sartorius, Germany) the vaccine strain (VCUSM14P) is cultivated in the vaccine growth medium (LB broth supplemented with 1% starch and 0.3% cellulose) in a laboratory fermenter at 37° C., 0.6 vvm aeration and 150 rpm agitation for 48 hours. The cells are recovered by centrifugation at 10,000 rpm for 20 min at 4° C. The recovered bacterial cells are suspended in 200 ml of phosphate buffer saline (pH 7.2).

[0032] A sterile solution of excipients is prepared in 1000 ml of phosphate buffer saline (pH 7.2) supplemented with 5% starch, 1.5% cellulose, 20% dextrose and 0.05% yeast extract. The Live Attenuated Cholera Vaccine (LACV) formulation is done by aseptically mixing together 200 ml of the saline suspension of bacterial cells (6×108 CFU/ml) and 800 ml of the sterile solution of excipients. The formulation undergoes homogenously mixing and incubation at 37° C. for 48 h. After 48 hours, 5 ml aliquots of vaccine formulation is dispensed aseptically in 10 ml glass vials, closed with rubber stopper and stored at room temperature (25±2° C.).

[0033] Further, the storage stability of the formulation undergoes evaluation for its purity, potency and viability. The evaluation can be done by phenotypic and genotypic methods over an extended storage period of 180 days at 25° C.+2° C. and 60%+5% humidity.

[0034] The viability of the strain in the formulation is 2 logs lower after 180 days of storage as compared to its storage at room temperature (25° C.+2° C.) ie; 6×108 CFU/ml and the reduction of colonies may be attributed to the high humidity.

[0035] To identify the intact VCUSM14P culture in the formulation after 180 days of storage at 25° C.±2° C. and 60%+5%, PCR can be performed by using two different primer sets. PCR and gel electrophoresis ascertain the genetic purity of VCUSM14P culture in the cold chain free formulation.

[0036] The first PCR reaction is the Mctx reaction using ctxA112MS-F and ctxBCDS-R primer for the detection of the presence of the mutated ctxA gene in the samples to verify that the mutated ctxA gene does not revert back to the toxigenic form. All the samples containing culture in the formulations as well as the positive control which is the VCUSM14P strain from the glycerol stock (unformulated strain) have shown the band at 700 bps region, while no band is observed in the negative control column. Such a result indicates that mutated ctxA gene is present in the culture and it is free from contamination.

[0037] The second PCR reaction includes KanFse reaction. The KanFse reaction involves KanFse-2F and KanFse-R primers to detect the presence of the genetic marker inserted into VCUSM14P, which is the truncated aphA gene. aphA gene is a kanamycin resistance gene. All the samples containing culture in the formulations as well as the positive control show the band at 500 bps region. The presence of aphA gene in the formulation culture indicates it is a VCUSM14P strain which comparable to the positive control (VCUSM14P from glycerol stock (unformulated) strain.

[0038] During the extended storage at 25° C.±2° C. and 60%±5% humidity, the bacterial colonies from the LACV formulation are isolated periodically and streaked on TCBS agar. The colonies of VCUSM14P in formulation show 2-4 mm in diameter flat yellow colonies with opaque centers on TCBS agar. Gram staining shows the presence of Gram negative “comma” (curved rod) shaped cells under a light microscope. Besides, a series of biochemical tests are performed with HiVibrio™ identification kit to validate the identity of Vibrio cholerae (VCUSM14P) strain. All the above test results confirm the presence of a pure culture of VCUSM14P strain in the LACV formulation.

[0039] Since colonization is critical for elicitation of the immune response, the LACV formulation is examined for its colonization in the infant mouse model. The LACV formulation stored at 25° C.±2° C. and 60%±5% humidity for 180 days undergoes evaluation for its colonization potential in infant mouse model. In such an assay, 7×10.sup.7 CFU/ml of VCUSM14P is recovered after inoculation of 5×106 CFU/ml of formulation. The colonization potential of formulated VCUSM14P has one log higher recovery rate.

[0040] Experiment

[0041] Reactogenicity studies in rabbit can be carried out. In ligated ideal loop assay, loops injected with 10.sup.4, 10.sup.5 and 10.sup.6 CFU of LACV formulation can be recorded with significant decrease in fluid accumulation (0.2 Fluid Accumulation Ratio). Whereas, loops injected with 10.sup.4, 10.sup.5 and 10.sup.6 CFU of WT Vibrio cholerae 0139 record with 4 fold increase in fluid accumulation (0.8 Fluid Accumulation Ratio) and show the presence of hemorrhage. The LACV formulation is non-reactogenic at doses 10.sup.4-10.sup.6 in rabbit ideal loop model.

[0042] The vaccine formulation undergoes further evaluation for its protective capability in rabbit models. The protective capability can be determined using Reversible Intestinal Tie Adult Rabbit Diarrhoea (RETARD) model. RITARD Assay is performed on normal rabbits and rabbits immunized with LACV formulation by challenging with 10.sup.9 CFU/ml WT Vibrio cholerae 0139. Rabbits immunized with 10 ml of LACV formulation (5×10.sup.6 CFU/ml) do not show any sign of diarrhea or mortality up to 5 days at observation, whereas 100% mortality is observed on normal rabbits after 18 hours in RETARD model as shown in FIGS. 2A, 2B, and 2C. 2 male unvaccinated rabbits each of 3 kg are challenged with 1×10.sup.9 CFU/ml WT V. cholerae 0139 Bengal strain. After 18.sup.th hour, fluid accumulation in small intestine thereof is found as shown in FIG. 2A. 3 male rabbits each of 3.1 kg vaccinated with an oral 10 mL of unformulated VCUSMP14P show no signs of diarrhea after 6.sup.th day of vaccination. As shown in FIG. 2B, no fluid accumulates in small intestine thereof. Similarly, when 3 male rabbits each of 3.2 kg undergo vaccination with an oral 10 mL of formulated VCUSMP14P, the rabbits show no signs of diarrhea after 6.sup.th day of vaccination. As shown in FIG. 2C, no fluid accumulates in small intestine thereof.

[0043] The immune response of rabbits immunized with LACV formulation is evaluated by measuring anti-CT IgG antibodies. The anti-CT IgG is induced in rabbits vaccinated with LACV formulation when compared to pre-immune sera. An increase in IgG antibody response start at week 2 with increase of 6 fold and highest IgG response is recorded at week 4 with 17 fold in rabbits vaccinated with the LACV formulation.

[0044] Therefore, the LACV formulations are cold chain free, stable at 25° C.±2° C. and 60%+5% humidity for 180 days, non-reactogenic and immunogenic in vivo, and protect animals from lethal WT V. cholerae 0139 challenge.

[0045] While the preferred embodiment of the present invention and its advantages has been disclosed in the above Detailed Description, the invention is not limited there to but only by the scope of the appended claim.

[0046] As will be readily apparent to those skilled in the art, the present invention may easily be produced in other specific forms without departing from its essential characteristics. The present embodiments are, therefore, to be considered as merely illustrative and not restrictive, the scope of the invention being indicated by the claims rather than the foregoing description, and all changes which come within therefore intended to be embraced therein.