WO2020008475A1

WO2020008475A1 - Novel shigella vaccine formulation and process to prepare thereof

Info

Publication number: WO2020008475A1
Application number: PCT/IN2019/050492
Authority: WO
Inventors: Tarun Sharma; Nerraj JOSHI; Ashwini Kumar MANDYAL; Saugata ROY; Shushruta BHUNIA; Hemanta Koley; Shanta DUTTA; Davinder Gill
Original assignee: Msd Wellcome Trust Hilleman Laboratories Pvt. Ltd.
Priority date: 2018-07-02
Filing date: 2019-07-02
Publication date: 2020-01-09
Also published as: KR20210069622A; IL279878A; BR112020027067A2

Abstract

The present invention relates to novel Shigella vaccine formulation and process to prepare said vaccine formulation. More particularly, the present invention relates to inactivated whole cell Shigella vaccine formulation comprising whole cell inactivated Shigella strain selected from Shigella flexneri 2a, Shigella 3a, Shigella 6 and Shigella sonnei, alone or in combination with or without adjuvant and a process to prepare thereof.

Description

NOVEL SHIGELLA VACCINE FORMULATION AND PROCESS TO

PREPARE THEREOF

FIELD OF THE INVENTION

The present invention relates to novel Shigella vaccine formulation and process to prepare said vaccine formulation. More particularly, the present invention relates to inactivated whole cell Shigella vaccine and process to prepare thereof.

BACKGROUND OF THE INVENTION

Shigellosis is an important cause of morbidity and mortality among preschool- aged children, older children, and adults. Recent studies in sub-Saharan Africa and South Asia conducted under the Global Enteric Multicenter Study (GEMS) reaffirmed the importance of Shigella as a major cause of moderate-to-severe diarrhea (MSD). Shigella was among the top four causes of potentially life- threatening diarrheal illness among the children less than five years old who were brought to a center for treatment of diarrhea in both said regions.

Shigellosis is caused by the ingestion of bacteria of the genus Shigella. Three species of Shigella are responsible for most of infections: S. flexneri is the most frequently isolated species worldwide, accounting for most cases in the least- developed countries; S. sonnei is more common in low- and middle-income countries; and S. dysenteriae has historically caused epidemics of dysentery, particularly in confined populations such as refugee camps. A fourth species, S. boydii, a cause of infection in less-developed countries, accounts for 6 percent or less of Shigella cases.

In 2016, Shigella was responsible for 75,000 deaths among children under-5 and 270,000 deaths among all ages. Shigella ranked second with regard to pathogen contributions to global diarrheal deaths. Therefore, there is an urgent need of effective licensed Shigella vaccine to protect humans against the significant worldwide morbidity and mortality caused by this microorganism.

The vaccine development is biologically feasible is evident from naturally acquired immunity, also from animal models or in vitro data. Any successful diarrheal disease control strategy against Shigella needs to employ all effective prevention and treatment interventions, including vaccines, improved sanitation/ hygiene, and breastfeeding to help ensure long-term success and maximum impact. However, in more short-term cost-benefit analyses, many public health stakeholders view vaccination as one of the most equitable and cost-effective preventive interventions since more people can receive the benefit for every dollar invested.

Unfortunately, there are currently no licensed vaccines available for Shigella, but human challenge study results and a sharp decline in agent-specific attack rates with increasing age indicate that solid protective immunity does develop. Studies also indicate that protective immunity is directed against the O-specific polysaccharide (O-SP) antigen of the Shigella cell wall lipopolysaccharide (LPS), and is type-specific. Furthermore, animal studies with experimental vaccines have shown that immune protection against Shigella infection is possible.

The Shigella vaccines under development span a spectrum of approaches and antigens. Almost all Shigella vaccines include the O-specific lipopolysaccharide (LPS) whether in a cellular or subunit format, which is considered a protective antigen, but this antigen restricts vaccine efficacy to only homologous or cross- reactive serotypes. In theory, broad coverage with an LPS-based vaccine can be achieved by including LPS from the five serotypes (Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, S. sonnei, and S. dysenteriae 1) that are the most prevalent and demonstrate some level of cross-reactivity with other prevalent serotypes. Conserved proteins such as the invasion plasmid antigens (IpaB, IpaC, and IpaD) or OmpA are also dominant antigens recognized by the immune system after natural infection and are attractive vaccine components because of inherent structural similarities within all Shigella species. A vaccine that stimulates an immune response, presumably a mucosal response, to both LPS and the conserved Ipa proteins would mimic the specificity of the immune response observed after natural infection. Two categories of Shigella vaccine candidates that have the potential to stimulate such a comprehensive immune response are live-attenuated and inactivated whole cell vaccines. Inactivated whole-cell Shigella vaccines including heat-killed, acetone-killed, and formalin- inactivated bacteria have been evaluated in several studies encompassing small animals, nonhuman primates, and humans.

Protection is consistently observed in various animal models for all inactivation methods, which has justified interest in this approach as a promising vaccine for shigellosis. Furthermore, current good manufacturing practices (cGMP) manufacture of formalin-inactivated S. sonnei with an uncomplicated manufacturing process permitted clinical evaluation in human volunteers in which both a mucosal and a systemic immune response to Shigella antigens is induced after oral immunization.

The formulation of the present invention comprises one or more composition, one or more pharmaceutically acceptable components/excipients. The pharmaceutically acceptable excipients can be buffer, preservative, stabilizer, surfactant, either alone or in combination. The formulation of the present invention may or may not contain adjuvants. The formulation of present invention is liquid or lyophilized or a combination with mono- or multi-dose regimen with or without a preservative. Although the four (4) Shigella species are divided into more than 47 serotypes, only certain types are considered to be important causal agents of human Shigellosis. Since S. dysenteriae, associated in the past with epidemic outbreaks, is now rare, a Shigella vaccine targeting protective antigens from these types, including S. flexneri 2a, 3a, and 6, as well as S. sonnei should cover a large majority of Shigella infections.

Mucosal immunity is required for optimal protection, so most experimental vaccines, with the exception of conjugates, have been given by the mucosal route.

There are number of patent and non-patent literature which discloses the vaccine against shigella, For instance, one such patent application is WOUS08005342 titled "Combinations Of Gene Deletions For Live Attenuated Shigella Vaccine Strains" discloses live attenuated Shigella vaccine whose primary attenuating feature is deletion of the virG(icsA) gene and additional two or more deletions in setAB(shETl), senA(shET2), senB(shET2- 2), stxAB, and msbB2 genes. In contrast to above, the present invention provides a whole cell inactivated vaccine which is relatively easy to manufacture, in-expensive, safer due to inactivation and its administration is patient compliant.

Also, there are non-patent literature titled "Development and Preclinical Evaluation of a Trivalent, Formalin-Inactivated Shigella Whole-Cell Vaccine" by Kaminski et al discloses trivalent Inactivated whole cell Shigella vaccine, whereas the present invention is a tetravalent vaccine providing wide immunity or protection against four most prevalent species of Shigella.

The leading candidate vaccines for shigellosis thus far are polysaccharide conjugates, synthetic conjugates, ribosomal subunits, invasion complex-based, outer membrane vesicles and live attenuated vaccines. Oral administration of inactivated whole cells is another strategy for developing an effective vaccine against shigellosis. Present invention discloses potential inactivated whole-cell vaccines providing the advantages of being relatively inexpensive also its administration does not require needles. The delivery of oral shigella vaccines would invariably require buffer administration to neutralize stomach acid. The present invention overcomes such requirement and provides a vaccine with ease of administration.

OBTECT OF THE INVENTION

In order to obviate the drawbacks in the existing state of art, the main object of present invention is to provide a novel shigella vaccine formulation.

Another object of the present invention is to provide novel inactivated whole cell shigella vaccine formulation.

Yet another object of the present invention is to provide a novel shigella vaccine formulation which is efficacious, cost effective and easy to administer/ deliver.

Yet another object of the invention is to provide high biomass for all Shigella strains by optimized fermentation.

Yet another object of the invention is to provide shigella vaccine formulation with enhanced immune response achieved by the use/not use of selective adjuvants.

Yet another object of present invention is to present the vaccine formulation which may or may not be enteric coated.

Yet another object of the present invention is to provide a process of preparing novel shigella vaccine formulation. Yet another object of the present invention is to provide a process of preparing a novel shigella vaccine formulation which is simple, efficient, improved and commercially scalable method.

Yet another object of the invention is to provide a process of preparing shigella vaccine formulation with enhanced immune response achieved by the use of selective adjuvants.

Yet another object of the present invention is to produce high quality vaccine that meet the relevant WHO specifications.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a novel shigella vaccine formulation and process to prepare thereof. More particularly, the present invention relates to inactivated whole cell shigella vaccine formulation and process to prepare thereof.

The formulation of the present invention comprises one or more composition, one or more pharmaceutically acceptable components/excipients. The pharmaceutically acceptable excipients can be buffer, preservative, stabilizer, surfactant, either alone or in combination. The formulation of the present invention may or may not contain adjuvants. The formulation of present invention is liquid or lyophilized or a combination with mono- or multi-dose regimen with or without a preservative.

Shigella strains, namely S. flex 2a, S. Flex 3a, S. Flex 6, a single congo red positive colony is transferred to 50 ml TSB media in Erlenmeyer flask incubating at 37°C±2°C temperature and 200±20 rpm till the OD600nm is reached 0.9-1.5/ml. for about 4 to 6 hours. For Shigella strain, namely S. sonnei multiple single congo red positive colonies are picked from Congo red TSB agar plate, mixed with 5ml of TSB media and used directly to inoculate the fermenter. This 50 ml of the inoculum for Shigela Flex 2a, 3a 6 and 5 ml of the inoculum for S. sonnie is used to inoculate 2.5-liter fermenter with TSB media separately in different fermenter. pH of the fermenter is maintained at 7.2±0.2, temperature at 37°C±2°C and aeration of 2 reactor volumes/ min, stirring at 350-1000 and a feed containing TSB media with 1M Glucose is started after 2 to 3 hours post transfer of inoculum. The feed is provided at rate of 0.2 ml/ min to 0.5 ml/ min.

After inoculum transfer to the fermenter, once culture attains the required optical density, the feeding is initiated with feeding agent. After 8 to 11 hours the culture optical density at 600nm of 25-30 is achieved. At this stage fermenter culture is harvested and centrifuged at 7000 to 7500 rpm at a temperature of 2°C±8°C for 20 to 30 mins. The pellets so obtained after centrifugation are resuspended in a sterile Hanks' Balanced Salt Solution (HBSS) solution and adjusted to OD 600nm at 16-20. An inactivating agent 0.1% to 1.0% v/ v formalin is added to the culture and kept for 20 to 36 hours. The resultant inactivated culture is washed three to four times with a PBS buffer solution and the optical density is adjusted. Gram staining is done to check the purity of the culture. Expression of Ipa proteins is confirmed in the inoculum and fermentation culture before and after inactivation by western blot.

In one of the embodiments of the invention, the novel shigella vaccine is in liquid state, solid state or a combination of both. In one of the embodiments of the invention, the novel shigella vaccine can be administered to adults, children and other age groups of population.

In one of the embodiments of the invention is to formulate a vaccine which eliminates the necessity of buffer administration at the time of delivery. In one of the embodiments of the invention, the vaccine is administered orally or parenterally or by any other suitable mode of administration.

Therefore, the present invention provides a novel inactivated whole cell shigella vaccine which is cost effective, efficacious and easy to administer and provides protection against the significant worldwide morbidity and mortality caused by Shigella species.

BRIEF DESCRIPTION OF DRAWINGS:

Figure 1 depicts Fermentation growth curve of 2 batches of Shigella flexneri 2a in

Tryptic Soya broth media, Animal origin, BD

Figure 2 depicts Fermentation growth curve of 2 batches of Shigella flexneri 3a in

Tryptic Soya broth media, animal origin, BD

Figure 3 depicts Fermentation growth curve of 2 batches of Shigella flexneri 6 in

Tryptic Soya broth media, Animal Origin, BD

Figure 4 depicts Fermentation growth curve of 2 batches of Shigella sonnei in

Tryptic Soya broth media, Animal origin, BD

Figure 5 depicts Western blot with Ipa D monoclonal antibody, showing preserved conserved ipaD protein post formalin inactivation Figure 6 depicts the presence of Virulence genes in the fermentation harvest of

Shigella strains flex.2a, 3a, 6 and sonnei

DETAILED DESCRIPTION OF INVENTION:

Accordingly, the present invention provides a novel shigella vaccine formulation and process to prepare thereof. More particularly, the present invention relates to inactivated whole cell shigella vaccine and process to prepare thereof.

Before the preferred embodiment of the present invention is described, it is understood that this invention is not limited to the particular materials described, as they may vary. It is also understood that the terminology used herein is for the purpose of describing the particular embodiment only and is not intended to limit the scope of the invention in any way.

It must be noted that as used herein, the singular forms "a", "an" and "the" include plural reference unless the context clearly dictates otherwise.

The terms "vaccine" and "vaccine formulation" have been used interchangeably throughout the specification.

Shigella strains, namely S. flex 2a, S. Flex 3a, S. Flex 6, a single congo red positive colony is transferred to 50 ml TSB media in Erlenmeyer flask incubating at 37°C and 180 rpm till the culture OD600nm reaches 0.9-1.0/ml in about 4 to 6 hours. For Shigella strain, namely S. sonnei multiple single Congo red positive colonies are picked from Congo red TSB agar plate, mixed with 5ml of TSB media and used directly to inoculate the fermenter.

This 50 ml of the inoculum for Shigella Flex 2a, 3a 6 and 5 ml of the inoculum for S. sonnie is used to inoculate 2.5-liter fermenter with TSB media separately in different fermenter. pH of the fermenter is maintained at 7.2 at 37°C and aeration of 2 reactor volumes/ min, stirring at 350-1000 and a feed containing TSB media with 1M Glucose is started after 2 to 3 hours post transfer of inoculum. The feed is provided at rate of 0.2 ml/ min to 0.5 ml/ min.

Fermentation continues for approximately 8 to 11 hrs, i.e. after reaching the final culture OD at 600nm of 25-30. The harvest culture is collected and centrifuged at 7000-7500rpm for 20 to 30 min at 2-8°C. In case of commercial manufacturing scale the centrifugation step can be replaced for buffer exchange/ dialysis by tangential flow microfiltration or hollow fiber filter technology.

The use of glucose in the feed and the feeding strategy used results in high biomass production i.e. optical density of 25-30 with intact virulence plasmid in whole cell shigella bacteria resulting in expression of virulence protein (ipaBCD) on the surface of bacteria in the fermenter culture and harvest. Presence of virulence gene and its expression is confirmed at each process step by PCR and western blot using monoclonal antibody. This high biomass process directly reduces the overall production cost of the vaccine.

The cell pellet so obtained from the centrifugation of fermenter culture are resuspended in a sterile HBSS solution and OD 600nm is adjusted to 16-20. An inactivating agent 0.1% to 1.0% v/ v formalin is added to the culture and kept for 20 to 36 hours at 25°C with shaking at 150-200rpm. The resultant inactivated culture is three to four times washed by repeated centrifugation and resuspending the pellet in PBS. Finally, the cell pellet is resuspended with PBS buffer solution, alternatively by performing tangential flow microfiltration dialysis using PBS buffer. The centrifugation followed by buffer exchange or diafilteration using microfilters is performed to reduce the residual formalin content at per the EU pharmacopeial requirement for other oral vaccines of less than 6.7mM. The individual inactivated bulk is stored at 2-8°C prior to mixing for preparation of adjuvanted or non-adjuvanted tetravalent vaccine. The genes VirF and IpaBCD are virulence genes of Shigella and are present on the 213Kb virulence plasmid of Shigella. A positive PCR of these two genes confirms that the virulence plasmid is present in Shigella. The bulk is tested for LPS content, presence of conserved proteins Ipa BCD, residual formalin and sterility.

In one of the embodiments of the invention, the novel shigella vaccine is in liquid state, solid state or a combination of both.

In one of the embodiments of the invention, the novel shigella vaccine can be administered to adults, children and other age groups of population.

In one of the embodiments of the invention is to formulate a vaccine which eliminates the necessity of buffer administration at the time of delivery. In one of the embodiments of the invention, the vaccine is administered orally or parenterally or by any other suitable mode of administration. The above detailed High-level process flow for four shigella bulk and adjuvant production in shown below in a flowchart manner:

Formulation:

Each of the four monovalent bulk with or without adjuvant are mixed at equal quantity based on analytical results. The mice dose of final formulated cocktail vaccine is tested in animal challenge and immunogenicity study.

The above detailed description of process is illustrated by non-limiting experiments as described below: Fermentation process: (Shigella flexneri 2a)

Fermentation and Flask Media -TSB

Note: dissolved in 1000 ml of

pH-7.2 with 5M NaOH, Autoclave

121°C for 20 minutes. Fermentation Feed -TSB Animal Origin-BD with 1M Glucose

Note: dissolved TSB in 500 ml of MQW & Autoclave

Dissolved D Glucose in 500 ml of MQW & 0.2um filter. Mix Both in 1 Ltr sterile Glass Bottle. The working cell bank vial of Shigella Flexneri 2a, kept at -80°C, is thawed and this is then streaked onto the Congo Red TSB agar plate using sterile loop. After steaking the plate, it is incubated at 37°C overnight.

Following day Inoculum for fermentation of the strain flexneri 2a is made by inoculating 50 ml TSB media in Erlenmeyer flask by single red positive S. flexneri 2a colony and incubating at 37°C, with shaking at 180 rpm till the OD600nm reached 0.9-1.0/ ml. Approximately 50 ml of the inoculum is used to inoculate 2.0-2.5 liter of TSB medium in a 5-liter fermenter. pH of the fermenter is maintained at 7.2 to 7.4, temperature at 37°C, aeration of 2 reactor volumes / min, stirring at 350-1000 and a feed containing TSB media with 1M Glucose is started after 2 to 3 hours at rate of 0.2 ml/min to 0.5 ml/min. Antifoam 204 (Sigma) diluted to 10% in water is used to control foaming.

At the completion of fermentation for approximately 8 to 11 Hrs, i.e. after reaching the final OD at 600nm of 25-30. The harvest culture pellet is collected by centrifugation at 7000-7500rpm for 20 to 30 min at 2-8°C. The pellet is resuspended in 500-1000 ml Hanks balanced salt solution (HBSS, without phenol red, with calcium and magnesium chloride) and the OD at 600nm is adjusted to 16-18. 0.2% to 1% v/v formaldehyde solution (37%) is added to the cell suspension for inactivation. After addition of formalin the flask or bottle is placed in shaker incubator at 25°C with agitation at 200rpm for 20 - 36 hrs. After 20-36 hours inactivated bacteria are centrifugation at 7500 rpm for 20-30 min at 2-8°C, cells are resuspended in PBS and washed 3-4 times with PBS. Post reconstitution with PBS complete inactivation is checked by plating on TSA plate. TSA plates is incubated at 37°C, no growth is first confirmed at 24 hours of incubation and then verified again at 48 hours.

The growth curve as obtained by Shigella flexneri 2a is as shown in Figure 1.

Antigen expression: Western blot assays is used to monitor the expression of IpaB, IpaC, IpaD in the formalin treated final bulk sample. The expression of IpaD in the formalin treated final bulk sample is confirmed and is shown in Figure 5.

Two consistency fermenter runs with same media and parameters

Fermentation process: (Shigella flexneri 3a)

Fermentation and Flask Media -TSB - Animal Origin Media (BD)

Note: dissolved in 1000 ml of

pH-7.2 with 5M NaOH, Autoclave

121°C for 20 minutes.

Fermentation Feed -TSB Animal Origin-BD with 1M Glucose

Note: dissolved TSB in 500 ml of MQW & Autoclave

Dissolved D Glucose in 500 ml of MQW & 0.2um filter. Mix Both in 1 Ltr sterile Glass Bottle.

The working cell bank vial of Shigella flexneri 3a, kept at -80°C is thawed and this is then streaked onto the Congo Red TSB agar plate using sterile loop. After steaking the plate, it is incubated at 37°C overnight.

Following day Inoculum for fermentation of the strain flexneri 3a is made by inoculating 50 ml TSB media in Erlenmeyer flask by single red positive S. flexneri 2a colony and incubating at 37°C, with shaking at 180 rpm till the OD600nm reached 0.9-1.0/ ml. Approximately 50 ml of the inoculum is used to inoculate 2.0 - 2.5 liter of TSB medium in a 5-liter fermenter. pH of the fermenter is maintained at 7.2 to 7.4, temperature at 37°C, aeration of 2 reactor volumes / min, stirring at 350-1000 and a feed containing TSB media with 1M Glucose is started after 2 to 3 hours at rate of 0.2 ml/min to 0.5 ml/min. Antifoam 204 (Sigma) diluted to 10% in water is used to control foaming.

At the completion of fermentation for approximately 8 to 11 hrs, i.e. i.e. after reaching the final OD at 600nm of 25 - 30. The harvest culture pellet is collected by centrifugation at 7000-7500rpm for 20 to 30 min at 2-8°C. The pellet is resuspended in 500-1000 ml Hanks balanced salt solution (HBSS, without phenol red, with calcium and magnesium chloride) and the OD at 600nm is adjusted to 16-18. 0.1% to 1% v/v formaldehyde solution (37%) is added to the cell suspension for inactivation. After addition of formalin the flask or bottle is placed in shaker incubator at 25°C with agitation at 200rpm for 20 - 36 hrs. After 20 - 36 hours inactivated bacteria are centrifugation at 7500 rpm for 20-30 min at 2-8°C, cells are resuspended in PBS and washed 3-4 times with PBS. Post reconstitution with PBS complete inactivation is checked by plating on TSA plate. TSA plates is incubated at 37°C, no growth is first confirmed at 24 hours of incubation and then verified again at 48 hours.

The growth curve as obtained by Shigella flex 3a is as shown in Figure 2.

Two consistency fermenter runs with same media and parameters

Fermentation process: (Shigella flexneri 6)

Fermentation and Flask Media -TSB Animal Origin - BD

Note: dissolved in 1000 ml of

pH-7.2 with 5M NaOH, Autoclave

121°C for 20 minutes.

Fermentation Feed -TSB Animal Origin-BD with 1M Glucose

Note: dissolved TSB in 500 ml of MQW & Autoclave

The working cell bank vial of Shigella flexneri 6, kept at -80° C, is thawed and this is then streaked onto the Congo Red TSB agar plate using sterile loop. After steaking the plate is incubated at 37°C overnight. Following day Inoculum for fermentation of the strain flexneri 6 is made by inoculating 50 ml TSB media in Erlenmeyer flask by single red positive S. flexneri 6 colony and incubating at 37°C, with shaking at 180 rpm till the OD600nm reached 0.9-1.0/ ml. Approximately 50 ml of the inoculum is used to inoculate 2.0-2.5 liter of TSB medium in a 5-liter fermenter. pH of the fermenter is maintained at 7.2 to 7.4, temperature at 37°C, aeration of 2 reactor volumes / min, stirring at 350-1000 and a feed containing TSB media with 1M Glucose is started after 2 to 3 hours at rate of 0.2 ml/min to 0.5 ml/min. Antifoam 204 (Sigma) diluted to 10% in water is used to control foaming.

At the completion of fermentation for approximately 8 to 11 Hrs, i.e. after reaching the final OD at 600nm of 25 - 30. The harvest culture pellet is collected by centrifugation at 7000-7500rpm for 20-30 min at 2-8°C. The pellet is resuspended in 500-1000 ml Hanks balanced salt solution (HBSS, without phenol red, with calcium and magnesium chloride) and the OD at 600nm is adjusted to 16-18. 0.1% to 1% v/v formaldehyde solution (37%) is added to the cell suspension for inactivation. After addition of formalin the flask or bottle is placed in shaker incubator at 25°C with agitation at 200rpm for 20 - 36 hrs. After 20 - 36 hours inactivated bacteria are centrifugation at 7500 rpm for 20-30 min at 2-8°C, cells are resuspended in PBS and washed 3-4 times with PBS. Post reconstitution with PBS complete inactivation is checked by plating on TSA plate. TSA plates is incubated at 37°C, no growth is first confirmed at 24 hours of incubation and then verified again at 48 hours.

The growth curve as obtained by Shigella flex 6 is as shown in Figure 4.

Antigen expression: Western blot assays is used to monitor the expression of IpaB, IpaC, IpaD in the formalin treated final bulk sample. The expression of IpaD in the formalin treated final bulk sample is confirmed as shown in Figure 5.

Two consistency fermenter runs with same media and parameters

Fermentation process: ( Shigella sonnei)

Fermentation and Flask Media -TSB Animal Origin - BD

Note: dissolved in 1000 ml of

pH-7.2 with 5M NaOH, Autoclave

121°C for 20 minutes.

Fermentation Feed -TSB Animal Origin-BD with 1M Glucose

Note: dissolved TSB in 500 ml of MQW & Autoclave

The working cell bank vial of Shigella Sonnei, kept at -80°C, is thawed and this is then streaked onto the Congo Red TSB agar plate using sterile loop. After steaking the plate is incubated at 37°C overnight. Following day Inoculum for fermentation of the strain Shigella Sonnei is prepared by dissolving multiple positive Congo red colonies in 5ml TSB media.

Approximately 5 ml of the inoculum is used to inoculate 2.0 -2.5 liter of TSB medium in a 5-liter fermenter. pH of the fermenter is maintained at 7.2 to 7.4, temperature at 37°C, aeration of 2 reactor volumes / min, stirring at 350-1000 and a feed containing TSB media with 1M Glucose is started after 2 to 3 hours at rate of 0.2 ml/min to 0.5 ml/min. Antifoam 204 (Sigma) diluted to 10% in water is used to control foaming.

The growth curve as obtained by Shigella sonnei is as shown in Figure 4.

Antigen expression: Western blot assays is used to monitor the expression of IpaB, IpaC, IpaD in the formalin treated final bulk sample. The expression of

IpaD in the formalin treated final bulk sample is confirmed and is shown in Figure 5.

Two consistency fermenter runs with same media and parameters

Process for Inactivation:

Formalin Inactivation:

After 8 to 11 hours of fermentation with final optical density at 600 nm of 25-30, the fermenter culture is harvested into sterile, 5-liter screw-cap bottles.

The harvested culture centrifugated at 7500 rpm for 30 min at 4°C and cell pellet is suspended in 500 ml Hanks balanced salt solution (HBSS, without phenol red, with calcium chloride and magnesium chloride) at 25°C.

The HBSS-suspended cells are placed in sterile 1-liter screw-cap flasks for formalin inactivation. A control consisting of cells treated with HBSS only is included to monitor the stability of the antigens during the inactivation process.

For S. flexneri 2a and 3a, 6 and sonnei the formalin (37% formaldehyde) concentration to be used is determined for each serotype during the inactivation experiments by using final formalin concentration of 0.1%, 0.2%, 0.6% and 1% (v/ v). During inactivation, the flasks are placed in a shaking water bath at 25°C with agitation at 200 rpm for 48 h. Samples were removed for determination of viability and antigen stability at 0, 1, 2, 3, 4, 6, 8, 10, 12, 16, 24 and 36 hours.

At the end of the 36 hours inactivation period, the treated cell suspensions are centrifuged at 7500 rpm for 30 min at 4°C.

Bacteria collected at the end of the fermentation period and during the various steps of inactivation are checked for viability by plating serial 10-fold dilutions of the bacteria on tryptic soya agar (TSA) plates in triplicate. Cultures and treated bacteria are also plated on TSA-Congo red to determine the stability of the Congo red phenotype. Plates are incubated at 37°C and read for viable bacteria at 24 and 48 hrs.

Sterility test is performed with final formalin inachvated bulk and vaccine formulation is done by direct 0.1ml inoculation of 10 tubes each of TSB and thioglycolate broth. The tubes were incubated for 14 days at 37°C, and the OD600nm of each tube is measured daily with a spectrophotometer.

Following are the analysis done and results as obtained to derive quantity of formalin and duration of inactivabon incubation for each of the four Shigella serotypes, i.e. Shigella flexneri 2a, Shigella flexneri 3a, Shigella flexneri 6, Shigella sonnei.

Shigella flexneri 2a:

Shigella flexneri 3a:

Shigella flexneri 6:

Shigella sonnei:

The final inactivation condition for fermenter harvest of each Shigella Serotype is determined. For Shigella flexneri 2a, 3a, 6 final formalin concentration to be used is 0.2% with 24hrs incubation at 25°C. For Shigella sonnei 0.6% final formalin concentration is selected with 24hrs incubation at 25°C.

Shigella tetravalent Formulation

The four inactivated Shigella strains, flexneri 2a, 3a, 6 and sonnei are mixed in equal volumes, i.e. 10⁷ to 10⁹ cfu/ml before inactivation of each strain for preparation of mice dose. Human dose will approximately contain 10⁹ to 10¹³ cfu/ml before inactivation of each four serotype in final vaccine formulation. Formulations are made with or without adjuvants. The adjuvanted formulation contain either rCTB (recombinant cholera toxin B) (20pg/200pl mice dose, lmg/ dose as Human dose) or mmCT (multi mutant cholera toxin) (1 Opg/ 200m1 mice dose, appropriate human dose will have to be determined).

Shigella vaccine immunogenicity studies and animal challenge studies

Oral immunization

Seven- week-old BALB/c mice were immunized orally at days 0, 7 and 14 days with 200m1 of Formalin killed tetravalent suspension (10⁷cfu/ml). One bolus of sodium bicarbonate (300 pL of a 5% solution; SRL, India) is introduced directly into the stomach through a mouse feeding needle (Harvard Apparatus). After five minutes, 200 mΐ of immunogen for the experimental mice and the same volume of PBS for the non-immunized group were administered followed by an immediate second bolus of sodium bicarbonate given orally. All immunized and non-immunized group of mice were returned to their cages and given limited amounts of sterile food and water.

Challenge study On 35th Day of animal challenge study. Six mice per group which had received three doses of adjuvanted or non-adjuvanted vaccine formulations along with and mice which were administered PBS as control, were challenged by intraperitoneal injection containing lethal dose of circulating Shigella strains (1.0 x 10⁹ CFU) of either S. dysenteriael (HK811), S. flexneri2a (2457T), S. flexneri3a (NK3758), S. flexneri6 (NK4025), S. sonnei (NK3918) or S. boydii type 2 (NK4023) and monitored every 1 h for 2 days for survival.

Said Immunization and Challenge Study for BALB/c C mice animal studies pattern is depicted as a flow diagram as shown below:

Results of animal Challenge studies.

In multiple extensive mice animal challenge studies with tetravalent inactivated whole cell Shigella vaccine with and without adjuvant provides 80% to 100% protective efficacy is achieved against homologous and heterologous challenge with all six serotypes of shigella circulating virulent strains. All control group mice immunized with PBS died post challenge with virulent shigella serotypes.

The immunized mice with whole cell inactivated adjuvanted or non-adjuvanted tetravalent formulation survived the challenge with all six virulent shigella serotypes dose. There is more than four-fold increase in the serum IgG, IgA and

IgM antibodies against whole cell shigella strains. The adjuvant effect of both rCTB and mmCT is observed with heightened immune response quantified by increase in antibodies titer in comparison to non-adjuvanted formulations. The IpaD is a conserved protein across species and thus it was qualitatively determined at different time intervals to ensure the cross species protective ability of vaccine formulation as shown in figure 5.

Claims

We Claim:

1. A Shigella vaccine formulation wherein said formulation comprises of whole cell inactivated Shigella strain is selected from Shigella flexneri 2a, Shigella 3a, Shigella 6 and Shigella sonnei, alone or in combination with or without adjuvant.

2. The Shigella vaccine formulation as claimed in claim 1 wherein said Shigella strains, S. flexneri 2a, 3a, 6 and sonnei are mixed in equal volumes either based on Cfu estimated before inactivation or LPS content, wherein the Cfu content before inactivation is 10⁷ to 10¹³ cfu/ ml of each strain.

3. The Shigella vaccine formulation as claimed in claim 1 wherein said whole cell inactivated tetravalent formulation is made with or without adjuvants.

4. The Shigella vaccine formulation as claimed in claim 1 wherein said adjuvanted formulation contain either rCTB (recombinant cholera toxin B) or mmCT (multi mutant cholera toxin).

5. The Shigella vaccine formulation as claimed in claim 1 wherein said formulation is administered orally with or without buffer administration as liquid formulation or freeze-dried solid dosage form to be reconstituted before administration.

6. The Shigella vaccine formulation as claimed in preceding claims wherein said tetravalent inactivated whole cell Shigella vaccine with and without adjuvant provides 80% to 100% protective efficacy for six Shigella strains, namely S. dysenteriael, S. flexneri2a, S. flexneri3a, S. flexneri6, S. sonnei and S. boydii type.

7. A process of preparing Shigella vaccine formulation wherein said process comprises the steps of

(a) inoculating whole cell Shigella bacterial strain in fermenter to obtain fermenter broth,

(b) harvesting of the fermenter broth to obtain fermenter harvest and examining the expression of virulence protein of bacterial strain in the fermenter harvest,

(c) centrifugation of the fermenter harvest to obtain cell pellet,

(d) resuspension of pellets obtained in step (c) in sterile solution to obtain a suspension with desired optical density 600nm at 16-20,

(e) addition of inactivating agent to the suspension of step (d), to obtain inactivated culture of Shigella strain,

(f) washing of the inactivated culture of Shigella strains with PBS buffer solution to obtain inactivated bulk of Shigella strain with intact virulence plasmid in the whole cell Shigella bacteria, wherein said Shigella bacterial strain is selected from Shigella flexneri 2a, Shigella 3a, Shigella 6, Shigella sonnei, either alone or in any combination.

8. The process of preparing shigella vaccine formulation as claimed in claim 7 wherein the fermenter is maintained at following process conditions to obtain the high yield biomass:

a. pH 7.2±0.2,

b. temperature at 37°C±2°C,

c. aeration of 2 reactor volumes / min,

d. stirring at 350-1000 rpm,

e. feed containing TSB media with 1M Glucose at rate of 0.2 ml/ min to 0.5 ml/min resulting in high biomass production with intact virulence plasmid in whole cell Shigella bacterial strain resulting in expression of virulence protein on the surface of bacteria in the fermenter harvest, fermentation is aborted after final OD at 600nm of the culture reaches 25-30

9. The process of preparing shigella vaccine formulation as claimed in claim 7 wherein the fermenter harvest is centrifuged, and cell pellet is suspended in Hanks' Balanced Salt Solution (HBSS) to preserve ipaBCD conserved antigens on the surface of bacteria after inactivation with formalin.

10. The process of preparing novel shigella vaccine formulation as claimed in claim 7 wherein said inactivating agent is formalin in the concentration range of 0.1% to 1.0% v/ v.

11. The process of preparing novel shigella vaccine formulation as claimed in preceding claims wherein high biomass production would directly reduce the overall production cost of the vaccine.