WO2021005623A1

WO2021005623A1 - A vaccine formulation for protection against enterotoxigenic e. coli (etec) and cholera

Info

Publication number: WO2021005623A1
Application number: PCT/IN2020/050593
Authority: WO
Inventors: Tarun Sharma; Nidhi Shukla; Neeraj Joshi; Ashwani Kumar MANDYAL; Davinder Gill
Original assignee: Msd Wellcome Trust Hilleman Laboratories Pvt. Ltd.
Priority date: 2019-07-08
Filing date: 2020-07-08
Publication date: 2021-01-14

Abstract

The present invention relates to a novel vaccine formulation for Enterotoxigenic E. coli (ETEC) and Vibrio cholerae vaccine and a process to prepare Enterotoxigenic E. coli (ETEC) and Vibrio cholerae vaccine. The formulation provides protection against diarrhea caused by Enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae. The present invention also relates to a process of producing novel vaccine formulation for Enterotoxigenic E. coli (ETEC) and Vibrio cholerae vaccine resulting in high yield.

Description

TITLE OF THE INVENTION

A VACCINE FORMULATION FOR PROTECTION AGAINST ENTEROTOXIGENIC E. COLI (ETEQ AND CHOLERA

FIELD OF THE INVENTION

The present invention relates to a novel vaccine formulation for Enterotoxigenic E. coli (ETEC) and Vibrio cholerae vaccine and a process to prepare Enterotoxigenic E. coli (ETEC) and Vibrio cholerae vaccine. More particularly, present invention relates to formulation for Enterotoxigenic E. coli (ETEC) and Vibrio cholerae vaccine comprising four inactivated whole cell strains wherein three inactivated whole cell strains are recombinant Vibrio cholerae strains of the Hikojima serotype and the fourth one is either a recombinant Vibrio cholerae Hikojima strain or a recombinant ETEC strain. The formulation provides protection against diarrhea caused by Enterotoxigenic Escherichia coli (ETEQ and Vibrio cholerae. The present invention also relates to a process of producing novel vaccine formulation for Enterotoxigenic E. coli (ETEQ and Vibrio cholerae vaccine resulting in high yield.

BACKGROUND OF THE INVENTION

Diarrheal diseases are a constant global threat to public health. Major causes of diarrhea include certain bacteria, viruses or parasites, food intolerances etc. One of the major causes of infant and adult diarrhea is Enterotoxigenic E.Coli (ETEC).

Enterotoxigenic Escherichia coli (ETEQ is a pathogenic variant of Escherichia coli and belong to the family Enterobacteriaceae. The bacteria are gram negative, rod shaped, non-spore forming and motile. ETEC is transmitted in the humans by the fecal-oral route. It is among the leading causes of diarrhoea in children and adults in low- and middle-income countries, and among travellers and military personnel from high-income countries. ETEC, a major cause of travellers' diarrhoea, was detected in 30.4% of cases of diarrhoea in travellers, with the highest rates seen in those travelling to areas with a high prevalence of ETEC. ETEC is one of the first symptomatic enteric illnesses for many children. According to the Global burden of disease 2018, ETEC was the eighth leading cause of diarrhoea mortality in 2016 among all age groups, accounting for ~51,000 deaths which is about 3 -2% of diarrhoea deaths. ETEC was responsible for about 4.2% of diarrhoea deaths (an estimated 8,00,000 deaths) in children younger than 5 years in 2016. The attack rate for ETEC illness appears to be highest during the first 2 years of life in endemic areas, however, the incidence of symptomatic illness decreases with age, suggesting that protective immunity develops following infection. Repeated infections due to ETEC can induce stunting and other forms of malnutrition, reduce immune function and increase the propensity for subsequent chronic inflammatory bowel disease.

ETEC expresses more than 25 types of fimbrial or non-fimbrial adhesins known as colonization factors or surface coh antigens which attach the bacteria to host receptors for colonisation in human small intestines. ETEC produces special toxins, a heat-stable toxin (known as ST) and a heat-labile toxin (LT) in small intestine epithelia. These toxins stimulate the lining of the intestines thus disrupting homoeostasis and causing fluid hypersecretion leading to abrupt onset of watery diarrhoea which does not contain blood, pus or mucus (nondysenteric) as the bacteria is non-invasive. The diarrhea is usually mild to moderate in severity, but some patients may have severe fluid loss. Low-grade fever, nausea, and abdominal pain may also be present ETEC infections are typically self-limiting with resolution of symptoms 3-7 days after initial onset and a recommended treatment strategy of rest and oral rehydration therapy. Sometimes, dehydration may become severe or life threatening in neonates and children. Treatment includes intravenous rehydration, antisecretory drugs and antibiotics. Initial treatment has historically been administration of first-line beta- lactams and quinolones. However, unregulated antibiotic use in Low Middle Income Countries (LMICs) has led to an increase of multidrug resistant ETEC bacteria that are not only insensitive to first-line antibiotics but also to Extended-spectrum b-lactamases (ESBLs). With the increase of anti-microbial resistance in ETEC, vaccination is the only effective method to prevent ETEC diarrheoa. Also, implementation of sanitation systems, supply of safe drinking water, which can prevent diarrhoea caused by ETEC are not quickly achievable for many resource-limited countries. Developing effective vaccines for ETEC is a priority for many public health institutes, including WHO.

There is no licensed vaccine for ETEC yet, although there are many vaccine candidates being pursued which are in pre-clinical and clinical stages of development A leading vaccine candidate among them is the whole cell inactivated ETVAX vaccine being developed by Scandinavian Biopharma AB, Sweden, which has successfully completed phase 1 trial. Whole cell inactivated vaccines have the advantage of stimulating a comprehensive mucosal and systemic immune response by delivering multiple surface antigens to the gut mucosa, which works very well for the vaccines whose correlates of protection are not established.

There are a number of patents and non-patents disclosures that describe the vaccine formulation for protection against enterotoxigenic E. coli (ETEC) and cholera. One such disclosure is US Application no. US 14/744,874 which discloses an oral vaccine for immunization against ETEC-induced diarrhoea, comprising inactivated Escherichia coli cells expressing an ETEC colonization factor antigen and dmLT protein adjuvant The vaccine formulation of the present invention uses a different strain of Enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae involving the use of rCTB and mmCT proteins. The formulation is efficacious and provides broader range of protection against ETEC-induced and cholera induced diarrhoea. The vaccine disclosed by present application is cost effective, less reactogenic.

OBTECT OF THE INVENTION

The main object of the present invention is to provide a novel vaccine formulation which provides protection against diarrhea caused by Enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae.

Another object of the invention is to provide process for preparing a novel vaccine which provides protection against diarrhea caused by Enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae.

Yet another object of the invention is to provide a novel vaccine formulation for Enterotoxigenic E. coli (ETEC) and Vibrio cholerae vaccine which comprises of four inactivated whole cell strains.

Yet another object of the invention is to provide a novel vaccine formulation for Enterotoxigenic £. coli (ETEC) and Vibrio cholerae vaccine with or without adjuvants. Yet another object of the invention is to provide a novel vaccine formulation in dosage form with acceptable mode of administration.

Yet another object of the invention is to provide low cost novel vaccine formulation rendering improved and synergistic effects.

Yet another object of the invention to provide an efficacious vaccine against cholera, ETEC diarrhoea with a formulation which ideally produces protective immunity after administration.

SUMMARY OF INVENTION

Accordingly, present invention provides a novel vaccine formulation for Enterotoxigenic E. coli (ETEC) and Vibrio cholerae vaccine which comprises of inactivated whole cell strains of Vibrio cholerae and a process to prepare thereof.

More particularly, present invention relates to a formulation for Enterotoxigenic E. coli (ETEC) vaccine comprising four inactivated whole cell strains wherein three inactivated whole cell strains are recombinant Vibrio cholerae strains of the Hikojima serotype and the fourth one is either a recombinant Vibrio cholerae strains of the Hikojima serotype or a recombinant ETEC (Enterotoxigenic E. coli ) strain and a process to prepare thereof.

The oral tetravalent whole cell inactivated ETEC vaccine of the present invention comprises of three Igt gene deleted V. cholerae O1 classical Hikojima strains, each overexpressing ETEC colonization factor CFA1, CSS, CS6, individually and an LT deleted ETEC strain of 06 serotype which expresses CS3, CS1 and CS21 naturally. This tetravalent vaccine also contains purified rCTB protein or mmCT (multiple mutated cholera toxin) to provide protection against LT enterotoxin and also act as an adjuvant

The above invention is disclosed below in a non-limiting manner:

The oral tetravalent vaccine formulation of the instant invention comprises of four inactivated whole cell strains, three of which are recombinant classical or El Tor O1 Vibrio cholerae strains of the Hikojima serotype from which the lipoprotein glyceryl transferase gene (lgt gene) and Cholera toxin subunit A (ctxA gene) have been deleted and which heterologously express Enterotoxigenic E. coli (ETEQ surface coli antigens CFA1, CS6, CSS individually. The fourth strain in the formulation is either selected from a recombinant classical or El Tor O1 Vibrio cholerae strain of the Hikojima serotype (lgt gene and ctxA gene deleted) expressing ETEC surface coli antigen CS3 or a recombinant LT (heat labile enterotoxin gene ) deleted ETEC strain of 06 serotype which expresses ETEC surface coli antigens CS3, CS1 and CS21 naturally. The formulation further contains a mucosal adjuvant multiple mutated cholera toxin (mmCT) protein or recombinant cholera toxin B subunit (rCTB) protein. The recombinant Hikojima strains are developed by transforming an expression plasmid which expresses the ETEC surface coli antigen selected from any one of CFA1/CS6/CS5/CS3 under the tac promoter. The expression vector also harbours the Lipoprotein glyceryl transferase ( lgt gene) as the non-antibiotic selection marker because antibiotic selection for the maintenance of expression plasmids is discouraged in the production of recombinant proteins for pharmaceutical or other human uses due to the risks of antibiotic residue contamination of the final products and the release of DNA encoding antibiotic resistance into the environment lgt gene encodes a (pro) lipoprotein glyceryl transferase protein essential for the biosynthesis of bacterial lipoprotein. Mutations in Igt are lethal in Vibrio cholerae and other Gram-negative organisms. The Igt gene is deleted in the Hikojima strains in the vaccine formulation and is complemented by the Igt gene carried by the expression vector. This makes the expression vector indispensable for the Hikojima strain and thus it keeps expressing the ETEC surface coli antigen as well. The Hikojima strain also has the ctxA gene (gene encoding a subunit gene of cholera toxin) deleted from its chromosome. This deletion does not allow the production of functional cholera toxin as the subunit A protein of cholera toxin is not produced, thus making the strain non-pathogenic and safe for use as a vaccine.

Besides providing protection against ETEC, the Hikojima strains also provide protection against Vibrio cholerae as the Hikojima strains have a site specific mutation in the wbeT gene coding for methyl transferase at position 158, wherein Serine is substituted with Phenyl alanine which results in reduced LPS perosamine methylation and thereby give only partial transformation of Inaba LPS into Ogawa LPS on the cell surface due to which the Hikojima strain has both Inaba and Ogawa LPS on its cell surface. Applicant holds a patent with US patent no. 9,511,133 which relates to a vaccine comprising Vibrio cholerae O1 cell characterized in that said cell comprises O1 antigens of both Ogawa and Inaba serotypes.

The fourth strain in the formulation is either a recombinant Hikojima strain expressing CS3 or a recombinant ETEC strain of 06 serogroup. The 06 serogroup is most prevalent worldwide and therefore will provide broad coverage to the vaccine. In order to make the vaccine safe for human use, the heat labile enterotoxin (LT) gene has been deleted from the strain as LT protein is not completely secretory, some of it stays in the periplasm after inactivation. However, the strain still harbors ST gene (Heat stable enterotoxin) but as the protein is completely secretory it does not have safety issues in an inactivated vaccine. This strain naturally expresses CS3 along with CS1 and CS21. Including a wild type ETEC strain in the vaccine formulation, will generate antibodies not only against the ETEC LPS but also against some unknown proteins which may have a role in protection.

The adjuvants mmCT or rCTB to be used in the vaccine formulation, are very similar to the LT protein of ETEC. Therefore, the antibodies against mmCT or rCTB can provide protection against LT secreting ETEC. mmCT is fully resistant to proteolytic cleavage and lacks detectable enterotoxicity yet mmCT has potent adjuvant activity on mucosal and systemic antibody as well as cellular immune responses to various antigens and vaccines. rCTB is only the B subunit pentamer of cholera toxin. rCTB is absolutely non-toxic and a good adjuvant Applicant holds a pending patent application no. PCT / IB2016 / 057460 which relates to process for production of purified recombinant cholera toxin B.

One of the main advantages of present invention over prior art is that this vaccine is capable of providing around 80% coverage of ETEC strains worldwide due to inclusion of CFA1, CS6, CSS CS3, CS1 and CS21 antigens in this vaccine candidate.

Another advantage of the present formulation is that formulation does not cause reactogenecity in pediatric population as the LPS of the Hikojima strains is not that reactogenic as the Enterotoxigenic E. colt LPS. This result is confirmed by phase I clinical trials for the Hikojima based cholera vaccine of the applicant Therefore, the present invention describes a formulation and a method of preparing a formulation which has improved acceptability, reduced dosage requirement, reduced footprint, low cost of manufacturing, enhanced stability, easy to administer hence, highly cost-effective vaccine.

BRIEF DESCRIPTION OF DRAWINGS

Figure-1 depicts the Immunization, blood collection and challenge schedule.

Figure-2 details the sequence of the Igt region of strain 1915 (Hikojima strain expressing CFA1). The arrowed protein sequence shows the start and end of the native Igt sequence. The blue region shows the DNA replacing the bulk of the Igt gene. The underlined region is the FRT site recognized by the FPL recombinase.

Figure-3 depicts the vector map of pML-CFAl/lgtEc plasmid.

Figure-4 depicts growth curve of Strain 1915 and V. cholerae O1 classical Hikojima strain.

Figure-5 explains the LT deletion and confirmation of LT deletion from ETEC strain 718279 of 06 serogroup by PCR.

Figure-6 depicts the Western blot showing expression of CFA1 in strain 1915 in comparison to wild type ETEC strain H10407.

Figure-7 depicts the Western blot showing native expression of CS3 in ETEC strain of serogroup 06.

Figure-8 depicts the Western blot of CFA1 and CS3 after inactivation of strain 1915 and LT deleted ETEC of serogroup 06.

Figure-9 depicts the Dot blot of (A) CFA1 in induced inactivated strain 1915.

(B) CS3 in inactivated LT deleted ETEC strain.

Figure-10 depicts the Gram staining of the inactivated strains 1915 and LT deleted ETEC strain.

Figure-llA depicts theAnti-CFAl serum IgG titres. Figure-llB depicts the Anti-CFAl serum IgA titres.

Figure-12A depicts the Anti-CI'B serum IgG titres.

Figure-12B depicts the Anti-CI'B serum IgA titres.

Figure-13A depicts the IgA titres in fecal pellet against CFA1

Figure-13B depicts the IgA titres in fecal pellet against CTB.

Figure-14 depicts the serum IgG titres against LFS of Hikojima strain.

DETAILED DESCRIPTION OF THE INVENTION:

Accordingly, the present invention relates to a novel vaccine formulation which comprises of inactivated whole cell strains of Vibrio cholerae to provide protection against diarrhea caused by Enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae. The formulation comprises of four inactivated whole cell strains wherein three inactivated whole cell strains are recombinant Vibrio cholerae strains of the Hikojima serotype and the fourth one is either a recombinant Vibrio cholerae strains of the Hikojima serotype or a recombinant ETEC (Enterotoxigenic E. colt).

The recombinant Vibrio cholerae strains of the Hikojima serotype is a novel Igt gene deleted V. cholerae 01 classical Hikojima strain that over-expresses ETEC colonization factor CFA/I, CS5, CS6, individually, is capable of inducing serum and mucosal immune response to CFA1 and provides protection against wild type ETEC challenge. The recombinant ETEC is a LT deleted ETEC strain of 06 serotype which expresses CS3, CS1 and CS21 naturally.

The advantage of an ETEC strain in the vaccine formulation is highlighted as it seems to improve the protective efficacy. The vaccine formulation also comprises rCTB protein or mmCT (multiple mutated cholera toxin) to provide protection against LT enterotoxin and also act as an adjuvant

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 invention is explained hereinbelow with non-limiting embodiments and examples for the strain 1915. Accordingly, the below mentioned process can be replicated for the other recombinant Hikojima strains expressing ETEC colonization factors CSS, CS6 and CS3 as well.

BACTERIAL STRAIN AND CULTURE CONDITIONS

The V. cholerae O1 classical Hikojima strain expressing ETEC colonization factor CFA1 (strain 1915) is developed at the University of Gothenburg, Sweden. These strains were constructed as part of a collaborative partnership between Hilleman laboratories (India) and Biokonsult AB, University of Gothenburg (Sweden). The ETEC strain of serogroup 06 is obtained from National Institute of Communicable disease (NICD), Johannesburg, South Africa. ETEC strain H10407, used for challenge studies, is obtained from Wellcome Trust Sanger Institute, Cambridge, UK. All the strains were obtained as glycerol stocks. Strain 1915, LT deleted ETEC 06 and H10407 are grown on CFA agar plates or CFA media. All the chemicals are procured from Sigma Aldrich except for casamino acid and yeast extract which are procured from Becton and Dickinson (BD).

EXAMPLE CONSTRUCTION OF STRAIN 1915

(lgt deleted V. cholerae O1 classical Hikojima strain expressing CFA1 antigen)

a) Construction of lgt deleted Hikojima strain;

V. cholerae O1 classical Hikojima strain expressing approximately equal amounts of Ogawa and Inaba O1 LPS is developed at the University of Gothenburg, Sweden. This Hikojima strain is the predecessor of the strain 1915 (Hikojima strain expressing CFA1). Lgt gene, which encodes a (pro)hpoprotein glyceryl transferase, is essential for the biosynthesis of bacterial lipoprotein. Mutations in the lgt gene are lethal in Vibrio cholerae and other Gram negative organisms. The lgt gene is used as a non-antibiotic selection marker in the strain 1915, as antibiotic selection for the maintenance of expression plasmids is discouraged by regulatory authorities worldwide for pharmaceutical or other human uses due to the risks of antibiotic residue contamination of the final products and the release of DNA encoding antibiotic resistance into the environment The lgt gene is deleted from the Hikojima strain for the maintenance of expression plasmid carrying the CFA1 gene in strain 1915. Briefly, the Hikojima strain is transformed with the temperature sensitive complementation plasmid pMT-lgtEC(ts) which confers ampicillin resistance and carries the lgt gene from the £. colt. The resulting strain is maintained thereafter at 30°C. The chromosomal lgt deletion is introduced by conjugating the Hikojima pMT- lgtEC(ts) with a strain carrying the suicide plasmid pMT-ssB-AlgtVc/Km^r. The resulting strain carried the suicide plasmid integrated into the chromosome conferring kanamycin and chloramphenicol resistance. The kanamycin in this case is flanked by FRT sites. In order to remove the plasmid and retain the Igt deletion, the strain is passaged extensively in medium containing ampicillin and kanamycin only. Cultures are then plated out onto medium containing sucrose. Qones which had lost the plasmid are able to grow since they had lost the suicide plasmid due to homologous recombination and thereby also lost the sacB gene from Bacillus subtilis that generates a metabolite from sucrose that is lethal to gram negative organisms. The kanamycin ensured that the Igt deleted clones are selected rather than the wild type since the kanamycin resistance gene had been inserted in place of the Igt gene. Qones are selected that are chloramphenicol sensitive but still resistant to kanamycin and ampicillin. The presence of the deletion is then confirmed by PCR amplification and sequencing of the relevant region of the chromosome. The strain is now Igt- deleted but retained the kanamycin resistance gene in the chromosome. The strain is now dependent upon the pMT-lgtEC(ts) plasmid to survive and therefore the presence of ampicillin in the growth medium is not necessary but could be used from time to time to confirm the phenotype of the strain. The strain must be maintained at temperature ranging from 30°C ± 2°C. The kanamycin resistance gene is removed by introduction of a plasmid pMT- FLP(tac) carrying the FLP recombinase under the control the of tac promoter regulated by the lacli repressor. Expression of the recombinase is induced by addition of Isopropyl b- d-l-thiogalactopyranoside (IPTG) to the medium in a concentration of 1 mM. Thus, liquid cultures are grown up in the presence of chloramphenicol in medium supplemented with IPTG. Cultures are plated out to single colonies and screened for sensitivity to kanamycin. The final strain is then passaged in the absence of any antibiotics in order to lose the pMT-FLP(tac) plasmid which is inherently unstable in the absence of antibiotic selection. The strain without the pMT- FLP(tac) plasmid is retained. The generated strain is Igt gene deleted Hikojima strain.

The deletion of Igt gene from the chromosome has no effect on the growth kinetics of Hikojima strain, as contrary to the deletion of metabolic genes used as non-antibiotic selection markers which generally reduce the growth of the strain thereby generating less biomass in large scale fermentation, finally impacting the overall cost of production. Hikojima strain with Igt deletion shows the same growth kinetics as the naive Hikojima strain and the same is shown in Fig.4. b) Construction of expression plasmid harboring CFA1 and Igt gene:

The plasmid vector for expression of CFA1 in Igt deleted Hikojima strain, is based on the vector pML-CTB::p45. In this vector the protein expression is driven by the powerful synthetic tac promoter and controlled by the lac repressor. The Igt gene cloned in the plasmid pML-CTB::p45 is derived from E. coZi as after the deletion of the Igt gene from Hikojima there would be little chance for reversion through recombination. CFA1 operon is cloned into this plasmid by digesting the operon with restriction enzymes from another CFA1 clone which is constructed earlier. The pMT-CTB/lgtEc vector is also digested with the same enzyme as the CFA1 operon and then dephosphorylated in order to prevent religation of the vector. CFA1 operon and the pMT-CTB/lgtEc vector are ligated to generate pML-CFAl/lgtEc plasmid (refer Fig. 3) which is the final expression vector harboring CFA1 and Igt gene. Sequencing of the expression vector confirmed the presence of Igt gene and CFA1 operon in the expression plasmid. c) Construction of strain 1915: pMT-CF A1 / lgtEc plasmid is transformed into the Igt deleted Hikojima strain. Transformants were initially selected by growth at 39°C ± 2°C in the absence of antibiotic and then screened for sensitivity to ampicillin. Clones that are sensitive to ampicillin are then induced with IPTG and screened for expression of CFA1 by agglutination with specific antibodies (antibody against CFA1 raised by University of Gothenburg). Clones giving good agglutination are saved as glycerol stocks and the generated strain is termed as strain 1915. The novel Igt deleted V. cholerae O1 classical Hikojima strain thus constructed is able to express CFA1 surface protein of ETEC from a recombinant temperature-insensitive expression vector without any antibiotic selection marker. The plasmid is maintained by the complementation of the chromosomal deletion of Igt gene. Whole genome Sequencing of the strain 1915 is done which confirmed Igt deletion in the Hikojima strain as shown in Fig.2.

EXAMPLE CONSTRUCTION OF LT DELETED ETEC 06 STRAIN:

ETEC strain 718279 is modified by the deletion of LT gene (heat labile enterotoxin gene) to make the strain safe for use as an immunogen. The deletion is done by a company named Bgene Genetics based at Grenoble, France, using their proprietary technology UNBELT®. Whole genome Sequencing of the LT deleted ETEC 06 strain, is done.

The deletion of heat labile enterotoxin (LT) gene from ETEC strain 718279 is confirmed by PCR as shown in Fig.5 and sequencing. Contrary to the cholera toxin protein, which is quite homologous to LT protein and is completely secreted out of the Vibrio cholerae bacteria, the LT protein is a soluble periplasmic protein which is not completely secretory. So even after inactivation of the bacteria, the protein still stays in the cell. Therefore, for safety and regulatory reasons, the LT gene was deleted from ETEC strain. CELL BANK PREPARATION

Strain 1915, ETEC 718279 and H10407 are revived from a glycerol stock tube by streaking onto a 1.5% CFA agar plate with overnight incubation at 37°C. A single colony is transferred to an Erlenmeyer flask containing sterile CFA medium and cultured at 37°C ± 2°C with shaking at 180 rpm ± 20 rpm. The growth of the culture is recorded by taking optical density readings at wavelength 600nm (OD₆₀₀). A master cell bank is prepared by making multiple 12% glycerol stock aliquot vials of the strain in mid-log growth phase and freezing them at -70°C. A working cell bank is prepared in the same way as described above by using a vial from the master cell bank as starting material. Cell morphology and purity of both the master and working cell banks were confirmed by gram staining followed by microscopic examination of multiple vials of each strain. Biochemical tests for identity were done according to Bacteriological Analytical Manual (BAM) of USFDA. Serological testing of strain 1915 is done by agglutination using polyclonal Ogawa and Inaba antibodies from Abeam (Cat.No.: ab79794) and of ETEC strain 718279 and H10407 is done using polyclonal antibodies (CatNo.45745, SSI Diagnostica, Denmark).

EXPRESSION OF COLONIZATION FACTORS CFA1 AND CS3

Expression in Flask:

CFA1 operon carrying the structural genes cfaA, cfaB, cfaC, cfaE is cloned in the plasmid pMT-CTB/lgtEc to produce expression vector pMT- CFAl/lgtEc which is transformed in Igt deleted Hikojima strain to obtain strain 1915. Overnight culture of strain 1915 is diluted 1/100 in CFA liquid medium. Resulting culture is incubated at 37°C ± 2°C with shaking at 190 rpm ± 10 rpm. The culture was induced at an OD₆₀₀ of 0.7-0.8 with IPTG which is added to a final concentration of 1 mM. Incubation continued under the same conditions for another 4 hrs after which the culture is proceeded for inactivation. CFA/I is successfully expressed at 14Kda by addition of IPTG to the growth medium, whereas no expression is observed in the absence of the inducer. The expression of CFA1 in CFA medium is observed to be at-least 4-fold higher than the wild type ETEC strain H10407 by western blot as shown in Fig.6.

LT deleted ETEC 06 strain expresses CS3 naturally. This strain is not transformed by any recombinant plasmid. For the native expression of CS3, overnight culture of the strain was diluted 1/100 in CFA liquid medium. Resulting culture is incubated at 37°C ± 2°C with shaking at 190 ± 10 rpm till the culture reached an OD600 of 1-1.2 after which the culture is proceeded for inactivation. The native expression of CS3 was observed at 14 Kda in LT deleted ETEC 06 strain by western blot as shown in Fig.7.

Western blot and dot blot of CFA1 and CS3 is done according to standard procedures. A 1:1000 dilution of primary antibody was used for CFA1 (Cat.No. LS-C394367; LS Biosciences) and CS3 (Cat No. CSB- PA323362XA01ENL; CusaBio) in both western and dot blot Anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody is used at a dilution of 1:20,000 for both CFA1 and CS3 in western blot and dot blot The blots are developed with DAB (3,3'-Diaminobenzidine; Sigma) and H₂O₂ (Sigma).

Expression in Fermenter The immunogenicity and protection studies described are done with the flask culture of strain 1915 and LT deleted

ETEC. Fermentation of both the strains is done to see whether the flasks studies could be scaled up to 2.5 litres. Inoculum for fermentation of strain 1915 is prepared by inoculating a working cell bank vial in an Erlenmeyer flask containing 250 ml sterile CFA media and incubating the flask at 37°C ± 2°C with shaking at 180 ± 20 rpm until the culture OD₆₀₀ reached 0.9-1.5. The resulting culture is used to inoculate 2.5 litre of sterile CFA medium in a 5 litre glass fermenter connected to a controlling unit The pH of the fermenter culture is maintained at 7.4, temperature at 37°C ± 2°C, aeration at 2 reactor volumes/min, p02 above 35% (using air and pure oxygen) and stirring at 350-1000 rpm. Antifoam 204 (Sigma) diluted to 10% in water is used to control foaming and is added as and when required. Culture samples are withdrawn at regular intervals, and OD₆₀₀ is recorded along with gram staining followed by microscopic examination. The culture is induced with ImM IPTG at an OD₆₀₀ of 2 -3 and is harvested 5 - 7 hrs post induction as there is no increase in OD₆₀₀ values for subsequent samples. Fermentation of LT deleted ETEC strain is done just as described above. The only difference is that this culture is not induced. The fermentation of ETEC strain is continued for 8-10 hrs where after the culture is harvested.

INACTIVATION OF BACTERIA BY FORMALIN

Inactivation of flask culture: Inactivation of strain 1915 and LT deleted ETEC strain, is done by adding formalin to the culture to a final concentration of 0.1 M and incubating the suspension at 4°C for 2 days without agitation. After washing the suspension with double the volume of PBS, the culture is checked for inactivation by spreading 0.1ml aliquots on CFA agar and LB agar plates and incubating the plates at 37°C for 72hrs.

Inactivation of fermenter culture : The fermenter cultures of Strain 1915 and LT deleted ETEC strain are put in separate 5 litre blue cap bottles and formaldehyde solution (37%) is added to the fermenter culture to a final concentration of 0.1M formalin, it is allowed to stand for 2 days at 4°C without agitation where after the inactivated culture is harvested by centrifugation at 10,000xg ± 2000rpm for 25± 5 min at a temperature range of 2°C to 7° C . Post centrifugation supernatant is discarded, and the cell pellet is washed with double the volume of PBS. The cell pellet is finally suspended in PBS and is checked for inactivation.

The inactivation of strain 1915 and LT deleted ETEC by formalin resulted in complete killing of bacteria with no colonies seen on heavily inoculated CFA agar/ LB agar plates. Residual formaldehyde in the inactivated cell suspension is assayed at room temperature by adding 5 ml of 0.05% 3- Methyl-2-benzothiazolinone hydrazone (MBΊΉ) to 0.5 ml of 1:200 inactivated cell suspension diluted in MilliQ water and incubating the mixture for 1 hr after which 1 ml of Ferric chloride (1%)-Sulphamic acid (1.6% ) reagent is added to the solution and incubation continued for 15 min. Absorbance is measured at 628 run after spinning down the cells. A standard plot of formaldehyde (2 g/L, 1 g/L, 0.5 g/L, 0.25 g/L & 0.125 g/L) is also prepared by the same procedure, and residual formaldehyde is estimated by comparing the absorbance of the inactivated sample with the standard plot The residual formaldehyde in the inactivated suspensions is well within the limit of <6.7 nmoles/ml as per the pharmacopeial requirements for other oral inactivated licensed similar vaccines specifications.

Western blot of the inactivated strains 1915 and LT deleted ETEC gave a sharp band of CFA1 and CS3 indicating that CFA1 and CS3 are preserved after inactivation as shown in Fig.8. The presence of CFA1 and CS3 in native conformation is checked by dot blot using primary and secondary antibody concentrations as described above. Dot blot of the inactivated strains 1915 and LT deleted ETEC showed that both CFA1 and CS3 are present in correct conformation after the inactivation as shown in Fig.9. Purity and morphology of inactivated cultures is checked by Gram staining followed by microscopic examination. Both inactivated cultures are pure and showed no cell disruption and also their morphological characteristics matched with their respective master and working cell banks as shown in Fig.10. Serological identity test for strain 1915 and LT deleted ETEC is done by agglutination using Ogawa, Inaba specific antibodies (Abeam) as previously described and ETEC 06 polyclonal antibodies respectively. The agglutination of the cells with respective serotype antisera confirmed that the bacterial cells had maintained their phenotype after fermentation and inactivation.

IMMUNOGEN FORMULATION

Immunogen formulation for mice: Immunogenicity and protective efficacy studies are done in three groups of mice. Group G1 is the placebo group, group G2 and G3 are administered immunogen and are further divided into two subgroups, a and b. The immunogen given to each group/subgroup is detailed in Table 1 below:

Table 1: Immunogen given to each group/subgroup: Dose Volume, Route and Regimen for Immunization.

The immunogen for mice immunogenicity and protection studies is prepared by the flask cultures of strain 1915 and ETEC strain. Strain 1915 is the immunogen for group G2a and G2b which is made by adjusting the OD₆₀₀ of the inactivated strain 1915 to 8 and 70 respectively. The immunogen for G2a and G2b had 1x10⁹ cfu and 1x10¹⁰ cfu of the bacteria respectively in each 200ul dose. rCTB (recombinant cholera toxin B-subunit) is added to the immunogen of both groups at a dose of 40mg/200ml/mouse dose.

There are two immunogens used in group 3, strain 1915 and LT deleted ETEC 06 strain. The immunogen for group G3a, comprised of inactivated strain 1915 with OD₆₀₀ adjusted to 8 (1x10⁹ cfu in each 200ul dose) and inactivated LT deleted ETEC strain with OD₆₀₀ adjusted to 2 (1x10⁸ cfu in each 200ul dose). The immunogen for group G3b, comprised of inactivated strain 1915 with OD₆₀₀ adjusted to 70 (1x10¹⁰ cfu in each 200ul dose) and inactivated LT deleted ETEC strain with OD₆₀₀ adjusted to 2 (1x10⁸ cfu in each 200ul dose). rCTB is added to the immunogen of both groups, G3a and G3b, at a dose of 40mg/200ml/mouse dose. The process optimization and purification of rCTB used in this study was done at Hilleman Labs. The strain producing recombinant CTB is developed at the University of Gothenburg and is licensed to Hilleman Labs as part of a collaborative partnership between Hilleman laboratories (India) and Gotovax AB, University of Gothenburg (Sweden).

Immunogen formulation for humans: A vaccine formulation with the inactivated fermenter culture for humans is prepared, although no human trails are done for the same yet Each human dose of 2ml consists of 8X10¹⁰ to 2X10¹³ inactivated ETEC bacteria (2X10¹⁰ to 5X10¹² bacteria/ strain of Hikojima expressing CFA1, CSS, CS6 recombinantly and LT deleted ETEC expressing CS3 naturally) and mixed with lmg of rCTB or/ and 15ug -250ug of mmCT. Each dose will be administered along with 5 to 150ml of 5% sodium bicarbonate. Two to three doses of the vaccine to be administered orally at an interval of two weeks.

REPRESENTATIVE SAMPLE IMMUNOGENICITY STUDIES

(a) Animals

25 female and 13 male, specific pathogen free BALB/ c mice, 6-7 weeks old, with weight in the range of 18-20 gm are procured from Hysalco Biotechnology (India) Pvt Ltd., Hyderabad, India. Male and female mice are caged separately, male in a group of 4 or 5 and female in a group of two. At the time of mating, one male was put into the cage with two females. During gestation there was just one female per cage. The mice are maintained at 25°C with 75% environmental humidity. All the animal experiments were conducted at Dabur Research Foundation (DRF), Sahibabad, Delhi, following the standard operating procedures as outlined by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India. The animal experimental protocol was approved by the Institutional Animal Ethical Committee of DRF (Ref. No. IAEC/52/928).

(b) Oral immunization of mice

Twenty five female mice are divided into three groups. Group G1 is the placebo group, group G2 and G3 are administered immunogen and are further divided into two subgroups a and b. The number of mice in each group/ subgroup and the immunogen given to them is detailed in Table 1. All the mice are immunized orally on day 0, 7, 14 and 28 as shown in Fig.l using a mouse feeding needle (Harvard apparatus). The mice are starved for 3-4 hrs before oral immunization but water is given ad libitum. Fifteen minutes before the oral immunization, each mouse is anaesthetized by intramuscular injection of a mixture of ketamine (35 mg/kg body weight, Sterfil Laboratories Pvt Ltd, India) and xylazine (5 mg/kg body weight AstraZeneca Pharma India Ltd, India). Group G1 mice are administered orally with 200 mL of 5% sodium bicarbonate, followed by a combination of 200 mL PBS and 100 mL 5% sodium bicarbonate, followed by a combination of 200 mL PBS and 200 mL sodium bicarbonate. Group G2 mice are administered orally with 200 mL sodium bicarbonate followed by a combination of 200 mL sodium bicarbonate and 200 mL strain 1915 at a dose of 10⁹ cfu + 40mg rCTB per mouse for group G2a and 10¹⁰ cfu of strain 1915 in 200ul + 40mg rCTB per mouse for group G2b. Group G3 animals are administered orally with prior 200 mL of sodium bicarbonate followed by a combination of 100 mL sodium bicarbonate and 200 mL of strain 1915 at a dose of 10⁹ cfu in 200ul+ 40mg rCTB per mouse for group G3a and 10¹⁰ cfu in 200ul + 40mg rCTB per mouse for group G3b, followed by a combination of 200 mL sodium bicarbonate and 200 mL of strain ETEC 06 strain 10⁸ cfu in 200ul. All immunized and non-immunized group of mice are returned to their cages and are given food and water. Body weight is recorded daily. Blood samples are collected by retro-orbital bleeding from (n=3) mice per group/subgroup on Day 0, Day 7, Day 14, Day 21, Day 28 & Day 53 after first oral immunization from both immunized and non-immunized mice. Sera is separated and stored at -80° C till further analysis. Fecal pellets are collected from each group from (n=3) mice, on Day 14 & Day 28. Fecal extracts are prepared and stored at -80° C. Both immunized and non- immunized female mice are kept for mating with age-matched males at a 2:1 ratio, on day 21 post the start date of the study. (c)Immune response in mice

IgA and IgG antibodies against CFA1 and CTB in serum and fecal extracts is determined by ELISA. IgG antibodies to V. cholerae O1 Hikojima LPS in serum is also done by ELISA. Sera from all the mice is pooled for group G1 and each subgroup of G2 and G3 for analysis. Sera from each group is tested at an initial dilution of 1/100, followed by serial twofold dilution. The antibody titers are expressed as the reciprocals of the sample dilutions which are log2 transformed and these values are used for graphs and statistical analyses.

Strain 1915 induced a robust serum IgG and IgA antibody response against CFA1 and CTB in group G2 and G3 (Fig.ll and 12). There is a significant difference (P < 0.001) between the pre-immunization and postimmunization titers of serum antibodies in all immunized mice. No significant effect on the IgG and IgA antibody titres against CFA1 and CTB is seen by increasing the immunogen dose in group G2b and G3b. This clearly demonstrates that a dose of 10⁹ for strain 1915 and a dose of 10⁸ for ETEC is good enough to generate a robust immune response. However, this may also be due to the adjuvant effect of CTB as we did not evaluate an immune response of either strain without CTB. Fecal pellets contain significant amounts of secreted immunoglobulin, mainly IgA produced locally along the intestinal tract, and fecal pellets extracted with a buffer containing protease inhibitors are therefore a useful source for measuring intestinal-mucosal IgA responses after oral immunization. IgA titres in fecal pellets was estimated only on day 14 and 28. Strain 1915 induced significant levels of mucosal-intestinal IgA antibody response against CFA1 and CTB in group G2 and G3, as evident from the analysis of the fecal pellets. No significant effect on the mucosal-intestinal IgA antibody titres against CFA1 and CTB was seen by increasing the immunogen dose in group G2b and G3b as shown in Fig.13.

The antibody titres, IgG or IgA, whether in serum or fecal pellet, are higher for CTB than CFA1. Additionally, strain 1915 also induced significant serum IgG response against the Hikojima LPS as shown in Fig.14. No significant change was observed in the body weight

REPRESENTATIVE SAMPLE PROTECTIVE EFFICACY STUDY IN

MICE

The protective efficacy of two ETEC vaccine candidate strains, strain 1915 and LT deleted ETEC 06, is assessed using the neonatal mouse challenge model. This indirect protection assay is used since adult mice are only successfully colonized by V. cholerae/ETEC after pretreatment with antibiotics to decrease the bacterial gut flora. Wild type H10407 ETEC strain is used for the challenge of neonatal pups of mice. H10407 expresses CFA1 surface coli antigen which is confirmed by CFA1 dot blot and western blot It also expresses LT/ST (heat labile/heat stable) enterotoxins. Expression of LT enterotoxin by H10407, was confirmed by GM1 ELISA. For preparing the challenge dose, 10ml of CFA media is inoculated with 50ul of working cell bank of H10407 and kept overnight at 37°C incubator shaker with shaking at 190rpm. Next morning, 100ml of CFA media is inoculated with lml of overnight culture and kept at 37°C with shaking at 190rpm till the OD₆₀₀ reached 0.9-1. The culture is then pelleted, washed once with PBS and finally resuspended in PBS. The OD of the culture is adjusted to 70 so that each pup gets a challenge dose of lx 10⁹cfu.

3 days old neonatal mice of both the immunized and non-immunized group of dams, were challenged orally with the wild type ETEC strain H10407 at a dose of 1x10⁹ cfu/ mouse in 30m1 using the mouse feeding needle (Harvard apparatus). Infected mice are given back to their respective dams. After oral inoculation, the neonatal mice are monitored up to 6 days for survival. Some neonatal mice are euthanized at 24hr, and their intestine is removed by dissection, to observe inflammation and water retention. Protective efficacy was calculated using the formula:

Protective Efficacy = (percent deaths of controls) - (percent deaths of immunized mice)/ (percent deaths of controls) x 100.

The protective efficacy of strain 1915, with and without LT deleted ETEC 06 strain is shown in Table 2. The table shows explicitly that although strain

1915 alone can provide protection, best results were observed when it was given along with the ETEC strain where the protective efficacy seen is 100% . No significant effect on the protective efficacy is observed by increasing the dose of strain 1915 in group G2b and G3b. During the study, not all of the five dams in each group got pregnant, around 40% of the dams didn't conceive. Of the pups which got delivered, some died within 24hrs of delivery due to unknown reason. Remaining live pups were challenged and observed for survival for 6 days. Pups from one or two dams in each group were sacrificed 24hr post challenge to observe for inflammation and water retention in the small intestine. The placebo group showed inflammation and water retention whereas pups from immunized mice did not show these effects. Table 2: Number of pregnant dams, pups delivered, sacrificed and challenged.

Table 3: Protective efficacy results,

wherein P.E. stands for Protective efficacy. It was calculated following the formula {[(percent of deaths of controls) - (percent of deaths of immunized mice)]/ [percent of deaths of controls]} x 100. L: Live. D: Dead.

Accordingly, the said oral tetravalent vaccine of whole cell inactivated ETEC vaccine comprising of three Igt gene deleted V. cholerae O1 classical Hikojima strains, each overexpressing ETEC colonization factor CFA1, CSS, CS6, individually and an LT deleted ETEC strain of 06 serotype which expresses CS3, CS1 and CS21 naturally. This tetravalent vaccine will also contain purified rCTB protein or/and mmCT (multiple mutated cholera toxin) to provide protection against LT enterotoxin and also act as an adjuvant In order to demonstrate the feasibility and effectiveness of our ETEC vaccine approach, two of the four vaccine strains, Hikojima strain overexpressing CFA1 (strain 1915) and LT deleted ETEC strain of serogroup 06, were evaluated for their immunogenicity and protective efficacy.

Strain 1915 is constructed by deleting the Igt gene from the V. cholerae O1 classical Hikojima strain which was confirmed by whole genome sequencing. The expression of CFA1 in this Igt deleted Hikojima strain was observed to be at-least 4-fold higher than the wild type ETEC strain H10407 by western blot Native expression of only CS3 is confirmed in the LT deleted ETEC strain by western blot The presence of CS1 and CS21 is confirmed by PCR. An ETEC strain of 06 serogroup is chosen for the vaccine as 06 serogroup is most prevalent worldwide. All the six colonization factors included in said vaccine (CFA1, CS3, CS5, CS6, CS1, CS21) can together provide a coverage of 80% of the ETEC strains globally. The colonization factors CFA1, CS3, CS4, CSS and CS6 along with a LT component are considered important for development of vaccine candidates.

Strain 1915 (Hikojima expressing CFA1) induced a good serum, mucosal immune response and provided good protection against challenge by H10407 ETEC strain. The protective efficacy of strain 1915 is observed to be better when administered along with the LT deleted ETEC strain (Group 3). Although immune response to the LPS of ETEC strain and its CS antigens (CS3, CS1, CS21) is not checked due to unavailability of some required reagents, the protective efficacy result confirms that ETEC surface antigens (besides CS antigens), also play an important role in providing protection. A good serum IgG response is observed against Hikojima LPS as well which suggests that the tetravalent ETEC vaccine can be used as a combined vaccine against both cholera and ETEC diarrhea since these infections often occur simultaneously and in the same settings. Another advantage of using the Hikojima strain is that the toxicity of V. cholerae LPS is lower than that of E. coli LPS, which may render the recombinant Hikojima strains less reactogenic. A V. cholerae strain is also best suited for delivery of antigens to the intestines due to the selective adherence of V. cholerae to the M cells of the gastrointestinal tract and other epithelial cells, thus generating a better immune response. Hikojima strain with Igt deletion shows the same growth kinetics as the naive Hikojima strain, it generates a high biomass in large scale fermentation as compared to the deletion of metabolic genes from genome which eventually reduce the growth of strain.

Claims

We Claim;

1. An oral tetravalent vaccine formulation for immunization against ETEC and Cholera induced diarrhea, comprising

- four inactivated whole cell strains wherein three inactivated whole cell strains are recombinant Vibrio cholerae strains of the Hikojima serotype and the fourth one is either a recombinant Vibrio cholerae strains of the Hikojima serotype or a recombinant ETEC (Enterotoxigenic E. coli ),

- purified rCTB protein or mmCT (multiple mutated cholera toxin) to provide protection against LT enterotoxin, characterized in that

- said recombinant Vibrio cholerae strains of the Hikojima serotype is a novel Igt gene deleted V. cholerae O1 classical Hikojima strain that over-expresses ETEC colonization factors CFA/I, CS5, CS6, CS3 individually,

- said recombinant ETEC is a LT deleted ETEC strain of 06 serotype which expresses colonization factors CS3, CS1 and CS21 naturally.

- wherein the vaccine formulation ideally produces protective immunity after administration.

2. The vaccine formulation as claimed in claim 1 wherein the vaccine comprises of less than 10¹⁴ bacterial cells per unit dose.

3. The vaccine formulation as claimed in claim 1 wherein the vaccine comprises of 0.5mg to 1.5mg rCTB or 15ug to 250ug mmCT along with the bacteria.

4. The vaccine formulation as claimed in claim 1 wherein Vibrio cholerae O1 classical Hikojima strain has the Igt (pro-lipoprotein glyceryl transferase) and ctxA (cholera toxin subunit-A) gene deleted from its genome.

5. The vaccine formulation as claimed in claim 1 wherein:

a. the Igt gene is used as a non-antibiotic selection marker in the Hikojima strain,

b. the genes CFA1, CS5, CS3, CS6 are expressed recombinantly under tac promoter.

6. The vaccine formulation as claimed in claim 1 wherein the vaccine is inactivated by either formalin in the concentration range of 0.05 M to 0.5M or phenol in the concentration range of 0.5 to 3 percent

7. The vaccine formulation as claimed in claim 1 wherein the vaccine is administered 2 to 3 times orally, each dose separated by 7-15 days.

8. The vaccine formulation as claimed in claim 1 wherein the vaccine is used with or without adjuvants rendering improved and synergistic effect

9. The oral vaccine formulation for immunization against ETEC and Cholera induced diarrhea, said vaccine is obtained by a process comprising the steps of:

- construction of the Igt deleted V. cholerae O1 classical Hikojima strain expressing approximately equal amounts of Ogawa and

Inaba O1 LPS, by

• conjugating the Hikojima strain harbouring pMT-lgtEC(ts) plasmid with a strain carrying a suicide plasmid pMT-ssB- AlgtVc/Km^r to obtain a Igt gene deleted hikojima strain carrying kanamycin gene in place of Igt gene, then removal of kanamycin gene by FLP recombinase by transforming Hikojima strain by pMT-FLP(tac) plasmid

- construction of Igt deleted V. cholerae O1 classical Hikojima strain expressing ETEC colonization factors CFA1, CSS, CS6 and CS3 by

• constructing of an expression plasmid harboring said colonization factors and Igt gene, • constructing of strain by transforming the expression plasmid into Igt deleted Hikojima strain capable to express said colonization factors of ETEC from a recombinant temperature-insensitive expression vector without any antibiotic selection marker

- construction of LT deleted ETEC 06 strain by known methods,

- expression of said colonization factors,

- inactivation of the strain and the LT deleted ETEC strain by formalin/ phenol.