TECHNICAL FIELD

The present invention relates to the field of vaccines, in particular whole cell vaccines against cholera, shigellosis and enterotoxigenic E. coli (ETEC) diarrhea.

BACKGROUND TO THE INVENTION

Cholera and shigellosis remain major health problems in large parts of the world. This is also true for ETEC, which is the main cause of diarrheal disease in developing countries as well as in travellers to these countries. In many developing countries effective water and sanitary measures for control of cholera and other enteric infections are currently impossible, and in this context, vaccines have an important role to play. In order to do so however, they need to be effective, readily accessible and affordable for public health use. There is also a medical need and a very substantial commercial market for use of cholera, Shigella and especially ETEC vaccines in travellers.

One approach has been the development of oral killed whole cell vaccines. Dukoral™ is an oral cell vaccine (OCV) with demonstrated up to 90% efficacy against cholera and also a significant efficacy against ETEC-induced diarrhea. It comprises S different V. cholerae strains comprising the two V. cholerae 01 serotypes Ogawa and Inaba and the two biotypes Classical and El Tor, in four different formulations (two heat-killed and two formalin-killed) and in addition recombinantly produced cholera toxin B subunit (rCTB). The rCTB component contributes significantly to the efficacy against cholera and is solely responsible for the observed protection against ETEC diarrhea due to its ability to induce cross- neutralizing antibodies against the cholera toxin (CT)-like E. coli heat-labile toxin (LT). However, rCTB is acid-labile and thus the vaccine (which needs to be given in two doses) must be administered with a bicarbonate buffer.

While Dukoral™ was the first internationally licensed and WHO-prequalified OCV (meaning that it was approved for purchase through the United Nation system), copies of this vaccine with or without the CTB component are currently being marketed in many developing countries, two of which Shanchol™ (produced in India) and Euvichol™ (produced in R. South Korea) have also been WHO-prequalified. These OCVs like the mOrcVax™ OCV made in Vietnam (which omit the CTB component mainly for cost reasons) contain the same 4 bacterial components as in Dukoral™ plus a fifth formalin-killed V. cholerae strain of serogroup 0139 (which latter component is currently irrelevant since >99% or all cholera in the world is caused by V. cholera of the 01 serogroup). A nationally licensed vaccine in China has in a dry capsule formulation the same composition as Dukoral including cholera toxin B subunit.

Protective immunity against cholera elicited by OCVs is mainly if not exclusively based on mucosal production in the intestine of secretory IgA (slgA) antibodies against cell wall lipopolysaccharide 01 (01 LPS) of Inaba and Ogawa types and for the CTB-containing Dukoral™ vaccine also of slgA antitoxin antibodies.

From the above it is evident that the present state of the art for production of cholera/ETEC vaccine is far from simple (especially considering the extreme cost constraints required for wide-spread use in developed countries) which adds to cost and to limitations in the global capacity of OCV production. Thus, although the already available OCVs are effective, a real contribution to making a cholera vaccine more accessible would be to rationalize the composition of the formulation and the methods of making it at several levels.

A cholera vaccine with V. choleroe cells engineered to simultaneously express 01 antigens of both Ogawa and Inaba serotypes has been disclosed in WO 2011/034495 Al.

WO2013/037397 Al discloses an oral ETEC vaccine comprising dmLT adjuvant.

Thus, an object of the present invention is the provision of an improved or alternative method of manufacturing immunogenic compositions; and/or improved or alternative immunogenic compositions.

DEFINITIONS

The term isogenic refers in the present context to at least two microbial strains sharing the essentially same genetic makeup (preferably being genetically identical) except for the presence of a specific difference in 1-10 genes between the isogenic strains. Preferably, there are differences in less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2 or only 1 of the genes. Most preferably, there is a single gene with a difference between the strains. The term immunogenic antigen refers in the present context to an antigen suitable for use in an immunogenic composition (preferably a vaccine) to elicit an immune response (preferably protective immunity) in a subject (animal or human) upon administration.

The term prevention in the present context refers to preventive measures resulting in any degree of reduction in the likelihood of developing the condition to be prevented, including a minor, substantial, major or total reduction in likelihood of developing the condition. Preferably, the degree of likelihood reduction is at least a minor reduction.

The term cholera toxin A-subunit, abbreviated "CTA" refers to the toxic-active ADP- ribosylating A subunit of cholera toxin. Wild-type CTA has an amino-acid sequence according to SEQ ID NO: 1. The term may preferably also encompass sequence variants having at least 80%, preferably at least 90%, most preferably at least 95% sequence identify to SEQ ID NO:

1.

The term cholera toxin B-subunit, abbreviated "CTB" refers to the cell-binding B subunit, present as a pentamer in the native cholera toxin. Wild-type CTB has an amino-acid sequence according to SEQ ID NO: 2. The term may preferably also encompass sequence variants having at least 80%, preferably at least 90%, most preferably at least 95% sequence identify to SEQ ID NO: 2.

The term heat-labile enterotoxin A-subunit, abbreviated "LTA" refers to the toxic-active ADP-ribosylating A subunit of E. coli heat-labile enterotoxin. LTA is highly homologous to

CTA. Wild-type LTA has an amino-acid sequence according to SEQ ID NO: 3. The term may preferably also encompass sequence variants having at least 80%, preferably at least 90%, most preferably at least 95% sequence identify to SEQ ID NO: 3.

The term heat-labile enterotoxin B-subunit, abbreviated "LTB" refers to the cell-binding B subunit present as a pentamer in the heat-labile enterotoxin. LTB is highly homologous to

CTB. Wild-type CTB has an amino-acid sequence according to SEQ ID NO: 4. The term may preferably also encompass sequence variants having at least 80%, preferably at least 90%, most preferably at least 95% sequence identify to SEQ ID NO: 4.

The term dmLT refers to a non-toxic mutant of the LT toxin from E. coli. This molecule has been mutated in two different positions; a substitution of G for R at position 192, and L for A at position 211 and has been charaterized by Norton et al. 2011 (Norton EB, Lawson LB, Freytag LC, Clements JD. Characterization of a mutant Escherichia coli heat-labile toxin, LT(R192G/L211A), as a safe and effective oral adjuvant. Clin Vaccine Immunol. 2011 Apr;18(4):546-51; SEQ ID NO: 5). The term may preferably also encompass sequence variants having at least 80%, preferably at least 90%, most preferably at least 95% sequence identify to SEQ ID NO: 5. mmCT refers to a cholera toxin A-like polypeptide when associated with CTB, disclosed in W02015/044105 Al, useful as adjuvant component. The term may preferably also encompass sequence variants having at least 80%, preferably at least 90%, most preferably at least 95% sequence identify to SEQ ID NO: 6.

The synonymous terms LCTBA, LCTBA-protein and LCTBA hybrid protein refer to a hybrid protein between the B-subunit of the E. coli heat-labile enterotoxin (LTB) and the B-subunit of the cholera toxin (CTB). Seven amino acids in the CTB molecule have been replaced by amino acids at corresponding positions of the LTB molecule. For details, see Lebens et al. 1996 (Lebens M, Shahabi V, Backstrom M, et al. 1996. Synthesis of hybrid molecules between heat-labile enterotoxin and cholera toxin B subunits: potential for use in a broad spectrum vaccine. Infect Immun 64:2144-2150; SEQ ID NO: 7). The term may preferably also encompass sequence variants having at least 80%, preferably at least 90%, most preferably at least 95% sequence identify to SEQ ID NO: 7.

In the above, the "sequence variants" are preferably functionally similar or equivalent to the reference sequence.

The term sequence identity expressed in percentage is defined as the value determined by comparing two optimally aligned sequences over a comparison window, wherein a portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Unless indicated otherwise, the comparison window is the entire length of the sequence being referred to. In this context, optimal alignment is the alignment produced by the BLASTP algorithm as implemented online by the US National Center for Biotechnology Information (see The NCBI Handbook [Internet], Chapter 16), with the following input parameters: Word length=3, Matrix=BLOSUM62, Gap cost=ll, Gap extension cost=l.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Direct comparison of growth of Ogawa and Inaba isogenic strains in different growth media. Two set of isogenic strains were grown in under different conditions. No differences were detected. Ogawa strains in solid and Inaba strains in dotted line. A) MS1571 (Ogawa) vs MS1712 (Inaba) in LB at 37°C for 20h, B) A493 (Ogawa) vs MS1843 (Inaba) in LB at 37°C for 20 hours, C) MS1571 vs MS1712 in LB high salt for 20h, D) MS1571 vs MS1712 in AKI for 20h.

Figure 2: Ratio of Inaba to Ogawa in co-cultures grown in different M9 medium with different carbon sources A: Circle marker line; maltose, square marker line; casamino acids, triangle marker line 0.1% glycerol. B: Cultures were grown with different concentrations of glycerol as sole carbon and energy source. Circle marker line; 0.3% glycerol, square marker line; 0.2% glycerol, triangle marker line; 0.1% glycerol.

SUMMARY OF THE INVENTION

The present invention relates to the following items. The subject matter disclosed in the items below should be regarded disclosed in the same manner as if the subject matter were disclosed in patent claims.

1) A method for the production of an immunogenic composition, comprising: a) providing cells of a microbial strain expressing at least two different immunogenic antigens, wherein the cells further express a soluble immunogenic antigen secreted into the culture medium, the expression of the soluble antigen preferably being conferred by a recombinant DNA sequence introduced into the cells; b) culturing said strain in a culture medium to obtain whole cells expressing the at least two different immunogenic antigens and culture medium containing the soluble antigen; c) inactivating the cells (preferably by heat, formalin or phenol) and formulating the inactivated whole cells containing said at least two different immunogenic antigens and the secreted soluble antigen into an immunogenic composition, wherein said antigens are included in immunogenic amounts in the immunogenic composition.

2) A method for the production of an immunogenic composition, comprising: a) providing cells of at least two different microbial strains expressing immunogenic antigens, wherein the strains are isogenic apart from that they express different immunogenic antigens, and optionally further express different intracellular fluorescent markers; b) culturing said strains in a single vessel in a liquid culture medium to obtain a mixture of whole cells expressing the different immunogenic antigens; c) formulating the mixture of whole cells into an immunogenic composition containing said different immunogenic antigens in immunogenic amounts.

3) The method according to item 2, wherein the cells further express a soluble immunogenic antigen secreted into the culture medium and wherein the soluble antigen is included in an immunogenic amount in the immunogenic composition formulated in the formulation step, the expression of the soluble antigen being preferably conferred by a recombinant DNA sequence introduced into the cells.

4) The method according to any of items 1 or 3, wherein the soluble antigen is a bacterial toxin, or a derivative of a bacterial toxin.

5) The method according to any of items 1, 3 or 4, wherein the soluble antigen is dmLT, mmCT, CTB, LTB or a hybrid CTB/LTB protein such as LCTBA, preferably CTB.

6) The method according to any of items 1 or 3-5, wherein the soluble antigen is isolated prior to the formulation step.

7) The method according to any of the preceding items, wherein the method involves concentrating the culture medium from the culture step including the whole cells and the soluble antigen into a concentrate and using the concentrate in the formulation step.

8) The method according to any of the preceding items, wherein the formulation step includes inactivating the cells, preferably by heat inactivation, formalin inactivation or phenol inactivation. 9) The method according to any of the preceding items, wherein the formulation step includes inactivating the cells by heat inactivation.

10) The method according to any items 1-8, wherein the formulation step includes inactivating the cells by formalin inactivation.

11) The method according to any of the preceding items, wherein the method is performed without isolating the bacterial cells from the culture medium.

12) The method according to any of items 1-10, wherein the method involves separation and differential treatment of the cells and the culture medium.

13) The method according to any of the preceding items, wherein the culture medium is concentrated using ultrafiltration, preferably with 10 kDa cut off.

14) The method according to any of the preceding items, wherein the cells belong to the genus of Vibrionacae, Escherichia, Salmonella, Shigella, Campylobacter or Helicobacter.

15) The method according to any of the preceding items, wherein the cells are Escherichia coli, V. cholerae or Shigella strains expressing at least two antigens selected from the list consisting of E. coli colonization factor 1, coli surface antigen CS1, coli surface antigen CS2, coli surface antigen CS3, coli surface antigen CS4, coli surface antigen CS5 and coli surface antigen CS6.

16) The method according to any of the preceding items, wherein the cells are Escherichia coli, V. cholerae or Shigella strains expressing at least two antigens selected from the list consisting of Shigella sonnei O antigen, Shigella flexneri 2a O-antigen, Shigella flexneri 3a O-antigen and Shigella flexneri 6 O antigen, wherein the strain is preferably a Shigella strain.

17) The method according to any of the preceding items, wherein the cells comprise at least one Vibrio cholerae strain expressing each of the 01 Ogawa and Inaba antigens, and wherein the cells optionally further express the 0139 lipopolysaccharide antigen.

18) The method according to any of the preceding items, wherein the strains comprise two different Vibrio cholerae strains, one expressing the 01 Ogawa antigen and the other expressing the Inaba antigen, and wherein the cells optionally further express the 0139 lipopolysaccharide antigen.

19) The method according to item 18 wherein at least one of the strains further express CTB secreted into the culture medium. ) The method according to item 18, wherein both strains further express CTB secreted into the culture medium. ) The method according to any of the preceding items, wherein the immunogenic composition is formulated for mucosal administration, preferably oral, sublingual, nasal or rectal administration. ) The method according to any of the preceding items, wherein the immunogenic composition is formulated for parenteral administration to a subject, preferably for being injected subcutaneuously, intramuscularly or intradermally. ) The method according to any of the preceding items, wherein the immunogenic composition is formulated for oral administration to a subject. ) The method according to any of the preceding items, wherein the immunogenic composition is formulated as a vaccine. ) The method according to any of the preceding items, wherein the intracellular fluorescent markers are expressed, and the method comprises determining the amount of the intracellular fluorescent markers. ) A method for the production of an immunogenic composition according to item 1 or any item dependent thereon, comprising: a) providing Vibrio choleroe 01 cells expressing 01 antigens of both Ogawa and Inaba serotypes, wherein on average, 10-90 % of the 01 antigens expressed by the cells are of the Ogawa serotype, and wherein the cells further comprise a recombinant DNA sequence conferring to the cells a capability for expressing and secreting the cholera toxin B subunit (CTB) to the growth medium during culture; b) culturing said cells under conditions permissive for secretion of the CTB to the growth medium; c) concentrating the cell culture medium, such that a substantial amount of the secreted CTB is retained in the concentrated medium; d) inactivating the cells by heat, formalin or phenol; e) formulating the inactivated whole cells and the concentrated cell culture medium containing CTB into an immunogenic composition containing immunogenic amounts of the cells and the CTB. 27) The method according to item 26, wherein in the inactivation step is performed with heat while the cells are contained in the concentrated cell culture medium also comprising the CTB.

28) The method according to item 26, wherein the cells are separated from the concentrated medium and the separated cells are inactivated with formalin.

29) The method according to any of items 26 or 28, wherein the soluble antigen is purified from the concentrated cell culture medium prior to the formulation step.

30) The method according to any of the preceding items, wherein the formulation step involves freeze-drying or spray-drying the cells and the soluble antigen in the presence of a stabilizing sugar, preferably sucrose or trehalose.

31) The method according to any of the preceding items, wherein the formulation step involved packaging the freeze-dried or spray-dried cells and the soluble antigen in an enterocoated unit dose, preferably an enterocoated capsule, granulate or tablet formulation.

32) The method according to any of the preceding items, wherein the immunogenic composition is formulated as an oral vaccine.

33) The method according to any of the preceding items, wherein immunogenic composition is formulated as a liquid formulation, comprising 10 ⁸ -10 ¹⁴ cells/ml, more preferably 10 ¹⁰ -10 ¹² cells/ml, and most preferably about 10 ¹¹ cells/ml.

34) The method according to any of the preceding items, for the production of an immunogenic composition according to any of items 35-65, preferably items 59-65.

35) An immunogenic composition obtainable by the method of any of the preceding items.

36) An immunogenic composition obtainable according to item 35, for use in the prevention of cholera, enterotoxigenic E. coli diarrhea or shigellosis, preferably as an oral vaccine.

37) An immunogenic composition comprising: a) a mixture of inactivated whole cells from at least two different microbial strains, wherein the strains are isogenic apart from that they comprise different immunogenic antigens, and optionally further comprise different intracellular fluorescent markers; and wherein the cells further comprise a recombinant DNA sequence conferring capability to the cells for expressing and secreting a soluble immunogenic antigen into the culture medium during culture, and optionally a recombinant DNA sequence conferring capability to the cells for expressing different intracellular fluorescent markers during culture; and b) concentrated cell culture medium comprising the soluble antigen which was secreted into the culture medium during culture, wherein the composition is formulated for oral administration to a subject.

38) The immunogenic composition according to item 37, wherein the soluble antigen is a bacterial toxin, or a derivative of a bacterial toxin.

39) The immunogenic composition according to item 38, wherein the soluble antigen is dmLT, mmCT, CTB, LTB or a hybrid CTB/LTB protein such as LCTBA, preferably CTB.

40) The composition according to any of items 37-39, wherein the strains comprise two different Vibrio choleroe strains, one expressing the 01 Ogawa antigen and the other expressing the Inaba antigen.

41) The composition according to any of items 37-40, wherein the soluble antigen is CTB

42) .The composition according to any of items 37-41, wherein the strains express different intracellular fluorescent markers.

43) The immunogenic composition according to any of items 37-42, wherein the cells have been inactivated by heat inactivation, formalin inactivation or phenol inactivation, preferably heat inactivation.

44) The immunogenic composition according to any of items 37-43, wherein the concentration of the cell culture medium was performed using ultrafiltration, preferably 10 kD cut off.

45) The immunogenic composition according to any of items 37-44, wherein the cells belong to the genus of Vibrionacae, Escherichia, Salmonella, Shigella, Campylobacter or Helicobacter.

46) The immunogenic composition according to any of items 37-44, wherein the cells are Escherichia coli, V. cholerae or Shigella strains expressing at least two antigens selected from the list consisting of f. coli colonization factor 1, coli surface antigen CS1, coli surface antigen CS2, coli surface antigen CS3, coli surface antigen CS4, coli surface antigen CS5 and coli surface antigen CS6.

47) The immunogenic composition according to any of items 37-46, wherein the cells are Escherichia coli, V. cholerae or Shigella strains expressing at least two antigens selected from the list consisting of Shigella sonnei O antigen, Shigella flexneri 2a O-antigen, Shigella flexneri Sa O-antigen and Shigella flexneri 6 O antigen, wherein the strain is preferably a Shigella strain.

48) The immunogenic composition according to any of items 37-47, wherein the cells comprise at least two different Vibrio choleroe strains expressing each of the 01 Ogawa and Inaba antigens, and wherein the cells optionally further express the 0139 lipopolysaccharide antigen.

49) The immunogenic composition according to any of items 37-48, wherein the cells comprise at least two different intracellular fluorescent markers.

50) The immunogenic composition according to any of items 37-49, wherein the immunogenic composition is formulated for mucosal administration, preferably oral, sublingual, nasal or rectal administration.

51) The immunogenic composition according to any of items 37-50, wherein the immunogenic composition is formulated for parenteral administration to a subject, preferably for being injected subcutaneuously, intramuscularly or intradermally.

52) The immunogenic composition according to any of items 37-51, wherein the immunogenic composition is formulated for oral administration to a subject.

53) The immunogenic composition according to any of items 37-52, wherein the immunogenic composition is formulated as a vaccine.

54) The immunogenic composition according to any of items 37-53, for use as a vaccine.

55) The immunogenic composition according to any of items 37-54, for use as a vaccine for the prevention of cholera, enterotoxigenic E. coli diarrhea or shigellosis, preferably as an oral vaccine.

56) The immunogenic composition according to any of items 37-55 being a liquid formulation, comprising 10 ⁸ -10 ¹⁴ cells/ml, more preferably 10 ¹⁰ -10 ¹² cells/ml, and most preferably about 10 ¹¹ cells/ml.

57) The immunogenic composition according to any of items 37-55 being a freeze-dried or a spray-dried formulation comprising a stabilizing sugar, preferably sucrose or trehalose.

58) The immunogenic composition according to any of items 37-55 or 57 comprising freeze- dried or spray-dried cells and the soluble antigen in an enterocoated unit dose, preferably an enterocoated capsule, granulate or tablet formulation. ) An immunogenic composition comprising inactivated whole Vibrio cholerae 01 cells comprising 01 antigens of both Ogawa and Inaba serotypes, wherein on average, 10-90 % of the 01 antigens of the cells are of the Ogawa-serotype; wherein the cells comprise a recombinant DNA sequence conferring a capability to the cells for expressing and secreting the cholera toxin B subunit (CTB) to the growth medium during culture; wherein the cells in the composition are suspended in a vehicle comprising concentrated cell culture medium derived from culture of said cells, said concentrated culture media comprising the CTB secreted by the cells during culture into the culture medium; and wherein the composition is formulated as a vaccine for oral administration to a subject.) The immunogenic composition according to item 59, wherein the cell culture medium was concentrated using ultrafiltration, preferably lOkDa cut-off. ) The immunogenic composition according to any of items 59-60, wherein the cells were present in concentrated cell culture medium comprising the secreted CTB during inactivation performed with heat, phenol or formalin, preferably heat. ) The immunogenic composition according to any of items 59-61, for use in the preventive immunization against cholera. ) The immunogenic composition according to any of items 59-62, being a liquid formulation, comprising 10 ⁸ -10 ¹⁴ cells/ml, more preferably 10 ¹⁰ -10 ¹² cells/ml, and most preferably about 10 ¹¹ cells/ml. ) The immunogenic composition according to any of items 59-62 being a freeze-dried or a spray-dried formulation comprising a stabilizing sugar, preferably sucrose or trehalose.) The immunogenic composition according to any of items 59-62 or 64 comprising freeze- dried or spray-dried cells and the soluble antigen in an enterocoated unit dose, preferably an enterocoated capsule, granulate or tablet formulation. ) An immunogenic composition comprising: a) a mixture of inactivated whole cells from at least two different microbial strains comprising immunogenic antigens, wherein the strains are isogenic apart from that they comprise different cell-associated immunogenic antigens; and b) a soluble antigen which is a bacterial toxin, or a derivative of a bacterial toxin, such as dmLT, mmCT, CTB, LTB or a hybrid CTB/LTB protein such as LCTBA, preferably CTB, IB isolated from the culture medium of the same culture as the isogenic cells or from a separate bacterial strain and/or culture.

67) An immunogenic composition according to item 60, wherein the strains comprise two different Vibrio choleroe strains, one expressing the 01 Ogawa antigen and the other expressing the Inaba antigen, and the soluble antigen is CTB.

DETAILED DESCRIPTION

It has been surprisingly found that it is possible to culture two different microbial strains expressing two different antigens in a single vessel while substantially maintaining the original proportions of cells of the two strains, provided that the strains are isogenic apart from that they express two different immunogenic antigens (Example 1). The generally held view would have been that culturing two different microbial strains in the same culture vessel would inevitably lead to one strain outcompeting the other.

These cells can be used to formulate an immunogenic composition i.e. vaccine (Example 3). The inventors also demonstrate simultaneous production of whole cell and soluble antigens for an immunogenic composition (Example 2). The immunogenicity is demonstrated in Example 4. The immunogenic compositions may be prepared in solid form oral vaccines (Example 5), having advantages to more traditional liquid formulations.

Methods for the production of an immunogenic composition

In a first aspect, the present invention provides a method for the production of an immunogenic composition, comprising: a) providing cells of a microbial strain expressing at least two different immunogenic antigens associated (i.e. physically bound to) with the cells, wherein the cells further express a soluble immunogenic antigen secreted into the culture medium, the expression of the soluble antigen preferably being conferred by a recombinant DNA sequence introduced into the cells; b) culturing said strain in a culture medium to obtain whole cells expressing the at least two different cell-associated immunogenic antigens and culture medium containing the soluble antigen; c) inactivating the cells (preferably by heat, formalin or phenol) and formulating the inactivated whole cells containing said at least two different immunogenic antigens and the secreted soluble antigen into an immunogenic composition.

In a second aspect, the present invention provides a method for the production of an immunogenic composition, comprising: a) providing cells of at least two different microbial strains expressing immunogenic antigens associated with the cells, wherein the strains are isogenic apart from that they express different immunogenic antigens, and optionally further express different intracellular fluorescent markers; b) culturing said strains in a single vessel in a liquid culture medium to obtain a mixture of whole cells expressing the different cell-associated immunogenic antigens; c) formulating the mixture of whole cells into an immunogenic composition containing said different immunogenic antigens.

The cells of the second aspect preferably further express a soluble immunogenic antigen secreted into the culture medium and wherein the soluble antigen is included in the immunogenic composition formulated in the formulation step, the expression of the soluble antigen being preferably conferred by a recombinant DNA sequence introduced into the cells. The cell-associated immunogenic antigens are preferably cell surface antigens.

Said antigens (cell-associated and/or soluble) are present in the final immunogenic composition in relevant i.e. immunogenic amounts. In this context, immunogenic amounts are amounts that are sufficient for eliciting an immune response in a subject when properly administered. Circumstances such as the type of subject, the manner of administration and the presence or absence of adjuvants affect the amounts required in practice.

The soluble antigen may be a bacterial toxin, or a derivative of a bacterial toxin. The soluble antigen is preferably dmLT, mmCT, CTB, LTB or a hybrid CTB/LTB protein such as LCTBA, most preferably CTB.

The soluble antigen may be isolated prior to the formulation step. Preferably, in the method of the second aspect, the intracellular fluorescent markers are expressed, and the method comprises determining the amount of the intracellular fluorescent markers, e.g. for quality control. This allows a simple way of ascertaining that the proportion and amount of each isogenic strain are within the desired range.

The method according to the first and second aspects may be for the production of an immunogenic composition according third to sixth aspects.

Process features for the methods of the first and second aspects

There are several possible variants of performing the method, which are illustrated by way of the following examples: a) Preparations 1 are obtained by simply heating the cultures (e.g. to 58C for lh) to kill all cells, optionally after initial 2- to 3-fold concentration by tangential ultrafiltration through a 10 kDa cut-off membrane. b) Preparations 2 are obtained by in parallel:

(i) isolating the bacteria (e.g. by tangential filtration through a 300 kDa cut-off membrane or by centrifugation at 3000xg for 1 hour) and then resuspending the bacteria e.g. in phosphate buffered saline (PBS) to OD=30 (corresponding to ca 7.5xl0 ¹⁰ cells/ml) followed by heat or formalin killing of the cells as described (Lebens et al. Vaccine. 2011 Oct 6;29(43):7505-13; Karlsson et al. PLoS One. 2014 Nov 14;9(ll):el08521);

(ii) heat-treating the culture medium containing a soluble antigen (e.g. at 58C for lh) to inactivate any contaminating cells, optionally after having concentrated the culture medium e.g. by ultrafiltration through a 10 kDa cut-off membrane. The killed bacteria and the treated culture medium with the soluble antigen are then mixed to yield the immunogenic composition. c) Preparations 3 : Bacteria are isolated and killed in the same way as described for preparations 2; however, the culture medium instead of being heated is ultrafiltered e.g. through a 300 kDa cut-off filter to remove contaminating cells (unless this was already done in the initial separation from the bacteria - see prep. 2 above) and then concentrated 5-10 times (e.g. through ultrafiltration through a 10 kDa cut-off membrane), whereafter the soluble antigen is isolated from the concentrated culture medium. For instance for CTB, this could be by first precipitation with hexametaphosphate followed by gel filtration or ion exchange chromatography on a Resource Q column as described (Lebens et al. Nature Bio/Technology 11: 1574- 1578,

The methods of the first and second aspects may involve concentrating the culture medium from the culture step including the whole cells and the soluble antigen into a concentrate and using the concentrate in the formulation step.

The formulation step of the first and second aspects may include inactivating the cells, preferably by heat inactivation, formalin inactivation or phenol inactivation. The formulation step may include inactivating the cells by heat inactivation. The formulation step may include inactivating the cells by formalin inactivation.

The methods may be performed without isolating the bacterial cells from the culture medium.

Preferably, the methods involve separation and differential treatment of the cells and the culture medium.

Preferably, the culture medium is concentrated using ultrafiltration. The cut-off for the ultrafiltration is selected such that the soluble antigen is retained in the concentrate. Preferably, ultrafiltration is performed using a 10 kDa cut off, which is appropriate for bacterial toxins and their derivates such as CTB. Preferably, the cell culture medium is concentrated using ultrafiltration, most preferably with a lOkDa cut-off and optionally applying this after a BOOkDa initial filtration to remove cells.

When recombinant CTB is isolated this may preferably be done with acid precipitation and chromatographic methods as described (M Lebens et al. Large-Scale Production of Vibrio cholerae Toxin B Subunit for Use in Oral Vaccines. Bio/Technology volume 11, pages 1574- 1578; 1993)

The cells in the methods of the first and second aspects

The cells in the methods of the first and second aspects may belong to the genus of Vibrionaceae, Escherichia, Salmonella, Shigella, Campylobacter or Helicobacter.

Preferably, the cells are Escherichia coli, Vibrio cholerae or Shigella strains expressing at least two antigens selected from the list consisting of E. coli colonization factor 1, coli surface antigen CS1, coli surface antigen CS2, coli surface antigen CS3, coli surface antigen CS4, coli surface antigen CS5 and coli surface antigen CS6.

Preferably, the cells are Escherichia coli, V. cholerae or Shigella strains expressing at least two antigens selected from the list consisting of Shigella sonnei O antigen, Shigella flexneri 2a O-antigen, Shigella flexneri 3a O-antigen and Shigella flexneri 6 O antigen, wherein the strain is preferably a Shigella strain.

Preferably, the cells comprise at least one Vibrio cholerae strain expressing each of the 01 Ogawa and Inaba antigens, and wherein the cells optionally further express the 0139 lipopolysaccharide antigen.

More preferably, the strains comprise two different Vibrio cholerae strains, one expressing the 01 Ogawa antigen and the other expressing the Inaba antigen, and wherein the cells optionally further express the 0139 lipopolysaccharide antigen. Most preferably, both Vibrio cholerae strains further express CTB secreted into the culture medium.

Formulation of the composition in the methods of the first and second aspects

In the methods of the first and second aspects, immunogenic composition may be formulated for mucosal administration, preferably oral, sublingual, nasal or rectal administration.

The immunogenic composition may also be formulated for parenteral administration to a subject, preferably for being injected subcutaneuously, intramuscularly or intradermally.

Preferably, the immunogenic composition is formulated for oral administration to a subject. Preferably, the immunogenic composition is formulated as a vaccine, most preferably an oral vaccine.

The formulation step may involve freeze-drying the cells and the soluble antigen in the presence of a stabilizing sugar, preferably sucrose or trehalose. The formulation step may further involve packaging the freeze-dried cells and the soluble antigen in an enterocoated unit dose, preferably an enterocoated capsule, granulate or tablet formulation.

The immunogenic composition may also be formulated as a liquid formulation, preferably comprising 10 ⁸ -10 ¹⁴ cells/ml, more preferably 10 ¹⁰ -10 ¹² cells/ml, and most preferably about 10 ¹¹ cells/ml with a human dose for oral administration being 0.5-5ml, preferably l-2ml of said formulation.

Method for making a cholera vaccine

Preferably, the method of the first aspect comprises: a) providing Vibrio cholerae 01 cells expressing 01 antigens of both Ogawa and Inaba serotypes, wherein on average, 10-90 % of the 01 antigens expressed by the cells are of the Ogawa-serotype, and wherein the cells further comprise a recombinant DNA sequence conferring to the cells a capability for expressing and secreting the cholera toxin B subunit (CTB) to the growth medium during culture; b) culturing said cells under conditions permissive for secretion of the CTB to the growth medium; c) concentrating the cell culture medium, such that a substantial amount of the secreted CTB is retained in the concentrated medium; d) inactivating the cells by heat, formalin or phenol; e) formulating the inactivated whole cells and the concentrated cell culture medium containing CTB into an immunogenic composition containing immunogenic amounts of the cells and the CTB.

Preferably, the inactivation step is performed with heat while the cells are contained in the concentrated cell culture medium also comprising the CTB. Preferably, the inactivation step is performed with heat at 57-59°C while the cells are contained in the concentrated cell culture medium also comprising the CTB. The CTB survives the heat-inactivation. As the cells and the medium are prepared in the same culture and optionally need not be separated, this simplifies the production process.

In an alternative preferable variant, the cells are separated from the concentrated medium and the separated cells are inactivated with formalin. Formalin inactivation is more traditional and may therefore present an easier regulatory path to market. It may also be preferred in production at industrial scale. In this case, the CTB is purified from the concentrated cell culture medium prior to the formulation step and subsequently mixed with the formalin inactivated cells into the final vaccine formulation. Still, the process is advantageous in that the CTB production does not require a separate strain and/or separate fermentation.

In yet another alternative preferable variant, the cells are separated from the concentrated medium and the separated cells are inactivated with formalin. In this case, the CTB is purified from the concentrated cell culture medium of another bacterial strain prior to the formulation step and subsequently mixed with the formalin inactivated cells into the final vaccine formulation. Still, the process is advantageous in that the killed bacteria expressing the Ogawa and Inaba antigens can be produced in a single process and that the processes for obtaining maximal yields of the bacterial and CTB components can be optimized.

Preferably, the immunogenic composition is formulated as an oral vaccine.

Immunogenic compositions

In a third aspect, the present invention provides an immunogenic composition obtainable by the method of the first or second aspects.

The immunogenic composition of the third aspect may be for use in the prevention of cholera, enterotoxigenic E. coli diarrhea or shigellosis, preferably as an oral vaccine.

In a fourth aspect, the present invention provides an immunogenic composition comprising: a) a mixture of inactivated whole cells from at least two different microbial strains comprising cell-associated immunogenic antigens, wherein the strains are isogenic apart from that they express different immunogenic antigens, and optionally further comprise different intracellular fluorescent markers; and wherein the cells further comprise a recombinant DNA sequence conferring capability to the cells for expressing and secreting a soluble immunogenic antigen into the culture medium during culture, and optionally a recombinant DNA sequence conferring capability to the cells for expressing different intracellular fluorescent markers during culture; and b) concentrated cell culture medium comprising the soluble antigen which was secreted into the culture medium during culture of the whole cells prior to inactivation, wherein the composition is formulated for oral administration to a subject.

The immunogenic composition according to the fourth aspect may be for use as a vaccine, preferably for use as a vaccine for the prevention of cholera, enterotoxigenic E. coli diarrhea or shigellosis, most preferably as an oral vaccine.

The soluble antigen of the fourth aspect may be a bacterial toxin, or a derivative of a bacterial toxin. The soluble antigen is preferably dmLT, mmCT, CTB, LTB or a hybrid CTB/LTB protein such as LCTBA, most preferably CTB.

The cells of the fourth aspect may have been inactivated by heat inactivation, formalin inactivation or phenol inactivation, preferably heat inactivation, most preferably heat at 57- 59 °C.

Alternatively, cells were separated from the culture medium before being inactivated with heat, phenol or formalin, preferably formalin.

Preferably, the concentration of the cell culture medium was performed using ultrafiltration, preferably with 10 kDa cut off. Preferably, the cell culture medium was concentrated using ultrafiltration, most preferably with a lOkDa cut-off and optionally applying this after a BOOkDa initial filtration to remove cells, and when rCTB was isolated this was done with acid precipitation and chromatographic methods as described (M Lebens et al. supra).

Preferably, the cells of the fourth aspect belong to the genus of Vibrionaceae, Escherichia, Salmonella, Shigella, Campylobacter or Helicobacter.

Preferably, the cells of the fourth aspect are Escherichia coli, V. cholerae or Shigella strains expressing at least two antigens selected from the list consisting of E. coli colonization factor 1, coli surface antigen CS1, coli surface antigen CS2, coli surface antigen CS3, coli surface antigen CS4, coli surface antigen CS5 and coli surface antigen CS6.

Preferably, the cells of the fourth aspect are Escherichia coli, V. cholerae or Shigella strains expressing at least two antigens selected from the list consisting of Shigella sonnei O antigen, Shigella flexneri 2a O-antigen, Shigella flexneri 3a O-antigen and Shigella flexneri 6 O antigen, wherein the strain is preferably a Shigella strain. Preferably, the cells of the fourth aspect comprise at least two different Vibrio cholerae strains expressing each of the 01 Ogawa and Inaba antigens, and wherein the cells optionally further express the 0139 lipopolysaccharide antigen. Most preferably, the Vibrio choleroe strains further express the soluble antigen CTB.

Preferably, the cells of the fourth aspect comprise at least two different intracellular fluorescent markers.

The immunogenic composition of the fourth aspect may be formulated for mucosal administration, preferably oral, sublingual, nasal or rectal administration.

The immunogenic composition of the fourth aspect may be formulated for parenteral administration to a subject, preferably for being injected subcutaneuously, intramuscularly or intradermally.

The immunogenic composition of the fourth aspect may be a liquid formulation, comprising 10 ⁸ -10 ¹⁴ cells/ml, more preferably 10 ¹⁰ -10 ¹² cells/ml, and most preferably about 10 ¹¹ cells/ml.

The immunogenic composition of the fourth aspect may be a freeze-dried or spray-dried formulation comprising a stabilizing sugar, preferably sucrose or trehalose.

The immunogenic composition of the fourth aspect may comprise freeze-dried or spray- dried cells and the soluble antigen in an enterocoated unit dose, preferably an enterocoated capsule, granulate or tablet formulation.

The immunogenic composition of the fourth aspect is preferably formulated for oral administration to a subject.

The immunogenic composition of the fourth aspect is preferably formulated as a vaccine.

Cholera vaccine composition

In a fifth aspect, the present invention provides an immunogenic composition comprising inactivated whole Vibrio choleroe 01 cells comprising 01 antigens of both Ogawa and Inaba serotypes, wherein on average, 10-90 % of the 01 antigens of the cells are of the Ogawa-serotype; wherein the cells comprise a recombinant DNA sequence conferring a capability to the cells for expressing and secreting the cholera toxin B subunit (CTB) to the growth medium during culture; wherein the cells in the composition are suspended in a vehicle comprising concentrated cell culture medium derived from culture of said cells, said concentrated culture media comprising the CTB secreted by the cells during culture into the culture medium; and wherein the composition is formulated as a vaccine for oral administration to a subject.

Preferably, the cell culture medium was concentrated using ultrafiltration, preferably using a lOkDa cut-off ultrafiltration membrane.

Preferably, the cells were present in concentrated cell culture medium comprising the secreted CTB during inactivation performed with heat, phenol or formalin, preferably heat.

The composition according to the fifth aspect may be for use in the preventive immunization against cholera.

The composition according to the fifth aspect may be a liquid formulation, comprising 10 ⁸ - 10 ¹⁴ cells/ml, more preferably 10 ¹⁰ -10 ¹² cells/ml, and most preferably about 10 ¹¹ cells/ml. Such liquid formulations are preferably for use as an oral vaccine administered in a volume of 0.5-5 ml, preferably 1-2 ml per dose and preferably in two doses given at a suitable interval such as 1-6 weeks, preferably ca 2 weeks, between the doses to a human being.

The composition according to the fifth aspect may be a freeze-dried or spray-dried formulation comprising a stabilizing sugar, preferably sucrose or trehalose. The freeze-dried (or spray-dried) formulation may preferably comprise freeze-dried (or spray-dried) cells and soluble antigen in an enterocoated unit dose, preferably an enterocoated capsule, granulate or tablet formulation.

Preferably, the cell culture medium was concentrated using ultrafiltration, most preferably with a lOkDa cut-off and optionally applying this after a BOOkDa initial filtration to remove cells. In cases where rCTB is isolated this may be done with acid precipitation and chromatographic methods as described (M Lebens et al. supra)

In a sixth aspect, the present invention provides the immunogenic composition according to fifth aspect, for use in the preventive immunization against cholera. Vaccine compositions

A vaccine of the invention may be a vaccine composition comprising one or more pharmaceutically acceptable, excipients, carriers, diluents and adjuvants.

The formulation of vaccine compositions according to the invention is well known to persons skilled in the art. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA.

Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The vaccine composition for oral use may preferably comprise 10 ⁸ -10 ¹⁴ cells/ml, more preferably 10 ¹⁰ -10 ¹² cells/ml, and most preferably about 10 ¹¹ cells/ml; with a human dose typically comprising 0.5 - 2ml of such composition in liquid or desiccated form.

The vaccine composition for oral use may be formulated as a foodstuff, beverage or a feed supplement (when for use in immunizing animals).

The vaccine composition may comprise an adjuvant known in the art or may lack any adjuvants. The formulation of a vaccine for oral use may comprise a buffer, such as a carbonate buffer to protect the vaccine antigens from inactivation in the acidic environment of the stomach, or other means to achieve such protection such as the enterocoating of whole or part of the vaccine.

Use of the vaccine

The present invention discloses use of the above immunogenic composition or vaccine in preventive immunization, preferably against cholera and/or enterotoxic Escherichia coli- infection (ETEC). Preferably, the immunogenic composition or vaccine is administered orally or sublingually. The present invention also discloses a method for inducing preventive immunity, comprising administering a vaccine according to the above to a subject to be immunized. Preferably, the preventive immunity is against cholera and/or enterotoxic Escherichia coil-infection (ETEC). Preferably, the administration is done orally or sublingually. The administration may also be done by injection. The vaccine is preferably used to immunize humans and other mammals, such as pets (cats, dogs and the like) or farm animals (such as cows, horses, sheep, goats, pigs and the like).

Preferably, the vaccine is administered orally at comprise 10 ⁸ -10 ¹⁴ cells per dose, more preferably 10 ¹⁰ -10 ¹² cells per dose, and most preferably about 10 ¹¹ cells per dose.

The immunization protocol may consist of a single administration or may comprise two or more administrations. In a preferred embodiment, the initial immunization protocol to induce protective immunity comprises a first administration and a second administration, separated in time by at least 7 days but by no more than about 2 months (preferably about 2 weeks). After the initial immunization protocol, protective immunity may be maintained as long as desired by booster administrations occurring with up to several year intervals, preferably less than 3 year intervals in adults and children above 5 years of age and less than 1-2 years in younger children. It is may be preferable that a booster administration does not take place before at least 6 months has elapsed from the first administration.

Further immunogenic composition

The present invention also relates to an immunogenic composition comprising: a) a mixture of inactivated whole cells from at least two different microbial strains comprising immunogenic antigens, wherein the strains are isogenic apart from that they comprise different cell-associated immunogenic antigens; and b) a soluble antigen which is a bacterial toxin, or a derivative of a bacterial toxin, such as dmLT, mmCT, CTB, LTB or a hybrid CTB/LTB protein such as LCTBA, preferably CTB, isolated from the culture medium of the same culture as the isogenic cells or from a separate bacterial strain and/or culture.

Preferably, the strains comprise two different Vibrio cholerae strains, one expressing the 01 Ogawa antigen and the other expressing the Inaba antigen, and the soluble antigen is CTB. General statements relating to the present disclosure

The term "comprising" is to be interpreted as including, but not being limited to. All references are hereby incorporated by reference. The arrangement of the present disclosure into sections with headings and subheadings is merely to improve legibility and is not to be interpreted limiting in any way, in particular, the division does not in any way preclude or limit combining features under different headings and subheadings with each other.

EXAMPLES

The following examples are not to be regarded as limiting. For further information on the experimental details, the skilled reader is directed to a separate section titled Materials and Methods.

Example 1. Similar growth rates in co-culture of two isogenic V. cholerae bacteria expressing different antigens

In currently licensed oral cholera vaccines comprising inactivated V. cholerae 01 bacterial cells of the Ogawa and Inaba serotypes these cells are always produced separately in different cultures of the different strains. It would be an advantage if the Ogawa and Inaba bacteria could be manufactured together from the same culture. We show in this example that this is feasible provided that the Ogawa and Inaba strains are isogenic except for the serotype determining gene.

As detailed below, two pairs of isogenic strains of 01 Vibrio cholerae were constructed, each pair comprising one Inaba and one Ogawa strain. The first set was derived from strain Phil6973 isolated from a patient in India in 1973 and currently a component in all prequalified killed oral cholera vaccines. Phil6973 is of Inaba serotype due to a stop codon at position 252 in the wbeT gene and it was converted to Ogawa serotype by replacing the mutant wbeT gene with a wild-type gene from the Ogawa strain VX44945 resulting in the strain MS1571. Subsequently, the wbeT gene in MS1571 was deleted in order to produce the Inaba strain MS1712, in which the wbeT gene was entirely absent. A second pair of isogenic strains was generated from the clinical Ogawa isolate A493 isolated in Bangladesh in 2012. The wbeT gene was deleted in a similar fashion to generate the Inaba strain MS1843. Using these strains we can show that isogenic bacteria expressing different immunoprotective antigens have indistinguishable growth rates in liquid media when grown separately and multiply to the same extent when co-cultured together.

Materials and methods:

Constructing Isogenic strains.

In order to construct isogenic V. cholerae 01 strains that were Ogawa or Inaba, gene replacement was achieved using the suicide vector pMT- suicide/socS ²⁵ (Genebank accession: KF188719.1). Two strains were made isogenic: the well-known V. cholerae clinical isolate Phil6973 (isolated in Bangladesh in 1963) which is a component in all currently licensed OCVs and A493, a V. cholerae El Tor strain from Bangladesh isolated in 2012.

Two fragments flanking the wbeT gene were amplified by PCR using primer pairs wbeT5 (5'- GGCTTTAGTGAATCGCGATTTGTCGG-3'; SEQ ID NO. 8) with wbeT_deletion_linker_rev (5'- GTCGACGCGGCCGCGATATCACAGAATCAACTTGCAGATGCAGGTTTG-3', SEQ ID NO: 9) and wbe\Z/(5'-GGCGTATTACGGTACTACAAGGGTCTAG-3'; SEQ ID NO: 10) with wbeT_deietion_Hnker_fwd{ 5'-

GATATCGCGGCCGCGTCGACTGCAAGTTCAACAGACATTTCCGAAGAG-3'; SEQ ID NO: 11). The two resulting fragments were combined using primerless PCR, finally amplifying with the primer pair wbeT5/wbeVf to generate a fragment in which the wbeT gene was deleted. This fragment was inserted into the suicide pMT- suicid e/sacB and a Km ^R gene flanked by lox sites was inserted between Sail and EcoV sites. The final plasmid was used to transform E. coli strain MFDp/r and the resulting strain was used to introduce the plasmid into the recipient strains Phil6973 and A493 conferring resistance to both kanamycin and chloramphenicol. The two strains were then passaged for two days in LB broth supplemented with kanamycin before plating out onto LB agar plates containing no salt but supplemented with sucrose (6% w/v). Individual colonies were picked onto duplicate LB agar plates supplemented with kanamycin and chloramphenicol respectively in order to screen for loss of the plasmid. The resulting strains were Kanamycin resistant and chloramphenicol sensitive. Deletion of the wbeT gene was confirmed by sequencing DNA fragments obtained by PCR amplification using the primers wbeT5 and wbeVf. In both generated strains the Km ^R gene was removed by Cre-mediated recombination. The ere gene was introduced using an expression plasmid conferring chloramphenicol resistance in which the Cre expression was induced by addition of IPTG to the growth medium. Cells were grown overnight in LB broth supplemented with chloramphenicol and IPTG to a final concentration of 1 mM. The cells were then serially diluted and spread on LB agar plates to obtain single colonies. These were then picked onto duplicate plates to check for sensitivity to kanamycin. A kanamycin sensitive colony was taken and streaked out onto LB agar. The Cre plasmid was lost due to its inherent instability in the absence of selection with chloramphenicol. The phenotype of the resulting strains was checked by agglutination with an Ol-specific and Ogawa-specific antisera (Fitzgerald, United States). In the case of Phil6973 the original strain is Inaba and in order to obtain an Ogawa derivative we used the same suicide plasmid-based procedure to introduce a wild-type wbeT gene back into the strain in which the gene had been deleted. In this case selection was based solely on the acquisition and loss of chloramphenicol resistance combined with a change in phenotype from Inaba to Ogawa.

Comparing growth rates.

Bacteria were revived from -80°C glycerol stock on LB-Agar plates at 37°C for 16h. 3 colonies were used for inoculation in 5 ml LB medium for 4h in 37°C at 180 rpm. Strains were set to the same OD by diluting with PBS and inoculated either in LB broth, LB broth high salt ²⁶ or AKI ²⁷ . High salt medium was used to maximize growth owing to the halophilic nature of V. cholerae. The inoculated media were distributed in a 24 or 48 well plate and were incubated in a Synergy™ 2 (Biotek, United States) plate reader at 30°C or 37°C, measuring optical density every 15 min at l=600hiti.

Comparing growth rates during competitive co-culture.

The growth of isogenic strains in competition experiments was tested using strains carrying plasmids pML-GreenFP /clssz and pML-BlueFP /clssz that express the green fluorescent protein and blue fluorescent protein respectively when induced by incubation at 42°C. The plasmids are essentially identical except for small differences in the structural genes of the fluorescent proteins. Furthermore, the fluorescent proteins are not expressed during the competition experiments since they were performed at 30°C.

Briefly, isogenic pairs of strains, one carrying the pML-GreenFP /clssz plasmid and pML- BlueFP /cls57 were grown up overnight in 5 ml LB broth supplemented with ampicillin (100 pg/ml). The cells were washed and resuspended in PBS and the Oϋeoo was adjusted to 0.5. The cells were then mixed in at a ratio of 1:1. Serial dilutions of the suspension were then spread onto LB agar plates supplemented with ampicillin (100 pg/ml) and incubated at 30°C overnight in order to determine the actual number of colony forming units (cfus). In order to determine the ratio of Inaba to Ogawa cells in the suspension after growth overnight at 30°C the plates were transferred to 42°C in order to express the green and blue fluorescent proteins. The actual ratio of one serotype to the other was determined by counting the number of colonies expressing each of the fluorescent proteins.

50 pi of the mixed cell suspension was used to inoculate 5 ml LB broth supplemented with ampicillin and the resulting culture was incubated at 30°C for 14h with shaking (180 rpm). Serial dilutions of the culture were again spread onto LB agar plates in order to determine the total number of cfus and the ratio of Inaba to Ogawa cells was determined by transferring the plates to 42°C and counting colonies with different fluorescence.

The cultures were then passaged every 14 hours over a period of five days and the ratio of Inaba to Ogawa cells determined.

Results

Growth comparison in rich medium.

Each set of isogenic strains was grown in 20 replicates in LB broth at 37°C for 20 hours and the OD _6OO was measured every 15 min. No difference in growth between the Ogawa and Inaba strains could be detected, figure 1A and B. The first set of isogenic strains was also tested with high salt and AKI medium and again showing no detectable difference in growth between the Ogawa and Inaba variants, figure 1C and D.

Competition experiments.

In order to check whether the two cell types compete with each other in mixed culture we introduced plasmids carrying fluorescent proteins with different colours. The blue and green proteins are almost identical in sequence and the plasmids of 3.5 Kb differ in only 12 bases. Thus the overall effect of the plasmids on growth should be the same. Furthermore, protein expression is induced by growth of the cells at 42°C. The cells of each serotype were mixed at a ratio of approximately 1:1 and grown up in different media at 30°C. The cultures were then serially diluted and spread onto selective agar plates and incubated overnight at 30°C. The total number of cells in the culture was determined and the ratios of cells of each serotype determined by transferring the plates to 42°C and incubating for four hours to induce expression of the fluorescent proteins. Results are shown in figure 2 and demonstrate that the ratios remain stable. Further experiments were performed in which cultures where passaged over a longer period, which also showed that the ratio of the two serotypes did not change with time.

Example 2: Preparation of immunogenic vaccine formulations comprising bacterial cells and secreted soluble antigen produced in the same culture.

In vaccines that comprise a mixture of bacterial cells and a secreted soluble antigen, such as in the example of the oral cholera vaccine Dukoral™, the bacteria and the soluble cholera toxin B subunit antigen (CTB) are always produced by separately culturing different bacterial strains for providing the cells and the soluble antigen which antigens are then mixed together for preparing the immunogenic vaccine formulation.

It would be a significant advantage reducing both the labour and cost of vaccine production if the immunogenic vaccine formulation could be prepared from a single bacterial culture providing both the cells and the soluble antigen for the immunogenic vaccine formulation.

We describe below a method for the preparation of an immunogenic composition, comprising:

(i) providing cells of a microbial strain expressing at least one and preferably two different cell-associated immunogenic antigens, wherein the cells further express a soluble antigen secreted into the culture medium and wherein the soluble antigen is included in the immunogenic composition formulated, the expression being preferably conferred by a recombinant DNA sequence introduced into the cells;

(ii) culturing said strain in a single vessel in a culture medium to obtain whole cells expressing the cell-associated immunogenic antigen(s) and culture medium containing the soluble antigen;

(iii) and formulating the whole cells containing said immunogenic antigen(s) and the secreted soluble antigen into an immunogenic composition. BO

Material and methods:

Strains.

Two different V. cholerae strains are used : 1) Strain 1012, a high-yield CTB producing derivative of V. cholerae JS1569 Inaba harbouring the high copy-number

CTB-encoding plasmid pML-LCTBtac2 and generated as described by Lebens et al. (Nature Bio/Technology 11: 1574-1578, 1993); and 2) MS1568/CTB, an El Tor Hikojima vaccine strain engineered to express approximately equal amounts of the Ogawa and Inaba O antigens as described by Karlsson et al. (PLoS One. 2014 Nov 14;9(ll):el08521) into which the CTB-encoding plasmid pML-LCTBtac2 (Lebens et al. 1993, see above) is introduced by electroporation.

Vaccine preparations.

These strains are cultured over night at 37C by fermentation in modified syncase medium under conditions detailed in Lebens et al. (Nature Bio/Technology 11: 1574-1578, 1993). Cultures are grown to an ODeoo _nm of ca 10 [corresponding to ca 2.5xl0 ¹⁰ bacteria/ml] when, as examined by GM1-ELISA as described by Lebens et al. (Nature Bio/Technology 11: 1574- 1578, 1993), they have a CTB content between 0.5-1.2 mg/ml. From each culture three different vaccine preparations are made: a) Preparations 1 are obtained by simply heating the cultures to 58°C for lh to kill all cells, optionally after initial 2- to 3-fold concentration by tangential ultrafiltration through a 10 kDa cut-off membrane. b) Preparations 2 are obtained by in parallel: (i) isolating the bacteria by tangential filtration through a 300 kDa cut-off membrane (or by centrifugation at 3000xg for 1 hour) and then resuspending the bacteria in phosphate buffered saline (PBS) to OD=30 (corresponding to ca 7.5xl0 ¹⁰ cells/ml) followed by heat or formalin killing of the cells as described (Lebens et al. Vaccine. 2011 Oct 6;29(43):7505-13; Karlsson et al. PLoS One. 2014 Nov 14;9(ll):el08521); and (ii) heat-treating the CTB-containing culture medium at 58C for lh to inactivate any contaminating cells, optionally after having concentrated the culture medium by ultrafiltration through a 10 kDa cut-off membrane. The killed bacteria and the treated CTB-containing culture medium are then mixed to yield the immunogenic preparation. c) Preparations 3 : Bacteria are isolated and killed in the same way as described for preparations 2; however, instead of being heated the culture medium is ultrafiltered through a 300 kDa cut-off filter to remove contaminating cells (unless this was already done in the initial separation from the bacteria - see prep. 2 above) and then concentrated 5-10 times through ultrafiltration through a 10 kDa cut-off membrane, whereafter CTB is isolated from the concentrated culture medium by precipitation with hexametaphosphate followed by gel filtration (or ion exchange chromatography on a Resource Q column) as described (Lebens et al. Nature Bio/Technology 11: 1574-1578, 1993).

Results:

Retention of immunoprotective LPS and CTB antigens in vaccine preparations.

The defined immunoprotective antigens in oral cholera vaccines of the types described are, from strain 1012 the 01 Inaba lipopolysaccharide (LPS) antigen on the bacterial cells and the rCTB secreted into the culture medium; for vaccine preparations from strain MS1568/CTB they are both the cell-associated 01 Ogawa and 01 Inaba LPS antigens and the secreted rCTB. These antigens are quantified by methods described in detail in Karlsson et al. (PLoS One. 2014 Nov 14;9(ll):el08521) and found to be fully preserved in the different vaccine preparations both in quantity and quality. When adjusted for differences in OD, the amounts of 01 LPS antigen measured by a monoclonal antibody based Inhibition ELISA method are within the range of the method variation for each of the preparations 1-3 from both V. cholerae strains and no different from the level measured in a pretreatment culture sample: 01 LPS levels within 0.8 -1.1 mg/ml 01 LPS are found in all preparations adjusted to OD=30. The proportion of Ogawa and Inaba LPS also remains intact in the different preparations from the Hikojima strain MS1568/CTB as examined by Inhibition ELISA using an Ogawa LPS-specific monoclonal antibody as well as by a dot blot method using the same antibody being 40-60% Ogawa; in accordance with this vaccine preparations from MS1568/CTB agglutinates well with serotype specific antisera against both Ogawa and Inaba (while preparations from strain 1012 only agglutinates with anti-lnaba antiserum).

The amounts of CTB in the preparations are measured with a GM1-ELISA method measuring both the binding to the GM1 ganglioside receptor and the reactivity with a specific monoclonal antibody (LT39) specifically recognizing immunoprotective oligomeric CTB. The CTB levels in preparations 1 and 2 after adjustments for ultrafiltration concentration of the culture medium are indistinguishable from those in culture medium before treatment, i.e. the CTB remains intact and functional after heat inactivation as described. The levels of purified CTB used for formulating preparations 3 are consistent with a 30-50% loss of CTB during the purification process. Purity of the CTB is determined by SDS-PAGE of boiled material using densitometric quantitation of the percentage of CTB of total Comassie Blue stained soluble protein (Lebens et al., Nature Bio/Technology 11: 1574-1578, 1993). By this approach CTB accounts for ca 50% of total soluble protein in preparations 1 and 2, and for >95% in preparations 3.

Example 3: Preparation of immunogenic vaccine formulations comprising isogenic bacterial cells expressing different antigens and secreted soluble antigen produced in the same culture.

The methods described in Example 2 for preparing immunogenic vaccine formulations comprising bacterial cells and secreted soluble antigen produced in the same culture are not limited to using a single strain for producing the immunoprotective cell-associated and secreted soluble antigens. As shown here they also apply for preparing immunogenic vaccine formulations comprising bacterial cells and secreted soluble antigen from two co cultured isogenic strains expressing different cell-associated antigens and the same secreted soluble antigen.

The CTB-encoding plasmid pML-LCTBtac2 (Lebens et al. 1993, see above) is introduced by electroporation into the isogenic V. cholerae strains MS1571 (Ogawa) and MS1572 (Inaba) described in Example 1 above. The modified strains are then mixed in equal proportions and co-cultured in modified Syncase medium for ca 24 h at 37C in a vessel under vigorous shaking to reach an OD of ca 5. The culture is divided in three parts and treated as described for preparing material analogous to the Preparations 1, 2 and 3 described in Example 2. These vaccine preparations are examined for their contents of Ogawa and Inaba bacteria as described in Example 1, and for their contents of total 01 LPS and proportions of Ogawa and Inaba LPS as well as of their contents of CTB by the same methods as used in Example 2 above. Similar to the results in example 1, the proportion of Ogawa and Inaba bacteria in the three vaccine preparations remains stable resulting in approximately 50% (±20%) of cells of each serotype. Consistent with this the preparations show a similar proportion of Ogawa LPS (around 50%) and CTB at similar levels and purity in preparations 1 and 2 as well as in preparation 3 as those described in Example 2 (as adjusted for the lower growth rate/OD in shake culture in Example 3 compared to the optimized fermentor cultures in Example 2).

Example 4. Immunogenic efficacy in preclinical models

Since the exemplified methods for preparing immunogenic vaccine formulations comprising isogenic bacterial cells expressing different antigens and secreted soluble antigen produced in the same culture refer to vaccine preparations mainly intended for use as oral vaccines against cholera, it is most relevant to assess their immunogenic efficacy in preclinical models after oral administrations resembling oral cholera vaccination in humans (two oral vaccine doses given ca 2 weeks apart) and measuring those components of the immune response that best predict the immunoprotective efficacy in humans, i.e. the vibriocidal antibody and anti-CTB response in serum and most important the intestinal-mucosal IgA anti-LPS and anti-CTB response in fecal extracts.

To this end groups of Balb/c mice are immunized in two rounds by the oral/intragastric route at 2-week interval with the various vaccine preparations. The vaccine dose administered in each round is aimed at corresponding to l/20th of a human dose, i.e. around 5xl0 ⁹ bacterial cells and 50 micrograms of CTB. The licensed oral cholera vaccine Dukoral is included at the same dosage as a positive reference given in one group of mice in each experiment and an unimmunized group also included as a negative reference group. Fecal samples are collected 10-11 days after the last vaccine administration and serum collected the next day in connection with the sacrifice of the animals. The experimental design and procedures of immunization and the analytical methods used are described in detail in Karlsson et al. ((PLoS One. 2014 Nov 14;9(ll):el08521).

The differently prepared vaccine preparations described in Examples 2 and 3 all are found to exhibit strong immunogenicity being non-inferior (equivalent or higher) than that of the concurrently tested Dukoral vaccine with regard to serum vibriocidal antibody responses and serum IgG and IgA anti-CTB responses as well as to fecal IgA anti-LPS and anti-CTB responses. Geometric mean vibriocidal antibody titers after two rounds of oral immunization typically range between 1:500 -1:3000, and serum anti-CTB titers between 1/3,000 - 1/20,000 for IgG and 1/500 -1/3,000 for IgA. Mean IgA antibody titers in fecal extracts range between 1:30-1:100 for anti-Ol LPS and 1:50-1:200 for anti-CTB.

Example 5: Preparation of dry vaccine formulations While current oral cholera vaccines are being formulated as liquid vaccines it could be advantageous to formulate oral vaccines as dry vaccines in the form of e.g. an enterocoated capsule or tablet or as enterocoated granulate. To achieve and assess the antigen stability and immunogenicity of such dry formulation of the currently exemplified oral cholera vaccine formulations, the vaccine preparations 1-3 of Example 2 are lyophilized in presence of 25 mg/ml of sucrose or trehalose and stored at 4°C, 25°C or 40°C for up to 3 months. The stability of the LPS and CTB antigens are determined by the methods described in the above examples in comparison with the liquid preparations. Results show that stability of both types of antigens is excellent in the dry formulations with no evidence of deterioration at any temperature between 4-40°C over the tested time period. There is also no indication of decline in immunogenicity as examined in orally immunized mice using the same design and methods as described in Example 4 above.