IMMUNOGENIC COMPOSITION COMPRISING CYAA-DERIVED POLYPEPTIDE PROMOTING A TH1/TH17-ORIENTED IMMUNE RESPONSE

Abstract

The invention relates to the use of a polypeptide derived from the adenylate cyclase of a Bordetella sp. (CyaA-derived polypeptide) by deletion of a segment of at least 93 amino acid residues, in particular a polypeptide derived from CyaA of Bordetella pertussis, as an immunomodifying antigen of the TH1/TH17-oriented immune response in an immunogenic composition. The invention relates to a vaccine candidate comprising such CyaA-derived polypeptide, either in an acellular immunogenic composition for active immunization against a condition causally related to the infection of a host by Bordetella sp. or in a combination composition encompassing said acellular immunogenic composition.

Claims

1. An immunogenic composition which is chosen in the group of: a) an acellular immunogenic composition suitable for the protection against a condition causally related to the infection by a Bordetella strain, in particular Bordetella pertussis in a human host, or b) a combination immunogenic composition comprising said acellular immunogenic composition suitable for the protection against a condition causally related to the infection by a Bordetella strain, in particular Bordetella pertussis in a human host wherein said acellular immunogenic composition comprises: a non-toxic polypeptide, in particular a non-cytotoxic polypeptide, derived from the adenylate cyclase protein (CyaA-derived polypeptide) of a Bordetella strain and, optionally an adjuvant of the TH1 immune response or an adjuvant of the TH2 immune response or a combination of both.

2. An immunogenic composition according to claim 1, wherein the non-cytotoxic polypeptide, derived from the adenylate cyclase protein (CyaA-derived polypeptide) of a Bordetella strain is (i) a polypeptide the amino acid sequence of which is obtained from CyaA of Bordetella pertussis and contains a deletion of a continuous segment of at least 93 amino acid residues from position 227 to position 321 in the amino acid sequence of the native CyaA of Bordetella pertussis or (ii) a polypeptide which is a variant of the polypeptide in (i) the amino acid sequence of said variant polypeptide being obtained from CyaA of Bordetella parapertussis, Bordetella bronchiseptica or Bordetella hinzii, and containing the deletion of a continuous segment of at least 93 amino acid residues defined by the positions in the native CyaA sequence of Bordetella parapertussis, Bordetella bronchiseptica or Bordetella hinzii matching positions 227 and 321 in CyaA of said continuous segment of at least 93 amino acid residues of Bordetella pertussis.

3. An acellular immunogenic composition according to claim 1 or 2 suitable for the protection against a condition causally related to the infection by a Bordetella strain, in particular Bordetella pertussis, in a human which comprises: at least two antigens from a Bordetella strain wherein one antigen is (i) a non-toxic polypeptide, in particular a non-cytotoxic polypeptide, derived from the adenylcyclase protein (CyaA-derived polypeptide) of a Bordetella strain, said polypeptide being in particular (i.1) either a polypeptide the amino acid sequence of which is obtained from CyaA of Bordetella pertussis and contains a deletion of a continuous segment of at least 93 amino acid residues from position 227 to position 321 in the amino acid sequence of the native CyaA of Bordetella pertussis, (i.2) or a polypeptide which is a variant of the polypeptide in (i) the amino acid sequence of said variant polypeptide being obtained from CyaA of Bordetella parapertussis, Bordetella bronchiseptica or Bordetella hinzii, and containing the deletion of a continuous segment of at least 93 amino acid residues defined by the positions in the native CyaA sequence of Bordetella parapertussis, Bordetella bronchiseptica or Bordetella hinzii matching positions 227 and 321 in CyaA of said continuous segment of at least 93 amino acid residues of Bordetella pertussis, and wherein further antigen(s) is (are) selected in the group of (ii) Pertussis toxoid (PT), (iii) Filamentous Haemagglutinin (FHA), (iv) Pertactin (PRN) and (v) Fimbriae (Fim), wherein a protective immune response against the Bordetella strain is raised against said antigens and, optionally an adjuvant of the TH1 immune response or an adjuvant of the TH2 immune response or a combination of both.

4. A Combination immunogenic composition according to claim 1 or to claim 2, comprising further antigens as active ingredients for the elicitation of an immune protection against determined pathogens which are (vi) at least one antigen of Clostridium tetani consisting of the Tetanus toxin in detoxified form, (vii) an antigen of Corynebacterium complex consisting of the diphtheria toxin in detoxified from, and optionally (viii) further antigens of different pathogen(s).

5. A combination immunogenic composition according to any one of claims 1 to 4 which comprises further antigens as active ingredients for the elicitation of an immune protection against determined pathogens which are selected in the group of Hepatitis B surface antigen (HBs), inactivated poliovirus (IPV) of one or several virus strains, Haemophilus influenza type b polysaccharide.

6. An acellular immunogenic composition according to any one of claims 1 to 3 or a combination immunogenic composition according to any one of claim 1, 4 or 5, wherein the antigens selected in the group of Bordetella toxin in detoxified form (PT), Filamentous Haemagglutinin (FHA), Pertactin (PRN) and Fimbriae (Fim) are independently of each other from Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica or Bordetella hinzii, preferably from B. pertussis, and preferably are all from the same Bordetella strain.

7. Combination immunogenic composition according to any one of claims 1, 2 or 4 to 5 wherein one dose of 0.5 ml of the immunogenic composition contains: Bordetella toxin in detoxified form, in particular Pertussis toxoid: 1 to 50 micrograms; Filamentous Haemagglutinin: 1 to 50 μg; Pertactin: 1 to 20 μg; optionally Fimbriae: 2 to 25 μg; Tetanus toxoid: at least 0.25 and less than 25 Lf; Diphtheria toxoid: at least 0.25 and less than 50 Lf; and optionally, Hepatitis B surface antigen: 5 μg to 25 μg inactivated poliovirus: from 5 to 45, in particular from 8 to 40 D-antigen unit per strain; Haemophilus influenza type b polysaccharide: 1 to 20 μg.

8. Combination immunogenic composition according to any one of claims 1, 2 or 4 to 6, wherein one dose of 0.5 ml of the immunogenic composition comprises: Bordetella toxin in detoxified form in particular Pertusssis toxoid: 8 μg; Filamentous Haemagglutinin: 8 μg; Pertactin: 2.5 μg; Tetanus toxoid: 5 Lf; Diphteria toxoid: 2.5 Lf.

9. An immunogenic composition, in particular a combination immunogenic composition, according to any one of claims 1 to 8 wherein one dose of the immunogenic composition comprises 2.5 to 600 micrograms, 2.5 to 500 μg, 2.5 to 400 μg, 2.5 to 300 μg, 2.5 to 200 μg or 2.5 to 100 μg and preferably comprises 25 μg of the CyaA-derived polypeptide of Bordetella pertussis.

10. An immunogenic composition, in particular a combination immunogenic composition, according to any one of claims 1 to 9 wherein the adjuvant of the TH2 response is an aluminum salt, in particular is aluminum hydroxide, aluminum hydroxyphosphate sulfate or aluminum phosphate.

11. An immunogenic composition, in particular a combination immunogenic composition according to any one of claims 1 to 10 wherein the adjuvant of the TH1 response is selected: i. in the group of MPL-containing adjuvant, in particular AS15, AS01B, AS01D, or AS01E; CpG-containing adjuvant, in particular AS15, QS21-containing adjuvant, in particular AS01B, D, and E or AS15, immune stimulating complexes (ISCOMs), IC31-containing adjuvant, chitosan-containing adjuvant, liposomal formulation-based adjuvant, in particular AS01B D, E or AS15 or ISCOMs, lipid-based emulsions such as MF59, or ii. In the group of adjuvants which are or contain a ligand of a toll-like receptor selected among: a ligand of toll-like receptor 4 (TLR-4), in particular monophosphoryl lipid A (MPL) or Glucopyranosyl Lipid A (GLA) or, a ligand of toll-like receptor 9 (TLR-9), in particular a synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs and more particularly HBsAg-1018 or, a ligand of toll-like receptor 3 (TLR-3), in particular a synthetic analog of double-stranded RNA (dsRNA) and more particularly a composition containing Poly(I:C) such as polyinosinic-polycytidylic acid plus poly-L-lysine double-stranded RNA in the presence of carboxymethylcellulose (Poly (ICLC). or, a ligand of toll-like receptor 2 (TLR-2), in particular synthetic lipopeptides or a recombinant lipoprotein and, a ligand of toll-like receptor 5 (TLR-5), in particular a recombinant bacterial flagellin.

12. An immunogenic composition, in particular a combination immunogenic composition, according to any one of claims 1 to 11 wherein the CyaA-derived polypeptide of Bordetella consists of: 1) a segment or a fragment of Bordetella pertussis CyaA protein as set forth in SEQ ID No. 2, the sequence of said segment or fragment beginning with the first residue of SEQ ID No.2 and ending with a residue located from position 183 to position 227 of SEQ ID No.2 or a polypeptide variant obtained from Bordetella parapertussis, Bordetella bronchiseptica or Bordetella hinzii and having at least 95% identity in amino acid residues with the segment or fragment consisting of residues 1 to 183 or 1 to 227 in SEQ ID No.2, fused to 2) a segment or a fragment of Bordetella pertussis CyaA protein as set forth in SEQ ID No. 2, the sequence of said segment or fragment beginning with a residue located from position 321 to position 387 of SEQ ID No.2 and ending with the last residue of SEQ ID No.2 or a polypeptide variant obtained from Bordetella parapertussis, Bordetella bronchiseptica or Bordetella hinzii, and having at least 95% identity in amino acid residues with the segment or fragment consisting of residues 321 to final residue or 387 to final residue in SEQ ID No.2.

13. An immunogenic composition, in particular a combination immunogenic composition according to any one of claims 1 to 12 wherein the CyaA-derived polypeptide of Bordetella consists of: 1) a polypeptide comprising or consisting of the sequence as set forth in SEQ ID NO:10 or a polypeptide variant obtained from Bordetella parapertussis, Bordetella bronchiseptica or Bordetella hinzii and characterized by an amino acid sequence matching the amino acid residue positions of SEQ ID No.10; 2) a polypeptide comprising or consisting of the sequence as set forth in SEQ ID NO:12 or a variant obtained from Bordetella parapertussis, Bordetella bronchiseptica or Bordetella hinzii, and characterized by an amino acid sequence matching the amino acid residue positions of SEQ ID No.12, 3) a polypeptide comprising or consisting of the sequence as set forth in SEQ ID NO:19 or a variant obtained from Bordetella parapertussis, Bordetella bronchiseptica or Bordetella hinzii, and characterized by an amino acid sequence matching the amino acid residue positions of SEQ ID No.19, 4) a polypeptide comprising or consisting of the sequence as set forth in SEQ ID NO:20 or a variant obtained from Bordetella parapertussis, Bordetella bronchiseptica or Bordetella hinzii and characterized by an amino acid sequence matching the amino acid residue positions of SEQ ID No.20.

14. An immunogenic composition, in particular a combination immunogenic composition according to any one of claims 1 to 13, wherein the CyaA-derived polypeptide is the expression product in E. coli cells of the nucleic acid molecule gtCyaad93-CyaC-opt borne by plasmid pGTP-gtCyaad93-CyaC-opt of SEQ ID No. 25.

15. A medicinal composition for administration to a human host which comprises an immunogenic composition according to any one of claims 1 to 14 and which is provided as an administration form selected among a powder, a powder and solution for reconstitution, a liquid, in particular a suspension or a solution, a lyophilized component and a combination of such administration forms.

16. A medicinal composition according to claim 15 wherein one dose for administration to a human host contains 0.25 ml to 1 ml of liquid, in particular of reconstituted product.

17. A medicinal composition according to claim 15 or 16, for administration to a human host which is provided as a pharmaceutical form formulated for administration by injection, in particular for intramuscular injection.

18. A method for preparing a vaccine comprising as active ingredients for protection against determined pathogens or condition causally related to such pathogens, antigens of said pathogens and furthermore a polypeptide derived from the adenylate cyclase protein (CyaA-derived polypeptide) of a Bordetella strain as defined in any of claim 1 or 13, wherein the method comprises the step(s) of: a) providing antigens selected in the group of (i) at least one of the antigens from Bordetella selected in the group of Pertussis toxin in detoxified form (PT), Filamentous Haemagglutinin (FHA), Pertactin (PRN) and Fimbriae (Fim) and preferably the first three antigens or all of these antigens, (ii) at least one antigen of Clostridium tetani consisting of the Tetanus toxin in detoxified form, (iii) an antigen of Corynebacterium complex consisting of the diphtheria toxin in detoxified form, and (iv) the CyaA-derived polypeptide as defined in any one of claim 1 or 13 to 15 and optionally (v) further antigens of different pathogen(s) as active ingredients for the elicitation of an immune response against said determined pathogens formulated as one or several component(s) and, b) admixing said component(s) with one or more compounds that enhance the immune response when the vaccine is administered to a host such as adjuvant(s) of the TH2-oriented immune response, adjuvant of the TH1-oriented immune response, adjuvant of the TH17-oriented immune response or a combination of such adjuvants, wherein steps a) and b) are optionally carried out as a single step.

19. A method according to claim 18 wherein the active ingredients for protection are as defined in any one of claims 2 to 14.

20. A combination immunogenic composition according to any one of claims 1 to 14 or a medicinal composition according to any one of claims 15 to 17 for use in active immunization of a human host against diphtheria, tetanus and condition causally related to pertussis infection, and optionally against hepatitis B, poliomyelitis and/or disease caused by Haemophilus influenza type b.

21. A combination immunogenic composition according to any one of claims 1 to 13 or a medicinal composition according to any one of claims 14 to 16 for use in active immunization of a human host against the persistence of Pertussis bacteria after infection of a human host, or against the transmission and/or against the colonization of airways of the human host infected by a Bordetella strain, in particular B. pertussis.

22. A combination immunogenic composition according to any one of claims 1 to 13 or a medicinal composition according to any one of claims 15 to 17 for use according to claim 19 or 20 as a dose for administration after a first dose, in particular (i) as a second or as a further dose of a multiple-dose setting or (ii) as a booster dose, in individuals who previously received first vaccination dose or primary vaccination with a monovalent or a combination vaccine against at least one of the diseases selected in the group of tetanus, diphtheria and a condition causally related to infection by a Bordetella strain, in particular Bordetella pertussis, and optionally hepatitis B, poliomyelitis and disease caused by Haemophilus influenza type b.

23. A combination immunogenic composition according to any one of claims 1 to 14 or a medicinal composition according to any one of claims 16 to 18 for use according to any one of claims 20 to 22 as a dose for administration as a second or as a further dose, in particular as a booster dose and wherein said second, further, or in particular booster dose, is different from the first or from the previously administered dose(s) in that said first or previous doses is(are) devoid of non-toxic CyaA derived polypeptide as defined in claims 1, 2 or 12 to 14, and preferably is (are) devoid of any CyaA polypeptide or CyaA-derived polypeptide.

24. A combination immunogenic composition or a medicinal composition for use according to any one of claims 20 to 23, wherein the host is a child at birth or later, in particular until 18 months of age

25. A combination immunogenic composition for use according to claim 22 or 23, as a booster dose in a prime/boost vaccination setting wherein the host is a Child over 4 years of age, an adolescent over 11 or an adult, in particular an elderly person, a pregnant woman or relatives close to a pregnant woman, said host having previously received primary vaccination with a different acellular vaccine against a condition causally related to infection by Bordetella strain, in particular Bordetella pertussis either administered as a monovalent pertussis vaccine or a combination vaccine against whopping cough.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0159] FIGS. 1A-B:

[0160] Cytokine responses after vaccination with Tdap+/−GTL003: Intramuscular route The results in histograms are representative of mean recall response of 4 animals. The splenocytes of 4 animals were pooled together and were cultured in duplicates. Each result is a mean response of duplicate wells. Levels of cytokines were analyzed using multiplex bead based ELISA and % variation (CV) between duplicates were below 20%. Error bars representative of % CV of duplicate wells.

[0161] FIG. 1A:

[0162] Cytokine response of animals primed with Tdap and boosted either with Tdap or with Tdap+GTL003

[0163] White bars: Tdap prime+Tdap boost

[0164] Grey bars: Tdap prime+Tdap+GTL003 boost

[0165] FIG. 1B:

[0166] Cytokine response of animals primed with Tdap and boosted either with WCV or with Tdap+GTL003

[0167] White bars: Tdap prime+tdap+GTL003 boost

[0168] Grey bars: Tdap prime+WCV boost

[0169] FIGS. 2A-B:

[0170] Cytokine responses after vaccination with Tdap+/−GTL003: Intraperitoneal route

[0171] The results in histograms are representative of mean recall response of 4 animals. The splenocytes of 4 animals were pooled together and were cultured in duplicates. Each result is a mean response of duplicate wells. Levels of cytokines were analyzed using multiplex bead based ELISA and % variation (CV) between duplicates were below 20%. Error bars representative of % CV of duplicate wells.

[0172] FIG. 2A:

[0173] Cytokine response of animals primed with Tdap and boosted either with Tdap or with Tdap+GTL003

[0174] White bars: Tdap prime+Tdap boost

[0175] Grey bars: Tdap prime+tdap+GTL003 boost

[0176] FIG. 2B:

[0177] Cytokine response of animals primed with Tdap and boosted either with WCV or with Tdap+GTL003

[0178] White bars: Tdap prime+tdap+GTL003 boost

[0179] Grey bars: Tdap prime+WCV boost

[0180] FIG. 3:

[0181] Cytokine responses after immunization with Tdap or Tdap+GTL00: intraperitoneal INF-g immune response measured in animals vaccinated either with adjuvanted-Tdap alone or with adjuvanted-Tdap+GTL003

[0182] In vitro restimulations were performed with antigens contained in the Tdap or antigens contained in the Tdap+GTL003

[0183] FIG. 4:

[0184] Cytokine responses after immunization with Tdap or Tdap+GTL00: intraperitoneal IL-17 immune response measured in animals vaccinated either with adjuvanted-Tdap alone or with adjuvanted-Tdap+GTL003

[0185] FIGS. 5A-D:

[0186] Isotyping Results (from intramuscular vaccination) [0187] Results are from two pools of two animals each [0188] Histograms represent mean GMT [0189] Error bars represent 95% CI of these titers [0190] Pool to Pool variation observed as expected [0191] In IM route, the effects were mixed Th1/Th2

[0192] FIG. 5A:

[0193] Pertussis toxin-IgG1 antibodies after a prime with Tdap and a boost with either Tdap, Tdap+GTL003 or WCV

[0194] FIG. 5B:

[0195] Pertussis toxin-IgG2a antibodies after a prime with Tdap and a boost with either Tdap, Tdap+GTL003 or WCV

[0196] FIG. 5C:

[0197] Pertussis toxin-IgG2b antibodies after a prime with Tdap and a boost with either Tdap, Tdap+GTL003 or WCV

[0198] FIG. 5D: Pertussis toxin-IgG3 antibodies after a prime with Tdap and a boost with either Tdap, Tdap+GTL003 or WCV

[0199] FIGS. 6A-D:

[0200] Isotyping Results (from intramuscular vaccination) [0201] Results are from two pools of two animals each [0202] Histograms represent mean GMT [0203] Error bars represent 95% CI of these titers [0204] Pool to Pool variation observed as expected [0205] In IM route, the effects were mixed Th1/Th2

[0206] FIG. 6A:

[0207] FHA-IgG1 antibodies after a prime with Tdap and a boost with either Tdap, Tdap+GTL003 or WCV

[0208] FIG. 6B:

[0209] FHA-IgG2a antibodies after a prime with Tdap and a boost with either Tdap, Tdap+GTL003 or WCV

[0210] FIG. 6C:

[0211] FHA-IgG2b antibodies after a prime with Tdap and a boost with either Tdap, Tdap+GTL003 or WCV

[0212] FIG. 6D:

[0213] FHA-IgG3 antibodies after a prime with Tdap and a boost with either Tdap, Tdap+GTL003 or WCV

[0214] FIG. 7:

[0215] Plasmid maps of GTL003 (gtCyaad93)

[0216] FIG. 8:

[0217] Plasmid sequence (SEQ ID No.25)

EXAMPLES

Abbreviations

[0218]

TABLE-US-00002 aa Aminoacid ACT GTL003 aP Acellular pertussis vaccine APC Antigen-presenting cells CD Cluster designation CTL Cytotoxic T lymphocytes CyaA Adenylate cyclase DC Dendritic cells DTaP Diphtheria and tetanus toxoids combined with acellular pertussis vaccine DTwP Diphteria and tetanus toxoids combined with whole cell pertussis inactivated bacteria vaccine FHA Filamentous hemagglutinin FIM Fimbriae GTL003 recombinant genetically detoxified CyaA described in WO 2014/016310 id Intradermal IL-4 Interleukin 4 INF-γ Interferon gamma im Intramuscular ip Intraperitoneal KLH Keyhole Limpet hemocyanin LPS Lipopolysaccharide PRN Pertactin PT Pertussis toxin Tdap* Tetanus and diphtheria toxoids combined with acellular pertussis reduced vaccine TH1 T-helper l cells TH2 T-helper 2 cells TH17 T-helper 17 cells TLR Toll like receptor WCV Whole cell pertussis vaccine WHO World health organization wP Whole cell pertussis vaccine *Tdap distinguishes over DTap vaccine in that it contains a lower concentration of antigens: Tdap indeed reflects the concentration of antigens which is used for booster doses in conventional vaccines (such as Boostrix ™-GSK Biologicals S.A)

[0219] 1. Material and Methods

[0220] 1.1. Mice

[0221] 13-23 grams female/male NIH mice were in bred at Serum Institute of India animal house. Mice were housed under pathogen-free conditions with water and food ad libitum. Procedures involving animals and their care were conformed to SIIL's guidelines that comply with national and international laws and policies and that are reviewed by the local ethical committee.

[0222] 1.2. Vaccine

[0223] Manufacturing and Formulation of Tdap Vaccine

[0224] The TdaP Vaccine is a blend of individually adsorbed antigens viz; Diphtheria Toxoid, Tetanus toxoid, Pertussis toxoid, Filamentous haemagglutinin and Pertactin. The tetanus component is purified toxoid manufactured by the chemical detoxification of toxin produced by Clostridium tetani. Tetanus toxoid is prepared from the toxin produced by the growth of this strain of Clostridium tetani in Semi-synthetic (meat free) medium using fermentation technology. The diphtheria toxoid is produced using Corynebacterium diphtheriae. Toxin is concentrated, partially purified and subsequently detoxified. And the toxoid is further purified to produce purified diphtheria toxoid.

[0225] The bulk Acellular pertussis is non-infectious and made up of sterile combination of three purified pertussis antigens i.e. Pertussis toxoid, Filamentous haemagglutinin and Pertactin. The fermentation is followed by chromatography based purifications followed by chemical detoxification of Toxoids. The Pertussis Toxin (PT) and Filamentous Haemagglutinin (FHA) are produced as extra cellular proteins which are secreted during fermentation process where as Pertactin (69 kDa outer membrane protein) is extracted from cells.

[0226] Tdap is a reduced antigen version of Acellular pertussis based DTaP vaccine. On Dec. 17, 1991 the first Acellular pertussis vaccine viz; Acel-imune by lederle was licensed as a 4th and 5th Booster vaccine. These vaccines are classical vaccines.

[0227] Construction and Purification of GTL003

[0228] The DNA sequence of wild type CyaA (CyaAwt: GeneBank: CAE41066.1) was optimized and synthetized (GeneCust) for the expression in E. coli. The optimized DNA sequence is named gtCyaA.

[0229] The gtCyaA was then inserted in the pGTPc608 plasmid that contains a pTAC inducible promoter (plasmid provided by GTP Technology, Labége, France).

[0230] The deletion of 93 aa in gtCyaA was generated between aa 227 and 321 by enzymatic restriction/ligation. The obtained amino acid sequence is derived from the sequence of SEQ ID No.2 wherein the segment from amino acid residue 227 to amino acid residue 321 has been deleted.

[0231] Purification protocol was already described in EP1 576 967 B1.

[0232] 1.3. Vaccine Formulation and Administration

[0233] Three vaccines were used to vaccinate mice against pertussis. All vaccines were adjuvanted with aluminum hydroxide in buffer. WCV was not adjuvanted.

TABLE-US-00003 TABLE 1 Vaccine antigen and adjuvant doses injected per immunization Antigen and adjuvant injected per immunization/per mouse DT TT PT FHA PRN GTL003 Alum Vaccines (Lf) (Lf) (mcg) (mcg) (mcg) (mcg) (mg) Tdap(1:10) 0.25 0.5 0.8 0.8 0.25 — 0.39 Tdap(1:10) + 0.25 0.5 0.8 0.8 0.25 25 0.39 GTL003

[0234] WCV used in the present experiments corresponds to 0.5 IU/dose of B. pertussis (without alum) injected in mice.

[0235] In GTL003-containing vaccines, 25 μg of GTL003 were added to the Tdap vaccines preparation before injection. In prime boost protocol, mice were vaccinated twice via the same route, i.e. im, ip or sc, at day 0 and day 28.

TABLE-US-00004 TABLE 2 summary of the vaccination schedule, In vitro stimulation was performed aP antigens +GTL003 In vitro 1st Dose 2nd Dose Stimulus at Groups at day 0 at day 28 day 42 Group 1 Tdap (1:10) Tdap (1:10) aP antigens + Group 2 Tdap (1:10) Tdap (1:10) + GTL003 25 μg GTL003 Group 3 Tdap (1:10) WCV

[0236] 1.4. Cytokine Response Profiling

[0237] Spleen cells from control and treatment groups were isolated using reported procedures. Briefly, Splenocytes were obtained by crushing the spleen using syringe plunger. The resulting Splenocytes were lysed using (0.15 M NH4Cl, 10 mM NaHCO3, 0.1 mM EDTA, pH 7.3). Splenocytes were cultured at 4×10.sup.6 cells/well in RPMI 1640 (2 mM L-Glutamine, 10% FCS) for 96 hours in presence of Pertussis antigens in presence and absence of GTL003. The cell supernatants were collected and cytokine levels were analyzed using commercial multiplex bead-based immunoassay kits according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, Calif.).

[0238] 1.5. Humoral Immune Response Profiling

[0239] Antibody titres and isotyping was done using multiplex bead based immunoassay. Color-coded carboxylated microspheres representing distinct bead regions were obtained from Luminex Laboratories. Purified pertussis antigens (PT toxin, pertactin and FHA) were coupled to distinct activated beads essentially as described by van Gageldonk et al (25). Commercially available kit from Luminex technologies were used to couple the antigens to the beads. The kit is based on activation of beads by EDC and sulfo-NHS chemistry. Phycoerythrin (RPE)-conjugated goat anti-mouse total IgG, IgG1, IgG2a, IgG2b and IgG3 antibodies from Santa cruz were used for the assay. Purified pertussis antigens were obtained from Serum Institute of India Ltd. Humoral responses to GTL003 was analyzed using conventional ELISA. Briefly, optimized concentration of GTL003 was used for coating the plates. The sera samples were added and captured antibodies were detected using anti-mouse enzyme conjugated antibodies. The titre was determined as highest dilution of antibody showing the response higher than the cut-off OD.

[0240] 1.6. Respiratory Challenge

[0241] Six mice each group were inoculated intraperitoneally with 0.5 ml from the test and reference vaccines (Mills et al. 1998, Xing et al. 1999). On day 21, B. pertussis suspension (2×10.sup.8 CFU/ml) was instilled in each mouse using intranasal challenge. Infected mice were sacrificed 7 days post-challenge. After ablation, the lung of each animal was homogenized and plated on BG and incubated at 37° C. for five days for determination of viable counts (Colony Forming Units—CFU).

[0242] 1.7 Evaluation of Impact of GTL003 on Tetanus and Diphtheria Antibody Titres in Tdap Vaccines

[0243] The experiments were conducted in accordance with the following design:

TABLE-US-00005 Control group Test group Experimental Tdap Vaccine alone Tdap Vaccine + 25 μg groups of GTL003 immunization Day 0, 14, 28 Bleed on Day 42 Number of animals 10 per group Assay Titre estimation using Luminex assay Antibody titres Geometric Mean Titres reported as

[0244] The obtained results were as follows:

TABLE-US-00006 GTL003 in prime Diphtheria GMT Tetanus GMT and boost at Day 42 at Day 42 Tdap Vaccine alone 19401 235253 Tdap Vaccine + 25401 235253 GTL003

[0245] 1.8 Effect of GTL003 on Th1 Cytokine (IFN-Gamma) Levels when Formulated with Tdap Vaccine and Administered as Booster Vaccine

[0246] In order to assay the effect of repeated administration of Tdap vaccine and Tdap+GTL003 on IFN-gamma levels, the following experiments were carried out:

TABLE-US-00007 Day 0 Booster 1 Booster 2 Stimulus IFNGamma Route Immunization (Day 28) (Day 56) Stimulus time Median value IM Tdap (1:10) aP 96 hrs 2282 (10 μg/ml) (day 28) IM Tdap (1:10) Tdap(1:10) aP 96 hrs  484 (10 μg/ml) (day 56) IM Tdap (1:10) Tdap(1:10) Tdap(1:10) aP 96 hrs  200 (10 μg/ml) (day 72) GTL003 Treatment groups IM Tdap (1:10) Tdap(1:10) + aP 96 hrs 1900 25 μg (10 μg/ml) (day 56) GTL003 IM Tdap (1:10) Tdap(1:10) + Tdap(1:10) + aP 96 hrs 1600 25 μg 25 μg (10 μg/ml) (day 72) GTL003 GTL003

[0247] Median values are representative of data from 6 animals (spleen processed in 3 different pools of 2 spleen each). Splenocytes were stimulated (4 million cells/well) with mixture of aP antigens (10 μg each) and at 96 hours the supernatants were collected for cytokine analysis.

[0248] 2. Results

[0249] 2.1. Mice Immune Response after Prime with Tdap and Boost with Tdap.sup.+ GTL003

[0250] To assess the impact of GTL003 in the booster vaccination on the immune response induced in primed mice, animals were primed with one injection of alum adjuvanted-Tdap at day 0 and received the boost injection containing alum adjuvanted-Tdap with or without GTL003 added to the formulation at day 28. In another group animals were primed with one injection of alum adjuvanted-Tdap at day 0 and received the boost injection containing Whole cell Vaccine without Alum at day 28. Three injection routes where tested: intra-muscular (IM), intra-peritoneal (IP) and subcutaneous (SC).

[0251] 2.1.1. Cytokine Immune Response: Towards a Th1/Th17 Immune Response

[0252] Aluminium hydroxide adjuvanted-Tdap vaccinated people develop a more pronounced Th2 and lower Th1 immune response than induced by wP vaccination. This is thought to be the reason why immunity wanes more rapidly compared to immunity in people vaccinated with the WCV that is more prone to induce a TH1 and TH17 types of immune response (10, 15). (Bancroft)

[0253] IM Immunization

[0254] Animals primed by IM injection of Tdap/Alum and boosted with Tdap/Alum+GTL003 developed a higher antigen-specific IFN-g immune response as well as a higher antigen-specific IL-17 immune response compared to animals primed with Tdap and boosted with Tdap alone. Ratios were calculated between the level of IL-4 or IL5 vs IFN-g secreted by antigen-stimulated spleen cells to assess the impact of the booster of this TH1/TH2 ratio.

TABLE-US-00008 TABLE 3 Cytokine response measured after mice immunization either with Tdap (prime) + Tdap (boost) or with Tdap (prime)/Tdap + GTL003 (Boost) (pg/ml). Ratio of IFN-g/IL-4 and IFNg/IL5 secreted cytokines is also calculated. Splenocytes restimulations were performed with Ap antigens + GTL003 Group IFN Prime Boost Stimulus Route Gamma IL4 IL5 IL-17 IFNg/IL4 IFNg/IL5 TdaP TdaP (1:10) Ap + IM 388 210 610 389 1.85 0.64 (1:10) GTL003 TdaP TdaP(1:10) + Ap + IM 1800 753 278 969 2.39 6.47 (1:10) GTL003 GTL003

[0255] As shown in Table 3, surprisingly, the addition of GTL003 in the booster skewed the immune response towards a Th1 immune response, i.e. increased IFNg/IL-4 and IFNg/IL-5 ratio compared to the TdaP boost without GTL003. Of note, the IL-17 response was also increased.

[0256] When we then compared the effect of GTL003 on the Th1 immune response when added to a Tdap+GTL003 boost vs the WCV boost, the IFNg immune response was higher in animals that have been boosted with Tdap+GTL003 (Table 4) whereas the Il-17 is equivalent.

TABLE-US-00009 TABLE 4 Cytokine response measured after mice immunization either with Tdap (prime) + Tdap (boost) or with Tdap (prime) + WCV (Boost) (pg/ml). Ratio of IFN-g/IL-4 and IFNg/IL5 secreted cytokines is also calculated. Splenocytes restimulations were performed with Ap antigens + GTL003. Group IFNg/ IFNg/ Prime Boost Stimulus Route IFNg IL4 IL5 IL-17 IL4 IL5 TdaP TdaP + Ap + IM 1800 753 278 969 2.39 6.47 (1:10) GTL003(1:10) GTL003 TdaP WCV Ap + IM 811 435 729 914 1.86 1.11 (1:10) GTL003

[0257] When looking at the IFNg/IL-4 ratio and IFNg/IL-5 ratio, they were more-Th1 oriented response in the Tdap+GTL003 boost compared to the WCV boost.

[0258] IP Immunization:

[0259] The same immunizations were performed via the intraperitoneal route. Surprisingly, the results were not the same as via the IM route. The Tdap prime/Tdap boost induced a better TH1 profile compared to the Tdap prime/Tdap+GTL003 boost. No skew of the response towards a TH1 is observed when adding GTL003 in the Tdap boost (Table 5) or WCV in the boost (table 6)

TABLE-US-00010 TABLE 5 Cytokine response measured after mice immunization either with Tdap (prime) + Tdap (boost) or with Tdap (prime)/Tdap + GTL003 (Boost) (pg/ml). Ratio of IFN-g/IL-4 and IFNg/IL5 secreted cytokines was also calculated. Splenocytes restimulations were performed with Ap antigens + GTL003. Group Prime Boost Stimulus Route IFNg IL4 IL5 IL-17 IFNg/IL4 IFNg/IL5 TdaP TdaP Ap + IP 838 1207 1816 1810 0.69 0.46 (1:10) (1:10) GTL003 TdaP TdaP + Ap + IP 341 2207 1492 209 0.15 0.23 (1:10) GTL003 GTL003 (1:10)

[0260] Finally, when comparing the WCV boost with Tdap+GTL003 boost via the IP route, WCV boost allowed a slightly better IFNg and IL-17 immune response (Table 6). The overall response was nevertheless Th2 oriented.

TABLE-US-00011 TABLE 6 Cytokine response measured after mice immunization either with Tdap (prime)/Tdap GTL003 (boost) or with Tdap (prime) + WCV (Boost) (pg/ml). Ratio of IFN-g/IL-4 and IFNg/IL5 secreted cytokines was also calculated. Splenocytes restimulations were performed with Ap antigens + GTL003. Group Prime Boost Stimulus Route IFNg IL4 IL5 IL-17 IFNg/IL4 IFNg/IL5 TdaP TdaP + Ap + IP 341 2207 1492 209 0.15 0.23 (1:10) GTL003 GTL003 (1:10) TdaP WCV Ap + IP 751 827 2719 1637 0.91 0.28 (1:10) GTL003

[0261] SC Immunization:

[0262] No significant difference was observed between the Tdap prime/Tdap boost vs Tdap prime/tdap+GTL003 boost in terms of cytokine responses.

[0263] 2.1.2. Humoral Immune Response

[0264] The antibody response to FHA, and PT was measured in the sera of vaccinated animals with Tdap and boosted either with Tdap+GTL003 or WCV.

[0265] FHA-Specific Antibodies:

[0266] Animals boosted with Tdap+GTL003 had a slight decrease in IgG1 antibodiy response with almost no IgG2a, an increase in IgG2b and IgG3 levels allowing to say that the Ab response is a mixed Th1/Th2 profile but with a trend towards a Th1 IgG isotype response in the presence of GTL003.

[0267] Animals boosted with WCV developed a similar pattern of response except that IgG1 Ab level did not change while IgG2b ab level increased dramatically.

[0268] PT-Specific Antibodies:

[0269] A decrease of Th2 IgG1 isotype response post-Tdap+GTL003 boost was measured. IgG2a Ab were the same and IgG2b and IgG3 Ab decreased. The same pattern of responses was obtained in animals boosted with WCV.

[0270] PRN-Specific Antibodies:

[0271] The levels of PRN-specific IgG were below the limit of detection of our assays at the dilutions used for all vaccine conditions

[0272] GTL003-Specific Antibody:

[0273] The anti-GTL003 IgG titers of are significantly higher after boosting with Tdap+GTL003 than with WCV. This is suggesting that the GTL003-containing vaccine boost will provide a better anti-CyaA mediated protection against whooping cough than WCV.

TABLE-US-00012 TABLE 7 Serum Anti-Cya antibody levels in animals immunized with GTL003 or WCV. Animals were primed with Tdap Route 1st dose 2.sup.nd dose GMT IM Tdap Whole cell vaccine 800 IM Tdap (1:10) Tdap (1:10) + GTL003 12800

[0274] 2.2. Protection of Mice Vaccinated with Tdap+Gtl003 Compared to wP or Tdap Alone

[0275] BalbC mice were vaccinated at day 0, intraperitoneally, with either wP, Tdap alone or Tdap with increasing doses of GTL003 (10 μg, 25 μg, 50 μg and 100 μg). Three dilutions were tested as shown in Table 8. Animals were challenged 21 days after vaccination with Bordetella pertussis strain. Seven days after challenge, lungs were analyzed for the presence of pertussis colonization.

TABLE-US-00013 TABLE 8 Intranasal Challenge assay post vaccination Lung CFU distribution Vaccine 2 10 50 dilution Whole Cell 508 4383 86000 vaccine Tdap (Control 188 16931 62167 group) Tdap + GTL003 37 52 1935 (10 μg)

[0276] Tdap+GTL003 vaccine induces overall a better protection against B. pertussis than Tdap alone and equivalent or better than WCV. For vaccine dilution 10, protection was observed with Tdap+10 μg GTL003 but also 25 (1028 CFU) and 50 μg (2625 CFU) GTL003 compared to Tdap alone and WCV.

[0277] Analysis of the immune response after a single intra-peritoneal injection of Tdap+GTL003 was performed to understand the protection gain in the presence of GTL003.

[0278] 2.3. Mice Immune Response after Single Dose Vaccination with Tdap+GTL003

[0279] 2.3.1 Humoral Immune Response

[0280] Four groups of mice were vaccinated either with the Aluminium hydroxide adjuvanted-Tdap alone or adjuvanted-Tdap mixed with increasing doses of GTL003 as presented in the table hereafter.

TABLE-US-00014 TABLE 9 Serum anti-aP antibody levels in vaccinated mice Vaccine composition Total IgG titers GTL003 Anti-PT Anti-FHA Anti-PRN Vaccinated Groups μg Ab Ab Ab Tdap (ref) 0 49.2 117.2 6.7 Tdap + GTL003 4 79.7 127 10.54 Tdap + GTL003 40 96.8 142.9 14.38 Tdap + GTL003 120 73 194.2 5.03

[0281] A slight increase of antibodies against PT, FHA and PRN was detected only in groups of mice immunized with pertussis vaccine containing GTL003.

[0282] When comparing the production of antibodies against CyaA, the response was higher with adjuvanted-Tdap+GTL003 vaccinated animals compared with those vaccinated with the WCV. Of note, WCV contains wild type CyaA.

TABLE-US-00015 TABLE 10 Serum anti-CyaA antibody levels in vaccinated mice. Anti-cyaA Route Prime Boost IgG Titer IP Tdap (1:10) Whole cell vaccine 800 IP Tdap (1:10) Tdap (1:10) + 12800 GTL003

[0283] 2.3.2 Cytokine Production by Spleen and Macrophages

[0284] The secreted cytokines by spleen cells restimulated in vitro with Ap antigens from mice vaccinated with either adjuvanted-Tdap+GTL003 or Tdap alone or WCV were analyzed in unchallenged mice, i.e. mice that have not been challenged in vivo by live B. pertussis post vaccination.

TABLE-US-00016 TABLE 11 Cytokine immune response measured in spleens and macrophages of vaccinated, but unchallenged mice, after in vitro stimulation with vaccine antigens. Groups according to GTL003 dose (10, 25, 50, 100 μg) injected IP with Tdap +/− GTL003 or WCV (Tdap + GTL003 dose in μg) Readout Tdap Tdap + 25 Tdap + 50 Tdap + 100 WCV Th1 cytokines  IL-12 Reference No No No − (p70) change change change IFN-g Reference + − − − Th2 cytokines IL-4 Reference − − − NA IL-5 Reference No No − NA change change  IL-13 Reference No + No − change change IL-9 Reference No − − No change change Th17 cytokines IL-17A Reference ++ + ++ NA Macrophage activity Nitric Reference ++ +++ + NA Oxide +, ++, +++: 1-2×, 2-4× and >4× increase in cytokine level compared to cytokine induced by Tdap alone (reference), respectively; −: decrease

[0285] The addition of GTL003 at the concentration of either 25 or 50 μg to the Tdap vaccine induces an increase of the TH1 and TH17 immune responses. Unexpectedly, GTL003 induces the same antigen-specific effect on nitric oxide (NO) in stimulated spleen cells while the injection has been performed intra-peritonealy compared to mice vaccinated with Tdap alone. In previous experiments, NO has been shown to increase after peritoneal macrophages stimulation after intra-peritoneal injection that could be expected because of the recruitment of macrophages at injection site. The same conclusions on the effect of the addition of GTL003 to Tdap vaccine are drawn in vaccinated mice followed by an intranasal challenge with live B. pertussis.

TABLE-US-00017 TABLE 12 Analysis of the cytokine profile after challenge of mice vaccinated with adjuvanted-Tdap, adjuvanted Tdap + GTL003 or WCV. Tdap + GTL003 dose in μg Groups according to GTL003 dose (10, 25, 50, 100) groups injected IP with Tdap +/− GTL003 or WCV Tdap + Tdap + Tdap + Readout Tdap 25 50 100 WCV Th1 cytokines IL-12 (p70) Reference ++ ++ ++ = ++ IFN-gamma Reference +++ +++ +++ = +++ Th2 cytokines IL-4 Reference + +++ +++ ≥ + IL-5 Reference +++ +++ +++ = +++ IL-13 Reference ++ +++ +++ ≥ ++ IL-9 Reference + + + > No change Th17 cytokines IL-17A Reference ++ +++ +++ ≥ ++ Pro-inflammatory cytokines IL-1alpha Reference ++ +++ ++ ≥ + IL-1beta Reference + + + = + IL-6 Reference +++ +++ +++ = +++ TNFalpha Reference + + + = + Macrophage activity Nitric NA ++ − − > Reference Oxide +, ++, +++: 1-2x, 2-4x and >4x increase in cytokine level compared to cytokine induced by Tdap alone (reference), respectively; −: decrease

[0286] A dramatic overall increase in the immune response in the groups vaccinated with GTL003-containing adjuvanted-Tdap+GTL003 compared to adjuvanted Tdap without GTL003 was observed. This response was equivalent or superior to WCV. A strong increase of IFN-g, IL17 and Nitric Oxide were observed.

[0287] The analysis of the IFN-g after stimulation either with Tdap antigens or Tdap+GTL003 showed that the response against all antigens was increased in the groups immunized with GTL003-containing vaccine.

[0288] For IL-17, the level of cytokine measured was higher in the presence of GTL003 and increased between 10 and 25 μg of GTL003 (FIG. 3A). Unexpectedly, the increased response in those animals vaccinated with Tdap+GTL003 was GTL003-specific (FIG. 4 B).

Conclusion

[0289] These results show for the first time the ability of GTL003 to enhance the Th1 immune response against pertussis antigens when included in a Tdap vaccine containing aluminium, a Th2 adjuvant. [0290] The effect is observed after intramuscular injection when GTL003 is in the Tdap boost which is unexpected following what the literature reports; [0291] The effect is observed after a single intraperitoneal injection (standard route of vaccination in a challenge assay) allowing a better protection of animals, which is supported by the cytokine and humoral responses that are superior to the WCV; this is also unexpected in the presence of aluminium.

[0292] 2.4 Evaluation of the Impact of GTL003 on Tetanus and Diphtheria Antibody Titres in Tdap Vaccine

[0293] From the obtained data, no interfering effect of GTL003 on Tetanus and Diphtheria antibody titres was observed. Titres of GTL003 treatment groups were similar to titres observed in control Tdap Vaccine.

[0294] 2.5 Effect of GTL003 on Th1 Cytokine (IFN-Gamma) Levels when Formulated with Tdap Vaccine and Administered as Booster Vaccine

[0295] Administration of multiple doses of Tdap results in excessive Th2-bias wherein a significant reduction of IFN-gamma levels was noted. By contrast, administration of GTL003 with Tdap at the time of boosting prevented a significant drop in IFN-gamma levels in treatment groups even when the number of doses of Tdap increases. Accordingly, this confirms that the administration of GTL003 in the booster dose of vaccine skews the response toward a Th1 response.

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