Bacterial vaccine

Abstract

The disclosure relates to a composition comprising one, two or more immunogenic bacterial polypeptides and multivalent and monovalent vaccine compositions comprising the immunogenic bacterial polypeptides.

Claims

1. An immunogenic composition, comprising an effective amount of an adjuvant and two or more different isolated polypeptides selected from: i) a polypeptide comprising or consisting of the amino acid sequence as represented in SEQ ID NO: 119 or 149; ii) an immunogenic polypeptide variant having at least 90% identity to the amino acid sequence represented in SEQ ID NO:119 or 149, which immunogenic polypeptide variants have retained immunogenicity as compared to the polypeptide having the amino acid sequence represented in SEQ ID NO: 119 or 149 respectively; and iii) any combination of isolated polypeptides from (i) and/or (ii).

2. The immunogenic composition according to claim 1, wherein the immunogenic composition consists essentially of 3, 4 or 5 different polypeptides.

3. The immunogenic composition according to claim 1, wherein the composition further comprises at least one carrier.

4. The immunogenic composition according to claim 3, wherein the composition further comprises at least one additional anti-bacterial agent.

5. A method to immunize a subject from a Clostridium difficile infection comprising administering an effective amount of an immunogenic composition according to claim 1 to the subject.

6. An immunogenic composition, comprising (a) an effective amount of an adjuvant, (b) a first polypeptide comprising or consisting of the amino acid sequence as represented in SEQ ID NO: 119, and (c) a second polypeptide comprising or consisting of the amino acid sequence as represented in SEQ ID NO: 149.

7. An immunogenic composition of claim 6, wherein the first polypeptide consists of the amino acid sequence as represented in SEQ ID NO: 119, and the second polypeptide consists of the amino acid sequence as represented in SEQ ID NO: 149.

8. The immunogenic composition according to claim 6, wherein the immunogenic composition consists essentially of 3, 4, or 5 different polypeptides.

9. The immunogenic composition according to claim 1, wherein the adjuvant is a gel-type adjuvant.

10. The immunogenic composition according to claim 1, wherein the adjuvant is an aluminium based adjuvant.

11. The immunogenic composition according to claim 10, wherein the aluminium based adjuvant is aluminium hydroxide.

12. The immunogenic composition according to claim 10, wherein the aluminium based adjuvant is aluminium phosphate.

13. The immunogenic composition according to claim 6, wherein the adjuvant is a gel-type adjuvant.

14. The immunogenic composition according to claim 6, wherein the adjuvant is an aluminium based adjuvant.

15. The immunogenic composition according to claim 14, wherein the aluminium based adjuvant is aluminium hydroxide.

16. The immunogenic composition according to claim 14, wherein the aluminium based adjuvant is aluminium phosphate.

17. The immunogenic composition according to claim 1, wherein the immunogenic polypeptide variant has at least 95% identity to the amino acid sequence represented in SEQ ID NO: 119 or 149.

18. The immunogenic composition according to claim 1, wherein the immunogenic polypeptide variant has 99% identity to the amino acid sequence represented in SEQ ID NO: 119 or 149.

19. The method according to claim 5, wherein the immunogenic composition consists essentially of 3, 4, or 5 different polypeptides.

20. The method according to claim 5, wherein the two or more different isolated polypeptides of the immunogenic composition comprise a first polypeptide comprising or consisting of the amino acid sequence as represented in SEQ ID NO: 119, and a second polypeptide comprising or consisting of the amino acid sequence as represented in SEQ ID NO: 149.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the sequences of SEQ ID NOS.: 1-111.

(2) FIG. 2 shows the sequences of SEQ ID NOS.: 112-143.

(3) FIG. 3 shows the sequences of SEQ ID NOS.: 144-157.

(4) FIG. 4 shows the sequences of SEQ ID NOS.: 158-177.

MATERIALS AND METHODS

(5) Construction of Plasmids for the Overexpression in E. coli of the Extramembranous Fragments of the Proteins

(6) The PheP selected peptides were synthesized and conjugated through a cysteine at its C terminal to the carrier protein KLH to undertake as a chimeric protein used in vaccinations. The extramembranous fragments of YdiE, DivlB, DivlC and FtsL were PCR amplified using oligonucleotide pairs indicated according to the following PCR reaction conditions: 1 initial denaturation cycle of 94 C. for 4 min; 30 amplification cycles of denaturation 94 C. for 30 seconds, annealing 45 C. for 30 seconds, and extension at 30 72 C. for up to 2.5 minutes; finally, ongoing amplification rounds were allow to complete at 72 C. for 4 min.

(7) The restrictions sites were engineered within the oligonucleotides. The amplified fragments were digested with the corresponding restriction enzymes (NcoI for the 5 end, 35 and XhoI for the 3 end) and cloned into the equivalent sites of the pET-21d(+) expression vector from Novagen (Cat. No. 69743-3) and resulting in the overexpression plasmids generating a T7-tagged (partial, at the N-terminal) and 6xHis-tagged (at the C-terminal end) form of the extramembranous fragments. The over expression plasmids were transferred into E. coli BL21 for over expression of the recombinant protein fragment.

(8) The cloning of the PCR amplified fragment indicated above into the recipient pET21d(+) recipient plasmid vector at the NcoI and XhoI sites entailed the addition of hinge amino acids between the T7-tag and the extramembranous fragment, and between the latter and the His-tag.

(9) Over Expression of Polypeptide Sequences

(10) SEQ IDs were over expressed from plasmids pGL597, pGL601, pALB26, and pALB27 in E. coli BL21 strain using Brain Heart Infusion Broth (CMO225, Oxoid, United Kingdom) in the presence of 100 ^g/ml ampicillin and the Plac promoter gratuitous inducer IPTG (Isopropyl p-D-1-thiogalactopyranoside, 1 mM) for 4 to 6 hours at 37 C. and vigorous shaking. Following harvesting of the cells by centrifugation (5,000g for 15 minutes at 4 C.) and subsequent lysis with 1 mg/ml lysozyme in phosphate buffer (Buffer A; 0.1 M pH7.2) containing 0.5M NaCl) for one hour and subsequent sonication (3 cycles of 10 second pulses in sonicating water bath) the soluble and insoluble forms of the proteins of interest were separated by centrifugation at 13,000g for 10 minutes. The precipitate was then resuspended in Buffer A containing 8M urea by freeze/thawing (3 cycles of freezing at 80 C. for 10 minutes and subsequent thawing to room temperature) and sonication (3 cycles of 10 second pulses in sonicating water bath), and subsequent centrifugation for 25 minutes at 18,000g). The over expressed proteins of interest in the supernatant and the solubilised pellet were purified by initial specific binding (through their His-tag) to a nickel (NiSO4)-bound Sepharose chromatography column (Ni Sepharose) and elution with an imidazole solution run through the column in the following stepwise manner: 5% for 5 minutes, 30% for 60 minutes, 35% for 60 minutes, 50% for 100 minutes and 55% for 100 minutes. Fractions from this stepwise elution were analysed in acrylamide denaturing gels with a 4% acrylamide/bis-acrilamide stacking layer and a 12% acrylamide/bis-acrylamide separating layer. The fractions containing the over expressed proteins of interest were pooled and dyalized against sterile phosphate buffer (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO, 0.24 g KH.sub.2PO.sub.4, per liter of distilled H.sub.2O, pH 7.4).

(11) All the proteins of interest were successfully over expressed from the indicated strains and under the indicated conditions. They were also subsequently extracted from the total cellular protein content of the over expressing E. coli strains with more than 95% purity. Examples of the purification obtained for each of the proteins are indicated below.

(12) Vaccination: Generic Protocol for Polyvalent Vaccines

(13) Combination (or polyvalent) vaccines including variations of the antigens (conjugated selected PheP peptide, YdiE, DivlB, DivlC and FtsL) will follow an identical protocol with the following modifications. The vaccine priming and boost mixtures will contain rather than a single component 2 or more of the components. The total volume of mixed vaccine used for priming and boosting injections will fluctuate in a range of 50-100 microliters per animal. Similarly the total amount in each of those injections may vary between 50-100 micrograms. The amount of each antigen to contribute to the total amount of vaccine in the priming or boosting mix will vary between 20% to 80% of the total.

(14) Vaccination: Generic Protocol for Monovalent Vaccines

(15) The generic protocol followed for the vaccinations is as follows. Each animal was primed with 100 microliters of a solution made up of a mixture 50 micrograms of recombinant antigen in 50 microliters endotoxin-free PBS (Phosphate Buffer Saline pH 7.4) and 50 microliters of Complete Freund's adjuvant. Two weeks later the animals were boosted 25 with 100 microliters of a solution made up of a mixture 50 micrograms of recombinant protein in 50 microliters of endotoxin-free PBS and 50 microliters of Incomplete Freund's adjuvant. A week later the animals received an identical boost. In each experiment, a control group of 10 animals were treated following an identical protocol except for the fact that instead of the recombinant protein component the mixture contained commercially available KLH protein (Keyhole limpet hemocyanin). Priming and boost injections were performed intradermally in the scruff of the neck of the animals.

(16) TABLE-US-00001 Bacterial PheP PheP GCP GCP Div1B DIV1B Div1C Div1C FtsL FtsL Species FL ECD FL ECD FL ECD FL ECD FL ECD Bacillus 1 2, 3, 4, 5, 6, 7 112 144 145 158 159 168 169 Anthracis Burkholderia 8 9, 10, 11, 12, 113 114 Mallei 13, 14 Burkholderia 15 16, 17, 18, 19, 115 116 pseudomallei 20, 21 Chlamydia 117 trachomatis Clostridium 22 23, 24, 25, 118 146 147 160 161 Botulinum 26, 27, 28 Clostridium 29 30, 31, 32, 119 148 149 Difficile 33, 34, 35 Enterococcus 36 37, 38, 39, 120 121 150 151 162 163 170 171 Faecalis 40, 41, 42 Escherichia 43 44, 45, 46, 122 123 Coli 47, 48, 49 Haemophilus 50 51, 52, 53, 54, 124 125 influenza 55, 56 Helicobacter 57 58, 59, 60, 126 Pylori 61, 62, 63 Mycobacterium 64 65, 66, 67, 68, 127 128 tuberculosis 69, 70 Pseudomonas 71 72, 73, 74, Aeruginosa 75, 76, 77 Neisseria 130 gonorrhoeae Neisseria 131 meningitidis Pseudomonas 132 133 aeruginosa Shigella 78 79, 80, 81, 134 135 flexneri 82, 83, 84 Streptococcus 85 86, 87, 88, 136 137 152 153 174 175 pneumonia 89, 90 Yersinia pestis 91 92, 93, 94, 95, 138 139 96, 97 Streptococcus 98 99, 100, 140 141 154 155 164 165 172 173 agalactiae 101, 102, 103, 104 Streptococcus 105 106, 107, 142 143 156 157 166 167 176 177 pyogenes 108, 109, 110, 111 Table 1 discloses SEQ ID NOs corresponding to antigenic polypeptides [FL = full length sequence; ECD = extracellular domain