Vaccines comprising enhanced antigenic helicobacter spp.

• US 5,897,475 A
• Filed: 10/03/1995
• Issued: 04/27/1999
• Est. Priority Date: 10/05/1994
• Status: Expired due to Fees

• Current Assignee: Bioport Company Limited
• Original Assignee: Antex Biologics Inc.
• Inventors: Pace, John Lee, Frey, Steven Michael, Walker, Richard Ives
• Primary Examiner(s): Wityshyn, Michael G.
• Assistant Examiner(s): WARE, DEBORAH K
• Application Number:
  US08/538,544
• Time in Patent Office: 1,302 Days
• Field of Search: 435/252.2, 435/822, 435/252.1, 424/184.1, 424/93.4, 424/282.1
• US Class Current: 435/252.1
• CPC Class Codes: A61K 2039/55555 Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
• CPC Class Codes: A61K 39/105 Delta proteobacteriales, e.g. Lawsonia; Epsilon proteobacteriales, e.g. campylobacter, helicobacter
• CPC Class Codes: A61K 39/107 Vibrio
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12Q 1/045 Culture media therefor
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: C12R 2001/01 Bacteria or Actinomycetales ; using bacteria or Actinomycetales
• CPC Class Codes: G01N 2333/25 Shigella (G)
• CPC Class Codes: G01N 2469/20 Detection of antibodies in sample from host which are directed against antigens from microorganisms
• CPC Class Codes: G01N 2474/20 Immunohistochemistry assay
• CPC Class Codes: G01N 33/56916 Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
• CPC Class Codes: G01N 33/56922 Campylobacter
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
• CPC Class Codes: Y10S 424/802 Antibody or antigen-binding fragment thereof that binds gram-positive bacteria
• CPC Class Codes: Y10S 435/822 using bacteria or actinomycetales
• CPC Class Codes: Y10S 435/975 Kit

Abstract

Methods using in vitro processes are disclosed for inducing or enhancing expression of enteric bacterial antigens or virulence factors. The methods, therefore, produce antigenically enhanced enteric bacteria. Also methods for using the antigenically enhanced bacteria are also disclosed, as well as vaccines containing the enteric bacteria. Specifically, a whole enteric bacteria or components thereof are provided by Helicobacter species. Also there are other enteric bacteria which are useful for the disclosed invention; such as Campylobacter jejuni.

Claims

1. A vaccine comprising a Helicobacter bacterium having an enhanced antigenic property or an immunogenic fragment of said bacterium, which bacterium is harvested from a liquid culture of a Helicobacter species grown in vitro in a culture medium with a combination of conditions comprising:

a) about 0.05% to about 3% bile or about 0.025% to about 0.6% of one or more bile acids or salts thereof;

b) at a temperature between about 30°

C. and about 42°

C.;

c) in air or a gas mixture, wherein the gas mixture comprises i) about 5% to about 20% CO₂ with about 80% to about 95% air;

or ii) about 5% to about 10% O₂ with about 10% to about 20% CO₂ with about 70% to about 85% N₂ ; and

d) a divalent cation chelator selected from the group consisting of 0 to about 25 μ

M of 1,2-bis(2-aminophenoxy)ethane-N,N,N'"'"',N'"'"'-tetraacetic acid/acetoxymethyl ester, 0 to about 10 mM of ethylene-bis(oxyethylenenitrilo)-tetraacetic acid, and 0 to about 100 μ

M of ethylene-bis(oxyethylenenitrilo)-tetraacetic acid/acetoxymethyl ester,

wherein said Helicobacter culture is at about early log phase, between early log phase and stationary phase, or at about stationary phase and the enhanced antigenic property is a higher level of a virulence factor associated with a higher level of adherence to a host cell when compared to the adherence ability of bacteria from a culture of the Helicobacter species grown in brain heart infusion broth supplemented with bovine calf serum.

3. The vaccine according to claim 1 or 2, further comprising a pharmaceutically acceptable carrier or diluent.

4. The vaccine according to claim 1 or 2, wherein said Helicobacter bacterium is inactivated.

5. The vaccine according to claim 4, wherein said Helicobacter bacterium is inactivated by formalin treatment.

6. The vaccine according to claim 1 or 2, wherein said vaccine is suitable for mucosal or parenteral administration.

7. The vaccine according to claim 1 or 2, further comprising an adjuvant.

8. The vaccine according to claim 1, wherein the Helicobacter species is Helicobacter pylori or Helicobacter felis.

9. The vaccine according to claim 8, wherein the Helicobacter species is Helicobacter pylori strain NB3-2 (ATCC 55714) or G1-4 (ATCC 55713).

10. The vaccine according to claim 1, wherein the combination of conditions comprises:

a) about 0.05% bile salt which is glycocholate or about 0.1 to about 0.2% bile;

b) the temperature is about 37°

C.;

c) in about 10% to about 20% CO₂ with about 80% to about 90% air, or about 10% CO₂ with about 5% O₂ with about 85% N₂ ; and

the culture is at about log phase.

11. The vaccine according to claim 10, wherein the Helicobacter species is Helicobacter pylori or Helicobacter felis.

2. A vaccine comprising a Helicobacter bacterium having an enhanced antigenic property or an immunogenic fragment of said bacterium, which bacterium is harvested from a liquid culture of a Helicobacter species grown in vitro in a culture medium with a combination of conditions comprising:

a) a divalent cation chelator selected from the group consisting of about 1.0 to about 25 μ

M of 1,2-bis(2-aminophenoxy)ethane-N,N,N'"'"',N'"'"'-tetraacetic acid/acetoxymethyl ester, about 0.5 to about 10 mM of ethylene-bis(oxyethylenenitrilo)-tetraacetic acid, and about 1.0 to about 100 μ

M of ethylene-bis(oxyethylenenitrilo)-tetraacetic acid/acetoxymethyl ester;

b) at a temperature between about 30°

C. and about 42°

C.; and

c) in air or a gas mixture, wherein the gas mixture comprises i) about 5% to about 20% CO₂ with about 80% to about 95% air;

or ii) about 5% to about 10% O₂ with about 10% to about 20% CO₂ with about 70% to about 85% N₂,

wherein said Helicobacter culture is at about early log phase, between early log phase and stationary phase, or at about stationary phase and the enhanced antigenic property is a higher level of a virulence factor associated with a higher level of adherence to a host cell when compared to the adherence ability of bacteria from a culture of the Helicobacter species grown in brain heart infusion broth supplemented with bovine calf serum.