WO1990011088A1 - Preparation de vaccins - Google Patents

Preparation de vaccins Download PDF

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Publication number
WO1990011088A1
WO1990011088A1 PCT/GB1990/000435 GB9000435W WO9011088A1 WO 1990011088 A1 WO1990011088 A1 WO 1990011088A1 GB 9000435 W GB9000435 W GB 9000435W WO 9011088 A1 WO9011088 A1 WO 9011088A1
Authority
WO
WIPO (PCT)
Prior art keywords
bacterial
suspension
vaccine
anyone
bacterial cells
Prior art date
Application number
PCT/GB1990/000435
Other languages
English (en)
Inventor
Afshan Ahmad
Alexander Buchan
Gordon Robert Bruce Skinner
Original Assignee
Medical Research International Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Medical Research International Limited filed Critical Medical Research International Limited
Priority to BR909007246A priority Critical patent/BR9007246A/pt
Publication of WO1990011088A1 publication Critical patent/WO1990011088A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus

Definitions

  • This invention relates to a process for the preparation of vaccines.
  • Vaccines prepared in this way have cross-reactivity against other bacteria.
  • the vaccine In the preparation of vaccines a general problem exists in that the vaccine has to be efficacious in terms of immunogenicity and the production of protective immunity but at the same time the vaccine or vaccine preparation should not contain infectious micro-organisms and should preferably be substantially free of bacterial DNA.
  • a vaccine is prepared by incubating a suspension of peptidoglycan containg bacteria with Lysostaphin for a period of time sufficient to digest a substantial proportion of the bacterial cells, subjecting the Lysostaphin treated material to further disruption of the bacterial cells by mechanical means, separating the resulting material into a solid phase containing any remaining live or killed bacterial cells and a supernatant liquid phase containing bacterial antigens but substantially no bacterial cells and collecting the vaccine preparation thus prepared.
  • the bacterial suspension prior to the Lysostaphin treatment the bacterial suspension is subjected to mechanical agitation, for example ultrasonic vibration, to disaggregate the bacterial suspension and possibly to disrupt bacterial cells although on account of the accompanying disaggregation, there is little or no reduction in the bacterial colony count.
  • mechanical agitation for example ultrasonic vibration
  • the further disruption step following the Lysostaphin treatment comprises ultrasonic vibration although other mechanical agitation can be used.
  • Separation of the liquid phase from the solid phase is preferably effected by ultracentrifugation of the suspension to remove the live and killed bacterial cells and other large bacterial debris followed by filtration using an appropriately sized millipore filter to remove any remaining bacterial cells and other cell debris.
  • the filtrate is again subjected to mechanical agitation, preferably ultrasonic vibration to degrade any residual DNA which mitght remain in the preparation.
  • the vaccine preparation can be stored as a liquid or can be freeze dried for prolonged storage.
  • the process of the invention is particularly useful for preparing vaccines against Staphylococci and has been used to prepare vaccines against 22 strains of Staphylococcus epidermidis and 6 strains of Staphylococcus aureus.
  • Staphyloccus epidermidis ii usually a commensal of human subjects it can give rise to a number of infections, for example bacterial endocarditis and in recent years an increasing number of infections has been encountered in association with catheterisation, peritoneal dialysis or insertion of prosthetic implants in human subjects.
  • Staphylococcus epidermidis is cultivated in nutrient broth under aerobic conditions at 37°C over a period of 18 hours. The bacterial concentration is then estimated spectro-photometrically and by colony count following which the bacteria are then washed by three centrifrugations with resuspension in tris buffer at a concentration of 10 10 CFU per ml.
  • the sample was then subjected to a second ultrasonic vibration for 5 minutes at 4°C further to disrupt the bacterial cells, typically with a five fold decrease in number.
  • This second ultrasonic vibration degrades the bacterial nucleic acid which has been released from the cells by the Lysostaphin treatment and the first ultrasonic vibration.
  • Colony counts are low-typically less than 10 4 cfu per ml.-in the supernatant fluid following the ultracentrifugation but seme bacterial debris or inactivated bacteria may remain in the supernatant liquid. These can be identified by Gram staining or electronmicroscopy.
  • the sample was filtered using a 0.22 ⁇ m millipore filter and then the filtrate was subjected to further ultrasonic vibration for sufficient time to degrade any residual DNA that might remain in the preparation.
  • the vaccine preparation thus obtained was stored either as a solution at -70° C or was freeze dried.
  • the protein concentration of the vaccine solution is approximately 20mg/ml
  • FIG. 1 The polypeptide profile of this preparation is shown in Figure 1; (track 3).
  • Fig.1 also shows the polypetide profile of bacterial suspensions which have been treated in a number of different ways each of which represents one or more of the procedures in the vaccine preparative process, as follows:
  • Track 1 filtered extracts of cells exposed to ultrasound for 5 mins; track 2 cells exposed to ultrasound followed by lysis with lysostaphin; track 4 lysostaphin only; and track 5 marker tracks.
  • the immunoprecipitating antigens were investigated in Ouchterlony gel diffusion against hyper-immune antisera raised in rabbits against crude whole bacterial cell suspensions. There was evidence of at least four immunoprecipitins with this hyper-imune antisera and the vaccine and one immunoprecipitin line was identified with a serum for a sero-positive human subject. In addition two immunoprecipitin lines were identified when the vaccine was tested against Pseudornonas aeruginosa and one immunoprecipitin line was identified when the vaccine was tested against proteus mirabilis and also against a serum from a sero-positive human subject.
  • the total antigenicity of the vaccine was tested by indirect Elisa assay and had a titre of 1/10000 against the above hyper-immune antiserum, and 1/3000 against the sero-positive human serum.
  • Western blotting at least 20-25 polypeptides were identified with the hyper-immune sera and approximately 8 with the human serum.
  • a rabbit was hyper-immunised with the vaccine and the serum tested at various stages in the imm unisation programme for reactivity against Staph. epidermidis antigen. In Ouchterlony gel diffusion, there were at least four immunoprecipitins against Staph.epidermidis extract and against the vaccine preparation. The serum also gave high titres by indirect Elisa technique against the vaccine and 1/300 against Pseudomonas aeruginosa and at least 20-25 polypeptides were detected by Western blotting. 2. Protection studies in Mice
  • CBA mice were given five immunisations of vaccine containing 10 9 cell equivalents per vaccine dose at 4 day intervals and challenged intraperitoneally 5 days following the last imm unisation with a dose of 8 x 10 8 Staph. epidermidis cells.
  • the vaccine was given with an equal volume of 'Alhydrogel' adjuvant. There was evidence of protection against morbidity and mortality. Control animals were given injections of saline solution.
  • mice were vaccinated with four weekly doses of the vaccine preparation containing the equivalent of 5 x 10 8 cfu/dose and challenged with Pseudomonas aeruginosa. The results are shown in Table 2.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un vaccin dont la préparation consiste à incuber une suspension de bactéries contenant une peptidoglycane avec une lysostaphine, pendant une période de temps suffisante pour digérer une proportion substantielle des cellules bactériennes, à soumettre la matière traitée à une rupture mécanique par exemple ultrasonique, à séparer la matière ainsi obtenue, de préférence par centrifugation en une phase solide contenant n'importe quelles cellules bactériennes vivantes ou tuées, ainsi qu'une phase liquide surnageante contenant des antigènes bactériens, puis à récupérer le vaccin préparé.
PCT/GB1990/000435 1989-03-23 1990-03-21 Preparation de vaccins WO1990011088A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
BR909007246A BR9007246A (pt) 1989-03-23 1990-03-21 Preparacao de vacinas

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8906794.6 1989-03-23
GB898906794A GB8906794D0 (en) 1989-03-23 1989-03-23 Preparation of vaccines

Publications (1)

Publication Number Publication Date
WO1990011088A1 true WO1990011088A1 (fr) 1990-10-04

Family

ID=10653942

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1990/000435 WO1990011088A1 (fr) 1989-03-23 1990-03-21 Preparation de vaccins

Country Status (4)

Country Link
AU (1) AU5273890A (fr)
BR (1) BR9007246A (fr)
GB (2) GB8906794D0 (fr)
WO (1) WO1990011088A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0514199A2 (fr) * 1991-05-17 1992-11-19 Retroscreen Limited Vaccins et méthodes de production
US7943352B2 (en) * 2006-03-29 2011-05-17 Bacoustics, Llc Apparatus and methods for vaccine development using ultrasound technology

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0302781A1 (fr) * 1987-08-03 1989-02-08 Institut Pasteur Procédé d'obtention de polyosides capsulaires de staphylocoques, applications de ces polyosides et souches pour la mise en oeuvre du procédé

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0302781A1 (fr) * 1987-08-03 1989-02-08 Institut Pasteur Procédé d'obtention de polyosides capsulaires de staphylocoques, applications de ces polyosides et souches pour la mise en oeuvre du procédé

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Infection and Immunity, Volume 56, No. 5, May 1988, (Washington, DC, US), A.L. CHEUNG et al.: "Variation in the Expression of Cell Wall Proteins of Staphylococcus Aureus Grown on Solid and Liquid Media", pages 1061-1065 *
The Merck Index, An Encyclopedia of Chemicals, Drugs, and Biologicals, Tenth Edition, 1983, Merck & Co. Inc., (Rahway, N.J., US), "Lysostaphin", see page 5459* Abstract 5456, & Proc. Nat. Acad. Sci. USA, Vol.51, Nr. 414 (1964) *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0514199A2 (fr) * 1991-05-17 1992-11-19 Retroscreen Limited Vaccins et méthodes de production
EP0514199A3 (en) * 1991-05-17 1993-11-10 Retroscreen Ltd Vaccines and methods for their production
US7943352B2 (en) * 2006-03-29 2011-05-17 Bacoustics, Llc Apparatus and methods for vaccine development using ultrasound technology

Also Published As

Publication number Publication date
GB9120825D0 (en) 1991-12-04
GB8906794D0 (en) 1989-05-10
BR9007246A (pt) 1992-02-25
GB2248186A (en) 1992-04-01
AU5273890A (en) 1990-10-22
GB2248186B (en) 1993-04-28

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