WO2006002502A1

WO2006002502A1 - Process for obtention of new cellular pertussis vaccine

Info

Publication number: WO2006002502A1
Application number: PCT/BR2004/000172
Authority: WO
Inventors: Isaias Raw; Flávia Saldanha KUBRUSLY; Waldely De Oliveira Dias; Maria Izabel Esteves; Noemi Furuyama; Denise Silvina Piccini Quintas Horton; Ednilse Leme; Wagner Quintilio; Maria Aparecida Sakauchi; Sally Muller Affonso Prado; Elizabeth Mendes; Hisako Gondo Higashi
Original assignee: Fundação Butantan
Priority date: 2004-07-05
Filing date: 2004-09-13
Publication date: 2006-01-12
Also published as: BRPI0402630B1; BRPI0402630A; EP1809323A1; BRPI0402630B8

Abstract

process for obtention of new cellular pertussis vaccine, which foresees the use of Bordetella pertussis cells, obtained by culture in fermentation unit, detoxified by formaldehyde, which after treatment with organic solvent originate vaccine preparations that are less toxic and as potent as the traditional pertussis cellular vaccine.

Description

TITLE OF THE INVENTION

PROCESS FOR OBTENTION OF NEW CELLULAR PERTUSSIS VACCINE

FIELD OF THE INVENTION

This invention refers to the development of a cellular pertussis vaccine, obtained by the process referred to, showing to be less toxic and as potent as the traditional cellular pertussis vaccine.

BACKGROUND OF THE INVENTION

Whooping Cough, also known as pertussis or "long cough", is an infectious disease of the upper respiratory tract, caused by bacteria, the Bordetella pertussis. According to the World Health Organization, this disease attacks about 60 million children every year, being responsible for 600.000 deaths, mainly in underdeveloped or developing countries, with precarious sanitary conditions or lack of vaccination programmes.

The cellular pertussis vaccine was described in the decade of 1940, by Kendrick and collaborators. It is a vaccine prepared with bacterial cells obtained by centrifugation or filtration of the culture in liquid media, submitted to inactivation by formaldehyde, thimerosal or thermal treatment. The immunization with such vaccine has been accepted worldwide, with high efficiency in the control of the disease. In spite of being very efficient and leading to the vaccine coverage, the detoxification of the vaccine preparations is one of the crucial points of the production process, with undesirable adverse events being described, sometimes with serious danger.

It led to the development of acellular, less toxic vaccines, composed by the main antigenic elements of the bacteria. Most of these preparations differ between themselves qualitatively and/or quantitatively as to the antigenic components used, since the final formulation has not yet been completely defined. Whichever the formulation chosen by the producer is, most of the times the added value of the final product is high, since several steps of purification are usually required, by several chromatography processes, from the supernatants or filtrates of culture, for obtention of each of the vaccine's components, mostly excreted in the culture media.

SUMMARY OF THE INVENTION

This invent refers to the "PROCESS FOR OBTENTION OF NEW

CELLULAR PERTUSSIS VACCINE", which foresees the use of Bordetella pertussis cells, obtained by culture in fermentation unit, detoxified by formaldehyde, which after treatment with organic solvent originate vaccine preparations that are less toxic and as potent as the traditional pertussis cellular vaccine.

DETAILED DESCRIPTION OF THE INVENTION

This invent refers to the "PROCESS OF OBTENTION OF NEW CELLULAR PERTUSSIS VACCINE", from Bordetella pertussis cells treated with organic solvent.

For this, the process mentioned foresees the following steps: 1) lyophilized strain of Bordetella pertussis 137 is re-suspended in Saline Solution

0,85%, cultivated in Bordet-Gengou tubes and incubated at 35⁰C for 72 hours; 2) the cultures are transferred to other tubes containing Bordet-Gengou medium and are incubated at 35⁰C for additional 24 hours; 3) the bacterial growths are inoculated in 50OmL erlenmeyers with 10OmL of Stainer-Scholte modified medium and are kept at 35⁰C with stirring at 150 rpm; 4) after 24 hours, this culture is inoculated in a pre-fermentation unit (150 liters) containing 60 liters of culture media; 5) after 20 hours at 35⁰C, 40 liters of the culture are transferred to the fermentation unit of 750 liters with 400 liters of medium, or 60 liters to a fermentation unit of 1000 liters with 600 liters of medium; 6) after 20 hours at 35⁰C the culture of Bordetella pertussis is submitted to a tangential filtration (0,22μm) in order for the biomass of Bordeiella pertussis to be obtained, which is further detoxified with formol 0,2%; 7) the Bordetella pertussis cells detoxified by formaldehyde (100 - 250 opalescence units/mL) are centrifuged at 8000 rpm, during 1 hour at 4% or concentrated by tangential filtration (0,22 μm); 8) the wet cellular mass, at the concentration of 1g/20mL of solvent 9%, is kept under stirring for 1 hour at room temperature; 9) the suspension is centrifuged at 8000 rpm, for 1 hour at 4°C or concentrated by tangential filtration; 10) the precipitated is rinsed with 0,9% saline solution (1g precipitated/1 OmL saline solution), followed by a new centrifugation as described above or by tangential filtration (0,22μm); 11) the final precipitated is re-suspended in a saline solution buffered with phosphate, pH 6.8, and this suspension constitutes the pertussis cellular vaccine treated with organic solvent (VPL).

Evaluations were performed in three experimental batches of VP_L, :

UOp = opalescence units. The Petitioner evaluated these experimental VP_L batches for

Pyrogen, Potency, Specific Toxicity and Dermonecrotic Tests, shortly described below.

Pyrogen Test, performed to determine and quantify the presence of bacterial endotoxins in Vaccine samples by the Qualitative Gel Clot Method Potency Test, performed in Swiss mice, weighting 14-16g each , which were immunized by intraperitoneal route , (0,5 mL/dose), with dilutions of the test vaccine or with Reference Pertussis Vaccine. After 14 days, the animals were challenged, intracerebrally, with virus strains of Bordetella pertussis ATCC 18323 (0,03mL/dose; in the dilution of 1/3000 of a bacterial suspension of 10UOp/mL). The animals which died up to 72 hours after the challenge were excluded from the test. The animals were observed on a daily basis for 14 days, and the number of deaths in the test groups was recorded, in relation to animals that were injected with dilutions of the reference pertussis vaccine.

Specific Toxicity Test, where the sample was inoculated intraperitoneally (0,5mL, 20UOp/mL), in Swiss mice. The product was considered satisfactory when : at the end of the 72h, the total weight of every group of Swiss albino mice, susceptible, was not lower than its initial weight; at the end of the 7^th day, the mean weight gain of the group inoculated with the sample was not lower than 60% of the mean weight gain of the negative control group, injected with physiological saline solution; more than 95% of the animals inoculated with the sample survived.

Dermonecrotic Toxicity Test, performed by inoculation of the test sample in suckling mice (0,05 ml_ of the sample at 40 UOp/mL), subcutaneously. The animals were observed after 24h and 48h. The product is approved in this test when none of the inoculated animals presents dark stains at the site of the inoculation. The number of doses of experimental vaccine was estimated, comparatively to the doses obtained with the same number of cells of the traditional cellular pertussis vaccine.

The results of the pyrogen, potency, specific toxicity and dermonecrotic tests, in Pertussis Cellular Vaccine treated with organic solvent (VPL) and in Traditional Pertussis Cellular Vaccine are in the table below, as well as the overview of the experiments 63/03, 05/04 and 72/03 of the Inactivated Individual Collection of Bordetella pertussis treated with organic solvent.

Ul = international units EU = endotoxin units - DNT = dermonecrotic test

5 VT = Traditional Vaccine VP_L = New Vaccine NT = not tested

This vaccine was also re-evaluated, by the Petitioner, in mice, as for its immunogenic activity, by induction of seric antibodies anti-pertussis toxin and io protective effect against intracerebral challenge with virus strain of Bordetella pertussis 18323.

Groups of Swiss mice (14-16g) were immunized, intraperitoneal^. (0,5ml_/dose) with VP_L (3UOp/dose); bacterial triple vaccine (Diphtheria - Tetanus - Pertussis), produced by the Petitoner (DTP - 1/8 of the human dose)

15 or physiological solution. After 14 days, the animals were bleeded, by retro- orbital puncture and their serum was evaluated, by immune-enzymatic assay (ELISA), for the title of antibodies anti-toxic pertussis. This evaluation of IgG antibodies anti-toxic pertussis (PT) in serum of immunized animals is represented in Figure 1.

^"26 On the following day of the bleeding, the animals were challenged, intracerebrally, with virus strain of Bordetella pertussis 18323, as described above, in the potency test. The test vaccine (VP_L) was compared to DTP and to the negative control group, which had saline injected. The cerebral challenge test with virus strain of Bordetella pertussis, in animals immunized with cellular pertussis vaccine treated with organic solvent (VP_L) is illustrated in Figure 2. The new vaccine, obtained through the process subject of the present patent, showed to be atoxic, as potent as the traditional vaccine, as well as having a reduced rate of pyrogen in relation to this one. It also showed to be immunogenic, unleashing a humoral response in mice, with high titles of seric antibodies anti -toxin pertussis and with high in vivo protective effect. Additionally, it showed to be compatible with other products aiming at the preparation of the quadruple vaccine (diphtheria, pertussis, tetanus and haemophylus or diphtheria, pertussis, tetanus and hepatitis) and pentavalent (diphtheria, pertussis, tetanus, hepatitis and haemophylus).

The tests performed in laboratory by the Petitioner attest that the pertussis vaccine obtained by the present process, is a potent and innocuous preparation, containing the same antigenic characteristics of the traditional cellular vaccine produced also by the Petitioner.

The technical simplicity of this process implies in reduction of the pyrogen of the cellular vaccine and maintenance of the vaccine's potency, without implying in price raise or a more difficult process, which is already standardized for the traditional vaccine. The production of cellular vaccine against whooping cough, less toxic and with low cost maintenance, will be of great impact to the National Public Health System.

Claims

1^a) "PROCESS FOR OBTENTION OF A NEW PERTUSSIS CELLULAR VACCINE", characterized by the fact that using Bordetella pertussis cells obtained from cultures in fermentation units, detoxified by formaldehyde and treated with organic solvent, and due to the fact of foreseeing these steps: 1) lyophilized strain of Bordetella pertussis 137 is re-suspended in Saline solution 0,85%, sowed in Bordet-Gengou tubes and incubated at 35⁰C for 72 hours; 2) the cultures are transferred to other tubes containing Bordet-Gengou media and incubated at 35⁰C for 24 hours more; 3) the bacterial growths are inoculated in 50OmL erlenmeyers with 10OmL of Stainer-Scholte modified medium and kept at 35⁰C under stirring at 150 rpm; 4) after 24 hours, this culture is inoculated in a pre-fermentation unit (150 liters) containing 60 liters of culture media; 5) after 20 hours at 35⁰C, 40 liters of the culture are transferred to the fermentation unit of 750 liters with 400 liters of the medium, or 60 liters to a fermentation unit of 1000 liters with 600 liters of the medium; 6) after 20 hours at 35⁰C the culture of Bordetella pertussis is submitted to a tangential filtration (0,22μm) in order for the biomass of Bordetella pertussis to be obtained, which is further detoxified with formaldehyde solution 0,2%; 7) the Bordetella pertussis cells detoxified by formaldehyde (100 - 250 opalescence units/mL) are centrifuged at 8000 rpm, during 1 hour at 4% or concentrated by tangential filtration (0,22 μm); 8) the wet cellular mass, at the concentration of 1g/20mL of solvent 9%, is kept under stirring for 1 hour at room temperature; 9) the suspension is centrifuged at 8000 rpm, for 1 hour at 4°C or concentrated by tangential filtration; 10) the precipitated is rinsed with 0,9% saline solution (1g precipitated/1 OmL saline), followed by a new centrifugation as described above or by tangential filtration (0,22μm); 11) the final precipitated is re-suspended in a saline solution buffered with phosphate, pH 6.8, and this suspension constitutes the pertussis cellular vaccine treated with organic solvent (VPL). 2^a) "PROCESS FOR OBTENTION OF NEW CELLULAR PERTUSSIS VACCINE", according to claim 1 , characterized by the fact of minimizing the concentration of bacterial lipopolysaccharide (LPS) in the final preparation, resulting in a less toxic vaccine. 3^a) "PROCESS FOR OBTENTION OF NEW CELLULAR PERTUSSIS VACCINE", according to claim 1 , characterized by the fact of maintaining the immunogenic activity of the traditional vaccine preparation, resulting in a vaccine as potent as the traditional cellular vaccine. 4^a) "PROCESS FOR OBTENTION OF NEW CELLULAR PERTUSSIS VACCINE", according to claim 1 , characterized by the fact of maintaining the immunogenic activity and being compatible with other products aiming at the preparation of a quadruple vaccine (diphtheria, pertussis, tetanus and haemophylus or diphtheria, pertussis, tetanus and hepatitis) and pentavalent (diphtheria, pertussis, tetanus, hepatitis and haemophylus).