Oral cholera vaccine – Dukoral; Vibrio cholerae, inactivated with recom-binant cholera toxin B (rCTB) subunit
Status: marketed in Europe, Canada and other countries
Organizations involved:
SBL Vaccine AB – Manuf.; R&D; Tech.; Europe mark.
Crucell N.V. – Intl. mark.; Parent
Chiron Vaccines – Europe mark.
Novartis AG – Parent
Active Biotech AB – Former
Cross ref.: See the entry for Cholera Vaccine/Wyeth, the only cholera vaccine ever approved in the U.S.
Description: Dukoral is an aqueous formulation of five antigens—four heat or formalin/formaldehyde inactivated strains of Vibrio cholerae, a total of 1 x 1010 bacteria, plus recombinant cholera (Vibrio cholerae) toxin B (rCTB). Each dose of vaccine contains 2.5 x 109 each of inactivated Vibrio cholerae O1 Inabo, classic (heat inactivated); Vibrio cholerae O1 Inabo, El Tor (formalin inactivated); Vibrio cholerae O1 Oqawa, classic (heat inactivated); and Vibrio cholerae O1 Oqawa, El Tor (formalin inactivated); plus 1 mg recombinant cholera toxin B (rCTB) subunit [presumably, expressed by transformed Escherichia coli (E. coli)]. The bicarbonate buffer taken with Dukoral serves to neutralize the stomach acid that otherwise destroys the rCTB.
rCTB is a 102-amino acid protein with one disulfide bridge between cysteine residues at positions 9 and 86. rCTB consists of five identical monomers with a molecular weight of ~11.6 kDa. The monomers are tightly linked into a trypsin-resistant pentameric ring-like structure with a molecular weight of ~58 kDa. Both Vibrio cholerae-derived CTB (formerly used in the vaccine) and rCTB have been shown to be comparable, except for six short peptide extensions at the N-terminal in rCTB (an artifact of the vector construct and expression cassette used). rCTB (other than cleavage of the extra leader sequence) is stable against protease, and no cleavage products shorter than native CTB has been found in Dukoral.
Dukoral containing the same inactivated V. cholerae but with purified V. cholerae-derived cholera toxin B (CTB) had previously been marketed in Sweden since 1991. The current formulation with recombinant CTB was approved in Sweden in 1992.
Dukoral is the first and only commercial vaccine that, besides providing protection against cholera from Vibrio cholerae, also safely provides protection for travelers against enterotoxigenic Escherichia coli (ETEC), the most common cause of Travellers’ diarrhea.
Dukoral is packaged as a suspension in vials containing vaccine, along with a 5.6 g packet/sachet of raspberry-flavored effervescent powder (buffer). These are dissolved in 150 mL (~5 ounces) of cool water and consumed by drinking within 2 hours. Excipients (in the vaccine component) are sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride. Each sachet 5.6 gram sachet of buffer contains sodium hydrogen carbonate, citric acid, sodium carbonate, saccharin sodium, sodium citrate, and raspberry flavor. The dissolution of the acids and bases in the buffer results in effervescence (bubbles). Dukoral has a shelf life of three years, when stored at 2-8°C (refrigerated).
Biological.: Enterotoxigenic E. coli (ETEC) causes diarrhea by first colonizing the small intestine and producing heat-labile (LT) and/or heat-stable (ST) enterotoxins. The LT enterotoxin is structurally, functionally and immunologically similar to the cholera (Vibrio cholerae) toxin, with an amino acid homology of ~80%. The two toxins cross-react immunologically, and crossprotection has been demonstrated in animal models challenged with ETEC after immunization with CT. Based on these findings, protective efficacy of Dukoral against LT-producing ETEC was studied in its clinical trials.
Dukoral stimulates mucosal (IgA antibody-based) and humoral (IgG antibody-based) immune responses in the intestinal tract, providing protection against cholera (Vibrio cholerae) and ETEC-diarrhea. The vaccine induces antibodies against both its bacterial components and CTB. Bacteria-neutralizing IgA intestinal antibodies prevent V. cholerae and ETEC bacteria from attaching to the intestinal wall, impeding colonization. rCTB IgA intestinal antibodies prevent cholera toxin from binding to the intestinal mucosal surface, preventing toxin-mediated diarrheal symptoms. Antibodies are primarily against V. cholerae outer membrane lipopolysaccharides (LPS). Bacterial Toxin Coregulated Pili (TCP) and mannose sensitive hemagglutinin (MSHA) antibodies have been suggested as factors in the establishment of immunity to cholera.
Mucosal IgA, rather than IgG humoral antibodies, provide primary protection against V. cholerae. Humoral antibody levels are a surrogate marker for intestinal secretory IgA antibodies against V. cholerae LPS and ETEC.
Naturally occurring cholera toxin belongs to the AB class of bacterial toxins. It consists of a pentameric B oligomer that binds to GM-1 receptors (e.g., on the surface of intestinal epithelial cells), and an enzymatically active A subunit that is responsible for toxicity. Recombinant cholera toxin B-subunit (rCTB) consists only of the nontoxic B part of the cholera enterotoxin, with the A-subunit genetically deleted from the production strain.
Nomenclature: Cholera Vaccine (rDNA)/SBL [BIO]; Dukoral [TR]; Oral cholera vaccine [INN]; WC/rBS [SY]
Companies.: Dukoral was developed and is manufactured by SBL Vaccine AB. Formerly Active Biotech AB, SBL was acquired by Chiron Corp. in 2001, and was divested in May 2004 through a management buyout. SBL was acquired by Crucell N.V. in Nov. 2006. [See the SBL entry in the Organizations Index for more complete history of the company]. Under terms of managment buyout agreement, Chiron Vaccines (merged into Novartis AG in spring 2006) markets Dukoral in some of the most important markets in Europe. The reason that Chiron sold SBL was “that its operations are not aligned with our strategic pursuits...and its main cooperation partners are companies that compete with Chiron Vaccines.” SBL (Crucell) markets Dukoral (and its IPV and vaccines from other manufacturers) in Sweden and Norway. Crucell and other companies market Dukoral in various other countries worldwide.
Manufacture: The whole cell bacteria are cultured conventionally in 550 L fermenters, with each strain separately cultured and concentrated. The formalin-inactivated strains are exposed to 0.5% formalin (formaldehyde). The heat-inactivated strains are exposed to 56˚C. Formalin-inactivated bacteria are subjected to another concentration step to remove residual formaldehyde. Levels of residual cholera toxin (other than that added) later is <20 ng/dose. The amounts (doses) or each strain are based on assay of the live bacteria prior to inactivation.
rCTB is cultured in plasmid-transformed bacteria (presumably, E. coli) in 500 Liter scale fermentor(s) at 36˚C with aeration and agitation to keep pO2 (oxygen levels) at 30%. rCTB is fermented without antibiotics. Hexametaphosphate and the anti-foam agent Adekanol LG-109 are used during fermentation. Hexameta-phos-phate is a common food additive, and levels of the Ade-kanol in the vaccine are below detection levels. Fermentation is halted after ~18 hours by cooling. The suspension is harvested and concentrated by ultrafiltration (MWCO 1,000 kDa). Concentrate is precipitated by addition of sodium hexametaphosphate (2 g/L) and adjustment of pH to 4.9. Precipitate is stored at 2-8˚C for 14 hours-5 days. Solution containing rCTB is centrifuged, filtered to remove bacterial lipopolysaccharides (LPS), lipids, and other proteins and non-dissolved matter; and purified by hydroxyapatite chromatography. Eluted fractions are pooled and buffer is changed to 0.02 M phosphate buffer. rCTB solution is filtered through a 0.22 µm membrane. This process results in “very pure protein” (according to EMEA/EU).
Few components of animal origin are used in the production of the vaccine. Most are either from species where no TSE issue exists (pig, horse) or from milk of bovine origin where the process is such that it falls outside of the European Union’s current TSE guidelines.
Status: Over 20 countries have approved Dukoral, including the European Union (EU) and individual approvals in most European countries (before EU-wide approval); Canada; and New Zealand. Canada granted approval on May 13, 2003.
On April 28, 2004, SBL received a MAA for European Union (EU)-wide approval of Dukoral for “active immunization against disease caused by Vibrio cholerae serogroup O1 in adults and children from 2 years of age.” Note, this approval was for cholera, not E. coli diarrhea (although SBL’s marketing, e.g., its Web site, concentrates more on ETEC than cholera prevention). EMEA/EU concluded that the existence of only one pivotal trial in the target population for travellers’ diarrhea with results of borderline statistical significance was not sufficient to recommend approval for this indication.
Although not formally approved for traveler’s diarrhea, European product labelling acknowledges the cross reactivity between the rCTB component of Dukoral and ETEC-LT toxin, the cause of traveller’s diarrhea.
Dukoral containing the same inactived V. cholerae but with purified V. cholerae-derived cholera toxin B (CTB) had been approved in Sweden since 1991. The current formulation with recombinant CTB was approved in Sweden in 1992 for protection against cholera; and supplemental approval for E. coli diarrhea was granted in 1995.
SBL Vaccin AB/Crucell has a Biologics Master File with CBER, FDA, with information about manufacturing and characterization of rCTB.
In Oct. 2000, the World Health Organization (WHO) officially recognized (approved) Dukoral as the only vaccine useful for protection against cholera or ETEC. Dukoral is also the only vaccine recommended by WHO for preemptive use in emergency situations, such as the areas affected by the Dec. 2004 Indian Ocean tsunami, where there was imminent risk of a cholera epidemic. WHO recognition allows the United Nations (UN) to purchase the vaccine and build up stocks for emergency use. In addition, WHO approval is recognized by a number of developing countries, and allows distribution of the vaccine in those territories.
Tech. transfer: SBL has received patents covering aspects of Dukoral including EP1444987, “Oral vaccine against diarrhea,”
Trials: EU-wide approval was based on three randomized, placebo-controlled trials in which the vaccine was immunogenic in children and adults, including those from areas where cholera is endemic and non-endemic areas. The clinical trial program ran from 1980 to 1997, with several Phase III trials (Bangladesh and Morocco) using Dukoral (WC/BS) containing non-recombinant CTB, which was later concluded to be comparable to vaccine with rCTB (WC/rBS), which was used in a Phase III study in the Peruvian military (study 27) and in the Pampas field trial (study 30). Both the Peru and Pampas studies tested a two-dose regimen.
In its primary randomized double-blind study efficacy trial (in Bangladesh, 1985-1988), data were reported for 63,498 adults and children aged 2 years and older having received three doses of vaccine at six week intervals. Efficacy was followed-up for five years. Overall, protective efficacy was 85% (95 CI, 56-95%) for the first six months, regardless of age and severity of cholera. Long-term protection differed by age and type of the infecting V cholerae, lasting only six months for children, with best efficacy against El Toro cholera. Sustained protection against classic cholera was also observed in children. Dukoral conferred 67% protection against episodes of diarrhea caused by enterotoxigenic E. coli expressing heat-labile toxin (LT-ETEC), and 86% protection against clinically severe episodes of LT-ETEC. The most common adverse events were gastrointestinal symptoms, including abdominal pain or discomfort, diarrhea, loose stools, nausea, and vomiting—observed with equal frequency in both active and placebo groups.
Two Phase III trials (Peru and Pampas) supported a 2-dose schedule, while the main study (Bangladesh) used three doses. Memory responses induced by two doses were of the same magnitude as after three doses. Dukoral is considered to have improved efficacy against cholera, in comparison with the Wyeth vaccine.
In postmarketing surveillance, serious adverse events including headache, dizziness and dyspnea have been reported very rarely (<1/100,000 doses distributed).
Medical: Dukoral is primarily intended for travelers, particularly those visiting lesser-developed countries with high incidence of cholera and/or E. coli-associated diarrhea. Dukoral is suitable for adults and children two years of age and older. Two doses of Dukoral, taken one week apart prior to the trip, provide protection for three months or more. The last dose should be taken at least one week before departure (potential exposure). If taken previously, only a single booster dose may be required.
Dukoral has been sold in Sweden and Norway for over a decade, where it is currently the leading traveler’s vaccine. Dukoral is recommended and used by the World Health Organization (WHO).
Dukoral is suitable both for individual as well as for population immunization. For Europe, it current primary market, the intended target group is travellers to endemic areas. Immunogenicity studies were performed in naive EU subjects, but no pivotal efficacy trial was conducted in this population. The risk of cholera for regular tourists is minor, although the disease is serious. The vaccine is particularly useful for immunization of health care workers in cholera epidemics.
Disease: Traveller’s (ETEC) diarrhea occurs with a high-incidence in many parts of the world. Traveller’s diarrhea is the most common medical problem affecting travellers to Mexico, the Caribbean, Africa, Latin America, the Middle East, and most parts of Asia. Factors that may be associated with a higher probability of acquiring traveller’s diarrhea include adventurous eating habits, gastric hypochlorhydria, gastrectomy, history of repeated severe traveller’s diarrhea, immunodeficiency diseases, and the relative lack of gut immunity seen in younger persons. Antibiotics are not generally recommended for prophylactic protection against traveler’s diarrhea.
Enterotoxigenic E. coli (ETEC) is the most common cause of bacterial diarrhea in developing countries, responsible for up to 700 000 deaths yearly in children <5 years of age. ETEC has been estimated to cause 5-18 million cases each year. The highest incidence of ETEC infection is in children less than 2 years of age, decreasing with age due to acquired immunity. The clinical spectrum includes mild self-limiting diarrhea to severe cholera-like illness. The incidence of TD varies substantially with destination and season. Approximately 30-50% of travellers to Latin America, Africa and Asia experience diarrhea, and in 50% (20 to 75%) of these cases ETEC are isolated. Widespread use of antibiotics for prophylaxis and treatment of traveller’s diarrhea has resulted to the emergence of multiresistant ETEC.
Market: The price in the U.K. has been reported to be £23.42 (~$42 in 4/2006) for two doses.
In June 2008, Crucell reported over 6 million doses had been distributed to date in the Nordic region, with 400,000 doses sold in Sweden in 2007.
In 2005, total sales of Dukoral were over EUR 14 million ($18.7 million in mid-June 2007). The author roughly estimated (guesses) 2006 total sales of $25 million.
The World Health Organization (WHO), United Nations (UN), is a major purchaser of Dukoral. WHO distributes the vaccine, primarily for its utility against cholera, in African and Latin American countries with high incidence of cholera.
Index Terms:
Companies involvement:
Full monograph
415 Cholera Vaccine (rDNA)/SBL
Nomenclature:
Cholera Vaccine (rDNA)/SBL [BIO]
Dukoral [TR]
Oral cholera vaccine [INN]
cholera toxin B subunit, recombinant (rCTB) [SY]
Vibrio cholerae, inactivated vaccine [SY]
FDA Class: Biologic BLA
biopharmaceutical products
recombinant DNA
vaccines, bacterial
vaccines, bacterial
vaccines, subunit
acute respiratory distress syndrome (ARDS)
bacterial culture <!-- bacterialculture -->
Escherichia coli (E. coli)
fermenters, 1,000 liter
fermenters, 1,000 liter
hexadecyltrimethylammonium bromide
sodium glutamate
vesicular stomatitis virus (VSV)
vesicular stomatitis virus (VSV)
vesicular stomatitis virus (VSV)
vesicular stomatitis virus (VSV)
cholera prophylaxis
citric acid
disodium hydrogen phosphate
First International Reference Preparation for human menopausal gonadotropins (code 70/45)
formaldehyde
heat treatment (pasteurization)
radioimmune conjugates<!-- radioconjugates -->
S-(carboxymethyl)-homocysteine
sodium bromide
sodium chloride
sodium citrate
sodium dihydrogen phosphate
sodium glutamate
North American coral snake
EU002 EU application pending
UM999 Not Available/Not Marketed in US
US000 never filed/no plans
EM001 Marketed Product in EU
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