Mucosal DTPa vaccines, especially intranasal vaccines, comprising (a) a diphtheria antigen, a tetanus antigen and an acellular pertussis antigen, and (b) a detoxified mutant of cholera toxin (CT) or E. coli heat labile toxin (LT). Component (b) acts as a mucosal adjuvant. The acellular pertussis antigen preferably comprises pertussis holotoxin (PT) and filamentous haemagglutinin (FHA) and, optionally, pertactin. The mucosally-delivered combined DTPa formulation is capable of generating a level of protection against B. pertussis infection equivalent to that observed by alum-adjuvanted parenteral administration.

FIELD OF THE INVENTION

This application relates to mucosal DTP vaccines, especially intranasal vaccines.

BACKGROUND TO THE INVENTION

Bordetella pertussis is the causative agent of whooping cough. A highly effective inactivated whole cell vaccine has been available since the 1940s but concern over its safety, due to the presence of toxic cellular components, has limited its uptake [1]. Acellular pertussis vaccines(Pa) comprising a small number of defined B. pertussis antigens have therefore been produced, and have been approved for use in humans [2].

Pertussis vaccines are usually administered intramuscularly to children in the form of atrivalent DTP combination (diphtheria, tetanus, pertussis) on alum adjuvant. Intramuscular vaccination is not, however, the ideal route of administration. Mucosal vaccines (oral, intranasal etc.) are preferred for two reasons [3]. Firstly, they are easier to administer on a large scale, avoiding the need for specialized equipment and the problems associated with needles. Secondly, they stimulate mucosal immunity, mediated by secretory IgA. As most pathogens enter the body across mucous membranes, mucosal immunity is desirable.

Attempts to make acellular mucosal pertussis vaccines have been described [e.g. 4,5,6,7,8,9], but the levels of protection reported were either not compared with conventional vaccine, or did not approach that observed the alum-adjuvanted antigens given parenterally.

There is therefore a need for an effective mucosal DTP combination vaccine.

DISCLOSURE OF THE INVENTION

The invention provides a mucosal DTPa vaccine comprising (a) a diphtheria antigen (D), a tetanus antigen (T), an acellular pertussis antigen (Pa), and (b) a detoxified form of either cholera toxin (CT) or E. coli heat labile toxin (LT).

The detoxified form of cholera toxin (CT) or E. coli heat labile toxin (LT) acts as a mucosal adjuvant [10]. CT and LT are homologous and are typically interchangeable. Detoxification of the CT or LT may be by chemical or, preferably, by genetic means. Suitable examples include LT having a lysine residue at amino acid 63 ["LT-K63"--ref. 11], and LT having an arginine residue at amino acid 72 ["LT-R72"--ref. 12], both of which have been found to enhance antigen-specific serum IgG, sIgA, and local and systemic T cell responses to DTPa, LT-K63 is preferred, as this has been found in a reliable animal model of B. pertussis infection to result in a high level of protection, equivalent to that generated with a parenterally-delivered DTPa vaccine formulated with alum. Other suitable mutants include LT with a tyrosine at residue 63 ["Y63"--ref. 13] and the various mutants disclosed in reference 14, namely D53, K97, K104 and S106, as well as combinations thereof (e.g. LT with both a D53 and a K63 mutation).

The mucosal vaccine of the invention is preferably an intranasal vaccine. In such an embodiment, it is preferably adapted for intranasal administration, such as by nasal spray, nasal drops, gel or powder [e.g. 15].

The acellular pertussis antigen preferably comprises pertussis holotoxin (PT) and filamentous haemagglutinin (FHA). It may further comprise pertactin and, optionally, agglutinogens 2 and 3 [16, 17].

PT is a toxic protein and, when present in the pertussis antigen, it is preferably detoxified. Detoxification may be by chemical and/or genetic means. A preferred detoxified mutant is the 9K/129G double mutant [2], referred to herein as "rPT".

The diphtheria antigen (D) is preferably a diphtheria toxoid, more preferably the CRM197 mutant [10]. The tetanus antigen (T) is preferably a tetanus toxoid [18].

Non-DTP antigens, preferably ones that do not diminish the immune response against the DTP components, may also be included [e.g. ref. 19, which includes a HBV antigen, and ref. 20].

The invention also provides a method of raising an immune response in a patient, comprising administering to a patient a vaccine according to the invention. The immune response is preferably protective against whooping cough, diphtheria and tetanus. The patient is preferably a child.

The method may raise a booster response, in a patient that has already been primed against B. pertussis. The primer vaccination may have been by a mucosal or parenteral route.

The invention also provides the use of a detoxified mutant of cholera toxin (CT) or E. coli heat labile toxin (LT) in the manufacture of an intranasal medicament for vaccinating a patient against whooping cough, diphtheria and tetanus, or for boosting an primer immune response previously raised against B. pertussis.

The invention also provides an immunogenic composition comprising (a) a diphtheria antigen (D), a tetanus antigen (T), an acellular pertussis antigen (Pa), and (b) a detoxified form of either cholera toxin (CT) or E. coli heat labile toxin (LT).

It will be appreciated that references in the above text to particular proteins (e.g. pertactin, PT, etc.) encompass their allelic variants and functional mutants. They also encompass proteins having significant sequence identity to the wild-type proteins. The degree of identity is preferably greater than 50% (e.g. 65%, 80%, 90%, or more) calculated using, for instance, the Smith-Waterman homology search algorithm as implemented in the MPSRCH program(Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1. Immunogenic fragments of these proteins may also be used, as may longer proteins incorporating the proteins, variants or fragments (e.g. fusion proteins). In all cases, however, the protein (whether wild-type, variant, mutant, fragment or fusion) will substantially retain the wild-type immunogenicity.

The proteins can, of course, be prepared by various means (e.g. recombinant expression, purification from cell culture, chemical synthesis etc.) and in various forms (e.g. native, fusions etc.). They are preferably prepared in substantially pure or isolated form (i.e. substantially free from other bacterial or host cell proteins with which they are normally associated in nature).

The vaccines of the invention may comprise nucleic acid for "genetic immunization" [e.g. 21]. The nucleic acid will encode a protein component of the vaccine and may replace individual protein components, or may supplement them. As an example, the vaccine may comprise DNA that encodes a tetanus toxin.

Vaccines according to the invention will typically be prophylacetic (i.e. to prevent infection), but may also be therapeutic (i.e. to treat disease after infection).

The vaccines of the invention will, in addition to components (a) and (b), typically comprise "pharmaceutically acceptable carriers," which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylacetic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. The vaccines may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present.

Immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen, as well as any other of the above-mentioned components, as needed. By "immunologically effective amount," it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.

Claim 1 of 9 Claims

1. An intranasal vaccine comprising: a diphtheria toxoid, a tetanus toxoid, and an acellular pertussis antigen (DTPa) comprising detoxified pertussis holotoxin; and a detoxified E. coli heat labile toxin, wherein the detoxified E. coli heat labile toxin is LT-K63 or LT-R72.

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