Link:  Pharm/Biotech Resources

Title:  Vaccines containing attenuated bacteria

United States Patent:  6,905,691

Issued:  June 14, 2005

Inventors:  Chatfield; Steven Neville (Berkshire, GB); Dougan; Gordon (London, GB); Sydenham; Mark (London, GB)

Assignee:  Celltech Pharma Europe Limited (GB)

Appl. No.:  591447

Filed:  June 9, 2000

The invention relates to a vaccine comprising a bacterium attenuated by a non-reverting mutation in a gene encoding a protein which promotes folding of extracytoplasmic proteins. Such mutations were initially identified as being useful in vaccines from a bank of randomly inserted, transposon mutants in which attenuation was determined as a reduction in virulence of the organism in the mouse model of infection. Site directed mutation of the gene results in a strain which shows at least 4 logs of attenuation when delivered both orally and intravenously. Animals vaccinated with such a strain are protected against subsequent challenge with the parent wild type strain. Finally, heterologous antigens such as the non-toxic and protective, binding domain from tetanus toxin, fragment C, can be delivered via the mucosal immune system using such strains of bacteria. This results in the induction of a fully protective immune response to subsequent challenge with native tetanus toxin.

BACKGROUND OF THE INVENTION

The principle behind vaccination is to induce an immune response in the host thus providing protection against subsequent challenge with a pathogen. This may be achieved by inoculation with a live attenuated strain of the pathogen (i.e. a strain having reduced virulence such that it does not cause the disease caused by the virulent pathogen).

Classically, live attenuated vaccine strains of bacteria and viruses have been selected using one of two different methodologies. Mutants have been created either by treatment of the organism using mutagenic chemical compounds or by repeated passage of the organism in vitro. However, use of either method gives rise to attenuated strains in which the mode of attenuation is unclear. These strains are particularly difficult to characterize in terms of possible reversion to the wild type strain as attenuation may reflect single (easily reversible) or multiple mutation events.

Using modem genetic techniques, it is now possible to construct genetically defined attenuated bacterial strains in which stable attenuating deletions can be created. A number of site directed mutants of Salmonella have been created using this type of technology (2, 5, 6, 12, 22, 35, 36, 37). Amongst the most comprehensively studied attenuating lesions are those in which mutations in the biosynthetic pathways have been created, rendering the bacteria auxotrophic (e.g. aro genes). Mutations in these genes were described as early as 1950 (1) as responsible for rendering Salmonella less virulent for mice. Several different auxotrophic mutations such as galE, aroA or purA have also been described previously (6, 12). Salmonella aroA mutants have now been well characterised and have been shown to be excellent live vaccines against salmonellosis in several animal species. In addition, in order to reduce the chances of a reversion to virulence by a recombination event mutations have now been introduced into two independent genes such as aroA/purA and aroA/aroC Identical mutations in host adapted strains of Salmonella such as S. typhi (man) and S. dublin (cattle) has also resulted in the creation of a number of single dose vaccines which have proved successful in clinical (11, 17) and field trials (15).

In animal studies, attenuated S. typhimurium has been used as a vehicle for the delivery of heterologous antigens to the immune system (3, 8, 32). This raises the potential of the development of multivalent vaccines for use in man (9).

SUMMARY OF THE INVENTION

The original aim of the work that led to the invention was the identification of novel genes that are involved in the virulence pathways of pathogenic bacteria, the identification and deletion of which may render the bacteria avirulent and suitable for use as vaccines. To identify attenuating lesions, random mutations were introduced into the chromosome of S. typhimurium using the transposon TnphoA (18). This transposon is unique in that it is engineered to identify proteins that are expressed in or at the bacterial outer membrane; such proteins may be those involved in interaction with and uptake by host tissues: By using the natural oral route of infection to screen these mutants, those with important, in vivo induced, attenuat lesions in genes were identified.

One such gene identified through this work is surA. The surA gene product is known to promote the folding of extracytoplasmic proteins. Accordingly, the invention provides a vaccine comprising a pharmaceutically acceptable carrier or diluent and a bacterium attenuated by a non-reverting mutation in a gene encoding a protein which promotes the folding of extracytoplasmic proteins. The vaccine has the ability to confer protection against a homologous wild type oral challenge with the virulent bacterium. In addition, the bacterium used in the vaccine can act as a carrier for heterologous antigens such as fragment C of tetanus toxin.

DETAILED DESCRIPTION OF THE INVENTION

Proteins that Promote the Folding of Extracytoplasmic Proteins

Periplasmic and outer membrane proteins are secreted across the cytoplasmic (inner) membrane in a mostly unfolded state, and they then fold after secretion. The folding often has enzymatic assistance to catalyse the formation of bonds necessary for the protein to reach its folded state. For example, the folding often requires the participation of enzymes that catalyse the formation of disulphide bonds or enzymes that catalyse the isomerisation of prolyl bonds (peptidyl-prolyl cis-tans isomerases or PPiases).

One known PPiase is SurA. The inventors have now shown that mutation of the surA gene causes attenuation of virulent bacteria and that the attenuated bacteria are useful as vaccines.

SurA was first described as being essential for the survival of E.coli in the stationary phase (33). It is a periplasmic protein. More recently, SurA has been described as belonging to a third, new family of PPiases (30), the parvulin family. Further studies have shown SurA to be involved in the correct folding of outer membrane proteins such as OmpA, OmpF, and LamB (16, 24, 29).

PPiases are divided into three families, the cyclophilins, FK506-binding proteins (FKBPs) and parvulins. Members of all three families have been found in E.coli. Apart from SurA, the parvulin family includes several proteins such as NifM, PrsA and PrtM.

Bacteria Useful in the Invention

The bacteria that are used to make the vaccines of the invention are generally those that infect via the oral route. The bacteria may be those that invade and grow within eukaryotic cells and/or colonise mucosal surfaces. The bacteria are generally Gram-negative.

The bacteria may be from the genera Salmonella, Escherichia, Vibrio, Haemophilus, Neisseria, Yersinia, Bordetella or Brucelia Examples of such bacteria are Salmonella typhimurium —the cause of salmonellosis in several animal species; Salmonella typhi —the cause of human typhoid; Salmonella enteritidis —a cause of food poisoning in humans; Salmonella choleraesuis —a cause of salmonellosis in pigs; Salmonella dublin —a cause of both a systemic and diarrhoel disease in cattle, especially of new-born calves; Escherichia coli —a cause of diarrhoea and food poisoning in humans; Haemophilus influenzae —a cause of meningitis; Neisseria gonorrhoeae —a cause of gonnorrhoeae; Yersinia enterocolitica —the cause of a spectrum of diseases in humans ranging from gastroenteritis to fatal septicemic disease; Bordetella pertussis —the cause of whooping cough; or Brucella abortus —a cause of abortion and infertility in cattle and a condition known as undulant fever in humans.

Salmonella bacteria are particularly useful in the invention. As well as being vaccines in their own right against infection by Salmonella, attenuated Salmonella can be used as carriers of heterologous antigens from other organisms to the immune system via the oral route. Salmonella are patent immunogens and are able to stimulate systemic and local cellular and antibody responses. Systems for driving expression of heterologous antigens in Salmonella in vivo are known; for example the nirB and hrrA promoters are known to be effective drivers of antigen expression in vivo.

The invention is also particularly applicable to E.coli, especially exterotoxigenic E.coli ("ETEC"). ETEC is a class of E.coli that cause diarrhoea. They colonise the proximal small intestine. A standard ETEC strain is ATCC H10407.

Infections of ETEC are the single most frequent cause of travellers diarrhoea, causing 3-9 million cases per year amongst visitors to developing countries. In endemic areas, ETEC infections are an important cause of dehydrating diarrhoea in infants and young children, resulting in 800,000 deaths a year in the under fives world-wide. In developing countries, the incidence of ETEC infections leading to clinical disease decreases with age, indicating that immunity to ETEC infection can be acquired. In contrast, naive adults from industrialized countries who visit endemic areas are highly susceptible to ETEC infections. However, with prolonged or repeated visits to endemic areas susceptibility to ETEC infections diminishes, suggesting that a live attenuated approach to ETEC vaccination may prove is successful.

Seq. Id. No. 1 shows the sequence of the surA open reading frame in Salmonella typhimurium, and Seq. Id. No. 2 shows the corresponding amino acid sequence. Seq. Id. No. 3 shows the sequence of the surA open reading frame in E.coli, and Seq. Id. N. 4 shows the corresponding amino acid sequence.

Second Mutations

The bacteria used in vaccines of the invention preferably contain a mutation in one or more genes in addition to the mutation in the gene encoding a protein which promotes folding of extracytoplasmic proteins. This is so that the risk of the bacterium reverting to the virulent state is minimised which is clearly important for the use of the bacterium as a human or animal vaccine. Although bacteria containing only a mutation in a protein which promotes folding of extracytoplasmic proteins are attenuated and the risk of reversion is small, it will generally be desirable to introduce at least one further mutation so as to reduce the risk of attenuation yet further

A number of genes that are candidates for second and further mutations are known (see e.g. ref 39). These include the aro genes (35), the pur genes, the htrA gene (37), the ompR gene (36), the galE gene, the cya gene, the crp gene or the phoP gene. The aro gene may be aroA, aroC, aroD or aroE. The pur gene may be purA, purB, purE or purH. The use of aro mutants, especially double aro mutants, is preferred because such mutants have been shown to be particularly effective as vaccines. Suitable combinations of aro mutations are aroAaroC, aroAaroD and aroAaroE.

The Nature of the Mutation

The mutations introduced into the bacterial vaccine generally knock-out the function of the gene completely. This may be achieved either by abolishing synthesis of any polypeptide at all from the gene or by making a mutation that results in synthesis on non-functional polypeptide. In order to abolish synthesis of any polypeptide, either the entire gene or its 5′-end may be deleted. A deletion or insertion within the coding sequence of a gene may be used to create a gene that synthesises only non-functional polypeptide (e.g. polypeptide that contains only the N-terminal sequence of the wild-type protein). In the case of mutations in genes encoding proteins which promote the folding of extracytoplasmic proteins, the mutation generally abolishes the ability of the protein to promote such protein folding.

The mutations are non-reverting mutations. These are mutations that show essentially no reversion back to the wild-type when the bacterium is used as a vaccine. Such mutations include insertions and deletions. Insertions and deletions are preferably large, typically at least 10 nucleotides in length, for example from 10 to 600 nucleotides.

The bacterium used in the vaccine preferably contains only defined mutations, i.e. mutations which are characterised. It is clearly undesirable to use a bacterium which has uncharacterised mutations in its genome as a vaccine because there would be a risk that the uncharacterised mutations may confer properties on the bacterium that cause undesirable side-effects.

The attenuating mutations may be constructed by methods well known to those skilled in the art (see ref 31). One means for introducing non-reverting mutations into extracytoplasmic proteins is to use transposon TnphoA. This can be introduced into bacteria to generate enzymatically active protein fusions of alkaline phosphatase to extracytoplasmic proteins. The TnphoA transposon carries a gene encoding kanamycin resistance. Transductants are selected that are kanamycin resistant by growing colonies on an appropriate selection medium.

Alternative methods include cloning the DNA sequence of the wild-type gene into a vector, e.g. a plasmid or cosmid, and inserting a selectable marker into the cloned DNA sequence or deleting a part of the DNA sequence, resulting in its inactivation. A deletion may be introduced by, for example, cutting the DNA sequence using restriction enzymes that cut at two points in the coding sequence and ligating together the two ends in the remaining sequence. A plasmid carrying the inactivated DNA sequence can be transformed into the bacterium by known techniques. It is then possible by suitable selection to identify a mutant wherein the inactivated DNA sequence has recombined into the chromosome of the bacterium and the wild-type DNA sequence has been rendered non-functional in a process known as homologous recombination.

Expression of Heterologous Antigens

The attenuated bacterium used in the vaccine of the invention may be genetically engineered to express an antigen from another organism (a "heterologous antigen"), so that the attenuated bacterium acts as a carrier of the antigen from the other organism. In this way it is possible to create a vaccine which provides protection against the other organism. A multivalent vaccine may be produced which not only provides immunity against the virulent parent of the attenuated bacterium but also provides immunity against the other organism. Furthermore, the attenuated bacterium may be engineered to express more than one heterologous antigen, in which case the heterologous antigens may be from the same or different organisms.

The heterologous antigen may be a complete protein or a part of a protein containing an epitope. The antigen may be from another bacterium, a virus, a yeast or a fungus. More especially, the antigenic sequence may be from tetanus, hepatitis A, B or C virus, human rhinovirus such as type 2 or type 14, herpes simplex virus, poliovirus type 2 or 3, foot-and-mouth disease virus, influenza virus, coxsackie virus or Chlamydia trachomatis. Useful antigens include E.coli heat labile toxin B subunit (LT-B), E.coli K88 antigens, P.69 protein from B. pertussis and tetanus toxin fragment C.

The DNA encoding the heterologous antigen is expressed from a promoter that is active in vivo. Two good promoters are the nirB promoter (38, 40) and the htrA promoter (40).

A DNA construct comprising the promoter operably linked to DNA encoding the heterologous antigen may be made and transformed into the attenuated bacterium using conventional techniques. Transformants containing the DNA construct may be selected, for example be screening for a selectable marker on the construct. Bacteria containing the construct may be grown in vitro before being formulated for administration to the host for vaccination purposes.

Formulation of the Vaccine

The vaccine may be formulated using known techniques for formulating attenuated bacterial vaccines. The vaccine is advantageously presented for oral administration, for example in a lyophilised encapsulated form. Such capsules may be provided with an enteric coating comprising, for example, Eudragate "S" (Trade Mark), Eudragate "L" (Trade Mark), cellulose acetate, cellulose phthalate or hydroxypropylmethyl cellulose. These capsules may be used as such, or alternatively, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is advantageously effected in a buffer at a suitable pH to ensure the viability of the bacteria. In order to protect the attenuated bacteria and the vaccine from gastric acidity, a sodium bicarbonate preparation is advantageously administered before each administration of the vaccine. Alternatively, the vaccine may be prepared for parenteral administration, intranasal administration or intramuscular administration.

The vaccine may be used in the vaccination of a host, particularly a human host but also an animal host An infection caused by a microorganism, especially a pathogen, may therefore be prevented by administering an effective dose of a vaccine prepared according to the invention. The dosage employed will be dependent on various factors including the size and weight of the host and the type of vaccine formulated. However, a dosage comprising the oral administration of from 10⁷ to 10¹¹ bacteria per dose may be convenient for a 70kg adult human host.

Claim 1 of 27 Claims

1. A composition which invokes an immune response to a pathogenic bacterium comprising an immunogenically effective amount of a pathogenic bacterium attenuated by a non-reverting, defined mutation in the surA gene and a pharmaceutically acceptable carrier or diluent.

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