The present invention relates to acellular pertussis vaccines, components thereof, and their preparation.
Whooping cough or pertussis is a severe, highly contagious upper respiratory tract infection caused by Bordetella pertussis. The World Health Organization estimates that there are 60 million cases of pertussis per year and 0.5 to 1 million associated deaths (ref. 1. Throughout this specification., various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately following the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). In unvaccinated populations, a pertussis incidence rate as high as 80% has been observed in children under 5 years old (ref. 2). Although pertussis is generally considered to be a childhood disease, there is increasing evidence of clinical and asymptomatic disease in adolescents and adults (refs. 3, 4 and 5).
The introduction of whole-cell vaccines composed of chemically- and heat-inactivated B. pertussis organisms in the 1940""s was responsible for a dramatic reduction in the incidence of whooping cough caused by B. pertussis. The efficacy rates for whole-cell vaccines have been estimated at up to 95% depending on case definition (ref. 6). While infection with B. pertussis confers life-long immunity, there is increasing evidence for waning protection after immunization with whole-cell vaccines (ref. 3). Several reports citing a relationship between whole-cell pertussis vaccination, reactogenicity and serious side-effects led to a decline in vaccine acceptance and consequent renewed epidemics (ref. 7). More recently defined component pertussis vaccines have been developed.
Antigens for Defined Pertussis Vaccines
Various acellular pertussis vaccines have been developed and include the Bordetella pertussis antigens, Pertussis Toxin (PT), Filamentous haemagglutonin (FHA), the 69 kDa outer membrane protein (pertactin) and fimbrial agglutinogens (see Table 1 below. The Tables appear at the end of the specification).
Pertussis Toxin
Pertussis toxin is an exotoxin which is a member of the A/B family of bacterial toxins with ADP-ribosyltransferase activity (ref. 8). The A-moiety of these toxins exhibit the ADP-ribosyltransferase activity and the B portion mediates binding of the toxin to host cell receptors and the translocation of A to its site of action. PT also facilitates the adherence of B. pertussis to ciliated epithelial cells (ref. 9) and also plays a role in the invasion of macrophages by B. pertussis (ref. 10).
All acellular pertussis vaccines have included PT, which has been proposed as a major virulence factor and protective antigen (ref. 11, 12). Natural infection with B. pertussis generates both humoral and cell-mediated responses to PT (refs. 13 to 17). Infants have transplacentally-derived anti-PT antibodies (refs. 16, 18) and human colostrum containing anti-PT antibodies was effective in the passive protection of mice against aerosol infection (ref. 19). A cell-mediated immune (CMI) response to PT subunits has been demonstrated after immunization with an acellular vaccine (ref. 20) and a CMI response to PT was generated after whole-cell vaccination (ref. 13). Chemically-inactivated PT in whole-cell or component vaccines is protective in animal models and in humans (ref. 21) Furthermore, monoclonal antibodies specific for subunit S1 protect against B. pertussis infection (refs. 22 and 23).
The main pathophysiological effects of PT are due to its ADP-ribosyltransferase activity. PT catalyses the transfer of ADP-ribose from NAD to the Gi guanine nucleotide-binding protein, thus disrupting the cellular adenylate cyclase regulatory system (ref. 24). PT also prevents the migration of macrophages and lymphocytes to sites of inflammation and interferes with the neutrophil-mediated phagocytosis and killing of bacteria (ref. 25). A number of in vitro and in vivo assays have been used to asses the enzymatic activity of S1 and/or PT, including the ADP-ribosylation of bovine transducin (ref. 26), the Chinese hamster ovary (CHO) cell clustering assay (ref. 27) , histamine sensitization (ref. 28), leukocytosis, and NAD glycohydrolase. When exposed to PT, CHO cells develop a characteristic clustered morphology. This phenomenon is dependent upon the binding of PT, and subsequent translocation and ADP-ribosyltransferase activity of S1 and thus the CHO cell clustering assay is widely used to test the integrity and toxicity of PT holotoxins.
Filamentous Haemagglutinin
Filamentous haemagglutinin is a large (220 kDa) non-toxic polypeptide which mediates attachment of B. pertussis to ciliated cells of the upper respiratory tract during bacterial colonization (refs. 9, 29). Natural infection induces anti-FHA antibodies and cell mediated immunity (refs. 13, 15, 17, 30 and 31). Anti-FHA antibodies are found in human colostrum and are also transmitted transplacentally (refs. 17, 18 and 19). Vaccination with whole-cell or acellular pertussis vaccines generates anti-FHA antibodies and acellular vaccines containing FHA also induce a CMI response to FHA (refs. 20, 32). FHA is a protective antigen in a mouse respiratory challenge model after active or passive immunization (refs. 33, 34). However, alone FHA does not protect in the mouse intracerebral challenge potency assay (ref. 28).
69 kDa Outer Membrane Protein (Pertactin)
The 69 kDa protein is an outer membrane protein which was originally identified from B. bronchiseptica (ref. 35). It was shown to be a protective antigen against B. bronchiseptica and was subsequently identified in both B. pertussis and B. parapertussis. The 69 kDa protein binds directly to eukaryotic cells (ref. 36) and natural infection with B. pertussis induces an anti-P.69 humoral response (ref. 14) and P.69 also induces a cell-mediated immune response (ref. 17, 37, 38). Vaccination with whole-cell or acellular vaccines induces anti-P.69 antibodies (refs. 32, 39) and acellular vaccines induce P.69 CMI (ref. 39). Pertactin protects mice against aerosol challenge with B. pertussis (ref. 40) and in combination with FHA, protects in the intracerebral challenge test against B. pertussis (ref. 41). Passive transfer of polyclonal or monoclonal anti-P.69 antibodies also protects mice against aerosol challenge (ref. 42).
Agglutinogens
Serotypes of B. pertussis are defined by their agglutinating fimbriae. The WHO recommends that whole-cell vaccines include types 1, 2 and 3 agglutinogens (Aggs) since they are not cross-protective (ref. 43). Agg 1 is non-fimbrial and is found on all B. pertussis strains while the serotype 2 and 3 Aggs are fimbrial. Natural infection or immunization with whole-cell or acellular vaccines induces anti-Agg antibodies (refs. 15, 32). A specific cell-mediated immune response can be generated in mice by Agg 2 and Agg 3 after aerosol infection (ref. 17). Aggs 2 and 3 are protective in mice against respiratory challenge and human colostrum containing anti-agglutinogens will also protect in this assay (refs. 19, 44, 45).
Acellular Vaccines
The first acellular vaccine developed was the two-component PT+FHA vaccine (JNIH 6) of Sato et al. (ref. 46). This vaccine was prepared by co-purification of PT and FHA antigens from the culture supernatant of B. pertussis strain Tohama, followed by formalin toxoiding. Acellular vaccines from various manufacturers and of various compositions have been used successfully to immunize Japanese children against whopping cough since 1981 resulting in a dramatic decrease in incidence of disease (ref. 47). The JNIH 6 vaccine and a mono-component PT toxoid vaccine (JNIH 7) were tested in a large clinical trial in Sweden in 1986. Initial results indicated lower efficacy than the reported efficacy of a whole-cell vaccine, but follow-up studies have shown it to be more effective against milder disease diagnosed by serological methods (refs. 48, 49, 50, 51). However, there was evidence for reversion to toxicity of formalin-inactivated PT in these vaccines. These vaccines were also found to protect against disease rather than infection.
A number of new acellular pertussis vaccines are currently being assessed which include combinations of PT, FHA, P.69, and/or agglutinogens and these are listed in Table 1. Several techniques of chemical detoxication have been used for PT including inactivation with formalin (ref. 46), glutaraldehyde (ref. 52), hydrogen peroxide (ref. 53), and tetranitromethane (ref. 54).
Poliomyelitis
Both inactivated (IPV) and live attenuated (OPV) poliovirus vaccines have been effective in controlling poliomyelitis worldwide. A combined DPT-IPV vaccine is currently licensed in Europe and in Canada and has been shown to be safe and effective in millions of children worldwide.
Haemophilus influenzae type b
Prior to the availability of effective vaccines, Haemophilus influenzae type b was a major cause of meningitis invasive bloodborne infections in young children and was the main cause of meningitis in the first 2 years of life (ref. 80). Approximately 10% of Haemophilus influenzae meningitis victims die despite medical care. Permanent sequelae are common in survivors. Immunization against Haemophilus influenzae began in Canada in 1987 with a polysaccharide vaccine (polyribose ribitol phosphate Haemophilus influenzae type b [PRP]). Improved immunogenicity was achieved in children 18 months of age and older with the introduction in 1988 of a vaccine consisting of PRP conjugated to diphtheria toxoid (PRP-D). Since 1992, infant immunization has been possible with the licensure of PRP conjugate vaccines immunogenic in infants under 1 year of age (PRP conjugated with tetanus toxoid or PRP-T). The use of these Haemophilus influenzae conjugate vaccines has been associated with a dramatic decrease in the incidence of invasive Haemophilus infection in Canada and elsewhere (ref. 81). Two Canadian clinical studies involving nearly 900 children in British Columbia and Alberta demonstrated that lyophilized PRP-T may be reconstituted with DPT (COMBIPAC) (ref. 82) or with DPT-Polio Adsorbed (PENTA(trademark)) (Ref. 83) in addition to the usual saline diluent. Clinical studies involving more than 100,000 children around the world have demonstrated the efficacy of lyophilized PRP-T (ActHib(trademark)). Over 90% achieve anti-PRP levels considered to be protective (xe2x89xa70.15 xcexcg/ml) after 3 doses of PRP-T starting at 2 months or after a single dose of PRP-T given after 12 months of age. The proportion achieving levels indicative of long term protection ( greater than 1.0 xcexcg/ml) varies from 70 to 100% depending on the study. Millions of doses of PRP-T have been sold in Canada since 1992. Breakthrough cases of invasive haemophilus infection after vaccination with PRP-T are rare and may be associated with diseases such as immunodeficiency (ref. 84).
Combination Vaccines
Although there are many actual and potential benefits of vaccines that combine antigens to confer protection against multiple pathogens, these combinations may have a detrimental effect on the immunogenicity of the individual components. Combinations of diphtheria and tetanus toxoids with whole cell pertussis vaccine (DTP) have been available for over 50 years and the antibody response to the combination is superior to the individual components, perhaps as a result of an adjuvant effect of the whole cell pertussis vaccine. DTP combinations that also include inactivated poliovirus vaccine are licensed in many jurisdictions, although the antibody response to the pertussis antigens may be diminished by this combination (ref 69-71). The effect of combining DTP vaccines with Hib conjugate vaccine have been variable. Studies with a French DTP and PRPT demonstrated similar safety but a decreased antibody response to PRP (ref. 72-73) whereas studies with a Canadian DTP and PRPT vaccine showed no effect on the PRP response but lower pertussis agglutinins and increased injection site tenderness in the combined immunization group (ref 74, 75).
Data are now becoming available on the effect of combining APDT vaccines with Hib conjugate vaccine. In two month old infants given three doses of an acellular pertussis-diphtheria-tetanus vaccine (APDT) combined with a Hib conjugate vaccine (PRPT), the antibody, response to PRP was significantly lower than in the group given separate injections on the same day (ref. 76). Similar results were reported with another acellular pertussis-diphtheria-tetanus vaccine combined with PRPT given for the first three doses (ref 77).
In contrast to other reported studies, children immunized with the combined vaccine had a superior antibody response to PRP, diphtheria, and several of the pertussis antigens when compared to children given PRP at a separate visit. There may be several reasons for the equivalent or better immunogenicity for these vaccines when given as a combined injection rather than the decreased immunogenicity reported with other products. All acellular pertussis vaccines are not identical in their antigenic content, method of toxoiding, adjuvant or preservative. However, decreased immunogenicity has been reported with acellular pertussis vaccines containing PT, FHA, and 69K (ref. 77) and with containing PT, FHA, 69K and fimbriae (ref. 76).
The five component APDT examined in this study was found to have a protective efficacy of 85% (example 5) (95% CI 81/89) in a phase III clinical trial recently completed in Sweden under the auspices of the National Institutes of Health (ref. 78). This study demonstrated that this vaccine can be combined with Hib-tetanus toxoid conjugate vaccine as a single injection for the fourth dose in children between 17 and 21 months of age.
Current commercially-available combination vaccines may not contain appropriate formulations of appropriate antigens in appropriate immunogenic forms to achieve a desired level of efficacy in a pertussis-susceptible human population.
It would be desirable to provide efficacious combination vaccines comprising acellular pertussis components containing selected relative amounts of selected antigens.
The present invention is directed towards combination vaccines containing acellular pertussis vaccine components, and methods of use thereof.
In accordance with one aspect of the present invention, there is provided a multi-valent immunogenic composition for conferring protective in a host against disease caused by infection by Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, poliovirus and/or Haemophilus influenzae, comprising:
(a) pertussis toxoid, filamentous haemagglutinin, pertactin and agglutinogens in purified form,
(b) tetanus toxoid,
(c) diphtheria toxoid,
(d) inactivated polio virus, and, optionally,
(e) a conjugate of a carrier molecule selected from tetanus toxoid and diphtheria toxoid and a capsular polysaccharide of Haemophilus influenzae type b.
The immunogenic compositions may be formulated as a vaccine for in vivo administration to the host wherein the individual components of the composition are formulated such that the immunogenicity of individual components is not impaired by other individual components of the composition.
In immunogenic composition may further comprise an adjuvant, particularly aluminum hydroxide or aluminum phosphate.
Such vaccine composition may contain about 5 to about 30 xcexcg nitrogen of pertussis toxoid, about 5 to about 30 xcexcg nitrogen of filamentous haemagglutinin, about 3 to about 15 xcexcg nitrogen of pertactin and about 1 to about 10 xcexcg nitrogen of agglutinogens.
In one specific embodiment, the vaccine may comprise pertussis toxoid, fimbrial haemagglutinin, the 69 kDa protein and filamentous agglutinogens of Bordetella pertussis at a weight ratio of about 10:20:5:3 as provided by about 20 xcexcg of pertussis toxoid, about 20 xcexcg of filamentous haemagglutinin, about 5 xcexcg of fimbrial agglutinogens and about 3 xcexcg of fimbrial 69 Kda protein in a single human dose. In one embodiment of the vaccine provided herein, the vaccine contains about 15 Lfs of diphtheria toxoid and about 5 Lfs of tetanus toxoid.
The inactivated poliovirus employed in the immunogenic composition of the invention generally comprises a mixture of inactivated poliovirus types 1,2 and 3. In one formulation, such mixtures of inactivated poliovirus types may comprise:
about 40 D antigen units of piliovirus type 1
about 8 D antigen units of poliovirus type 2
about 32 D antigen units of poliovirus type 3 in a single human dose.
The conjugate molecule may comprise a conjugate of toxoid or diphtheria toxoid and polyribose ribitol phosphate (PRP) of Haemophilus influenzae type b. Such conjugate molecule may be provided in a hydrolyzed form, which is reconstituted for administration by combination with the other components. In one formulation, the conjugate is employed in the form of about 10 xcexcg of PRP conjugate to about 20 xcexcg of tetanus toxoid.
In addition, the vaccine may also comprise an adjuvant, particularly aluminum phosphate.
In such particular embodiments, the immunogenic compositions provide an immune response profile to each of the pertussis antigens contained therein and the response profile provided by the acellular components is substantially equivalent to that produced by a whole cell pertussis vaccine.
In a further aspect of the invention, there is provided a method of immunizing a host against multiple diseases, comprising administering to the host, which may be human, an immunoeffective amount of the immunogenic composition or vaccine as provided herein.
Advantages of the present invention include a multi-valent vaccine which can confer protection against a range of common pediatric diseases in a safe and efficacious manner. The ability to provide a single vaccination against multiple diseases without interference between the immunogenic responses to the various immunogens is beneficial.