Title: Vaccine compositions for mucosal delivery
United States Patent: 6,562,352
Issued: May 13, 2003
Inventors: Roberts; Mark (London, GB); Dougan; Gordon
Assignee: Medeva Holdings, B.V. (Amsterdam, NL)
Appl. No.: 963129
Filed: October 28, 1997
The invention provides the use of an antigen which is a mucosally
immunogenically active substance comprising the 50 kD C fragment of tetanus
toxin, an immunogenic fragment thereof, or a derivative thereof formed by
amino acid deletion, substitution or insertion for the manufacture of a
vaccine composition for administration to a mucosal surface to induce an
immune response in the mucosal surface against tetanus infection. The
Vaccine composition preferably contains the P.69 outer membrane protein of
B. pertussis, and B. pertussis filamentous haemaglutiuin. The invention also
provides vaccine compositions per se and a method of treating tetanus and
optionally whooping cough using the vaccine compositions.
SUMMARY OF THE INVENTION
We have now found that certain molecules make excellent mucosal immunogens
and such components can be utilised in the development of a mucosally (intranasally
or orally) delivered vaccine against the diseases whooping cough and
tetanus. In particular, we have found that the P69 outer membrane protein
(P69--also known as pertactin) from B. pertussis and the non-toxic
immunogenic 50 Kd portion of tetanus toxin (C-Fragment) from C. tetanii
are highly immunogenic when given intranasally. C-Fragment and P.69 were
generated by DNA recombinant technology. Recombinant C-Fragment and P.69
produced from E. coli and yeast have been demonstrated to be immunogenic
and protective in mice, see M. Roberts et al, Recombinant P.69/pertactin:
imminogenicity and protection of mice against Bordetella pertussis
infection; Vaccine 10, 43 (1992); and see also N. F. Fairweather et al,
Infection and Immunity, 55, 2541 (1987).
In a first aspect, the invention provides the use of a mucosally
immunogenically active substance comprising the 50 kD C fragment of
tetanus toxin, an immunogenic fragment thereof, or a derivative thereof
formed by amino acid deletion, substitution or insertion, for the
manufacture of a vaccine composition for immunising a patient against
In one particular embodiment of the invention, there is provided the use
of a mixture of antigens for the manufacture of a vaccine composition for
administration to a mucosal surface to induce an immune response in the
mucosal surface against each of the said antigens, the mixture of antigens
(a) a mucosally immunogenically active substance comprising the 50 kD C
fragment of tetanus toxin, an immunogenic fragment thereof, or a
derivative thereof formed by amino acid deletion, substitution or
(b) a mucosally immunogenically active substance comprising the P.69 outer
membrane protein of B. pertussis;
an immunogenic fragment thereof, or a derivative thereof formed by amino
acid deletion, substitution or insertion.
In a preferred embodiment the invention provides the use of a mixture of
antigens as hereinbefore defined but wherein said mixture comprises in
addition to (a) and (b);
(c)a mucosally immunogenically active substance comprising B. pertussis
filamentous haemaglutinin, an immunogenic fragment thereof, or a
derivative thereof formed by amino acid deletion, substitution or
In a further aspect, the invention provides a vaccine composition for
application to a mucosal surface, the composition comprising antigen (a)
or a mixture of antigens as hereinbefore defined and a pharmaceutically
In another aspect the invention, provides a method of immunising a host
such as a mammal, (e.g. human) against infection, which method comprises
administering an effective amount of antigen (a), or a mixture of antigens
as hereinbefore defined, directly to a mucosal surface in the host to
induce in said mucosal surface an immune response to each said antigen.
The mucosal delivery compositions of the present invention can be
formulated, for example, for delivery to one or more of the oral,
gastro-intestinal, and respiratory (e.g. nasal and bronchial) mucosa.
Where the composition is intended for delivery to the respiratory (e.g.
nasal or bronchial) mucosa, typically it is formulated as an aqueous
solution for administration as an aerosol or nasal drops, or as a dry
powder, e.g. for inhalation.
Compositions for administration as nasal drops may contain one or more
excipients of the type usually included in such compositions, for example
preservatives, viscosity adjusting agents, tonicity adjusting agents,
buffering agents and the like.
The antigenic preparations of the present invention may also take the form
of compositions intended to deliver the mixture of antigen to mucosal
surfaces in the gastrointestinal tract. It is preferred that such
compositions are provided with means for preventing degradation of the
antigens by the gastric juices. For example, the compositions may take the
form of capsules, e.g. microcapsules, in which the antigens are retained
within a protective matrix or coating formed from an appropriate
protective polymer such as a poly (glycolide), poly (lactide-co-glycolide),
polyactyl starch, or pH-dependent coatings such as the polyacrylates or
hydroxypropylmethyl cellulose phthalate.
The antigens may take the form of the tetanus toxin C fragment per se, the
P.69 protein per se, or the B. pertussis haemaglutinin per se. Or it may
take the form of a larger molecule containing one or more of the aforesaid
antigens, immunogenically active fragments thereof, or derivatives formed
by amino acid deletion, substitution and insertion, provided that the
larger molecule is immunogenically active when administered directly to
The antigen or mixture of antigens typically is selected such that it is
non-toxic to a recipient thereof at concentrations employed to elicit an
In one embodiment, two more of the antigens forming the mixture may be
presented in a single molecule. Such a molecule may be prepared by
recombinant methods by preparing a DNA construct containing genes coding
for two or more of the antigens and expressing in a suitable host in
accordance with known methods.
The individual antigenic substances making up the compositions of the
invention may each also act as carriers for one or more other antigens.
For example, an antigen such as the P.69 outer membrane protein or
C-Fragment may be coupled to another antigen, and examples of such "other"
antigens include Haemophilus group B and meningococcal polysaccharide
In order to enhance the mucosal immunogenicity of the mixture of antigens
or any component antigen thereof or appropriate immunogenic fragments
thereof, they may be incorporated into appropriate carriers, for example
virosomes, or the antigens or immunogenic fragments thereof may be
expressed in suitable attenuated carrier strains of Salmonella.
Immunogenicity may also be enhanced by incorporating appropriate mucosal
adjuvants such as cholera toxin or E. coli heat-labile toxin, genetically
detoxified variants thereof or their binding (B) sub-units in the vaccine.
The vaccine composition may optionally contain another mucosally
immunogenically active portion of the tetanus toxin molecule. In addition,
the mixed vaccine may contain one or more further mucosally
immunogenically active antigens.
In one embodiment, the vaccine composition may, in addition to non-toxic
immunogenic forms of tetanus toxin and pertussis antigens, contain
non-toxic immunogenic forms of diphtheria toxin and immunogenic forms of
Haemophilus influenzae group B polysaccharide (HiB), thereby providing a
mucosal diphtheria-tetanus-pertussis (DTP) vaccine or DTPHiB vaccine.
The P.69 outer membrane protein of B. pertussis is a protein of
approximately 51 KD molecular weight; see A. J. Makoff et al, "Protective
surface antigen P.69 of Bordetella pertussis: its characteristics and very
high level expression in Escherichia coli", Bio-Technology, 8, 1030
It can be prepared and isolated according to the method disclosed in P.
Novotny et al: The Journal of Infectious Diseases, 164, 114 (1991), or
recombinant material prepared from E. coli by the method given in the
article by A. J. Makoff et al referred to above. It can bind to eukaryotic
Purified B. pertussis filamentous haemaglutinin usually contains
polypeptides of differing molecular weight ranging from 98-220 KD, and can
be isolated and purified from cell culture supernatants of B. pertussis,
for example as described in the article by P. Novotny et al referred to
above. The filamentous haemaglutinin is able to bind to eukaryotic cells
and cause haemaglutination of sheep erythrocytes.
The C fragment of tetanus toxin is a peptide of approximately 50 KD
molecular weight which can be isolated and purified form E. coli by the
method described in A. J. Makoffet al., Bio/Technology, 7, 1043 (1989).
The C fragment is characterised by an ability to bind the eukaryotic cells
possessing the trisialoganglioside GT 16 and by an ability to elicit
protection in mice against lethal challenge with tetanus toxin.
The antigenic molecules of the present invention can be prepared by
isolation and purification from the organisms in which they occur
naturally, or they may be prepared by recombinant techniques and expressed
in a suitable host such as E.coli in known manner. When prepared by a
recombinant method or by synthesis, one or more insertions, deletions,
inversions or substitutions of the amino acids constituting the peptide
may be made. Each of the aforementioned antigens is preferably used in the
substantially pure state. The quantity of the mixture of antigens
administered will depend, in part, upon the purity of the individual
antigens. Thus, for a substantially pure form of the P.69 outer membrane
protein, a dose in the range from about 1-100 microgrammes/dose typically
would be administered to a human, the actual amount depending on the
immunogenicity of the preparation in humans when applied to mucosal
For a substantially pure form of the B. pertussis filamentous
haemaglutinin, and the 50 KD C fragment of tetanus toxin, a typical dose
range would be of the order given above in respect of P.69 protein. In a
typical immunisation regime employing the antigenic preparations of the
present invention, the vaccine may be administered in several doses (e.g.
1-4), each dose containing 1-100 microgrammes of each antigen. The
immunisation regime may involve immunisation purely by the mucosal route
or by a combination of mucosal and parenteral immunisation. The dosage
will in general depend upon the immunogenicity of the different antigens
when applied to the respiratory or gastrointestinal tracts of humans.
Claim 1 of 16 Claims
What is claimed is:
1. A method for immunizing a host against tetanus infection, which method
consists of administering directly to a mucosal surface of said host, by
intranasal administration, in an amount effective to induce an immune
response in said surface, an acellular vaccine composition comprising:
(a) a mucosally immunogenically active substance consisting of the 50 kD C
fragment of tetanus toxin, or an immunoprotective fragment thereof having
the ability to bind eukaryotic cells possessing the trisialoganlioside G16
and the ability to elicit protection in mice against lethal challenge with
tetanus toxin, and
(b) a pharmaceutically acceptable carrier.
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