Note: Descriptions are shown in the official language in which they were submitted.
CA 02352617 2001-05-25
WO 00/30678 PCT/FR99/02913
_ 1 _
Multivalent T.d.Polio vaccine against at least
diphtheria, poliomyelitis and tetanus
The present invention relates to a novel
multivalent vaccine against at least diphtheria,
poliomyelitis and tetanus, intended to be used in
primary immunization and as an immunization booster in
an individual who has already undergone primary
immunization or is sensitized.
State of the art
Diphtheria is due to Corynebacterium
diphtheriae. The transmission is essentially direct via
the respiratory pathways. The clinical symptoms of the
respiratory form are a product of two mechanisms. The
proliferation of the bacteria at the portal of entry
determines the local symptomatology, conventionally
pharyngeal (pseudomembranous angina), sometimes
laryngeal (croup). The diffusion of the exotoxin of
certain strains is responsible for malignant angina
and/or for visceral complications (polyneuritis,
myocarditis, malignant Marfan syndrome). Many
serological studies, carried out recently in most
developed countries, have shown that, depending on age,
sex and local epidemic or immunization history, 20 to
800 of inhabitants are no longer, today, correctly
immunized; and that individuals over the age of 50 and
females are particularly vulnerable (Rappuoli et al.,
Vaccine 11, 576-577, 1993. Simonsen et al., Acta
Pathol. Microbiol. Immunol. Scand, 95, 225-231, 1987.
Wirz et al., Vaccine, 13, 771-773, 1995). This
vulnerability has been revealed over the last twenty
years by the sometimes dramatic resurgence of
diphtheria in developed countries (Galazka et al., Eur.
J. Epidemiol, 11, 107-117, 1995).
Tetanus is due to Clostridium tetani. The spore
form, which is essentially telluric, introduced by
breaching the organism (wound, bite/sting, burn or
minor lesion), gives rise to the vegetative form. This
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form secretes an endotoxin, tetanospasmin. Bacterial
lysis causes diffusion thereof. The toxin, which is
neurotropic, determines the symptomatology, i.e. an
initial trismus and muscle contractions. Without
treatment, the evolution is fatal through asphyxia or
syncope. Immunization with toxoid remains the only
riposte. The immunity acquired with primary
immunization decreases over time and requires boosters.
The absence or loss of the immunity with age has been
noted in the American population for decades, by
several serological investigations (Hilton et al., Ann.
Intern. Med., 115, 32-33, 1991). In Europe, serological
investigations have given equivalent results (Kjeldsen
et al., Scared. J. Infect. Dis., 20, 177-185, 1988). It
is therefore clear that many adults, particularly those
over 50 years old and women, have become, or have
always been, vulnerable to tetanus, and require
immunization with toxoid.
Poliomyelitis is due to the poliovirus,
belonging to the enterovirus group. Three serotypes
exist, named type l, type 2 and type 3. The
transmission is essentially fecal-oral, direct or
indirect, and sometimes oral-aral. The virus has an
intestinal, muscular, meningeal and nervous tropism.
The infection is, in general, inconspicuous or mild.
This "poliomyelitis infection" is immunizing, but
without cross-immunity between types, and is contagious
for several weeks. It gives rise exceptionally (10o to
to of infections depending on age and type) to
meningeal or paralytic (acute flaccid paralysis) forms.
This "poliomyelitis disease" is always serious since
there is no etiological curative treatment. When it is
not lethal through asphyxia (2 to 10o depending on age
and type), it is followed by disabling ventilatory or
muscular after-effects. Human exchanges, which are
increasingly numerous, between countries endemic for
poliomyelitis and countries free of poliomyelitis
expose the latter to the risks of importing and of
reintroducing wild-type strains. In the last few years,
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despite a high level of immunization coverage and a
very low worldwide incidence of the disease, imported
cases and epidemics have appeared in Europe, in North
America and in the Middle East (WHO, Releve
Epidemiologique Hebdomadaire [Weekly Epidemiological
Record], 29, 220-221, 1996). While waiting for the
disease to be eliminated, the many human exchanges
between the countries endemic for the disease and the
countries free of the disease therefore risk causing,
in any area of the world, outbreaks due to imported
wild-type strains . In order to reduce to a minimum the
propagation of the disease during exacerbations of this
type, it is important that all countries, including
those in which there are no longer any reports of a
single case of acute poliomyelitis, maintain a high
level of immunization coverage throughout the
population.
The diphtheria and tetanus vaccines, which were
developed by Ramon in the 1930s, are based on toxoids .
They can be obtained through the detoxifying action of
formaldehyde on a concentrate of Corynebacterium
diphtheriae or Clostridium tetani culture, and then
purification. The respective immunizing activities
thereof are assessed in vitro and in vivo. A
floculation assay measures the amount of toxoid,
expressed in floculation units (Lf) per dose (Lyng et
al., J. Biol. Stand., 15 27-37, 1987: J. Lyng.
Biologicals, 18, 11-17, 1990).
The tetanus toxoid is sometimes administered
alone. However, historically this toxoid has always
been combined, at least with the diphtheria toxoid. In
general, primary tetanus and diphtheria immunization is
carried out during the first year of life in 3 doses
(WHO, WHO/EP/GEN, 95.3, 1995). According to country, a
booster dose is administered during the second year
and/or between 4 and 10 years of age. Sometimes, a
booster is also carried out between 11 and 18 years of
age. In addition, since 1987 the WHO has recommended
immunizing women of child-bearing age in developing
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countries with tetanus toxoid, using 3 primary
immunization doses and then 1 booster dose 1 and 5
years afterwards.
The amount of tetanus toxoid (T) per
immunization dose varies, according to country, from 5
to 20 Lf for one dose of 0.5 ml as a primary
immunization or as a booster. The European Pharmacopeia
recommends an activity of at least 20 IU.
The most widely and the oldest administered
amount of diphtheria toxoid (D} per immunization dose
is that which exists in the pediatric primary
immunization combinations: it ranges from 12 to 50 Lf
for one dose of 0.5 ml (Galazka et al., Vaccine, 14,
845-857, 1996) .
I5 For an immunization booster, the decrease in
the amount of diphtheria toxoid (d) has now also become
generalized. The amount is generally about 1/10 of the
pediatric amount. It is thus set at 2 Lf for one dose
of 0.5 ml in the US and in Canada, and is recommended
as a booster from the age of 7 (Edsall et a1. , Am. J.
Hyg, 53, 283-295, 1951). In Europe, the amount of
toxoid and the youngest administration age change
according to country. The amount is not set because the
only condition imposed by the European Pharmacopeia
relates not to the amount but to the activity: it must
be at least 2 IU per dose of 0.5 ml.
For booster vaccines intended for an adult
population, the diphtheria toxoid is generally also
combined with the tetanus toxoid. It is known that this
combination slightly decreases the tolerance occurring
for the tetanus toxoid alone (Palmer et al., Br. Med.
J., 286, 624-626, 1983). In the United States, the
reports of undesirable events linked to the T and Td
vaccines administered to individuals over 7 years old
have recently been analyzed (Haber et al., ICAAC,
1996) . The Td vaccine exposed the individuals to local
reaction, dyspnea, loss of consciousness or a
convulsion more than the T vaccine did.
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With regard to poliomyelitis, two vaccines were
developed in the 1950s: the injectable vaccine (IPV),
which is inactivated with formaldehyde, developed by
Salk (Plotkin et al., E.A. Vaccines, ed. Raven Press,
1994), and the oral vaccine (OPV), which is a live
attenuated vaccine, developed by Sabin (Plotkin et al.,
above). The IPV has enabled all countries to eliminate
poliomyelitis, both wild-type and post-immunization
poliomyelitis (Murdin et al., Vaccine, 14, 735-746,
20 1996). The method of production and the composition of
the IPV vary according to country. The three types of
poliovirus are thus cultured on the VERO continuous
cell line, and then purified and inactivated with
formaldehyde. The respective immunizing activity
I5 thereof is assessed in vitro and in vivo. An ELISA
measures the viral antigen content, expressed in
international units: the WHO recommends, per dose of
0.5 ml, 40 IU, 8 IU and 32 IU (as determined by the
Sigmoid method), respectively, for the D antigens of
20 types 1, 2 and 3.
The IPV offers two pharmaceutical advantages:
it is stable and does not require any specific
logistic. It can sometimes be combined with other
antigens.
25 Simultaneous immunization during infancy
against poliomyelitis, tetanus and diphtheria has been
common practice for some years throughout the world,
indeed for decades in most developed countries. This
practice has greatly contributed to considerably
30 decreasing the number of cases and of deaths due to
these three diseases. However, they have not
disappeared. In developed countries, when they occur,
the later in life they do occur, the more serious they
are. Now, the immunity acquired by immunization
35 attenuates with time. Since they do not benefit from a
natural maintenance of this immunity, the teenagers and
adults of these countries become vulnerable again.
Maintaining the immunization coverage against each of
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the three diseases throughout life is, from now on, an
epidemiological obligation.
A need therefore exists for a novel vaccine
against at least diphtheria, poliomyelitis and tetanus,
which can be used in primary immunization and as an
immunization booster, which would aim to confer
protection against tetanus, poliomyelitis and
diphtheria, and/or to prolong protection conferred
initially during a primary immunization or a
sensitization, and which would minimize, on this
population, the undesirable effects induced by existing
vaccines.
The injectable pediatric vaccines of the type
TDPolio (T: tetanus toxoid; D: conventional dose of
diphtheria toxoid; Polio: inactivated type 1, 2 and 3
polioviruses), for example the D.T.Polio~ vaccine (PMsv
S.A., France), cannot satisfy this need. Specifically,
these types of vaccine are used mainly in pediatric
primary immunization. They lack aluminum salts. They
contain amounts of diphtheria toxoid which are too
high, about 100 Lf/ml, for example, and they trigger
undesirable reactions in adults (Bjorkholm et al., Eur.
J. Clin. Microb., 6, 637-640, 1987).
It is also not possible to envisage simply
modifying an existing TDPolio vaccine in order to
minimize the undesirable effects thereof on a
population having undergone primary immunization.
Specifically, a target of choice would, for example, be
to reduce the amount of diphtheria toxoid therein. In
this case, selecting the amounts of toxoid used
conventionally in a Td vaccine (which is the only
current reference), of the order of at least 4 Lf per
ml, for example, would not be envisaged, but rather
adding more thereof, probably more than 20 Lf/ml, for
example. In fact, in a conventional Td vaccine, the
diphtheria toxoid is always adjuvanted with an aluminum
salt, which reinforces the immunogenicity thereof. In a
TDPolio vaccine, the fact of not having any adjuvant
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implies reinforcing the toxoid dose with respect to
that used in a Td vaccine.
For the same reasons, simply combining an
existing vaccine of the injectable Td type, for example
the vaccinol~ or Td-Pur~ vaccines (Chiron-Behring GmbH,
Germany) or the Diftavax~ vaccine (PMsv S.A., France),
with a conventional vaccine against poliomyelitis
(PMsv, France), could not produce a satisfactory
solution either. Specifically, the choice of each
constituent in such a vaccine, and also the dose
thereof, are determinant in producing an optimal immune
response and minimizing the undesirable effects.
Thus, for example, if novel antigens (Polio)
are added to a Td. vaccine, the amount of aluminum
salts is then likely to be insufficient to play an
optimal adjuvant role. There would thus be a risk of
the immunogenicity of such a vaccine being decreased.
On the other hand, if the intention is to overcome this
problem by increasing the amount of aluminum salts,
there would be the risk, in parallel, of worsening the
undesirable reactions linked to these salts (1.2 to
3 mg per ml in the Td vaccines; Gupta et a1. , Vaccine,
13, 1263-1276, 1995) .
Similarly, if novel antigens (Polio) are added
to a Td vaccine, the relative load of each T or d
antigen is also decreased. There would thus be a risk
of the immunogenicity of such a vaccine also being
decreased. On the other hand, if the intention is to
overcome this problem by increasing the dose of each
antigen, in particular that of the diphtheria toxoid,
there would be a risk, in parallel, of worsening the
undesirable reactions linked to this toxoid (Bjorkholm
et al., Eur. J. Clin. Microb., 6, 637-640, 1987).
The choice of each constituent of a vaccine of
the type TdPolio, and also their dose, are therefore
difficult to determine, and cannot be easily deduced
from existing vaccines.
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In addition, there is no immunization
combination, against diphtheria, tetanus, poliomyelitis
and whooping cough, or even also against hepatitis A
and/or hepatitis B which is suitable for use as an
immunization booster in adults or teenagers.
The present invention is thus aimed toward
providing a vaccine having at least the basic Td Polio
combination, which, while differing from the previous
TDPolio, Td and Polio vaccines, has an immunogenicity
comparable to these vaccines and, in addition,
minimizes the undesirable effects thereof. The vaccine
according to the invention has the advantage that it
can be used in primary vaccination and in booster
immunization.
Summary of the invention
To this end, the invention relates to a vaccine
comprising:
- less than 1.2 mg per ml of aluminum salt,
expressed with respect to the A13+ atom,
- immunogenic antigens originating at least
from the poliovirus, from Corynebacterium
diphtheriae and from Clostridiurri tetani, and
- an amount of diphtheria toxoid used as an
immunogenic antigen of Corynebacterium
diphtheriae of between 4-16 Lf per ml.
The vaccine according to the present invention
can be used in primary immunization and as a booster
vaccine in a population which has undergone primary
vaccination or is sensitized. The Applicant has
demonstrated that the vaccine as defined above is
particularly suitable for use as an immunization
booster.
The Applicant has demonstrated, surprisingly,
that the vaccine as defined above makes it possible to
minimize the reactogenic and/or allergic effects
induced by the constituent antigens.
According to another subject, the present
invention relates to a method for immunizing against at
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least the poliovirus, Corynebacterium diphtheriae and
Clostridium tetani, comprising the administration of a
vaccine as defined above.
Finally, the invention also relates to a
pharmaceutical kit comprising at least 2 injectable
doses of a vaccine according to the present invention.
Detailed description of the invention
In the context of the present invention, the
expression "a population which has already undergone
primary immunization or is already sensitized" is
intended to mean adult, teenage or young individuals
having already been immunized against at least the
poliovirus, Corynebacterium diphtheriae and/or
Clostridium tetani, or individuals having already been
in contact with one at least of the poliovirus,
Corynebacterium diphtheriae and Clostridium tetani
microorganisms; the preferred population consisting of
teenagers and adults, and more particularly elderly
individuals.
Preferably, the origin of the Corynebacterium
diphtheriae and Clostridium tetani antigens is the
toxins thereof, which are detoxified with formaldehyde
and then purified. The techniques for detoxifying and
for purifying these toxins have been well known for
decades and are incorporated into the description of
the present invention by way of reference, such as
those described by Leong et al., Science, 220, 515-517,
1983; Ramon G., Ann. Inst. Pasteur, 38, 1-105, 1924; or
Raynaud et al., Ann. Inst. Pasteur, 96, 60-71, 1959; or
by Bizzini et al., Eur. J. Biochem., 17, 100-105, 1970,
for example. The detoxified analogs which can be
produced by genetic engineering are also included in
the present invention.
Similarly, the poliovirus antigens can simply
consist of one or more types of inactivated poliovirus
(see hereinafter) and/or of purified immunogenic
antigens of the poliovirus, such as those described by
Delpeyroux et al., 70, 1065-73, 1988; EP323861 (Pasteur
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Institut); EP85707 (Pasteur Institut); or in EP65924
(Pasteur Institut), for example.
In order to obtain inactivated polioviruses, it
is possible to culture them on VERO cell lines, purify
them and then detoxify them with formaldehyde, for
example. The techniques for culturing and for
detoxifying the poliovirus have been well known for
decades and are also incorporated into the description
of the present invention by way of reference, such as
those described in W09800167 (Connaught); Dulbecco,
Nature, 376, p.216, 1995; and Cohen, Acta Leiden, 56,
65-83, 1987, and by Salk, Dev. Biol. Stand., 47, 247-
55, 1981, for example.
Preferably, inactivated type 1 (MAHONEY
strain), 2 (MEF 1 strain) and 3 (Saukett strain)
polioviruses are used.
The amount of diphtheria toxoid used per
immunization dose as an immunogenic antigen of
Corynebacterium diphtheriae should be between 4 and
16 Lf per ml, preferably 6-14 Lf, in particular about
10 Lf per ml, for example. This amount makes it
possible to ensure optimal immunogenicity, while at the
same time minimizing the undesirable effects, such as
the reactogenic or allergic reactions to the antigens,
for example.
Similarly, the amount of tetanus toxoid per
immunization dose as immunogenic antigen of Clostridium
tetani is between 6 and 30 Lf per ml, preferably 8-
20 Lf, in particular about 10 Lf per dose, for example.
This amount makes it possible to ensure optimal
immunogenicity, while at the same time minimizing the
undesirable effects.
In order to measure the amount of diphtheria or
tetanus toxoid (Lf), the floculation assay described by
Lyng (J. Biol. Stand., 15, 27-37, 1987; or Biologicals,
18, 11-17, 1990), or the WHO (Manual for the production
and the control of vaccines, BLG/UNDP/77.1 and
BLG/UNDP/77.2) is used, taking into account, however,
that the purity of the toxoid used is related to
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1500-1800 Lf per mg of nitrogen for diphtheria, or
1000-1300 Lf per mg of nitrogen for tetanus.
By way of indication, in most manufacturings,
the purity of the purified diphtheria toxoid is
generally about 1700-1800 Lf per mg of nitrogen
(internal source). Similarly, for the tetanus toxoid,
this purity is about 1200-1400 Lf (internal source).
With regard to the inactivated polioviruses
used as immunogenic antigens, an immunization dose
according to the invention can comprise 60-120 IU/ml of
D antigen of the type 1 poliovirus, 8-30 IU/ml of D
antigen of the type 2 poliovirus and/or 16-80 IU/ml of
D antigen of the type 3 poliovirus, for example. The
International Units are determined according to the
SigmoYd method.
In a particular embodiment of the present
invention, the vaccine according to the invention can
comprise, in addition to the basic TdPolio combination
described above, 0.1 to 60 ~.g/ml of purified B.
pertussis antigens, preferably B. pertussis toxoid (PT)
and F-HA; 0.1 to 40 ~g/ml of the hepatitis B HBs
antigen; and/or 0.1 to 40 ~g/ml of inactivated
hepatitis A virus or an antigen thereof, for example.
The Bordetella pertussis toxin, including the
detoxified analogs produced by genetic engineering, can
be produced in different ways. For example, a B.
pertussis strain can be cultured according to
conventional methods (Sekura et al., J. Biol. Chem.,
258, 14647-14651, 1993), the toxoid can be isolated by
adsorbing the culture medium onto an Affi-Gel Blues
(BioRad Lab, US) column and then eluting it with a
solution rich in salts (for example, 0.75 M of
magnesium chloride), and then, after having removed the
salts, this eluent can then again be adsorbed onto a
fetuin-Sepharose affinity column (composed of fetuin
linked to cyanogen bromide) and then eluted with a 4M
solution of a magnesium salt. The B. pertussis toxin
can then be detoxified using glutaraldehyde according
to a modified method from Munoz et a1. (Infect. Immun.,
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33, 820-826, 1981), as described in patent application
PCT/EP97/05378 (PMsv). Many other methods are also
available to those skilled in the art, such as those of
Irons et a1. (Biochem. Biophys. Acta. 580, 175-185,
1979), or those described in patents US 4705686 and
EP 336736, and are incorporated into the description of
the present invention by way of reference.
The F-HA can be purified from the culture
supernatant essentially by the process described by
Cowell et a1. (Infect. And Immun., 41, 1, 313-320,
1983). Growth promoters, such as methylated beta-
cyclodextrins, can be used to increase the F-HA yield
in the supernatant. The culture supernatant is loaded
onto a hydroxyapatite column. The F-HA is adsorbed onto
the column, but not the PT. The column is very
thoroughly washed with Triton X-100 in order to
eliminate the endotoxin. The F-HA is then eluted using
0.5M NaCl in O.1M sodium phosphate and, if necessary,
passed over a fetuin-Sepharose column in order to
eliminate the residual PT. A further purification can
comprise being passed through a Sepharose CL-6B column.
An alternative can comprise purifying the F-HA through
the use of monoclonal antibodies directed against the
antigen, in which the antibodies are attached to a
CNBr-activated affinity column. The F-HA can also be
purified using chromatography on perlite, as described
in patent EP 336 736.
A method suitable for purifying the F-HA is
also described in Example 3 of patent EP 0242 302. In
the context of the present invention, the F-HA is
prepared according to this method.
A suspension of the hepatitis B antigen can be
produced according to the method described in patent
EP 273811 (Pasteur Vaccins), in which antigenic
hepatitis B surface particles are produced by
expression using a culture of CHO cells transfected
with a plasmid carrying the HBsAg gene so as to release
the antigenic surface particles into the culture
medium. Other techniques are well known to those
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skilled in the art, and are incorporated into the
description of the present invention by way of
reference, such as those described in EP 864649, UE
56711 or IE 48665, for example.
Similarly, an inactivated hepatitis A virus can
be produced according to the protocol of Flehmig et a1.
(Viral Hepatitis and Liver Disease, 87-90, 1988: J.
Med. Virol., 22, 7-16, 1987). Other techniques are well
known to those skilled in the art, and are incorporated
into the description of the present invention by way of
reference, such as those described by Wang et al.,
Vaccine, 23, 835-40, 1995; Shevtsova et al., Zh
Mikrobiol Epidemiol Immunobiol., 2, 55-90, 1995; and
Richtmann et al., J. Med. Virol., 48, 147-50, 1996; or
in EP 199480; IE 48399; or IE 50191, for example.
The vaccine according to the invention
comprises one or more adjuvants chosen from adjuvants
recognized as such, in particular all aluminum salts,
such as aluminum phosphates and hydroxides; N-
acetylmuramyl-L-alnanyl-D-isoglutamyl-L-alanine-2-[1,2-
dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy)) (see
Sanchez-Pescador et al., J. Immu., 141, 1720-1727,
1988); molecules derived from Quillaja saponaria, such
as Stimulon~ (Aquila, US); Iscoms~ (CSL Ltd., US); all
molecules based on cholesterol and analogs, such as
DC Chol~ (Targeted Genetics); the glycolipid Bay 81005~
(Bayer, Germany); Leishmania brasiliensis antigens,
such as LeIF (technical name), available from Corixa
Corp. (US), and polymers of the polyphosphazene family,
such as Adjumer (technical name), available from the
"Virus Research Institute" (US), for example. The
vaccine according to the present invention contains
less than 1.2 mg/ml, preferably 0.70 mg/ml, of aluminum
salt, expressed with respect to the A13+ atom.
It may be noted that, with respect to
conventional TDPolio vaccines, such as D.T. Polio
(PMsv, France), the present invention proposes, for the
first time, the addition of an adjuvant, and in
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particular an aluminum salt such as aluminum hydroxide
for example.
Against all expectations, the vaccine according
to the present invention can comprise an amount of
aluminum salt which is lower than those encountered in
all the conventional Td vaccines, such as Td-Pur~ or
Diftavax°, whereas it might have been considered
necessary to increase the load thereof subsequent to
the addition of the inactivated polioviruses. A vaccine
according to the invention comprises less than
1.2 mg/ml of an aluminum salt, preferably less than
0.8 mg/ml. The amount of aluminum salt is always
expressed with respect to the aluminum atom (A13+),
which corresponds to the only method which can be used
in the vaccines field. All the amounts of aluminum
salts to which reference is made in the present
application are therefore expressed in this way.
A vaccine according to the present invention
can comprise other constituents, for instance
preserving agents such as 2-phenoxyethanol and/or
formaldehyde, etc:, for example.
The various formulations selected can be one of
those described above, for example, in particular those
comprising other antigens originating from Bordetella
pertussis, from hepatitis A or from hepatitis B.
The vaccine according to the invention can be
in the form of an injectable suspension, slightly
opalescent due to the presence of an insoluble aluminum
salt. The immunization dose is preferably about 0.5 ml,
contained in a prefilled glass syringe. The
administration is carried out via the deep subcutaneous
route or via the intramuscular route, preferably via
the intramuscular route, for example in one of the
deltoid muscles.
In a particular embodiment of the present use,
several doses of vaccine are intended to be injected
separately into the same individual in a period of time
of between 10 days and 3 months, so as to promote an
optimal immune reaction and so as to minimize the
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reactogenic and/or allergic effects of these antigens.
For a primary immunization, the vaccine according to
the present invention is preferably administered in 3
doses, the first two doses being administered 1 to 2
months apart, the third dose being separated from the
second injection by a period of 6 to 12 months. For a
booster immunization which can be used on a population
which has undergone primary immunization or is
sensitized, the vaccine according to the present
invention is administered in one dose, or 2 doses
separated by at least 1 month.
The vaccine according to the invention can
advantageously be used in a booster immunization in
which the primary immunization has been carried out
using an oral vaccine against poliomyelitis.
Preferably, a dose of 0.5 ml comprising 4 to
15 Lf/ml of diphtheria toxoid, preferably 10 Lf/ml; 6
to 30 Lf/ml of tetanus toxoid, preferably 20 Lf/ml and
preferably 40 IU, 8 IU and 32 IU (Sigmoid method),
respectively, of the D antigens of the polioviruses 1,
2 and 3 are used, both in primary immunization and in
booster immunization. The amount of aluminum salt
present is preferably 0.70 mg/ml, expressed with
respect to the A13+ atom.
The vaccine according to the invention makes it
possible to decrease the undesirable effects induced by
the existing TDPolio and Td vaccines when they are
injected. It is thus possible to observe a decrease in
local reactions such as numbness of the limbs,
sweating, fever, pain associated with red blotches,
nodules, indurations and/or ecchymoses, and a decrease
in dyspnea, losses of consciousness or convulsions, for
example. These reactions can be classified among
allergic, or even reactogenic, reactions to the
antigens and/or to certain other compounds of the
vaccine, such as the aluminum salt. The Applicant has
thus shown that the vaccine according to the present
invention is particularly suitable for booster
immunization, in particular in adult individuals.
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The present invention is described in greater
detail in the examples given hereinafter. The
percentages are given by weight unless otherwise
indicated. It goes without saying, however, that these
examples are given by way of illustration of the
subject of the invention, of which they in no way
constitute a limitation.
Example 1 Immunogenicity of the TdPolio with various
doses of diphtheria toxoid
Diphtheria toxoid (d) is prepared by culturing
the IM 1514 N3S strain in an IMD medium for 15 to 18 h
at 36°C, centrifuging the medium, clarifying it,
concentrating it by ultrafiltration, detoxifying it at
37°C for 4 weeks in the presence of 6/1000 of formalin,
and then purifying the toxin to reach a purity of about
1700 Lf per mg of nitrogen.
In parallel, the tetanus toxoid (T) is prepared
by culturing the Harvard strain No. 49205 IM 1472C in a
Massachusetts medium at 35°C for 4 days, adding NaCl
and sodium citrate thereto, centrifuging the medium,
concentrating it by ultrafiltration, detoxifying it for
2 weeks at 35°C in the presence of 5.5/1000 of formalin
and 5 g/1 of sodium bicarbonate, and then purifying the
toxin to reach a purity of about 1200 Lf per mg of
nitrogen.
Similarly, the inactivated type 1 (MAHONEY
strain), 2 (MEF 1 strain) and 3 (Saukett strain)
polioviruses are prepared according to the Salk method.
The 5 immunogenic antigens above are then mixed
with aluminum hydroxide, 2-phenoxyethanol,
formaldehyde, Hanks medium 199 without phenol red, and
water. To do this, the aluminum gel is sterilized in
the presence of water, the pH is adjusted between 5.6
and 6, the PDT (2 or 8 Lf/dose) and PTT (10 Lf/dose)
and the Hanks medium 199 are added sequentially, the pH
is adjusted to 6-6.9, the three types of poliovirus are
added, and then the 2-phenoxyethanol and formaldehyde
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are added and, optionally, the pH is adjusted between
6.8 and 7.
The final product of 0.5 ml will be used as
TdPolio vaccine and contains a minimum of 2 IU of
diphtheria toxoid, a minimum of 20 IU of purified
tetanus toxoid, 40 IU, 8 IU and 32 IU of D antigen
(values as measured by the Sigmoid method),
respectively, for the inactivated type 1, 2 and 3
polioviruses, 0.35 mg of aluminum hydroxide, expressed
with respect to the aluminum atom A13+, 2.5 ~,1 of 2-
phenoxyethanol and 12.5 dug of formaldehyde, the rest
consisting of Hanks medium 199 without phenol red, and
of water.
The main objective of a phase I study has been
to validate the clinical and biological tolerance of
the first administration of an adsorbed TdPolio
vaccine. With this aim, 31 healthy volunteer adults
were recruited. Three batches of the vaccine, of 0.5 ml
each, were used. They differed only in the amount of
purified diphtheria toxoid described above: 2 Lf, 5 Lf
and 8 Lf per dose. They were attributed sequentially in
a proportion of 10 individuals per batch. Each
individual was immunized with one dose from one batch,
injected into a deltoid muscle.
No general reaction was reported in the
immunized groups, with the batches at 2, 5 and 8 Lf. At
least one local reaction was reported during the first
week in 8 individuals of the group immunized with the
batch at 2 Lf of PDT, in 6 individuals of the group
immunized with the batch at 5 Lf of PDT and in 8
individuals of the group immunized with the batch at
8 Lf of PDT; it always involved pain associated with
some red blotches, nodules, indurations and/or
ecchymoses. All the reactions disappeared without
treatment and did not alter the every-day life of the
individuals. No reaction occurred beyond the first
week. No serious undesirable event was declared by the
investigators.
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The secondary objective of this study was to
evaluate the immunogenicity of the first three batches
of the adsorbed vaccine. The results show that the
immune response to the five antigens was excellent for
the three batches. Despite high initial titers due to
the young age of the individuals and to recent
immunizations, a booster effect was obtained for each
antigen.
Example 2 Immunogenicity in young adults
The immunogenicity and the innocuity of a
TdPolio vaccine was determined during a clinical trial
in 508 young adults.
For each individual, a dose of 0.5 rnl of a Td
(Diftavax~) or TdPolio vaccine was injected into the
left deltoid muscle, and a dose of 0.5 ml of the
vaccine against poliomyelitis (VPI~, PMsv) or a placebo
was injected into the right deltoid muscle. A dose of
adsorbed Td vaccine contains tetanus toxoid (activity
>_ 20 IU), diphtheria toxoid (activity >_ 2 IU) and
aluminum hydroxide (_< 1.25 mg). The IPV vaccine
contains the D antigens of the type 1 (40 IU), 2 (8 IU)
and 3 (32 IU) polioviruses. The placebo has the same
composition as the IPV with the only difference being
that it does not contain any D antigens. The TdPolio
vaccine has the same composition as that described in
example 1 (5 Lf of PDT per dose).
The anti-diphtheria, tetanus and poliomyelitis
antibodies were determined from the sera of the
individuals, using the ELISA assay.
The results show that, before immunization,
virtually all the individuals were seropositive with
regard to the PDT (99.2a have a titer >_ 0.01 IU/ml:
possible protection; and 92.60 have a titer
> 0.1 IU/ml: assured protection). One month after
immunization with the TdPolio, 99.60 of the individuals
had an antibody titer ? 0.1 IU/ml and 82.40 had a titer
>_ 1 IU/ml (long term protection). In total, 17.40 of
the individuals showed seroconversion.
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Similarly, the majority of the individuals were
seropositive with regard to the tetanus toxoid (99.60
having a titer >_ 0.01 IU/ml: possible protection; and
98.40 having a titer >_ 0.1 IU/ml: assured protection).
One month after immunization with the TdPolio, 100% of
the individuals have an antibody titer >_ 0.1 IU/ml, and
99.20 have a titer >_ 1 IU/ml (long term protection). In
total, 25.20 of the individuals showed seroconversion.
With regard to protection against
poliomyelitis, the majority of the individuals were
seropositive with regard to the three types of
poliovirus. In total, 99.20 of the individuals had an
antibody titer >_ 5 for type 1, 100% for type 2 and
97.60 for type 3. One month after immunization with the
TdPolio, 1000 of the individuals have a titer >_ 5 for
all the poliovirus types, with a minimum titer of 120
for type 1, 160 for type 2 and 80 for type 3. In total,
63.10 of the individuals already having a high antibody
titer show seroconversion.
When the results obtained with the TdPolio and
those obtained when combining the Td and VPI° reference
vaccines are compared, an equivalent antibody response
i s c~hi-a i nc~ci _
Example 3 Innocuity and tolerability in young adults
The innocuity and tolerability of a TdPolio
vaccine were determined in 1742 young adults. Each one
of these individuals received a dose of 0.5 ml of the
vaccine shown in example 1 (PDT: 5 Lf per dose),
injected into the left deltoid muscle. The effects were
evaluated 15 minutes after immunization. The local,
regional and systemic effects were evaluated during the
month following immunization.
The common immediate effect is the appearance
of a red blotch at the point of injection (0.340 of the
individuals ) and pain ( 0 . 11 o ) .
The common local effects during the months
following immunization are as follows for 660 of the
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individuals: pain (64.41%), red blotch at the point of
injection (9.13%) and subcutaneous nodules (3.33%).
These undesirable effects appear on the first 3 days
following immunization and last 2 to 3 days. 0.860 of
the individuals also reported events of edema, of
inflammation, of migraine, of numbness of the arm, of
involuntary muscle contractions and paraesthesia.
The common systemic effects during the month
following immunization are as follows for 18% of the
individuals: headaches (10.50), nausea or vomiting
(2.75%) and malaise (2.410). No event of generalized
urticaria or itching was reported. These events appear
in the first 3 days following immunization and last 2
to 3 days. Only 0.230 of the individuals have a
temperature which exceeds 40°C during the first 3 days
following immunization.
Example 4 Innocuity and tolerability of the TdPolio
versus Td + Polio in young individuals
The TdPolio vaccine described in example 2
shows excellent tolerability during the trial on 508
young adults (example 2). The results are, moreover,
comparable to those obtained during the trial described
in example 3.
In subtracting the percentage of individuals
having had at least one local or regional sign at the
site of injection of the placebo (14.80) from those
observed at the site of injection of the Polio vaccine
(36.50), and then adding this percentage to that
observed at the site of injection of the Td vaccine
(66.70), a difference of approximately 8o in the
appearance of undesirable events (88.40 vs 80.5%) is
observed. The TdPolio vaccine is therefore tolerated
better than the simultaneous administration of the
existing vaccines.
Example 5 Immunogenicity in individuals over 40 years
old
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The immunogenicity and innocuity of a TdPolio
vaccine were determined during a clinical trial on 113
individuals over 40 years old (40 to 78 years old). All
the individuals had received primary immunization (3
doses in one year) against diphtheria, tetanus and
poliomyelitis, the last immunization dating back 32
years (minimum 15 years), 28 years (minimum 10 years)
and 28 years (minimum 10 years), respectively.
The individuals received an injection of a dose
of 0.5 ml of the TdPolio described in example 1 (PDT
5 Lf per dose) in the right deltoid muscle, and 28 days
later, a second injection in the left muscle. The anti-
diphtheria, tetanus and poliomyelitis antibodies were
evaluated from the sera of the individuals, using the
ELISA assay.
The results show that only 500 of the
individuals were initially protected against tetanus,
thus demonstrating the decline in the immunity. 830 of
the individuals were initially seropositive against the
type 1, 2 and 3 polioviruses. On day 28 following the
first immunization, the proportion of individuals
protected against diphtheria, tetanus and the type 1, 2
and 3 polioviruses had risen to 80 . 5%, 97 . 3 o and 100 0,
respectively. On day 56 following the administration of
the second dose, the proportion of individuals
protected against diphtheria, tetanus and the type l, 2
and 3 polioviruses had risen to 93.70, 1000 and 1000,
respectively.
Example 6 Innocuity and tolerability of the TdPolio
versus Td + Polio in individuals over 40
years old
The innocuity and tolerability of the TdPolio
vaccine described in example 5 were evaluated. The
results show a tolerability profile similar to that
obtained with the Td + Polio combination. However, the
:o
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TdPolio vaccine induces fewer serious undesirable
events (less than 1.8 per 1000 injections).
Example 7 d.T.Polio.PT.F-HA booster vaccine for adults
An immunization suspension of 0.5 ml is
prepared, with or without- preserving agent; consisting
of 5 Lf of diphtheria toxoid (having a purity of
1700 Lf per mg of nitrogen), 5 Lf of tetanus toxoid
(having a purity of 1200 Lf per mg of nitrogen), 40 IU,
8 IU and 32 IU of D antigen (values as determined by
the Sigmoid method) respectively, for the inactivated
type 1, 2 and 3 polioviruses, 6 ~g/ml of purified
Bordetella pertussis toxoid (PT) and 6 ~g/ml of
Bordetella pertussis F-HA, and 0.35 mg of aluminum
hydroxide, the rest consisting of Hanks medium 199
without phenol red, and of water.
2.5 ~l of 2-phenoxyethanol and 12.5 ~g of
formaldehyde can also be added as a preserving agent.
The diphtheria and tetanus toxoids, and also
the inactivated polioviruses, were prepared as
described in example 1.
The B. pertussis toxoid is prepared according
to the method of Sekura et a1. (J. Biol. Chem., 258,
14647-14651, 1993), detoxified according to a modified
protocol from Munoz et a1. (Infect. Immun., 33, 820-
826, 1981), and then preadsorbed on an aluminum gel.
The B. pertussis F-HA is prepared using the method
described in example 3 of EP 0242 302, and then
preadsorbed on an aluminum salt.
The immunogenic antigens above are then mixed
with aluminum hydroxide and water, and where
appropriate, with the preserving agents. To do this,
the aluminum gel is sterilized in the presence of
water, the pH is adjusted between 5.6 and 6, the PDT,
the PTT and the B, pertussis PT and F-HA are added
sequentially, the pH is adjusted to 6.8-7, the Hanks
medium 199 and the three types of poliovirus are added,
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and then, where appropriate, the 2-phenoxyethanol and
the formaldehyde are added.
Example 8 d.T.Polio.PT.F-HA.HBs booster vaccine for
adults
An immunization suspension of 0.5 m1 is
prepared, without preserving agent, consisting of 5 Lf
of diphtheria toxoid (having a purity of 1700 Lf per mg
of nitrogen), 5 Lf of tetanus toxoid (having a purity
of 1200 Lf per mg of nitrogen), 40 IU, 8 IU and 32 IU
of D antigen, respectively, for the inactivated type 1,
2 and 3 polioviruses, 6 ~,g/ml of purified Bordetella
pertussis toxoid (PT), 6 ~,g/ml of B. pertussis F-HA,
5 ~g/ml of hepatitis B HBs antigen and 0.35 mg of
aluminum hydroxide, the rest consisting of Hanks medium
199 without phenol red, and of water.
The diphtheria and tetanus toxoids, and also
the inactivated polioviruses, were prepared as
described in example 1. The B. pertussis PT and F-HA
were prepared as described in example 7.
The HBs antigen was prepared according to the
method described in patent EP 273 811 (Pasteur
Vaccins). It is stabilized by preadsorption on an
aluminum gel.
The immunogenic antigens above are then mixed
with aluminum hydroxide and water. To do this, the
aluminum gel is sterilized in the presence of water,
the pH is adjusted between 5.6 and 6, the PDT, the PTT
and the B. pertussis PT and F-HA are added
sequentially, the pH is adjusted to 6.8-7, the Hanks
medium 199 and the three types of poliovirus are added,
the pH is adjusted to 6.8 if necessary and the HBs is
added.
Example 9 d.T.Polio.PT.F-HA.HBs.HA booster vaccine for
adults
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An immunization suspension of 0.5 ml is
prepared, without preserving agent, consisting of 5 Lf
of diphtheria toxoid (having a purity of 1700 Lf per mg
of nitrogen), 5 Lf of tetanus toxoid (having a purity
of 1200 Lf per rng of nitrogen), 40 IU, 8 IU and 32 IU
of D antigen, respectively, for the inactivated type 1,
2 and 3 polioviruses, 6 ~,g/ml of purified Bordetella
pertussis toxoid (PT), 6 ~g/ml of B. pertussis F-HA,
5 ~g/ml of hepatitis B HBs antigen, 5 ~,g/ml of
inactivated hepatitis A virus and 0.35 mg of aluminum
hydroxide, the rest consisting of Hanks medium 199
without phenol red, and of water.
The diphtheria and tetanus toxoids, and also
the inactive polioviruses, were prepared as described
in example 1. The B, pertussis PT and F-HA were
prepared as described in example 7. The HBs antigen was
prepared as described in example 8.
The inactivated hepatitis A virus is prepared
according to the method of Flehmig et a1. (above).
The immunogenic antigens above are then mixed
with aluminum hydroxide, the preserving agents and
water. To do this, the aluminum gel is sterilized in
the presence of water, the pH is adjusted between 5.6
and 6, the PDT, the PTT and the B. pertussis PT and
F-HA are added sequentially, the pH is adjusted to
6.8-7, the Hanks medium 199 and the three types of
poliovirus are added, the pH is adjusted to 6.8 if
necessary, and then the HBs and the inactivated
hepatitis A virus are added sequentially.
