Note: Descriptions are shown in the official language in which they were submitted.
WO 95/05194 2168885 PCT/GB94/01646
VACCINE COMPOSITIONS
The present invention relates to vaccine compositions
for delivery to mucosal surfaces, and to a method of
inducing, in a mammal, an immune response to an antigen by
delivering the antigen to a mucosal surface of the mammal.
More particularly , the present invention relates to
vaccine compositions for inoculating a mammal such as a
human against picornavirus infection and particularly
Hepatitis A infection.
Hepatitis A is an acute disease caused by infection
with a small picornavirus closely related to the
poliovirus. Infection is spread by the faecal/oral route
and consequently the disease in endemic in areas where
hygiene and sanitation standards are low. The risk of
travellers to developing countries acquiring Hepatitis A is
far greater than that of contracting typhoid and cholera
(40 and 800 times respectively).
The virus itself is not directly cytopathic. The
liver damage resulting from Hepatitis A virus (HAV)
infection arises from destruction of virally infected cells
by the host's cytotoxic T-lymphocytes. There is only a
single serotype of HAV and infection results in long-term
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immunity, characteristics that are ideal for developing
Hepatitis A prophylaxis. Protection is mediated by
neutralising antibodies that prevent entry of hepatitis A
virus into hepatocytes. Passive immunisation with purified
human serum y-globulin provides short term protection
against the disease and until recently this was the only
means of preventing hepatitis A.
In recent years, HAV vaccines have been developed but
development has focused on inactivated and live attenuated
vaccines. Both types of vaccines are prepared from HAV
propagated in tissue culture cells. HAV replication is
slow and the majority of the virus remains cell associated,
and consequently the viral yields are low and relatively
commercially unattractive. The problem of low viral yield
could be overcome by using recombinant techniques which
allow for the production of large quantities of proteins.
However, it is important to ensure correct processing and
folding of HAV proteins because the known neutralising
epitopes are conformationally dependent. It has proved
difficult so far to obtain recombinant HAV antigens that
elicit appropriate immune responses.
One recombinant HAV antigen that has proved successful
in inducing protection against HAV when injected
parenterally is the HAV capsid preparation developed by
American Biogenetic Sciences. American Biogenetic Sciences
have succeeded in producing empty HAV capsids in eucaryotic
cells using vaccinia and baculovirus expression vectors.
The recombinant capsids are recognised by neutralising
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monoclonal antibodies, induce protection against HAV in
chimpanzees when injected parenterally and are produced in
considerably larger quantities than that obtained by
conventional means. The HAV capsids are disclosed in
International Patent Application W0-A-9301279 .
A disadvantage with many vaccination regimens is that
it is frequently necessary to administer the vaccine
composition by means of injection, a factor which has a
potential deterrent effect to many people, particularly
when follow-up or booster injections are required to
complete a course of treatment. One way of overcoming this
problem would be to administer the vaccine composition to
the oral or nasal mucosa, but although immunisation by the
oral or intranasal route has been explored with certain
other antigens, it has been found usually to be less
effective in evoking serum antibodies than parenteral
immunisation. For example, the article by M.H. Sjogren et
al, Vaccine, Vol. 10, Suppi. 1, S135-S137, 1992, describes
the administration of a live attenuated hepatitis A vaccine
by either the oral route or the intramuscular route.
Whereas intramuscular administration elicited a good serum
antibody response, an antibody response to oral
administration was not dbserved at any dose.
The fact that there are very few mucosal vaccines
commercially available indicates that there are problems
with developing such vaccines. May non-living soluble
antigens, particularly those used traditionally by
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immunologists, such as ovalbumin (OVA) and Keyhole Limpet
Haemocyanin (KLH) are poor mucosal immunogens. Large doses
of such antigens are necessary to induce any responses but
large doses can also cause tolerance in the individual to
subsequent parenteral exposure to antigen, a condition
known as Oral Tolerance. Although some microbial
components such as the cholera toxin (CT) or E.coli heat
labile toxin (LT) or the non-toxic binding portions of
these toxins (CT-B and LT-B) have been found to be potent
mucosal immunogens eliciting strong secretory and
circulating antibodies, the reasons why such molecules are
good mucosal immunogens has not been fully elucidated. One
property that may be important is the ability of these
molecules to bind to mucosal epithelial cells via certain
surface receptors, although it has been found in studies by
others that there is not necessarily a correlation between
the ability of an antigen to bind to eucaryotic cells and
its mucosal immunogenicity. In the present case, it is not
known, at the molecular or cellular level, how HAV enters
the body, nor is it known whether specific receptors are
involved.
Thus, as far as we are aware, there is currently no
way of predicting with any certainty whether a given
antigen will possess good mucosal immunogenicity.
It has now been found that the recombinant empty HAV
capsid referred to hereinabove, when administered
mucosally, and in particular intranasally, is efficient at
inducing serum anti-HAV antibodies. Thus, when the HAV
WO 95/05194 21~ 8 1 885 PCT/GB94/01646
capsid preparation was administered intranasally, following
first and second booster doses, seroconversion to anti-HAV
was observed in the majority of animals, and this compared
favourably with the administration of the antigen in the
presence of an alum adjuvant by the subcutaneous route.
Accordingly, in a first aspect, the invention provides
the use of Hepatitis A virus capsid, or mucosally
immunogenic fragments or epitopes thereof, for the
manufacture of a mucosal vaccine composition for
administration to a mucosal surface of a patient to induce
the production of serum Immunoglobulin G antibody against
Hepatitis A virus.
In a second aspect, the invention provides a vaccine
composition for application to a mucosal surface, the
composition comprising Hepatitis A virus capsid, or a
mucosally immunogenic fragment or epitope thereof, and a
pharmaceutically acceptable carrier.
In a still further aspect of the invention, there is
provided a method of inducing the production of serum
Immunoglobulin G antibody against Hepatitis A virus in a
host such as a mammal (eg. human), which method comprises
administering an effective amount of a Hepatitis A virus
capsid antigen, or a mucosally immunogenic fragment or
epitope thereof, directly to a mucosal surface in the host.
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
(eg. nasal and bronchial) mucosa.
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Where the composition is intended for delivery to the
respiratory (eg. 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 eg. 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 vaccine compositions of the
present invention may also take the form of compositions
intended to deliver the antigen to mucosal surfaces in the
gastro intestinal tract. Such compositions can be provided
with means for preventing degradation of the antigens by
the gastric juices, for example by encasing the vaccine
preparation in a capsule within a protective matrix or
coating of known type.
The quantity of Hepatitis virus A capsid administered
to the patient typically is selected such that it is non-
toxic to the patient at concentrations employed to elicit
an immune response. For example, the concentration of
capsid administered may lie in the range 0.1mg to 100mg per
kg/host.
The invention will now be illustrated in more detail
by reference to the specific embodiments described in the
following examples, and illustrated in the accompanying
drawings. The examples are intended to be purely
illustrative of the invention and are not intended to limit
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its scope in any way.
BRIEF DESCRIPTION OF THS FIGURES
Figure 1 illustrates the individual and mean serum
anti-HAV titres following three doses of HAV capsids by
each of the intranasal, oral and subcutaneous routes.
A sample of recombinant HAV capsids was obtained from
American Biogenetic Sciences, Reyniers Germ Free Building,
P.O. Box 1001 Notre Dame, Indianna 46556, USA. The salTtple
was prepared from vaccinia-HAV infected Vero cells. Cells
were lysed with NP40 and then extracted with
trichlorotrifluroethane. The aqueous phase was
concentrated and then chromatofocussed on Biogel* A-15
column. Fractions containing empty capsids were pooled and
concentrated. Formalin was added to inactivate any
remaining vaccinia virus. The content of HAV capsids in
the sample as given below is expressed in terms of ELISA
units (EU). The EU values have been standardised on a
sample of Hepatitis A virus obtained from SmithKline
Beecham. The sample received contained 52 ELISA units (EU)
HAV capsids per l. The protein content of the sample was
30 mg/ml of which 100ng/=ml was estimated to be HAV antigen.
Recombinant HAV capsids of the aforementioned type can
be prepared in accordance with the methods as set out in
International Patent Application W0-A-9301279
(PCT/US92/05714).
* r_radE-tna: k
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Mice were immunised orally and intranasally (I/N) with
different doses of HAV. A small quantity (1 ugm) of
cholera toxin (CT) was included in the material given to
some groups of mice to act as an adjuvant. CT was used
because it is the most potent mucosal adjuvant known. A
separate group of mice were immunised parenterally with HAV
adsorbed to aluminium hydroxide as an adjuvant as a
positive control. The groups were as set out in Table 1
below:
TABLE 1
Group Number Dose Adjuvants
of Elisa
Mice Units
(EU)
1. Parenteral 5 250 x 3 Alum
2. Parenteral Control 5 - Alum
3. Oral 10 500 x 3 -
4. Oral 10 250 x 3 -
5. Oral 10 125 x 3 -
6. Oral 10 250 x 3 CTa
7. Oral Control 5 - CT
8. I/Nb 10 -
9. I/N 10 500 x 3 -
10.I/N 10 250 x 3 -
11.I/N 10 125 x 3 CT
12.I/N Control 5 250 x 3 CT
aCT, Cholerae toxin
(1ug/dose)
bI/N, intranasal
Day Procedure
0 Primary immunisation
20 Sample bleed
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24 lst booster immunisation
31 Bleed, gut and nasal washes
47 2nd booster immunisation
54 Sample bleed
The immunisation was carried out by the method set out
in Table 2 below:
TABLE 2
IMMTNISATION DETAILS
Route Volume Diluent Apparatus Anaesthetic
Delivered (Halothane)
(uIl
Intranasal 30 PBS micro iight
pipette
Oral 200 5% gavage light
Bicarbonate needle
sol.
Subcutaneous 100 PBS needle nil
Anti-HAV responses were analysed using a capture ELISA
technique as follows. Human convalescent polyclonal serum
from an individual with known Hepatitis A was coated on 96
well plastic plates. The polyclonal serum captures HAV
capsids binding them to the plate. Mouse serum was then
incubated with the HAV capsid bound plates and the mouse
serum reactivity to HAV determined using labelled anti-
mouse antibodies. The protocol for the assay was as
follows:
PROTOCOL ( All volumes 50 uL/well unless otherwise stated)
1) Coat Costar EIA plates (Cat no: 3590) overnight, 4 C,
with 1:25000 human capture antibody diluted in PBS.
2) Wash plate x3 with phosphate buffered saline/Tween*
(0.05%) (PBST).
* tr. ade-m~.r. k
WO 95/05194 PCT/GB94/01646
3) Block lhr, 37 C, 1%BSA(Sigma, Cat no: A7888) in
PBST ( 200}il/well ) .
4) Wash plate x3 PBST.
5) Coat plates with sample containing HAV (0.1EU/pl) in
PBST (0.1$BSA), 2-3 hours 37 C.
6) Wash plate x3 PBST.
7) Incubate plate with mouse serum diluted PBST, 2-3
hours, 37 C.
8) Wash plate x3 PBST.
9) Incubate, 1-2 hours, 37 C, with anti-mouse IgG, 1:1000
(goat, Sigma, Cat No: B7022) in PBST.
10) Wash plate x3 PBST.
11) Incubate,1-2 hours, 37 C, with Streptavidin-peroxidase
1:1000 (Dako, Cat No: P397) in PBST.
12) Wash plate x3 PBST.
13) Add substrate, OPD in phosphate-citrate buffer (Sigma,
Cat no: P8287), incubate for up to 30 mins 37 C.
14) Read colour development after stopping substrate
reaction ( 3MHZS04) .
Human sera and HAV capsid sample supplied by ABS.
Optimum conditions of capture Antibody and HAV capsid
sample were selected to minimise the S/N ratio.
Problems of high background noise, attributed to reactivity
of the mouse serum against the human sera capture antibody
were encountered. Reduction of the background was obtained
by diluting out the capture antibody, beyond the dilution
recommended by ABS.
Similar results for capsid capture were obtained using,
1:1000-1:25000 dilutions of the capture Antibody.
plf
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The serum IgG anti-HAV responses were as shown in
Table 3 below:
TABLE 3
Table 3. Mean serum IgG anti-HAV response following a
primary and booster immunisations
Titre
Intranasal Oral Sub/cut
Dose 125 250 500 250+CT 125 250 500 250+CT 250+Alum
1 <50 <50 50 6000 <50 <50 <50 <50 6000
2 <250 250 9462 8772 <250 <250 <250 <250 10438
3 7650 3777 23030 46250 <250 250 480 2170 54000
Mean titre calculated from responding mice only
As can be seen from the Table, after a single dose,
anti-HAV antibodies could be detected in the sera of mice
immunised subcutaneously with 250EU and intranasally with
500EU. There was no detectable response in any of the
other groups. Boosting greatly enhanced the response in
the subcutaneous and intranasal 500 groups, giving titres
of approximately 10500 and 9500 respectively. Also, a very
similar response was seen in mice receiving 250EU plus CT
I/N and a low but measurable response was detected in the
sera of half the mice receiving two doses of 250EU I/N. No
serum response was detectable in any of the orally
immunised mice at this point. Further boosting did result
in seroconversion of some of the orally immunised mice into
250EU, 500EU and 250EU + CT groups. The response was
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greatest in the later group. After three doses, all of the
I/N immunised mice had seroconverted, the magnitude of the
response being dose dependant in the absence of CT. I/N
immunisation with 500EU, 250EU+CT produced high serum
titres that were slightly lower but comparable to those
produced by S/C immunisation with 250EU, HAV adsorbed to
alum.
Figure 1 illustrates the individual and mean serum
anti-HAV titres following three doses of HAV capsids. As
can be seen, CT greatly augmented the serum anti-HAV
response of I/N and orally administered antigen. The
titres in mice receiving 250EU+CT I/N were more than 10-
fold rated at each time point than those evoked by I/N
immunisation with 250EU alone. There was a similar
difference in anti-HAV titre in the mice immunised orally
with 250EU with and without CT. CT also increased the
number of mice seroconverting. Moreover, the titres in the
I/N 250EU+CT group follow each immunisation were very
similar to those of mice given 250EU adsorbed to alum and
given subcutaneously.
Table 2 below shows the number of mice in the
different groups seroconverting following the first and
second booster doses. Seroconversion was dose and route
dependant. Subcutaneous immunisation with 2 doses of 250EU
led to seroconversion in all the mice, whereas the same
dose, only half of the mice to whom the antigen had been
administered intranasally, and none of the mice to whom
antigen had been administered orally, had seroconverted.
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After the second boost, all of the mice in the I/N groups
had seroconverted, including those in the 125EU dose group
that exhibited no response after two doses. Likewise, the
orally immunised mice started a response after the second
boost although even the addition of CT did not result in
the seroconversion of all of the mice.
TABLE 4
Rate of seroconversion to anti-HAV following the first and
second booster immunisations
Intranasal Oral Sub/cut
Dose 125 250 500 250+ 125 250 500 250+ 250+Alum
CT CT
2 0/4* 2/4 4/4 4/4 0/4 0/4 0/4 0/4 5/5
3 6/6 5/5 5/5 5/5 0/4 1/4 2/4 3/4 5/5
* No. of mice responding/No. of mice tested
Table 4 shows the number of mice in the different groups
seroconverting following the first and second booster
doses. Seroconversion was dose and route dependant.
subcutaneous immunisations with two doses of 250EU led to
seroconversion in all the mice, whereas at the same dose
only half of the mice treated intranasally and none of the
mice treated orally had seroconverted. After the second
boost all of the mice in the intranasal groups had
seroconverted, including those in the 125EU dose group that
exhibited no response after 2 doses. Likewise the orally
immunised mice started to respond after the second boost
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although even the addition of CT did not result in the
seroconversion of all of the mice.
The aforementioned examples are given by way of
illustration only and are not intended the scope of the
application, which is limited only by the claims appended
hereto.
