U.S. patent application number 10/067870 was filed with the patent office on 2003-04-17 for processes for production of immunoglobulin a in milk.
This patent application is currently assigned to AGRESEARCH LIMITED. Invention is credited to Hodgkinson, Alison Joy, Hodgkinson, Steven Charles.
Application Number | 20030074676 10/067870 |
Document ID | / |
Family ID | 19926267 |
Filed Date | 2003-04-17 |
United States Patent
Application |
20030074676 |
Kind Code |
A1 |
Hodgkinson, Alison Joy ; et
al. |
April 17, 2003 |
Processes for production of immunoglobulin a in milk
Abstract
The present invention provides a process for producing
immunoglobulin A in the milk of hyperimmunised ruminants using a 3
route immunisation protocol and uses for the resultant product.
These products are useful in producing formulations useful for
passive immunisation against selected pathogens, especially in
preparations for food products and dietary preparations.
Inventors: |
Hodgkinson, Alison Joy;
(Hamilton, NZ) ; Hodgkinson, Steven Charles;
(Hamilton, NZ) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W.
SUITE 600
WASHINGTON
DC
20004
US
|
Assignee: |
AGRESEARCH LIMITED
|
Family ID: |
19926267 |
Appl. No.: |
10/067870 |
Filed: |
February 8, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10067870 |
Feb 8, 2002 |
|
|
|
09424246 |
Feb 29, 2000 |
|
|
|
Current U.S.
Class: |
800/7 ;
424/130.1 |
Current CPC
Class: |
A61K 2039/54 20130101;
A61P 31/12 20180101; A61K 35/20 20130101; C12N 2770/20034 20130101;
A61K 2039/5252 20130101; A61K 39/15 20130101; A61K 2039/70
20130101; C07K 16/04 20130101; A61K 2039/552 20130101; A61P 31/04
20180101; Y02A 50/474 20180101; A61K 39/215 20130101; A61K 39/0258
20130101; C12N 2720/12334 20130101; Y02A 50/30 20180101; A61K
2039/55566 20130101; A61K 2039/545 20130101; A61K 39/12
20130101 |
Class at
Publication: |
800/7 ;
424/130.1 |
International
Class: |
A61K 039/395; C12P
021/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 29, 1997 |
NZ |
314959 |
Claims
1. A process for the induction of immunoglobulin A (IgA) in a
mammal which process comprises: (a) actively immunising a pregnant
mammal with an antigen by any two routes of administration selected
from intramammary (IMM), intraperitoneal (IP) and intramuscular
(IM); and (b) actively immunising said mammal with an antigen by a
third administration route selected from intramammary (IMM),
intraperitoneal (IP) and intramuscular (IM); with the proviso that
all three administration routes are different.
2. A process according to claim 1 wherein in step (a) the two
routes of administration selected are IP and IM and in step (b) the
third route of administration is IMM.
3. A process according to claim 1 or claim 2 wherein the two active
immunisations of step (a) are effected sequentially,
discontinuously or concurrently.
4. A process according to claim 3 wherein the two active
immunisations of step (a) are effected concurrently.
5. A process according to any one of claims 1 to 4 wherein steps
(a) and (b) are effected sequentially, discontinuously or
concurrently.
6. A process according to any one of claims 1 to 5 wherein steps
(a) and (b) are repeated once or twice prior to parturition.
7. A process according to any one of claims 1 to 5 wherein step (a)
is repeated twice, prior to parturition.
8. A process according to claim 7 wherein each step (a) is effected
at 2 to 8 week intervals.
9. A process according to claim 8 wherein each step (a) is effected
at 2 to 4 week intervals.
10. A process according to any one of claims 6 to 9 wherein step
(a) is effected 6 to 14 weeks prior to parturition, first repeat
step (a) at 2 to 10 weeks prior to parturition, and the final step
(a) at 1 to 4 weeks prior to parturition.
11. A process according to claim 10 wherein step (a) is effected 8
to 12 weeks prior to parturition, first repeat step (a) at 4 to 8
weeks prior to parturition, and the final step (a) at 1 to 4 weeks
prior to parturition.
12. A process according to claim 11 wherein step (a) is effected 8
weeks prior to parturition, first repeat step (a) at 4 weeks prior
to parturition, and the final step (a) at 1 week prior to
parturtion.
13. A process according to any one of claims 6 to 12 wherein step
(b) is repeated once prior to parturition.
14. A process according to any one of claims 6 to 13 wherein the
(b) steps are effected at 1 to 6 week intervals.
15. A process according to claim 14 wherein the (b) steps are
effected at 2 week intervals.
16. A process according to claim 13 or claim 14 wherein step (b) is
effected 3 to 12 weeks prior to parturition, and repeat step (b) at
1 to 10 weeks prior to parturition.
17. A process according to claim 16 wherein step (b) is effected 4
to 8 weeks prior to parturition and repeat step (b) 2 to 4 weeks
prior to parturition.
18. A process according to claim 17 wherein step (b) is effected 4
weeks prior to parturition and repeat step (b) at 2 weeks prior to
parturition.
19. A process for the production of mammalian milk containing
immunoglobulin A (IgA), which process comprises: (a) induction of
IgA according to the process of any one of claims 1 to 18; and (b)
collecting milk containing IgA from said mammal.
20. A process according to any one of claims 1 to 19 wherein the
antigen comprises at least one of the group of bacteria, yeasts,
viruses, mycoplasmas, proteins, haptens, animal tissue extracts,
plant tissue extracts, spermatozoa, fungi, pollens, dust and a
complex of antigens.
21. A process according to claim 20 wherein the antigen is a
bacterial antigen.
22. A process according to claim 21 wherein the bacterial antigen
is selected from the group consisting of Escherichia,
Staphylococcus, Salmonella, Pneumonococcus, Helicobacter,
Cryptosporidiosus, Campylobacter and Shigella.
23. A process according to claim 22 wherein the bacterial antigen
is E.coli.
24. A process according to claim 20 wherein the antigen is a yeast
antigen.
25. A process according to claim 24 wherein the yeast is Candida
albicans.
26. A process according to claim 20 wherein the antigen is a
protein antigen.
27. A process according to claim 26 wherein the protein antigen is
tumour necrosis factor.
28. A process according to claim 20 wherein the antigen is a
complex of antigens
29. A process according to claim 28 wherein the complex of antigens
comprises E. coli, rotavirus and coronavirus.
30. A process according to any one of claims 1 to 29 wherein the
antigen is formulated as a suspension.
31. A process according to any one of claims 1 to 30 wherein the
antigen is administered together with an acceptable carrier,
diluent, buffer, and/or adjuvant.
32. A process according to claim 31 wherein the antigen is
administered together with an adjuvant.
33. A process according to claim 32 wherein the adjuvant is
selected from Freund's complete adjuvant (FCA), Freund's incomplete
adjuvant (FIC) adjuvant 65, cholera toxin B subunit, alhydrogel; or
bordetella pertussis, muramyl dipeptide, cytokinins and saponin.
Oil based adjuvants and in particular FCA and FIC are preferred
34. A process according to claim 33 wherein the adjuvant is Freunds
incomplete adjuvant.
35. A process according to any one of claims 1 to 34 wherein the
antigen is administered together with an antibiotic.
36. A process according to any one of claims 1 to 35 wherein the
antigen administered in each immunising process, and at each site,
is the same or different.
37. A process according to claim 36 wherein the antigen
administered in each immunising process, and at each site, is the
same.
38. The process according to any one of claims 1 to 37 wherein the
mammal immunised is selected from the group consisting of cows,
goats and sheep.
39. A process according to claim 38 wherein the mammal is a diary
cow.
40. IgA produced in accordance with the process of any one of
claims 1 to 39.
41. A process for the production of mammalian milk containing IgA,
which process comprises: (a) induction of IgA according to the
process of any one of claims 1 to 39; and (b) collecting milk
containing said IgA from said mammal.
42. IgA containing mammaliam milk produced in accordance with the
process of claim 41.
43. IgA isolated from the mammalian milk of claim 42.
44. The IgA of claim 43 which is purified IgA.
45. Use of the IgA of claim 40 or claim 44 as, or in the
preparation of, pharmaceutical, cosmetic, and/or veterinary
compositions.
46. Use of the IgA of any one of claims 42 to 44 as, or in the
preparation of, food products and/or dietary supplements.
Description
TECHNICAL FIELD
[0001] This invention relates to processes for producing
immunoglobulin A in mammals, processes for producing milk
containing immunoglobulin A and to the uses of the immunoglobulin A
and milk produced.
BACKGROUND
[0002] Immunoglobulin A (IgA) is a well documented immunoglobulin
present in almost all body fluids. It is thought to play a major
role in the protection of the host from infection by pathogenic
organisms invading via the mucosal surfaces of the respiratory,
gastrointestinal and urogenital tracts. IgA participates in the
clearance of pathogenic bacterial, viral or parasitical organisms
and a variety of ingested or inhaled antigens from the mucosal
surfaces by neutralising toxins and viral particles, inhibiting
adherence of bacterial pathogens and preventing colonisation and
penetration of mucosal surfaces by pathogenic microorganisms. The
key role of immunoglobulins including IgA in milk therefore is to
provide local protective immunity in the gastrointestinal tract of
the offspring during the suckling period.
[0003] Immunoglobulins have come to be recognised as useful in the
pharmaceutical and veterinary fields for treating bacterial or
viral infections of the gut, and more generally in the treatment of
disease and inflammation. Over the years various techniques for
producing immunoglobulins have been proposed. A particularly
popular method is for the induction and harvesting of
immunoglobulins from ruminant milk. This approach has particular
advantages in that the immunoglobulin produced in the milk is in a
form suitable for immediate consumption, or may be processed into
appropriate formulae or products. It is safe to use and the
industry infrastructure for producing milk containing antibodies is
already in place.
[0004] The ruminant immune system appears to differ from its human
counterpart in that the immunoglobulin dominant in bovine mammary
secretions is IgG.sub.1. Accordingly, the main focus of antibody
production in milk by active immunisation has been on
immunoglobulin G's, although theoretically, the preferred
immunoglobulin would be IgA for the reasons outlined above.
[0005] Some attempts have been made to produce increased levels of
IgA in ruminant milk. Proposals for vaccination by a single
administration route such as parenteral, subcutaneous, intravenous,
systemic, oral, intraperitoneal, intramuscular, intramammary and
the like have been suggested. Generally, these routes of
administration have resulted in the predominant production of IgG,.
Systemic immunisation produced both IgA and IgM in milk, but only
at low concentrations. The response was enhanced when
intramuscular/sub-cutaneous (IM) and intramammary (IMM)
immunisation processes were combined (Am. J. Vet. Res.sup.1).
Combinations of intraperitoneal (IP) and intramammary (IMM)
infusion have also been shown to produce IgA and IgG.sub.1
(Immunology.sup.7; Res. in Vet.Sci.sup.8, Res. in Vet.Sci.sup.11,
The Ruminant Immune System in Health and Disease.sup.10. It is
noted that this route leads to limited enhancement of IgA
production (The Ruminant Immune System in Health and
Disease.sup.10). A combination of IM and IMM immunisation gave rise
to a predominance of IgG.sub.1 in the milk (Aus. J. Dairy
Technology.sup.6), as well as increasing generally the levels of
IgG.sub.2, IgA, and IgM (Am. J. Vet. Res.sup.1). Significant
between animal variability in the antibody titres produced was also
noted.
[0006] The predominance of the production of IgG.sub.1 is
consistent with the findings that IgG's produced are the major
immunoglobulins in ruminant mammary secretions.
[0007] Intramammary immunisation techniques have generally not been
preferred as a route for vaccination under field conditions due to
the high chance of mammary infection (Aus. J. Dairy
Technology.sup.6). However, other work suggests that this may not
be the case (Am. J. Vet. Res.sup.1).
[0008] It should be noted that much of the published literature
concerning immunoglobulin production in mammary gland secretions is
directed to disease prevention in animals or their offspring. Few
are directed to the production of immunoglobulin enriched milk for
the purposes of obtaining the immunoglobulins themselves.
[0009] An exception to this is a process for the production of a
protein concentrate containing immunological factors of lactic
origin in Swiss Patent No. 1,573,995. Nearly 20 years ago, this
patent disclosed a process for producing milk with a high antibody
titre, by intracisternal instillation into the mammary gland,
parental injection (subcutaneous, intravenous), injection into the
retromammary ganglionic system by scarification, by oral ingestion
or by a combination of several of these modes. The only specific
immunisation protocol for obtaining colostral and transition milk
disclosed involved some 11 immunisation steps over a period of 8
weeks prior to calving. This protocol comprises multiple parental
(including intravenous) administration steps, with several IMM
administration steps interspersed and requires 2 oral
administration steps in the week prior to calving.
[0010] This protocol is not in widespread use today. The
immunisation plan is onerous in the number of steps involved and is
not in fact optimised for immunoglobulin A production. Indeed, the
patent is misleading in suggesting that IgA's are preponderant in
ruminant maternal milk; a misconception that may have resulted from
the knowledge that IgA is predominant in human milk As established
in other teachings (see for example, Aus. J. Dairy
Technology.sup.6) IgG is the predominant immunoglobulin produced in
the maternal milk.
[0011] It has also been shown in the intervening years that oral
delivery of antigens results in little or no increase of IgA titres
in mammary secretions when compared with non-inoculated controls
(Am. J. Vet. Res.sup.1). It is presumed that the presence of the
rumen may preclude the antigen reaching the small intestine.
Accordingly, the oral administration step called for by Hilpert is
now contraindicated.
[0012] Similarly, intravenous injection would not generally be
recommended for immunisation purposes because of the possible
adverse effects such as anaphylactic shock (Cold Spring
Harbour.sup.18, ILAR Journal.sup.19).
[0013] There is currently a need for a process for inducing, and
producing IgA in milk at higher levels than have previously been
obtained by known antigen administration processes. A process which
additionally reduces between animal variability in the production
of IgA is also desirable. A commercial process which optimises
production of IgA while simplifying the immunisation protocol is
also sought.
[0014] It is therefore an object of this invention to provide a
process for the induction and production of immunoglobulin A in
milk which goes some way towards overcoming the above disadvantages
or at least provides the public with a useful choice.
[0015] Accordingly, the present invention can broadly be said to
consist in a process for the induction of immunoglobulin A (IgA) in
a mammal which process comprises:
[0016] (a) actively immunising a pregnant mammal with an antigen by
any two routes of administration selected from intramammary (IMM),
intraperitoneal (IP) and intramuscular (IM); and
[0017] (b) actively immunising said mammal with an antigen by a
third administration route selected from intramammary (IMM),
intraperitoneal (IP) and intramuscular (IM);
[0018] with the proviso that all three administration routes are
different.
[0019] In a further aspect the present invention provides a process
for the production of mammalian milk containing immunoglobulin A
(IgA), which process comprises:
[0020] (a) induction of IgA according to the process set out above;
and
[0021] (b) collecting milk containing IgA from said mammal.
[0022] Preferably, the initial immunisation protocol is followed by
a programme of booster immunisations over the preparturition
period.
[0023] In a preferred process of the present invention the antigen
administered is the same for each route of administration.
[0024] Preferably, the antigen administered is emulsified in an
adjuvant. A particularly preferred adjuvant is Freunds incomplete
adjuvant (FIC).
[0025] In one embodiment of the invention IgA may be isolated from
the mammalian milk collected The isolated IgA may be purified if
desired.
[0026] In a further aspect, the present invention provides
mammalian milk containing IgA produced in accordance with the
processes of the invention.
[0027] In a still further aspect, the present invention provides
IgA produced in accordance with the processes of the invention.
[0028] Preferred mammals for use in the processes of the present
invention are ruminants, especially diary cows.
[0029] The present invention further provides for the use of
immunoglobulin A produced in accordance with the processes of the
invention in pharmaceutical, cosmetic and veterinary compositions
as well as in food products including functional foods and dietary
supplements.
[0030] Although the present invention is broadly as defined above,
it will be appreciated by those persons skilled in the art that the
invention is not limited thereto and that it also includes
embodiments of which the following description gives examples. In
particular, preferred aspects of the invention will be described in
relation to the accompanying drawings in which:
[0031] FIG. 1 shows a typical immunoglobulin dilution curve for a
positive control sample in the ovine E.coli IgA enzyme linked
immunoabsorbent assay (ELISA).
[0032] FIG. 2 shows the right (immunised) gland milk anti-E.coli
IgA titres for all groups on day 0, day 5, week 2 and week 4 after
parturition.
[0033] FIG. 3 contrasts anti-E. coli IgA milk responses of the
right (immunised) gland and the left (untreated) gland on day
0.
[0034] FIG. 4 shows the anti-E.coli IgA responses for individual
sheep with right (immunised) glands on day 1 post-partum. The
effect of the immunisation route is depicted.
[0035] FIG. 5 shows the anti-E.coli responses for individual sheep
with right (immunised) glands on day 2 postpartum.
[0036] FIG. 6 shows the anti-3K scour guard right (immunised) gland
milk IgA antibody titres for all groups on days 0 and 5.
[0037] FIG. 7 shows the anti-3K scour guard right (immunised) gland
and left gland milk IgA antibody titres for all groups on day
0.
[0038] FIG. 8 shows a typical dilution curve for positive and
negative control samples in the ovine TNF IgA ELISA.
[0039] FIG. 9 shows individual sample analyses of anti-TNF IgA
titre responses in the right (immunised) gland day 1 postpartum
milk samples for each of the immunisation groups.
[0040] FIG. 10 shows the data for anti-TNF titres from day 1
post-partum samples from the right (immunised) and left (untreated)
gland.
[0041] FIG. 11 shows the relationship of anti-TNF IgA to stage of
lactation.
[0042] FIG. 12 shows a typical dilution curve for positive and
negative control samples in the C.albicans IgA ELISA.
[0043] FIG. 13 shows the right (IMM immunogen in FIC) gland milk
anti-C.albicans IgA antibody titre for all groups on Day 1, 2, 7,
14 and 60.
[0044] FIG. 14 contrasts the anti-C.albicans IgA response in the
left gland (aqueous IMM immunogen) and right (FIC IMM immunogen)
glands over the lactation period.
DETAILED DESCRIPTION
[0045] The term "milk" used herein refers to both milk and
colostrum in the form in which it is produced by the mammal.
[0046] The term "antigen" as used herein refers to any material
capable of inducing an antigenic response in a treated mammal.
[0047] In a first aspect the present invention relates to a process
for the induction of immunoglobulin A (IgA) in a mammal. As a first
step the method comprises actively immunising a pregnant mammal
with an antigen by any two administration routes selected from
intramammary (IMM), intraperitoneal (IP) and intramuscular (IM). As
a second step the mammal is again actively immunised by a third
administration route selected from those routes given above. The
proviso to this process is that all three administration routes
selected are different.
[0048] The applicants have surprisingly found that the use of three
routes of administration increases IgA antibody titre levels above
what might be expected by simply combining two known administration
route processes with a third route of administration, or at least
decreases the between animal variability in IgA antibody titre
response.
[0049] It will be appreciated by the reader that the ordering of
the routes and timing of administration is not crucial to the
process for inducing immunoglobulin A. Moreover, the immunisations
by the different routes may be carried out sequentially,
discontinuously or concurrently. A presently preferred immunisation
protocol is for concurrent IM and IP immunisation followed by IMM
immunisation. The IM and IP immunisations effectively act to prime
the immune system response. The IMM immunisation is a localised
challenge to induce IgA production in that immunised region.
[0050] In a further preferred embodiment the initial immunisation
protocol is followed by a number of booster doses of antigen over
the pre-parturition period. The amounts of antigen introduced, the
frequency (time interval), and the number of booster doses may vary
widely. For example, from a single booster shot via a single route
of administration on one occasion through to multiple shots via
each of three administration routes on many different
occasions.
[0051] Booster shots are generally spaced to suit the convenience
of the operator. To avoid local irritation and congestion, it is
usually preferred that booster shots to the same site not be given
more frequently than every other week.
[0052] One regimen preferred requires concurrent IM and IP
immunisation on two separate occasions, followed by IMM
immunisation on one occasion. That is, effectively two priming
steps followed by local challenge. The first priming step is
generally carried out 2 to 8 weeks before the second priming and
challenge steps. These latter steps are desirably carried out
concurrently. A convenient protocol is for the first priming step
to be effected 6 to 14, preferably 8 to 12, and most desirably, 8
weeks before parturition and the second priming/local challenge
step to be effected 2 to 10 weeks, preferably 4 to 8, and most
desirably, 4 weeks before parturition. However, as noted above
timing is not crucial.
[0053] A second preferred regimen is for initial immunisation 6 to
14, preferably 8 to 12 and most desirably, 8 weeks before
parturition followed by 1 or 2 booster shots via each of the three
administration routes on 1 to 3 occasions before parturition. The
final immunisation will generally be given 1 to 2 weeks before
parturition.
[0054] Particularly preferred is a regimen requiring one additional
priming and local challenge step such that at 8 weeks prior to
parturition (minus 8 weeks) there is a concurrent IM and IP
immunisation, followed by concurrent IM/IP and IMM immunisations at
minus 4 weeks, a second IMM immunisation at minus two weeks, and a
final concurrent IM and IP immunisation at minus one week.
[0055] A further preferred regimen is for the initial IM/IP
immunisations to be effected at 12 weeks before parturition and the
second priming/local challenge step at 8 weeks before parturtion, a
second IMM immunisation at minus 6 weeks and a final concurrent
IM/IP immunisation at minus 4 weeks.
[0056] It will be appreciated from the above that a wide variation
in the timing of the immunisations is feasible generally starting
14 weeks prior to parturition, but preferably 12 or 8 weeks prior
to parturition.
[0057] Subsequent to parturition, declining antibody concentrations
can be increased by periodically introducing boosters shots of the
selected antigen into the mammal during the lactation period
according to equivalent preparurition protocols outlined above.
Generally, this involves between 1 to 6, preferably 2 to 4, and
most preferably 2 or 3, concurrent IM and IP immunisations in the
lactation phase following parturition together with 1 IMM
immunisation at the involution stage of lactation.
[0058] In a further embodiment of the invention the process of the
invention further comprises a preselection step. In this step
individual animals are tested and selected for their ability to
produce IgA.
[0059] As noted above, considerable between animal variability
exists for the production of immunoglobulins. This preselection
step wherein the animals showing the best IgA antibody titre
responses are selected assists in decreasing the between animal
variability factor. This process may similarly be used to build
groups of animals particularly suited to IgA production.
[0060] Processes for IM, IP and IMM administration are well known
in the art. For IM immunisation it is generally preferred that more
than one site be used for administration by this process. Preferred
sites for IM administration are the left and right sides of the
brachio chepalic muscle (that is, two sites in one muscle). For IP
immunisation, administration into the peritoneal cavity, generally
at only one site is presently preferred. Desirably, administration
is at the sub lumbar fossa. The precise sites of administration for
these routes may of course vary according to known administration
protocols. The amount and form of the antigen administered will
also vary according to the antigen used and the mammal to be
immunised in accordance with known vaccine formulations.
[0061] Generally, the antigen is injected using the syringe and
needle for IM and IP routes and fine-bore polyethylene surgical
tubing fitted to a syringe for the IMM route or alternatively a
conventional sterile intra mammary applicator. For the IMM
immunisation, the antigen is generally administered via the major
lactiferous duct or the supramammary lymph node. Preferably, via
the teat orifice into the teat canal. For best results it is also
preferred that each mammary gland be immunised on each occasion.
This maximises the localised IgA response mounted in the
mammal.
[0062] The volume of antigen injected will vary according to the
mammal and the route of immunisation. Table 1 below is a summary of
the injection volumes for sheep and cows immunised via the IM, IP
and IMM routes.
1 TABLE 1 IM IP IMM Sheep volume 1.0 ml (per site) 1.0 ml 1.0 ml
(per gland) maximum volume 5.0 ml 2.5 ml 2.0 ml (per gland) Cows
volume 2.0 ml (per site) 4.0 ml 2.0 ml (per gland) maximum volume
8.0 ml 10.0 ml 5.0 ml (per gland)
[0063] Typically for immunising the bovine, antigen is administered
at 2 ml per site and 2 sites for IM, 4 ml at 1 site for IP and 2 ml
into each of the four glands for IMM.
[0064] Contrary to conventional wisdom, field trials show that
there is no significant risk of infection using intramammary
immunisation provided appropriate precautions are taken. For
example, care must be taken to sterilise glands prior to
immunisation. Appropriate sterilation methods are known in the art.
For example, ethanol/iodine washes will serve this purpose. A
further precaution is to ensure that the antigen is administered in
a solution containing antibiotic. Suitable antibiotics include
dupocillin and ampicillin and clavuox L.C.
[0065] The mammals selected for use in the process of the invention
will generally be economically useful mammals such as ruminants.
Examples of the ruminants preferred for use are cows, goats, and
sheep.
[0066] The term "antigen" as used herein refers to any material
capable of inducing an antigenic response in the treated mammal.
Antigens may be selected according to the ultimate utility of the
IgA formulation. That is, if the formulation is to be used for
generating passive immunity, the antigen against which such
immunity is sought should be used. Antigenic substances which may
be employed in the process of the invention include bacteria,
viruses, yeasts, mycoplasmas, proteins, haptens, animal tissue
extracts, plant tissue extracts, spernatozoa, fangi, pollens, dust,
chemical antigens and mammalian cells.
[0067] Where haptens are to be used as antigens these should first
be conjugated to carrier substances such as proteins using
chemistry well known to people versed in the art. (ILAR
Journal.sup.19).
[0068] Useful bacterial antigens include species of Escherichia,
Staphylococcus, Streptococcus, Salmonella and Pneunonococcus.
Particularly preferred bacterial antigens are Escherichia coli,
clostridium difficile, vibriocholerae and helicobacter pylori.
[0069] Preferred yeast antigens include species of Candida.
[0070] A particularly preferred yeast antigen is Candida
albicans.
[0071] Useful viral antigens include rotavirus, herpes, fowlpox,
rhinopneumonitis, coronavirus, parvovirus and influenza. Protein
antigens include tumour necrosis factor, insulin-like growth
factors, and somatostatin, viral or bacterial cell surface proteins
and conjugated protein antigens. Chemical antigens include pollens,
pesticides, insecticides, fungicides and toxins. Complex antigens
comprising a combination of two or more antigens of the types
identified are also feasible. One such preferred complex antigen is
3K Scourguard (SmithKline Beecham, Royal Oak, Auckland, New
Zealand). The vaccine contains pathogenic E. coli, bovine
rotavirus, and coronavirus.
[0072] Useful mycoplasma antigens include mycoplasma pneumoniae and
cryptosporidiurn parvum.
[0073] Generally, the antigenic substances are suspended in liquid
medium for infusion or injection according to known protocols. Any
appropriate carriers, diluents, buffers, and adjuvants known in the
art may be used. Suitable suspension liquids include saline
solution, water, and physiologic buffers.
[0074] The use of adjuvants is also desirable. Suitable adjuvants
for use with the antigens of the invention include Freund's
complete adjuvant (FCA), Freund's incomplete adjuvant (FIC),
adjuvant 65, cholera toxin B subunit, alhydrogel; or bordetella
pertussis, muramyl dipeptide, cytokines and saponin. Oil based
adjuvants and in particular FCA and FIC are preferred.
[0075] Prior to injection antigens in appropriate carriers are
typically emulsified with oil-based adjuvant (FIC is preferred)
using a laboratory homogeniser. Aqueous antigen is typically mixed
with 3 volumes of oil adjuvant and emulsified until a stable water
in oil emulsion is formed as demonstrated using tests well known in
the art.
[0076] Conventional wisdom also taught that the use of oil based
adjuvants with direct intramammary immunisation was not feasible
because of the risk of adverse reactions. The present applicants
have found that not only is the administration with oil based
adjuvants feasible under appropriate care conditions, but is also
desirable. The use of FIC may significantly enhance the immunogenic
response obtained for some antigens, particularly when administered
by the IMM route. It is therefore presently preferred that antigens
be emulsified in FIC for all immunisations except for small
polypeptides where FCA may be preferable for the first IM/IP
immunisation. However, FCA has not been used for IMM.
[0077] As noted above, the size and concentration of the antigen
doses are not critical and it is known in the art that there is a
dose range known as the window of immunogenicity for antigen and
that this is generally relatively broad.
[0078] However, too much or too little antigen may induce
suppression, tolerance or immune deviation towards cellular
immunity and away from humoral immune response. Typically, for
protein antigens optimal doses are of the order of 5 to 25 .mu.g/kg
live weight in ruminants and for dead, lyophilised bacterial or
viral antigens doses in the range of 1.times.10.sup.8 to
4.times.10.sup.10 organisms per ml are typical.
[0079] As also noted above, in IMM immunisation it is preferred
that the antigen additionally be formatted in suspension with an
antibiotic.
[0080] Regarding the specific form of the antigen, it will be
appreciated by the reader that both live and killed vaccines are
possible. Studies have shown that killed vaccines will stimulate
IgG.sub.1 responses while live vaccines stimulate
IgG.sub.2responses. For the production of IgA either alternative is
possible.
[0081] The antigen administered by each of the immunisation routes
may be the same or different. Accordingly, several different
antigens may be administered by the three different immunisation
routes for each of the initial and booster immunisations. However,
it is presently preferred that the same antigen or combination of
antigens be administered via the three routes on each immunisation
occasion.
[0082] In a further aspect the present invention relates to a
process for the production of mammalian milk containing IgA which
method comprises induction of IgA antibodies according to the
process detailed above and then collecting the IgA containing milk
from the mammal. The collection of milk may be effected using
normal milking processes.
[0083] IgA titre responses are generally highest on the first day
following parturition. After this antibody levels drop to between 5
to 20% of the initial level. This subsequent level is usually
maintained for two to three months or until drying off or
involution. IgA levels may be raised in this period through booster
immunisations as discussed above. Milk containing IgA may usefully
be collected throughout this period.
[0084] This milk is useful in the form obtained directly from the
mammal but may be processed if required. Examples of processing
steps include heat treatment, ultra violet radiation,
concentration, supplementation with food additives, drying into
concentrates, milk powders and the like.
[0085] As a further step to the process of the invention, the IgA
may be isolated from the milk. Isolation may be effected using
separation techniques known in the art. For example, isolation of
immunoglobulin rich fractions from whey in Can. J. Vet. Res.sup.21,
EP 0320152, WO 97/27757, GB2 179947, from milk in
Milchwissenschaft.sup.22, U.S. Pat. No. 4,229,342, from colostrum
in Agric. Biol. Chem.sup.20, French Patent No. 2520235, New Zealand
Patent No. 239466 and U.S. Pat. No. 4,582,580, and from milk and
colostrum in U.S. Pat. No. 4,644,056.
[0086] The isolated IgA may subsequently be purified if desired.
Purification may be carried out according to known techniques such
as precipitation and ion exchange chromatography. Suitable
techniques are disclosed in the journals and patents referenced
above. Both the isolated and purified immunoglobulin A produced in
accordance with the additional process steps also form part of the
present invention.
[0087] In a further aspect the present invention relates to
mammalian milk containing IgA produced in accordance with the
process of the invention.
[0088] Processes for producing protein concentrates containing
immunoglobulins on a commercial scale are disclosed in Swiss Patent
No. 1,573,995 incorporated herein by reference. Briefly, the
process comprises collecting the milk of hyperimmunised
milk-bearing females; separating the cream and the impurities,
coagulating the clarified and skimmed milk, separating the casein,
filtering, ultrafiltering and sterilising the proteins of the whey
by filtration, evaporating and drying the product under conditions
which do not denature the immunoglobulins and which maintain
sterility.
[0089] In a further aspect, the present invention provides for the
use of IgA in the form of milk, processed milk products,
concentrates, isolated IgA and purified IgA produced in accordance
with the process of the invention. IgA has potentially broad
applications in the fields of pharmaceutical, veterinary and
cosmetic compositions as well as in foods and dietary supplements.
Such compositions, food and supplements can be administered to
patients (including human patients) having need of same.
[0090] More specifically, in the human health care field passive
oral immunisation using milk immunoglobulins from specifically
vaccinated cows has long been known. Given the significant role
that IgA plays in preventing enteric infections, formulations
containing IgA may be effective in treating patients susceptible to
such enteric infections. Formulations containing IgA antibodies
against enterotoxigenic gastric pathogens including pathogenic
E.coli, Rotavirus, Staphylococcus, Streptococcus, Aerobacter,
Salmonella, Pseudomonas, Haemophilus influenza, proteus vulgaris,
shigella dysenteriae, Diplococcus pneumonae, coronavirus and
Corynebacterium acne are all possible.
[0091] Formulations containing high levels of IgA specific for
infants is one application. Infants are often very susceptible to
enteric gastric disorders. Specific formulations containing
anti-cryptosporidiosis IgA for protection against cryptosporidiosis
infection in HIV and AIDS patients is a further possibility.
General formulations for protection of travellers against diarrhoea
and general gastric disorders are contemplated. Valuable
formulations containing antibodies against Helicobacter pylori for
protection against stomach ulcers are feasible.
[0092] Appropriate formulations can be produced based on known art
formulations. For example, formulations for treating the following
disorders are provided for in the art:
2 Treatment of Reference Gastroenteritis Swiss Patent No. 1,573,995
Infantile E.coli gastroenteritis Eur. J. Pediatr.sup.14 Enteric
infections Advances in Exp. Med. & Biol..sup.15 Enteric disease
U.S. Pat. No. 5,066,491 Campylobacter jejuni J. Applied
Bacteriology.sup.25 Shigella Flexneri Am. J. Tropical medicine and
Hygiene.sup.27 Rotavirus diarrhoea Indigenous Antimicrobial
Agents.sup.23 Dental Caries Infection and Immunity.sup.28
Cryptosporidial diarrhoea Lancet.sup.24, Gastroenterology.sup.29
Cryptosporidiosis in AIDS Archives of Disease in Childhood.sup.30
Rotavirus gastroenteritis J. Infectious Diseases.sup.16, J.
Clinical Microbiology.sup.17 H.pylori U.S. Pat. No. 5,260,057
Respiratory disease U.S. Pat. No. 5,066,491 Cryptosporidlosis U.S.
Pat. No. 5,066,491
[0093] A comprehensive review of the use of bovine immunoglobulins
to treat or prevent certain human diseases caused by H.pylori,
C.parvum, E.coli, S.flexneri, C.difficile, V.cholerae and rotavirus
is provided in the proceedings of the IDF seminar on Indigenous
antimicrobial agents of milk.sup.23.
[0094] In a more general context, pharmaceutical formulations
containing IgA tailored to the needs of the young, old, medically
impaired, and terminally ill are all desirable.
[0095] The formulations of the invention similarly have
applications in the veterinary field. For example in the
preparation of formulations containing specific IgA antibodies
against pathogenic microbiologics such as E.coli, rotavirus,
coronavirus and other scour causing microbes for the prevention and
treatment of gastric disorders in neonatal livestock.
[0096] Formulations containing specific IgA antibodies against
mycotoxins, phytotoxins, aflotoxins, herbicides, pesticides and
fungicides to block absorption of these following oral ingestion
are possible.
[0097] More generally, formulations may be prepared containing IgA
against undesirable food ingredients to block their absorption.
[0098] As well as pharmaceutical and veterinary formulations, IgA
antibodies produced in accordance with the present invention have
applications in the nutritional fields. This may range from the use
of the milk per se to specific formulations produced containing
high IgA levels for well-being, for applications such as
nutritional beverages and sports nutrition.
[0099] Formulations containing specific IgA against common
allergens such as pollens, dust, and mites for allergy protection
are possible. Contemplated herein are formulations containing
specific antibodies against mycotoxins, phytotoxins, aflotoxins,
pesticides, herbicides, environmental pollutants such as dioxins,
polychlorinated biphenyls and fungicides to block absorption of
these compounds. Formulations against undesirable food ingredients
such as cholesterol to block absorption of these would be
particularly useful. In a further aspect the specific IgA may be
complexed to probiotics or growth factors for the preparation of
formulations for gastric well-being.
[0100] In the veterinary corollary, formulations containing IgA for
nutritional support particularly of economically important animal
offspring such as lambs, piglets, calves, foals and chickens are
possible. Formulations consisting of specific IgA directed against
undesirable food ingredients such as .beta. carotene to block
absorption of these may also be useful.
[0101] A further area of application for the IgA product of the
present invention is in formulations containing the antibodies
against skin or hair protein antigens for topical applications,
against skin antigens complexed to UV absorbing compounds such as
zinc for long-lasting protection against sunburn and with specific
IgA antibodies complexed to growth factors for skin repair.
[0102] The formulations may be prepared in the form of drinks,
lotions, powders, creams and the like according to principles well
known in the art. The formulations may be for oral, intravenous,
intramuscular, subcutaneous, rectal, topical, parenteral
administration or such other routes as may be desired.
[0103] The formulations may include pharmaceutically acceptable
carriers or, in the case of nutritional supplements, nutritionally
acceptable carriers. Such carriers include aqueous solutions,
non-toxic excipients, including salts, preservatives, and buffers.
The formulations may include additives such as minerals, vitamins,
flavouring agents, scenting agents and the like.
[0104] General assistance in the preparation of such formulations
may be obtained from Remingtons Pharmaceutical Sciences, 16th
Edition. Easton: Mac Publishing Company (1980); the National
Formulary XIV, 14th Edition. Washington: American Pharmaceutical
Association (1975); and Goodman and Gillmans The Pharmacological
basis for Therapeutics (7th Edition) the contents of which are
hereby incorporated by reference.
[0105] Specific non-limiting examples of the invention will now be
described.
EXAMPLE 1
[0106] 64 pregnant ewes were selected and divided into eight groups
and immunised by intra muscular (IM), intra peritoneal (IP) or
intra mammary (IMM) routes or combinations of these three. For the
IMM immunisation the right gland only was immunised while the left
gland was untreated and acted as the control.
3 Immunisation Protocol Group (n = 8) Immunisation Route 1 IM 2 IP
3 IMM 4 IM/IP 5 IM/IMM 6 IP/IMM 7 IM/IP/IMM 8 NIL (Control)
[0107] Animals were immunised according to the following
schedule.
4 Immunisation Schedule I II III IV V VI VII -8 wk -4 wk -2 wk -1
wk 0 +4 wk +8 wk .div.12 wk .dwnarw. .dwnarw. .dwnarw. .dwnarw.
Par- .dwnarw. .dwnarw. .dwnarw. IM-1 IM-2 IMM-2 IM-3 turi- IM-4
IM-5 Wean- IP-1 IP-2 IP-3 tion IP-4 IP-5 ing IMM-1 IM-6 IP-6
IMM-3
[0108] Antigen
[0109] A commercially available pathogenic E. coli vaccine
(Suvaxyn, Maternafend-4; J&H Pacific Ltd, NZ) was used as
antigen. This vaccine is know to have high concentrations of K88,
K99, 987P and F41 pili antigens.
[0110] Details of the immunisation protocols are as follows:
[0111] Immunisation I.
[0112] IM/IP
[0113] Stock vaccine was emulsified with Freund's Incomplete
Adjuvant (FIC; 1 part vaccine: 3.parts FIC).
[0114] IM; 1 ml per site; 2 sites
[0115] IP; 1 ml per site; I site
[0116] Immunisation II.
[0117] IM/IP
[0118] Repeat IM/IP as for Immunisation I.
[0119] Stock vaccine was diluted in sterile saline (1 part vaccine:
1 part saline). Antibiotic (Dupocillin) was added to the IMM
immunogen in the ratio of 1 ml antibiotic: 30 ml immunogen.
[0120] IMM; 1 ml per bland; right gland only
[0121] Immunisation III
[0122] Repeat Immunisation II for IMM only.
[0123] Immunisation IV, V and VI
[0124] IM/IP
[0125] Repeat Immunisation I
[0126] Immunisation VII
[0127] IM/IP & IMM
[0128] Repeat Immunisation II
[0129] Animal Health Status
[0130] The general health of the ewes in the trial was monitored by
regular weight checks and veterinary inspections. There was no
discernible difference observed in weight gain/loss between the
treatment groups. No adverse effects of immunisation were observed.
Two of the 64 ewes were treated for mastitis, one ewe from Group 6
(IP/IMM) and one ewe from Group 7 (IM/IP/IMM). Overall, no
deleterious effects of IMM immunisation were noted.
[0131] Samples
[0132] The ewes were bled before Immunisation I, II, and IV, prior
to lambing. Post parturition samples of blood and colostrum/milk
(left and right mammary glands separately) were collected at Day 0
(parturition), 1, 2, 3, and 5, then Week 1, 2 and 3, then Month 1,
2 and 3.
[0133] Bloods were collected on ice into EDTA vacutainers.
Separated plasma was stored at -20.degree. C. for antibody
analysis. Left (untreated) and right (immunised) mammary gland
colostrum/milk samples (20-30 ml) were kept on ice until
centrifuged (4.degree. C.; 20 minutes; 2,000 g.sub.max) to remove
fat. Skimmed supernatant was re-centrifaged (4.degree. C.; 1 hour;
40,000 g.sub.max) to separate milk whey/plasma and casein.
Supernatant was stored at -20 .degree. C. for antibody
analysis.
[0134] Sample analysis
[0135] All samples and reagents were diluted with 0.01M phosphate
buffered saline (pH 7.5) containing 0.05% v/v Tween-20 (PBS-T) and
1% w/v Bovine Serum Albumin ISA; type A7030, Sigma Chem. Co., USA)
and all washes were carried out by an automated plate washer
(ELP-35, BioTek Instruments, USA) using PBS-T, unless otherwise
stated. ELISA plates (Maxisorp F-96 immunoplates, Nunc, Denmark)
were coated with 100 .mu.l of E. coli antigen (Suvaxyn
Maternafend4; J & H Pacific Ltd, NZ) diluted 1:1K in 0.05M
carbonate buffer (pH 9.6), incubated overnight at 4.degree. C. and
washed three times. Remaining activated sites on immunoplates were
blocked by incubating 2 hours at 22.degree. C. with 250.mu.l PBS-T
containing 1% w/v BSA. After washing plates 2 times, 100 .mu.l of
10-fold serial dilutions of test samples (primary antibody; 1:100,
1:1K, 1:10K, 1:100K) were added to duplicate wells. Plates were
incubated 2 hours at 22.degree. C. then washed 3 times. 100 .mu.l
of second antibody consisting of heavy-chain specific rabbit
anti-sheep IgA (1:200K; Bethyl Laboratories, USA) were added to the
plates. Plates were incubated overnight at 4.degree. C., then
washed 3 times prior to the addition of 100 .mu.l of the enzyme
conjugate, goat anti-rabbit Ig conjugated to horse radish
peroxidase (1:8K; Dako, Denmark). After a 2 hour incubation at 22
.degree. C., the plates were-washed 2 times with PBS-T then 2 times
with PBS containing no Tween-20 and filled with 1 00 .mu.l of
freshly prepared substrate solution. The substrate solution
consisted of 0.1 g/l 3,3'.5,5'-tetramethylbenzidine (Boehringer
Mannheim, Germany) in 0.1M sodium acetate buffer (pH 5.5)
containing 1.3 mmol/l hydrogen peroxide. Following a 30 minute
incubation at 22.degree. C., 50 .mu.l of stopping solution, 2M
H.sub.2SO.sub.4, were added and the optical density (OD) was
measured at 450 nm by an automated plate reader (EL311s, BioTek
Instruments, USA).
[0136] With each ELISA microplate a positive quality control sample
(assayed at 1:100, 1:1K, 1:10K, 1:100K and 1:1000K) and a negative
quality control sample (assayed at 1:1K) was run with the samples.
Absorbance values from these control samples were used in
calculations to determine sample antibody titres. The median
absorbance between the maximum absorbance of the positive control
and the absorbance of the negative control gives a 50% figure. The
reciprocal dilution of sample antibody equivalent to this 50%
absorbance figure is classified as the antibody titre for the
sample.
[0137] FIG. 1 shows a typical dilution curve for the positive
control sample in the E. coli IgA ELISA.
[0138] Results
[0139] Samples were initially assayed as group pools to obtain an
overview of the group responses to the different immunisation
regimens.
[0140] FIG. 2 shows the right (immunised) gland milk anti-E. coli
IgA titres for all groups on Day 0, Day 5, Week 2 and Week 4. In
all groups, IgA milk antibody titres were highest on Day 0
(parturition) with levels falling over the first week. Group 7
(IM/IP/MM) gave the best IgA response with a milk antibody titre of
105K. This was followed by Group 5 (IM/IMM) with a titre of 50K and
Group 6 (IP/IMM) with a titre of 22K. The other groups gave a
minimal response including Group 3 (IMM). By Day 5, the IgA milk
antibody titres in Groups 5, 6 and 7 had fallen to about 20% of Day
0 titres but by Week 4 the titres were still significant being
approximately 3K.
[0141] FIG. 3 contrasts anti-E. coli IgA responses of the right
(immunised) gland and the left (untreated) gland on Day 0. IgA milk
antibody titres showed a marked difference in response between
samples from the right (immunised) gland and the left (untreated)
gland. While high titres were measured in the right milk samples of
Groups 5, 6 and 7, the titres in the corresponding left milk
samples were, at best, only 20% of these levels.
EXAMPLE 2
[0142] Individual samples analysis of anti-E. coli IgA titre
responses was conducted for each of the immunisation groups of
Example 1. Samples were analysed using the ELISA assay according to
the process of Example 1. The results for Day 0 and 1 are shown in
FIGS. 4 and 5, respectively.
[0143] Results
[0144] In general agreement with the earlier pooled data, titres
were low in animals immunised by IM, IP or IMM routes alone and
much higher in animals treated by the combination IM/IMM, IP/IMM
and IM/IP/IMM routes (Mean.+-.s.e.m. antibody titres;
1/32,100.+-.1/13,500; 1/29,000.+-.1/12,000; 1/30,000.+-.1/7,300,
respectively).
[0145] No significant differences were observed in mean IgA titre
responses resulting from immunisation by each of the three
combination routes. However, substantial within-group variability
in titre response was observed and notably the standard error of
the mean for the IM/IP/IMM group (1/7,300) was much lower than that
calculated for the IM/IMM (1/13,500) and IP/IMM (1/12,500)
routes.
[0146] This pattern of titre response was maintained in milk
samples from Day 2 and subsequently. The data appear to indicate
that immunisation by the three site IM/IP/IMM procedure does not
increase the magnitude of the response above those obtained with
the IM/IMM and IP/IMM combinations but serves to decrease the
between animal variability in IgA response.
EXAMPLE 3
[0147] 64 pregnant ewes were selected and divided into eight groups
and immunised by intra muscular (IM), intra peritoneal (IP) or
intra mammary (IMM) routes or combinations of these three. For the
IMM immunisation the right gland only was immunised while the left
gland was untreated and acted-as the control.
5 Immunisation Protocol Group (n = 8) Immunisation Route 1 IM 2 IP
3 IMM 4 IM/IP 5 IM/IMM 6 IP/IMM 7 IM/IP/IMM 8 NIL - Control
[0148] Animals were immunised according to the following
schedule.
6 Immunisation Schedule I II III IV V VI VII -8 wk -4 wk -2 wk -1
wk 0 +4 wk +8 wk .div.12 wk .dwnarw. .dwnarw. .dwnarw. .dwnarw.
Par- .dwnarw. .dwnarw. .dwnarw. IM-1 IM-2 IMM-2 IM-3 turi- IM-4
IM-5 Wean- IP-1 IP-2 IP-3 tion IP-4 IP-5 ing IMM-1 IM-6 IP-6
IMM-3
[0149] Antigen
[0150] A commercially available vaccine, 3K Scourguard (SmithKline
Beecham, Royal Oak, Auckland, New Zealand) was used as immunogen.
The vaccine contains pathogenic E. coli, bovine rotavirus and
coronavirus.
[0151] Details of tie immunisation protocols are as follows:
[0152] The immunisation protocol of Example 1 was repeated with 3K
Scourguard used as stock vaccine in place of Maternafend.
[0153] Animal Health Status
[0154] The general health of the ewes in the trial was monitored by
regular weight checks and veterinary inspections. And as for
Example 1, there was no discernible difference observed in weight
gain/loss between the treatment groups and no adverse effects of
immunisation were observed.
[0155] Samples
[0156] Samples of blood and colostrum/milk were- taken according to
the protocol of Example 1.
[0157] Sample analysis
[0158] The ELISA assay was performed according to the method of
Example 1 with the exception that 3K Scourguard (1:1K) was used for
microplate coating in place of Maternafend. Blood plasma and
colostrum/milk were pooled to obtain an initial indication of group
antibody responses.
[0159] Results
[0160] FIG. 6 shows the right (immunised) gland milk anti-3K
Scourguard IgA titres for all groups on Day 0 and 5. In all groups,
IgA milk antibody titres were highest on Day 0 (parturition) with
levels falling over the first week. Group 7 (IM/IP/IMM) gave the
best IgA response with a milk antibody titre of 210K. This was
followed by Group 5 (IM/IMM) with a titre of 70K and Group 3 with a
titre of 27K. Group 6 (IP/IMM) gave a milk antibody response of 20K
The other groups gave a minimal response. By Day 5, the IgA milk
antibody titres in Group 3, 5 and 7 had fallen to about 10% of Day
0 titres.
[0161] FIG. 7 shows the right (immunised) and left (untreated)
gland milk anti-3K Scourguard IgA titres for all groups on Day 0.
The right land had a much greater response than that of the left
gland. There was no significant antibody titre response for the
untreated left gland except for Group 3.
EXAMPLE 4
[0162] 32 pregnant ewes were assigned to four treatment groups and
immunised by combinations of intra muscular (IM), intra peritoneal
(IP) or intra mammary (IMM) routes. IMM immunogen for Group 3 was
in aqueous solution while IMM immunogen for Group 4 was emulsified
in FIC. For the IMM immunisations the right gland only was
immunised while the left gland was untreated and acted as the
control.
7 Immunisation Protocol Group (n = 8) Immunisation Route 1 IM/IMM 2
IP/IMM 3 IM/IP/IMM 4 IM/IP/IMM(FIC)
[0163] Animals were immunised according to the following
schedule.
8 Immunisation Schedule I II III IV -8 wk -4 wk -2 wk -1 wk 0
.dwnarw. .dwnarw. .dwnarw. .dwnarw. Parturition IM-1 IMM-1 IMM-2
IM-3 IP-1 IMM(FIC)-1 IMM(FIC)-2 IP-3 IM-2 IP-2
[0164] Antigen
[0165] A TNF preparation, commercially available from R & D
Systems, 614 McKinley Place, New England, USA, was used as antigen.
A stock solution (1 mg/ml) was prepared by reconstituting the
freeze dried TNF in sterile saline.
[0166] Details of the immunisation protocols are as follows:
[0167] Immunisation I.
[0168] IM/IP
[0169] Stock antigen solution was diluted to 0.16 mg/ml then
emulsified with FIC (1 part saline: 3 parts FIC).
[0170] IM; 1 ml per site; 2 sites
[0171] IP; 1 ml per site; 1 site
[0172] Immunisation II.
[0173] IM/IP
[0174] Repeat IM/IP as for Immunisation I.
[0175] IMM
[0176] For right glands: Stock antigen solution was diluted to 0.1
mg/ml in sterile saline. Antibiotic (Dupocillin) was added in the
ratio of 1 ml antibiotic: 40 ml immunogen. IMM; 1 ml per night
gland
[0177] IMM(FIC)
[0178] For right glands: Stock antigen solution was diluted to 0.32
mg/ml in sterile saline and emulsified with FIC (1 part saline: 3
parts FIC). Antibiotic (Dupocillin) was added in the ratio of 1 ml
antibiotic: 40 ml immunogen.
[0179] IMM (FIC); 1 ml per right gland
[0180] Immunisation III
[0181] IMM/IMM(FIC)
[0182] Repeat Immunisation II for IMM/IMM(FIC) only.
[0183] Immunisation IV
[0184] IM/IP
[0185] Repeat Immunisation I
[0186] Animal Health Status
[0187] The general health of the ewes in the trial was monitored by
regular weight checks and veterinary inspections. The animals
maintained weight during pregnancy and lactation and no between
group effects of treatment were observed. Two animals died from
unrelated causes during pregnancy/lambing (both ewes from Group 1,
IM/IMM). One animal was withdrawn from the trial. due to mastitis
in the left untreated gland. No adverse effects on immunisation
were noted Evidence of ulceration at IM and IP immunisation sites
was minimal. No significant differences were observed in mammary
function between the left and right glands and between glands
immunised with immunogen in sterile saline or FIC.
[0188] Samples
[0189] The ewes were bled before Immunisation I, II, and IV, prior
to lambing. Post lambing, samples of blood and colostrum/milk (left
and right mammary glands separately) were collected at Day 1
(parturition), 2, 3, 6, 14 and 28, and Month 2 and 3.
[0190] Samples were collected and treated according the format used
in Example 1.
[0191] Sample Analysis
[0192] The ELISA assay for TNF was performed according to the
format used for Example 1, with the exception that TNF was used for
plate coating (2 mg/ml). FIG. 8 shows a typical dilution curve for
the positive and negative control samples in the TNF IgA ELISA.
Inter assay precision was calculated from 10 repeat analysis of the
positive control and the coefficient of variation was 10.2%.
[0193] Results
[0194] IgA milk responses
[0195] Individual sample analyses of anti-TNF IgA titre responses
in the right (immunised) gland Day 1 postpartum milk samples for
each of the immunisation groups are shown in FIG. 9. Titres were
low in animals immunised by the IM/IMM or IP/IMM or IM/IP/IMM
routes where the immunogen was administered in saline solution.
(Mean.+-.s.e.m. antibody titres: 1/3,600.+-.1/2,600;
1/3,300.+-.1/1,500; 1/900.+-.1/500, respectively.) By contrast,
titres were some 20-fold higher in animals treated by the
combination IM/IP/IMM routes where the IMM immunogen was emulsified
in FIC (1/61,900.+-.1/29,600). Considerable variation was observed
in the responses of the individual Group 4 animals with titres
ranging from 1/4,000 to 1/250,000.
[0196] Right and Left Gland Milk Antibody Responses
[0197] Significant differences were seen in anti-TNF IgA milk
titres from right (immunised) and left (untreated) glands with the
left gland IgA titre being almost undetectable, in agreement with
earlier findings for E. coli. FIG. 10 depicts the data for anti-TNF
IgA titres for Day 1 post-partum milk samples from the left and
right glands.
[0198] Milk Antibody Response Over Lactation
[0199] The relationship of anti-TNF IgA to stage of lactation is
shown in FIG. 11. Data are mean=s.e.m. right (immunised) gland milk
titres from Group 4 animals treated by the combination
IM/IP/IMM(FIC) route. Anti-TNF IgA titres were highest in initial
post-partum mammary secretions and found to decline to
approximately 10% of peak levels by Day 6 and approximately 5% by
Month 1 (equivalent to an IgA titre of 1/3,500). The overall
pattern of response was similar to that seen in the E. coli trial,
the antibody decline coinciding with the onset of fall lactation
and increasing milk volumes.
EXAMPLE 5
[0200] 35 pregnant cows were divided into four groups and immunised
by either two routes, three routes or none according to the
protocol below Immunogen was emulsified in Freund's Incomplete
Adjuvant (FIC) for right sided glands intra mammary (IMM)
immunisations and for intra muscular (IM) and intra peritoneal (IP)
immunisations. Left sided glands utilised immunogen in aqueous
solution.
9 Immunisation Protocol Group Immunisation Route 1a (n = 10) IM/IMM
1b (n = 10) IP/IMM 1c (n = 10) IM/IP/IMM 1d (n = 5) NIL
[0201] Animals were immunised according to the followings
schedule.
10 Immunisation schedule I II III IV -8 wk -4 wk -2 wk -1 wk 0
.dwnarw. .dwnarw. .dwnarw. .dwnarw. Parturition IM-1 IMM-1 IMM-2
IM-3 IP-1 IM-2 IP-3 IP-2
[0202] Antigen
[0203] Antigen for immunisation was the yeast, Candida albicans.
The yeast cells were cultured in medium, harvested by
centrifugation, washed and heat killed then freeze dried A stock
solution of C. albicans (7 mg protein per ml) was prepared by
reconstituting the freeze dried C. albicans in phosphate
buffer.
[0204] Details of the immunisation protocols are as follows:
[0205] Immunisation I.
[0206] IM/IP
[0207] Stock antigen solution was diluted to 1 mg/ml in sterile
saline and emulsified with FIC (1 part saline: 3 parts FIC).
[0208] IM; 2 ml per site; 2 sites
[0209] IP; 4 ml per site; 1 site
[0210] Immunisation II.
[0211] IM/IP
[0212] Repeat IM/IP as for Immunisation I.
[0213] IMM
[0214] For right glands: Stock antigen solution was diluted to 1
mg/ml in sterile saline and emulsified with FIC (1 part saline: 3
parts FIC).
[0215] For left glands: Stock antigen solution was diluted to
0.25mg/ml in sterile saline. IMM; 2 ml per gland; 4 glands
[0216] Immunisation III
[0217] IMM
[0218] Repeat Immunisation II for IMM only.
[0219] Immunisation IV
[0220] IM/IP
[0221] Repeat Immunisation I
[0222] Animal Health Status
[0223] The general health of the cows in the trial was monitored by
regular weight checks and veterinary inspections. Immunisation
sites were inspected at regular intervals to assess effects of the
immunisation procedure. No clinical grade site reactions were
observed at any of the sites immunised. In addition, milk volume
data collected indicated that treatment of the mammary gland did
not effect the overall lactation performance of the animals.
[0224] Samples
[0225] The cows were bled before Immunisation I, II, and IV, prior
to calving. Post parturition samples of blood and colostrum/milk
were collected at Day 1, 2, 3, 5, 7, 14, 28 and 60. On sample days,
cows were quarter milked (ie. samples were collected from
individual glands) AM and PM, milk volumes were recorded and 100 ml
sample retained. AM and PM quarter milk samples were pooled for
laboratory analyses.
[0226] Blood and colostrum/milk samples were treated according to
the format used for Example 1.
[0227] Sample Analysis
[0228] The ELISA assay for C albicans was performed according to
the format used for Example 1, with the exception that: C albicans
(5 mg/ml) was used for plate coating; second antibody used to
identify class specificity was rabbit anti-bovine IgA (1:40K;
Bethyl Laboratories, USA); enzyme conjugated antibody was goat
anti-rabbit (1:12K; Dako, Denmark). The end point detection system
was the same as in Example 1.
[0229] FIG. 12 shows a typical antibody dilution curve for the
positive control sample.
[0230] Results
[0231] For Groups 1a, 1b and 1c, the anti-C. albicans IgA milk
antibody titres were highest on Day 1 (parturition) with levels
falling over the lactation period, as observed with the earlier
sheep trials (Examples 1-4).
[0232] Two site versus three site immunisation
[0233] FIG. 13 shows the right (IMM immunogen in FIC) gland milk
anti-C. albicans IgA antibody titres for all groups on Day 1, 2, 7,
14 and 60. Animals immunised at three sites (Group 1 c; IM/IP/IMM)
had a much higher response then animals immunised at two sites
(Group 1a; IM/IMM and Group 1b; IP/IMM). The mean.+-.s.e.m.
antibody titres were 11,700.+-.3,700,2,500.+-.700 and 3,000.+-.900,
respectively. Group 1d (Control) had a very low antibody titre on
Day 1 (titre of 200.+-.70). The higher titres for animals immunised
at three sites was maintained over the lactation period.
[0234] Freund's versus saline for IMM immunisations
[0235] FIG. 14 shows the milk anti-C. albicans IgA titre comparison
of the left (aqueous IMM immunogen) and right (FIC IMM immunogen)
glands in Group 1c over the lactation period. Glands immunised with
immunogen emulsified in FIC gave a higher response than glands
immunised with aqueous immunogen and this difference increased with
time. On Day 1 the right gland titre was 11,700.+-.3,700 compared
to the left gland titre of 8,700.+-.2,900. By Day 60, the left
gland titre had declined to 590.+-.200 while the right gland titre
was 4 fold higher (2,300 +800). This difference between left and
right gland response was also apparent in the groups immunised at
two sites (Group 1a and 1b).
[0236] Thus, in accordance with the present invention there is
provided a process for the induction of IgA in a mammal, and the
production of IgA in mammalian milk at levels higher than have
previously been obtained or might have been anticipated from
combining a third administration route for an antigen with known
two route administration protocols. Alternatively, the present
invention at least provides a method whereby the between animal
variability in IgA antibody titre response can be reduced. It will
be appreciated that these results represent an advantage where
products containing IgA are sought for use in pharmaceutical,
veterinary and cosmetic formulations as well as in nutritional and
dietary supplements.
[0237] It will be further appreciated by those persons skilled in
the art that the present description is provided by way of example
only and that the scope of the invention is not limited
thereto.
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[0269] All articles and patents referenced here and in the
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* * * * *