Production of antibodies using a mixture of strains of E. coli collectively expressing type I pili, CFA I pili, CFA II pili and K88 pili

Antibodies are produced by hyperimmunizing a mammal, such as cow, with a vaccine derived from E. coli bacteria. The bacterial strains in the vaccine are selected on the basis of their virulence characteristics, especially adhesion factors (pili), associated with gastroenteric disease in humans. The antibodies can be recovered from the mammal's milk or serum, and used in human foods.

The present invention relates to the production of antibodies. 
The mammalian defense mechanism against many diseases, including those 
caused by bacteria such as Escherichia coli, involves the production of 
antibodies known as immunoglobulins. Several distinct classes of 
immunoglobulins, of which the commonest are referred to as IgA, IgG, IgM, 
IgD, and IgE, have been identified. Not all mammalian species produce the 
same range of immunoglobulins, and some species apparently place greater 
reliance on one particular immunoglobulin rather than on another, type 
that may predominate in the defence mechanisms of other mammalian species. 
The immunoglobulins, which are complex protein structures, circulate in the 
mammal's bloodstream, and in the lactating female are important 
constituents of her milk, especially in the colostrum (first milk) 
produced during the first few days following the birth of an infant. The 
suckling infant ingests these immunoglobulins, and thereby derives passive 
immunity against, in particular, enteropathogenic bacteria. This is highly 
important, because it may be several days or even weeks before the 
infant's own immune mechanisms are sufficiently stimulated for it to 
generate its own antibodies in effective quantities. In this way, the 
breast-fed human infant derives passive immunity against gastro-entestinal 
infections because its mother's milk contains substantial quantities of 
appropriate immunoglobulins. 
As an alternative to breast-feeding, it is common practice to use bovine 
milk as a substitute for human milk, either consumed as such or in the 
form of synthetic milks based on bovine milk, i.e. skimmed milk powder. In 
many parts of the world, the milk from other mammals, such as goats, is 
used. 
Natural bovine milk also contains immunoglobulins designed by nature to 
protect the calf in a similar manner. However, the relative concentrations 
of immunoglobulins in bovine milk differ from those in human milk. The 
immunoglobulin (IgA) predominates in human milk and lines the intestinal 
mucosa of the infant and provides very efficient long-term protection, but 
bovine milk contains lower levels of IgA. Bovine milk, and in fact the 
milk of ruminant animals generally, is rich in the immunoglobulin 
IgG.sub.1 which is closely related to but not identical with the 
immunoglobulin IgG that occurs in minor proportions in human milk. 
IgG.sub.1 only remains in the lumen of the intestine, and provides 
comparatively short-term protection against gastro-intestinal infections 
in the human. Furthermore, the specific immunoglobulins present in bovine 
milk are raised by the cow against its own pathogens, and not against 
those that commonly infect the human. 
A further problem is that the normal processing to which bovine milk is 
subjected in Western countries, e.g. pasteurisation or sterilisation in 
the case of whole milk and also spray-drying in the case of milk powder, 
usually involves temperature conditions that are sufficiently extreme to 
destroy the beneficial activity of any immunoglobulins present in the 
original milk. Therefore the natural protection afforded by these 
immunoglobulins is usually lost. 
It has been proposed to supplement the immunoprophylactic activity of milk 
and milk substitutes for human use by the addition thereto of concentrates 
derived from bovine milk, containing the natural bovine immunoglobulins in 
active form. This will indeed provide some measure of passive immunity, 
but the concentrate will contain in addition to the desirable 
immunoglobulins many other trace ingredients of natural milk. Moreover, as 
has already been indicated, the immunoglobulins present in bovine milk are 
not those of prime importance to the human infant. 
To improve this situation it has been proposed to hyperimmunise 
milk-producing animals, especially cattle, by means of vaccines prepared 
from known human gut pathogens, with the objective of causing the animal 
to produce antibodies that are more specific to and hence more effective 
against the human pathogens. This approach is described in UK patent 
specification No. 1 573 995 (Societe des Produits Nestle SA; inventor 
different strains of E. coli, selected probably because they had been most 
commonly identified in the literature as being implicated in outbreaks of 
human gastroenteric disease. 
Gastro-enteric disorders in human adults and in human infants have been the 
subject of extensive research, but a study of the scientific literature 
reveals a wide divergence of views amongst the experts in this field as to 
which strains of micro-organisms are principally implicated in causing 
such disorders. In view of the large number of bacterial strains that are 
apparently implicated, and in view of the confusion that is generated by 
reading the diverse opinions on this topic as expressed in the scientific 
literature, it is no simple matter to identify a narrow selection of key 
bacterial strains that is likely to provide the basis of a vaccine that 
will impart immunity against a broad spectrum of gut micro-organisms. From 
the economic standpoiont, the number of stains involved in the selection 
should be kept to a minimum. From the standpoint of efficacy, the vaccine 
should impart immunity against as many gut-infective strains of 
micro-organisms as possible. No such simple selection can be derived from 
the present knowledge as recorded in the scientific literature. On the 
contrary, it would seem from the published data that a very large number 
of different strains would have to be involved in order to provide broad 
immunity in the human. This is in line with the approach adopted by 
Hilpert. 
We now provide the means for selecting appropriate stains, based on their 
virulence characteristics. 
The invention provides a process for the preparation of immunoglobulins 
useful in providing passive protection against E. coli bacteria implicated 
in causing gastroenteric disease in humans, in which process a host mammal 
is immunised with a vaccine comprising antigens of at least two strains of 
E. coli expressing collectively chromosome-mediated adhesion factors 
(pili) and plasmid-mediated adhesion factors (pili) to induce the host 
mammal to produce substantial quantities of immunoglobulins specific to 
the antigens, and the immunoglobulins are recovered in functional form. 
Preferably the chromosome-mediated adhesion factors expressed by at least 
one of the bacterial strains from which the vaccine is derived, include 
Type I pili. 
Preferably the plasmid-mediated adhesion factors expressed by the bacterial 
strains from which the vaccine is derived, include CFA I pili and/or CFA 
II pili. 
In a particularly preferred embodiment of the invention, the vaccine 
comprises antigens of a plurality of bacterial strains expressing 
collectively Type I pili, CFA I pili, CFA II pili and K88 pili. 
Preferably the vaccine used includes antigens of at least one bacterial 
strain that produces enterotoxins, ideally of both the heat-stable (ST) 
and heat-labile (LT) types. This requirement is secondary to the 
pili-expression criteria set out above. Preferably, at least one of the 
selected bacterial strains is also a producer of enterotoxins. 
An important product of the invention is non-human milk incorporating 
immunoglobulins active against a plurality of E. coli strains implicated 
in causing gastro-enteric disease in humans, expressing collectively the 
pili of the types Type I CFA I and CFA II. 
A further product of the invention is immunoglobulin material that has been 
prepared as described in the immediately proceeding paragraphs. Such 
immunoglobulin material can be added to human foodstuffs to provide 
passive immunity against gut infective bacteria. The immunoglobulin 
material can be recovered from immune milk by conventional methods 
involving concentration, precipitation or chromatographic techniques. 
Alternatively, although less desirably, the immunoglobulin material can be 
recovered from serum derived from the host animal, in which case a non 
milk producing (or indeed male) host animal can be used. 
A further embodiment of the invention is a food product for humans, and 
especially a milk substitute formulated for human infants, containing 
recovered immunoglobulin material as just described. Such milk substitutes 
can be manufactured and marketed in liquid form, but more commonly are 
provided in the form of dry powders requiring reconstitution in water. 
An important aspect of the invention is a vaccine for oral and/or 
parenteral administration comprising, in a pharmaceutically acceptable 
carrier or diluent, antigens of a plurality of E. coli strains 
collectively expressing at least one virulence characteristic selected 
from each of the following groups: 
(a) chromosome-mediated pili, 
(b) CFA I pili and CFA II pili, 
(c) K88 pili and plasmid-mediated pili of the antigenic type expressed by 
0159 E. coli strain E2985/76. 
A further important embodiment of the invention is a process for the 
preparation of an oral product for humans, capable of imparting passive 
protection against E. coli bacteria implicated in causing gastroenteric 
disease in humans, in which process a milk-producing host mammal selected 
from the group consisting of the Bovidae is immunised with a vaccine 
comprising antigens of at least one E. coli strain selected from each of 
the following groups: 
(a) a Type 1 pili expressing E. coli of the serogroups 018 and 0125, 
(b) a CFA I pili expressing E. coli of the serogroups 025 and 078, and 
(c) a CFA II pili expressing E. coli of the serogroups 06 and 08, 
to induce the host mammal to produce substantial quantities of 
immunoglobulins specific to the antigens, milk from the host mammal is 
collected, and the immunoglobulins are recovered in functional form from 
the milk and formulated into an orally ingestable product in an amount 
sufficient to provide passive protection. 
Pili are proteinaceous features on the exterior of bacterial cells that are 
in some way associated with the ability of the living bacteria to cling to 
the gut wall. Under the electron microscope, pili appear as spine-like 
projections on the surface of the bacterium. The expression of pili types 
CFA (Colonisation Factor Antigen) I and CFA II and K88 appear to be 
dictated by plasmid-born genetic information, and for this reason this 
characteristic appears to be transmissable from one strain to another. 
However, as far as we are aware a given E. coli strain will express CFA I 
pili, or CFA II pili or K88 pili but not two or more types simultaneously. 
Many E. coli strains do not express any of these types. In view of the 
transmissible nature of this characteristic, and hence the fact that a 
given strain of E. coli that may previously have been identified as 
expressing for example CFA I pili may later lose the relevant plasmid-born 
genetic information and therefore cease to express such pili, it is 
important to check positively by regular tests, e.g. using antisera as 
described below, that the relevant strains involved in vaccine production 
are indeed still exhibiting their essential characteristics. 
The expression of Type I pili appears to be dictated by chromosomal genetic 
information, and this characteristic is not transmissable from one strain 
to another. Some E. coli strains express Type I pili only and some E. coli 
strains that normally express CFA I, CFA II or K88 pili also express (or 
at least have the potential to express) Type I pili. In the art, Type I 
pili are sometimes referred to as "Common pili"; when first identified, 
they were found to be common to more than one E. coli serotype. 
These four classes of pili (Type I, CFA I/II and K88) are familiar 
bacterial characteristics and are fully described in the scientific 
literature. For example, Type I pili are described by Brinton, C.C. in 
Nature, 1959, Vol. 183, pages 782-786 and further by Brinton in Proc. 13th 
Joint US/Japan Conference on Cholera, (1978) NIH Bethesda 78-1590 pages 
33-70. CFA I pili are described by Evans, D. G. et al. in Infection and 
Immunology, 1975, Vol. 12, pages 656-667. CFA II pili are described by 
Evans D. G. and Evans D. J. in Infection and Immunology, 1978, Vol. 21, 
pages 638-647. K88 pili are described in Orskov, I. and Orskov, F. in J. 
Bacteriol, 1966, Vol. 91, pages 66-75 and by Stirm et al. in J. Bacteriol, 
1967, Vol. 93, pages 740-748. 
The term "antigen" is used herein to mean any antigenic material naturally 
generated by bacteria in the live state. Such material can be present on 
the exterior of the bacterium, excreted by the living organism, or can 
normally be present only within the body of the organism. It will be 
appreciated that the vaccine should not comprise viable pathogenic 
organisms, and hence the usual way in which an appropriate vaccine will be 
produced will include the step of killing, or at least attenuating, the 
pathogens so as to render them effectively harmless. This step will also 
often lead to substantial release of antigenic material from physical 
association with the bacterial cells. For example, killing the bacteria by 
means of heat causes the release of large quantities of pili and 
endotoxins from the bacterial cell. Such released antigenic material can 
be used as the sole active constituent of the vaccine if desired, but the 
separation of the killed or attenuated bacterial cells is not strictly 
necessary and indeed the cell debris will generally contribute usefully to 
the antigenic character of the vaccine. Whatever the composition of the 
vaccine, it is most preferable that it should include pili material. 
There are various options open for exposing the host animal to the antigens 
in order to promote the production of appropriate antibodies. One method 
is to infect the gastro-intestinal system of the host animal with one or 
more strains of E. coli that are implicated in causing gastro-intestinal 
infections in the human. However, in view of the species specificity of 
most bacterial strains, and for general health reasons, such a procedure 
is not particularly desirable. 
Alternatively, killed or inactivated bacteria, and/or antigens released 
from the bacteria, can be administered, in an appropriate carrier or 
diluent, orally to the host animal in order to promote an appropriate 
response by the host's immune system. For example, endotoxins can be 
released from the relevant bacteria when they are killed by means of heat, 
and the endotoxins can for example be incorporated in a feedstuff for the 
host animal. The killed bacteria can also be incorporated in the diet to 
enhance the immune response further. Alternatively, the vaccine can be 
presented in the form of an oral medicament such as a pill, capsule, 
powder or liquid. An aqueous solution or suspension of antigenic material 
can be used. Relatively large doses of vaccine can be administered orally 
without risk. 
A third alternative is to administer parenterally an injectable composition 
containing killed or inactivated bacteria and/or antigens released 
therefrom, in a pharmaceutically acceptable carrier or diluent, such as 
water. The presence of this composition in the body of the host will also 
promote an appropriate immune response and result in the production of the 
required antibodies. Injection tends to produce a more immediate and 
efficient response, although the magnitude of the dose may need to be 
limited due to sensitivity of the host animal. The injection can be 
effected by any convenient route, such as intravenous, intramuscular, 
subcutaneous or intramammary. The vaccine composition can include a wide 
variety of standard injectable vaccine adjuvants, such as gums and 
proteins, inorganic adsorbents such as aluminium hydroxide, and oil-water 
emulsions such as Freund's adjuvant (preferably in its incomplete form). 
These adjuvants can enhance the efficacy of the vaccine response or 
provide a delayed release to prolong the effect of the injection. The 
vaccine can also incorporate preservatives, such as phenol or formalin. 
When an injectable or oral vaccine is used to immunise a milk-producing 
mammal from whose milk it is intended to recover the required 
immunoglobulins, it is preferable that the immunisation should be 
performed, or at least begun, prior to parturition. Administration of the 
vaccine should be timed ideally such that the host mammal produces a high 
level (titre) of specific antibodies during colostrum formation. An 
optimum immunisation schedule in the cow will include giving at least one 
parenteral administration prior to parturition, preferably about 2-3 weeks 
in advance. 
A useful manner of administering the vaccine to the host animal is by 
feeding the host animal on a diet containing the vaccine and periodically 
boosting the immune response by supplementary parenteral administration. 
The bacterial strains used in preparing the vaccine of the invention can 
conveniently and economically be selected from the many strains that have 
been implicated in causing actual instances of gastroenteric disease. Many 
samples of such strains are held by hospitals, research institutions and 
public health laboratories throughout the world, and bona fide workers in 
this field can have access to such samples readily. Hence there is no 
difficulty whatsoever in obtaining appropriate bacterial strains from 
which to prepare the vaccine. However, the invention is not necessarily 
limited to the use of such naturally-occurring disease-causing organisms, 
and the current advances in bacterial fermentation and genetic 
manipulation have made it possible for "synthetic" micro-organisms to be 
prepared possessing the essential criteria needed for the invention. This 
would be particularly easy as far as the plasmid-born pili expression 
criteria are concerned. Nevertheless, in terms of vaccine efficacy, it is 
still preferable to use naturally-occurring bacterial strains possessing 
the required criteria, because by so doing the vaccine is likely to induce 
the host to generate antibodies having specificities to other 
characteristics of the bacteria that are also related to their 
disease-causing properties, especially enterotoxins. Such additional 
characteristics might be lacking in "synthetic" organisms. 
It is therefore a preferred feature of the invention that at least one, and 
more preferably more than one, of the bacterial strains used to prepare 
the vaccine are naturally-occurring strains that have been implicated in 
causing gastro-enteric disease in humans. 
A preferred embodiment of the invention is an injectable or oral vaccine 
comprising, in a pharmaceutically acceptable carrier or diluent, antigens 
of at least one strain of Type I pili-expressing E. coli of the serogroups 
018 and/or 0125. 
Preferably, the vaccine also includes antigens of at least one bacterial 
strain from one (more preferably both) of the following groups: 
(a) CFA I pili-expressing E. coli of the serogroups 025 and/or 078; 
(b) CFA II pili-expressing E. coli of the serogroups 06 and/or 08. 
A particularly preferred embodiment of the invention is an injectable or 
oral vaccine comprising, in a pharmaceutically acceptable carrier or 
diluent, antigens of an 018 E. coli expressing Type I pili, an 078 E. coli 
expressing CFA I pili and an 06 E. coli expressing CFA II pili. 
Preferably, the vaccine includes antigens of an 0149 E. coli expressing K88 
adhesion factor. An additional benefit to be obtained from including such 
antigens is that 0149 E. coli are generally observed to be strong 
producers of both LT and ST toxins, and hence such a vaccine should lead 
to antibodies that are particularly effective against toxin-producing 
bacterial strains. 
It is also preferable that the vaccine should include antigens of a human 
gut adherent 0159 E. coli or another E. coli serotype expressing an 
antigenically-identical pili type. This serogroup exhibits a mode of gut 
adhesion that cannot be antigenically associated with any of the above 
pilus types, although such surface features are evident on the bacterium. 
This previously unidentified pilus type is also apparently 
plasmid-mediated and hence probably transmissable. The particular strain 
of 0159 E. coli with which we have worked was obtained from the Central 
Public Health Laboratory, London, and is described by McConnell, M.M. et 
al. in J. Bacteriol, 1979, Vol. 139, pages 346-355. According to 
McConnell, the strain was isolated in Canada and reference is made to 
Gurwith M. J. et al. in Arch. Intern. Med., 1977, Vol. 137, pages 
1461-1464. It is identified by the Central Public Health Laboratory 
designation E2985/76. This particular strain has been deposited by 
McConnell in the National Collection of Type Cultures (NCTC), Central 
Public Health Laboratory, 175 Colindale Avenue, London NW 9 5HT, UK. The 
NCTC deposition number for this strain is 11602. 
Any mammal is a potential host animal for the purposes of the invention, 
but it is most preferable that members of the Bovidae, especially cows, 
and to a lesser extent other domesticated animals whose milk is 
conventionally used as human food, such as goats, should be employed. 
The immune milk from the host mammal can be fed directly to a human infant 
or adult in order that the recipient can benefit from the immunoglobulins 
therein. The milk can be in its natural state, or can be processed prior 
to consumption provided that such processing does not destroy the 
essential functionality of the immunoglobulins. Controlled pasteurisation 
and concentration (evaporation) are examples of conventional milk 
processing techniques that can be used. The milk can be whole milk, 
skimmed milk, or whey. If serum from the host mammal is recovered as the 
source of the immunoglobulins, the immune serum can also be fed directly 
to a human infant or adult. The immune milk or immune serum can be fed in 
admixture with other materials, and especially with other food 
ingredients, if desired. Indeed, subject to the proviso that the essential 
functionality of the immunoglobulins be maintained, the immune milk or 
immune serum can be incorporated in any human foodstuff in which milk is 
traditionally an ingredient. 
In general, however, it is envisaged that the immunoglobulins will be 
recovered in concentrated form from the immune milk or immune serum, and 
that such recovered immunoglobulins will then be used to provide passive 
immunity. A variety of techniques are now available in the art, by means 
of which recovery of the immunoglobulins can be effected. One such 
technique is to separate an immunoglobulin-rich concentrate from the bulk 
of the milk components, and an example of such a procedure is described in 
UK patent specification No. 1 573 995. An alternative technique is to 
separate immunoglobulins from milk or serum by means of chromatographic 
techniques. Chromatography can provide immunoglobulin-rich fractions in 
which the immunoglobulins are present in relatively pure (or sometimes 
completely pure) form. Affinity chromatography in which the 
immunoglobulins are recovered by being bound to insolubilised antibodies, 
especially mono-specific antibodies (e.g. so-called "monoclonal" 
antibodies) is preferred. Such a procedure is described in European patent 
application No. 0059598. After recovery, the immunoglobulin material 
should be carefully stored prior to use, to preserve its essential 
functionality. Freeze-drying is an example of a useful technique for 
rendering the recovered immunoglobulin material storage-stable. 
The recovered immunoglobulins can be incorporated in a human foodstuff. 
Potentially, any foodstuff that does not require subsequent processing 
(e.g. cooking) which would denature the functional immunoglobulins, can be 
used as a carrier. A particular embodiment of the invention is an 
artificial "milk" product, especially such a product intended for 
consumption by human infants. In general such products are marketed in the 
form of dry powders and require reconstitution with water to yield a 
milk-like liquid ready for consumption. Apart from the incorporation of 
the immunoglobulin material, the composition of the foodstuff need not 
differ in any way from conventional compositions. By way of example only, 
such compositions can be based on milk solids, e.g. skimmed milk powder 
and/or whey powder, together with non-milk materials, or can be 
formulation entirely from non-milk materials. An example of the latter 
type of formulation is set out in Example 7. The quantity of functional 
immunoglobulin material incorporated in the food product is not critical, 
as long as sufficient is provided in the digestive tract to cause a 
protective benefit. The minimum effective content in the food product will 
depend on the functionality of the immunoglobulin material and the 
quantity of the foodstuff that is likely to be consumed. The minimum 
effective content can readily be ascertained by one skilled in the art. As 
the immunoglobulin material itself is proteinaceous and entirely harmless 
to the human consuming it, there is no upper limit on the inclusion level 
in the foodstuff, other than the constraints imposed by economics. 
As an alternative to artificial milk products, the immunoglobulins can, for 
example, be incorporated in powdered beverage bases such as soft drink 
products. Such products will be reconstituble with water to provide, for 
example, fruit-flavoured beverages. Typical formulations will be based on 
flavourings such as orange or lemon, plus maltodextrins and sugars. 
The immunoglobulins can also be used to provide passive immunity against 
"traveller's diarrhoea" illnesses acquired whilst visiting foreign 
countries, for example. Indeed, in this context an 
immunoglobulin-containing product of the invention can provide a valuable 
therapeutic benefit in mitigating the effects of any such infection. It is 
envisaged that an oral product containing the immunoglobulins, for example 
in the form of pills or capsules, if ingested according to a prescribed 
schedule, will maintain a protective level of immunoglobulins in the 
digestive tract of the traveller. Any conventional medicinal encapsulation 
method can be used, e.g. sugar pills or gelatin capsules. 
The bacterial antigen vaccine of the invention can also be used to promote 
active immunity in the human by being administered directly, so 
stimulating the natural immune system of the human recipient. In this 
context, the vaccine can have several modes of application, depending on 
its precise purpose. For example, as a measure against endemic or epidemic 
gastro-enteric diseases, the vaccine can be generally administered orally 
and/or parenterally to adults and infants. If used as a safeguard against 
gastro-enteric diseases encountered by an individual when travelling to an 
unusual location, the vaccine can be administered as a single or multiple 
injection and/or oral inoculation suitably timed prior to the journey. If 
used as a means of reducing the incidence of neonatal infection in human 
infants, the vaccine can be administered to the expectant mother on one or 
more occasions suitably timed during pregnancy so that at birth the mother 
is producing enhanced amounts of antibodies and hence the colostrum will 
contain unusually high levels of antibody. In this last embodiment of the 
invention, the unborn child will also be receiving enhanced antibody 
levels because in the human the antibodies are transmitted to the foetus 
via the placenta. 
The following procedures can be used to identify strains of 
enteropathogenic bacteria appropriate for use as the basis of a vaccine in 
accordance with the invention. These are given by way of example only, as 
the skilled reader will recognise that such procedures can be modified 
readily in detail to suit individual laboratory practice and the 
availability of equipment and other facilities. 
Expression of pili 
In the first instance the presence or absence of pili on any given strain 
of bacteria can be determined by examining specimens of the bacteria by 
means of an electron microscope. At a magnification of about 20,000.times. 
any pili expressed by a bacterium will be clearly visible and will give 
the bacterium a characteristically "spiny" or "hairy" appearance. In 
contrast, at such magnification a smooth exterior on the bacterium will be 
indicative that the specimen is not expressing any pili. 
Having established that pili are present, it is necessary to determine 
whether any of the required types is being expressed. 
Various crude methods for differentiating between known pili types, such as 
mannose sensitivity tests and other erythrocyte agglutination procedures, 
are described in the literature, but for the present purposes we do not 
consider such procedures to be sufficiently accurate. The natural 
occurrence, for example, of bacterial strains possessing previously 
unidentified adhesins introduces ambiguity into such procedures. Instead, 
we recommend the positive identification of pili types by means of 
antisera.

Indeed, most workers will prefer to develop antisera to pili types in order 
that the identification of subsequent strains can be performed more 
readily. Suitable antisera can be easily prepared once definitive samples 
of the pili types have been obtained. The following Example illustrates 
the basic procedures entailed. 
EXAMPLE 1 
Purified pili were prepared as follows from known pili-bearing strains of 
E. coli that had been obtained from external reference collections or 
other reputable sources. 
The bacteria were grown in roux flasks for 48 hours at 37.degree. C. on CFA 
agar. CFA agar consists of 1% casamino acids (Difco), 0.15% yeast extract 
(Difco), 0.005% magnesium sulphate and 0.0005% manganese chloride plus 2% 
agar at pH 7.4. This medium is described in Evans et al. (Infection and 
Immunology, 1977, Vol 23 p 330). The bacteria were harvested and washed in 
sterile phosphate buffered saline (PBS). Pili were heat-stripped from the 
bacteria at 60.degree. C. for 45 minutes in the case of CFA I, CFA II, K88 
pili and pili expressed by 0159 E. coli strain E2985/76 referred to 
earlier. In the case of Type I pili the bacterial suspension was 
additionally disrupted for 1 minute using a homogeniser. The whole 
bacteria and cell debris were centrifuged at 1000.times. g, leaving pili 
in the supernatant liquor. The supernatant liquor was adjusted to pH 4.5 
by the addition of acetic acid and left for several days at 4.degree. C. 
to precipitate the pili. The resultant precipitates were recovered by 
centrifugation at 35,000.times.g and resuspended in PBS. Electron 
microscopic examination revealed the presence of large numbers of pili. 
Antisera were prepared in rabbits against the purified samples of pili. 
Taking the CFA I pili as an example, a total of 2 mg of purified pili were 
injected subcutaneously in multiple sites in each rabbit using Freund's 
complete adjuvant. After 4 weeks a boosting injection of 1 mg of pili, 
again in multiple sites in each rabbit using Freund's incomplete adjuvant, 
was made. Bleeding was performed after a further two weeks. Absorption to 
yield monospecific anti-pili sera was carried out using roux flask 
cultures of non-piliated variants of the three bacterial strains from 
which the original purified pili samples had been obtained. In this 
procedure equal volumes of centrifuged, washed bacteria and the sera 
obtained from the rabbits were incubated together for 15 minutes at 
ambient temperature and then spun to collect the supernatant. 
The resulting mono-specific anti-pili can be used to identify the pili 
types on further bacterial strains by means of standard bacterial slide 
agglutination tests. 
Enterotoxin production 
The identification of strains of enteropathogenic bacteria that produce an 
abundance of toxins can be effected by obtaining a cell-free preparation 
of enterotoxins from the bacteria under test and then examining for toxin 
activity using the suckling mouse assay and the Chinese hamster ovary cell 
assay. 
(a) Preparation of enterotoxins 
ST toxins are usually obtained from culture supernatants and LT toxins from 
culture supernatants or whole cell lysates. There are also published 
procedures available for the purification of LT and ST toxins, but for the 
determination of the enterotoxicity of E. coli strains by the suckling 
mouse and the Chinese hamster ovary cell assays separation and 
purification of the enterotoxins is unnecessary and, as the following 
example shows, 18-hour culture supernatants of strains grown in media such 
as Brain Heart Infusion (Oxoid CM225) or synyeast can be used for both 
tests. 
EXAMPLE 2 
Synyeast is a semi-synthetic medium comprising 20 g casamino acids, 6.0 g 
yeast extract, 2.5 g sodium chloride, 8.71 g dipotassium hydrogen 
phosphate, (0.05 M), and 1.0 ml of trace salts solution dissolved in 
almost a liter of distilled water, adjusted to a pH of 8.5 with 0.1 N 
sodium hydroxide and brought to a final volume of 1 liter. The trace salts 
mixture consists of 5.0% magnesium sulphate, 0.5% manganese chloride, and 
0.5% ferric chloride dissolved in 0.001 N sulphuric acid. 
Appropriate volumes of the media were dispensed into Erlenmeyer flasks and 
sterilised at 121.degree. C. for 15 minutes. The flasks were inoculated 
with starter broth cultures and the bacteria grown aerobically at 
37.degree. C. in a shaking water bath for 18 hours. The cultures were then 
centrifuged to remove the bacteria, and the supernatants containing the 
enterotoxins poured off and sterilised by millipore filtration. 
(b) Heat-stable (ST) toxins 
The production of ST toxins by a strain of bacteria can be identified by 
the following procedure. This technique is based on that of Dean et al. 
(J. Infect. Dis., 1972, Vol 125 p 407). 
EXAMPLE 3 
3-day old mice were separated from their mothers shortly before use and 
divided randomly into groups of 4. The infant mice were injected with 0.1 
ml of test material through the body wall directly into the milk filled 
stomach. Any necessary dilutions of the samples to be tested were done 
using PBS. One drop of 1% pontamine blue made up in PBS was added to each 
0.6 ml of the inoculum and results from mice with no dye in the intestinal 
tract at autopsy were discarded. After injection the mice were kept for 4 
hours and then killed with chloroform. The abdomen of each mouse was 
opened, the intestines were examined for distention and then removed. The 
intestines of the 4 mice in each group were weighed together and the ratio 
of total gut weight to total remaining body weight calculated. A ratio of 
greater than 0.09 was considered positive, less than 0.07 negative and 
between 0.07 and 0.09 questionably positive. 
(c) Heat-labile (LT) toxins 
The production of LT toxins can be determined by the following procedure. 
This is an adaptation of the procedure described by Guerrant et al. 
(Infection and Immunology, 1977, Vol 10 p 320) which in turn was based on 
the observation by Hsie et al. (Proc. Nat. Acad. Sci. USA, 1971, Vol 68 p 
358) that the Chinese hamster ovary clonal cell line CHO-Kl responds with 
distinct morphological and biochemical changes after treatment with cyclic 
adenosine monophosphate (AMP). 
EXAMPLE 4 
Stock cultures of CHO-Kl were grown in Ham's F12 medium supplemented with 
10% foetal calf serum and 1% glutamine in an atmosphere of 5% carbon 
dioxide in air at 37.degree. C. The cell line was passaged by 
trypsinization with 10% (v/v) trypsin solution in Earles Balanced Salt 
Solution (BSS) for 15 minutes at 37.degree. C. after washing the monolayer 
with Earles BSS without calcium and magnesium. The trypsinized cells were 
resuspended in growth medium. CHO-Kl cells, Ham's F12 medium and Earles 
BSS are obtainable from Flow Laboratories. For assay, cell suspensions 
containing approximately 1,000 cells per 0.02 ml in F12 medium plus 1% 
foetal calf serum were added to each well of a 96-well micro culture 
plate. Enterotoxin solution (10 micro liters per well) was added 
immediately after plating. The plates were incubated for 20 hours in an 
atmosphere of 5% carbon dioxide in air at 37.degree. C. and then fixed 
with methanol for 2 minutes and stained with Giemsa diluted 1:1 with 
distilled water. The action of LT enterotoxin induces over 40% of the 
cells to transform from an epithelial-like cell to a fibroblast-like cell, 
and hence by counting the number of cells elongated an estimate can be 
made of the toxins produced by the strain. 
Vaccine production 
The following example illustrates the production of a vaccine for use in 
accordance with the invention. 
EXAMPLE 5 
A considerable number of strains of E. coli implicated in causing 
gastro-enteric diseases were obtained from numerous sources. Such strains 
can be readily obtained by bona fide workers in this field from hospitals, 
public health laboratories and academic institutions. The various strains 
were subjected to the test procedures already described to determine 
whether they expressed particular pili types and were good toxin 
producers. The following strains were selected as the basis for a vaccine. 
TABLE 1 
______________________________________ 
E. coli 
Serogroup 
Adhesion Enterotoxin 
______________________________________ 
018 Expresses Type I pili 
-ve 
078 Expresses CFA I pili 
+ve(ST + LT) 
06 Expresses CFA II pili 
+ve(ST + LT) 
0149 Expresses K88 pili +ve(ST + LT) 
0159 Expresses human gut 
+ve(ST + LT) 
adhesive pili antigenically 
distinct from the above types. 
______________________________________ 
All 5 strains were cultured on a standard broth and then subjected to a 
heat strip at 60.degree. C. for 45 minutes to release pili and other 
useful antigenic factors and hence to maximise response to the vaccine 
when administered. The vaccine was preserved by the addition of 0.5% 
formalin. 
The vaccine was administered continuously to pregnant cows as part of their 
diet for a period of six weeks immediately prior to the expected calving 
date. The oral daily dose was 50 gms of a premix spread onto a standard 
feed concentrate. The premix comprised by weight: 
57% wheat flour (9% moisture) 
47% cheese whey powder 
4% citric acid 
2% centrifuged bacterial slurry containing 20 HI units of each strain per 
gm of premix. 
During the same period each cow was injected intramuscularly in the hip 
region on three occasions (6 weeks, 4 weeks and 2 weeks prior to 
parturition) with an injectable vaccine containing a total of 200 
Haemagglutination Inhibition units of bacterial matter. 
The antibody titre of the milk from each cow over the first four days of 
lactation was assayed and a clear O-antigen response to each strain 
injected was observed, indicating that the specificity of the natural 
antibodies in the bovine milk had been altered by the vaccine. The results 
are given in Table 2 below: 
TABLE 2 
______________________________________ 
Antibody titres engengered in the early 
milk of a cow immunized multiparenterally 
______________________________________ 
E.coli serotype 
06 018 078 0149 0159 
Vaccinate 1000 256 512 4000 64 
titre 
Control 8 4 16 4 4 
titre 
The immune milk was also demonstrated to be 
bacteriostatic, and to inhibit bacterial adhesion 
in vitro. 
______________________________________ 
EXAMPLE 6 
An antibody-rich concentrate was prepared as follows: 
Immune bovine milk obtained as in Example 5 was adjusted to pH 3.5 by the 
addition of hydrochloric acid in order to precipitate casein. Following 
centrifugation, the resulting supernatant liquor was adjusted to neutral 
pH by the addition of sodium hydroxide, and an immunoglobulin fraction 
precipitated by the addition of 40% aqueous ammonium sulphate. The 
precipitate was centrifuged, resuspended in PBS, subjected to exhaustive 
dialysis to remove ammonium sulphate, and freeze dried. 
EXAMPLE 7 
An edible product capable of imparting passive immunity against human 
enteropathogens was prepared as follows: 
An antibody concentrate obtained as in Example 6 was added to a 
commercially-available powdered milk substitute for human infants having 
the following composition: 
______________________________________ 
Ingredient Parts by weight 
______________________________________ 
Corn syrup solids 26 
Sucrose 26 
Soy protein isolate 
17.5 
Corn oil 13.9 
Coconut oil 13.9 
Calcium phosphate tribasic 
1.5 
Potassium citrate 0.7 
Potassium chloride 0.6 
Magnesium chloride 0.3 
Ascorbic acid 0.1 
Trace elements, vitamins, etc. 
0.1 
______________________________________ 
The antibody concentrate was included in the formulation at a level 
sufficient to impart to the milk substitute (when reconstituted with water 
to a drinkable form) and O-antigen titre of 1 in 256 serotype. 
EXAMPLE 8 
Evaluation of the therapeutic effect of passively administered bovine 
antibody in the mouse infection model 
As many human pathogenic E. coli will colonise and proliferate in the mouse 
intestine, it is possible to use the mouse as a protection model for the 
human infant. The 018 serogroup E. coli adhere very strongly to mouse 
enterocytes, so these were chosen for study, as there seemed to be little 
chance that they would clear spontaneously. Infections were established in 
the mice, and bovine serum antibodies were then administered in an attempt 
to clear this. The antibodies were administered orally as neat serum. 
Materials and Methods 
Bovine antisera 
The calf was used to raise hyperimmune serum separately to each of the E. 
coli strains selected in Example 5. Calves of approximately 8 weeks of age 
were given 6 intramuscular injections of 0.1 ml of killed bacteria 
(dose=approximately 200 HIU), one injection being given weekly for 6 
weeks. Blood was collected 2 weeks later by jugular venupuncture. Sera 
were separated and stored at -20.degree. C. 
Mice 
6-8 weeks old germ-free Balb-C mice were used throughout. 
Procedure 
Groups of mice were each orally inoculated with 0.1 ml of a 10.sup.8 /ml 
overnight nutrient broth culture of individual strains, and kept in a 
monocontaminated state in sealed cages for 10 days, to allow the bacteria 
to become established in the intestine. 
Faecal samples were taken and viable counts made during this time to verify 
the monocontaminated state. The mice were kept in sealed cages until day 
7, when samples were taken and the cages left unsealed. Antibody treatment 
began on day 8. 
Control Group - the mice were given no bovine antibodies. 
Test Group - each mouse was given 0.1 ml of bovine anti-serum three times a 
day. 
Further faecal samples were taken and viable counts made to monitor the 
progress of the infection. 
Dose rates 
The bovine anti-serum had haemagglutination titres of 1000 HIU. The group 
of mice receiving three 0.1 ml oral doses daily were thus each ingesting 
300 HIU/day. 
Results 
It can be seen from Table 3 that the group of mice being dosed three times 
daily with bovine anti-sera, cleared of infection within 5 days in each 
case. The control group maintained a high level of infection throughout 
the experiment. 
TABLE 3 
______________________________________ 
Days after 
Days after 
commencement 
infection 
of dosing Group 018 Count 
______________________________________ 
10 2 Control 9.0 .times. 10.sup.7 
Test 1.5 .times. 10.sup.8 
12 4 Control 6.0 .times. 10.sup.6 
Test 3.0 .times. 10.sup.3 
15 7 Control 1.5 .times. 10.sup.6 
Test 0 
______________________________________ 
Bovine anti-sera had been shown to block adhesion to mouse enterocytes in 
vitro, and it seems probable that this is an important factor in clearing 
the infection. Adult mice are not sensitive to the two toxins ST and LT, 
so any anti-toxic activity the sera may have is irrelevant in the mouse 
infection model. The sera had been shown to have anti-O and anti-pilus 
activity (by haemagglutination assay and slide agglutination tests 
respectively); as the anti-sera were raised against a slurry of whole 
bacteria it would seem likely that there are also antibodies to various 
other bacterial components. The clearing phenomenon was probably caused by 
a combination of these effects. 
EXAMPLE 9 
Evaluation of the therapeutic effect of passively administered bovine 
antibody in the pig infection model 
One of the human pathogenic serogroups of E. coli, 0149, is also pathogenic 
in pigs. This serogroup is one of those included in the vaccine of Example 
5, and the pig provides a useful animal model in which to evaluate bovine 
antibody products raised in response to this vaccine. The following 
experiment was an attempt to protect neonatal piglets from 0149 infection 
by oral immunisation with bovine antibodies. 
Materials and Methods 
Bovine anti-0149 serum 
The calf was used to raise hyperimmune serum to 0149. Calves at 
approximately 8 weeks of age were given 6 intramuscular injections of 0.1 
ml of killed bacterial (dose=approximately 200 HIU) one injection weekly 
for 6 weeks. Blood was collected 2 weeks later by jugular venupuncture. 
Sera were separated and stored at -20.degree. C. 
A haemagglutination assay performed on the serum showed it to have an 
anti-0149 titre of 1000. One piglet dose consisted of 5 ml of serum, i.e. 
5000 H.A. units. 
Procedure 
Samples of serum and colostrum were taken from a gilt and a 
haemagglutination assay performed to determine their anti-0149 activity. 
Both samples had an anti-0149 titre of 16 HIU, and this was deemed to be 
sufficiently low to allow the piglets to suckle, without raising their 
serum antibody titre to a level high enough to invalidate the experiment. 
The piglets were left on the gilt for 6 hours. 8 piglets were selected 
and marked into two groups of four, one group being a control group, and 
the other receiving anti-0149 serum. This group was dosed with serum every 
2 hours during the suckling period. 
All piglets were then removed from the gilt and each was given an oral 
infecting dose of 10.sup.8 0149 E. coli. Serum dosing of one group 
continued at two hourly intervals for a further two days. The interval 
between doses was then gradually increased over the following. 3 days, 
after which time it was stopped altogether. Faecal swabs were taken daily, 
and were plated out onto blood agar. The piglets were weighed daily. 
Results 
The groups being dosed with bovine anti-0149 serum gained weight from the 
onset of the experiment, even on the first day after the shock of 
separation and the change to a new diet. The control group, however, lost 
weight on day 2, the day after separation, and all were dead on day 3. The 
swabs showed that all of the dead piglets intestines were colonised by 
large numbers of 0149, whereas at this time, the group being dosed showed 
very little 0149 on swabs taken. A post-mortem performed on one of the 
dead piglets showed the intestines to be swollen and fluid-filled, with 
some haemorrhaging having occurred; symptoms typical of gastro-enteritis. 
The group being dosed with antibodies continued to gain weight and had all 
the appearance of normal healthy piglets. Swabs showed the infection to 
have cleared altogether 4 days after the infection date. Weighing and 
swabbing were then discontinued: however, weekly inspection of the pigs 
showed them to be healthy and progressing well. 
TABLE 4 
______________________________________ 
Piglet weights in kg during 0149 protection experiment 
______________________________________ 
Dose Group Control Group 
(Piglets A-D) (Piglets E-H) 
Day A B C D E F G H 
______________________________________ 
1 1.55 1.6 1.45 1.30 1.1 1.3 1.3 1.45 
2 1.60 1.7 1.50 1.25 1.1 1.3 1.15 * 
3 1.65 2.15 1.75 1.35 0.9* 1.1* 0.95* 
4 1.90 2.30 1.80 1.50 
5 2.20 2.45 2.00 1.70 
______________________________________ 
The symbol * denotes a death. 
TABLE 5 
______________________________________ 
Presence of 0149 in faecal swabs 
______________________________________ 
Dose Group Control Group 
(Piglets A-D) (Piglets E-H) 
Day A B C D E F G H 
______________________________________ 
1 - - - - - - - - 
2 - ++ - + + ++ ++ * 
3 - + - + 
4 + + - - 
5 - - - - 
______________________________________ 
+ = 10-20 0149 colonies 
++ = confluent 0149 growth 
* = a death 
- = no 0149 colonies 
This experiment demonstrated that bovine antibodies afford protection to 
the neonatal pig. The serum may have anti-toxic activity; this would 
explain the survival of the pigs, but not the clearing of the infection. 
Anti-adhesive activity would prevent colonisation of the intestine, as 
would the ability to agglutinate the bacteria. Previous work had shown 
that bovine anti-serum raised against 0149 has both anti-K88 and anti-0149 
activity (shown by slide agglutination and haemagglutination assay 
respectively). It had also been shown to block adhesion to pig enterocytes 
in vitro, and to be bacteriostatic. It has also been shown that 
oral/parenteral immunisation with antigens from heat-inactivated E. coli 
can give rise to anti-enterotoxin antibodies. It would seem probable that 
a combination of all of these factors is responsible for protection of the 
neonatal pig from infection. In a similar control experiment, piglets 
which were dosed with non-immune bovine serum rapidly succumbed to 
infection and died within three days, thus clearly demonstrating that 
normal bovine serum has no protective action in the piglet.