WO2008148149A1

WO2008148149A1 - Method of treatment or inhibition of enterovirus 71 infection

Info

Publication number: WO2008148149A1
Application number: PCT/AU2008/000786
Authority: WO
Inventors: Grant Thomas Rawlin; Roy Michael Robins-Browne; Zeil Rosenberg
Original assignee: Anadis Ltd
Priority date: 2007-06-05
Filing date: 2008-06-03
Publication date: 2008-12-11
Also published as: TW200918552A

Abstract

A composition for oral dosage for passive immunization of a subject against EV71 comprising polyclonal antibodies and a method for raising polyclonal antibodies against EV71 antigen in hyperimmune ungulate colostrum or hyperimmune avian eggs.

Description

METHOD OF TREATMENT OR INHIBITION OF ENTEROVIRUS 71 INFECTION

Field

[0001 ] This patent relates to a method of treatment or inhibition of infection by Enterovirus 71 and a method of inhibiting the spread of Enterovirus 71 infection within a community. The invention further relates to a composition for inhibition of Enterovirus 71 infection and a method of preparing such as composition.

Background

[0002] Enterovirus 71 belongs to the human Enterovirus A species of the Enterovirus genus within the family Picornaviridae. It is important to note that EV71 is not a zoonotic agent. EV71 is the causative agent of a number of neurological diseases including aseptic meningitis, encephalitis and polio-like paralysis. This virus is also associated with large outbreaks of hand foot and mouth disease in humans. Outbreaks of EV71 associated illness are common in Asian countries such as Taiwan, Malaysia, Singapore, Japan, Korea, Thailand, Vietnam, Hong Kong and even Australia.

[0003] The largest EV71 outbreaks have occurred in Bulgaria in 1975 with 44 deaths; Hungary in 1978 with 45 deaths; Malaysia in 1997 with 30 deaths; and in Taiwan in 1998, 2000 and 2001 with 78, 25 and 26 deaths respectively. The 1998 outbreak in Taiwan involved over 130,000 reported cases, including over 400 severe neurological cases and 78 deaths, of which 91 % were children under five years of age. There have also been more recent outbreaks in early 2005 in Hong Kong, Vietnam and Taiwan in which a combined total of 31 people died and in Malaysia in early 2006 with 8 deaths. Considerable public concern has been raised about EV71 because of its high incidence of mortality and because it's victims are commonly young children. [0004] The methods which have previously been used to control clinical disease from epidemic pathogens include:

o Isolation of infected individuals o Encouraging handwashing and toy washing o Avoiding crowds o Palliative care o closing schools

(Ref: Communicable Diseases Centre, Taiwan, public relations brochure, 1999.)

[0005] US Patent Publication 2006/0292693 provides a monoclonal antibody for veutralising EV71 which is used in passive vaccination by parenteral administration to patients. In order to reduce immune response in patient bloodstream the monoclonals are humanised. The vaccine preparation is therefore relatively expensive and the requirement for parenteral administration makes it cumbersome to ensure that making it difficult to provide economic population control. Also as EV71 infection originates in the gut US 2006/0292693 addresses the more severe systemic implication of EV71 infection rather than the source of infection.

[0006] However it is preferable to develop population immunity to the pathogen while avoiding clinical cases. It is important to decrease transmission rates between people, in order to avoid a widespread epidemic. The use of bioactive agents which a) decrease the rate of infection within the healthy population and b) decrease the contamination of the environment by infected people, is highly desirable.

[0007] Further often vaccines are not recommended to be used in children under 13 months of age due to a paucity of acceptable safety data. Likewise for most vaccines, correlates of immunity develop gradually and achieve adequate protection only after multiple doses. [0008] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Summary

[0009] We provide, in one embodiment, a composition for oral dosage for passive immunization of a subject against EV71 comprising polyclonal antibodies, from ungulate colostrum or avian egg, raised against EV71 antigen.

[0010] In one embodiment we provide a method of passive immunisation of subjects against EV71 comprising: providing a hyperimmune material comprising polyclonal antibodies raised by inoculation of a bovine animal or bird with EV71 and orally administering the polyclonal antibodies to the subjects.

[0011 ] The EV71 vaccine used to inoculate the animals will generally be selected from the group consisting of inactivated EV71 vaccine or an antigenic capsid protein of EV71 such as capsid proteins VP1.

[0012] In another embodiment we provide a method of preparing a passive oral vaccine for immunising subjects against EV71 infection the method comprising: immunising animals selected from bovine and avian animals with an EV71 antigen ;and collecting hyperimmune material selected from bovine colostrum and avian eggs from the animals said hyperimmune material comprising polyclonal antibodies to an EV71 antigen. The polyclonal antibodies are preferably used in an oral dosage form such as a food or food additive, a tablet, syrup, capsule or the like. [0013] In another embodiment there is provided use of polyclonal antibodies raised against an EV71 antigen in preparation of an oral vaccine for passively immunising subjects against EV71 infection by forming an oral dosage form of the polyclonal antibodies, said oral dosage form preferably containing bovine colostrum, for oral administration to the subject.

[0014] In another embodiment there is provided use of EV71 in preparation of a passive vaccine comprising polyclonal antibodies for oral administration for immunising a subject against EV71 infection wherein the polyclonal antibodies are prepared by innoculating animals selected from bovine and avian animals with EV71 antigen and collecting material selected from bovine colostrum and avian eggs said material being hyperimmune with respect to polyclonal antibodies to an EV71 antigen.

[0015] The subject of the passive vaccination is preferably a human subject.

[0016] In one embodiment of the invention the passive vaccine is for administration concurrently with active vaccination. Generally speaking active vaccination of an EV71 antigen will require a period of time for immunity to be conferred by the body raising antibodies to the active vaccine. The above described passive vaccine may be used as a measure of inhibiting the infection and spread of EV71 infection during the critical period after infection first becomes apparent.

[0017] In one embodiment, the oral dose form is taken periodically for at least 20 days in a high risk period, where the high risk period may include the period in which the pathogen is abundant, or the period prior to the completion of a course of active vaccines. Preferably the oral dosage form is taken periodically for 40 days. In one preference the oral dosage form is taken at least once a day. In one preference the oral dosage form is taken before during or immediately after a main meal. Detailed Description

[0018] The composition comprises EV71 antigen. The EV71 antigen may, for example, include one or more selected from the group consisting of be inactivated whole virus and one or more EV71 viral capsid proteins such as such as capsid protein VP1.

[0019] The term ungulate refers to four-footed, hoofed, grazing mammal. The biological subfamily bovine includes a diverse group of 10 species of medium-sized to large ungulates, including domestic cattle, Bison, the Water Buffalo, the Yak, and the four-horned and spiral-horned antelopes.

[0020] The term colostrum where used herein includes colostral milk ; processed colostral milk such as colostral milk processed to partly or completely remove one or more of fat, cellular debris, lactose and casein; and colostral milk or processed colostral milk which has been dried by for example, freeze drying, spray drying or other methods of drying known in the art. Colostral milk is generally taken from ungulate mammals such as a cow within five days after parturition. In one embodiment the composition for administration to the patient comprises colostrum in dried form. Other components such as selected from the group consisting of adjutants, carriers , drugs, and other actives may be present in the composition and may be intimately mixed before, during or after the drying process. The composition comprising colostrum may be dried by lyophilisation or other method know in the art for drying colostrum.

[0021 ] The composition for administration to the subject may be the hyperimmune material but may and preferably will be derived from the hyperimmune material. For example in the case of colostrum the composition for administration to the subject may have been processed using a detailing operation, more preferably using a defatting operation and an operation to remove cellular debris, more preferably a defatting operation, an operation to remove cellular debris and an operation to remove salts, sugars, other low molecular weight entities and some water. [0022] Throughout the description and the claims of this specification the word

"comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

[0023] This invention relates to the provision of compositions comprising polyclonal antibodies raised against EV71 antigen. The composition is preferably derived from a hyperimmune material selected from the group consisting of colostrum from ungulate animals and yolk from bird eggs.

[0024] The oral dosage form comprises polyclonal antibodies or binding fragments thereof, where the polyclonal antibody is taken from the colostrum of ungulates or from the yolk of an egg. Preferably the oral dosage form comprises at least partially purified bovine colostrum taken from cows or buffalo which have been vaccinated against at least one antigen from EV 71.

[0025] The vaccine for raising the polyclonal antibodies may be administered to a bovine animal and may contain at least one of inactivated EV71 virus and EV71 capsid proteins such as capsid protein VP1. The composition may contain one or more adjuvants. The adjuvants may be an immunostimulant composition containing esters of octadecenoic acid and anhydromannitol. Examples of other adjuvants include aluminium hydroxide or incomplete Freunds adjuvant or ISCOMS. Preferably the vaccine comprises the Montanide adjuvant, or a similar multiphase adjuvant. For example, the adjuvant may be "Montanide ISA 206 VG", an APVMA approved veterinary adjuvant made by Seppic and supplied by Tall Bennett in Australia. Montanide ISA 206 VG is an immunostimulant composition containing esters of octadecenoic acid and anhydromannitol in an oily solution.

[0026] When the polyclonal antibodies are derived from hyperimmune colostrum it is preferred that the colostrum is collected within 3 days postpartum, preferably one day post-partum and most preferably the first milking.

[0027] Each oral dose form may, for example, comprise the colostrum equivalent of less than 800 mg (dry weight basis), preferably less than 400 mg, more preferably less than 200 mg. By colostrum equivalent we mean the amount of raw colostrum, howsoever purified, which is processed to provide the contents of a dose form.

[0028] In one embodiment the polyclonal antibodies are present in the composition for oral administration in an amount sufficient to provide from at least 1.5% by weight of the composition of immunoglobulin specific to EV71 antigens such as at least 2% or at least 3% up to an amount such as no more than 25%, or no more than 20% by weight of the composition.

[0029] The oral dose form preferably comprises colostrum derived from the hyperimmune colostrum and/or colostrum which has been added to the polyclonal antibodies in accordance with the teaching of PCT/AU03/00348 (Pub. No.: WO/2003/080082). The oral dosage form may also comprise a buffer system such as that disclosed in PCT/AU2005/001746 (Pub. No.: WO/2006/053383). The contents of these patents are incorporated by reference.

[0030] In one preference, the oral dose form is used in conjunction with an active vaccination program involving the use of an active vaccine against the pathogen. Preferably the oral dose form is used in the period prior to the completion of the active vaccination protocol and response.

[0031 ] In one preference the oral compositions comprise polyclonal antibodies to at least one surface antigen of a virus taken from the enterovirus 71 group.

[0032] Preferably the oral dose form is administered periodically for at least 20 days over the period of highest risk eg the summer period, more preferably periodically for at least 40 days, even more preferably periodically for at least 80 days.

[0033] The polyclonal antigen is raised by inoculation of animals selected from ungulate animals and birds with a vaccine comprising an EV71 antigen such as whole inactivated EV71 and/or EV71 capsid protein such as VP1 capsid protein. The antigen may comprise one or more selected from the group consisting of whole inactivated EV71 type B, capsid protein (such as VP1 ) from EV71 type B, whole inactivated EV71 type C and capsid protein (such as VP1 ) from EV71 type C. Preferably the polyclonal antibody is raised against at least one antigen of each of enterovirus 71 type B antigen and one enterovirus 71 type C antigen.

[0034] Preferably the vaccine comprises whole inactivated EV71 virus.

[0035] In another preferment the vaccine comprises inactivated virus and further comprises the surface antigen VP1.

[0036] Preferably the antibodies comprise antibodies raised individually against antigens EV71 type B antigens and EV71 type C antigens.

[0037] The composition may be in a form selected from the group consisting of a food, a food additive, a tablet, syrup, capsule and caplet.

[0038] A composition according to any one of the previous claims in the form of a particulate material for mixing with food or drink.

[0039] A composition according to any one of the previous claims wherein the polyclonal antibodies are present in a carrier derived from colostrum.

[0040] The colostrum used to provide polyclonal antibodies is preferably collected in a separate vessel from each animal and colostrum from each vessel is individually tested before pooling. Preferably the colostrum from each animal is separately frozen. The colostrum may be collected using the method and apparatus described in Pub. No.: WO/2004/107851 (International Application No.: PCT/AU2004/000773) the contents of which are herein incorporated by reference. [0041 ] In one embodiment the neutralisation titre of the pooled dried colostrum against at least one of EV71 B and EV71 C is at least 1 in 20, preferably 1 in 40, more preferably 1 in 100, even ore preferably 1 in 200.

[0042] The neutralisation test involves the "TCI D50" neutralisation assay (TCI D50 = 50% Tissue Culture Infectious Dose). In this assay, a 10% solution of the anti-EV71 colostrum powder is mixed at several dilutions with 100 TCID50 of EV71 virus. For the purpose of reporting neutralisation titre this 10% solution constitutes the zero dilution reference solution (i.e. a 1 in 20 neutralisation titre means that a 1 in 20 dilution (or less) of the 10% solution is required to achieve neutralisation). After a set incubation time, these colostrum and virus mixtures are added to a culture plate that contains living kidney cells, which act as host cells to support the growth of virus. When active virus is in contact with the host cells, it readily infects the cells by attaching to the cell surface and causing changes called cytopathic effect (CPE) that eventually causes the cell to die. CPE are any detectable changes in the cell (due to infection with the virus) and include cell rounding, swelling or shrinking, death or detachment from the surface.

[0043] Preferably the oral dose form of the antibody is presented as a finely divided powder for mixing with food or drink.

[0044] In another preferment the oral dose form of the antibody is presented inside an inclusion which is incorporated in a food preferably a non heated food. The size of the inclusion incorporated in a food is greater than 10 micron and more preferably greater than 100 microns, even more preferably greater than one millimetre.

[0045] Preferably the inclusion comprising the antibody is provided in a non heated food eg ice cream or jelly or milk drink made from a powder.

[0046] Preferably the inclusion comprising the antibody further comprises another palatable moiety such as chocolate, candy or some other food component. [0047] The invention is particularly advantageous for controlling an outbreak of EV71 infection as a disease cluster (particularly a cluster involving children). The method in one embodiment thus comprises inhibiting an outbreak of EV71 in a disease cluster comprising orally administering the polyclonal antibodies to children and family members in direct contact with infected individuals.

[0048] Preferably the composition is administered to at least 30% preferably at least 50% of the individuals within the cluster and more preferably all members of the disease cluster.

[0049] The invention will now be described with reference to the following examples. It is to be understood that the examples are provided by way of illustration of the invention and that they are in no way limiting to the scope of the invention.

Example 1

[0050] An antibody formulation was prepared with activity against EV71 C (Taiwan strain). A cattle vaccine is prepared by growing EV71 virus in cell culture flasks within Vera cells in MEM growth media. The growth media is harvested, particulate matter is removed by centrifugation step and the resultant supernatant is digested with a Trypsin solution. The liquid is ultrafiltered in a cross-flow filtration system, removing fragment less than 90 KDa. The remaining larger moieties are in activated with first a formalin treatment step and then a BEI treat step.

[0051 ] The cattle vaccine was prepared using the following steps in sequence:

1) Expansion of Vero cells (pre-production)

Serial Vero cultures on Cytodex 1 microcarrier beads using Viral Production Serum Free Media

2) Initiation of Production Vero cell culture - Using Cytodex 1 microcarriers and VP-SFM. 3) Infect confluent cells with EV71 - Infect with working seed virus at

MOI=0.001

4) Harvest media SN, rinse beads and clarify by filtration -

At 48 hours post infection, allow beads to settle and collect media SN through stainless steel sieve (~100um), depth filter (~5um) and 0.8/0.2um filter. Wash beads with 2x VA volume PBS washes, filter and pool with media SN

5) Test of Virus Titre, contamination (mycoplasma / bacteria / fungi) and sterility -

6) Digest with Trypsin -

7) Inactivate with BEI and Formaldehyde -

8) Tangential flow filtration using 100KDa filter -

9) Collect TFF retentate of purified and concentrated EV71 antigen -

10) Test for purity, sterility and inactivation

Purity - SDS-PAGE gel, Western blot ; Antigen Potency - ELISA Inactivation - check of virus inactivation by inoculation of Vero cells

11) Sterile filtration using a 0.2um filter - Test of Bacterial and Fungal Sterility and Mycoplasma contamination

12) Combine EV71 Antigen with Montanide 206 adjuvant -

Final Sterility Check / Free Formalin Test / Safety Test / Vaccine Release

[0052] The resultant antigen mixed with a Montanide Adjuvant was injected into cattle in the last trimester of their pregnancy at least twice at 2 week intervals.

[0053] The colostrum produced by the cows directly after calving is collected using the system as described in US Pub No. 20060249084.

[0054] The liquid colostrum is further processed in a system as described in patent US Pub No. 20070053917 to yield a freeze dried powder, with at least 30% IgG. There are at least 5% preferably at least 10% (on a solids weight basis) of specific immunoglobulins. Example 2

Neutralisation Titre of Polyclonal antibodies made according to the method of Example 1

[0055] The neutralisation test used to analyse the colostrum powder was the "TCI D50" neutralisation assay (TCI D50 = 50% Tissue Culture Infectious Dose). In this assay, a 10% solution of the anti-EV71 colostrum powder was mixed at several dilutions with 100 TCI D50 of EV71 C virus (Taiwan Strain). After a set incubation time, these colostrum and virus mixtures are added to a culture plate that contains living kidney cells, which act as host cells to support the growth of virus. When active virus is in contact with the host cells, it readily infects the cells by attaching to the cell surface and causing changes called cytopathic effect (CPE) that eventually causes the cell to die. CPE are any detectable changes in the cell (due to infection with the virus) and include cell rounding, swelling or shrinking, death or detachment from the surface. The addition of colostrum containing specific antibodies to the virus was found to neutralise the EV71 and reduce its ability to infect the cell.

[0056] For the colostrum powder of Example 1 a dilution of 1 in 256 was found to neutralise the EV71 and inhibit at least 50% of the typical CPE of the virus.

[0057] The neutralisation assays (see table below) showed that only 10 mg of the colostrum powder of Example 1 was required to neutralise up to10⁶ pfu of EV71 C.

EV71 C Taiwan Strain with EV71 antibodies from Example 1

'N" means not detectable Example 3

Antibody Efficacy in an Animal Model:

[0058] As human efficacy challenge clinical trials in children would not be ethically acceptable to undertake, because they would require exposing children to harmful virus and because natural disease only occurs in geographically unpredictable epidemic settings, an animal model was developed using a strain of EV-71 to challenge mice. Groups of adult Balb/c mice were given oral doses of 20mg of the colostrum powder of Example 1 (20OuI of a 100mg/ml suspension) before a challenge dose with 10⁶ TCID₅₀ of EV-71 C.

[0059] 3 groups of 5 Balb/c adult mice were treated as in table below:

[0060] Group 3 was inoculated with 20mg of EV71 -immune colostrum by oral gavage and, one hour later, groups 1 and 3 were inoculated with 20OuI of EV71 C Taiwan (containing 10⁶ TCID₅₀). Group 2 was given with 20OuI of pre- neutralised EV71 C (pre-incubated mixture containing 10⁶ TCID₅₀ and 20mg of EV71 -immune colostrum) by oral gavage. Every faeces dropping from each individual mouse was collected over a 6-hour period. Each dropping was collected separately and allocated to an hourly period. Faeces samples/dropping were resuspended in media and 10OuL of chloroform was added to each sample. Supernatants were collected after centrifugation and diluted for analysis for presence of virus. Results: Adult Mouse Experiment

"+" virus was identified in the faeces "-" no virus identified in faeces

[0061 ] This adult mouse study demonstrated that a challenge dose of 10⁶ EV71 C could be pre-neutralised with only 20mg of EVProtec colostrum powder and remained neutralised, in all except for 1 mouse, following passage through their gastrointestinal tract. All mice in group 1 (infected only - no colostrum) were shown to have EV71 in their faeces. Most importantly, pre-inoculation (by gavage) of the mice in group 3, using 20mg of EVProtec colostrum powder given 1 hr before challenge with EV71 C, also neutralised the EV71 challenge in the gastrointestinal tract of the mice.

[0062] Finally, it is understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

Claims

1. A composition for oral dosage for passive immunization of a subject against EV71 comprising polyclonal antibodies raised against EV71 antigen said antibodies being derived from hyperimmune ungulate colostrum or hyperimmune avian eggs.

2. A composition according to any one of the previous claims in the form selected from the group consisting of a food, a food additive, a tablet, syrup, capsule and caplet.

3. A composition according to any one of the previous claims in the form of a particulate material for mixing with food or drink.

4. A composition according to any one of the previous claims wherein the polyclonal antibodies are present in a carrier derived from colostrum.

5. A composition according to any one of the previous claims wherein the composition further comprises at least one adjuvant selected from the group consisting of Montanide adjuvant, aluminium hydroxide, incomplete Freunds adjuvant and ISCOMS.

6. A composition according to any one of the previous claims wherein the subject of the passive vaccination is a human subject.

7. A composition according to any one of the previous claims wherein the polyclonal antibodies are derived from hyperimmune colostrum collected within 3 days post-partum, preferably one day post-partum and most preferably the first milking.

8. A composition according to any one of the previous claims comprising polyclonal antibodies in an amount sufficient to provide from 1.5% to 25% by weight of the composition of immunoglobulin specific to EV71 antigens.

9. A composition according to any one of the previous claims wherein the polyclonal antibodies are present in dried colostrum and the neutralisation titre of the dried colostrum (using a 10% solution of the composition as the zero dilution reference) against at least one of EV71 B and EV71 C is at least 1 in 20, preferably 1 in 40, more preferably 1 in 100, even ore preferably 1 in 200.

10. A composition according to any one of the previous claims wherein the oral dosage form comprises unit doses comprising the colostrum equivalent of less than 800 mg (dry weight basis), preferably less than 400 mg, more preferably less than 200 mg.

1 1. A composition according to any one of the previous claims wherein the EV71 antigen comprises one or more selected from the group consisting of whole inactivated EV71 type B, capsid protein (such as VP1 ) from EV71 type B, whole inactivated EV71 type C, and capsid protein (such as VP 1 ) from EV71 type C.

12. A composition according to any one of the previous claims wherein the polyclonal antibody is raised against at least one antigen of each of enterovirus 71 type B antigen and one enterovirus 71 type C antigen.

13. A composition according to any one of the previous claims wherein the oral dose form is for administration periodically for at least 20 days in a high risk period, where the high risk period may include the period in which the pathogen is abundant, or the period prior to the completion of a course of active vaccines. Preferably the oral dosage form is taken periodically for 40 days.

14. A method of treatment or inhibition of infection by EV7 in a subject comprising orally administering to the subject a composition according to any one of the previous claims.

15. A method of preparing a passive oral vaccine for immunising subjects against EV71 infection the method comprising: immunising animals selected from bovine and avian animals with EV71 antigen ;and collecting hyperimmune material selected from bovine colostrum and avian eggs from the animals said hyperimmune material comprising polyclonal antibodies to EV71 antigen.

16. A method according to claim 15 wherein the polyclonal antibodies are derived from hyperimmune colostrum collected within 3 days postpartum, preferably one day post-partum and most preferably the first milking.

17. A method according to any one of claims 15 or claim 16 wherein the EV71 antigen comprises inactivated virus and further comprises the surface antigen VP 1.

18. A method according to any one of claims 15 to 17 wherein the EV71 antigen comprises one or more selected from the group consisting of whole inactivated EV71 type B, capsid protein (such as VP1 ) from EV71 type B, whole inactivated EV71 type C, and capsid protein (such as VP1 ) from EV71 type C.

19. A method according to any one of claims 15 to 18 wherein the polyclonal antibody is raised against at least one antigen of each of enterovirus 71 type B antigen and one enterovirus 71 type C antigen.

20. A method according to any one of claims 15 to 19 wherein the oral dosage form comprises unit doses comprising the colostrum equivalent of less than 800 mg (dry weight basis), preferably less than 400 mg, more preferably less than 200 mg.

21. A method according to any one of claims 15 to 20 wherein the oral dose form is used in conjunction with an active vaccination program involving the use of an active vaccine against the pathogen.

22.A method according to any one of claim 21 wherein the oral dose form is used in the period prior to the completion of the active vaccination protocol and response.

23.A method according to any one of claims 15 to 22 wherein the polyclonal antibodies are formed into an oral dosage form such as a food or food additive, a tablet, syrup, capsule or the like.

24.A method according to any one of claims 15 to 23 wherein an inactivated EV71 vaccine is used to inoculate the animals.

25.A method according to any one of claims 15 to 24 wherein the neutralisation titre of the colostrum against at least one of EV71 B and EV71 C is at least 1 in 20, preferably 1 in 40, more preferably 1 in 100, even ore preferably 1 in 200 based on the dry weight of colostrum.

26.A method according to any one of claims 15 to 25 wherein the method is for inhibiting an outbreak of EV71 in an EV71 disease cluster comprising orally administering the polyclonal antibodies to children and family members in direct contact with infected individuals.

27. Use of (i) a composition according to any one of claims 1 to 14 and (ii) an active vaccine in preparation of one or more medicaments for treatment or prophylaxis of infection by EV71.

28.A use according to claim 27 wherein the use is for inhibiting an outbreak of EV71 in an EV71 disease cluster by orally administering the polyclonal antibodies to children and family members in direct contact with infected individuals.

29.A use according to claim 27 or claim 28 wherein the composition is for administration to at least 30% (preferably at least 50%) of the individuals within the cluster.

30. A use according to any one of claims 27 to 29 wherein the composition is for administration to all members of the disease cluster.

31. A kit for immunization against Infection by EV71 comprising (i) an oral dosage form of a composition according to any one of claims 1 to 7 and (ii) an active vaccine comprising at least one selected from a whole neutralized pathogen a killed pathogen and portion of one or more thereof.

32.A kit according to claim 31 with instructions for use of the oral dosage form prior to use of said active vaccine.

33. A method of passive oral vaccination of subjects against EV71 infection the method comprising: collecting hyperimmune material selected from bovine colostrum and avian eggs from animals selected from bovine and avian animals immunised with EV71 antigen said hyperimmune material comprising polyclonal antibodies to EV71 ;and orally administering said polyclonal antibodies to the subjects.

34.A method according to claim 33 wherein the composition is in a form selected from the group consisting of a food, a food additive, a tablet, syrup, capsule and caplet.

35.A method according to claim 33 or claim 34 wherein the composition is in the form of a particulate material for mixing with food or drink.

36.A method according to any one of claims 33 to 35 wherein the polyclonal antibodies are present in admixture with a material derived from colostrum.

37.A method according to any one of claims 33 to 36 wherein the composition further comprises at least one adjuvant selected from the group consisting of Montanide adjuvant, aluminium hydroxide, incomplete Freunds adjuvant and ISCOMS.

38.A method according to any one of claims 33 to 36 wherein the subject of the passive vaccination is a human subject.

39. A method according to any one of claims 33 to 37 wherein the polyclonal antibodies are derived from hyperimmune colostrum collected within 3 days post-partum, preferably one day post-partum and most preferably the first milking.

40. A method according to any one of claims 33 to 38 wherein the polyclonal antibodies are present in dried colostrum and the neutralisation titre of the dried colostrum against at least one of EV71 B and EV71 C is at least 1 in 20, preferably 1 in 40, more preferably 1 in 100, even ore preferably 1 in 200.

41. A method according to any one of claims 33 to 40 wherein the oral dosage form comprises unit doses comprising the colostrum equivalent of less than 800 mg (dry weight basis), preferably less than 400 mg, more preferably less than 200 mg.

42.A method according to any one of claims 33 to 41 wherein the oral dose form is for administration periodically for at least 20 days in a high risk period, where the high risk period may include the period in which the pathogen is abundant, or the period prior to the completion of a course of active vaccines. Preferably the oral dosage form is taken periodically for 40 days.

43.A method according to any one of claims 33 to 42 wherein the oral dose form is used in conjunction with an active vaccination program involving the use of an active vaccine against the pathogen.

44.A method according to claim 42 wherein the oral dose form is used in the period prior to the completion of the active vaccination protocol and response.

45.A method according to any one of claims 33 to 44 wherein the composition is administered to members of an EV71 disease cluster.

46. Use of polyclonal antibodies raised against EV71 in preparation of an oral vaccine for immunising subjects against EV71 infection by forming an oral dosage form of the polyclonal antibodies, said oral dosage form preferably containing bovine colostrum, for oral administration to the subject.

47. Use of EV71 in preparation of a passive vaccine comprising polyclonal antibodies for oral administration for immunising a subject against EV71 infection wherein the polyclonal antibodies are prepared by innoculating animals selected from bovine and avian animals with EV71 and collecting material selected from bovine colostrum and avian eggs said material being hyperimmune with respect to polyclonal antibodies to EV71.

48.A use according to claim 46 or claim 47 wherein the subject of the passive vaccination is a human subject.

49.A use according to claim 47 or claim 48 wherein the passive vaccine is for administration concurrently with active vaccination.

50. A method for inhibiting an outbreak of EV71 in an EV71 disease cluster by orally administering the polyclonal antibodies to children and family members in direct contact with infected individuals.