HEV vaccine

Abstract

The present invention relates to a vaccine comprising Open Reading Frame 2 (ORF2) protein of Swine Hepatitis E virus (HEV) and a pharmaceutically acceptable carrier, for use in the protection of anti-HEV MDA-positive piglets against HEV fecal shedding.

Claims

1. A method for protecting anti-HEV MDA-positive piglets against HEV fecal shedding, the method comprising administering to the piglet a vaccine comprising an immunogenically effective amount of a protein fragment of Hepatitis E Virus Open Reading Frame 2 (HEV ORF2) spanning at least the region from amino acid 125 to amino acid 607 and at most the region from amino acid 112 to amino acid 660, and a pharmaceutically acceptable carrier.

2. A method according to claim 1, characterized in that said vaccine comprises at least 5 microgram/dose of said protein fragment.

3. A method according to claim 1, characterized in that said vaccine comprises at least 20 microgram/dose of said protein fragment.

4. A method according to claim 1, characterized in that said composition comprises an adjuvant.

5. A method according to claim 4, characterized in that said adjuvant is an oil-in-water adjuvant.

6. A method as described in claim 1, wherein the method additionally comprises a booster vaccination for the protection of the anti-HEV MDA-positive piglets that have been vaccinated with a priming vaccine against HEV shedding no longer than 15 weeks prior to being vaccinated with the boost vaccine.

7. A method according to claim 6, where said priming vaccine is a vaccine comprising at least 10 microgram/dose of the fragment of HEV ORF-2 protein spanning at least the region from amino acid 125 to amino acid 607 and at most the region from amino acid 112 to amino acid 660, and a pharmaceutically acceptable carrier.

8. A method according to claim 1, characterized in that said vaccine comprises at least one other pig-pathogenic microorganism or pig-pathogenic virus and/or at least one other immunogenic component and/or genetic material encoding said other immunogenic component, of said pig-pathogenic microorganism or pig-pathogenic virus.

9. A method according to claim 8, wherein the virus or microorganism pathogenic to pigs is selected from the group consisting of Brachyspira hyodysenteriae, African Swine Fever virus, Nipah virus, Porcine Circovirus, Porcine Torque Teno virus, Pseudorabies virus, Porcine influenza virus, Porcine parvovirus, Porcine respiratory and Reproductive syndrome virus (PRRS), Porcine Epidemic Diarrhea virus (PEDV), Foot and Mouth disease virus, Transmissible gastro-enteritis virus, Rotavirus, Escherichia coli, Erysipelo rhusiopathiae, Bordetella bronchiseptica, Salmonella cholerasuis, Haemophilus parasuis, Pasteurella multocida, Streptococcus suis, Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae.

Description

LEGEND TO THE FIGURES

(1) FIG. 1: Protein expression HEV 112-607 in SF9 culture supernatant and SF9 cells at different harvesting time points after baculovirus infection:

(2) A) Western Blot results after staining of the blot with HIS antibody. After blotting of the SDS-PAGE gel, the membrane was stained for 1 hour with HIS-MCA diluted at 1:500 in blocking buffer. Next, the membrane was washed with wash buffer and incubated for 1 hour with the secondary antibody GAM/IgG(H+L)/PO diluted at 1:1000 in blocking buffer. The membrane was washed again with wash buffer and incubated with Vector SG substrate diluted in 15 ml of 3M Sodium Acetate.

(3) B) Instant blue protein staining on the SDS-PAGE gel. For instant blue staining, the SDS page gel was incubated for 2 hours in 10 ml of InstantBlue protein stain.

(4) The Molecular weight marker with protein size in kDa is loaded in left lane. The size marker of 50 kDa is indicated. Expected size of HEV 112-607 is 55 kD.

(5) Loading of the samples is indicated with numbers:

(6) 1. harvest 3 days medium

(7) 2. harvest 3 days cell

(8) 3. harvest 4 days cell

(9) 4. harvest 4 days medium

(10) 5. harvest 5 days cell

(11) 6. harvest 5 days medium

(12) 7. harvest 6 days cell

(13) 8. harvest 6 days medium

(14) FIG. 2: Instant blue staining on SDS-PAGE gel of eluate fractions 1-14 of HEV 112-607 after AKTA Avant purification of protein harvested from culture supernatant. The eluate was collected in 2 ml fractions. Lanes 15, 16 correspond to the unbound and wash fractions, respectively. For instant blue staining, the SDS page gel was incubated for 2 hours in 10 ml of InstantBlue protein stain.

(15) The molecular weight marker with protein size in kDa is loaded in the far left and right lanes. Expected size of HEV 112-607 is 55 kD.

(16) Eluates 1 to 7 were pooled after purification.

(17) FIG. 3: HEV specific antibodies measured at various time points in the vaccination study as determined in ELISA. Data are presented with error bars (Standard error of the mean, SEM)

(18) A: 3-week-old piglets at the start of the experiment carried significant maternal antibody titers against HEV

(19) B: 10-week-old pigs at the start of the experiment carried reduced (maternal) antibody titers against HEV, which is in line with reduced maternal immunity at this age.

(20) C: Vaccination with the 112-607 SUP and 112-607 AKTA vaccine resulted in seroconversion with specific antibodies to HEV ORF2 in the 3 week age groups at T=4 weeks after vaccination. Maternal antibodies have not disappeared entirely.

(21) D: Vaccination with the 112-607 SUP and 112-607 AKTA vaccine resulted in seroconversion with specific antibodies to HEV ORF2 in the 10 week age groups at T=4 weeks after vaccination.

(22) E: Vaccination with the 112-607 SUP and 112-607 AKTA vaccine resulted in seroconversion with specific antibodies to HEV ORF2 in the 3 week age groups at T=9 weeks after vaccination (subgroups marked with A). A booster vaccination at 5 weeks after primo vaccination resulted in increased antibody titers at T=9 weeks after primo vaccination (subgroups marked with B) compared to primo vaccination only. Maternal antibodies have dropped below detection level.

(23) F: Vaccination with the 112-607 SUP and 112-607 AKTA vaccine resulted in seroconversion with specific antibodies to HEV ORF2 in the 10 week age groups at T=9 weeks after vaccination (subgroups marked with A). A booster vaccination at 5 weeks after primo vaccination resulted in increased antibody titers at T=9 weeks after primo vaccination (subgroups marked with B) compared to primo vaccination only.

(24) FIG. 4: HEV specific antibodies measured at various time points in the vaccination-challenge experiment as determined in ELISA. Data are presented with error bars (Standard error of the mean, SEM)

(25) FIG. 5: this figure shows the amino acid sequence of ORF 2 from a.a. sequence 112-607 (SEQ ID NO: 1).

(26) FIG. 6: this figure shows the codon-optimised DNA sequence encoding the amino acid sequence of FIG. 5 (SEQ ID NO: 2).

EXAMPLES

Example 1

HEV ORF2 Expression in BAC to BAC Baculo Expression System

(27) A domain of the ORF2 capsid protein of HEV genotype 3 [Swine hepatitis E virus, based on GenBank: AFJ06417.1), as depicted in FIG. 5 (SEQ ID NO: 1) was expressed in the BAC to BAC baculo expression system (Invitrogen). The protein was designed with a C-terminal HIS tag for purification.

(28) The protein fragment expressed contains the core structural domain (amino acid 112-367) and the predicted neutralizing epitope (amino acid 456-607) but lacks the RNA binding domain of ORF2, the N-terminal 111 amino acids of the HEV ORF2.

(29) The coding sequence of the HIS-tagged protein as depicted in FIG. 6 (SEQ ID NO: 2) was cloned into the pFastbac1 plasmid (Invitrogen). The coding sequence was designed with a flanking 5 BamHI restriction site, a start codon, the codon optimized nucleotide sequence of the protein, a HIS tag, a stop codon and a 3 EcoRI restriction site. This information was sent to Genscript (Piscataway, N.J., USA) where the nucleotide sequence was synthesized, digested with BamHI and EcoRI restriction enzymes and cloned into the pFastbacl plasmid that was digested with the same restriction enzymes.

(30) Subsequently, the protein was expressed using the Bac-to-Bac Baculovirus Expression System according to the corresponding Instruction manual of Invitrogen Bac-to-Bac Baculovirus Expression System. An efficient site-specific transposition system to generate baculovirus for high-level expression of recombinant proteins. Invitrogen. Catalog Numbers 10359-016, 10360-014, 10584-027, 10712-024. Document Part Number 10359, Publication Number MAN0000414).

(31) Briefly, competent DH10Bac E. coli cells were transformed with plasmid DNA containing the nucleotide sequence as described above. Subsequently, the inserted sequence was transposed in the Bacmid sequence that is present in the DH10Bac cells. Next, bacteria were spread on agar plates containing antibiotics and IPTG. Selection was done by a white-blue screening since the white colonies contain the recombinant Bacmid. From a white colony a liquid culture was set up. Then, a miniprep isolation was performed to isolate the Bacmid DNA from the E. coli cells. SF9 insect cells were transfected (Cellfectin, Invitrogen) with this recombinant Bacmid DNA, resulting in formation of recombinant baculovirus particles. These particles were harvested and used to infect fresh monolayers of SF9 insect cells. The infection induces recombinant gene expression and the production of the HIS-tagged HEV-ORF2 protein 112-607.

(32) To determine the time point of optimum protein yield, protein was harvested at different time points, 3, 4, 5 and 6 days after infection. In all these infection experiments the optimized MOI of 0.1 was used.

(33) The recombinant protein that was expressed in SF9 cell culture medium or SF9 cells was collected. The cells were released from the culture flask by tapping and the cell suspension was centrifuged at 3.000g for 10 minutes. Medium was harvested and stored until analysis at 70 C. Cell pellets were stored until analysis at 20 C. Both the supernatant and cell harvest were analyzed for protein expression. The pelleted cells were resuspended in PBS before analysis of protein expression. Samples of both harvests were run on a SDS-PAGE together with a protein prestained all blue marker (Biorad) and analyzed by Instant Blue staining (Expedeon) and Western Blot (HIS-MCA).

(34) For instant blue staining, the SDS page gel was incubated for 2 hours in 10 ml of InstantBlue protein stain.

(35) For Western Blot, the Trans Blot Turbo Transfer Pack (Biorad) was used according to corresponding instruction manual. After blotting, the membranes were blocked for one hour with blocking buffer (1% skimmed milk in PBS 0.04 M, Polysorbate 20 0.05%). Subsequently, the membrane was stained for 1 hour with HIS-MCA (MSD Animal or commercially available equivalent), diluted at 1:500 in blocking buffer. Next, the membrane was washed with wash buffer (PBS 0.04 M, Polysorbate 20 0.5%) and incubated for 1 hour with the secondary antibody GAM/IgG(H+L)/PO (Nordic) diluted at 1:1000 in blocking buffer. The membrane was washed again with wash buffer and incubated shortly with 3 drops of Vector SG substrate (Vector Laboratories) diluted in 15 ml of 3M Sodium Acetate.

(36) Recombinant protein 112-607 was secreted in the culture medium as a soluble protein, but also present in the cell pellet (FIG. 1, A: Western blot, B: protein stain). The highest protein yield was achieved after 5 days of infection in culture medium. The culture medium containing the soluble protein was used for preparation of vaccine after inactivation of the baculovirus by gamma-irradiation=112-607 SUP (see: Example 3, vaccine preparation). From culture medium, also a purified subunit protein fraction was prepared. The HIS tagged protein was purified from the cell culture medium using the AKTA Avant protein purification system=112-607 AKTA (see Example 2, Purification method AKTA).

Example 2

Purification Method AKTA Avant Protein Purification System

(37) HIS tagged protein 112-607 present in culture medium was purified using the AKTA Avant purification system (GE Healthcare, Germany).

(38) The HIS tagged protein in the culture supernatant was purified on an AKTA Avant purification system (GE Healthcare, Germany) using a HIStrap FF column (GE Healthcare art. 17-5255-01 (5 ml). Prior to purification, the fraction was filtered on a 0.45 M filter (Millipore).

(39) The following program was used:

(40) TABLE-US-00001 Purification step Buffer CV Flow speed Equilibration wash buffer AKTA non-denaturing 5 1 ml/min Sample Soluble fraction 1 ml/min application Wash wash buffer non-denaturing 4 1 ml/min Gradient wash buffer non-denaturing .fwdarw. 20 1 ml/min elution elution buffer AKTA non-denaturing 100% elution elution buffer AKTA non-denaturing 4 1 ml/min Equilibration wash buffer AKTA non-denaturing 5 1 ml/min Wash buffer AKTA, non-denaturing: 300 mM KCl, 50 mM TRIS, 5 mM Immidazole Elution buffer, non-denaturing: 300 mM KCl, 50 mM TRIS, 500 mM Immidazole

(41) The eluate was collected in 2 ml fractions and these were run on a SDS page gel followed by instant blue staining. The SDS page gel was incubated for 2 hours in 10 ml of InstantBlue protein stain (Expedeon) (FIG. 2). Eluates 1 to 7 were pooled after purification.

(42) After purification, dialysis was performed on purified protein. The purified protein was loaded into a Spectra Por3 Dialysis membrane, MWCO 3.5 kD (Spectrum Labs) and this was placed into an external chamber with dialysis buffer (150 mM KCl+150 mM TRIS) of about 400 times the volume of the purified protein. The buffer was constantly stirred for 2 days at 4 C. and after 1 day the dialysis buffer was changed.

Example 3

Formulation of Vaccines

(43) The X-solve formulation is an oil-in-water emulsion based on mineral oil combined with solubilized Vitamin E Acetate. Droplet size of the mineral oil based emulsion is about 0.5 m and the droplet size of Solubilized Vitamin E Acetate is about 150 nm. Viscosity of the final vaccine is below 15 mPa.Math.s.

(44) The protein concentration of the antigen was determined as follows: The water-phase used for vaccine formulation was run on a SDS-PAGE together with a protein prestained all blue marker (Biorad) and a BSA standard with known protein concentration (25-50-100 g/ml) and analyzed by Instant Blue staining (Expedeon). For instant blue staining, the SDS page gel was incubated for 2 hours in 10 ml of InstantBlue protein stain. The density of the signals was measured by GeneTools software from Syngene. The protein concentration of the 112-607 AKTA and 112-607 SUP were estimated based on linear regression on the BSA standard results.

(45) 112-607 AKTA: 40 g/ml protein in final vaccine formulation

(46) 112-607 SUP: 40 g/ml protein in final vaccine formulation

Example 4

Determination of (Maternal) Antibody Levels in Serum

(47) Individual blood samples (Vacuolette 8 ml Sep Clot Activator (Greiner-Bio One)) of pigs were collected. After clotting at room temperature and overnight at 4 C., serum was obtained from the blood samples. This was done by centrifugation of the tubes at 2,500g for 25 minutes at 4 C. The serum samples for ELISA were heat inactivated for 30 minutes at +56 C. and finally stored at 20 C. or lower until use. The sera were tested for the presence of antibodies against HEV using the commercial test HEV ELISA 4.0 kit from MP Biomedicals, Santa Ana, Calif., USA. This was done according to the corresponding protocol of HEV ELISA 4.0 kit.

(48) The cut-off value of the ELISA is OD (450 nm) 0.2+the average of the negative control samples (0.05). Samples with OD lower than 0.250 are considered HEV negative.

Example 5

Detection of HEV in Rectal Swabs, Bile and Serum

(49) Individual blood samples (Vacuolette 8 ml Sep Clot Activator (Greiner-Bio One)) of pigs were collected. After clotting at room temperature and overnight at 4 C., serum was obtained from the blood samples. This was done by centrifugation of the tubes at 2,500g for 25 minutes at 4 C. Serum samples for PCR were not heat inactivated and stored at 20 C. or lower until analysis.

(50) Bile was taken from the gall bladder at necropsy using a syringe and stored at 20 C. or lower until analysis.

(51) Rectal swabs were taken at different time points during the experiment. The swabs were placed in tubes containing 1 ml PBS and antibiotics. In the lab, tubes were vortexed and decanted in cryotubes. Samples were stored at 20 C. or lower until analysis.

(52) The rectal swabs, bile and serum samples were tested for the presence of HEV RNA by q-RT-PCR.

(53) RNA was extracted by the MagNA Pure 96 Instrument (Roche) using the external lysis protocol as described by the manufacturer. Briefly, 200 l of undiluted sample was mixed with 250 l external lysis buffer (Roche) and nucleic acids were extracted according to the related protocol.

(54) For quantitative detection of HEV RNA in these samples, the QuantiTect Probe RT-PCR Kit (Qiagen) was used. A qPCR reaction consisted of 4.5 l RNase free water, 12.5 l QuantiTect Probe RT-PCR mastermix, 400 nm of the forward and reverse primer, 300 nm of the Probe, 0.75 l QuantiTect Probe RT-mix (enzyme) and 5 l RNA.

(55) The following primers and probe were used:

(56) TABLE-US-00002 Forwardprimer; (SEQIDNO:3) GGTGGTTTCTGGGGTGAC Reverseprimer; (SEQIDNO:4) AGGGGTTGGTTGGATGAA 6FAMTAMRAlabeledprobe; (SEQIDNO:5) TGATTCTCAGCCCTTCGC

(57) Reverse transcription was carried out at 50 C. for 30 min, followed by denaturation at 95 C. for 15 min. DNA was amplified immediately with 40 PCR cycles at 94 C. (15 s), 56 C. (30 s) and 76 C. (30 s). The reactions were run on the CFX96 real time system (Biorad) and the software analysis was done with CFX manager Version 3.1. Data are presented in copies/ml sample. Quantification was based on analysis of a set of standard samples containing 10.sup.1-10.sup.8 HEV ORF2 copies/5 l in each q-RT-PCR reaction on the CFX96.

Example 6

Vaccination Experiment

(58) A total of 30 piglets of 3 weeks old that were seropositive for HEV maternal antibodies were used in this study. The piglets were randomly selected from different litters.

(59) A total of 30 pigs of 10 weeks old were used in this study. The piglets were randomly selected. At the age of 10 weeks, maternal antibody levels to HEV have reduced compared to 2-week-old piglets (FIG. 3A, B).

(60) The piglets of 3 weeks of age were assigned to 3 treatment groups of 10 piglets each (groups 1-2-3). Piglets of group 1 were vaccinated (IM vaccination, 1 ml, in the neck) at 3 weeks of age with HEV ORF2 vaccine 112-607 SUP formulated in X-Solve. Piglets of group 2 were vaccinated (IM vaccination, 1 ml, in the neck) at 3 weeks of age with HEV ORF2 vaccine 112-607 AKTA formulated in X-Solve. Piglets of group 3 were vaccinated (IM vaccination, 1 ml, in the neck) at 3 weeks of age with PBS.

(61) The pigs of 10 weeks of age were assigned to 3 treatment groups of 10 piglets each (groups 4-5-6). Piglets of group 4 were vaccinated (IM vaccination, 1 ml, in the neck) at 10 weeks of age with HEV ORF2 vaccine 112-607 SUP formulated in X-Solve. Piglets of group 5 were vaccinated (IM vaccination, 1 ml, in the neck) at 10 weeks of age with HEV ORF2 vaccine 112-607 AKTA formulated in X-Solve. Piglets of group 6 were vaccinated (IM vaccination, 1 ml, in the neck) at 3 weeks of age with PBS.

(62) Each group of 10 piglets was subdivided into A and B groups at 5 weeks post primo vaccination (8/15 weeks of age). 5 piglets received no booster vaccination (A subgroup), whereas the other 5 pigs received a booster vaccination identical to the primo vaccination (B subgroup).

(63) Serum was collected prior to vaccination (3/10 weeks of age), at 7/14 weeks of age (4 weeks after vaccination) and 12/19 weeks of age (9 weeks after vaccination). Antibody levels to HEV were determined in serum as described in Example 4. Absence of replicating HEV was confirmed in all serum samples by q-RT-PCR analysis.

(64) At 3 weeks of age, piglets had significant antibody titers against HEV, which had largely disappeared in pigs of 10 weeks of age (FIG. 3A, B). Vaccination with the 112-607 SUP vaccine (group 1) and 112-607 AKTA vaccine (group 2) resulted in seroconversion with specific antibodies to HEV ORF2 in both age groups at 4 (FIG. 3C, D) and 9 weeks after vaccination (FIG. 3E, F; subgroups A). A booster 5 weeks after primo vaccination resulted in increased antibody titers at 9 weeks after primo vaccination. (FIG. 3E, F; subgroups B)

Example 7

Vaccination-Challenge Experiment in Piglets

(65) A total of 12 piglets of 3 weeks old that were seropositive for HEV maternal antibodies were used in this study. The piglets were randomly selected from different litters and housed together in one pen.

(66) The piglets were assigned to 3 treatment groups of 4 piglets each (groups 1-2-3). Piglets of group 1 were vaccinated (IM vaccination, 1 ml, in the neck) at 3 weeks of age with HEV ORF2 vaccine 112-607 SUP formulated in X-Solve. Serum was collected prior to vaccination (3 weeks of age), at 7 weeks of age (4 weeks after vaccination) and prior to experimental challenge (11 weeks of age, 8 weeks after vaccination).

(67) At 8 weeks after vaccination (11 weeks of age), the 4 pigs of group 2 were experimentally challenged with a liver homogenate of a HEV-positive animal from a Dutch farm. The challenge material was 10% liver homogenate in 50/50 (vol/vol) William's E medium/F-12 Ham medium. Challenge liver homogenate was applied via an intramuscular (IM) injection (22.5 ml, left and right neck) and oral dose of 15 ml applied using a syringe. For oral injection, the homogenate was centrifuged at 3,200g for 1 hour at 4 C. and the supernatant was used for challenge. The material contained 2.1210.sup.6 HEV RNA copies/ml. For IM injection, this supernatant was centrifuged again at 10,000g for 1 hour at 4 C., and subsequently filtered on a 0.22 M filter (Millipore) and injected. This material contained 1.2210.sup.6 HEV RNA copies/ml. The HEV-positive material used contained a genotype 3i HEV which was determined based on RNA sequence analysis of the virus nucleotide sequence. The virus showed highest homology to Genbank accession number: KC618403.1. Pigs of group 3 served as contact sentinels.

(68) Excretion of HEV via the feces after experimental challenge was monitored twice per week (12-17 weeks of age) by q-RT-PCR analysis of rectal swabs. Presence of virus in serum was monitored weekly (13-17 weeks of age) by q-RT-PCR analysis. Necropsies were performed at 17 weeks of age. Bile and blood were collected at the time of necropsy, with the exception of the group 2 animals. Presence of HEV virus was analyzed using q-RT-PCR (Example 5) in rectal swabs, bile and serum.

(69) Antibody levels to HEV were determined in serum as described in Example 4. FIG. 4 shows the ELISA results. Vaccination of 3-week-old piglets induced a seroconversion with HEV specific antibodies in vaccinated animals which was detectable from 7 weeks of age onwards. Groups 2 and 3 showed decline in antibody titers due to disappearance of maternal antibodies prior to experimental challenge of group 2 at 11 weeks of age. Challenge of group 2 animals resulted in induction of HEV-specific antibodies in this group between 13 and 14 weeks of age, and also the Sentinel group 3 seroconverted as a consequence of infection with HEV due to direct contact with the experimentally challenged pigs. The sentinel group developed antibody titers between 15 and 16 weeks of age, with a delay of approximately 2 weeks compared to the experimental challenge group 2, which corresponds to the incubation time of the virus. The vaccinated group 1 reacted to exposure to HEV via experimentally challenged pigs (group 2) with an increase in HEV-specific antibody titers starting between week 14 and 15.

(70) Successful experimental challenge of group 2, and infection of sentinel pigs in group 3 were confirmed by performing q-RT-PCR analyses on bile and serum of experimental animals (Table A). Three out of four experimentally challenged pigs, and four out of four sentinel pigs showed detectable levels of HEV in serum or bile. None of the vaccinated pig had detectable levels of HEV in serum or bile. Also rectal swabs were analyzed by q-RT-PCR analysis, which are shown in Table B.

(71) The present experiment was set up to mimic field situations wherein HEV infects pigs during the finishing phase. Vaccination protected pigs from viremia in blood and bile (challenged animals, sentinel animals), which indicates that vaccinated pigs do not have a systemic infection with HEV. Furthermore, HEV vaccinated pigs react to exposure to the virus with increases serum antibody levels. Excretion of HEV in feces is reduced in vaccinated pigs compared to sentinel pigs in the same pen.

(72) More specifically, what can be seen in Table B is that the vaccinated pigs, with respect to the sentinel pigs have a substantially reduced fecal shedding at weeks 13, 14 and 15 (factor 10-50 lower amount of HEV DNA material present in the feces). Around week 16 the shedding seems to be stabilized at a low level which is essentially the same for the vaccinated animals as well as the control animals. Since the vaccinated animals show no systemic infection with HEV at all (see Table A), this must be virus that is shed due to the piglets eating HEV infected feces. Apparently, the level of shedding arrived at after week 15 is due to eating infected feces, but not due to an actual infection of the animal. In short, when combining the data of Table A with those of Table B it is clear that with the present vaccine, an anti-HEV MDA-positive piglet can be protected against HEV fecal shedding, in particular against fecal shedding that is a result of an infection of the animal itself.

(73) TABLE-US-00003 TABLE A q-RT-PCR analysis of serum and bile at various time points in the vaccination-challenge study (RNA copies/ml). week 17 week 11 week 13 week 14 week 15 week 16 week 17 Group Treatment Pig BILE SERUM SERUM SERUM SERUM SERUM SERUM 1 Vaccinated 1 0 0 0 0 0 0 0 1 Vaccinated 2 0 0 0 0 0 0 0 1 Vaccinated 3 0 0 0 0 0 0 0 1 Vaccinated 4 0 0 0 0 0 0 0 average 0 0 0 0 0 0 0 2 Experimental challenge 5 NA 0 1.5E+03 1.8E+03 0 0 NA 2 Experimental challenge 6 NA 0 1.3E+04 0 0 0 NA 2 Experimental challenge 7 NA 0 1.0E+03 3.7E+03 7.3E+04 1.2E+04 NA 2 Experimental challenge 8 NA 0 0 0 0 0 NA average NA 0 3.8E+03 1.4E+03 1.8E+04 2.9E+03 NA 3 Sentinel 9 6.5E+03 0 0 0 1.3E+03 0 0 3 Sentinel 10 0 0 0 0 6.2E+02 0 0 3 Sentinel 11 3.4E+03 0 0 0 0 0 0 3 Sentinel 12 8.8E+02 0 0 0 3.4E+02 0 0 average 2.7E+03 0 0 0 5.6E+02 0 0 NA: not analysed

(74) TABLE-US-00004 TABLE B q-RT-PCR analysis of rectal swabs at various time points in the vaccination-challenge study (RNA copies/ml). Age week 11 week 12 week 13 week 14 Group Treatment Pig challenge 1 2 1 2 1 2 1 Vaccinated 1 0 0 0 0 0 0 3.1E+02 1 Vaccinated 2 0 0 0 0 0 2.1E+02 0 1 Vaccinated 3 0 0 0 0 0 2.4E+02 1.1E+03 1 Vaccinated 4 0 0 0 0 0 1.2E+02 4.6E+02 average 0 0 0 0 0 1.4E+02 4.6E+02 2 Experimental challenge 5 0 0 0 1.0E+04 2.6E+04 4.7E+05 1.4E+04 2 Experimental challenge 6 0 0 2.2E+02 1.2E+04 1.8E+05 6.5E+04 0 2 Experimental challenge 7 0 0 0 1.7E+04 4.9E+04 1.1E+06 2.4E+06 2 Experimental challenge 8 0 0 0 0 6.2E+03 2.6E+04 1.9E+04 average 0 0 5.6E+01 9.6E+03 6.5E+04 4.2E+05 6.0E+05 3 Sentinel 9 0 0 0 0 0 0 3.5E+02 3 Sentinel 10 0 0 0 0 5.0E+02 3.9E+02 3.8E+03 3 Sentinel 11 0 0 0 0 0 0 2.5E+03 3 Sentinel 12 0 0 0 0 0 7.1E+03 1.3E+04 average 0 0 0 0 1.2E+02 1.9E+03 5.0E+03 Age week 15 week 16 week 17 Group Treatment Pig 1 2 1 2 1 1 Vaccinated 1 1.5E+03 1.8E+02 0 0 3.9E+02 1 Vaccinated 2 7.5E+02 1.9E+02 1.3E+03 6.5E+02 3.8E+02 1 Vaccinated 3 1.4E+04 2.6E+02 0 0 1.6E+03 1 Vaccinated 4 4.2E+03 1.5E+02 6.9E+03 0 3.9E+02 average 5.1E+03 2.0E+02 2.0E+03 1.6E+02 6.8E+02 2 Experimental challenge 5 2.6E+04 1.6E+02 0 0 4.0E+02 2 Experimental challenge 6 4.1E+03 3.1E+02 5.2E+01 0 0 2 Experimental challenge 7 5.2E+06 9.8E+05 4.0E+04 5.0E+04 5.8E+05 2 Experimental challenge 8 1.3E+03 2.8E+03 3.9E+02 0 7.1E+02 average 1.3E+06 2.4E+05 1.0E+04 1.2E+04 1.5E+05 3 Sentinel 9 4.5E+02 1.8E+03 7.3E+02 0 9.2E+02 3 Sentinel 10 2.9E+03 3.5E+03 2.0E+03 0 0 3 Sentinel 11 4.1E+03 1.9E+04 2.0E+03 1.2E+02 0 3 Sentinel 12 2.4E+04 3.9E+03 1.4E+03 0 3.5E+02 average 7.8E+03 7.0E+03 1.5E+03 3.1E+01 3.2E+02