Prime-Boost Vaccination Regimen

Abstract

The present invention relates to a method of vaccination. Specifically the invention regards to a prime-boost vaccination regimen for protecting a target animal against infection or disease caused by a virus, wherein the vaccination regimen comprises the administration to said target animal of a vaccine comprising a live attenuated form of said virus, followed by the administration to said target animal of a vaccine comprising an RP encoding one or more antigens from said virus.

Claims

1. A method of vaccination for protecting a target animal against infection or disease caused by a virus, wherein the method comprises administering a vaccine comprising a live attenuated form of said virus to the target animal, followed by administering a vaccine comprising replicon particle (RP) encoding one or more antigens from said virus to the target animal.

2. The method of claim 1, wherein the target animal is a porcine.

3. The method of claim 2, wherein said virus is selected from the group consisting of a porcine epidemic diarrhoea virus (PEDV) and a porcine reproductive and respiratory syndrome virus (PRRSV).

4. The method of claim 1, wherein the vaccine comprising the RP comprises at least one further RP encoding one or more antigens from said virus.

5. The method of claim 1, wherein at least one viral antigen encoded by the RP is from a virus that is a variant within the species of the live attenuated virus.

6. The method of claim 1, wherein one or both of the vaccines comprise an adjuvant.

7. A prime-boost vaccination regimen for protecting a target animal against infection or disease caused by a virus, wherein the vaccination regimen comprises the administration to said target animal of a vaccine comprising a live attenuated form of said virus, followed by the administration to said target animal of a vaccine comprising an RP encoding one or more antigens from said virus.

8-11. (canceled)

12. A kit of parts comprising at least two containers, wherein at least one container comprises a vaccine comprising the live attenuated form of said virus of claim 1, and at least one container that comprises a vaccine comprising the RP encoding one or more antigens from said virus.

13. The kit of claim 12, wherein said virus is selected from the group consisting of a porcine epidemic diarrhoea virus (PEDV) and a porcine reproductive and respiratory syndrome virus (PRRSV).

14. The kit of claim 13, wherein the vaccine comprising the RP comprises at least one further RP encoding one or more antigens from said virus.

15. The kit of claim 13, wherein at least one viral antigen encoded by the RP is from a virus that is a variant within the species of the live attenuated virus.

16. The kit of claim 13, wherein one or both of the vaccines comprise an adjuvant.

17. The kit of claim 12, wherein the vaccine comprising the RP comprises at least one further RP encoding one or more antigens from said virus.

18. The kit of claim 12, wherein at least one viral antigen encoded by the RP is from a virus that is a variant within the species of the live attenuated virus.

19. The kit of claim 12, wherein one or both of the vaccines comprise an adjuvant

Description

EXAMPLES

1. Example 1: Homologous Grime-Boost PRRSV Vaccination

[0171] The existing PRRSV live vaccine Porcilis® PRRS is based on a European isolate of PRRSV, strain DV, a genotype 1 PRRSV. This was found to sometimes lack in efficacy when tested against a heterologous challenge strain, which is a problem common to all vaccines of attenuated live PRRSV. Different ways were investigated to improve this vaccination efficacy, such as a homologous prime-boost vaccination regimen.

[0172] In a vaccination-challenge experiment, 3 groups were used with 16 pigs of approximately 5 weeks old, and PRRSV-antibody negative at the day of 1.sup.st vaccination. Two groups received a vaccination with live attenuated virus Porcilis PRRS, either once, or two times with a 2-week interval. One group remained unvaccinated. All pigs received a challenge infection with PRRSV of a recent German genotype 1 isolate, at 4.5 weeks after the vaccination, respectively after the first vaccination. Challenge dose was 5.3 Log 10 TCID50 of virus, in 2 ml per animal (1 ml in each nostril). Blood samples were taken at the days of vaccination and at 0, 4, 7, 10, 14 and 20 days post challenge. PRRSV viremia was determined as cpe on PAM cells, all as described below.

[0173] FIG. 1 presents the viremia results of the 3 groups. As can be clearly seen, there was no significant difference in the effect of using one or two vaccinations with an attenuated live PRRSV vaccine.

[0174] Consequently, for PRRSV, the use of a homologous prime-boost vaccination regimen cannot improve the immune-protection of the single vaccination.

2. Example 2: PRRSV—Prime-Boost Vaccination, Initial Experiments

[0175] 2.1. Introduction

[0176] This experiment applied the method of vaccination according to the invention, with a variant in the booster vaccination: the vaccine of the live attenuated virus used was Porcilis® PRRS, which is of PRRSV type 1, strain DV, and the vaccine comprising an Alphavirus RNA RP, as well as the challenge virus used, were based on PRRSV type 1 strain Sc3.

[0177] The Alphavirus RNA RP used were based on VEEV strain TC-83, and expressed more than one viral antigen: one type of RP (here: RP1) was made to encode the PRRSV strain Sc3 genes: E, GP2, GP3 and GP4, leading to the expression of the native E-GP2-GP3-GP4 tetramer. Another type of RP (here: RP2) was made to encode the PRRSV strain Sc3 genes: GP5 and M, leading to the expression of the native GP5-M dimer. In each case (RP1 and RP2), each of the heterologous genes were under the transcriptional control of their own subgenomic promoter sequence. In RP1, four separate subgenomic promoter sequences were present; in RP2, two subgenomic promoter sequences were present.

[0178] 2.2. Study Design

[0179] 40 piglets seronegative for anti-PRRSV antibodies were divided over 4 groups of 10 animals. When the piglets of groups 1 to 3 were about 5 weeks old they were given a priming vaccination via the intramuscular (IM) route with the live attenuated virus vaccine Porcilis® PRRSV; animals of group 4 served as non-vaccinated control animals. At 3 weeks after the priming vaccination, groups 2 and 3 received their booster vaccination with vaccine comprising 2 types of Alphavirus RNA RP. At about 4.5 weeks post priming vaccination, i.e. 11 days post booster vaccination, all animals were challenged with contemporary virulent genotype 1 strain Sc-3, by intranasal route. Blood samples were collected on the day of priming vaccination and 7, 15, and 23 days thereafter, as well as on the day of challenge (=day 32 dpv) and 4, 7, 11, 17 and 21 days thereafter. Serum was isolated from these samples, and the samples obtained after challenge were used to determine the vaccine efficacy of the different vaccinations by measuring the challenge strain's viremia.

[0180] 2.3. Materials and Methods

[0181] Treatment:

[0182] The recommended route of vaccination for Porcilis® PRRS was used, i.e. intramuscular administration, also for the vaccine comprising an Alphavirus RNA RP. For the challenge inoculation a natural route of infection was used, the nasal route. Blood samples were taken to monitor the relevant parameters of PRRSV infection: viremia and serology.

[0183] Test Articles:

[0184] Challenge virus was PRRSV strain Sc-3, diluted to 0.5×10{circumflex over ( )}5.0 TCID50/ml.

[0185] Diluent for the freeze dried Porcilis® PRRS vaccine was 10 mM PBS buffer. This was applied shortly before use to obtain a titer of 0.5×10{circumflex over ( )}5.0 TCID50/ml.

[0186] Titrations were done on primary porcine alveolar macrophages (PAMs) by reading cpe after 5-7 days.

[0187] Diluent for the RP was Xsolve™ 50 oil-in-water emulsion, also applied shortly before use, to arrive at a titer of 0.5×10{circumflex over ( )}8.0 TCID50/ml for each of the vaccines comprising Alphavirus RNA RP1 or RP2.

[0188] When the RP are categorized as GMO, all materials, samples, and animals need to be handled and kept in facilities at the appropriate level of biological containment.

[0189] Test Animals:

[0190] Standard piglets, about 5 weeks old at day of vaccination, of mixed sex, and negative for antibodies against PRRSV until vaccination. The piglets had 5 days of acclimatization between transportation to the test facility and day of vaccination. Only clinically healthy pigs were used; pigs were identified using numbered ear tags. All animals were observed daily during the experiment for general health. All observations were recorded, and the responsible veterinarian was consulted when required.

[0191] All vaccinations were given IM at 2 ml/animal, the challenge was given IN at 2 ml/animal.

TABLE-US-00001 TABLE 1 Experimental outline of Example 2 # of Vaccination (prime) Vaccination (boost at 3 wpv) Challenge (at ~4.5 wpv) Group pigs Vaccine Dose Adjuvant Vaccine Dose Adjuvant Strain Dose Route 1 10 Porcilis 1 .Math. 10{circumflex over ( )}5 — — Sc-3 1 .Math. 10{circumflex over ( )}5 IN PRRS TCID50 TCID50 2 10 Porcilis 1 .Math. 10{circumflex over ( )}5 — RP1 + RP2 Per RP: X-Solve50 Sc-3 1 .Math. 10{circumflex over ( )}5 IN PRRS TCID50 1 .Math. 10{circumflex over ( )}8 TCID50 TCID50 3 10 RP1 + RP2 Per RP: X-Solve50 RP1 + RP2 Per RP: X-Solve50 Sc-3 1 .Math. 10{circumflex over ( )}5 IN 1 .Math. 10{circumflex over ( )}8 1 .Math. 10{circumflex over ( )}8 TCID50 TCID50 TCID50 4 10 — — Sc-3 1 .Math. 10{circumflex over ( )}5 IN TCID50

[0192] Sampling

[0193] Blood samples were taken from the jugular vein at 0, 7, 15, and 23 days post priming vaccination (dpv), and at 0 (=32 dpv), 4, 7, 11, 17, and 21 days post challenge (dpc). The last blood sample was taken during euthanization at the end of the experiment. Blood was collected in a vacutainer without anti-coagulant. The blood samples were allowed to coagulate at room temperature for 1 hr., then centrifuged for 10 minutes at 3000×g. and serum was collected and stored frozen below −20° C. until use.

[0194] Viremia Detection

[0195] Viremia of PRRSV (both of vaccine comprising a live attenuated form of a virus and of challenge virus) was detected by quantification of PRRSV RNA in serum by real-time RT-qPCR using primer sets specific for PRRSV genotype 1, and the iTaq™ Universal SYBR® Green One-Step Kit (BioRad).

[0196] Serology

[0197] Sera were tested for antibodies against PRRSV using a commercial ELISA kit: IDEXX™ PRRS X3 (IDEXX Laboratories, Westbrook, Me., USA). Serum was incubated in recombinant PRRSV antigen-coated micro-titration wells. Bound PRRSV-specific antibodies were then detected using horseradish peroxidase-conjugated anti-porcine antibody and a chromogen. Results were calculated as sample to positive (S/P) ratio. Samples with S/P ratios ≥0.4 were classified as positive and samples with S/P ratios <0.4 were classified as negative for PRRSV-specific antibodies.

[0198] 2.4. Results and Conclusions

[0199] The experiment was considered valid as all serum samples prior to vaccination were PRRSV-Ab negative, and the serum samples of group 4 were PRRSV-Ab negative until challenge.

[0200] Results of viremia determinations are presented in FIG. 2, which represents the qPCR scores compiled per group.

[0201] Non-vaccinated pigs (group 4) showed the highest levels of PRRS virus in their serum, which was as expected. Also as expected, the prime-booster vaccination with the two Alphavirus RNA RPs at 21 dpv does not result in the detection of PRRSV viremia levels in the serum (e.g. see group 3, FIG. 2).

[0202] In animals receiving both a priming and a booster vaccination with vaccine comprising Alphavirus RNA RP1 and RP2 (group 3), the viremia levels observed were almost equally high as the levels found in the non-vaccinated animals.

[0203] Clearly a homologous prime-boost regimen with a vaccine comprising Alphavirus RNA RP did not provide adequate protection for PRRSV, even when these encoded multiple antigens of the challenge virus.

[0204] A good reduction in viremia was provided by the live attenuated vaccine (group 1). However, the group receiving vaccinations according to the heterologous prime-boost vaccination regimen of the invention (group 2) showed the best protection against challenge infection of all; i.e. had the lowest levels of detectable PRRSV in serum samples after challenge at all times.

3. Example 3: PRRSV—Prime-Boost Vaccination, Extended Experiment

[0205] 3.1. Introduction

[0206] Further experiments were arranged, in order to repeat and expand on the remarkable results found in the experiments described in Example 2. In this set-up, groups were added to test the effect of separate vaccines comprising Alphavirus RNA RP1 or RP2 as booster vaccination. In addition one group was added to test the reverse order of the vaccinations in the method of vaccination according to the invention.

[0207] 3.2. Study Design

[0208] The study design in this example was essentially the same as that in Example 2, except for the extra test groups. The experimental outline of Example 3 is summarized in Table 2. All groups received the challenge infection with PRRSV strain Sc-3 at 5 weeks after the priming vaccination, i.e. at 2 weeks after the booster vaccination.

TABLE-US-00002 TABLE 2 Experimental outline of Example 3 Vaccination Group priming booster 1 Porcilis PRRS — 2 RP1 + RP2 (Xsolve50) 3 RP2 (Xsolve50) 4 RP1 (Xsolve50) 5 RP1 + RP2 (Xsolve50) Porcilis PRRS 6 — —

[0209] 3.3. Materials and Methods Vaccinations and challenges were largely the same as applied in Example 2. Regarding timing the challenge was at 5 weeks after priming vaccination, instead of at 4.5 weeks in Example 2. Two groups were added, groups 3 and 4, receiving the single vaccines comprising Alphavirus RNA RP1 or RP2 as booster vaccination.

[0210] 3.4. Results and Conclusions

[0211] Results are presented in FIG. 3. The best time point to compare results is the viremia (in Ct values) at 4 days post challenge.

[0212] Again the test was valid, based on seronegativity for anti-PRRSV-antibodies prior to vaccination. Also, the unvaccinated group 6 responded as expected, post challenge. A similar viremia as in group 6, was detected in the groups receiving a single vaccination with attenuated live virus (MLV: modified live vaccine; group 1) and the group 4, receiving a boost vaccination with RP1 (E-GP2-3-4) vaccine. Slightly better than the unvaccinated group, was the group 5, receiving the vaccinations of the method of vaccination according to the invention but in reversed order.

[0213] However, best of all were groups receiving as booster a vaccine comprising Alphavirus RNA RP2 alone or in combination with RP1. Group 2, receiving as booster a vaccine comprising Alphavirus RNA RP1 and RP2 had much reduced viremia post challenge, but group 3, receiving as booster vaccination the vaccine comprising Alphavirus RNA RP2 after the vaccine comprising live attenuated PRRSV had the lowest viremia overall.

[0214] The difference in viremia at 4 days post challenge between groups 3 (method of vaccination according to the invention, with RP2) and 5 (reversed order) is about 5 Cq points, which corresponds with a difference in virus load of 2{circumflex over ( )}5. Thus challenge virus viremia could be reduced with a factor of 32-fold, when applying the correct method of vaccination according to the invention.

[0215] Similarly, when considering the improvement in vaccine protection between single vaccine of live attenuated virus (group 1), and group 3 (method of vaccination according to the invention), the difference in viremia detected is 8 Cq points, a factor 256 less viremia, which is highly significant for the field, in terms of control of disease and spread of PRRSV in the herd.

[0216] Also this represents a broadening of the immune protection from the PRRSV DV strain of the priming vaccination, to the PRRSV Sc3 strain of the challenge virus, by using vaccine comprising an Alphavirus RNA RP encoding Sc3 genes.

4. Example 4: PEDV—Prime-Boost Vaccination Experiments

[0217] In line with the experiments performed for PRRSV, similar experiments were performed using a method of vaccination according to the invention to protect swine against infection with PEDV.

[0218] 4.1. Introduction

[0219] In this experiment, the efficacy of vaccination of pregnant sows was tested, in order to protect their offspring after suckling, against PEDV infection and disease, using a method of vaccination according to the invention. For the vaccine comprising a live attenuated virus was used the PEDV strain S-INDEL Iowa 106. This is one of the two types of the emerging strains of PEDV (Lin et al., 2016, Virus Res., vol. 226, p. 20-39). The vaccine of the Alphavirus RNA RP comprised an RP encoding the spike protein of a virulent US isolate of PEDV, which is a non-S-INDEL strain.

[0220] 4.2. Study Design

[0221] 14 pregnant sows were randomized into four treatment groups including 3 vaccinated groups and one non-vaccinated control group (group 4). All vaccinated sows (groups 1-3) received three vaccinations at 6, 3 and 1 week prior to farrowing. The vaccine for the first and second vaccination for sows in groups 2 and 3 was a vaccine comprising a live attenuated virus of PEDV strain S-INDEL Iowa 106, blended with one of two different mucosal adhesives. Sows in group 1 received two priming vaccinations with an RP vaccine encoding the spike gene of a PEDV non-S-INDEL strain. As the 3.sup.rd vaccination, all sows in groups 1-3 received a booster vaccination with the vaccine of the Alphavirus RNA RP encoding the PEDV-spike gene. An overview of the experimental groups is given in Table 3.

[0222] Suckling piglets born from the different sow groups were challenged intra-gastrically with a virulent PEDV strain. Vaccine efficacy was measured by the ability of the vaccine to reduce piglet mortality and maintain weight gain in the piglets. The immune response in respect of PEDV-specific, maternally derived antibodies (MDA) in piglets was also assessed.

TABLE-US-00003 TABLE 3 Experimental outline of Example 4 Dose Mucosal Weeks to Group N Vaccine (TCID or copy) adhesive Vaccination Route farrow 1(PED-RP) 4 RP 10{circumflex over ( )}8 copies No 1.sup.st IM 6 2.sup.nd IM 3 3.sup.rd IM 1 2 (Gantrez) 4 MLV 10{circumflex over ( )}6 TCID50 Gantrez 1.sup.st Oral 6 2.sup.nd Oral 3 RP 10{circumflex over ( )}8 copies No 3.sup.rd IM 1 3 (PVP) 4 MLV 10{circumflex over ( )}6 TCID50 PVP 1.sup.st Oral 6 2.sup.nd Oral 3 RP 10{circumflex over ( )}8 copies No 3.sup.rd IM 1 4 (Control) 2 — — — — — —

[0223] 4.3. Materials and Methods

[0224] Test Articles:

[0225] Vaccine of Alphavirus RNA RP:

[0226] The vaccine comprising an Alphavirus RNA RP was based on a replicon construct from VEEV strain TC-83. This was constructed to comprise the coding sequence of the spike protein of US virulent PEDV strain AH2012 (see GenBank Accession number KC210145). This was formulated at 10{circumflex over ( )}8 RP/ml, for 1 ml doses, without adjuvant, for IM vaccination.

[0227] The RP was titrated on Vero cell monolayer cultures in 96 well plates, in 10-fold serial dilutions. The plates were incubated at 37° C. for 18-24 hr. Next the plates were washed, fixed and stained with primary antibody followed by a conjugated secondary antibody. The cells are examined using a fluorescence microscope. The individual antigen-positive cells are counted. The titer, expressed as RP/ml, is calculated from the known dilution and inoculation volumes, and averaged between the duplicate wells.

[0228] Attenuated Live Vaccine:

[0229] Group 2 Vaccine:

[0230] 10{circumflex over ( )}6 TCID50 per dose of PEDV S-INDEL Iowa 106, passage 36 was mixed with Gantrez™ (1:1 v/v ratio), 5% Sucrose, and 20 mM HEPES pH 7.3, shortly prior to vaccination orally.

[0231] Group 3 Vaccine:

[0232] 10{circumflex over ( )}6 TCID50 per dose of PEDV S-INDEL Iowa 106, passage 36 was mixed with polyvinyl pyrrolidone (PVP) (1:1 v/v ratio) 5% Sucrose and 20 mM HEPES pH 7.3, shortly prior to vaccination orally.

[0233] The PEDV S-INDEL Iowa 106 virus was titrated on 96 well plates with confluent Vero cells. Plates were incubated at 37° C. with 5% CO.sub.2 and observed daily for cytopathic effect post infection for 2-3 days and/or tested using IFA staining with swine anti-PEDV specific antibody (USDA-NVSL, Ames, Iowa) followed by a FITC conjugated secondary antibody at the end of the incubation period. PEDV titers were calculated as Log 10 TCID50 per mL, using the Spearman/Karber method.

[0234] Challenge Virus:

[0235] Challenge was given intra-gastrically to suckling pigs at 3 to 5 days of age, with PEDV strain Colorado 2013 isolate, at 10{circumflex over ( )}5 TCID50 per pig.

[0236] Challenge virus was titrated on Vero cells, similar to PEDV live attenuated virus.

[0237] Fecal Shedding

[0238] PEDV RNA extraction and detection of PEDV viremia was done using qRT-PCR, according to Chen et al. (2014, J. Clin. Microbiol., vol. 52, p. 234-243). MagMAX™ Pathogen RNA/DNA Kit (Life Technologies, Carlsbad, Calif., USA) and a Kingfisher 96 instrument (Thermo Scientific, Waltham, Mass., USA) were used following the instructions of the manufacturers. The qRT-PCR was performed using a Path-ID Multiplex One-Step RT-PCR kit (Life Technologies) and conducted on an ABI 7500 Fast Instrument™ (Life Technologies).

[0239] Serology

[0240] PEDV-specific neutralizing antibodies in serum, colostrum and milk in sows and passive antibodies in piglet sera was determined by fluorescent focus neutralization (FFN) test.

[0241] PEDV antibody in sera against the 51 part of the PEDV spike protein of the challenge PEDV strain were determined in an antibody ELISA: micro-titration plates were coated with 100 μl of a PEDV spike protein (recombinant S1 dimer from PEDV strain Colorado 2013; 0.25 μg/ml). Sera were diluted 1:900 in PBS/1 BSA and added to the coated wells, followed by incubation for 1 hour at 37° C. After two washes with PBS/0.05% Tween20, 100 μl of rabbit anti-swine IgG HRPO conjugate diluted 1:10.000 in PBS/1% BSA/0.5% Tween20 was added to the wells, and plates were incubated for 1 hour at 37° C. After three washes with PBS/0.05% Tween20, 100 μl/well of TMB “Super Slow”™ substrate was added to each well followed by incubation at room temperature for 10 min in the dark. Reactions were stopped by adding 100 μl of 25% sulfuric acid per well. The optical density (OD) at 450 nm was measured with an ELISA reader. Sera from naturally PEDV infected pigs were included as positive controls.

[0242] 4.4. Results and Conclusions

[0243] Results are presented in Tables 4-6 below.

[0244] Piglets born from pregnant sows in groups 2 and 3 that were vaccinated with the method of vaccination according to the invention had significantly reduced percent mortality compared to piglets born from non-vaccinated sows (group 4), and compared to the group receiving the homologous prime-boost RP vaccine (group 1).

[0245] Sows primed orally with the heterologous method of vaccination (groups 2 and 3) had significantly higher PEDV-specific antibody levels in serum compared to sows vaccinated with the homologous prime-boost RP vaccine (group 1) and non-vaccinated control sows (group 4).

[0246] Piglets born from sows of groups 2 and 3 had significantly higher PEDV-specific neutralizing MDA compared to piglets born from sows of groups 1 and 4. The IgA antibody levels in piglets from sows of group 2 were significantly higher than in all other vaccinated groups.

TABLE-US-00004 TABLE 4 Piglet morbidity, mortality and weight gain demonstrated by litter Number Number Number Number of Number Average weight of of Morbid of Dead Morbid of Dead gain (lb.) from Treatment SowID Piglets Piglets Piglets Piglets Piglets piglets that survived 1 723 7 0 7 11 (37%) 26 (87%) 2.4 (PED-RP) 724 10 1 7 725 6 4 6 726 7 6 6 2 715 10 4 10  4 (14%) 11 (38%) 3.8 (Gantrez) 716 5 0 1 717 9 0 0 718 5 0 0 3 719 7 0 6  2 (6%) 16 (48%) 5.7 (PVP) 720 10 2 7 721 9 0 0 722 7 0 3 4 727 7 0 7 12 (63%) 19 (100%) NA (Control) 728 12 12 12

TABLE-US-00005 TABLE 5 PEDV-specific antibody response by FFN and IgA ELISA following vaccination in sows Treatment Serum Mean Ln(FFN) Serum Mean IgA ELISA 1 (PEDV-RP) 5.25 0.0685 2 (Gantrez) 6.81 0.2135 3 (PVP) 7.08 0.2830 4 (Control) 2.30 0.0030

TABLE-US-00006 TABLE 6 PEDV-specific maternally derived antibodies in piglets prior to challenge Number of Ln(FFN) IgA ELISA Treatment SowID Piglets Average Average 1 723 7 4.54 0.0283 (PEDV-RP) 724 10 725 6 726 7 2 715 10 6.27 0.2954 (Gantrez) 716 5 717 9 718 5 2 719 7 6.28 0.3093 (PVP) 720 10 721 9 722 7 5 727 7 2.34 0.0384 (Control) 728 12

LEGEND TO THE FIGURES

[0247] FIG. 1

[0248] PRRSV viremia results of the groups tested in the experiment of Example 1.

[0249] FIG. 2

[0250] Results of PRRSV viremia determinations from the experiments described in Example 2, as qPCR scores per group.

[0251] FIG. 3

[0252] Results of PRRSV viremia determinations from the experiments described in Example 3, as qPCR scores per group.