Experimental quantification of transmission of genetically engineered pseudorabies virus

There is concern that live pseudorabies virus (PRV) vaccine or PRV vector vaccine strains may spread from vaccinated to unvaccinated pigs. Moreover, it is feared that recombining PRV vaccine strains with related vaccine or wild-type strains may lead to spread and survival of recombinant PRV. To learn more about to what extent different PRV vaccine strains could spread we used a previously described experimental model to study the transmission of intranasally inoculated PRV mutant strains under experimental conditions. We used PRV strains that lacked glycoprotein E (gE) or thymidine kinase (TK), and a PRV vector vaccine (gE⁻, TK⁻, gG⁻) that expresses the glycoprotein E1 (E1) of hog cholera virus. In addition, we investigated whether intranasally co-inoculated gE-negative and gE-positive PRV strains competed in transmission among pigs. The extent of transmission was estimated using the reproduction ratio R. This ratio has a threshold property; when R1, the infection can spread; when R<1, the infection will disappear. We found that R for a gE-negative strain was 10.1, and R for a TK-negative strain was 5. Furthermore, the R for the vector vaccine (gE⁻, TK⁻, gG⁻) expressing E1 was 0.18, and did not differ significantly from the R for the control strain without E1. The R of gE-negative strain was significantly 1 (P=0.0005). Co-inoculation with a gE-positive field strain did not prevent the transmission of a gE-negative strain. This study shows that a small-scale experiment can be used to estimate the transmission of genetically engineered organisms in their host species. The results of this study indicate that the deletion of ge alone or TK alone is not enough to prevent spread of PRV among susceptible pigs, and that transmission of gE-negative PRV is not firmly limited by co-presence of a gE-positive strain.     

An approach to a FMD vaccine based on genetic engineered attenuated pseudorabies virus: one experiment using VP1 gene alone generates an antibody responds on FMD and pseudorabies in swine

Foot-and-mouth disease (FMD) and pseudorabies (PR) are two important infectious diseases in swine. An attenuated pseudorabies virus (PRV) has been successfully used as a gene delivery vector for the development of live-viral vaccines. In this study, a recombinant PRV-VP1 virus was constructed by fusioning the VP1 gene of FMD virus in frame to the N-terminal sequence of the gG gene of PRV. To test the protective immunity, 15 FMDV sero-negative white swine were divided into three groups and immunized with the recombinant PRV-VP1 virus, commercial FMD vaccine and vector virus (TK(-)/gG(-)/LacZ(+)), respectively, and challenged intramuscularly with 20 minimal infecting doses (MID) of virulent type O FMDV 4 weeks after booster immunization. Swine vaccinated with PRV-VP1 acquired antibodies against both FMDV and PRV, however, anti-FMDV antibodies were much lower than those vaccinated with the commercial FMD vaccine. Our results suggested that the recombinant PRV-VP1 virus, which only expressed FMDV VP1 gene controlled by PRV gG promoter, could not protect swine from the challenge of 20 MID type O FMDV, but could delay and reduce the clinical symptoms of FMD.     

Comparison of immune responses after intra-typic heterologous and homologous vaccination against foot-and-mouth disease virus infection in pigs

This study compares the immune responses and protection induced by intra-typic heterologous vaccination with that induced by homologous vaccination against challenge with foot-and-mouth disease virus (FMDV). Humoral and cell-mediated immune responses and protection against challenge with FMDV O Taiwan were examined in a non-vaccinated group, a group vaccinated with O Taiwan FMD vaccine and a group vaccinated with O Manisa FMD vaccine. Five pigs from each group were challenged with FMDV type O Taiwan 14 days after vaccination and five other pigs were contact-exposed to the inoculated pigs. Both homologous and heterologous vaccination protected against challenge with FMDV O Taiwan at 2 weeks after vaccination. In the heterologous vaccinated group, cross-neutralizing antibody titres against O Taiwan could be detected although the ratio 'r(1)' was 0.4, which was significantly smaller than the critical r-value. Cell-mediated immune responses were detected after both homologous and heterologous vaccination. Virus-induced in vitro lymphocyte (cross-) proliferation and production of both a Th1-type (IFN-gamma) and a Th2-type (IL-10) cytokine response were demonstrated in cultures of peripheral blood mononuclear cells (PBMC). The findings show that heterologous (emergency) vaccination can prevent clinical disease and shedding of virus. The induction of the cell-mediated immune responses after (heterologous) vaccination needs more research but data on these responses might provide additional tools for both vaccine choice and vaccine development.     

Foot and mouth disease virus transmission among vaccinated pigs after exposure to virus shedding pigs

The aim of this study was to design a transmission experiment that enabled quantification of the effectiveness of vaccination against foot and mouth disease (FMD) virus in groups of pigs. Previous experiments showed that intradermal injection of pigs with FMD virus 14 days after vaccination was not suitable to start an infection chain, as inoculated vaccinated pigs resisted challenge. Therefore, we carried out two experiments in which we used direct contact to a non-vaccinated pig as route of infection. In the first experiment only the vaccine effect on susceptibility was quantified by exposing pigs, either vaccinated 14 days before or not vaccinated, each to a non-vaccinated seeder pig inoculated with FMD virus O/NET/2001. Since no significant differences were observed between contact infections in vaccinated or non-vaccinated pigs, we performed a second experiment in which both susceptibility and infectivity were subject to vaccination. We quantified virus transmission in homogenous groups of vaccinated or non-vaccinated pigs in which the infection chain was started by exposure to a third group of non-vaccinated infected pigs. Transmission occurred to all contact-exposed pigs in the non-vaccinated groups and to 9 out of 10 contact-exposed pigs in the vaccinated groups. The rate of transmission (beta) was significantly reduced in the vaccine group. Yet, the estimated reproduction ratio in both groups was still above 1. In conclusion, by adjusting our transmission study design and challenge method, we were able to quantify transmission of FMDV among vaccinated pigs. According to this study a single vaccination was not sufficient to stop pig to pig virus transmission. With these results major outbreaks may still be expected, even in groups of vaccinated pigs.     

Use of ENABL® adjuvant to increase the potency of an adenovirus-vectored foot-and-mouth disease virus serotype A subunit vaccine

A foot-and-mouth disease (FMD) recombinant subunit vaccine formulated with a lipid/polymer adjuvant was evaluated in two vaccine efficacy challenge studies in steers. The vaccine active ingredient is a replication-deficient human adenovirus serotype 5 vector encoding the FMD virus (FMDV) A24/Cruzeiro/BRA/55 capsid (AdtA24). In the first study, AdtA24 formulated in ENABL® adjuvant was compared to a fourfold higher dose of AdtA24 without adjuvant. Steers vaccinated with AdtA24 + ENABL® adjuvant developed a significantly higher virus neutralizing test (VNT) antibody titer and an improved clinical response following FMDV A24/Cruzeiro/BRA/55 intradermal lingual challenge at 14 days post-vaccination (dpv) than steers vaccinated with the active ingredient alone. In the second study, vaccination with AdtA24 formulated in ENABL® at the same dose used in the first study, followed by FMDV A24/Cruzeiro/BRA/55 challenge on 7 or 14 dpv, prevented clinical FMD in all steers and conferred 90% protection against viremia. In addition, post-challenge FMDV titers in nasal samples from vaccinated steers compared to unvaccinated steers were significantly reduced. In both studies, none of the AdtA24 vaccinated steers developed antibodies to the FMDV non-structural proteins prior to challenge with FMDV, indicative of the capacity to differentiate infected from vaccinated animals (DIVA). These results demonstrate that administration of AdtA24 formulated in ENABL® adjuvant lowered the protective dose and prevented clinical FMD following exposure of vaccinated steers to virulent FMDV at 7 or 14 dpv.     

Bartha-k61 vaccine protects growing pigs against challenge with an emerging variant pseudorabies virus

Since late 2011, pseudorabies (PR) has resurfaced in many large pig farms, causing great economic loss for the swine industry in China. The PRV variant strain with high virulence and antigenic variation has been considered to be the main cause, and much attention has been focused on how to prevent and control the reoccurrence of this disease in China. In this study, two kinds of vaccination strategy were employed to evaluate the protective effects of Bartha-k61vaccine against both variant PRV (XJ5) and classical PRV (Ra) strain challenge. Humoral immunity response, clinical signs, survival rate, body weight, virus shedding and pathology were assessed in commercial pigs. The results showed that Bartha-k61vaccine, administered either once or twice, was effective against the PRV variant (XJ5) challenge, while no significant differences were observed between single and prime-boost vaccinated pigs. However, pigs vaccinated twice had better body weight gains than those vaccinated once, following challenge with the classical PRV strain (Ra) (p < 0.01). Therefore, the Bartha-k61 vaccine appears to be an effective vaccine to control the spread of PRV variants in China in the absence of new powerful candidate vaccines specific to these PRV strains.     

Orally delivered foot-and-mouth disease virus capsid protomer vaccine displayed on T4 bacteriophage surface: 100% protection from potency challenge in mice

An orally delivered foot-and-mouth disease (FMD) vaccine has not previously been reported. By using a T4 bacteriophage nanoparticle surface gene-protein display system (T4-S-GPDS), we created a foot-and-mouth disease virus (FMDV) entire capsid protein vaccine candidate. On the T4 phage surface SOC site, a full length FMDV capsid precursor polyprotein (P1, 755 aa) and proteinase 3C (213 aa) derived from an infected pig of serotype O strain GD-10 (1999), were separately displayed on different T4 phage particle surfaces through inserting their coding region DNAs into the T4 phage genome, yielding phage strains T4-P1 and T4-3C. We also constructed a series of FMDV sub-full length capsid structural protein (subunit) containing T4 phage recombinant vaccines. Both sucking and young BALB/c mice were used as two kinds of FMDV vaccine potency evaluation models. Many groups of both model mice were vaccinated orally or by subcutaneous injection with varying FMDV-T4 phage recombinant vaccines, with and without addition of adjuvant, then challenged with a lethal dose of cattle source virulent FMDV. In the case of immunization with a mixture of phage T4-P1 and phage T4-3C particles without any adjuvant added, all mice were 100% protected following either oral or injection immunization, whereas 100% of the control, non-immunized mice and mice immunized with only T4 phage vector Z1/Zh(-) or wild-type T4(+)D phage died; in contrast, with FMDV subunit vaccine, less than 75% protection followed the same potency challenge in both mice model groups. In addition, two pigs immunized with a phage T4-P1 and phage T4-3C mix were protected upon housing together with infected pigs. This study represents a clear example of how FMD and other pathogenic disease vaccines can be prepared by a simple and efficient bacteriophage route.     

Protective immunity against foot-and-mouth disease virus induced by a recombinant vaccinia virus

We report the construction of a recombinant vaccinia virus expressing the precursor for the four structural proteins of FMD virus (FMDV) (P1) strain C3Arg85 using a procedure for isolation of recombinant vaccinia viruses based solely on plaque formation. Adult mice vaccinated with this recombinant vaccinia virus elicited high titers of neutralizing antibodies against both the homologous FMDV and vaccinia virus, measured by neutralization assays. Liquid phase blocking sandwich enzyme-linked immunosorbent assays (ELISAs) using whole virus as antigen showed high total antibody titers against homologous FMDV, similar to those induced by the conventional inactivated vaccine. When ELISAs were carried out with heterologous strains A79 or O1Caseros as antigens, sera from animals vaccinated with the recombinant virus cross-reacted. Mice boosted once with the recombinant vaccinia virus were protected against challenge with infectious homologous virus. These results indicate that recombinant vaccinia viruses are efficient immunogens against FMDV when used as a live vaccine in a mouse model.     

A foot-and-mouth disease SAT2 vaccine protects swine against experimental challenge with a homologous virus strain,irrespective of mild pathogenicity in this species

FMDV serotype SAT2 is most frequently associated with outbreaks in ruminants. However, the risk of it spreading from cattle to pigs cannot be excluded. To assess the efficacy of an SAT2-type FMD inactivated vaccine against homologous challenge in pigs, a suitable challenge strain adapted to pigs was produced. After two passages in two pigs each, a FMDV stock of SAT2 challenge strain was produced. This material was used to infect two groups of five pigs. The first group being vaccinated 28 days before challenge and the other one left as an unvaccinated control. Clinical signs were recorded, virus shedding was assessed on mouth swabs, and neutralising antibody titres were determined. At least 80% of the vaccinated pigs were protected against clinical disease. Furthermore, no virus shedding was observed in any of the vaccinated pigs. This study shows that experimentally inoculated pigs can become infected with a SAT2 serotype. Furthermore, vaccination offers protection against generalisation and viral excretion, confirming the potential of vaccination as an important tool in the control of FMD in pigs.     

Immunogenicity and protective efficacy of recombinant pseudorabies virus expressing the two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) infection still remains today as the most significant health threat to swine and poses a challenge to current vaccination strategies. To develop a new generation of vaccine against PRRSV, a live attenuated pseudorabies virus (PRV) was used as vaccine vector to express the two major membrane-associated proteins (GP5 or M) of PRRSV in various forms. Four PRV recombinants, rPRV-GP5 (expressing native GP5), rPRV-GP5m (expressing GP5m, a modified GP5), rPRV-GP5-M (co-expressing GP5 and M proteins), rPRV-GP5m-M (co-expressing GP5m and M proteins) were generated. Mouse immunized with all these recombinants developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. However, the highest level of PRRSV-specific neutralizing antibodies and lymphocyte proliferative responses was observed in mice immunized with rPRV-GP5m-M. The immunogenicity and protective efficiency of rPRV-GP5m-M were further evaluated in the piglets. Compared to commercial PRRSV killed vaccine, detectable PRRSV-specific neutralizing antibody and higher lymphocyte proliferative responses could be developed in piglets immunized with rPRV-GP5m-M before virus challenge. Furthermore, more efficient protection against a PRRSV challenge was obtained in piglets immunized with rPRV-GP5m-M, as showed by the balanced body-temperature fluctuation, shorter-term viremia, lower proportion of virus load in nasal and oropharyngeal scrapings and tissues, and milder lung lesions. These data indicate that the recombinant rPRV-GP5m-M is a promising candidate bivalent vaccine against both PRV and PRRSV infection.     

Expression of foot-and-mouth disease virus epitopes in tobacco by a tobacco mosaic virus-based vector

We expressed two immunogenic dominant epitopes of foot-and-mouth disease virus (FMDV) serotype O in tobacco plant using a vector based on a recombinant tobacco mosaic virus (TMV). The recombinant viruses TMVF11 and TMVF14 contained peptides of 11 and 14 amino acid residues, respectively, from FMDV VP 1 fused to the open reading frame of TMV coat protein (CP) gene between amino acid residues 154 and 155. TMVF11 and TMVF14 systemically infected tobacco plant and produced large quantities of stable progeny viral particles assembled with the modified CP subunits. Guinea pigs, mice and swine were used to test the protective effects of the recombinant viruses against FMDV infection. Most guinea pigs were protected against FMDV challenge after parenteral injection with TMVF11, TMVF14, or the mixture TMVF11/TMVF14, but not wtTMV. The TMVF11/TMVF14 mixture protected all animals when challenged with 150 guinea pig 50% infection dosage (GPID(50)) FMDV. Oral administration of the TMVF11/TMVF14 mixture (3mg total) protected 3/8 guinea pigs against the same FMDV challenge. Most of the suckling mice parenterally injected with antiserum from guinea pigs immunized with the TMVF11/TMVF14 mixture, but not with wtTMV, were also protected against FMDV challenge with 10 suckling mouse 50% lethal dosage (SMLD(50)), indicating that antibodies produced in guinea pigs immunized with the TMVF11/TMVF14 mixture specifically neutralized FMDV. Western blot analysis indicated that antiserum from those guinea pigs reacted with the FMDV VP1 protein. The protective effect of TMVF11 was also demonstrated in swine, where preliminary tests showed that nine pigs immunized with TMVF11 in three experiments were protected against FMDV challenge with 20 minimal infecting dose (MID).     

Immunopotentiation of a foot-and-mouth disease virus subunit vaccine by interferon alpha

The adjuvant effect of porcine interferon alpha (pIFN-alpha) was examined in swine vaccinated with a replication-defective adenovirus containing foot-and-mouth disease virus (FMDV) A24 capsid and 3C proteinase coding regions (Ad5-A24). Groups of swine were inoculated with either high or low doses of Ad5-A24 in the presence or absence of Ad5-pIFNalpha or with a control Ad5 and challenged by intradermal inoculation in the heel bulb with FMDV at 42 days post-vaccination. After challenge all control animals developed viremia and lesions. Animals receiving low-dose Ad5-A24 had similar clinical disease, but only three of five animals developed viremia, while addition of IFN resulted in a delayed onset of lesions in three animals and only one animal had detectable viremia. Animals vaccinated with high-dose Ad5-A24 had no viremia, significantly fewer lesions and delayed onset of disease compared to the control and low-dose vaccine groups. Four of five pigs vaccinated with high-dose Ad5-A24 plus IFN were completely protected from disease and only one animal had a lesion which was restricted to the site of challenge. Thus, pIFN-alpha enhances the long-term level of protection induced by the Ad5-FMD vaccine, supporting its use as a potential adjuvant in FMD vaccination strategies.     

Early protection against homologous challenge after a single dose of replication-defective human adenovirus type 5 expressing capsid proteins of foot-and-mouth disease virus (FMDV) strain A24

Previously we demonstrated that two doses of a replication-defective human adenovirus serotype 5 (Ad5) carrying the capsid (P1) and 3C protease coding regions of a laboratory strain of FMDV (A12) completely protected five of six swine challenged with homologous virus. The objective of the current study was to evaluate the efficacy of one dose of an Ad5-vectored vaccine expressing the P1 coding region of an FMDV field strain. A replication-defective Ad5 containing the P1 coding region of FMDV A24 and the 3C coding region of A12 (Ad5A24) was constructed and evaluated for its ability to induce neutralizing antibodies and protect swine against homologous challenge after a single vaccination. Animals were challenged 7, 14 or 42 days after vaccination. Control groups included animals inoculated with commercial vaccine or phosphate-buffered saline. All vaccinated swine were completely protected against homologous challenge at 7, 14 or 42 days after vaccination. Based on these results, we conclude that a single inoculation of Ad5-vectored vaccines could be used as a tool to control FMD in outbreak situations.     

Alpha interferon is a powerful adjuvant for a recombinant protein vaccine against foot-and-mouth disease virus in swine, and an effective stimulus of in vivo immune response

The adjuvant effect of porcine interferon-alpha (PoIFN-alpha) was examined in swine vaccinated with a recombinant FMD protein vaccine named IgG-FMDV, which contains the swine IgG single heavy chain constant region and an immunogenic peptide of serotype O FMDV. The PoIFN-alpha gene was cloned into pcDNA3 vector and the recombinant plasmid was incorporated into cationic liposomes by a dehydration and rehydration procedure to use as an adjuvant, injected together with low-dose IgG-FMDV. This procedure resulted in strong induction of FMDV-specific neutralizing antibody and significant T-cell-mediated immune responses, whereas only a modest humoral and cellular response was observed with low-dose vaccine alone. As an adjuvant for the protein vaccine, PoIFN-alpha induced strong inflammatory cytokines production in vivo and the results denoted that IFN-adjuvant and our vaccines could drive the immune response toward Th1 type responses. The data of ELISA suggests that the recombinant protein vaccine synergizes with the IFN-adjuvant to produce endogenous IFN in vivo. In response to viral challenge, all control animals developed viremia and lesions, whereas all animals received IFN-adjuvant+IgG-FMDV were protected and nonstructural protein antibody in this group could not be detected by 14 days post-challenge (dpc). Our studies indicate that porcine IFN-alpha is a powerful adjuvant for recombinant FMD protein vaccine and could aid in vaccination against FMDV in swine.     

Prospects,including time-frames,for improved foot and mouth disease vaccines

Inactivated foot and mouth disease (FMD) vaccines have been used successfully as part of eradication programmes. However, there are a number of concerns with the use of such vaccines and the recent outbreaks of FMD in disease-free countries have increased the need for improved FMD control strategies. To address this requirement, new generation FMD vaccines are being developed. Currently, one of the most promising of these vaccine candidates utilises an empty viral capsid subunit delivered to animals by a live virus vector. This candidate, a replication-defective recombinant human adenovirus containing the capsid and 3C proteinase coding regions of FMD virus (FMDV), induces an FMDV-specific neutralising antibody response in inoculated animals. Upon challenge with a virulent animal-passaged homologous virus, swine and cattle vaccinated with this recombinant adenovirus are protected from clinical signs of FMD as well as from FMDV replication. One inoculation of a high dose of this vaccine candidate protected swine from challenge as early as seven days after vaccination.     

Versatility of the adenovirus-vectored foot-and-mouth disease vaccine platform across multiple foot-and-mouth disease virus serotypes and topotypes using a vaccine dose representative of the AdtA24 conditionally licensed vaccine

We investigated the serotype- and topotype versatility of a replication-deficient human adenovirus serotype 5 vectored foot-and-mouth disease (FMD) vaccine platform (AdtFMD). Sixteen AdtFMD recombinant subunit monovalent vaccines targeting twelve distinct FMD virus (FMDV) serotype/topotypes in FMD Regional Pools I-VII were constructed. The AdtA24 serotype conditionally licensed vaccine served as the basis for vaccine design and target dose for cattle clinical trials. Several vaccines contained an additional RGD motif genetic insertion in the adenovector fiber knob, and/or a full-length 2B gene insertion in the FMDV P1 gene cassette. In 13 of the 22 efficacy studies conducted, naïve control and AdtFMD vaccinated cattle were challenged intradermolingually at 2 weeks post-vaccination using a FMDV strain homologous to the AdtFMD vaccine strain. Each of the 16 AdtFMD vaccines were immunogenic based on the presence of homologous neutralizing antibodies in the serum of approximately 90% of total vaccinates (n = 375) on the day of challenge. Importantly, for 75% of vaccines tested, the effective dose that conferred 100% protection against clinical FMD was identical to or in some cases lower than, the minimum protective dose for the conditionally licensed AdtA24 vaccine formulated with ENABL® adjuvant. Results also confirmed the capability of the AdtFMD vaccine platform to differentiate infected from vaccinated animals (DIVA) across the five FMDV serotypes evaluated. Collectively, this comprehensive set of FMD cattle vaccine dose ranging studies highlights the serotype- and topotype versatility of the AdtFMD vaccine platform for further development, licensure, and application in FMD outbreak control and disease eradication efforts.     

Effective synthetic peptide vaccine for foot-and-mouth disease in swine

We have designed a peptide-based vaccine for foot-and-mouth disease (FMD) effective in swine. The peptide immunogen has a G-H loop domain from the VP1 capsid protein of foot-and-mouth disease virus (FMDV) and a novel promiscuous T helper (Th) site for broad immunogenicity in multiple species. The G-H loop VP1 site was optimised for cross-reactivity to FMDV by the inclusion into the peptide of cyclic constraint and adjoining sequences. The incorporation of consensus residues into the hypervariable positions of the VP1 site provided for broad immunogenicity. The vaccine protected 20 out of 21 immunised pigs from infectious challenge by FMDV O1 Taiwan using peptide doses as low as 12.5 microg, and a mild adjuvant that caused no lesions. A safe chemically-defined product would have considerable advantages for vaccination against FMD.     

Immune responses of pigs to commercialized emulsion FMD vaccines and live virus challenge

The immune response to structural and non-structural proteins (NSPs) was studied on sequential serum samples in swine from O/Taiwan/97 FMDV challenge studies, outbreaks and after vaccination. The results showed that pigs vaccinated with a commercial vaccine prior to or after infection maintained high neutralizing antibody titers with gradual decline from peak titers over the duration of this study. However, neutralizing antibody titers in non-vaccinated pigs only reached moderate levels 2-4 weeks post infection and remained low thereafter. For the 3B and 3ABC NSP antibody ELISA responses, there were gradually decreasing levels of NSP antibody over time. In multiple vaccinations, all pigs showed significant increases in neutralizing antibodies after booster vaccination. For the 3B NSP antibody ELISA after vaccination, the mean S/P ratios for pigs vaccinated with all three FMD vaccines were all below the 0.23 cut-off value set by the manufacture, but some sera from individual vaccinated pigs gave results above this cut-off after primary or secondary vaccination. However, with the 3ABC NSP antibody ELISA, all sera from vaccinated pigs had negative results for NSP antibody for all time points.     

Early protection in sheep against intratypic heterologous challenge with serotype O foot-and-mouth disease virus using high-potency,emergency vaccine

In 2009–2011, spread of a serotype O foot-and-mouth disease virus (FMDV) belonging to the South East Asia topotype led to the culling of over 3.5 million cattle and pigs in Japan and Korea. The O1 Manisa vaccine (belonging to the Middle East-South Asian topotype) was used at high potency in Korea to limit the expansion of the outbreak. However, no data are available on the spread of this virus or the efficacy of the O1 Manisa vaccine against this virus in sheep. In this study, the early protection afforded with a high potency (>6 PD₅₀) FMD O1 Manisa vaccine against challenge with the O/SKR/2010 virus was tested in sheep. Sheep (n = 8) were vaccinated 4 days prior to continuous direct-contact challenge with donor sheep. Donor sheep were infected with FMDV O/SKR/2010 by coronary band inoculation 24 h prior to contact with the vaccinated animals, or unvaccinated controls (n = 4). Three of the four control sheep became infected, two clinically. All eight O1 Manisa vaccinated sheep were protected from clinical disease. None had detectable antibodies to FMDV non-structural proteins (3ABC), no virus was isolated from nasal swabs, saliva or oro-pharyngeal fluid and none became carriers. Using this model of challenge, sheep were protected against infection as early as 4 days post vaccination.     

Vaccination-challenge studies with a Port Chalmers/73 (H3N2)-based swine influenza virus vaccine: Reflections on vaccine strain updates and on the vaccine potency test

The human A/Port Chalmers/1/73 (H3N2) influenza virus strain, the supposed ancestor of European H3N2 swine influenza viruses (SIVs), was used in most commercial SIV vaccines in Europe until recently. If manufacturers want to update vaccine strains, they have to perform laborious intratracheal (IT) challenge experiments and demonstrate reduced virus titres in the lungs of vaccinated pigs. We aimed to examine (a) the ability of a Port Chalmers/73-based commercial vaccine to induce cross-protection against a contemporary European H3N2 SIV and serologic cross-reaction against H3N2 SIVs from Europe and North America and (b) the validity of intranasal (IN) challenge and virus titrations of nasal swabs as alternatives for IT challenge and titrations of lung tissue in vaccine potency tests. Pigs were vaccinated with Suvaxyn Flu^® and challenged by the IT or IN route with sw/Gent/172/08. Post-vaccination sera were examined in haemagglutination-inhibition assays against vaccine and challenge strains and additional H3N2 SIVs from Europe and North America, including an H3N2 variant virus. Tissues of the respiratory tract and nasal swabs were collected 3 days post challenge (DPCh) and from 0–7 DPCh, respectively, and examined by virus titration. Two vaccinations consistently induced cross-reactive antibodies against European H3N2 SIVs from 1998–2012, but minimal or undetectable antibody titres against North American viruses. Challenge virus titres in the lungs, trachea and nasal mucosa of the vaccinated pigs were significantly reduced after both IT and IN challenge. Yet the reduction of virus titres and nasal shedding was greater after IT challenge. The Port Chalmers/73-based vaccine still offered protection against a European H3N2 SIV isolated 35 years later and with only 86.9% amino acid homology in its HA1, but it is unlikely to protect against H3N2 SIVs that are endemic in North America. We use our data to reflect on vaccine strain updates and on the vaccine potency test.