Marker Vaccine Research Articles

Protection against transplacental transmission of moderately virulent classical swine fever virus using live marker vaccine “CP7_E2alf”

Classical swine fever (CSF) remains as one of the most important infectious diseases of swine. While prophylactic vaccination is usually prohibited in free countries with industrialized pig production, emergency vaccination is still foreseen. In this context, marker vaccines are preferred as they can reduce the impact on trade.The live-attenuated Suvaxyn® CSF Marker vaccine by Zoetis (based on pestivirus chimera “CP7_E2alf”), was recently licensed by the European Medicines Agency. Its efficacy for the individual animal had been shown in prior studies, but questions remained regarding protection against transplacental transmission. To answer this question, a trial with eight pregnant sows and their offspring was performed as prescribed by the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Six of the sows were intramuscularly vaccinated on day 44 of gestation, while the other two remained as unvaccinated controls. All sows were challenged with the moderately virulent CSFV strain ”Roesrath” and euthanized shortly before the calculated farrowing date. Sows and piglets were grossly examined and necropsied. Organs (spleen, tonsil, lymph node, and kidney), EDTA-blood and serum were collected from all animals. All samples were tested for antibodies against CSFV glycoproteins E2 and Erns as well as CSFV (virus, antigen and genome). It could be demonstrated that the vaccine complies with all requirements, i.e. no virus was found in the blood of vaccinated sows and their fetuses, and no antibodies were found in the serum of the fetuses from the vaccinated sows. All controls were valid.Thus, it was demonstrated that a single dose vaccination in the sows efficiently protected the offspring against transplacental infection with a moderately virulent CSFV strain.

Разработка и оценка эффективности живой маркированной вакцины «ВЕРРЕС-БАgE-» против болезни Ауески

Разработка и оценка эффективности живой маркированной вакцины «ВЕРРЕС-БАgE-» против болезни Ауески

Bovine herpesvirus 1 infection in cattle: a discussion on vaccination and control

Infectious bovine rhinotracheitis is a disease of significant financial importance in cattle systems throughout the world. The causal organism bovine herpesvirus 1 (BHV1) is recognised as one of the agents involved in the bovine respiratory disease complex and infection with the virus also has an adverse impact on reproductive performance and can result in fetopathy. Latent viral infection is an important feature, allowing the virus to persist successfully in herds infecting successive generations of animals. In naïve animals BHV1 interferes with the innate response to infection by inhibiting interferon production and by infecting leukocytes, slowing the development of the immune response. Nevertheless recovery from infection is associated with a strong and life long immunity. A range of approaches have been taken to vaccination to protect against this disease, including gene-deleted marker vaccines that have facilitated a differentiation of infection from vaccination (DIVA) approach to diagnosis and herd level eradication. In general the diagnostic tests available are adequate to support herd and regional control and eradication programmes.

Porcine reproductive and respiratory syndrome virus expressing E2 of classical swine fever virus protects pigs from a lethal challenge of highly-pathogenic PRRSV and CSFV

Porcine reproductive and respiratory syndrome (PRRS) and classical swine fever (CSF) are economically significant diseases that affect the swine industry worldwide. However, the current vaccination strategy, which uses two single live attenuated vaccines, can result in interference for each other. In addition, the universally used CSFV vaccine C-strain does not allow for differentiation of infected and vaccinated animals. In this study, rPRRSV-E2, PRRS virus (PRRSV) expressing CSF virus (CSFV) E2, was constructed by reverse genetics. The E2 gene of CSFV was inserted between ORF1b and ORF2 in the genome of the PRRS vaccine virus, HuN4-F112. A copy of transcriptional regulatory sequence 6 was inserted at the 3′ terminal of the exogenous gene to produce CSFV E2 as a unique subgenomic mRNA transcript. The rPRRSV-E2 was stable for at least 25 serial cell passages. Single-shot intramuscular immunization of rPRRSV-E2 into pigs induced PRRSV-specific and CSFV-specific antibodies and fully protected pigs from lethal challenge with highly-pathogenic PRRSV and CSFV. These results demonstrate that a novel strategy for recombinant PRRSV production is effective, and suggest that rPRRSV-E2 is a promising live, virus-vectored vaccine against PRRS and a marker vaccine against CSF.

A canine adenovirus type 2 vaccine vector confers protection against foot-and-mouth disease in guinea pigs

Vaccination is a key element in the control of foot-and-mouth disease (FMD). The majority of the antigenic sites that induce protective immune responses are localized on the FMD virus (FMDV) capsid that is formed by four virus-encoded structural proteins, VP1 to VP4. In the present study, recombinant canine adenovirus type 2 (CAV2)-based FMD vaccines, Cav-P1/3C R° and Cav-VP1 R°, respectively expressing the structural P1 precursor protein along with the non-structural 3C protein or expressing the structural VP1 protein of the FMDV strain O/FRA/1/2001, were evaluated as novel vaccines against FMD. A strong humoral immune response was elicited in guinea pigs (GP) following immunization with Cav-P1/3C R°, while administration of Cav-VP1 R° did not induce a satisfying antibody response in GP or mice. GP were then used as an experimental model for the determination of the protection afforded by the Cav-P1/3C R° vaccine against challenge with the FMDV strain O1 Manisa/Turkey/1969. The Cav-P1/3C R° vaccine protected GP from generalized FMD to a similar extent as a high potency double-oil emulsion O1 Manisa vaccine. The results of the present study show that CAV2-based vector vaccines can express immunogenic FMDV antigens and offer protection against generalized FMD in GP. This suggest that Cav-P1/3C R° FMDV vaccine may protect natural host species from FMD. In combination with an appropriate diagnostic test, the Cav-P1/3C R° FMDV vaccine may also serve as a marker vaccine to differentiate vaccinated from infected animals.

Phase I study of an active immunotherapy for asymptomatic phase Lymphoplasmacytic lymphoma with DNA vaccines encoding antigen-chemokine fusion: study protocol

BackgroundThere is now a renewed interest in cancer vaccines. Patients responding to immune checkpoint blockade usually bear tumors that are heavily infiltrated by T cells and express a high load of neoantigens, indicating that the immune system is involved in the therapeutic effect of these agents; this finding strongly supports the use of cancer vaccine strategies. Lymphoplasmacytic lymphoma (LPL) is a low grade, incurable disease featuring an abnormal proliferation of Immunoglobulin (Ig)-producing malignant cells. Asymptomatic patients are currently managed by a “watchful waiting” approach, as available therapies provide no survival advantage if started before symptoms develop. Idiotypic determinants of a lymphoma surface Ig, formed by the interaction of the variable regions of heavy and light chains, can be used as a tumor-specific marker and effective vaccination using idiotypes was demonstrated in a positive controlled phase III trial.MethodsThese variable region genes can be cloned and used as a DNA vaccine, a delivery system holding tremendous potential for streamlining vaccine production. To increase vaccination potency, we are targeting antigen-presenting cells (APCs) by fusing the antigen with a sequence encoding a chemokine (MIP-3α), which binds an endocytic surface receptor on APCs. Asymptomatic phase LPL is an excellent model to test our vaccine since patients have not received chemotherapeutics that interfere with innate immune function and have low tumor burden. We are evaluating the safety of this next-generation DNA vaccine in a first-in-human clinical trial currently enrolling asymptomatic LPL patients. To elucidate the mode of action of this vaccine, we will assess its ability to generate tumor-specific immune responses and examine changes in the immune profile of both the peripheral blood and bone marrow.DiscussionThis vaccine could shift the current paradigm of clinical management for patients with asymptomatic LPL and inform development of other personalized approaches.Trial registrationClinicalTrials.gov identifier NCT01209871; registered on September 24, 2010.

Effect of suckler cow vaccination against glycoprotein E (gE)-negative bovine herpesvirus type 1 (BoHV-1) on passive immunity and physiological response to subsequent bovine respiratory disease vaccination of their progeny

The study objectives were: 1) to characterise the development of immunocompetence in beef suckler calves from birth to three months of age, and 2) to trace glycoprotein E (gE)-negative bovine herpesvirus type 1 (BoHV-1) antibodies from dam to calf and subsequent vaccination against pneumonia. Thirty multiparous beef suckler, spring-calving cows, consisting of two genotypes were involved; Limousin×Friesian (LF) and Charolais×Limousin (CL). Cows were immunised against the inactivated antigen strain of BoHV-1 (gE- (IBR marker vaccine) at day −84 and received a booster at day −56 relative to the expected calving date (d 0). Calves were immunised at 14 and 42days of age against PI-3 virus, BRSV and Mannheimia (Pasteurella) haemolytica serotype A1 using a commercial vaccine administered subcutaneously. Additionally, calves were immunised against BoHV-1 at 42days of age, using 1 dose of a live commercial vaccine administered intranasally. Blood samples were collected from all calves (n=30) via jugular venipuncture at birth, prior to colostrum feeding (0h), at 12h (h), 24h, 72h and 168h after the initial feeding of colostrum, and at d 7, 14, 28, 42, 56 and 84 post birth. The mean ratio of gE negative antibodies circulating in the blood of LF and CL dams pre-partum scored negative to gE ab (S/N≥0.70). Antibody levels of BoHV-1 (wild type (wt)) peaked at 12h post-birth in calves and declined thereafter, as the maternal antibodies decayed. There was no difference in BoHV-1 and BRSV antibody levels in calves post vaccination.

Reduced specificity of Erns antibody ELISAs for samples from piglets with maternally derived antibodies induced by vaccination of sows with classical swine fever marker vaccine CP7_E2alf.

Successful implementation of marker vaccines against classical swine fever virus is dependent on a reliable accompanying diagnostic assay that allows differentiation of infected from vaccinated animals (DIVA) as well as the development of a testing scheme during emergency vaccination. In this context, special attention needs to be paid to breeding farms, because the offspring of marker vaccinated sows possess maternally derived antibodies (MDAs). So far, limited information is available on the influence of MDAs on serological testing in the context of a DIVA strategy. Therefore, two commercially available Erns antibody ELISAs were compared, using serum samples of piglets with a high-to-moderate titre of MDAs against marker vaccine CP7_E2alf. False-positive results were detected by both Erns antibody ELISAs for serum samples of piglets with an age of up to 4weeks. Interestingly, most samples tested false-positive in the first Erns antibody ELISA were identified correctly by the other Erns antibody ELISA and vice versa. In conclusion, in case of emergency vaccination of sows, the specificity of both ELISAs in newborn piglets younger than 4weeks may be relatively low. This could be addressed in a testing strategy by either not sampling piglets up to the age of 4weeks or using both ELISAs in a screening-confirmation set-up.

Effect of passive transfer status on response to a glycoprotein E (gE)-negative bovine herpesvirus type 1 (BoHV-1) and bovine respiratory syncytial virus (BRSV) vaccine and weaning stress in pre-weaned dairy calves

ABSTRACTThe study objectives were to: 1) examine how calves of divergent immune status respond to BRSV vaccination at 3 weeks of age; 2) trace glycoprotein E negative BoHV-1 antibodies from vaccinated dams to calf sera and to investigate how passive transfer affects response to live BoHV-1 vaccine at 6 weeks of age; 3) explore the impact of passive transfer status on blood metabolites around weaning. Thirty seven Holstein cows and their calves were included in the study. All cows were immunised with a commercial marker vaccine against BoHV-1(gE-) administered intra-muscularly at 4 month prior to the start of calving. Calves were assigned to 1 of 2 colostrum treatment groups: 1) 5% of BW in colostrum fed at birth, or 2) 10% of BW in colostrum fed at birth. Calves were also immunised at 3 weeks of age with a respiratory commercial vaccine, and a booster administered 4 weeks later. Calves were also immunised against BoHV-1 at 6 weeks of age, using one dose of a live commercial vaccine. The results demonstrated that level of passive immunity had no effect on immune response to vaccination and the importance of feeding colostrum from vaccinated BoHV-1 gE- dams to provide calves with passive protection against IBRV.

Persistent viral infections in farm and companion animals: mechanisms of establishment and maintenance of viral persistence

In persistent viral infections the responsible virus is not eliminated by the host's immune system, but it is maintained in infected cells for months, years or lifetime and it is excreted periodically or continuously. The carrier animal may either appear healthy or show clinical signs. The epizootiologic importance of persistent infections is of compelling interest, since the asymptomatic carriers become a permanent source of viral dissemination that can transport the virus across long-distances and reintroduce it into a given herd, region or country, where the disease had been eliminated. Moreover, a persistent viral infection may be reactivated and cause recrudescent episodes of disease, may lead to immunopathologic or neoplastic disease in the individual host and yet may be transmitted to other animals or to the humans via contaminated materials of animal origin. The economic losses are significant at both farm and national level. Fifty four RNA or DNA viruses induce persistent infections in farm and companion animals. In particular, 14 viruses cause persistent infections in bovines, 7 in small ruminants, 9 in swine, 11 in equines, 8 in cats and 5 in dogs. The majority of these infections are caused by retroviruses and herpesviruses. For a virus to establish and maintain persistent infection, it should have limited cytolytic action, it should be able to maintain its genome within host cells over time and to evade the immune defence mechanisms of the host. The preventive measures are largely based either on the immunization of susceptible animals or on the eradication of the disease by stamping-out policy. The currendy available vaccines are inactivated or live attenuated. Most of them are prepared by conventional methods, but they also exist those that are biotechnologicaUy engineered, such as vector vaccines marker vaccines or subunit vaccines. A major advantage related to the use of marker vaccines is that vaccinated animals can potentially be differentiated from the naturally infected ones by this process.

Long-term study (2005-2010) on the vaccination with BoHV-1 glycoprotein E-deleted marker vaccine in selected two dairy herds in Turkey.

A follow-up study from 2005 to 2010 was carried out in two herds where eradication programme for the bovine herpes virus-1 (BoHV-1) infection depends on the vaccination with inactivated glycoprotein E-deleted vaccine that was started in 2001 following the vaccination with inactivated conventional vaccine between 1999 and 2001. For serological screening, a total of 12,976 sera sampled over several sampling times approximately 6months of interval during 5years (2005-2010) were tested for glycoprotein E (gE)- and glycoprotein B-specific antibodies using ELISA. According to the serological evidence, the long-term persistence of BoHV-1 antibodies, success of marker vaccine, first vaccination time of the calves in herds regularly vaccinated, etc. were discussed in this paper. In conclusion, the vaccination programme using gE (-) marker vaccines, with making efforts to prevent the other factors about transmission of infection, was suggested for the eradication of BoHV-1 infection in Turkey as many EU countries. This is the first report on the BoHV-1 eradication programme in some dairy cattle in Turkey.

Mutational analysis of foot and mouth disease virus nonstructural polyprotein 3AB-coding region to design a negative marker virus

Inactivated purified whole virus vaccines are used for control of foot and mouth disease (FMD). ELISAs detecting antibodies to the nonstructural proteins (NSP), a marker of infection, are primarily used to differentiate FMD virus (FMDV) infected from vaccinated animals (DIVA). However, such DIVA assays have a limitation to their specificity since residual NSPs present in the relatively impure vaccines are suspected to induce an NSP-antibody response in the repeatedly vaccinated animals. Epitope-deleted negative marker vaccine strategy seems to have an advantage over the conventional vaccines in identifying the infected animals with accuracy. NSP 3AB contains an abundance of immunodominant B-cell epitopes of diagnostic importance. This study addresses the feasibility of producing 3AB-truncated FMDV mutant as a potential negative marker vaccine candidate. An infectious cDNA clone of FMDV serotype Asia 1 strain was used to engineer an array of deletion mutations in the established antigenic domain of 3AB. The maximum length of deletion tolerated by the virus was found to be restricted to amino acid residues 87–144 in the C-terminal half of 3A protein along with deletion of the first two copies of 3B peptide. The 3AB-truncated marker virus (Asia 1 IND 491/1997Δ3A87-1443B1,2+FLAG) demonstrated infectivity titres comparable to that of the parental virus in BHK-21 (log10 7.42 TCID50/ml) and LFBK-αVβ6 (log10 8.30 TCID50/ml) cell monolayer culture. The protein fragment corresponding to the viable deletion in the 3AB region was expressed in a prokaryotic system to standardize a companion assay (3A87-1533B1,2 I-ELISA) for the negative marker virus which showed reasonably high diagnostic sensitivity (96.9%) and specificity (100% for naïve and 97.1% for uninfected vaccinated samples). The marker virus and its companion ELISA designed in this study provide a basis to devise a marker vaccine strategy for FMD control.

Identification of two conserved B-cell epitopes in the gp90 of reticuloendothelial virus using peptide microarray

Since the gp90 protein of Reticuloendotheliosis virus (REV) plays vital roles in virus neutralization, so detailed analysis of REV-gp90 epitopes is important for the development of epitope based marker vaccines and diagnostic tools for REV infections. In this study, two monoclonal antibodies (mAbs) namely 6C12 and 09980 were used to map the epitopes in REVgp90 using peptide microarray and ELISA. Peptide microarray revealed that mAbs 6C12 and 09980 recognized 216YHPLA220 and 230DPQTSDILEA239 motifs, respectively. This result was confirmed by ELISA using synthetic peptides. Moreover, homology analysis indicated that mAbs defined epitopes are highly conserved among REV strains used in this study. The mAbs and their epitopes identified in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines for control of REV infections.

Epidemiology, diagnosis and control of classical swine fever: Recent developments and future challenges.

Classical swine fever (CSF) represents a major health and trade problem for the pig industry. In endemic countries or those with a wild boar reservoir, CSF remains a priority for Veterinary Services. Surveillance as well as stamping out and/or vaccination are the principle tools of prevention and control, depending on the context. In the past decades, marker vaccines and accompanying diagnostic tests allowing the discrimination of infected from vaccinated animals have been developed. In the European Union, an E2 subunit and a chimeric live vaccine have been licensed and are available for the use in future disease outbreak scenarios. The implementation of commonly accepted and globally harmonized concepts could pave the way to replace the ethically questionable stamping out policy by a vaccination-to-live strategy and thereby avoid culling of a large number of healthy animals and save food resources. Although a number of vaccines and diagnostic tests are available worldwide, technological advancement in both domains is desirable. This work provides a summary of an analysis undertaken by the DISCONTOOLS group of experts on CSF. Details of the analysis can be downloaded from the web site at http://www.discontools.eu/.

Update on infectious bovine rhinotracheitis

Infectious bovine rhinotracheitis (IBR) is the major disease caused by bovine herpesvirus 1 (BoHV-1), an important pathogen of cattle. Being an alphaherpesvirus, the virus establishes a lifelong latent infection in...

Early protection events in swine immunized with an experimental live attenuated classical swine fever marker vaccine, FlagT4G.

Prophylactic vaccination using live attenuated classical swine fever (CSF) vaccines has been a very effective method to control the disease in endemic regions and during outbreaks in previously disease-free areas. These vaccines confer effective protection against the disease at early times post-vaccination although the mechanisms mediating the protection are poorly characterized. Here we present the events occurring after the administration of our in-house developed live attenuated marker vaccine, FlagT4Gv. We previously reported that FlagT4Gv intramuscular (IM) administered conferred effective protection against intranasal challenge with virulent CSFV (BICv) as early as 7 days post-vaccination. Here we report that FlagT4Gv is able to induce protection against disease as early as three days post-vaccination. Immunohistochemical testing of tissues from FlagT4Gv-inoculated animals showed that tonsils were colonized by the vaccine virus by day 3 post-inoculation. There was a complete absence of BICv in tonsils of FlagT4Gv-inoculated animals which had been intranasal (IN) challenged with BICv 3 days after FlagT4Gv infection, confirming that FlagT4Gv inoculation confers sterile immunity. Analysis of systemic levels of 19 different cytokines in vaccinated animals demonstrated an increase of IFN-α three days after FlagT4Gv inoculation compared with mock infected controls.

Efficacy of bacteriophage Lysed Pasteurella marker vaccine in laboratory animal models with a novel DIVA for haemorrhagic septicaemia.

ObjectiveThe present study aimed at evaluating the efficacy of an improved phage lysate marker vaccine for haemorrhagic septicaemia in mice and rabbit model and development of a DIVA ELISA based on iron restricted outer membrane protein (IROMP).MethodThe experimental vaccine was prepared by lysing P. multocida B:2 grown under iron restricted conditions with a Pasteurella bacteriophage and addition of an alum adjuvant to enhance the immunogenicity. The vaccine was administered in mice and rabbits divided into two group each. Phage lysate vaccine (PL-VacI) was administered to group I mice and rabbits whereas group II mice and rabbits received alum precipitated HS vaccine (HS-VacII). Antibody titres were monitored 0, 30, 60, 90, 210 and 240 dpv. An IROMP (130 kDa) based indirect ELISA was also developed to differentiate between infected and vaccinated animals. The Pasteurella phage isolated in present study was sequenced at Georgia Genomic Facilty, Georgia.ResultThe sequence of PMP-GAD-IND (Pasteurella bacteriophage) was deposited in GenBank under no KY203335. The group I mice and rabbits vaccinated with Phage lysate vaccine (PL-VacI) group revealed significantly higher antibody titres than group II mice and rabbits receiving alum-precipitated bacterin (HS-VacII) by MAT, IHA and ELISA (P < 0.05 and P < 0.001). The peak log 10 values (3.46) in case of group I mice by ELISA were attained at 90DPI whereas in group II mice the peak values at 90DPI were 2.82. Mean log10 titres by ELISA in group I and II rabbits were 2.43 and 2.35 respectively at 30DPI whereas at 120DPI the titres were 3.29 and 2.75, respectively. The DIVA ELISA detected presence of a novel 137 kDa IROMP/siderophore antibody in sera of group I mice and rabbits (PL-VacI) absent in sera of mice and rabbits given HS-VacII.ConclusionThe bacteriophage based marker vaccine (PL-VacI) had a more effective and longer immune response against HS in mice and rabbit in comparison to the widely used alum precipitated HS vaccine (HS-VacII). Moreover, the development of a recombinant IROMP based indirect ELISA could serve as an excellent tool to differentiate between infected and vaccinated cattle and buffaloes for effective control of HS.

Efficacy of E2 glycoprotein fused to porcine CD154 as a novel chimeric subunit vaccine to prevent classical swine fever virus vertical transmission in pregnant sows

Here we evaluated the effect of double vaccination with a novel subunit marker vaccine candidate based in the CSFV E2 glycoprotein fused to the porcine CD154 to prevent CSFV vertical transmission. A lentivirus-based gene delivery system was used to obtain a stable recombinant HEK 293 cell line for the expression of E2 fused to porcine CD154 molecule. Six pregnant sows were distributed in two groups and at 64days of gestation animals numbered 1–4 (group 1) were vaccinated via intramuscular inoculation with 50μg of E2-CD154 subunit vaccine. Animals from group 2 (numbered 5 and 6, control animals) were injected with PBS. Seventeen days later sows from group 1 were boosted with the same vaccine dose. Twenty-seven days after the first immunization, the sows were challenged with a virulent CSFV Margarita strain and clinical signs were registered. Samples were collected during the experiment and at necropsy to evaluate immune response and virological protection. Between 14 and 18days after challenge, the sows were euthanized, the foetuses were obtained and samples of sera and tissues were collected. E2-CD154 vaccinated animals remained clinically healthy until the end of the study; also, no adverse reaction was shown after vaccination. An effective boost effect in the neutralizing antibody response after the second immunization and viral challenge was observed and support the virological protection detected in these animals after vaccination. Protection against CSFV vertical transmission was found in the 100% of serums samples from foetus of vaccinated sows. Only two out of 208 samples (0.96%) were positive with Ct value about 36 corresponding to one tonsil and one thymus, which may be non-infective viral particles. Besides, its DIVA potential and protection from vertical transmission, the novel CSFV E2 bound to CD154 subunit vaccine, is a promising alternative to the live-attenuated vaccine for developing countries.

Characterization of three commercial ELISA kits for detection of BOHV-1 gE specific antibodies in serum and milk samples and applicability of bulk milk for determination of herd status

Vaccination of animals with gE-deleted vaccine strains (gE- marker vaccines) and differential detection of vaccinated vs infected animals with antibody ELISA targeting the gE or the gB proteins have been proved to be useful tools in programs for control and eradication of the bovine herpesvirus 1 (BoHV-1) responsible for infectious bovine rhinotracheitis (IBR), a major pathogen of cattle.The diagnostic sensitivity (DSe) and specificity (DSp) of three commercial gE ELISA kits from IDEXX, IDVet and CIV-HIPRA were compared for serum and milk matrices. Limiting the analysis to 198 individual with concordant ELISA results in serum (91 naïve, 37 vaccinated and 70 infected) the DSe of gE kits was estimated to 0,97 for IDEXX, 0,93 for CIV-HIPRA and 0,53 for IDVet using milk samples and the DSp to 0,95 for IDEXX, 1,00 for IDVet and CIV-HIPRA.The applicability of gE ELISA for individual or bulk milk testing as an additional tool in control programs dedicated to the certification and control of vaccinated herds was evaluated.Two of the three evaluated gE ELISA kits presented substantial to good agreement individual milk and serum samples.The bulk-tank milk also proved to be suitable for the detection of BoHV-1 in vaccinated herds provided that gE prevalence is superior to 10% as false negative results are often observed at lower gE herd prevalence. This limitation could be reduced to 8% of prevalence when a prior concentration step was applied to bulk milk samples.

Vaccine Efficacy of Inactivated, Chimeric Hemagglutinin H9/H5N2 Avian Influenza Virus and Its Suitability for the Marker Vaccine Strategy.

In order to produce a dually effective vaccine against H9 and H5 avian influenza viruses that aligns with the DIVA (differentiating infected from vaccinated animals) strategy, we generated a chimeric H9/H5N2 recombinant vaccine that expressed the whole HA1 region of A/CK/Korea/04163/04 (H9N2) and the HA2 region of recent highly pathogenic avian influenza (HPAI) A/MD/Korea/W452/14 (H5N8) viruses. The chimeric H9/H5N2 virus showed in vitro and in vivo growth properties and virulence that were similar to those of the low-pathogenic avian influenza (LPAI) H9 virus. An inactivated vaccine based on this chimeric virus induced serum neutralizing (SN) antibodies against both H9 and H5 viruses but induced cross-reactive hemagglutination inhibition (HI) antibody only against H9 viruses. Thus, this suggests its compatibility for use in the DIVA strategy against H5 strains. Furthermore, the chimeric H9/H5N2 recombinant vaccine protected immunized chickens against lethal challenge by HPAI H5N8 viruses and significantly attenuated virus shedding after infection by both H9N2 and HPAI H5N8 viruses. In mice, serological analyses confirmed that HA1- and HA2 stalk-specific antibody responses were induced by vaccination and that the DIVA principle could be employed through the use of an HI assay against H5 viruses. Furthermore, each HA1- and HA2 stalk-specific antibody response was sufficient to inhibit viral replication and protect the chimeric virus-immunized mice from lethal challenge with both mouse-adapted H9N2 and wild-type HPAI H5N1 viruses, although differences in vaccine efficacy against a homologous H9 virus (HA1 head domain immune-mediated protection) and a heterosubtypic H5 virus (HA2 stalk domain immune-mediated protection) were observed. Taken together, these results demonstrate that the novel chimeric H9/H5N2 recombinant virus is a low-pathogenic virus, and this chimeric vaccine is suitable for a DIVA vaccine with broad-spectrum neutralizing antibody against H5 avian influenza viruses.IMPORTANCE Current influenza virus killed vaccines predominantly induce antihemagglutinin (anti-HA) antibodies that are commonly strain specific in that the antibodies have potent neutralizing activity against homologous strains but do not cross-react with HAs of other influenza virus subtypes. In contrast, the HA2 stalk domain is relatively well conserved among subtypes, and recently, broadly neutralizing antibodies against this domain have been isolated. Therefore, in light of the need for a vaccine strain that applies the DIVA strategy utilizing an HI assay and induces broad cross-protection against H5N1 and H9N2 viruses, we generated a novel chimeric H9/H5N1 virus that expresses the entire HA1 portion from the H9N2 virus and the HA2 region of the heterosubtypic H5N8 virus. The chimeric H9/H5N2 recombinant vaccine protected immunized hosts against lethal challenge with H9N2 and HPAI H5N1 viruses with significantly attenuated virus shedding in immunized hosts. Therefore, this chimeric vaccine is suitable as a DIVA vaccine against H5 avian influenza viruses.