Differentiating Infected From Vaccinated Animals Research Articles

Bluetongue virus vaccine: conventional to modern approach.

Bluetongue (BT) is an economically important, infectious and non-contagious disease of ruminant animals. BT disease is caused by bluetongue virus (BTV) of the genus Orbivirus (the family Reoviridae). BTV is transmitted by certain species of biting midges of the genus Culicoides. Although originally BT was restricted to African continent, now it is present in all the continents except Antarctica. Conventional BT vaccines such as live attenuated and inactivated vaccines showed different degree of success in BT control. However, conventional vaccines have certain disadvantages of reversion to virulent strain and frequent booster dose requirement. Several BT outbreaks in India and the rest of the world open a new insight for development of better vaccines. The development in molecular biology techniques allowed the development and validation of several modern vaccines such as subunit vaccine, recombinant vector vaccine, disabled infections single cycle (DISC) vaccine, differentiating infected from vaccinated animals (DIVA) approach etc. Most of these vaccines are considered as safer, having better protective immune response and provided cross-protective immunization against more than one serotype. Keywords: bluetongue virus; live vaccine; inactivated vaccine; DISC; recombinant vaccine.

Efficacy and Safety of BCG Vaccine for Control of Tuberculosis in Domestic Livestock and Wildlife.

Bovine tuberculosis (TB) continues to be an intractable problem in many countries, particularly where “test and slaughter” policies cannot be implemented or where wildlife reservoirs of Mycobacterium bovis infection serve as a recurrent source of infection for domestic livestock. Alternative control measures are urgently required and vaccination is a promising option. Although the M. bovis bacille Calmette-Guérin (BCG) vaccine has been used in humans for nearly a century, its use in animals has been limited, principally as protection against TB has been incomplete and vaccination may result in animals reacting in the tuberculin skin test. Valuable insights have been gained over the past 25 years to optimise protection induced by BCG vaccine in animals and in the development of tests to differentiate infected from vaccinated animals (DIVA). This review examines factors affecting the efficacy of BCG vaccine in cattle, recent field trials, use of DIVA tests and the effectiveness of BCG vaccine in other domestic livestock as well as in wildlife. Oral delivery of BCG vaccine to wildlife reservoirs of infection such as European badgers, brushtail possums, wild boar, and deer has been shown to induce protection against TB and could prove to be a practical means to vaccinate these species at scale. Testing of BCG vaccine in a wide range of animal species has indicated that it is safe and vaccination has the potential to be a valuable tool to assist in the control of TB in both domestic livestock and wildlife.

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.

Insertion site of FLAG on foot-and-mouth disease virus VP1 G-H loop affects immunogenicity of FLAG

The G-H loop of the foot-and-mouth disease virus (FMDV) virion contains certain dominant immunogenic epitopes, as well as an arginine-glycine-aspartic acid (RGD) motif that is recognized by cell surface integrin receptors. Previous experiments indicate that it is critical to maintain virus structural integrity when inserting an exogenous epitope into the surface of an FMDV structural protein. However, it remains to be determined how factors such as different insertion positions affect interactions among the virus, cells and host immune system. In this study, one infectious cDNA clone of the swine FMDV Cathay topotype strain O/CHA/90 was constructed. Then, a FLAG marker (DYKDDDDK) was inserted upstream (–4) or downstream (+10) of the RGD motif to generate tagged viruses vFLAG-O/CHA/90 or vO/CHA/90-FLAG, investigating the possibility of expressing foreign antigen and effect on its immunogenicity. Compared to the parental virus, both tagged viruses exhibited similar plaque phenotypes, suckling mouse pathogenicity and antigenicity. Additionally, the FLAG tag insertion position did not change the use of integrin-mediated cell entry by the tagged viruses. Interestingly, both tagged vaccines protected pigs against challenge with the parental virus O/CHA/90 and induced immune responses against FMDV in BALB/c mice and pigs, but only vaccination with vFLAG-O/CHA/90 generated anti-FLAG antibodies. Our findings demonstrated that two sites (RGD–4 and RGD+10) tolerated the insertion of an exogenous gene in the swine FMDV O/CHA/90 strain. However, only RGD–4 was a novel and appropriate inserting site which could tolerate exogenous FLAG. The resultant tagged virus is a promising candidate for FMD vaccine which can be differentiating infected from vaccinated animals (DIVA).

An improved, rapid competitive ELISA using a novel conserved 3B epitope for the detection of serum antibodies to foot-and-mouth disease virus.

The highly contagious foot-and-mouth disease virus (FMDV) afflicts cloven-hoofed animals, resulting in significant costs because of loss of trade and recovery from disease. We developed a sensitive, specific, and rapid competitive ELISA (cELISA) to detect serum antibodies to FMDV. The cELISA utilized a monoclonal blocking antibody specific for a highly conserved FMDV nonstructural 3B epitope, a recombinant mutant FMDV 3ABC coating protein, and optimized format variables including serum incubation for 90 min at 20–25°C. Samples from 16 animals experimentally infected with one FMDV serotype (A, O, Asia, or SAT-1) demonstrated early detection capacity beginning 7 d post-inoculation. All samples from 55 vesicular stomatitis virus antibody-positive cattle and 44 samples from cloven-hoofed animals affected by non-FMD vesicular diseases were negative in the cELISA, demonstrating 100% analytical specificity. The diagnostic sensitivity was 100% against sera from 128 cattle infected with isolates of all FMDV serotypes, emphasizing serotype-agnostic results. Diagnostic specificities of U.S. cattle (n = 1135) and swine (n = 207) sera were 99.4% and 100%, respectively. High repeatability and reproducibility were demonstrated with 3.1% coefficient of variation in percent inhibition data and 100% agreement using 2 kit lots and 400 negative control serum samples, with no difference between bench and biosafety cabinet operation. Negative results from vaccinated, uninfected cattle, pig, and sheep sera confirmed the DIVA (differentiate infected from vaccinated animals) capability. This rapid (<3 h), select agent–free assay with high sensitivity and specificity, DIVA capability, and room temperature processing capability will serve as a useful tool in FMDV surveillance, emergency preparedness, response, and outbreak recovery programs.

Characterisation of the antigenic epitopes in the subunit 2 haemagglutinin of avian influenza virus H5N1.

Monitoring avian influenza (AI) infection and detecting silent infection in vaccinated chickens has been challenging due to the lack of effective serological diagnostic assays to differentiate between vaccinated and infected animals. Very few studies have identified suitable proteins in AI virus that can be used in successfully differentiating infected from vaccinated animals (DIVA). An HA2 peptide: HA2 position 197-201 (HA position 488-516) described by Khurana et al. (J Virol 85(23):12455-12463, 2011), was shown to have DIVA ability by differentiating H5N1-infected human sera in ELISA. In order to explore the capacity of the HA2 protein, as a DIVA reagent in chickens, four overlapping recombinant HA2 proteins, were expressed in E. coli and tested for reaction with H5N1 sera obtained from infected and vaccinated chickens. Recombinant protein HA2_B2 (380-461) was able to generate a detectable reaction with both H5N1 infected and vaccinated chicken sera but recombinant protein HA2_B4 (483-565) reacted strongly only with sera obtained from chickens infected with live virus, confirming its suitability as a DIVA antigen. Further analysis of the HA2 using several overlapping peptides suggested that positions 380-461 and 483-565 were antigenic in mouse and chicken. This study, for the first time, identified novel antigenic epitopes on the H5N1 HA2 subunit. Two epitopes, found in the HA2 ectodomain, have never been described for AIV infection in any animal species. Also one HA2 epitope was found to have high potential as a DIVA antigen.

Engineering of a rough auxotrophic mutant Salmonella Typhimurium for effective delivery.

Live Salmonella vaccine vectors offer a remarkable platform for delivering immunogens and therapeutic molecules by mimicking natural intracellular infections; however, pre-existing anti-vector immunity can impede effective deployment. Measures to alleviate pre-existing immunity include the use of heterologous vectors, development of highly attenuated strain enabling greater payload, removal of major immunoreactive components from the vector, and/or augmentation of delivered antigens via increased presentation in antigen presenting cells. Here we report a Salmonella Typhimurium (ST) vector-JOL1800 that embodies these requisite properties. JOL1800 is a highly attenuated, auxotrophic, and O-antigen deficient rough-mutant strain. Heterologous bacterial and viral antigens were expressed and delivered using JOL1800 in mice, irrespective of the inoculation route successful inductions of the mucosal and systemic humoral responses were observed. Compared to smooth LPS vector delivery, we observed an increased fraction of delivered-antigen presenting dendritic cells and a higher frequency of delivered-antigen displayed per macrophage. Upon post-priming with JOL1800 delivery, efficacy of the delivery was minimally affected as indicated by insignificant decrease in colonization, humoral and cellular responses. Our results show that the generated vector is capable of remote antigen delivery, manifests higher antigen presentation, is Differentiating Infected from Vaccinated Animals (DIVA) capable, evades normal pre-existing immunity, and can be deployed for effective delivery.

The immunogenicity of recombinant vaccines based on modified Vaccinia Ankara (MVA) viruses expressing African horse sickness virus VP2 antigens depends on the levels of expressed VP2 protein delivered to the host

African horse sickness (AHS) is a lethal equine disease transmitted by Culicoides biting midges and caused by African horse sickness virus (AHSV). AHS is endemic to sub-Saharan Africa, but devastating outbreaks have been recorded periodically outside this region. The perceived risk of an AHS outbreak occurring in Europe has increased following the frequent epidemics caused in ruminants by bluetongue virus, closely related to AHSV.Attenuated vaccines for AHS are considered unsuitable for use in non-endemic countries due bio-safety concerns. Further, attenuated and inactivated vaccines are not compatible with DIVA (differentiate infected from vaccinated animals) strategies. All these factors stimulated the development of novel AHS vaccines that are safer, more efficacious and DIVA compatible.We showed previously that recombinant modified Vaccinia Ankara virus (MVA) vaccines encoding the outer capsid protein of AHSV (AHSV-VP2) induced virus neutralising antibodies (VNAb) and protection against AHSV in a mouse model and also in the horse. Passive immunisation studies demonstrated that immunity induced by MVA-VP2 was associated with pre-challenge VNAb titres in the vaccinates. Analyses of the inoculum of these MVA-VP2 experimental vaccines showed that they contained pre-formed AHSV-VP2.We continued studying the influence of pre-formed AHSV-VP2, present in the inoculum of MVA-VP2 vaccines, in the immunogenicity of MVA-VP2 vaccines. Thus, we compared correlates of immunity in challenged mice that were previously vaccinated with: a) MVA-VP2 (live); b) MVA-VP2 (live and sucrose gradient purified); c) MVA-VP2 (UV light inactivated); d) MVA-VP2 (UV light inactivated and diluted); e) MVA-VP2 (heat inactivated); f) MVA-VP2 (UV inactivated) + MVA-VP2 (purified); g) MVA-VP2 (heat inactivated) + MVA-VP2 (purified); and h) wild type-MVA (no insert). The results of these experiments showed that protection was maximal using MVA-VP2 (live) vaccine and that the protection conferred by all other vaccines correlated strongly with the levels of pre-formed AHSV-VP2 in the vaccine inoculum.

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.

New generation vaccines against bluetongue virus

Over the last three decades, a variety of approaches have been investigated to develop new types of bluetongue virus (BTB) vaccines, ranging from baculovirus-expressed subunit vaccines to live vector vaccines. DNA vaccines against BTV consist of DNA plasmid expressing different BTV proteins after inoculation of the animals. The recombinant viral vector vaccines against BTV are based on recombinant viruses that express desired BTV antigens in the host upon inoculation. Viruses such as vaccinia, modified vaccinia Ancara (MVA), capripox, canarypox, herpes, myxoma and fowlpox viruses have been used as vectors of BTV genes. The reverse genetics (RG) systems for BTV are useful tools for BTV vaccine development. Disabled infectious single-cycle (DISC) vaccines make it possible to restore virus replication and can be used for differentiating infected from vaccinated animals (DIVA). These vaccines are based on the production of a modified virus with a deletion in one or more genes that are essential for virus replication. Another approach for BTV vaccine development using RG is the disabled infectious single-animal (DISA) vaccine, generated by deletion of NS3/NS3a expression. DISC and DISA vaccines can mimic the natural tropism of the virus and can express BTV proteins at the site of infection. Important advantages of these new generation vaccines over the conventional BTV vaccines are their high efficacy as well as the possibility of applying them for DIVA. At present, there are a number of novel laboratory-scale BTV vaccines that could meet vaccine profiles required for different field situations. However, further development and licensing of these vaccine candidates for many BTV serotypes is needed in order to prepare for future BT outbreaks. To date, all novel BTV vaccines described in this paper are still under laboratory testing. They are not available commercially, and the time of their application in the field is still indefinite. .

Safety profile of a replication-deficient human adenovirus-vectored foot-and-mouth disease virus serotype A24 subunit vaccine in cattle.

The safety of a replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was evaluated in five independent safety studies. The target animal safety studies were designed in compliance with United States (U.S.) regulatory requirements (Title 9, U.S. Code of Federal Regulation [9CFR]) and international standard guidelines (VICH Topic GL-44) for veterinary live vaccines. The first three studies were conducted in a total of 22 vaccinees and demonstrated that the AdtA24 master seed virus (MSV) was safe, did not revert to virulence and was not shed or spread from vaccinees to susceptible cattle or pigs. The fourth safety study conducted in 10 lactating cows using an AdtA24 vaccine serial showed that the vaccine was completely absent from milk. The fifth safety study was conducted under typical U.S. production field conditions in 500 healthy beef and dairy cattle using two AdtA24 vaccine serials. These results demonstrated that the vaccine was safe when used per the product label recommendations. Additional data collected during these five studies confirmed that AdtA24 vaccinees developed FMDV A24 and the HAd5 vaccine vector serum neutralization antibodies that test negative in a FMDV non-structural protein antibody test, confirming AdtA24 vaccine's capability to differentiate infected from vaccinated animals (DIVA). In conclusion, results from this comprehensive set of cattle studies demonstrated the safety of the replication-deficient AdtA24 vaccine and fulfilled safety-related requirements for U.S. regulatory requirements.

A glycosylated recombinant subunit candidate vaccine consisting of Ehrlichia ruminantium major antigenic protein1 induces specific humoral and Th1 type cell responses in sheep.

Heartwater, or cowdriosis, is a tick-borne disease of domestic and wild ruminants that is endemic in the Caribbean and sub-Saharan Africa. The disease is caused by an intracellular pathogen, Ehrlichia ruminantium and may be fatal within days of the onset of clinical signs with mortality rates of up to 90% in susceptible hosts. Due to the presence of competent tick vectors in North America, there is substantial risk of introduction of heartwater with potentially devastating consequences to the domestic livestock industry. There is currently no reliable or safe vaccine for use globally. To develop a protective DIVA (differentiate infected from vaccinated animals) subunit vaccine for heartwater, we targeted the E. ruminantium immunodominant major antigenic protein1 (MAP1) with the hypothesis that MAP1 is a glycosylated protein and glycans contained in the antigenic protein are important epitope determinants. Using a eukaryotic recombinant baculovirus expression system, we expressed and characterized, for the first time, a glycoform profile of MAP1 of two Caribbean E. ruminantium isolates, Antigua and Gardel. We have shown that the 37–38 kDa protein corresponded to a glycosylated form of the MAP1 protein, whereas the 31–32 kDa molecular weight band represented the non-glycosylated form of the protein frequently reported in scientific literature. Three groups of sheep (n = 3–6) were vaccinated with increasing doses of a bivalent (Antigua and Gardel MAP1) rMAP1 vaccine cocktail formulation with montanide ISA25 as an adjuvant. The glycosylated recombinant subunit vaccine induced E. ruminantium-specific humoral and Th1 type T cell responses, which are critical for controlling intracellular pathogens, including E. ruminantium, in infected hosts. These results provide an important basis for development of a subunit vaccine as a novel strategy to protect susceptible livestock against heartwater in non-endemic and endemic areas.

Current Status of Rift Valley Fever Vaccine Development.

Rift Valley Fever (RVF) is a mosquito-borne zoonotic disease that presents a substantial threat to human and public health. It is caused by Rift Valley fever phlebovirus (RVFV), which belongs to the genus Phlebovirus and the family Phenuiviridae within the order Bunyavirales. The wide distribution of competent vectors in non-endemic areas coupled with global climate change poses a significant threat of the transboundary spread of RVFV. In the last decade, an improved understanding of the molecular biology of RVFV has facilitated significant progress in the development of novel vaccines, including DIVA (differentiating infected from vaccinated animals) vaccines. Despite these advances, there is no fully licensed vaccine for veterinary or human use available in non-endemic countries, whereas in endemic countries, there is no clear policy or practice of routine/strategic livestock vaccinations as a preventive or mitigating strategy against potential RVF disease outbreaks. The purpose of this review was to provide an update on the status of RVF vaccine development and provide perspectives on the best strategies for disease control. Herein, we argue that the routine or strategic vaccination of livestock could be the best control approach for preventing the outbreak and spread of future disease.

Exploitation of stable nanostructures based on the mouse polyomavirus for development of a recombinant vaccine against porcine circovirus 2.

The aim of this study was to develop a suitable vaccine antigen against porcine circovirus 2 (PCV2), the causative agent of post-weaning multi-systemic wasting syndrome, which causes significant economic losses in swine breeding. Chimeric antigens containing PCV2b Cap protein sequences based on the mouse polyomavirus (MPyV) nanostructures were developed. First, universal vectors for baculovirus-directed production of chimeric MPyV VLPs or pentamers of the major capsid protein, VP1, were designed for their exploitation as vaccines against other pathogens. Various strategies were employed based on: A) exposure of selected immunogenic epitopes on the surface of MPyV VLPs by insertion into a surface loop of the VP1 protein, B) insertion of foreign protein molecules inside the VLPs, or C) fusion of a foreign protein or its part with the C-terminus of VP1 protein, to form giant pentamers of a chimeric protein. We evaluated these strategies by developing a recombinant vaccine against porcine circovirus 2. All candidate vaccines induced the production of antibodies against the capsid protein of porcine circovirus after immunization of mice. The candidate vaccine, Var C, based on fusion of mouse polyomavirus and porcine circovirus capsid proteins, could induce the production of antibodies with the highest PCV2 neutralizing capacity. Its ability to induce the production of neutralization antibodies was verified after immunization of pigs. The advantage of this vaccine, apart from its efficient production in insect cells and easy purification, is that it represents a DIVA (differentiating infected from vaccinated animals) vaccine, which also induces an immune response against the mouse polyoma VP1 protein and is thus able to distinguish between vaccinated and naturally infected animals.

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.

Development of duplex dual-gene and DIVA real-time RT-PCR assays and use of feathers as a non-invasive sampling method for diagnosis of Turkey Meningoencephalitis Virus

ABSTRACTThe avian flavivirus Turkey Meningoencephalitis Virus (TMEV) causes a neuroparalytic disease of commercial turkeys, expressed in paresis, incoordination, drooping wings and mortality that is controlled by vaccination. The molecular diagnosis using brain tissue RNA has now been upgraded by the development of a diagnostic dual-gene multiplex real-time PCR targeting the envelope and the non-structural NS5 gene, increasing the sensitivity by 10–100-fold compared to the previously existing assays. Based on the recent complete sequences of five TMEV isolates we have now developed a Differentiating Infected from Vaccinated Animals (DIVA) assay, to distinguish between wild-type TMEV strains and the vaccine virus. The DIVA assay was evaluated on commercial vaccines produced by two manufacturers, on RNA purified from brains of experimentally infected turkeys with TMEV strains, and on clinical samples collected between the years 2009 and 2015. We also investigated turkey feather pulps for their suitability to serve for TMEV detection, to avoid invasive sampling and bird killing. The parallel TMEV diagnosis in brain and feather-pulp RNA were similarly useful for diagnosis, at least in experimentally infected turkeys and in three cases of disease encountered in commercial flocks.

Surveillance of Infectious Bovine Rhinotracheitis in marker-vaccinated dairy herds: Application of a recombinant gE ELISA on bulk milk samples

Infectious Bovine Rhinotracheitis (IBR) occurs worldwide, requiring significant resources for eradication programs or surveillance purposes. The status of infection is usually detected by serological methods using the virus neutralization test (VNT) or enzyme-linked immunosorbent assay (ELISA) on individual sera. The gE DIVA (Differentiating Infected from Vaccinated Animals) vaccines approach, adopted in order to reduce the virus circulation and prevent clinical signs, have tightened the range of available methods for the serological diagnosis. Different gE blocking ELISA could be performed to detect specific antibodies in sera of infected or whole virus-vaccinated animals but with less sensitivity if applied to bulk milk samples, especially in marker-vaccinated herds. A new rec-gE ELISA was recently developed in Italy and applied with good performances on blood serum samples. The present paper focuses on the application of a rapid protocol for purification/concentration of immunoglobulin G (IgG) from bulk milk and on the use of the new rec-gE indirect ELISA. The study involved three different partners and 225 herds (12,800 lactating cows) with different official IBR diagnostic statuses. The diagnostic specificity of the method was demonstrated closed to 100% while the diagnostic sensitivity was strictly related to the herd-seroprevalence. Considering 2.5% as the limit of detection of within-herd seropositivity prevalence, the diagnostic sensitivity showed by the proposed method was equal to 100%. A single reactivation of a whole strain vaccine in an old cow was detected inside a group of 67 lactating cows, showing the field applicability of the method.

Chimeric Bivalent Virus-Like Particle Vaccine for H5N1 HPAI and ND Confers Protection against a Lethal Challenge in Chickens and Allows a Strategy of Differentiating Infected from Vaccinated Animals (DIVA).

Highly pathogenic avian influenza (HPAI) and Newcastle disease (ND) are considered as the most devastating poultry infections, owing to their worldwide distribution and economical threat. Vaccines have been widely used to control these diseases in the poultry industry in endemic countries. However, vaccination policy without differentiating infected animals from vaccinated animals (DIVA) makes the virus surveillance difficult. In this study, we developed a bivalent virus-like particle (VLP) vaccine that is composed of the hemagglutinin (HA) and matrix 1 (M1) proteins of the H5N1 HPAI virus (HPAIV) and a chimeric protein containing the ectodomain of the ND virus (NDV) fusion (F) protein fused with the cytoplasmic and transmembrane domains of the HPAIV HA protein. A single immunization of chickens with the chimeric VLP vaccine induced high levels of hemagglutination inhibition (HI) antibody titers against H5N1 HPAI virus and anti-NDV antibody detected in ELISA and protected chickens against subsequent lethal HPAIV and NDV infections. Furthermore, we could easily perform DIVA test using the commercial NP-cELISA tests against HPAIV and HI assay against NDV. These results strongly suggest that utilization of chimeric VLP vaccine in poultry species would be a promising strategy for the better control of HPAI and ND simultaneously.

Engineering of a live Salmonella enterica serovar Choleraesuis negative-marker strain that allows serological differentiation between immunised and infected animals

The usefulness of Salmonella vaccine vehicles is limited by the fact that control programmes relying on Salmonella bacteriology and serology cannot differentiate infected animals from vaccinated ones, an ability referred to as DIVA (differentiating infected from vaccinated animals). As a first step towards Salmonella-based DIVA vaccines, the ompA gene was deleted in live attenuated ΔphoP and ΔrpoS vaccine strains. The ompA gene is present in all Salmonella enterica serovars and it encodes an abundant, highly immunogenic outer membrane protein.The double mutant ΔphoP ΔompA and ΔrpoS ΔompA strains showed similar virulence attenuation, safety and immunogenicity in a mouse model of infection as the parental ΔphoP and ΔrpoS strains. Sera from mice inoculated with the double mutant strains failed to recognise OmpA in Western blots of outer membrane extracts, whereas the protein was recognised by sera from mice inoculated with wild-type Salmonella or a mixture of double mutant and parental strains. These data suggest that OmpA can be a suitable negative marker for DIVA vaccines.

Use of vaccination in the eradication of strangles: the importance of differentiating infected from vaccinated animals (DIVA)

of the library enabled the simultaneous identification of essential regions over 94% of the S. equi genome. A total of 253 genes were identified as required for growth in standard laboratory conditions, providing a blue-print for basic cell survival. Amongst other findings, the 14 genes associated with the iron-sequestering molecule equibactin, were all found to be non-essential to S. equi in vitro with the exception of the regulatory gene, eqbA. This gene mediates iron uptake to prevent intracellular iron toxicity, which is reflected by its importance in this study. To reduce the impact of phage infections, bacteria have developed ‘cell suicide’ abortive infection mechanisms. In S. equi, the genes encoding toxin production for abortive infection were found to be non-essential, but a gene encoding an anti-toxin was required. In a pilot study, a library of mutants was used to determine if TraDIS is able to identify genes involved in the interaction between S. equi and equine phagocytes (equine macrophage-like cells (eCAS)). Mutants containing insertions in 11 genes involved in resistance to oxidative stress and into two genes involved in capsule formation were reduced in fitness. The identification of these genes highlights the importance of maintaining both the capsule and the ability to resist oxidative stress in S. equi, in the presence of eCAS. The ability of TraDIS to simultaneously allocate importance to genes under particular conditions makes it an attractive high-throughput method with which to explore and build on current knowledge of the functional genomics of S. equi to direct the design of safer and more effective vaccines.