Evaluation of vaccination against infectious laryngotracheitis (ILT) with recombinant herpesvirus of turkey (rHVT-LT) and chicken embryo origin (CEO) vaccines applied alone or in combination

ABSTRACT

The chicken embryo origin (CEO) infectious laryngotracheitis (ILT) live attenuated vaccines, although capable of protecting against disease and reducing challenge virus replication, can regain virulence. Recombinant ILT vaccines do not regain virulence but are partially successful at blocking challenge virus replication. The objective of this study was to evaluate the effect of rHVT-LT vaccination on CEO replication and how this vaccination strategy enhances protection and limits challenge virus transmission to naïve contact chickens. The rHVT-LT vaccine was administered at 1 day of age subcutaneously and the CEO vaccine was administered at 6 weeks of age via eye-drop or drinking water. CEO vaccine replication post vaccination, challenge virus replication and transmission post challenge were evaluated. After vaccination, only the group that received the CEO via eye-drop developed transient conjunctivitis. A significant decrease in CEO replication was detected for the rHVT-LT?+?CEO groups as compared to groups that received CEO alone. After challenge, reduction in clinical signs and challenge virus replication were observed in all vaccinated groups. However, among the vaccinated groups, the rHVT-LT group presented higher clinical signs and challenge virus replication. Transmission of the challenge virus to naïve contact chickens was only observed in the rHVT-LT vaccinated group of chickens. Overall, this study found that priming with rHVT-LT reduced CEO virus replication and the addition of a CEO vaccination provided a more robust protection than rHVT alone. Therefore, rHVT-LT?+?CEO vaccination strategy constitutes an alternative approach to gain better control of the disease.     

Long-term protection against a virulent field isolate of infectious laryngotracheitis virus induced by inactivated,recombinant, and modified live virus vaccines in commercial layers

Infectious laryngotracheitis (ILT) is an acute respiratory disease of chickens controlled through vaccination with live-modified attenuated vaccines, the chicken embryo origin (CEO) vaccines and the tissue-culture origin (TCO) vaccines. Recently, novel recombinant vaccines have been developed using herpesvirus of turkey (HVT) and fowl pox virus (FPV) as vectors to express ILTV immunogens for protection against ILT. The objective of this study was to assess the protection efficacy against ILT induced by recombinants, live-modified attenuated, and inactivated virus vaccines when administered alone or in combination. Commercial layer pullets were vaccinated with one or more vaccines and challenged at 35 (35 WCH) or 74 weeks of age (74 WCH). Protection was assessed by scoring clinical signs; and by determining the challenge viral load in the trachea at five days post-challenge. The FPV-LT vaccinated birds were not protected when challenged at 35 weeks; the HVT-LT and TCO vaccines in combination provided protection similar to that observed in chickens vaccinated with either HVT-LT or TCO vaccines when challenged at 35 weeks, whereas protection induced by vaccination with HVT-LT followed by TCO was superior in the 74 WCH group compared with the 35 WCH group. Birds given the inactivated ILT vaccine had fewer clinical signs and/or lower viral replication at 74 WCH when combined with TCO or HVT-LT, but not when given alone. Finally, the CEO-vaccinated birds had top protection as indicated by reduction of clinical signs and viral replication when challenged at 35 weeks (74 weeks not done). These results suggest that certain vaccine combinations may be successful to produce long-term protection up to 74 weeks of age against ILT.     

Comparison of the replication and transmissibility of an infectious laryngotracheitis virus vaccine delivered via eye-drop or drinking-water

Live attenuated vaccines have been extensively used to control infectious laryngotracheitis (ILT). Most vaccines are registered/recommended for use via eye-drop although vaccination via drinking-water is commonly used in the field. Drinking-water vaccination has been associated with non-uniform protection. Bird-to-bird passage of chick-embryo-origin (CEO) ILT vaccines has been shown to result in reversion to virulence. The purpose of the present study was to examine the replication and transmission of a commercial CEO infectious laryngotracheitis virus (ILTV) vaccine strain following drinking-water or eye-drop inoculation. Two groups of 10 specific-pathogen-free chickens were each vaccinated with Serva ILTV vaccine strain either via eye-drop or drinking-water. Groups of four or five unvaccinated birds were placed in contact with vaccinated birds at regular intervals. Tracheal swabs were collected every 4 days from vaccinated and in-contact birds to assess viral replication and transmission using quantitative polymerase chain reaction. Compared with eye-drop-vaccinated birds, drinking-water-vaccinated birds showed delayed viral replication but had detectable viral DNA for a longer period of time. Transmission to chickens exposed by contact on day 0 of the experiments was similar in both groups. Birds exposed to ILTV by contact with eye-drop vaccinated birds on days 4, 8, 12 and 16 of the experiment had detectable ILTV for up to 8 days post exposure. ILTV was not detected in chickens that were exposed by contact with drinking-water vaccinated birds on day 12 of the experiment or later. Results from this study provide valuable practical information for the use of ILT vaccine.     

Protection induced by commercially available live-attenuated and recombinant viral vector vaccines against infectious laryngotracheitis virus in broiler chickens

Viral vector vaccines using fowl poxvirus (FPV) and herpesvirus of turkey (HVT) as vectors and carrying infectious laryngotracheitis virus (ILTV) genes are commercially available to the poultry industry in the USA. Different sectors of the broiler industry have used these vaccines in ovo or subcutaneously, achieving variable results. The objective of the present study was to determine the efficacy of protection induced by viral vector vaccines as compared with live-attenuated ILTV vaccines. The HVT-LT vaccine was more effective than the FPV-LT vaccine in mitigating the disease and reducing levels of challenge virus when applied in ovo or subcutaneously, particularly when the challenge was performed at 57 days rather than 35 days of age. While the FPV-LT vaccine mitigated clinical signs more effectively when administered subcutaneously than in ovo, it did not reduce the concentration of challenge virus in the trachea by either application route. Detection of antibodies against ILTV glycoproteins expressed by the viral vectors was a useful criterion to assess the immunogenicity of the vectors. The presence of glycoprotein I antibodies detected pre-challenge and post challenge in chickens vaccinated with HVT-LT indicated that the vaccine induced a robust antibody response, which was paralleled by significant reduction of clinical signs. The chicken embryo origin vaccine provided optimal protection by significantly mitigating the disease and reducing the challenge virus in chickens vaccinated via eye drop. The viral vector vaccines, applied in ovo and subcutaneously, provided partial protection, reducing to some degree clinical signs, and challenge VIRUS replication in the trachea.     

The route of inoculation dictates the replication patterns of the infectious laryngotracheitis virus (ILTV) pathogenic strain and chicken embryo origin (CEO) vaccine

Infectious laryngotracheitis virus (ILTV) has a high proclivity to replicate in the larynx and trachea of chickens causing severe lesions. There is a lack of knowledge on the ability of ILTV to replicate in other respiratory associated tissues apart from in the trachea. The objective of this study was to investigate how tissues that first encounter the virus dictate further sites of viral replication during the lytic stage of infection. Replication patterns of the pathogenic strain 63140 and the chicken embryo origin (CEO) vaccine in the conjunctiva, the Harderian gland, nasal cavity and trachea were evaluated after ocular, oral, intranasal or intratracheal inoculation of specific pathogen-free chickens. Viral replication was assessed by detection of microscopic cytolytic lesions, detection of viral antigen and viral genome load. The route of viral entry greatly influenced virus replication of both strain 63140 and CEO vaccine in the conjunctiva and trachea, while replication in the nasal cavity was not affected. In the Harderian gland, independently of the route of viral entry, microscopic lesions characteristic of lytic replication were absent, whereas viral antigen and viral genomes for either virus were detected, suggesting that the Harderian gland may be a key site of antigen uptake. Findings from this study suggest that interactions of the virus with the epithelial-lymphoid tissues of the nasal cavity, conjunctiva and the Harderian gland dictate patterns of ILTV lytic replication.     

Evaluation of the protection elicited by direct and indirect exposure to live attenuated infectious laryngotracheitis virus vaccines against a recent challenge strain from the United States

In a recent study (Oldoni & García, 2007), some field strains of infectious laryngotracheitis viruses (ILTV) were characterized as genotypically different (group VI) from ILT vaccine strains. The objective of this study was to evaluate the protection elicited by one chicken embryo origin (CEO) and one tissue culture origin (TCO) vaccine against a field isolate from group VI after direct and indirect exposure to ILTV live attenuated vaccines. In phase 1 of the experiment, non-vaccinated chickens were placed into contact with the eye drop vaccinates for a period of four weeks after vaccination. Transmission of the vaccine virus to these in-contact birds was demonstrated by real time PCR and antibody production, although the in-contact birds did not become protected against disease when subsequently challenged in phase 2 of the experiment. This emphasized the importance of uniform vaccination to obtain adequate protection, both to avoid the occurrence of susceptible chickens, and to minimize the potential for reversion to virulence of live-attenuated vaccines. In phase 2, protection against challenge with a group VI field virus was assessed four weeks after vaccination by scoring clinical signs and mortality, and quantifying weight gain. Sentinel birds were added to the groups one day after challenge to assess shedding of challenge virus, using real time PCR and virus isolation, during the period 2 to 12 days post challenge. The results showed that the CEO and TCO eye drop-vaccinated chickens were protected against challenge with the group VI virus, even though it was genetically different from the vaccine strains, and that challenge virus was not transmitted from these protected birds to the sentinels.     

The appearance of viral antigen and antibody in the trachea of naive and vaccinated chickens infected with infectious laryngotracheitis virus

In chickens vaccinated with SA-2 infectious laryngotracheitis (ILT) virus, viral antigen could no longer be detected in tracheal washings from day 7 post infection (pi). Total specific antibody was detected in tracheal washings from day 5 pi, IgA antibody appeared at day 6 pi, but neutralising antibody could not be detected until day 14. In the serum of vaccinated chickens, total antibody appeared on day 5 pi and neutralising antibody on day 7. However, no IgA antibody could be detected in serum. There was a substantial increase in the numbers of IgA- and IgG-synthesising cells in the trachea by day 3 pi, with a marked increase in the numbers of IgA-positive cells at day 7 pi. Following challenge with virulent CSW-1 ILT virus, no virus could be detected in the trachea of vaccinated chickens. There was also no evidence of an anamnestic antibody response in the trachea or in serum up to day 10 post challenge, and there was no significant change in the numbers of IgA- or IgG-synthesising cells in the tracheas of vaccinated chickens up to day 7 post challenge.     

Correlations between vaccinia-specific immune responses within a cohort of armed forces members

Widespread vaccination with vaccinia virus (VACV) resulted in the eradication of smallpox; however, the licensed VACV-containing vaccines are associated with adverse events (AEs), making them unsuitable for certain high-risk populations. A better understanding of the host immune response following smallpox vaccination could result in vaccines with similar immunogenicity profiles to pre-eradication vaccines with a lower incidence of AEs. To study the immune response to VACV, we recruited 1,076 armed forces members who had been vaccinated with one dose of Dryvax(?). We measured multiple VACV-specific immune responses: neutralizing antibody titer, the level of 12 secreted cytokines in peripheral blood mononuclear cell (PBMC) cultures (IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p40, IL-12p70, TNF-α, IFN-γ, IFN-α, IFN-β, and IL-18), and the number of IFN-γ- and CD8(+) IFN-γ-secreting cells. We analyzed these data to determine correlations between immune response measures. We detected a strong proinflammatory response in concert with a Th-1-like cytokine response pattern at a median time point of 15.3 mo following primary vaccination. We also detected correlations between neutralizing antibody titer and secreted IL-2, as well as secreted IFN-γ (p=0.009 and p=0.0007, respectively). We also detected strong correlations between the proinflammatory cytokines IL-1β, TNF-α, IL-6, and IL-12p40 (p<0.0001). These results further advance our knowledge of vaccinia-specific cellular immune responses. Notably, vaccine-induced proinflammatory responses were not correlated with neutralizing antibody titers, suggesting that further attenuation to reduce inflammatory immune responses may result in decreased AEs without sacrificing VACV immunogenicity and population seropositivity.     

Combined infectious bronchitis virus Arkansas and Massachusetts serotype vaccination suppresses replication of Arkansas vaccine virus

Polyvalent infectious bronchitis virus vaccination is common worldwide. The possibility of vaccine interference after simultaneous combined vaccination with Arkansas (Ark) and Massachusetts (Mass)-type vaccines was evaluated in an effort to explain the high prevalence of Ark-type infectious bronchitis virus in vaccinated chickens. Chickens ocularly vaccinated with combinations of Ark and Mass showed predominance of Mass vaccine virus before 9 days post-vaccination (DPV) in tears. Even when Mass and Ark vaccines were inoculated into separate eyes, Mass vaccine virus was able to outcompete Ark vaccine virus. Although Mass vaccine virus apparently had a replication advantage over Ark vaccine in ocular tissues, Ark vaccine virus appeared to have an advantage in spreading to and/or replicating in the trachea. When chickens vaccinated with Ark or Mass vaccine were housed together, Mass vaccine virus was able to spread to Ark-vaccinated chickens, but the Ark vaccine was not detected in Mass-vaccinated chickens. Only Mass vaccine was detected in tears of sentinel birds introduced into groups receiving both vaccines. Furthermore, Ark vaccine virus RNA was not detectable until 10 DPV in most tear samples from chickens vaccinated with both Ark and Mass vaccines at varying Ark vaccine doses, while high concentrations of Mass virus RNA were detectable at 3–7 DPV. In contrast, Ark vaccine virus replicated effectively early after vaccination in chickens vaccinated with Ark vaccine alone. The different replication dynamics of Ark and Mass viruses in chickens vaccinated with combined vaccines did not result in reduced protection against Ark challenge at 21 DPV. Further studies are needed to clarify if the viral interference detected determines differences in protection against challenge at other time points after vaccination.     

Comparative in vivo safety and efficacy of a glycoprotein G-deficient candidate vaccine strain of infectious laryngotracheitis virus delivered via eye drop

Infectious laryngotracheitis (ILT) is an acute respiratory disease in poultry that is commonly controlled by vaccination with conventionally attenuated virus strains. Despite the use of these vaccines, ILT remains a threat to the intensive poultry industry. Our laboratory has developed a novel candidate vaccine strain of infectious laryngotracheitis virus (ILTV) lacking glycoprotein G (ΔgG-ILTV). The aim of the present study was to directly compare this candidate vaccine with three currently available commercial vaccines in vivo. Five groups of specific-pathogen-free chickens were eye-drop inoculated with one of the three commercial vaccine strains (SA2-ILTV, A20-ILTV or Serva-ILTV), or ΔgG-ILTV, or sterile medium. Vaccine safety was assessed by examining clinical signs, weight gain and persistence of virus in the trachea. Vaccine efficacy was assessed by scoring clinical signs and conducting post-mortem analyses following challenge with virulent virus. Following vaccination, birds that received ΔgG-ILTV had the highest weight gain among the vaccinated groups and had clinical scores that were significantly lower than birds vaccinated with SA2-ILTV or A20-ILTV, but not significantly different from those of birds vaccinated with Serva-ILTV. Analysis of clinical scores, weight gain, tracheal pathology and virus replication after challenge revealed a comparable level of efficacy for all vaccines. Findings from this study further demonstrate the suitability of ΔgG-ILTV as a vaccine to control ILT.     

Vaccination with affinity-purified glycoproteins protects chickens against infectious laryngotracheitis herpesvirus

Glycoproteins of infectious laryngotracheitis virus (ILTV) were purified from detergent extracts of virus-infected cells by lectin affinity chromatography. In these preparations, glycoproteins of 205, 160, 115, 90, 85, 74, 60 and 50 kDa were recognized by immune chicken serum in Western blotting. Vaccination of chickens with these ILTV glycoproteins protected up to 83% of chickens against replication of the challenge virus as demonstrated by the absence of viral antigen in the trachea. Both neutralizing antibody and delayed-type hypersensitivity responses were induced by vaccination with glycoprotein preparations, but neither correlated with protection.     

Gp96 enhances the immunogenicity of subunit vaccine of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses in the pig industry worldwide. Currently available commercial vaccines provide limited protection due to delayed and weak cell-mediated immunity and neutralizing antibody production, thus the immunomodulators should be considered in order to improve the efficacy of PRRSV vaccines. Heat shock protein gp96 may be used as a modulator to enhance both innate and adaptive immune responses. In the present study, two multi-epitope subunit vaccines, named as Cp1 and Cp2, were designed based on the conserved B cell epitopes of viral proteins with the N-terminal 22-370 amino acids (aa) of porcine gp96 (Gp96N) chosen as the adjuvant. Immune responses elicited by the different combinations of Cp1/Cp2 and Gp96N were examined in mice and piglets. The results indicated that the group of Cp1/Cp2-Gp96N (CG) combination induced 3-4-fold higher titers of Cp1/Cp2-ELISA antibodies and neutralizing antibodies (NAs) in mice than the groups which received Cp1/Cp2 immunization alone or with Freund's adjuvant. Additionally, Gp96N significantly enhanced the levels of lymphocyte proliferative responses of splenocytes or peripheral blood mononuclear cells from vaccinated mice or piglets. The production of IFN-γ in mice splenocytes, TNF-α, IFN-γ, and IL-12 in sera of piglets were also remarkably increased with the treatment of Gp96N, while IL-4 was reduced by half and IL-10 was decreased to an undetectable level. These results suggest that the porcine Gp96N could effectively enhance the innate and adaptive immune responses of Cp1/Cp2 with a Th1-type bias. Therefore, the multi-epitope subunit vaccine Cp1/Cp2 co-administered with porcine Gp96N might potentially be a promising candidate vaccine for the prevention and control of PRRSV in pigs.     

Pre-existing neutralizing antibodies prevent CD8 T cell-mediated immunopathology following respiratory syncytial virus infection

Despite being a leading cause of severe respiratory disease, there remains no licensed respiratory syncytial virus (RSV) vaccine. Neutralizing antibodies reduce the severity of RSV-associated disease, but are not sufficient for preventing reinfection. In contrast, the role of memory CD8 T cells in protecting against a secondary RSV infection is less established. We recently demonstrated that high-magnitude memory CD8 T cells efficiently reduced lung viral titers following RSV infection, but induced fatal immunopathology that was mediated by IFN-γ. To evaluate the ability of RSV-specific neutralizing antibodies to prevent memory CD8 T cell-mediated immunopathology, mice with high-magnitude memory CD8 T cell responses were treated with neutralizing antibodies prior to RSV challenge. Neutralizing antibody treatment significantly reduced morbidity and prevented mortality following RSV challenge compared with IgG-treated controls. Neutralizing antibody treatment restricted early virus replication, which caused a substantial reduction in memory CD8 T cell activation and IFN-γ production, directly resulting in survival. In contrast, therapeutic neutralizing antibody administration did not impact morbidity, mortality, or IFN-γ levels, despite significantly reducing lung viral titers. Therefore, only pre-existing neutralizing antibodies prevent memory CD8 T cell-mediated immunopathology following RSV infection. Overall, our results have important implications for the development of future RSV vaccines.     

Comparison of the replication and transmissibility of two infectious laryngotracheitis virus chicken embryo origin vaccines delivered via drinking water

Infectious laryngotracheitis (ILT) is an acute infectious viral disease that affects chickens, causing respiratory disease, loss of production and mortality in severe cases. Biosecurity measures and administration of attenuated viral vaccine strains are commonly used to prevent ILT. It is notable that most recent ILT outbreaks affecting the intensive poultry industry have been caused by vaccine-related virus strains. The purpose of this study was to characterize and compare viral replication and transmission patterns of two attenuated chicken embryo origin ILT vaccines delivered via the drinking water. Two groups of specific pathogen free chickens were each inoculated with SA-2 ILT or Serva ILT vaccine strains. Unvaccinated birds were then placed in contact with vaccinated birds at regular intervals. Tracheal swabs were collected every 4 days over a period of 60 days and examined for the presence and amount of virus using a quantitative polymerase chain reaction. A rapid increase in viral genome copy numbers was observed shortly after inoculation with SA-2 ILT virus. In contrast, a comparatively delayed virus replication was observed after vaccination with Serva ILT virus. Transmission to in-contact birds occurred soon after exposure to Serva ILT virus but only several days after exposure to SA-2 ILT virus. Results from this study demonstrate in vivo differences between ILT vaccine strains in virus replication and transmission patterns.     

Genome sequence comparison of two United States live attenuated vaccines of infectious laryngotracheitis virus (ILTV)

This study was conducted to identify unique nucleotide differences in two U.S. chicken embryo origin (CEO) vaccines [LT Blen (GenBank accession: JQ083493) designated as vaccine 1; Laryngo-Vac^? (GenBank accession: JQ083494) designated as vaccine 2] of infectious laryngotracheitis virus (ILTV) genomes compared to an Australian Serva vaccine reference ILTV genome sequence [Gallid herpesvirus 1 (GaHV-1); GenBank accession number: HQ630064]. Genomes of the two vaccine ILTV strains were sequenced using Illumina Genome Analyzer 2X of 36 cycles of single-end reads. Results revealed that few nucleotide differences (23 in vaccine 1; 31 in vaccine 2) were found and indicate that the US CEO strains are practically identical to the Australian Serva CEO strain, which is a European-origin vaccine. The sequence differences demonstrated the spectrum of variability among vaccine strains. Only eight amino acid differences were found in ILTV proteins including UL54, UL27, UL28, UL20, UL1, ICP4, and US8 in vaccine 1. Similarly, in vaccine 2, eight amino acid differences were found in UL54, UL27, UL28, UL36, UL1, ICP4, US10, and US8. Further comparison of US CEO vaccines to several ILTV genome sequences revealed that US CEO vaccines are genetically close to both the Serva vaccine and 63140/C/08/BR (GenBank accession: HM188407) and are distinct from the two Australian-origin CEO vaccines, SA2 (GenBank accession: JN596962) and A20 (GenBank accession: JN596963), which showed close similarity to each other. These data demonstrate the potential of high-throughput sequencing technology to yield insight into the sequence variation of different ILTV strains. This information can be used to discriminate between vaccine ILTV strains and further, to identify newly emerging mutant strains of field isolates.     

Very Virulent Plus Strains of MDV Induce an Acute Form of Transient Paralysis in Both Susceptible and Resistant Chicken Lines

Abstract Marek's disease (MD) is a lymphoproliferative disease of domestic chickens caused by a highly cell-associated alpha herpesvirus, Marek's disease virus (MDV). Clinical signs of MD include depression, crippling, weight loss, and transient paralysis (TP). TP is a disease of the central nervous system that affects MD-susceptible chickens 8-11 days post-infection (dpi), normally resulting in recovery 1-3?d after the onset of clinical signs. In this study we inoculated chickens from lines 7(2) (MD-susceptible) and 6(3) (MD-resistant) with a very virulent plus strain of MDV at 2?wk of age, and collected brain samples from birds with and without TP at 5, 11, and 21?dpi for gene expression profiling and histological analysis. Data revealed that chickens inoculated with MDV had higher levels of IL-6, IL-10, IL-18, IFN-α, IFN-β, IFN-γ, MHC I, and CD18 in their brains at 11?dpi compared to the uninfected control birds. In addition, the expression levels of IL-6, IL-10, IFN-α, IFN-β, and IFN-γ were significantly higher in the brains of the birds showing clinical signs of TP than in asymptomatic chickens. Comparative analysis between the two chicken lines showed that the expression levels of IL-6, IL-10, IFN-β, IFN-γ, IL-18, CD18, and MHC I were significantly higher in the brains of the birds from line 6(3) with TP than those of line 7(2) exhibiting neurological disorders. A differential expression pattern was observed for some of the tested genes at different time points post-inoculation. Histological analysis showed lymphocytic meningitis, perivascular cuffing, and neuronal degeneration within the brains of birds from both susceptible and resistant lines exhibiting TP at 11?dpi. Vaccination prevented development of TP and other MD-associated clinical symptoms. These observations are suggestive of an underlying immunological mechanism for viral-induced neurological dysfunction, and the differential responses of the two chicken lines to MDV infection.