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ARS Home Southeast Area Athens, Georgia U.S. National Poultry Research Center Endemic Poultry Viral Diseases Research Research





- Spatz, Stephen
- Hearn, Cari
- Kim, Taejoong
- Dunn, John
- 2023
- 2022
- Publications
- Animal Health (103)

Research Project: Elucidation of Molecular Determinants of Avian Herpesviruses Virulence and Evolution to Inform the Development of Safe and Effective Vaccines **Location:
                            Endemic Poultry Viral Diseases Research** 2022 Annual Report **Objectives**
                                

1. Predict and characterize the evolution of virulent strains of Gallid alphaherpesvirus type 2 Marek's Disease Virus (MDV) in chicken production systems.
1.A. Detect and predict the evolution of MDV strains.
1.B. Conduct studies to identify and characterize mechanisms associated with the evolution of MDV.
1.C. Identify viral genes with mutations that are associated with MDV virulence and verify the effects on virus pathogenesis.

2. Elucidate mechanisms of immunity that can enhance Marek’s Disease vaccinal control.
2.A. Characterize virus-host interactions in innate immune pathways that contribute to MD susceptibility or immunity, to inform the development of highly effective vaccines against very virulent strains.
2.B. Define mechanisms of cellular immune evasion that contribute to MD pathogenicity, and which can be targeted through recombinant vaccines to improve vaccine efficacy.

3. Develop safe and highly effective MDV platforms that convey protection against very virulent strains.
3.A. Development and evaluation of novel recombinant MD vaccines.
3.B. Utilization of novel adjuvants for enhanced immunogenicity of live-attenuated vaccines against MD.

4. Develop a novel Infectious laryngotracheitis virus (ILTV) vaccine platform that is safe, efficacious, and cost-effective.  
4.A. Development of an infectious clone of ILTV.
4.B. Develop improved vaccines against ILTV. **Approach**
                                

We plan to investigate the role of innate immunity in Marek's Disease Virus (MDV) infection, identify host and viral determinants involved in transmission that undoubtedly plays a role in virus evolution, and define cellular immune evasion mechanisms that contribute to MD-induced pathogenicity. These effects will lead to (1) novel, more efficacious vaccines that include recombinants expressing cancer driver genes and a Newcastle disease virus vector expressing antigens of MDV and Infectious laryngotracheitis virus (ILTV) to create a trivalent vaccine, and (2) fundamental information that can be applied for more effective and sustainable MD control. Live attenuated ILT vaccines are also imperfect, and not only can they revert to virulence but are highly recombinogenic, making them capable of generating new virulent strains. An infectious clone of ILTV will be chemically synthesized and used to create novel vaccines with enhanced replicative fidelity, reduced capacity for reversion, and the inability to establish latent infections. Lipid nanoparticles encapsulating mRNAs expressing the prefusion conformation of the essential fusion proteins, glycoprotein B orthologues of ILTV and MDV, will also be developed. **Progress Report**
                                

For Objective 1, in collaboration with investigators at the Roslin Institute, Edinburgh, Scotland, and Freie Universitat, Berlin, Germany, a large experiment was conducted to determine if Marek’s disease virus (MDV) evolves to higher virulence in Marek’s disease (MD) vaccinated chickens versus unvaccinated birds. Specifically, six biological replicates of groups of 10 birds were either vaccinated or not, then infected with virulent MDV. At two weeks of age, these birds were used as donors to transmit to another set of MD-vaccinated or unvaccinated birds. This continued to 10 passages. For each bird, blood and feather samples were collected, which are being analyzed now for the amount of virus present. And the viruses recovered in the last passage are being characterized as to whether they are more virulent or not. This information is important for the sustained control of MD in commercial chickens as well as having implications for other diseases that rely on vaccines to control disease. In addition, we performed pilot studies this year evaluating different vaccine dilutions and challenge intervals to optimize our model for evaluating the practice of MD vaccine dilution on the acceleration of MDV evolution. This model will be used for the next phase of experiments conducting virus passage in chickens.

In Objective 2, we have identified top candidate genes for inhibition of type I interferon for further evaluation. Criteria included: 1) presence in only one copy in the MDV genome; 2) Not co-located with any other known or annotated genes; and 3) Known to be non-essential for viral replication in vitro. In collaboration with NYU Langone, we are designing an MDV clone able to replicate in yeast to enable fast and easy manipulation. Our approach targeted the terminal region near the loxP site on the pBeloBAC11 backbone with a gRNA to linearize the BAC. This CRISPR-mediated cut was done as part of a co-transformation in yeast, with a linear DNA fragment encoding the selectable marker/origin LEU2/ARS-CEN bearing appropriate terminal homologies to the linearized BAC. Transformation followed by selection on medium lacking leucine led to the isolation of ~100 colonies. PCR screening for novel BAC-Leu2 junctions highlighted ten clones that had potentially assembled the BAC-yeast DNA fragments. We attempted to verify all ten by WGS to ensure the entire infectious BAC insert remained. Unfortunately, all sequenced clones lacked reads mapping to the BAC sequence of interest; thus, the clones seemingly lost the infectious BAC DNA. We are now attempting to construct the base BAC clone using alternative strategies.

In Objective 3, a candidate MD vaccine was evaluated in collaboration with an investigator at the Simon Fraser University, Burnaby, Canada. Specifically, a recombinant MDV called G2M-IKZF1 was produced that expressed the wild-type (normal) allele of Ikaros, which is frequently found to be mutated in MD tumors. This candidate vaccine was compared to HVT and Rispens, which are the first generation and most effective MD vaccines. Our results show that G2M-IKZF1 is more protective compared to HVT and equally protective as Rispens. Furthermore, this result shows that expressing Ikaros in a virulent MDV strain can attenuate this virus in a single step and act as a potential next-generation MD vaccine.
 
The recombinant MD vaccine that expresses chicken IL-15 was successfully constructed using BAC recombineering, and the vaccinal efficacy was evaluated as a bivalent MD vaccine with HVT against very virulent MDV challenge. This vaccine expressing IL-15 exhibited comparable protective efficacy to that of bivalent MD vaccines composed of MDV-2 plus HVT, but, unfortunately, the expression of IL-15 in MD did not significantly increase the protection efficacy over existing MD vaccines. Additional modification strategies are ongoing to increase the efficacy of this IL-15 expressing vaccine candidate.

In Objective 3, the protective efficacy of the LaSota vaccine strain of Newcastle disease virus (NDV) recombinants, individually expressing the immunogenic envelope glycoproteins B, C E, and I of Marek’s disease virus (MDV) was investigated in early (5 dpv) and late (14 dpv) vaccine-challenge models for Marek’s disease in experimental chickens. Biological assessments showed that these recombinants were slightly attenuated in vivo yet retained similar growth kinetics and virus titers in vitro compared to the parental LaSota virus. Vaccination of leghorn chickens (Line 15I5x71) with these recombinant viruses via intranasal and eye drop routes conferred differing levels of protection against virulent MDV challenge. Vaccination with the recombinant NDV expressing glycoprotein (rLS/MDV-gB) gave the highest protective index with significant protection against MDV-induced tumor formation. The other three recombinants (rLS/MDV-gC, rLS/MDV-gI, and rLS/MDV-gE) provided little or no protection against MDV. Furthermore, the protective efficacy of the rLS/MDV-gB recombinant could not be enhanced by adding other NDV/MDV recombinants in cocktail formulations. All four recombinants did, however, confer complete protection against velogenic NDV challenge. Further stabilization of rLS/MDV-gB to express a prefusion gB conformation could lead to a better vectored vaccine candidate for Marek’s disease, with several advantages over the current live cell-associated vaccines: elimination of cold chain requirement for vaccine preparation and administration, no horizontal spread, reduced selection pressure for highly virulent virus, no recombination, and no possibility of virulence reversion.

In Objectives 3 and 4, generating mRNA-based vaccines against infectious laryngotracheitis virus (ILTV) and Marek’s disease virus (MDV) requires developing a plasmid-based vector containing transcription regulatory elements. These regulatory elements, known as the 5’ and 3’ untranslated regions (5’UTR and 3’UTR, respectively), flank the open reading frame (ORF) encoding the antigens. They are necessary to ensure the long-lasting expression and stability of the synthetic transcripts within the cell. In developing our veterinary mRNA-based vaccines, we will use regulatory elements of the chicken ß-globin (Ck) gene. This decision was solely based on using similar regulatory elements within the human ß-globin gene for mRNA vaccines against human pathogens. It is well documented that these genes are phylogenetically conserved among different taxa, from fish to mammals. However, the regulatory elements for the chicken ß-globin transcript are poorly defined.

We analyzed (or mined) GenBank for cDNA sequences of chicken ß-globin and aligned 23 chicken corresponding mRNAs. The chicken ß-globin gene contains three exons, the first containing the 5’ UTR before the AUG starts codon. This data suggests that the 5’ end of the Ck ß-globin transcript is shorter than that predicted and begins at the first C within the sequence CACGGGAGCA and not the A within the upstream AGCGTGCT sequence. To provide additional suppose for this hypothesis, we further examined the secondary structure of the two proposed 5’ UTRs using RNA folding algorithms. The results suggest the larger 5’ UTR is translationally disadvantaged. Minimizing steric hindrance is essential to allow easy access for the binding of ribosomal subunits to initiate translation. Therefore, we will use the shorter 5’ UTR in the in vitro transcripted (IVT) vector design. The 3’ UTR of the chicken ß-globin gene is far better defined with its aaa(u/t)aaa polyadenylation signal and well-defined termination after a CA box. A closer inspection of the region has identified 4 CA boxes and a GU-rich area. Therefore, the termination of the transcript likely occurs at the A residue within the last CA box (CA18). Defining the 5’ and 3’ UTRs has allowed the generation of the synthetic IVT vector, a first step to ensuring the designs (blueprints) of the mRNA vaccines are logical and likely to elicit protective immunity without inducing innate immune responses.

In Objective 4, the genome of the infectious laryngotracheitis virus was dissected into 94 regions consisting of overlapping 1,544 nucleotides using an in silico approach. Each region was designed to overlap the adjacent region by 50 nucleotides, necessary for homologous recombination (or assembly) purposes. Double-stranded DNA (synthons) representing these 94 regions were synthesized commercially. There were nine regions that could not be synthesized chemically due to their highly repetitive nature or gross secondary structures. Primer pairs were designed for these nine regions and successfully amplified using PCR and virus DNA as template. Using ten synthons representing the first 14,965 nucleotides of the ILTV genome, experiments were implemented to connect these pieces (synthons) in yeast successfully. A PCR-based screening protocol was used to identify the yeast recombinants. Restriction endonuclease profiling confirmed the authenticity of the assembled synthons. These experiments established the cloning conditions and screening procedures that will be used in future assembly “patch-work” experiments to generate the infectious clone of ILTV. **Accomplishments**
                                

1. Comparison of Marek’s disease virus (MDV) challenge strains in white leghorn chickens. ARS researchers in Athens, Georgia, and East Lansing, Michigan, recently reported on a comparison of Marek’s disease virus (MDV) challenge strains in white leghorn chickens for the purpose of meeting immunogenicity testing required by the Code of Federal Regulations for vaccine licensing. For testing a serotype-3 vaccine, a “virulent” label claim requires at least 80% of the unvaccinated chickens to develop lesions, however, multiple vaccine companies have reported inconsistency in the development of Marek’s disease in unvaccinated commercial chickens inoculated with standard challenge strains. ARS scientists expanded this evaluation of challenge strains to commercial broiler-type chickens, which are required to be used for licensing vaccines that will be labeled for broiler-type chickens in some countries. We evaluated a total of seven challenge viruses, consisting of both virulent MDV (vMDV) and very virulent MDV (vvMDV) isolates. Several vvMDV candidates were close to the required 80% disease incidence, whereas none of the vMDV strains were near 80% and should not be used for broiler vaccine evaluation. 2. A serotype 2 Marek’s disease (MD) vaccine (301B/1). ARS researchers in Athens, Georgia, successfully developed a serotype 2 Marek’s disease (MD) vaccine (301B/1) expressing chicken IL-15 which is equally as protective as the most widely used MD vaccine and favorite vector vaccine platform in the poultry industry, the serotype 3 HVT vaccine. This vaccine opens the door for the development of a novel vaccine vector that can be administered in combination with HVT vector vaccines, allowing protection against multiple poultry viruses. 3. A single vaccine that protects against two hideous pathogens of poultry. ARS researchers in Athens, Georgia, recently developed a single vaccine that protects against two hideous pathogens of poultry: Marek’s disease and Newcastle disease. The biological assessments of the dual vaccine showed that it was slightly attenuated in animal studies yet retained growth properties deemed desirable to the vaccine manufacturing community. Furthermore, in animal studies, vaccination of specific-pathogen-free leghorn chickens with the dual vaccine conferred significant protection against virulent Marek’s disease virus challenge and complete protection against Newcastle disease challenge. These results demonstrated that the dual vaccine is a safe, stable, and effective vaccine that can be mass-administered via aerosol or drinking water to large chicken populations at an extremely low cost. The U.S. PTO issued a patent publication entitled “Recombinant vaccine against Marek’s disease and Newcastle” on 06/16/2022 for our abovementioned research. **Review Publications**
                            *Krieter, A., Xu, H., Akbar, H., Kim, T.N., Jarosiniski, K.W. 2022. The conserved Herpesviridae protein kinase (CHPK) of Gallid alphaherpesvirus 3 (GaHV3) is required for horizontal spread and natural infection in chickens. Viruses. 14(3):586. https://doi.org/10.3390/v14030586.*
                                *Conrad, S.J., Oluwayinka, E.B., Mays, J.K., Heidari, M., Dunn, J.R. 2021. Deletion of the viral thymidine kinase in a meq-deleted recombinant Marek's disease virus reduces lymphoid atrophy but is less protective. Microorganisms. 10(1):7. https://doi.org/10.3390/microorganisms10010007.*
                                *Hein, R., Koopman, R., Garcia, M., Armour, N., Dunn, J.R., Barbosa, T., Martinez, A. 2021. Review of poultry recombinant vector vaccines. Avian Diseases. 65(3):438–452. https://doi.org/10.1637/0005-2086-65.3.438.*
                                *Kim, T.N., Hearn, C.J. 2022. Vaccinal efficacy of recombinant Marek’s disease vaccine 301B/1 expressing chicken interleukin-15. Avian Diseases. 66(1):79-84. https://doi.org/10.1637/21-00089.*
                                *Xu, H., Kriter, A.L., Ponnuraj, N., Tien, Y., Kim, T.N., Jarosinski, K.W. 2022. Coinfection in the host can result in functional complementation between live vaccines and virulent virus. Virulence. 13(1):980-989. https://doi.org/10.1080/21505594.2022.2082645.*
                                *Dunn, J.R., Mays, J.K., Hearn, C.J., Hartman, A. 2021. Comparison of Marek's disease virus challenge strains and bird types for vaccine licensing. Avian Diseases. 65(2):241-249. https://doi.org/10.1637/aviandiseases-D-20-00122.*
                                *Gerber, P.F., Spatz, S.J., Alfirevich, S., Walkden-Brown, S.W. 2022. Circulation and molecular characterization of hemorrhagic enteritis virus (HEV) in commercial turkey and meat chicken flocks in Australia. Avian Diseases. 66(1):53-59. https://doi.org/10.1637/21-00095.*
                                *Davison, A.J., Depledge, D.P., Trimpert, J., Stewart, J.P., Spatz, S.J., Schmid, S., Hartley, C.A., Szpara, M.L., Jarosinski, K.W., Vaz, P.K. 2021. Family Herpesviridae. Journal of General Virology. https://doi.org/10.1099/jgv.0.001673.*
                                *Santos, W.H., Spatz, S.J., Ecco, R., Wenceslau, R., Hergot, I.G., De Rocha, C.M., Ferreira, H.L., Resende, M., Martins, N.R., De Oliveira, L.B., Leão, P.A. 2022. A five-year surveillance study of vaccination schedules using viral-vectored vaccines against infectious laryngotracheitis in a quarantine high-density layer region. Avian Diseases. 42:Article e07037. https://doi.org/10.1590/1678-5150-PVB-7037.*


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