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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** 2023 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, several extensive studies involving natural viral transmissions were conducted over the past several years to determine whether Marek’s disease virus (MDV) evolves to higher virulence. Each experiment varies a specific factor, namely: (1) compare inbred, MD-susceptible experimental chickens to outbred, MD-resistant commercial chickens, and (2) using inbred, MD-susceptible experimental chickens and one-tenth dose of Turkey herpesvirus (commonly called HVT) compare MDV versus a recombinant MDV that has a 2-5 times higher mutation rate.

For this past year, bird experiments were performed to determine the influence of host  (chickens) genetics and the rate of viral mutation that accumulate over time during MDV  transmission from birds to birds. At one day of age, all birds were infected with virulent MDV. At two weeks of age, these birds were used as donors to transmit to another set of birds. This continued for ten natural passages of MDV in chickens using commercial layers to examine how host genetic resistance influences MDV transmission and evolutionary dynamics. The transmission design allows for a detailed investigation about the direct protective effects of host resistance as well as the indirect protective effects on resistant or susceptible contact birds. For each bird, biological samples (e.g., blood and feather samples) from chickens that were shedding the virus (called shedders) and recipient birds (called sentinel birds) at were collected at three time points at the (1) introduction of the recipients, (2) removal of the recipients, and (3) termination of the experiment. Statistical analyses of the data showed that the infection and transmission dynamics of birds inoculated with MDV differ substantially from those of birds that have become naturally infected through contact with infected shedder birds. This highlights the importance of mimicking modes of transmissions representative of field conditions in vaccination and other MDV challenge experiments. Overall, this information is important for the sustained control of MD in commercial chickens and has implications for other diseases that rely on vaccines to control disease.

Lastly, we compared the virulence and pathotype of the single nucleotide-modified very virulent plus (vv+) MDV with other known virulence/pathotyped MDV strains. Selective nucleotides within nine viral genes within the vv+ genome were modified in hopes of retrieving an attenuated phenotype. These recombinants were tested in pathotypic assays and demonstrated a correlation between the “changed” nucleotide variants and the virulence of the virus; in essence, a very virulent recombinant’s pathotype was validated.

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; 3) known to be non-essential for viral replication in vitro. In collaboration with NYU Langone, we now have the entire MDV genome cloned on two yeast assemblon vectors, enabling fast and easy manipulation. These two DNAs have been transfected into chicken cells to confirm infectious virus can be reconstituted. Three of the top five candidate genes have been knocked out with URA3, a yeast gene used as the selectable marker.

Additionally, for Objective 2, we are looking at mechanisms of MD immune evasion that can be overcome through improved vaccine engineering, such as incorporating immune checkpoint inhibitors into MD vaccine virus strains. Commercial antisera to chicken PD-1 and PD-L1 (immune checkpoint molecules which have been well-characterized in human cancers) is currently being prepared. This will be used for an initial trial to measure the effects of in vivo immune checkpoint blockade on MDV infection in chickens. 

Under Objective 3, in collaboration with an investigator at Simon Fraser University, Vancouver, Canada, we previously demonstrated that most Marek’s disease virus (MDV)-induced tumors had a somatic mutation in Ikaros, a known tumor suppressor gene in humans. One unexpected result was that the rMDV engineered to express the wild-type allele of Ikaros, which we called G2M/Ikaros, did not produce tumors in susceptible birds. Thus, experiments were conducted to test whether G2M/Ikaros could act as an MD vaccine. We find that G2M/Ikaros provides more and equal protection compared to HVT (first-generation MD vaccine) and CVI988 (the most protective commercial MD vaccine), respectively. This result suggests that insertion and expression of the wild-type Ikaros allele into a virulent MDV strain can attenuate it in a single step and act as a highly protective MD vaccine. Thus, this is a novel approach for producing the next generation of MD vaccines.

Also, in Objective 3, we continued examining the vaccinal efficacy of the recombinant MDV vaccine expressing the cell-surface-targeted chicken interleukin 15 (IL-15) in birds challenged with a very virulent + strain of MDV. The vaccinal efficacy of this recombinant was evaluated in a bivalent formulation that included HVT against a very virulent plus MDV challenge. Unfortunately, this formulation failed to improve vaccine efficacy. We additionally found that IL-15 expressed on the surface of infected cells led to increased MD tumor formation, despite early recruitment of NK cells to the spleen, which was identified via NK cell functional assays. This confirms that IL-15 is not an optimal adjuvant for MDV vaccines and indicates a potential role for NK cell signaling in MD tumor immune evasion. Additional strategies are ongoing to express different cytokines, i.e., IL-18 and GM-CSF, to create more efficacious MD vaccines. To rapidly insert these candidate cytokines and chemokines, the HVT genome was cloned as a bacterial artificial chromosome, and the infectious virus was successfully reconstituted from the recombinant DNA. IL-18 and GM-CSF will be inserted into the genome of HVT to investigate whether these additions can improve the delayed-onset of immunity typical of HVT recombinant vaccines. 

In Objectives 3 and 4, generating mRNA-based vaccines against infectious laryngotracheitis virus (ILTV) and MDV involved synthesizing various DNA fragments. These DNAs acting as templates for in vitro run-off transcription reactions are needed to make translational mRNAs. To optimize translation of foreign antigenic genes a commercial algorithm was chosen, and the codon-optimized sequences of the glycoprotein B genes of MDV and ILTV were chemically synthesized and cloned into our propriety vector, the chicken globin 5’UTR/3’UTR construct. A polymerase chain reaction scheme was then used to generate templates necessary to make mRNA that contained the necessary structures (5’ cap and a 3’ poly adenylated tail) at their termini for efficient translation into antigenic proteins. The quality of these in vitro transcribed mRNAs was confirmed and used in transient transfection assays. To determine the functionality of these RNAs, experiments were conducted to add these mRNAs to cells. Following transfection, the cells were stained with glycoprotein B-specific antisera and intense staining was observed when fluorescent-conjugated anti species antibodies were included in the assays. These results indicated that the mRNAs were efficiently translated into antigenic polypeptides. 

In Objective 4, regions of the genome of the infectious laryngotracheitis virus consisting of approximately 15.5 kilobases were cloned into a yeast vector using transformation-associated recombination (TAR). Although some regions were successfully cloned, others containing repetitive nucleotides and palindromic sequences were difficult to clone using TAR. However, using a polymerase chain reaction scheme, these regions were successfully cloned. Additional problems were encountered in transforming E. coli with DNA isolated from PCR-positive recombinant yeast clones. This transfer from yeast to E. coli is necessary to generate sufficient amounts of recombinant DNA for downstream assembly purposes. Currently, we are using different strains of E. coli to resolve this problem. 

An additional goal for Objective 4 involved cloning glycoproteins genes of ILTV and MDV into the Newcastle disease virus (NDV) vector known as rLS-IL4R. This vector contains an inverted interleukin four gene and has been proven safe when inoculated into embryonated eggs. The complete DNA sequence was determined for the rLS-IL4R, and a strategy was designed to convert this E. coli vector into a yeast shuttle vector so that recombination/cloning experiments could be easily performed in yeast. For this conversion, the origin of replication and centromere partitioning (ARS/CEN) and an auxotrophic selectable gene (trp) necessary for yeast propagation were cloned into rLS-IL4R using a battery of oligonucleotides known as stitches. A 633 base pair independent transcription unit (ITU) containing artificial 5’ and 3’ untranslated regions (UTRs) was synthesized commercially. This ITU will be cloned between the L and M genes of the NDV vector so that foreign genes can be inserted using a cassette format. **Accomplishments**
                                

1. Direct and indirection protection of vaccinates and sentinel chickens by Marek's Disease (MD) vaccination. ARS researchers in Athens, Georgia, and East Lansing, Michigan, recently generated three high-resolution empirical datasets for detailed analyses of the effects of vaccination and host genetics on Marek’s disease virus (MDV) transmission and virulence evolution over ten successive generations (passages) of virus transmission between naturally infected shedder birds and naïve contact birds. The transmission design allows for a detailed investigation about the direct protective effects that vaccines offer to vaccinated birds as well as the indirect protective effects on vaccinated or non-vaccinated contact birds. Analyses of the experimental data showed that the infection and transmission dynamics of birds that have been inoculated with MDV differ substantially from those of birds that have become naturally infected through contact with infected shedder birds, highlighting the importance of mimicking modes of transmissions representative of field conditions in vaccination and other MDV challenge experiments. Furthermore, experiments demonstrated that vaccination does not prevent MDV transmission within all ten subsequent passages. However, vaccination with the full recommended dose was found to provide not only direct protection from MD and death to the vaccinated birds but also indirect protection for non-vaccinated contact birds. 2. Generation of a novel herpesvirus-based vaccine against high pathogenic avian influenza virus. Current measures to prevent, control, and eradicate high pathogenic avian influenza devastating the United States poultry industry are mainly by stamping-out (culling millions of birds), which results in high economic losses and invokes growing public protest. Vaccination is the most efficient and cost-effective measure to combat highly pathogenic avian influenza (HPAI) outbreaks in support of prevention, control, and eradication measures. To this end, ARS researchers in Athens, Georgia, successfully created a novel vaccine candidate that expresses the hemagglutinin (HA) antigen of the 2022 highly pathogenic avian influenza virus (HPAIV) H5 strain. This was accomplished by first generating a bacterial artificial chromosome containing the complete genome of the turkey herpesvirus (HVT) and using this construct to insert the H5 hemagglutinin gene of HPAI H5N1. A high expression level could be demonstrated in cells in vitro infected with the HVT recombinant. Currently, this HVT-H5 recombinant is being tested in SPF layers chicks to examine the onset of immunity against AI challenge, breadth of protection, reduction of virus shedding, and induction of both antibody and cellular responses. Hopefully, this vaccine candidate can be used to mitigate the current HPAI outbreak. 3. Usage of a natural virus-host model system for studying avian herpesvirus transmission. Efficient transmission of herpesviruses is essential for dissemination in host populations; however, little is known about the viral genes that mediate transmission. ARS researchers in Athens, Georgia, working with scientists from the University of Illinois used a natural virus-host model system to investigate the transcripts and proteins essential for transmitting MDV among chickens. Infected feather follicle epithelial skin cells of live chickens, heavily enriched with fluorescent-tagged viral proteins, were used to measure both viral transcription and protein expression using both short- and long-read RNA sequencing technologies and mass spectrometry to define the viral proteome. This enrichment protocol produced a previously unseen breadth and depth of viral peptide sequencing. Eighty-four viral genes were identified at high confidence levels with correlated relative protein abundance and RNA expression levels. Using this proteogenomic approach, ARS scientists identified novel genes that may be important in virus transmission. This natural animal host model system to examine viral gene expression and protein profiles will provide a robust, efficient, and meaningful way of validating results gathered from cell culture systems. **Review Publications**
                            *Xing, N., Hofler, T., Hearn, C.J., Nascimento, M., Camps Paradell, G., Mcmahon, D.P., Kunec, D., Cheng, H.H., Trimpert, J., Osterrieder, N. 2022. Fast forwarding evolution – accelerated adaptation in a proofreading deficient hypermutator herpesvirus. Virus Evolution. 8(2):veac099. https://doi.org/10.1093/ve/veac099.*
                                *Conrad, S.J., Mays, J.K., Hearn, C.J., Dunn, J.R. 2023. Targeted ablation of exon 2 of the Avian Leukosis Virus-A (ALV-A)receptor gene in a chicken fibroblast cell line by CRISPR abrogates ALV-A infection. Avian Diseases. 67(1):102-107. https://doi.org/10.1637/aviandiseases-D-22-00072.*
                                *Sani, N., Abalaka, S., Ugochukwu, C., Saleh, A., Muhammad, S., Oladele, S., Abdu, P., Njoku, C., Dunn, J.R. 2022. Detection of Avian Neoplastic Disease viruses in formalin-fixed neoplastic livers of layers from farms in Kaduna and Plateau States, Nigeria. Comparative Clinical Pathology. https://doi.org/10.1007/s00580-022-03373-x.*
                                *Hearn, C.J., Cheng, H.H. 2023. Contribution of the TCR beta repertoire to Marek's disease genetic resistance in chicken. Viruses. 15(3):607. https://doi.org/10.3390/v15030607.*
                                *Spatz, S.J., Garcia, M., Fuchs, W., Loncoman, C., Volkening, J., Ross, T.A., Riblet, S., Kim, T.N., Likens, N., Mettenleiter, T. 2023. Reconstitution and mutagenesis of avian infectious laryngotracheitis virus from cosmid and yeast centromeric plasmid clones. Journal of Virology. 97(4). Article e01406-22. https://doi.org/10.1128/jvi.01406-22.*
                                *Volkening, J.D., Spatz, S.J., Ponnuraj, N., Akbar, H., Arrington, J.V., Vega-Rodriguez, W., Jaronsinski, K.W. 2023. Viral proteogenomic and expression profiling during productive replication of a skin-tropic herpesvirus in the natural host. PLoS Pathogens. 19(6):e1011204. https://doi.org/10.1371/journal.ppat.1011204.*


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