WO2025201383A1

WO2025201383A1 - A recombinant serotype 1 Marek's Disease Virus

Info

Publication number: WO2025201383A1
Application number: PCT/CN2025/084962
Authority: WO
Inventors: Qingshui ZHANG; Sidi JU; Ning Chen; Chengtai MA; Yifan Huangfu
Original assignee: Boehringer Ingelheim Vetmedica China Co Ltd
Current assignee: Boehringer Ingelheim Vetmedica China Co Ltd
Priority date: 2024-03-27
Filing date: 2025-03-26
Publication date: 2025-10-02
Anticipated expiration: 2026-09-27

Abstract

The present invention relates to the field of animal health. Particularly, the present invention relates to recombinant serotype 1 Marek's Disease Virus (rMDV-1) comprising an expression cassette in the genome thereof, wherein the expression cassette comprises a coding sequence of at least one antigenic protein of Infectious laryngotracheitis virus (ILTV) operably linked to a corresponding native ILTV promoter. Further, the present invention provides an immunogenic composition comprising the rMDV-1 of the present invention and the use of the immunogenic composition for preventing and/or treating diseases in an animal.

Description

A recombinant serotype 1 Marek′s Disease Virus

Cross-Reference to Related Application

This application claims the priority of PCT/CN2024/084058 filed on March 27, 2024 entitled by “A recombinant serotype 1 Marek's Disease Virus” , the entirety of which is incorporated by reference herein.

Technical Field

The present invention relates to the field of animal health. Particularly, the present invention relates to recombinant serotype 1 Marek's Disease Virus (rMDV1) comprising an expression cassette in the genome thereof, wherein the expression cassette comprises a coding sequence of at least one antigenic protein of Infectious laryngotracheitis virus (ILTV) operably linked to a corresponding native ILTV promoter. Further, the present invention provides an immunogenic composition comprising the rMDV1 of the present invention and the use of the immunogenic composition for preventing and/or treating diseases in an animal.

Technical background

Marek's Disease Virus (MDV) is classified in the genus Mardivirus, belonging to the subfamily Alphaherpesvirinae of Herpesviridae. The viral genome is a double-stranded linear DNA with a full length of approximately 180 kb, encoding 103 proteins. Marek's disease virus can be divided into 3 serotypes. Among them, serotype 1 virus is pathogenic and oncogenic to chicken hosts. According to the pathogenicity and virulence, the virus can be further divided into mild MDV (mMDV) , virulent MDV (vMDV) , and very virulent MDV (vvMDV) , and very virulent plus MDV (vv+MDV) . Serotype 2 Marek's disease virus is non-oncogenic, and serotype 3 Marek's disease virus is not pathogenic to chickens. The vaccines currently used for MD prevention are mainly serotype 1 attenuated vaccines, including the Dutch CVI988 strain (Rispens) and the Chinese 814 strain.

Recombinant herpes virus live vector vaccine is a genetically engineered vaccine that has been studied in depth and has broad application prospects. The principle is to insert the protective antigen gene of a certain pathogen through genetic engineering technology into a region not essential for replication of the viral vector, such that the antigen is continuously expressed as the vector replicates, inducing the body to produce corresponding antibodies to exert immune protection. The viral vector of the recombinant live virus vaccine can replicate itself, so usually a lower dose can produce sufficient exogenous proteins in the body to produce prolonged and good immune protection. Recombinant viral vector vaccines can not only induce humoral immunity, but also induce cellular immunity and mucosal immunity. Compared with traditional inactivated vaccines or live attenuated vaccines, different recombinant viruses have been proven to significantly reduce the level of shedding after infection and reduce the viral load in the environment.

As for MDV, Serotype 3 Herpesvirus of Turkeys (HVT) and serotype 1 MDV are considered as ideal viral vectors for construction of live vectored vaccines. These viruses have been used as vectors to construct different recombinant viruses. Li et al. used the MDV 814 vaccine strain as a vector to successfully construct a recombinant MDV expressing the VP2 gene of infectious bursal disease virus. Studies have shown that the recombinant virus is safe and stable, and can protect chickens well against both Marek's disease and infectious bursal disease. Recombinant HVT with H9HA inserted into US2 gene are shown to be capable of achieving 100%protection against H9 AIV (see e.g., CN106031793B, CN107142280A) ; however, unexpectedly, the inventor’s data (not provided) showed that recombinant serotype 1 MDV strain SC9-2 with H9HA inserted into US2 gene cannot be rescued or can only provide 30%protection.

Infectious Laryngotracheitis (ILT) is an acute, highly contagious upper respiratory tract infection caused by Infectious Laryngotracheitis virus (ILTV) . ILTV belongs to Alphaherpesvirinae, a double-stranded DNA enveloped virus. The viral genome is about 155kb and mainly contains a 120kb long unique region (UL) and a 17kb short unique region (US) . Envelope glycoproteins gB, gD, gI and gE are the main immunogenicity-related proteins of ILTV. They are located on the surface of the viral envelope and are related to virus adsorption and invasion of host cells. They can also induce the host to produce corresponding cellular immunity and humoral immune responses. At present, in addition to biosecurity measures in chicken farms, vaccination is the main preventive measure against ILT. However, the current attenuated ILT vaccine has shortcomings such as high virulence and difficulty in distinguishing it from wild strains. Moreover, there are reports that recombination may occur between different attenuated vaccine strains of ILTV and produce new virulent viruses. Therefore, there is an urgent need to develop a new generation of efficient and safe vaccines to prevent and control ILT.

Brief Description of the Invention

In one aspect, the present invention provides a recombinant serotype 1 Marek's Disease Virus (rMDV-1) comprising an expression cassette in the genome thereof, wherein the expression cassette comprises a coding sequence of at least one antigenic polypeptide of Infectious laryngotracheitis virus (ILTV) operably linked to a corresponding native ILTV promoter.

In one aspect, the present invention provides a host cell expressing the rMDV-1 of the present invention.

In one aspect, the present invention provides an immunogenic composition, comprising the rMDV-1 of the present invention, and optionally a pharmaceutical-or veterinary-acceptable carrier or excipient.

In one aspect, the present invention provides use of the rMDV-1 of the present invention in preparation of an immunogenic composition for inducing a protective immune response in a host animal against a pathogen, preferably said animal is an avian, more preferably, a poultry such as a chicken.

In one aspect, the present invention provides the rMDV-1 of the present invention or the immunogenic composition of the present invention for use in a method of inducing a protective immune response in a host animal against a pathogen, preferably said animal is an avian, more preferably, a poultry such as a chicken.

In one aspect, the present invention provides a method of inducing a protective immune response in a host animal against a pathogen, said method comprising the step of administering to the animal the rMDV-1 of the present invention or the immunogenic composition of the present invention, preferably said animal is an avian, more preferably, a poultry such as a chicken.

Brief Description of the Drawings

Figure 1. Detection of stability of inserted genes. a. rSC9-2/UL2-Native-ILTgD-gI-/gE/-UL3; b. rSC9-2/UL55-Native-ILTgD-gI-/gE/-Lorf10; c. rSC9-2/US2-Native-ILTgD-gI-/gE/; M is DNA marker; and N is negative control of parental virus.

Figure 2. Dual indirect immunofluorescence images for different recombinant viruses.

Detailed Description

Before the aspects of the present invention are described, it must be noted that as used herein and in the appended claims, the singular forms "a" , "an" , and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a gene" includes a plurality of genes, a reference to the "virus" is a reference to one or more viruses and equivalents thereof known to those skilled in the art, and so forth. The term “and/or” is intended to encompass any combinations of the items connected by this term, equivalent to listing all the combinations individually. For example, “A, B and/or C” encompasses “A” , “B” , “C” , “A and B” , “A and C” , “B and C” , and “A and B and C” . In contrast, “A or B” means either “A” or “B” , without including “A and B” . Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the virus strains, the cell lines, vectors, and methodologies as reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Recombinant Marek's Disease Virus

In one aspect, the invention provides a recombinant serotype 1 Marek's Disease Virus (rMDV-1) comprising an expression cassette in the genome thereof, wherein the expression cassette comprises a coding sequence of at least one antigenic polypeptide of Infectious laryngotracheitis virus (ILTV) operably linked to a corresponding native ILTV promoter.

The term “recombinant” used herein refer to an MDV that has been altered, rearranged, or modified by genetic engineering. However, the term does not refer to alterations in polynucleotide, amino acid sequence, or nucleotide sequence that result from naturally occurring events, such as spontaneous mutations. The terms “recombinant MDV” , and “rMDV” are used interchangeably herein.

The term "virus" designates in particular a viral particle comprising a nucleic acid molecule (e.g., a genome) encapsulated in a capsid or capsule. The term "virus" also designates an isolated viral genome.

The term “MDV” as used herein refers to all viruses belonging to the genus Mardivirus within subfamily Alphaherpesvirinae of the family Herpesviridae.

In some embodiments, the recombinant serotype 1 MDV is derived from an attenuated strain of serotype 1 MDV. Examples of attenuated strains of serotype 1 MDV include but are not limited to Dutch CVI988 strain (Rispens) (GenBank: DQ530348.1; Comparative full-length sequence analysis of oncogenic and vaccine (Rispens) strains of Marek's disease virus, Nair, 2007) , Chinese 814 strain (GenBank: JF742597.1; Comparative full-length sequence analysis of Marek's disease virus vaccine strain 814, Cheng, 2012) , or SC9-2 strain (Chinese Patent Publication No: CN102628053A) .

The term "attenuated" as used herein refers to a modified virus that is essentially not virulent in chicken, i.e. does not cause or causes reduced illness, especially does not cause death in a host animal, such as chicken, as compared to the non-modified wildtype parent virus. More particularly, an attenuated virus can typically replicate in a host animal, such as chicken, without causing death thereof. More particularly, an attenuated virus designates a virus that is not virulent in in a host animal, such as chicken, when injected at a dose of 10^4.0-10^7.0 TCID₅₀/animal, such as 10^6.0 TCID₅₀/animal. More particularly, an attenuated virus designates a virus that is not virulent in a chicken at a dose of 10^4.0-10^7.0 TCID₅₀/chicken, such as 10^6.0 TCID₅₀/animal in at least 10%injected chickens, in at least 20%injected animals, in at least 30%injected animals, in at least 40%injected chickens, in at least 50%injected animals, in at least 60%injected animals, in at least 70%injected animals, more preferably in at least 80%injected animals, even more preferably in at least 90%, 95%, 97%, 98%, 99%or more. In some embodiments, an attenuated virus more particularly designates a virus that is not virulent in an embryo when injected at a dose of 10^4.0-10^7.0 TCID₅₀/egg, such as 10^6.0 TCID₅₀/egg. Most preferred an attenuated virus designates a virus that is not virulent in an embryo at a dose of 10^4.0-10^7.0 TCID₅₀/egg, such as 10^6.0 TCID₅₀/egg in at least 10%injected eggs, in at least 20%injected eggs, in at least 30%injected eggs, in at least 40%injected eggs, in at least 50%injected eggs, in at least 60%injected eggs, in at least 70%injected eggs, more preferably in at least 80%injected eggs, even more preferably in at least 90%, 95%, 97%, 98%, 99%or more. The rMDV of the invention is also not virulent for injection post-hatch, including at Day 0, Day 1, Day 2, Day 3 post-hatch (i.e., between 0.1 and 72 hours post-hatch) .

In some embodiments, the recombinant virus is derived from the SC9-2 strain. In some embodiments, the SC9-2 strain is deposited according to Budapest Treaty on December 15, 2023 at CHINA CENTER FOR TYPE CULTURE COLLECTION (Wuhan University, Wuhan 430072, P. R. China) , under the accession number: CCTCC No: V2023114.

An "antigenic polypeptide" or “antigen” as used herein refers to, but is not limited to, components which elicit an immune response in a host.

In some embodiments, at least one antigenic polypeptide of ILTV is selected from the gB protein gD protein, gI protein, or gE protein of ILTV, or any combinations thereof.

In some embodiments, the at least one antigenic protein of ILTV is selected from the gD protein of ILTV, the gI protein of ILTV, and/or the gE protein of ILTV or a partial gE protein of ILTV.

In some embodiments, the expression cassette comprises a coding sequence of gD, gI, and partial gE of ILTV.

In some embodiments, the expression cassette may comprise a coding sequence of the gD protein of ILTV operably linked to a native ILTV gD promoter, and/or a coding sequence of the gI protein of ILTV and a partial gE protein of ILTV operably linked to a native ILTV gI promoter.

In some embodiments, the gD protein may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 13. In some embodiments, the complete gD coding sequence may have a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 14.

In some embodiments, the gI protein may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 15. In some embodiments, the complete gI coding sequence may have a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 16.

In some embodiments, the partial gE protein may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 17. In some embodiments, the complete gE protein may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 18. In some embodiments, the complete gE coding sequence may have a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 19.

In some embodiments, the native ILTV gD promoter comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 22.

In some embodiments, the native ILTV gI promoter comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 23.

In some embodiments, the expression cassette may have a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 20.

"Sequence identity" between two polypeptide/nucleotide sequences indicates the percentage of amino acids/nucleotides that are identical between the sequences. Methods for evaluating the level of sequence identity between amino acid or nucleotide sequences are known in the art. For example, sequence analysis software is often used to determine the identity of amino acid/nucleotide sequences. For example, identity can be determined by using the BLAST program in the NCBI database. For determination of sequence identity, see, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

As used herein, it is in particular understood that the term “sequence identity with the sequence of SEQ ID NO: X” is equivalent to the term “sequence identity with the sequence of SEQ ID NO: X over the length of SEQ ID NO: X” or to the term “sequence identity with the sequence of SEQ ID NO: X over the whole length of SEQ ID NO: X” , respectively. In this context, “X” is any integer, such as 1, 2 or 3, so that “SEQ ID NO: X” represents any of the SEQ ID NOs mentioned herein.

The expression cassette may be located at a position between UL55 and Lorf10, a position between UL2 and UL3, or a position within US2 gene. In some preferred embodiments, the expression cassette is located at a position within the US2 gene.

UL55, Lorf10, UL2, UL3 and US2 genes are highly conserved between different MDV strains such as Dutch CVI988 strain (Rispens) , Chinese 814 strain and SC9-2 strain. It is understood that the skilled artisan may easily identify the exact location of the UL55, Lorf10, UL2, UL3 and US2 genes in any MDV strain using the information contained in the present application and general common knowledge, or by sequence alignment.

Exemplary amino acid sequence of UL55 of SC9-2 strain is shown in SEQ ID NO: 1. Exemplary nucleotide sequence of UL55 of SC9-2 strain is shown in SEQ ID NO: 2. Exemplary amino acid sequence of Lorf10 of SC9-2 strain is shown in SEQ ID NO: 3. Exemplary nucleotide sequence of Lorf10 of SC9-2 strain is shown in SEQ ID NO: 4. Exemplary nucleotide sequence between UL55 and Lorf10 is shown in SEQ ID NO: 5.

Exemplary amino acid sequence of UL2 of SC9-2 strain is shown in SEQ ID NO: 6. Exemplary nucleotide sequence of UL2 of SC9-2 strain is shown in SEQ ID NO: 7. Exemplary amino acid sequence of UL3 of SC9-2 strain is shown in SEQ ID NO: 8. Exemplary sequence of UL3 of SC9-2 strain is shown in SEQ ID NO: 9. Exemplary nucleotide sequence between UL2 and UL3 is shown in SEQ ID NO: 10.

Exemplary amino acid sequence of US2 of SC9-2 strain is shown in SEQ ID NO: 11. Exemplary nucleotide sequence of US2 of SC9-2 strain is shown in SEQ ID NO: 12.

The expression cassette may be inserted into the intergenic region between UL55 and Lorf10, or the intergenic region between UL2 and UL3, or within the US2 gene. In some embodiments, the insertion will not impact the replication of the recombinant virus.

In some embodiments, the expression cassette is inserted into the intergenic region between UL55 and Lorf10, into the intergenic region between UL2 and UL3, or into the US2 gene.

In some embodiments, the expression cassette is inserted into the intergenic region between UL55 and Lorf10.

In some embodiments, the expression cassette is inserted into the intergenic region between UL2 and UL3.

In some embodiments, the expression cassette is inserted into the US2 gene.

In some embodiments, the expression cassette is inserted between the sequence encoding the amino acid sequence as shown in SEQ ID NO: 1 (UL55) or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto and the sequence encoding the amino acid sequence as shown in SEQ ID NO: 3 (Lorf10) or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.

In some embodiments, the expression cassette is inserted between the sequence encoding the amino acid sequence as shown in SEQ ID NO: 6 (UL2) or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto and the sequence encoding the amino acid sequence as shown in SEQ ID NO: 8 (UL3) or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.

In some embodiments, the expression cassette is inserted within the sequence as shown in SEQ ID NO: 5 (between UL55 and Lorf10) or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.

In some embodiments, the expression cassette is inserted within the sequence as shown in SEQ ID NO: 10 (between UL2 and UL3) or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.

In some embodiments, the expression cassette is inserted into the intergenic region between UL55 and Lorf10, and

(i) at least one downstream UL55 flanking region selected from the group consisting of: SEQ ID NO: 24, 25 and 26, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto, and

(ii) at least one upstream Lorf10 flanking region selected from the group consisting of: SEQ ID NO: 27, 28 and 29, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.

In some embodiments, the expression cassette is inserted into the intergenic region between UL2 and UL3, and

(i) at least one downstream UL2 flanking region selected from the group consisting of: SEQ ID NO: 30, 31 and 32, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto, and

(ii) at least one upstream UL3 flanking region selected from the group consisting of: SEQ ID NO: 33, 34 and 35, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.

The term “intergenic region” is well known by the person skilled in the art. The term encompasses a region between two genes. By using an intergenic region for the insertion of a heterologous polynucleotide, no essential gene (gene essential for example for viability, infectivity or replication) of the virus is inactivated. Accordingly, an intergenic region can be used for the insertion of a heterologous polynucleotide such as an antigen encoding sequence.

In some embodiments, the expression cassette is inserted within the sequence encoding the amino acid sequence as shown in SEQ ID NO: 11 (US2) or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.

In some embodiments, the expression cassette is inserted within the sequence as shown in SEQ ID NO: 12 (US2) or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.

By insertion of a heterologous nucleotide sequence, e.g. an expression cassette, one or more nucleotides at or near the insertion site may be deleted. For example, the insertion of a heterologous nucleotide sequence into an intergenic region may result in a partial sequence of the intergenic region being replaced by the inserted heterologous nucleotide sequence.

In some embodiments, insertion of the expression cassette into the US2 gene results in a deletion of a part of the US2 gene. In other words, the expression cassette is inserted to replace a part of the US2 gene.

In some embodiments, insertion of the expression cassette into the US2 gene results in a deletion of a sequence of about 1bp to about 600bp or more or even the full length of the US2 gene.

In some embodiments, insertion of the expression cassette into the US2 gene results in a deletion of 616bp of the US2 gene. In some embodiments, the sequence from position 15 to position 630 of the US2 gene is deleted or replaced by the expression cassette, numbering of nucleotide refers to SEQ ID NO: 12. In some embodiments, the expression cassette is inserted between position 14 to position 631 of the US2 gene, numbering of nucleotide refers to SEQ ID NO: 12.

In a particular embodiment, the rMDV of the present invention is a live virus vector. A “live virus vector” is virus (in the present case a MDV) that is competent to replicate in a host when such host is infected with the live virus or the genomic nucleic acid of such virus and wherein such virus encodes, delivers and express a heterologous nucleotide sequence in such host.

In one aspect, the present invention provides the rMDV of the present invention for use as vector vaccine in a host animal, such as chicken. The term “vector vaccine” is a vaccine that uses virus (in the present case a MDV) as vector to deliver and express a nucleotide sequence coding for an antigenic polypeptide, wherein such antigenic polypeptide provides protection against a pathogen. The virus that is used as vector shows no or only limited pathogenicity to the target species in which the virus is used as a vector.

Thus, in one aspect, the present invention also provides the rMDV of the present invention as a live vector vaccine in a host animal, such as chicken.

Virus construction and cloning may be accomplished by techniques known per se in the art. Gene cloning and plasmid construction are well known to one person of ordinary skill in the art and may be essentially performed by standard molecular biology techniques (Molecular Cloning: A Laboratory Manual. 4th Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA, 2012) . Typically, the recombinant viruses may be prepared by homologous recombination between the viral genome and a construct (e.g., a homology plasmid) comprising the nucleic acid to be inserted, flanked by nucleotides from the insertion site to allow recombination. Cloning can be made with or without the deletion of endogenous sequences. the recombinant viruses may be prepared by BAC technology.

Host cell

The invention also relates to a host cell, expressing the rMDV as defined above. The invention also relates to a host cell, expressing the rMDV and the heterologous polynucleotide as defined above. In some embodiments, the host cell is CEF cell (Liang Z., et. al, Animal (Basel) , 2022, 12 (24) : 3523) , DEF cell (Chenghuai Yang, Arch virol 2015, 160: 267-274) , embryonated egg, or chicken kidney cell (Andres Rodr₁′guez-Avila et. al, Avian diseases 2007, 51: 905-911) .

The rMDV of the present invention may be propagated in some competent cell cultures. After the required growth of the viruses is achieved, the cells may be detached from the wells using a scraper or with trypsin and the infected cells may be separated from the supernatant by centrifugation.

Examples of competent cell include CEF, DEF, embryonated egg, chicken kidney cells, and the like. The cells or viruses may be cultured in a culture medium such as MEM containing 5%FBS at about 37℃ for 1h to 6 days.

Composition

The invention also relates to a composition, e.g., an immunogenic composition, which comprises the rMDV of the present invention.

The term “composition” as used herein refers to a composition that comprises at least one antigen, which elicits an immune response in the host to which the composition is administered. Such immune response may be a cellular and/or antibody-mediated (humoral) immune response to the composition of the invention. The host is also described as a “subject” , “host animal” or “animal” . The host may be an avian, more preferably, a poultry such as a chicken.

An "immune response" to a composition is the development in the host of a cellular and/or antibody-mediated (humoral) immune response to a composition of interest. Usually, an "immune response" includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition of interest. Preferably, the host will display either a therapeutic or protective immune response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced.

A "protective immune response" or "protective response" will be demonstrated by either a reduction or lack of clinical signs normally displayed by an infected host, a quicker recovery time and/or a lowered duration of infectivity or lowered pathogen titer in the tissues or body fluids or excretions of the infected host.

In case where the host displays a protective immune response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced, the composition of the invention is described as a “vaccine” . In one aspect, the composition of the present invention is a vaccine.

In some embodiments, the composition of the present invention is a vector vaccine. In some embodiments, the composition of the present invention is a vector vaccine in chicken.

Compositions and vaccines of the invention may further comprise a pharmaceutically or veterinarily acceptable carrier, excipient, vehicle, or adjuvant.

The pharmaceutically or veterinarily acceptable carriers or adjuvant or vehicles or excipients are well known to the one skilled in the art. For example, a pharmaceutically or veterinarily acceptable carrier or adjuvant or vehicle or excipient includes, but is not limited to, 0.9%NaCl (e.g., saline) solution or a phosphate buffer, poly- (L-glutamate) , the Lactated Ringer's Injection diluent (sodium chloride, sodium lactate, potassium chloride, and calcium chloride) , or polyvinylpyrrolidone. The pharmaceutically or veterinarily acceptable carrier or vehicle or adjuvant or excipients may be any compound or combination of compounds facilitating the administration of the vector (or protein expressed from an inventive vector in vitro) , or facilitating transfection or infection and/or improving the preservation of the vector (or protein) .

In some embodiments, the composition of the invention comprises a lyoprotectant. In a particular embodiment, the composition of the invention comprises a preservative.

The composition of the invention may be liquid (solutions, suspensions, emulsions) or solid (powder, gel, paste, oil) . The composition of the invention may be formulated for any administration route. Preferably, the composition may be formulated for oro-nasal, eye drop, spray, drinking water, in ovo, intramuscular, subcutaneous, intradermal, or transdermal administration.

The composition of the invention may contain a suitable dose sufficient to elicit a protective response in a chicken. Doses and dose volumes are herein discussed in the general description and can also be determined by the skilled artisan from this disclosure in conjunction with the knowledge in the art, without any undue experimentation. The viral vector may be titrated based on any virus titration methods including, but not limited to, FFA (Focus Forming Assay) or FFU (Focus Forming Unit) , TCID₅₀ (50%Tissue Culture Infective Dose) , PFU (Plaque Forming Units) , and FAID₅₀ (50%Fluorescent Antibody Infectious Dose) , and the VLPs produced in vitro can be titrated by hemagglutination assay, ELISA, and electron microscopy. In some embodiments, the rMDV in the composition is present in a dose from 1×10² TCID₅₀/ml or TCID₅₀/g to 1x10⁷ TCID₅₀/ml or TCID₅₀/g. In some embodiments, the rMDV in the composition is present in a dose from 1x10⁴ TCID50/ml or TCID₅₀/g to 1x10⁶ TCID₅₀/ml or TCID₅₀/g. In some embodiments, the dose volumes can be between about 0.01 and about 10 ml, between about 0.01 and about 5 ml.

The composition of the invention can be administered in a single dose or in repeated doses, depending on the vaccination protocol. The medicament or vector vaccine of the invention can be formulated as single doses or in repeated doses, depending on the vaccination protocol.

Use and Method

In one aspect, the present invention provides the rMDV of the invention, or the composition of the invention, or the vector vaccine of the invention, for the use in a method for inducing a protective immune response in a host animal against a pathogen, wherein such method comprises or consists of one or more administration of the rMDV of the invention, or the composition of the invention, or the vector vaccine of the invention to the host animal.

In one aspect, the present invention provides the rMDV of the invention, the composition of the invention, or the vector vaccine of the invention, for use in vaccinating a host animal by inducing a protective immune response in a host animal against a pathogen.

In one aspect, the present invention provides a method of vaccinating a host animal by inducing a protective immune response in a host animal against a pathogen, comprising or consisting of at least one administration of the rMDV of the invention, the composition of the invention, or the vector vaccine of the invention.

In one aspect, the present invention provides use of the composition of the present invention in the manufacture of a medicament for vaccinating a host animal by inducing a protective immune response in a host animal against a pathogen.

The term "vaccinating" relates to an active immunization by the administration of an immunogenic composition to a chicken to be immunized, thereby causing a protective immune response against the antigen included in such immunogenic composition.

In some embodiments, the host animal is 0 day-old, 1 day-old, 2 day-old, 3 day-old, 4 day-old, 5 day-old, 6 day-old, or 7 day-old at the day of vaccination.

In some embodiments, the rMDV, the composition or the vector vaccine is administrated at Day 0 post-hatch, Day 1 post-hatch, Day 2 post-hatch, Day 3 post-hatch, Day 4 post-hatch, Day 5 post-hatch, Day 6 post-hatch, or Day 7 post-hatch.

As indicated in the experimental section, the rMDVs of the invention are particularly advantageous for vaccinating young host animals (at Day 0, Day 1, Day 2, or Day 3 post-hatch) . Such early administration, combined with the early onset of immunity caused by the rMDV, is particularly advantageous to induce early protective immunity, before the host animal can be substantially exposed to pathogens.

In some embodiments, the rMDV is administered in ovo. In case in ovo vaccination is used, preferably the administration is performed when embryos are between 15 to 20 days old, preferably at day 17, 18 or 19, most preferably at day 18 of age.

In some embodiments, the pathogen is an avian pathogen. In some embodiments, the pathogen is MDV, and/or Infectious laryngotracheitis virus (ILTV) .

In some embodiments, the pathogen is MDV.

In some embodiments, the pathogen is ILTV.

The administration or the rMDV, the composition or the vector vaccine of the invention results in lessening of the incidence of the particular pathogen infection in a host animal or in the reduction in the severity of clinical signs caused by or associated with the specific pathogen infection. It is to be understood that the administration, or the rMDV, the composition or the vector vaccine of the invention may not be effective in all host animals administrated, but there is a significant portion (for example, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%) of host animals effectively immunized.

In some embodiments, the rMDV, the composition or the vector vaccine is administered by oro-nasal, eye drop, spray, drinking water, in ovo, intramuscular, subcutaneous, intradermal, or transdermal. In some embodiments, the medicament, the rMDV, the composition or the vector vaccine may be formulated for oro-nasal, eye drop, spray, drinking water, in ovo, intramuscular, subcutaneous, intradermal, or transdermal administration. However, depending on the nature and mode of action of a compound, the immunogenic composition may be administered by other routes as well.

In one aspect of the invention, the rMDV, the composition or the vector vaccine is administered once and is efficacious by such single administration.

However, while a single dose administration is preferred, the rMDV, the composition or the vector vaccine can also be administered twice or several times, with a first dose being administered prior to the administration of a second (booster) dose. Preferably, the second dose is administered at least 15 days after the first dose. More preferably, the second dose is administered between 15 and 40 days after the first dose. Even more preferably, the second dose is administered at least 17 days after the first dose. Still more preferably, the second dose is administered between 17 and 30 days after the first dose. Even more preferably, the second dose is administered at least 19 days after the first dose. Still more preferably, the second dose is administered between 19 and 25 days after the first dose. Most preferably the second dose is administered at least 21 days after the first dose. In a preferred aspect of the two-time administration regimen, both the first and second doses of the immunogenic composition are administered in the same amount. In addition to the first and second dose regimen, an alternate embodiment comprises further subsequent doses. For example, a third, fourth, or fifth dose could be administered in these aspects. Preferably, subsequent third, fourth, and fifth dose regimens are administered in the same amount as the first dose, with the time frame between the doses being consistent with the timing between the first and second doses mentioned above.

The rMDV, the composition or the vector vaccine of the invention may be administrated in a suitable dose sufficient to elicit a protective response in a chicken. Doses and dose volumes are herein discussed in the general description and can also be determined by the skilled artisan from this disclosure in conjunction with the knowledge in the art, without any undue experimentation. In some embodiments, the rMDV in the composition or the vector vaccine is present in a dose from 1×10² TCID₅₀/ml or TCID₅₀/g to 1x10⁷ TCID₅₀/ml or TCID₅₀/g. In some embodiments, the rMDV in the composition or the vector vaccine is present in a dose from 1x10⁴ TCID₅₀/ml or TCID₅₀/g to 1x10⁶ TCID₅₀/ml or TCID₅₀/g. In some embodiments, the dose volumes can be between about 0.01 and about 10 ml, between about 0.01 and about 5 ml.

The present invention further relates to vaccination kits for vaccinating a host animal by inducing a protective immune response in a host animal against a pathogen, which comprises an effective amount of the rMDV, the composition or the vector vaccine as described above and a means for administering said rMDV, the composition or the vector vaccine to said host animal. For example, such kit comprises an injection device filled with the rMDV, the composition or the vector vaccine according to the invention and instructions for intradermic, subcutaneous, intramuscular, or in ovo injection. Alternatively, the kit comprises a spray/aerosol or eye drop device filled with the rMDV, the composition or the vector vaccine according to the invention and instructions for oro-nasal administration, oral or mucosal administration.

The following clauses are also described herein and part of disclosure of the invention:

1. A recombinant serotype 1 Marek's Disease Virus (rMDV-1) comprising an expression cassette in the genome thereof,

wherein the expression cassette comprises a coding sequence of at least one antigenic polypeptide of Infectious laryngotracheitis virus (ILTV) operably linked to a corresponding native ILTV promoter.

2. The recombinant virus of clause 1, wherein the recombinant virus is derived from a serotype 1 MDV strain, preferably, an attenuated serotype 1 MDV strain.

3. The recombinant virus of clause 2, wherein serotype 1 MDV strain is selected from the group consisting of Dutch CVI988 strain (Rispens) , Chinese 814 strain and SC9-2 strain.

4. The recombinant virus of clause 3, wherein the recombinant virus is derived from the SC9-2 strain.

5. The recombinant virus of clause 4, wherein the recombinant virus is derived from the SC9-2 strain deposited with CCTCC under the accession number: CCTCC No: V2023114 on December 15, 2023.

6. The rMDV-1 of any one of clauses 1-5, wherein the at least one antigenic polypeptide of ILTV is selected from the gD protein of ILTV, the gI protein of ILTV, and/or the gE protein of ILTV or a partial gE protein of ILTV.

7. The rMDV-1 of clause 6, wherein the expression cassette comprises a coding sequence of the gD protein of ILTV operably linked to a native ILTV gD promoter, and/or a coding sequence of the gI protein of ILTV and a partial gE protein of ILTV operably linked to a native ILTV gI promoter.

8. The rMDV-1 of any one of clauses 6-7, wherein

i) the native ILTV gD promoter comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 22; and/or

ii) the native ILTV gI promoter comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 23.

9. The rMDV-1 of any one of clauses 6-8, wherein

i) the gD protein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 13;

ii) the gI protein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 15; and/or

iii) the partial gE protein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 17.

10. The rMDV-1 of any one of clauses 6-9, wherein

i) the coding sequence of the gD protein comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 14;

ii) the coding sequence of the gI protein comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 16; and/or

iii) the coding sequence of the partial gE protein comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 21.

11. The rMDV-1 of any one of clauses 1-10, wherein the expression cassette comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 20.

12. The rMDV-1 of any one of clauses 1-11, wherein the expression cassette is located at a position between UL55 and Lorf10, a position between UL2 and UL3, or a position within US2 gene, preferably, the expression cassette is located at a position within the US2 gene.

13. The rMDV-1 of clause 12, wherein the expression cassette is inserted into the intergenic region between UL55 and Lorf10.

14. The rMDV-1 of clause 12, wherein the expression cassette is inserted into the intergenic region between UL2 and UL3.

15. The rMDV-1 of clause 12, wherein the expression cassette is inserted into the US2 gene.

16. The rMDV-1 of clause 12, wherein the expression cassette is inserted between the sequence encoding the amino acid sequence as shown in SEQ ID NO: 1 (UL55) or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto and the sequence encoding the amino acid sequence as shown in SEQ ID NO: 3 (Lorf10) or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.

17. The rMDV-1 of clause 12, wherein the expression cassette is inserted between the sequence encoding the amino acid sequence as shown in SEQ ID NO: 6 (UL2) or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto and the sequence encoding the amino acid sequence as shown in SEQ ID NO: 8 (UL3) or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.

18. The rMDV-1 of clause 12, wherein the expression cassette is inserted within the sequence as shown in SEQ ID NO: 5 (between UL55 and Lorf10) or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.

19. The rMDV-1 of clause 12, wherein the expression cassette is inserted within the sequence as shown in SEQ ID NO: 10 (between UL2 and UL3) or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.

20. The rMDV-1 of clause 12, wherein the expression cassette is inserted into the intergenic region between UL55 and Lorf10, and

21. The rMDV-1 of clause 12, wherein the expression cassette is inserted into the intergenic region between UL2 and UL3, and

22. The rMDV-1 of clause 12, wherein the expression cassette is inserted within the sequence encoding the amino acid sequence as shown in SEQ ID NO: 11 (US2) or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.

23. The rMDV-1 of clause 12, wherein the expression cassette is inserted within the sequence as shown in SEQ ID NO: 12 (US2) or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.

24. The rMDV-1 of clause 12, wherein the expression cassette is inserted to replace a part of the US2 gene.

25. The rMDV-1 of clause 12, wherein the expression cassette is inserted into the US2 gene resulting in a deletion of a sequence of about 1bp to about 600bp or more or even the full length of the US2 gene;

preferably, wherein insertion of the expression cassette into the US2 gene results in a deletion of 616bp of the US2 gene;

more preferably, wherein the sequence from position 15 to position 630 of the US2 gene is deleted or replaced by the expression cassette, numbering of nucleotide refers to SEQ ID NO: 12.

26. The rMDV-1 of clause 12, wherein the expression cassette is inserted between position 14 to position 631 of the US2 gene, numbering of nucleotide refers to SEQ ID NO: 12.

27. A host cell, expressing the rMDV-1 of any one of clauses 1-26.

28. The host cell of clause 27, which is CEF cell, DEF cell, embryonated egg, or chicken kidney cell.

29. An immunogenic composition, comprising the recombinant virus of any one of clauses 1-26, and optionally a pharmaceutical-or veterinary-acceptable carrier or excipient.

30. The immunogenic composition of clause 29, which is a vaccine, and optionally comprises an adjuvant.

31. The immunogenic composition of clause 29 or 30, which is formulated for oro-nasal, eye drop, spray, drinking water, in ovo, intramuscular, subcutaneous, intradermal, or transdermal administration.

32. The immunogenic composition of any one of clauses 29-31, wherein the rMDV in the composition is present in a dose from 1×10² TCID₅₀/ml or TCID50/g to 1x10⁷ TCID₅₀/ml or TCID₅₀/g.

33. The immunogenic composition of any one of clauses 29-32, wherein the composition is administered in a single dose or in repeated doses.

34. Use of the recombinant virus of any one of clauses 1-26 in preparation of an immunogenic composition for inducing a protective immune response in a host animal against a pathogen, preferably said animal is an avian, more preferably, a poultry such as a chicken.

35. The use of clause 34, wherein the pathogen is MDV, and/or Infectious laryngotracheitis virus (ILTV) .

36. The recombinant virus of any one of clauses 1-26 or the immunogenic composition of any one of clauses 29-33, for use in a method of inducing a protective immune response in a host animal against a pathogen, preferably said animal is an avian, more preferably, a poultry such as a chicken.

37. The recombinant virus or immunogenic composition for use according to clause 36, wherein the pathogen is MDV and/or Infectious laryngotracheitis virus (ILTV) .

38. The recombinant virus or immunogenic composition for use according to clause 36 or 37, wherein the host animal is 0 day-old, 1 day-old, 2 day-old, 3 day-old, 4 day-old, 5 day-old, 6 day-old, or 7 day-old at the day of vaccination.

39. The recombinant virus or immunogenic composition for use according to any one of clauses 36-38, wherein the recombinant virus or immunogenic composition is administrated at Day 0 post-hatch, Day 1 post-hatch, Day 2 post-hatch, Day 3 post-hatch, Day 4 post-hatch, Day 5 post-hatch, Day 6 post-hatch, or Day 7 post-hatch.

40. The recombinant virus or immunogenic composition for use according to any one of clauses 36-38, wherein the recombinant virus or immunogenic composition is administrated in ovo, and preferably the administration is performed when embryos are between 15 to 20 days old, preferably at day 17, 18 or 19, most preferably at day 18 of age.

41. A method of inducing a protective immune response in a host animal against a pathogen, said method comprising the step of administering to the animal the recombinant virus of any one of clauses 1-26 or the immunogenic composition of any one of clauses 29-33, preferably said animal is an avian, more preferably, a poultry such as a chicken.

42. The method of clause 41, wherein the pathogen is selected from MDV and/or infectious laryngotracheitis virus (ILTV) .

43. The method according to clause 41 or 42, wherein the host animal is 0 day-old, 1 day-old, 2 day-old, 3 day-old, 4 day-old, 5 day-old, 6 day-old, or 7 day-old at the day of vaccination.

44. The method according to any one of clauses 41-42, wherein the recombinant virus or immunogenic composition is administrated at Day 0 post-hatch, Day 1 post-hatch, Day 2 post-hatch, Day 3 post-hatch, Day 4 post-hatch, Day 5 post-hatch, Day 6 post-hatch, or Day 7 post-hatch.

45. The method according to any one of clauses 41-42, wherein the recombinant virus or immunogenic composition is administrated in ovo, and preferably the administration is performed when embryos are between 15 to 20 days old, preferably at day 17, 18 or 19, most preferably at day 18 of age.

Examples

The subsequent examples further illustrate the invention in an exemplified manner. It is understood that the invention is not limited to any of those examples as described below. A person skilled in the art understands that the performance, results and findings of these examples can be adapted and applied in a broader sense in view of the general description of the present invention.

Example 1: Construction of recombinant serotype 1 Marek's disease virus expressing infectious laryngotracheitis virus gD, gI and partial gE

The infectious laryngotracheitis virus gD gene, gI gene and partial gE gene sequences were inserted into the US2 gene (replacing the nucleotide sequence from positions 15 to 630 of the US2 gene) , between the UL55-Lorf10 genes or between UL2-UL3 genes of the serotype 1 Marek's disease virus SC9-2 strain. Recombinant serotype 1 Marek's disease viruses expressing infectious laryngotracheitis virus gD, gI and partial gE were finally successfully constructed: rSC9-2/US2-Native-ILTgD-gI-/gE/, rSC9-2/UL55-Native-ILTgD-gI-/gE/-Lorf10, and rSC9-2/UL2-Native-ILTgD -gI-/gE/-UL3.

Materials and reagents used in the construction process:

1. Cells, virus strains, plasmids

Primary chicken embryo fibroblasts (CEF) were prepared from 9～11-day-old specific pathogen free (SPF) chicken embryos (purchased from Beijing Boehringer Ingelheim Vital Biotechnology Co., Ltd. ) according to a routine method.

The serotype I Marek's disease virus SC9-2 parent strain was purchased from Shandong Agricultural University (Chinese Patent Publication No: CN102628053A) . The SC9-2 strain used in this study was obtained by continuously passaging and amplifying the original SC9-2 strain on chicken embryo fibroblasts (CEF) . Then safety and efficacy of this strain were tested, and the results showed that the SC9-2 strain can achieve ≥90%protection against Marek's disease virus very virulent strain Md5. Safety experiments on SPF chickens showed that this strain did not cause clinical signs, death or tumors in 1-day-old SPF chickens, proving that this strain is safe for chickens.

The SC9-2 virus strain used in this study was deposited according to Budapest Treaty on December 15, 2023 at CHINA CENTER FOR TYPE CULTURE COLLECTION (Wuhan University, Wuhan 430072, P.R. China) , under the accession number CCTCC No: V2023114.

pUC57-ILT-gD-gI-/gE/plasmid was synthesized by GenScript Biotechnology Co., Ltd. and preserved at Global Innovation China Center Laboratory of Boehringer Ingelheim Animal Health (China) Co., Ltd.

2. Molecular biology reagents

Plasmid midi kit QIAGEN Plasmid Midi Kit (purchased from QIAGEN) ; Gel Extraction Kit (purchased from QIAGEN) ; Viral DNA extraction kitDNA/RNA Virus Mini Kit (INVITEK, Germany) ; Kpn I (purchased from NEB Company) ; Xho I (purchased from NEB Company) ; PrimeSTAR Max (purchased from TaKaRa Company) .

Specific construction process

1. SC9-2 virus genomic DNA extraction

(1) Take out SC9-2 virus from the liquid nitrogen and thaw the virus at37℃ water bath quickly;

(2) Infect 3х10^7.0 primary CEF cells with 0.01 MOI SC9-2 virus, and incubate the infected cells at 37℃, 5%CO₂ incubator;

(3) Harvest the cells after about 60 hours post infection (cytopathic effect reached to approximately 60%) ;

(4) Extract the total SC9-2 genome using HIRT method, and aliquot the extracted viral DNA (20μl/vial) and stored in -80℃ refrigerator for later use.

2. Preparation of donor plasmid

(1) the pUC57-ILT-gD-gI-/gE/plasmid was amplified in bacteria cells, extracted and purified using a QIAGEN Plasmid Midi Kit;

(2) the extracted plasmid was double digested with Kpn I and Xho I endonucleases at 37℃ for 3 hours;

(3) the enzyme digestion products were subject to 0.8%agarose gel electrophoresis, then, the target fragment is cut and recovered using a Gel Extraction Kit.

3. Construction of recombinant virus by homologous recombination method

(1) primary CEF cells were plated into a T175 cell flask and cultured in a 37℃, 5%CO₂ incubator for 20 hours; the supernatant was discarded, and the cells were washed once with sterile PBS; trypsin was added to digest the adherent CEF cells to prepare secondary CEF cells;

(2) 1х10^7.0 secondary CEF cells, 10 μg SC9-2 viral genomic DNA, and 5 μg gel-recovered double-digested donor plasmid fragment were placed into a 1.5 ml EP tube and mixed, then the mixed solution was transferred into a pre-cooled 2mm electroporation cup for electroporation (150V, 950μF) ;

(3) the transfected cells were resuspended in 20ml of cell growth medium, shaken and mixed, then plated into a 96-well cell culture plate, 200μl/well, and placed in a 37℃, 5%CO₂ incubator for 5 days;

(4) the supernatant in the 96-well cell culture plate was discarded, trypsin (50μL/well) was added into the cell culture plate for digestion in a 37℃ cell culture incubator, and growth medium (150μL/well) was added to resuspend the cells after dispersed into single cells;

(5) the cell suspension in step (4) was added to two new 96-well cell culture plates seeded with secondary CEF cells (100 μL/well) , allowing the corresponding wells of the old plate and the new plates in one-to-one correspondence, respectively; the two new plates were labeled as plate "A" and plate "B" and placed in a 37℃, 5%CO₂ incubator for 3 days;

(6) the supernatant in the 96-well cell culture plate "A" was discarded, 96%cold ethanol (pre-cooled at -20℃) was added to fix the cells in the plate "A" (100 μL/well) at room temperature for 10 minutes; the solution was discarded, and the cell plate was dried naturally at room temperature;

(7) the dual immunofluorescence assay was used to stain the fixed cells; diluted anti-ILTV gD protein monoclonal antibody and anti-ILTV gI protein monoclonal antibody were added to the wells (gD protein monoclonal antibody diluted at 1: 500, gI protein monoclonal antibody diluted at 1: 200) , incubated at 37℃for 1 hour; the primary antibodies were discarded and the wells were washed 3 times with PBS; anti-human IgG secondary antibody and anti-pig IgG secondary antibody (Alexa Fluor 488 goat anti-Human IgG (H+L) , purchased from Invitrogen Company, and Dylight 594 goat anti-Pig IgG (H+L) , purchased from Abcam Company) , were added to each well at the same time, incubated at 37℃ for 1 hour; the two antibodies were discarded and the wells were washed 3 times with PBS, observed under a fluorescence microscope; wells that shown both green and red fluoresce were selected and labeled;

(8) the wells in the 96-well cell culture plate "B" corresponding to the positive wells in the plate "A" labeled in step (7) were selected; the cell supernatant was discarded, the wells were washed once with sterile PBS, and trypsin (50 μL/well) was added for digestion at 37℃ for 5 minutes; growth solution (150μL/well) was added to resuspend the digested cells.

4. Purification of recombinant viruses

The rescued recombinant virus was purified using the limiting dilution method.

(1) the recombinant virus suspension harvested in step 3- (8) was added to a 15ml centrifuge tube 1 containing 5ml of cell growth medium; shaken and mixed; 0.5 ml of virus solution centrifuge tube 1 was transferred to a new 15 ml centrifuge tube 2 containing 4.5 ml of cell growth medium, shaken and mixed; 0.5 ml of virus solution from tube 2 was transferred to a new 15 ml centrifuge tube 3 containing 4.5 ml of cell growth medium;

(2) the virus dilutions in centrifuge tubes 2 and 3 were used to infect a new 96-well cell culture plate inoculated with secondary CEF cells, respectively. Half of a 96-well plate (100μL/well) was inoculated for each dilution and placed in a 37℃, 5%CO₂ incubator for 5 days;

(3) the supernatant in the 96-well cell culture plate was discarded, trypsin (50μL/well) was added, and then the cell culture plate was placed in a 37℃ cell culture incubator for digestion; after the cells were dispersed into single cells, growth solution (150 μL/well) was added for resuspension.

(4) the cell suspension in step (3) was added into two new 96-well cell culture plates seeded with secondary CEF cells (100 μL/well) , allowing the corresponding holes of the old plate and the new plates in one-to-one correspondence; the two new plates were labeled as plate "A" and plate "B" and placed in a 37℃, 5%CO₂ incubator for 3 days.

(5) the supernatant in the 96-well cell culture plate "A" was discarded, 96%cold ethanol (pre-cooled at -20℃) was added to fix the cells in the plate "A" (100 μL/well) at room temperature for 10 minutes; the solution was discarded, and the cell plate was dried naturally at room temperature.

(6) the dual immunofluorescence assay was used to stain the fixed cells; diluted anti-ILTV gD protein monoclonal antibody and anti-ILTV gI protein monoclonal antibody were added to the wells (gD protein monoclonal antibody diluted at 1: 500, gI protein monoclonal antibody diluted at 1: 200) , incubated at 37℃for 1 hour; the primary antibodies were discarded and the wells were washed 3 times with PBS; anti-human IgG secondary antibody and anti-pig IgG secondary antibody (Alexa Fluor 488 goat anti-Human IgG (H+L) , purchased from Invitrogen Company, and Dylight 594 goat anti-Pig IgG (H+L) , purchased from Abcam Company) , were added to each well at the same time, incubated at 37℃ for 1 hour; the two antibodies were discarded and the wells were washed 3 times with PBS, observed under a fluorescence microscope; wells that shown both green and red fluoresce were selected and labeled;

(7) the wells in the 96-well cell culture plate "B" corresponding to the positive wells in the plate "A" labeled in step (6) were selected; the cell supernatant was discarded, the wells were washed once with sterile PBS, and trypsin (50 μL/well) was added for digestion at 37℃ for 5 minutes; growth solution (150μL/well) was added to resuspend the digested cells;

(8) 100 μL of cell suspension from the positive well in cell plate "B" was used to extract the viral genomic DNA according to the instructions of theDNA/RNA Virus Mini Kit, and the primers listed in Table 1 were used amplify recombinant virus insert gene fragments; after amplification, the product was added to a 0.8%agarose gel for electrophoresis at 100V voltage for 60 minutes; the gel was placed in a UV imager to observe the band distribution of the amplification product. If a single target band appears, it indicates that the recombinant virus has been completely purified. If there are two bands of the sizes of the target fragment and the parental virus fragment or a single band of the size of the parental virus fragment, it indicates that the recombinant virus has not been completely purified, and the remaining virus suspension in the positive well needs to be subjected to the next round of limiting dilution purification method according to the above-mentioned relevant operations.

Table 1. PCR Primers

Example 2. Preparation of recombinant virus seed batches and in vitro characterization

1. Preparation of seed batches

The recombinant serotype 1 Marek's disease viruses rSC9-2/US2-Native-ILTgD-gI-/gE/, rSC9-2/UL55-Native-ILTgD-gI-/gE/-Lorf10, and rSC9-2/UL2-Native-ILTgD-gI-/gE/-UL3 containing infectious laryngotracheitis virus gD, gI and partial gE genes constructed in Example 1 were continuously passaged and amplified on chicken embryo fibroblasts (CEF) . These amplified viruses were harvested at the fourth passage and stored in liquid nitrogen tanks for subsequent safety evaluation in animal experiments and efficacy studies in protecting against virulent infectious laryngotracheitis virus challenge.

Specifically, recombinant serotype 1 Marek's disease virus and approximately 3х10^7.0 freshly prepared primary or secondary CEF cells were co-inoculated into a T75 cell culture flask (MEM+5%FBS) , placed in a 37℃, 5%CO₂ incubator for about three days (the cell cytopathic effect reached to 80%) . The cell supernatant was discarded, the cells were washed once with sterile PBS, then 2 ml of trypsin was added to the flask and placed in a 37℃ incubator to digest the cells. After the adherent cells fell off the flask, 8 ml of cell growth medium was added to terminate the digestion reaction and resuspend the cells. The cell suspension was collected into a 50ml centrifuge tube, centrifuged at 1000rpm for 10 minutes. The supernatant was discarded, and cells at the bottom of the tube were resuspended with 3ml of cell cryopreservation solution (80%MEM+10%FBS+10%DMSO) , and then the cells were aliquoted into 1.8 ml cryovials (1 ml/tube) , and the harvest viruses were stored in liquid nitrogen. The same method was applied until the virus was passaged to the fourth passage on CEF cells as a seed batch of virus.

2. In vitro characterization of recombinant viruses

2.1 Identification of the stability of exogenously inserted gD, gI and partial gE genes in recombinant viruses

The recombinant serotype 1 Marek's disease virus containing gD, gI and partial gE genes of infectious laryngotracheitis virus was continuously passaged on CEF cells for 15 generations. The 10th and 15th passages were used to extract viral DNA of different generations using DNA/RNA Virus Mini Kit (purchased from INVITEK, Germany) , and specific primers on both sides of the insertion site (shown in Table 2) were used to amplify the inserted fragment, homology arms, and partial gene sequences flanking the homology arms by PCR amplification. The products were subjected to agarose gel electrophoresis analysis, and the target bands were observed (shown in Figure 1) . At the same time, the PCR amplification products were sequenced, and the results showed that the inserted gD, gI and partial gE genes can exist in the recombinant virus genome stably.

Table 2. Specific primers used for detection of gene stability

2.2 Sterility and Mycoplasma Testing

To conduct relevant testing on the harvested virus liquid, thioglycolate fluid medium culture method and casein agar medium culture method were used for sterility testing, and qPCR method was used for mycoplasma testing. The results showed that these recombinant viruses were free of any exogenous microbial contamination.

2.3 Determination of recombinant virus titer

The plaque counting method was used to calculate the titer of the recombinant viruses:

(1) Take out one tube of recombinant virus from the liquid nitrogen and thaw the virus in a 37℃ water bath quickly.

(2) Add 0.5 ml of the thawed virus suspension into 15 ml centrifuge tube 1 containing 4.5 ml of cell growth medium, shaken to mix; then absorb 0.5 ml of the virus diluent from tube 1 and transfer into a 15ml centrifuge tube 2 containing 4.5ml of cell growth medium; and so on, until the virus was diluted to 10⁵ times.

(3) Inoculate 10³-10⁵ virus dilution into 60mm cell culture plates containing secondary CEF cells, 3 plates for each dilution, 1ml of virus dilution into each plate, and placed in a 37℃, 5%CO₂ incubator for 5-6 days.

(4) Count the plaques under a microscope to calculate virus titers (titers of different recombinant viruses in this study are listed in Table 3) .

Table 2. Titers of different recombinant viruses

2.4 Expression of inserted ILTV-gD and ILTV-gI genes of the recombinant viruses

The serotype 1 Marek's disease virus containing infectious laryngotracheitis virus gD and gI genes was infected at a dose of 0.001 MOI into a 60mm cell culture plate containing secondary CEF cells. After inoculation, the culture plate was placed at 37℃ and 5%CO₂ for culture for about 3 days, and plaque formation was observed under a white light microscope. After obvious plaques were observed, the cell supernatant in the plate were discarded, 96%cold ethanol was added to the plate to fix the cells, placed at room temperature for 10 minutes; the ethanol was discarded. The plate was dried naturally at room temperature.

Dual immunofluorescence assay was used to detect the protein expression of infectious laryngotracheitis virus gD and gI genes in the recombinant virus:

diluted anti-ILTV gD protein monoclonal antibody and anti-ILTV gI protein monoclonal antibody were added to the wells (gD protein monoclonal antibody diluted at 1: 500, gI protein monoclonal antibody diluted at 1: 200) , incubated at 37℃ for 1 hour; the primary antibodies were discarded and the wells were washed 3 times with PBS; anti-human IgG secondary antibody and anti-pig IgG secondary antibody (Alexa Fluor 488 goat anti-Human IgG (H+L) , purchased from Invitrogen Company, and Dylight 594 goat anti-Pig IgG (H+L) , purchased from Abcam Company) , were added to each well at the same time, incubated at 37℃ for 1 hour; the two antibodies were discarded and the wells were washed 3 times with PBS, observed under a fluorescence microscope.

The results showed that the specific green and red fluorescence of gD and gI proteins could be observed for all recombinant viruses (shown in Figure 2) . It shows that all recombinant viruses can normally express infectious laryngotracheitis virus gD and gI proteins in CEF cells.

Example 3: Safety and efficacy evaluation of recombinant serotype 1 Marek's disease viruses expressing infectious laryngotracheitis virus gD, gI and partial gE

3.1 Experimental design

In this example, three recombinant serotype 1 Marek's disease virus live vector vaccine candidate strains expressing infectious laryngotracheitis virus gD, gI and partial gE were inoculated into 1-day-old SPF chickens through the subcutaneous route. After vaccination, a virulent challenge strain of infectious laryngotracheitis virus (ILT/13 strain, prepared by Boehringer Ingelheim Animal Health (China) Co., Ltd. ) was used for challenge at 28 days post immunization. The purpose is to evaluate the safety of vaccine strains after immunization and the efficacy of vaccine candidate strains against virulent virus challenge.

The recombinant viruses studied in this example include rSC9-2/US2-Native-ILTgD-gI-/gE/, rSC9-2/UL55-Native-ILTgD-gI-/gE/-Lorf10, rSC9-2/UL2-Native-ILTgD-gI-/gE/-UL3, prepared as mentioned above.

On the day the test started (i.e., the day the SPF test chickens hatched) , 48 1-day-old SPF chickens were randomly divided into 4 groups (SPF chicken embryos were purchased from Beijing Boehringer Ingelheim Vital Biotechnology Co., Ltd., and hatched in Animal Experiment Center, Boehringer Ingelheim Animal Health (China) Co., Ltd. ) , 12 animals per group. Groups 1 to 3 were vaccination groups, and group 4 was the challenge control group. As shown in Table 3, all chickens were immunized with the corresponding materials through subcutaneous route. After vaccination, all test chickens underwent clinical observation for 28 days. On 28 days post immunization, all chickens were challenged with 0.2 ml of the virulent ILT/13 strain through intratracheal route, and the challenge dose was 10^3.0 TCID₅₀/bird. The experimental design and grouping are shown in Table 3.

Table 3 Experimental design and grouping

After inoculation, general clinical observation was conducted on all test chickens once a day for 28 days consecutively.

On the 28 days post immunization (the day of challenge) , blood samples from each group were collected before challenge, and the serum was separated and used for ILTV antibody detection. After blood collection, all the chickens in groups 1 to 4 were challenged with 0.2 ml ILTV ILT/13 strain via the intratracheal route, the target dose of virus challenge was 10^3.0TCID₅₀/bird. After challenge, all test chickens were clinically observed twice daily (once in the morning and once in the afternoon) for 10 consecutive days. On the 10th day after challenge, all surviving test chickens in the test group should be euthanized on the 10th day after challenge (after clinical observation in the afternoon) . The gross pathology of the trachea was observed in the experimental chickens that died after the challenge and in the experimental chickens that were euthanized at the end of the experiment.

3.2 Safety of the recombinant viruses on experimental chickens

After the recombinant viruses were inoculated into SPF chickens, no clinical symptoms or deaths related to Marek virus or infectious laryngotracheitis virus infection were observed for 28 days, indicating that the recombinant viruses are safe for SPF chickens.

3.3 Protection rate of recombinant viruses against ILT/13 challenge

The morbidity of chickens in the different groups was calculated after challenge. The results showed that the morbidity of test chickens in the challenge control group was 92%, and the challenge control was effective. The morbidity of the other three groups and the protection rates of the corresponding vaccine candidate strains are shown in Table 4.

Table 4. Mortality, morbidity and vaccine protection rates of different groups of test chickens

*One chicken in group 3 died non-specifically after immunization.

The recombinant serotype 1 Marek's disease virus live vector vaccine candidate strains studied in this example expressing infectious laryngotracheitis virus gD, gI and partial gE can provide protection against virulent ILT/13 challenge.

Example 4: Efficacy evaluation of recombinant serotype 1 Marek's disease viruses expressing infectious laryngotracheitis virus gD, gI and partial gE against challenge with the Marek's disease virus hypervirulent strain rMd5 in Chickens

4.1 Experimental design

In this example, the candidate strain rSC9-2/US2-Native-ILTgD-gI-/gE/was inoculated into 1-day-old SPF chickens through the subcutaneous route. After vaccination, a hypervirulent challenge strain of Marek's disease virus strain rMd5 (purchased from Shandong Agricultural University) was used for challenge at 10 days post immunization. The purpose is to evaluate the efficacy of this vaccine candidate strain against challenge with the Marek's disease virus hypervirulent strain.

The recombinant virus rSC9-2/US2-Native-ILTgD-gI-/gE/studied in this example was prepared as mentioned above.

On the day the test started (i.e., the day the SPF test chickens hatched) , 60 1-day-old SPF chickens were randomly divided into 3 groups (SPF chicken embryos were purchased from Beijing Boehringer Ingelheim Vital Biotechnology Co., Ltd., and hatched in Animal Experiment Center, Boehringer Ingelheim Animal Health (China) Co., Ltd. ) , 20 animals per group. Group 1 was a positive control group, inoculated with SC9-2 P60 (SC9-2 passaged to the 60th generation) ; group 2 was a vaccination group, inoculated with rSC9-2/US2-Native-ILTgD-gI-/gE/, and group 3 was a challenge control group. As shown in Table 5, all chickens were immunized with the corresponding materials through subcutaneous route. After vaccination, all test chickens underwent clinical observation for 10 days. On 10 days post immunization, all chickens were challenged with 0.5 ml of Marek's disease virus hypervirulent strain rMd5 through intratracheal route, and the challenge dose was 500 PFU/bird. The experimental design and grouping are shown in Table 5.

Table 5 Experimental design and grouping

After inoculation, general clinical observation was conducted on all test chickens once a day for 10 days consecutively.

On the 10 days post immunization, all the chickens in groups 1 to 3 were challenged with 0.5 ml hypervirulent strain rMd5 via the intratracheal route, the target dose of virus challenge was 500 PFU/bird. After challenge, all test chickens were clinically observed once daily for 60 consecutive days. On the 60th day after challenge, all surviving test chickens in the test groups should be euthanized on the 60th day after challenge (after clinical observation in the afternoon) . All chickens that died post-challenge or were euthanized at the end of the experiment underwent necropsy to observe gross pathological changes associated with Marek’s disease.

4.3 Protection efficacy of the recombinant SC9-2 against rMd5 challenge

The morbidities in chickens post-challenge were statistically analyzed across different groups, and the corresponding morbidities were calculated. The results showed that the challenge control group had a 100%morbidity rate, confirming the validity of the challenge model. The morbidities and protection efficacy of the rest two vaccinated groups are shown in Table 6.

Table 6. Mortality, morbidity and vaccine protection rates of different groups of test chickens

The vaccine candidate strain rSC9-2/US2-Native-ILTgD-gI-/gE/studied in this example demonstrated excellent protection against challenge with the MDV-1 hypervirulent strain rMd5, achieving a 100%protection rate. This candidate strain effectively resisted the hypervirulent MDV challenge.

Furthermore, the result confirmed that the serotype 1 MDV (SC9-2) , when used as a vector, retained its intrinsic protective efficacy against MDV challenge even after the insertion of foreign genes. This dual-capacity design enables simultaneous full protection against both Marek’s disease virus and pathogens corresponding to the inserted foreign antigens (e.g., infectious laryngotracheitis virus, ILTV) .

Sequences involved in the present application:

Claims

A recombinant serotype 1 Marek's Disease Virus (rMDV-1) comprising an expression cassette in the genome thereof,

wherein the expression cassette comprises a coding sequence of at least one antigenic polypeptide of Infectious laryngotracheitis virus (ILTV) operably linked to a corresponding native ILTV promoter.
The recombinant virus of claim 1, wherein the recombinant virus is derived from a serotype 1 MDV strain, preferably, an attenuated serotype 1 MDV strain.
The recombinant virus of claim 2, wherein serotype 1 MDV strain is selected from the group consisting of Dutch CVI988 strain (Rispens) , Chinese 814 strain and SC9-2 strain.
The recombinant virus of claim 3, wherein the recombinant virus is derived from the SC9-2 strain.
The recombinant virus of claim 4, wherein the recombinant virus is derived from the SC9-2 strain deposited with CCTCC under the accession number: CCTCC No: V2023114 on December 15, 2023.
The rMDV-1 of any one of claims 1-5, wherein the at least one antigenic polypeptide of ILTV is selected from the gD protein of ILTV, the gI protein of ILTV, and/or the gE protein of ILTV or a partial gE protein of ILTV.
The rMDV-1 of any one of claims 1-6, wherein the expression cassette comprises a coding sequence of the gD protein of ILTV operably linked to a native ILTV gD promoter, and/or a coding sequence of the gI protein of ILTV and a partial gE protein of ILTV operably linked to a native ILTV gI promoter.
The rMDV-1 of any one of claims 1-7, wherein

i) the native ILTV gD promoter comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 22; and/or

ii) the native ILTV gI promoter comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 23.
The rMDV-1 of any one of claims 6-8, wherein

i) the gD protein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 13;

ii) the gI protein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 15; and/or

iii) the partial gE protein comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 17.
The rMDV-1 of any one of claims 6-9, wherein

i) the coding sequence of the gD protein comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 14;

ii) the coding sequence of the gI protein comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 16; and/or

iii) the coding sequence of the partial gE protein comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 21.
The rMDV-1 of any one of claim 1-10, wherein the expression cassette comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with SEQ ID NO: 20.
The rMDV-1 of any one of claims 1-11, wherein the expression cassette is located at a position between UL55 and Lorf10, a position between UL2 and UL3, or a position within US2 gene, preferably, the expression cassette is located at a position within the US2 gene.
An immunogenic composition, comprising the recombinant virus of any one of claims 1-12, and optionally a pharmaceutical-or veterinary-acceptable carrier or excipient.
The immunogenic composition of claim 13, which is a vaccine, and optionally comprises an adjuvant.
Use of the recombinant virus of any one of claims 1-12 in preparation of an immunogenic composition for inducing a protective immune response in a host animal against a pathogen, preferably said animal is an avian, more preferably, a poultry such as a chicken.
The use of claim 15, wherein the pathogen is MDV and/or Infectious laryngotracheitis virus (ILTV) .
The recombinant virus of any one of claims 1-12 or the immunogenic composition of claim 13 or 14, for use in a method of inducing a protective immune response in a host animal against a pathogen, preferably said animal is an avian, more preferably, a poultry such as a chicken.
The recombinant virus or immunogenic composition for use according to claim 17, wherein the pathogen is MDV and/or Infectious laryngotracheitis virus (ILTV) .
A method of inducing a protective immune response in a host animal against a pathogen, said method comprising the step of administering to the animal the recombinant virus of any one of claims 1-12 or the immunogenic composition of claim 13 or 14, preferably said animal is an avian, more preferably, a poultry such as a chicken.
The method of claim 19, wherein the pathogen is MDV and/or Infectious laryngotracheitis virus (ILTV) .