Infectious laryngotracheitis virus (ILTV), also known as Gallid herpesvirus 1
, causes economically devastating respiratory disease in chickens around the globe. ILTV is a prototypic member of the genus Iltovirus
of the family Herpesviridae
and primarily replicates in the epithelial cells of the upper respiratory tract, including conjunctiva, trachea, and larynx [1
], leading to the characteristics gasping, coughing, expectoration of bloody mucus, and conjunctivitis. Generally, ILTV remains latent in sensory neurons without any apparent clinical signs; however, due to various stresses, occasional reactivation can lead to virus shedding [2
Similar to mammalian herpesviruses, the genome of ILTV carries a number of unique long (UL) and unique short (US) sequences. However, the genomic organisation and genetic distances are distinctive for ILTV [3
]. Compared to other herpesviruses, iltovirus-specific genes include relocation of UL47
gene from UL to US regions, the inversion of a conserved gene cluster within the UL region (i.e., UL22
]. The ILTV carries 12 glycoproteins, and several of these proteins (e.g., gB, gC, gG, gJ, gM,
) have been functionally characterised. On the other hand, several glycoproteins (gG, gJ, gM,
) are not crucial for virus replication, and deletion of gG
leads to the attenuation of ILTV in chickens [8
]. The UL23
(encoding for thymidine kinase, TK
) is one of the well-characterised virulence factors in herpesviruses, and deletion of TK
gene attenuates the pathogenicity of ILTV, while maintaining the immunogenicity against challenged virus [10
]. The US4
) is a non-structural protein (not incorporated into mature virus particles) and is secreted from infected cells only [11
]. Based on studies on multiple herpesviruses, it has been proposed that US4
modulates the host immune response by binding to chemokines [13
], and gG
-negative bovine herpesvirus-1 (BHV-1) mutants were attenuated in cattle [14
]. Taken together, knocking out TK
from ILTV genome could propose a stable vaccine vector.
Owing to low mortality and moderate impacts on weight gain and egg production, the live attenuated strains of ILTV are widely exploited as a vector to develop recombinant and multivalent vaccines [15
]. Moreover, due to the replication in the upper respiratory tract, the vaccine can successfully be deployed for mass application through eye-drops, aerosol, or drinking water [18
]. The ILTV vector proposes additional advantages through potent induction of both cellular and humoral immune responses, and enabling differentiation between infected and vaccinated animals (DIVA) [18
]. In both of these applications, conventionally attenuated live-virus vaccines are applied, which can pose significant residual virulence or can potentially revert into virulent phenotypes after several passages in animals [19
]. To propose a safer vectored vaccine, efforts have been made to delete virulence genes from the ILTV genome, either by conventional homologous recombination in virus-infected cells or through recombineering techniques on full-length genomes [18
]. However, the generation of recombinant ILTV using these methods is time-consuming, requiring the construction of transfer vectors and several rounds of plaque purifications to obtain the recombinant vaccine candidate. These shortcomings warrant application of next generation genome-editing approaches for the generation of safer, stable, and efficient vaccine vectors to be deployed in avian disease (e.g., Newcastle disease virus (NDV), avian influenza, and infectious bronchitis virus) control programmes.
The recent discovery of CRISPR (clustered regularly interspaced short palindromic repeat)–Cas9 system has revolutionised genome editing and is opening new avenues of genetic manipulation. The functional components of type II CRISPR–Cas system include the RNA-guided Cas9 endonuclease, originally characterised in multiple bacterial species such as Streptococcus pyogenes
, a single guide RNA (sgRNA), and the trans-activating crRNA (tracrRNA) [22
]. The sgRNA direct the Cas9 protein to the 20 nucleotide target sequence adjacent to a 5′-NGG-’3 protospacer adjacent motif (PAM). Upon binding, the Cas9 endonuclease introduces a double strand break (DSB) in this target sequence. The DSBs can then be repaired by either an error-prone non-homologous end-joining (NHEJ) or the high-fidelity homology-directed repair (HDR) pathway. This approach has been applied for genome editing of mammalian cells [23
], for genetic modification in animal models [24
], and genomic manipulation of several DNA viruses such as herpes simplex virus type I, adenovirus, pseudorabies virus, vaccinia virus, Epstein–Barr virus, guinea pig cytomegalovirus, herpesvirus of turkey, and duck enteritis virus [25
]. Accompanied by CRISPR/Cas9, the Cre–Lox recombination system can be applied to excise a pre-defined LoxP sites (34 base-pair DNA sequence) to cleave the DNA surrounded by LoxP sites [33
In this study, we developed and applied a highly efficient, versatile, and rapid NHEJ-CRISPR/Cas9 and Cre–Lox-mediated genome-editing approach for simultaneous deletion of pre-determined virulence factors and insertion of viral antigen to generate recombinant, multivalent, and safer vaccine vectors. We demonstrated the use of this approach first by the generation of a reporter virus and deletion of TK gene from ILTV genome. Thereafter, we engineered an ILTV-vectored vaccine candidate harbouring fusion gene (F) of the velogenic NDV and deletion of another gene, US4, from the ILTV genome. The reporter marker gene was excised using Cre–Lox system without affecting the expression and stability of the F protein. This potential vaccine candidate was assessed for the stable expression of inserted protein, replication kinetics, and for comparative in vitro characteristics. We demonstrate that NHEJ-CRISPR/Cas9 accompanied by Cre–Lox is an efficient method for rapid generation of ILTV-based recombinant vaccines and proposes multiple advantages to traditional recombination and recombineering techniques
The ILTV vectors carry remarkable capacity to express heterologous antigens, and are consistently being applied in the poultry industry for immunisation against multiple viruses, including NDV, and influenza H5 and H7 strains [15
]. With these proven applications of ILTV, the generation of recombinant viruses to expression foreign genes has been achieved using conventional recombination strategies, which are tedious, time-consuming, and error prone [18
]. We have demonstrated here the feasibility of the CRISPR/Cas9 nucleases accompanied by the Cre–Lox recombinase in constructing a recombinant ILTV vector expressing either surface antigen of NDV or marker proteins (GFP and dsRED).
While ILTV proposes satisfactory induction of cellular and humoral immunity, and conferring protection against multiple strains of ILTV, latency, and reactivation are crucial for the permanence of ILTV in the poultry field. The molecular mechanisms in establishment and reactivation of ILTV latency are not fully understood. However, it has been proposed that the intimate viral–host interactions and the immune surveillance during latent infections are decisive factors [18
]. Therefore, several previous studies have been conducted to delete virulence-determining gene such as dUTPase, thymidine kinase [10
], secreted glycoprotein gG [35
], tegument protein pUL47 [39
], or iltovirus-specific pUL0 [7
]. These gene-deleted ILTV mutants showed attenuation in vivo
. However, these studies have only focused on the deletion of individual genes and thus limit its safety, and necessitating deletion of multiple non-essential genes to propose a better, safer and immune-competent vaccine vector.
This is the first study to demonstrate the application of CRISPR/Cas9 system in the development of recombinant ILTV vaccines. The presented pipeline proposes a straightforward, rapid, and efficient approach to develop ILTV-based novel recombinant vaccines to protect against major diseases of the poultry. Compared to traditional technologies for modification, the Cas9 endonuclease generates guided and targeted double stranded breaks, which are then repaired by either of the two cellular repair mechanisms; NHEJ or HDR [40
]. In contrast to the HDR pathway, which occurs in the S and G2 phases of cell division, NHEJ is efficient and occurs throughout the cell division cycle [42
]. Using single bait, red fluorescent protein (RFP) has been knocked-in to the pseudorabies virus with high efficacy through the NHEJ pathway [28
]; however, this may lead to undesirable, error-prone, and unpredictable insertions. To mitigate this complication, we introduced dual-baits at the 5′ and 3′ ends of the insert. Since the bait sequence is independent of the sequence of the targeted virus and host, and is devoid of specific sgRNA selection, our benefactor system proposes a universal donor, which would be a valuable source for generation of multivalent vaccines against poultry pathogens.
As a proof of principle, we applied this approach to first generate a reporter ILTV by successfully and efficiently knocking-in a GFP expression cassette. In addition, we diversified the system by instantaneously deleting the virulence factors from the ILTV as a dynamic approach to concurrently attenuate the virus and to generate a marker virus. Using existing information on the roles of TK
in ILTV attenuation [10
], the generated ΔTK-GFP+
ILTV replicated competitively and sustainability. While individual genes have been deleted from ILTV genome, no studies have been performed to delete multiple genes from the same ILTV strain, which may propose a better and safer solution to the immunisation in poultry.
To follow up this line of safety, we generated a universal and repair donor vector system with a feature to efficiently excise dsRED marker flanked with LoxP sites and unique restriction sites to insert and swap heterologous genes of interest. We next applied our validated CRISPR/Cas9 approach to insert the entire cassette of ~5kb into the ILTV genome using gRNA flanking the US4 virulence factor. The pipeline showed an efficient and rapid production of recombinant ILTV vaccine expressing heterologous antigen, fusion gene of NDV, and excisable dsRED. After successful rescue of the virus, in vitro characterisation and stability studies, the dsRED was deleted using Cre-recombinase enzyme. Insertion of the F gene expression cassette was stable and did not compromise the replication of ILTV, as was monitored until at least 15 passages in vitro. However, potential implications of inserted genes on the replication of recombinant ILTV “in vivo” and as “vaccine candidate” warrant further research. Moreover, this double genes-deleted recombinant ILTV will act as marker vaccine permitting differentiation of successfully immunised from infected chickens (DIVA). To facilitate the detection of TK-specific serum antibodies, competitive diagnostic assays can be devised utilising TK expression constructs and TK-specific monoclonal antibodies.
Taken together, we described a versatile and customisable pipeline for the development of NHEJ-CRISPR/Cas9 and Cre–Lox system in the development of innovative ILTV-vectored vaccines. While the approach has displayed the expression of the F protein of the NDV, it is feasible to develop future ILTV-vectored vaccines by insertion of multiple viral antigens at locations different than applied here. We have screened and identified additional sites, which appear safe and can accommodate longer genes without compromising the ILTV replication (Atasoy et al., unpublished data). Additionally, the same platform can be applied to engineer other avian DNA viruses to develop new multivalent-vectored vaccines for protecting multiple poultry diseases.