Virus-like particles in a new vaccination approach against infectious laryngotracheitis.

Gallid alphaherpesvirus 1 (syn. infectious laryngotracheitis virus; ILTV) is the causative agent of infectious laryngotracheitis, a respiratory disease of chickens causing substantial economic losses in the poultry industry every year. Currently, the most efficient way to achieve protection against infection is immunization with live-attenuated vaccines. However, this vaccination strategy entails the risk of generating new pathogenic viruses resulting from spontaneous mutations or from recombination with field strains. This work presents a new approach based on virus-like particles (VLPs) displaying ILTV glycoproteins B (gB) or G (gG) on their surface. The main focus of this pilot study was to determine the tolerability of VLPs delivered in ovo and intramuscularly (i.m.) into chickens and to investigate the nature of the immune response elicited. The study revealed that the new vaccines were well tolerated in hybrid layer chicks independent of the administration method (in ovo or i.m.). Upon in ovo injection, vaccination with VLP-gG led to an antibody response, while a cellular immune response in VLP-gB-immunized chickens was hardly detectable. Since the administration of VLPs had no visible side effects in vivo and was shown to elicit an antibody-based immune response, we anticipate that VLPs will become a valuable platform for the development of new safe vaccines for poultry.


INTRODUCTION
Infectious laryngotracheitis (ILT) is a worldwide respiratory disease of chickens. The causative agent is the Gallid alphaherpesvirus 1 also known as infectious laryngotracheitis virus (ILTV), which mainly infects chickens, but has also been detected in pheasants, peafowls and turkeys [1][2][3]. The clinical signs caused by ILTV range from reduced general conditions with mild nasal discharge, conjunctivitis and tracheitis, to serious clinical conditions with discharge of bloody mucus caused by severe laryngotracheitis, leading to dyspnoea and eventually to death by suffocation [4]. Depending on the virus strain, mortality among infected chickens varies from 10 to 70 %. Improvement of safety measures to prevent ILTV infections in chicken farms is urgently needed in order to address animal welfare issues and to reduce high annual economic loss. The latter is mainly due to milder forms of ILT resulting in egg drop in laying hens and decreased weight gain in broilers [4].
Currently, several vaccines are available. The most efficient ones are based on live attenuated viruses of tissue culture (TCO) or of chicken embryo origin (CEO). Despite providing robust protection in most instances, these vaccines bear the risk of reversion or recombination with other vaccine or field strains of ILTV, potentially resulting in novel and more virulent variants [5][6][7]. Recently, vectored vaccines were developed based on either the fowlpox virus or the herpesvirus of turkey, engineered to express ILTV glycoprotein B (gB) and U L 32 (CEVA, vectormune FP LT) or glycoproteins I (gI) and D (gD) (Intervet/Merck Animal Health, INNOVAX-ILT), respectively [8][9][10][11]. Because these vectored vaccines are based on low-pathogenicity or apathogenic chicken viruses, there is no risk of reversion or recombination to virulent variants. Nonetheless, these engineered vaccines are replication competent, and thus retain the potential of accumulating mutations, possibly leading to undesired effects. Furthermore, vectored vaccines reduce clinical signs, but are not able to suppress replication of challenge virus in the trachea of experimentally infected chickens [12]. Other approaches use affinity purified gB or bicistronic plasmid vectors expressing gB and chicken interleukin   [13,14]. Even though the published results look promising, no further developments have been reported to date.
Another possibility, not yet applied in ILT vaccination strategies, is the engineering of virus-like particles (VLPs). Membrane-enveloped VLPs can be specifically modified to present heterologous antigens in high densities on their surface. In contrast to the aforementioned vaccine strategies, VLPs are not infectious, do not replicate and do not contain any genetic material. VLPs are thus a very safe vaccine platform. In addition, VLPs have the potential to stimulate an immune response on various levels. Due to their origin, they resemble the highly organized and repetitive symmetry of the virus from which they are derived. These features are important for efficient activation of B cells [15] and allow efficient binding of natural IgM antibodies, which in turn activate the classic complement cascade [16]. Moreover, VLPs are efficiently taken up by antigen-presenting cells (APCs). VLP shape, surface charge, hydrophobicity and hydrophilicity, but also their size (20-100 nm), mediate improved uptake into APCs as compared to smaller (<10 nm) affinity-purified proteins used as subunit vaccines [17]. Soluble proteins are inefficiently taken up by APCs and are usually only presented on MHC class II molecules leading to the activation of CD4 + T cells. In contrast, VLPs are degraded into peptides in APCs and can be cross-presented on MHC class I and on MHC class II molecules, thus resulting not only in the stimulation of CD4 + T cells but also CD8 + T cells [18]. In summary, VLPs are a powerful tool to stimulate the innate immune system as well as a humoral and cellular immune response.
In this report, VLPs based on the murine leukaemia virus (MLV) were engineered to present either ILTV gB or ILTV gG on their surfaces. Among herpesviruses, gB is highly conserved [19] and was shown to elicit humoral as well as cell-mediated immunity [20,21]. ILTV gB has already been tested in various vaccination approaches and been shown to confer protective immunity [12][13][14][22][23][24]. Because cellular immunity is of particular importance to combat ILTV infection [25][26][27][28][29], VLPs-gB were further modified by maleylation. Maleylated proteins are directed to scavenger receptors present on APCs, and thus are able to induce a more dominant cellular immune response, which in turn leads to decreased levels of IgM and IgG [30][31][32]. To further boost the cell-mediated immunity in vaccinated chickens, either plasmids mediating expression of ChIL-18 were administered together with the VLPs, or in a second approach, VLPs displaying ILTV gG were administered together with ChIL-2. In the latter, it was anticipated to provoke a humoral immune response to counteract gG. This is important, because gG initiates an immune evasion mechanism by directing the cell-mediated immune response toward an antibody-mediated immune response [33,34].
In this pilot study, VLPs were used for the first time as vaccines against ILTV in layer chickens. Thus, the main focus was put on examining the tolerability of the VLPs in chickens upon different inoculation methods [in ovo or intramuscular (i.m.)]. For animal welfare considerations, no challenge experiments were performed at this stage. However, immune responses upon vaccination were measured and shed light on the mechanisms mediating potential protection against infection with ILTV.

Plasmid construction
For the construction of pDisplay (Invitrogen, Thermo Fisher Scientific)-based expression vectors, two different ILTV glycoprotein regions were chosen: amino acids (aa) 2 to 700 from gB and aa 30 to 293 from gG as illustrated in Fig. 1. Cloning of the gB expression construct (pDgB700) was outsourced to GeneArt Gene Synthesis Services (Thermo Fisher). To generate pDgGΔ1-29, an 848 bp DNA fragment  (PDGFR TM). Below is a scheme of the wild type proteins (gB, gG) and of the coding regions that were inserted into pDisplay (gB700, gGΔ1-29), both of which contained a His tag. Respective amino acid numbers are indicated. TM, transmembrane domain; SS, signal sequence.
The MLV gag expression construct pcGag was constructed by amplifying the 1617 bp gag encoding sequence using oligonucleotides gag(+) and gag(-) and subsequent insertion into the pcDNA3_3 TOPO vector (Invitrogen, Thermo Fisher Scientific). The plasmid pHit60 [37] served as a template. All primers were synthesized by Microsynth (sequences are shown in Table 1).

Gene transcription of ChIL-2 and ChIL-18
To detect ChIL-2 and ChIL-18, RNA was isolated from chicken cells [leghorn male hepatoma (LMH), Cell Lines Service] 48 h after transfection or from blood and organ samples from chicks using the NucleoSpin RNA Kit (Macherey Nagel).

Production of ILTV gB-and ILTV gG-displaying VLPs
LMH cells were cultivated in 10 cm dishes in Waymouth's medium supplemented with 10 % FBS at 38 °C and 5 % CO 2 . When cultured cells reached appropriate confluency, co-transfection with pcGag (7 µg per dish) and pDgB700 (8 µg per dish) or pDgGΔ1-29 (8 µg per dish), respectively, was conducted under serum-free conditions employing TransIT−2020 (Mirus) according to the manufacturer's instructions. The VLP-containing cell culture supernatants were harvested 48 h later and subsequently passed through a 0.45 µm pore-size polyethersulfone filter (Sarstedt). VLPs were concentrated by ultracentrifugation (R av 150 000 g, 3 h, 4 °C) through a 20 % sucrose cushion. The pelleted VLPs were resuspended in an appropriate amount of PBS.

Maleylation of VLP-gB
VLPs displaying ILTV gB were further modified by adding maleyl groups as previously described by Butler and Hartley  [40]. In brief, VLPs were dissolved in carbonate-bicarbonate buffer (CB; 0.1 M Na 2 CO 3 , 0.1 M NaHCO 3 , pH 8.5) and maleic anhydride was added under constant monitoring of pH values. If necessary, Na 2 CO 3 was added to stabilize the pH at 8.5-9.0. To detect successful maleylation, the free amino groups were determined as reported by Habeeb [41]. In brief, 100 µl of serial two-fold dilutions of the maleylated VLP-gB (mVLP-gB) and controls (non-maleylated VLP-gB, maleylated and non-maleylated BSA) were transferred to a 96-well plate. Subsequently, 60 µl of 0.05 % picrylsulfonic acid (in 0.1 M NaHCO 3 ) was added and incubated for 2 h at 37 °C. Reactions were stopped by adding 50 µl SDS (10 %) and 25 µl HCl (1 M) and absorbance was measured at 335 nm using the Epoch2 microplate reader (Biotek).

Electron microscopy
Concentrated VLPs were adsorbed to parlodion/carboncoated copper grids for 2 min. Grids were rinsed four times with PBS containing 10 % goat serum and subsequently incubated for 30 min at room temperature with the anti-Myc. A7 antibody (Pierce, Thermo Fisher Scientific) diluted 1 : 50 in PBS containing 1 % goat serum. Grids were rinsed five times with PBS containing 10 % goat serum and incubated for 30 min at room temperature with 10 nm diameter goldconjugated anti-mouse IgG (Sigma Aldrich, Merck) diluted 1 : 50 in PBS containing 1 % goat serum followed by five washes with PBS and four washes with double distilled H 2 O. For the additional negative stain, grids were treated with 3 % uranyl acetate acid for a maximum of 20 s. Imaging was done using a TEM10 microscope (Zeiss) at 85 kV.

Immunofluorescence microscopy
LMH cells were grown on glass cover slips in 24-well plates. Then, 48 h after transfection with pcGag and pDgB and pDgG, respectively, cells were transferred and incubated at 4 °C for 30 min with an anti-His antibody (SignalChem) diluted 1:300 in RPMI supplemented with 2 % BSA. Cells were washed and fixated with 3 % paraformaldehyde. After washing three times with PBS, coverslips were incubated for 45 min at room temperature with an Alexa Fluor 488-coupled secondary antibody (Thermo Fisher Scientific) diluted 1:1000 in PBS containing 10 % goat serum and DAPI. After washing three times with PBS, coverslips were mounted on glass slides. Microscopic analysis was performed using an Olympus IX81 inverted epifluorescence microscope.

Western blot analysis
Transfected LMH cells were lysed with RIPA buffer 48 h after transfection and protein concentration was determined employing a BCA protein assay kit (Pierce, Thermo Fisher Scientific). Same amounts of cell lysates and concentrated VLPs were loaded onto SDS gels containing 10 % polyacrylamide. A nitrocellulose membrane was used for blotting. Proteins gB and gG were detected using an anti-HA antibody (clone16B12; Covance) and Gag was detected employing a polyclonal anti-MLV-p30 antibody (Icosagen). HRP-coupled secondary antibodies directed against rabbit IgG (Cell Signaling Technology) and mouse IgG (Santa Cruz Biotechnology), respectively, were used for visualization together with Clarity Western ECL Substrate (BioRad) and a ChemiDoc Imager (BioRad).

Protein expression and purification
LMH cells were cultivated in 10 cm dishes in Waymouth's medium supplemented with 10 % FBS. Cells were transfected with pDgB or pDgG (each 15 µg per dish) using TransIT−2020 (Mirus) according to the manufacturer's instructions. HA-tagged gB or gG were purified employing the HA-tagged protein purification kit (MBL International Corporation). Cell lysates, samples from washing steps and the finally eluted protein were separated on an SDS gel containing 10 % polyacrylamide. A nitrocellulose membrane was used for blotting and was subsequently stained with colloidal gold (BioRad) to assess the purity of the recombinant protein preparations. Protein concentration was determined with the BCA protein assay kit (Pierce, Thermo Fisher Scientific).

Enzyme-linked immunosorbent assay
To quantify the displayed ILTV antigens, a 96-well plate was coated with serial dilutions of VLP-gB and VLP-gG, respectively, in coating buffer (CB; 50 mM NaHCO 3 , pH 9.6). Serial dilutions of purified gB and gG were used as references. The plate was incubated for 1 h at room temperature followed by an overnight incubation at 4 °C. Groups IV to VI comprised the study group gB without in ovo vaccination. Fertilized eggs were hatched, and chicks were transferred to chicken isolators, and randomly divided into three groups on day 14 p.h.: four chicks received injections of PBS (group IV), seven chicks received plasmid pcDNA-ChIL-18-His (group V), and seven chicks received plasmid pcDNA-ChIL-18-His and mVLP-gB into the musculus iliotibialis lateralis (group VI).
Immediately after hatching, animals were monitored every morning for signs of fitness. This visual examination was done by the same two people experienced in poultry health and behaviour in accordance with the recommendations of the Federation for Laboratory Animal Science Associations (FELASA) working group [42]. Three days post-hatch (DPH) and 2 days post-vaccination, animals were monitored twice a day (morning and afternoon). According to the observations, the vitality score was determined to enable an objective assessment of the animals' wellbeing. Vitality score criteria were as follows: 0, normal; 1, for each of the following clinical signs: closed eyelids, ruffled feathers, increased breathing, separation from the group, limping. Scores were added and interpreted as: 0, normal; 1, mild pain or distress; ≥2, severe pain or distress (with euthanasia upon inspection

Isolation of spleen lymphocytes
Spleen cells were isolated by removing the capsule and passing the spleen through a 70 µm pore size cell strainer (Fisher Scientific) into isolation medium (IM; RPMI 1640, 2 % FBS). The single cell suspension was transferred onto Histopaque 1077 columns (Sigma Aldrich, Merck) and centrifuged for 20 min at 600 g at room temperature. Cells at the interface were collected in PBS and centrifuged again for 10 min at 300 g. After repeating the last step, spleen lymphocytes were collected in cultivation medium (CM; RPMI 1640, 10 % FBS, 50 µM beta-mercaptoethanol, 100 U ml −1 penicillin/streptomycin), ready for further processing.

Fluorescence-activated cell scanning
Citrated blood samples (~500 µl) were mixed 1 : 2 with PBS and transferred onto Histopaque 1077 columns (Sigma Aldrich, Merck) and centrifuged for 20 min at 400 g at room temperature. Cells at the interface were collected into PBS and centrifuged again for 5 min at 400 g at 4 °C. Upon suspension of the cell pellet in FACS buffer (PBS, 2 % FBS, 0.05 % NaN 3 ), cells were counted and aliquoted at a concentration of 5×10 5 cells/50 µl. Cells were kept at 4 °C and were analysed for the surface expression of CD3 + , CD4 + and CD8 + , respectively. Unstained cells served as negative controls. To facilitate compensation for spectral overlap, single stains were also performed. The FITC-coupled anti-ChCD3 antibody (Abcam) was used at a final concentration of 5 µg ml −1 , the PE-coupled anti-ChCD4 antibody (Acris) was used at a final concentration of 0.2 µg ml −1 , and the PE-coupled anti-ChCD8 antibody ( antibodies-online. com) was applied at a final concentration of 0.5 µg ml −1 . Cells were incubated for 45 min at 4 °C followed by centrifugation for 5 min at 400 g at 4 °C. Cells were washed with FACS buffer and centrifuged again. Upon fixation with 1 % paraformaldehyde for 10 min and a final centrifugation, cells were resuspended in FACS buffer and filtered through a 35 µm cell strainer into a polystyrene round bottom tube (Fisher Scientific). Samples were analysed using a FACS Canto (BD Bioscience) at the flow cytometry facility of the University of Zurich by acquiring 10 000 events in the lymphocyte gate. Data analysis was performed using the BD FACSDIVA (BD Bioscience) and the FlowJo, LLC software.

Statistical analysis
To calculate the number of animals needed to reveal significant results, the software G*Power was used. Mean values, percentages, standard deviations and ANOVA variance test (single factor) results were determined using Excel.

Expression of ChIL-2 and ChIL-18 in chicken LMH cells
The sequences of each chicken interleukin (ChIL-2, ChIL-18) were cloned into expression vectors containing either the CMV promoter or the CAG promoter consisting of the CMV immediate early enhancer followed by the chicken beta actin (CBA) promoter and the rabbit beta globin intron. To examine potential variations in promoter-dependent expression, the respective plasmids were transfected into LMH cells and total RNA was analysed 48 h later using real-time RT-PCR. As shown in Table 3, C t values did not differ significantly between ChIL-2 or -18 expressed from the CMV promoter or from the CAG promoter. In all further experiments described here, the ChIL-expression plasmids containing the CMV promoter were used.

Generation and characterization of VLPs
To achieve the display of ILTV gB and gG on the surface of VLPs, respectively, the corresponding coding regions were inserted into the pDisplay vector, encoding the murine immunoglobulin chain leader sequence as well as the transmembrane domain of the platelet-derived growth factor receptor (PDGFR), thereby facilitating transport through the secretory pathway and anchoring of the respective fusion protein in the plasma membrane. For gB, the coding region for the first 700 aa was inserted, consequently depleting the transmembrane domain and the cytoplasmic tail at the C terminus. For gG, the N-terminal signal sequence was removed and only the coding region for amino acid positions 30 to 292 was inserted into the expression vector (Fig. 1). Besides the pDisplay-encoded HA and Myc tags, the expressed fusion proteins described here harboured a His tag, which was introduced with the oligonucleotides used to amplify the respective DNA fragments prior to insertion into the vector (compared in Table 1).
Surface expression of the resulting ILTV protein variants gB700 and gGΔ1-29 was verified by immunofluorescence staining of LMH cells transiently transfected with the respective expression constructs. To ensure surface staining, living cells were kept on ice during exposure to antibodies and thereafter fixed. As illustrated in Fig. 2 (a), both glycoproteins were readily detected.
To verify the expression of VLPs displaying gB700 and gGΔ1-29, respectively, LMH cells were either transiently transfected with the respective ILTV glycoprotein variant expression plasmids only or co-transfected together with pcGag. As illustrated in Fig. 2b, lysates and concentrated supernatants of transfected cells were subjected to Western blotting analysis.
Since pcGag encodes only the MLV Gag protein and not the viral protease, the anti-p30 antibody detected the Gag precursor protein p65 and not the capsid protein p30 in cell lysates and supernatants. Both ILTV glycoprotein variants were detected in cell lysates independent of the co-expression of Gag. As expected, gB700 and gGΔ1-29 revealed approximate molecular weights of 100 and 60 kDa, respectively. In contrast, both ILTV protein variants were detected only in supernatants that also contained Gag. This indicated incorporation of the ILTV protein variants into MLV Gag-formed VLPs. This observation was further supported by electron microscopy using immunogold-labelled antibodies specific for the Myc tag present in both viral protein variants. As shown in Fig. 2c, particle structures were detected in all VLP preparations. Gold particles located at the membrane of these particles were visualized in VLP-gB and VLP-gG preparations but not in controls consisting of 'naked' VLPs. This further substantiated the decoration of VLPs with both ILTV protein variants.
VLPs-gB were further modified by maleylation. To measure a successful reaction, the availability of free amino groups of VLPs-gB was analysed. As a control, BSA was maleylated in parallel. The relative amount of free amino groups of unmodified proteins decreased with increasing dilution. In contrast, the relative amount of free amino groups of the maleylated proteins was constant at a very low level, indicating a successful maleylation of the proteins, as shown in Fig. 2d.

Tolerability of the vaccines
To identify potential adverse effects of the vaccine on the development and wellbeing of the chicken embryos or hatched chicks, several factors were taken into consideration, i.e. hatching rate, vitality score (indicating general health and wellbeing), sex ratio and weight gain.
The hatching rate of all in ovo injected individuals was 95 % (18 chicks out of 19 hatched; 18/19), including 4/4 (100 %) in the negative control group, which received only PBS (group I), 6/7 (86 %) in the group being treated with plasmids encoding ChIL-2 (group II), and 7/7 (100 %) in animals receiving plasmids expressing ChIL-2 and VLPs (group III). As depicted in Fig. 3a and b, single chicks showed mild signs of distress mainly in the first few days after hatching reflected by a vitality score of 1. However, this was not related to a particular treatment but a result of post-hatching adaptation to a new environment and food uptake. On day 12 p.h., a mock treated chick (PBS) of the gB group was rated with a score of 1 due to peculiar sleepiness (closed eyelids) probably associated with blood sampling earlier that day. No unusual behaviour was detected on the following day. Score 1 was also attributed to a ChIL-18, mVLP-gB-treated chick as breathing difficulties were observed. Examination of the chick revealed fibre originating from the bedding material stuck at the entrance of the larynx. Upon removal of the fibre, breathing normalized again. In summary, none of the clinical signs observed in the chicks could be attributed to vaccination.
The sex ratio in each group had -as expected for a layer hen line -a slight tendency towards more males as shown in Fig. 3c and as previously described [43].
Within the respective study groups, the weight of the chicks increased over time with no statistical significance between the different treatments (PBS, plasmid, plasmid and VLP) as shown in Fig. 4a , b. However and as illustrated in Fig. 4c, a difference was noted between the two study groups gB and gG concerning average weight gain per week. Although weight at hatch was comparable between all groups (on average 39 g), weight gain during the first week was much higher for gB groups IV-VI, which almost doubling their weight compared to the gG groups, which only gained around 30 % of their initial weight. However, in week 2 the relative weight gain of the gG group reached the level of the gB group, and even exceeded them in weeks 3 and 4, leading to average weights within the gG group of 351 g that were about 10 % lower than the average weight of the gB group (393 g) after week 5.

Transcription of ChIL-2/-18 upon plasmid injection in ovo or i.m.
To promote a humoral or cellular immune response, plasmid encoded ChIL-2 or ChIL-18 were administered in addition to the respective VLPs. Expression of both interleukins was monitored by qRT-PCR at various time points after vaccination. No differences in ChIL-2 or ChIL-18 levels were detected in the blood (or any other investigated organ) of animals receiving plasmids expressing chicken interleukins compared to PBS-treated animals (data not shown).

Immune response to VLPs-gB
Application of ILTV gB-displaying VLPs -further modified by maleylation -and co-administration of plasmid encoded ChIL-18 aimed at inducing a cell-mediated immune response.
To examine this, the IFN-γ response of spleen lymphocytes upon stimulation with different antigens was conducted. Three weeks after vaccination, at the age of 35 days, chicks were euthanized and spleens were processed for further analysis using an ELISPOT assay performed in triplicate. The average number of spots is depicted in Fig. 5a. Lymphocytes from all animal groups treated with PBS, ChIL-18, ChIL-18 and mVLP-gB, respectively, responded to stimulation with ionomycin and PMA, used as a positive control. Nevertheless, no reaction was detected upon stimulation of the lymphocytes with ILTV or gB alone.
The IFN-γ response of spleen lymphocytes cannot be monitored at multiple time points in one individual animal. Thus, the ratio of CD4 + to CD8 + T lymphocytes was determined as an additional indicator of the cellular immune response. These values are depicted in Fig. 5b as box plots as well as the average of the three treatment groups including a corresponding trend line. A treatment-independent increase in the ratio of CD4 + to CD8 + cells 2 days after vaccination (DPH 16) was detected. At later time points, a decrease of the CD4 + to CD8 + ratio was observed. This decrease was slightly more pronounced in chicks vaccinated with ChIL-18 and mVLP-18.

Immune response to VLPs-gG
Vaccination with VLP-gG and plasmid-encoded ChIL-2 aimed at provoking a humoral immune response, which should prevent -in case of an ILTV infection -the gG-induced immune evasion mechanism. ILTV gG-specific antibodies were detected using ELISA. Results are depicted as box blots in Fig. 5c. At the first time point of measurement (DPH 8) 11 days after in ovo vaccination, gG-specific antibodies were detected. Titres decreased on DPH 16 and reached background levels on DPH 22.

DISCUSSION
The present pilot study describes the use of heterologous VLPs as a vaccination platform against ILTV in chickens. VLP-based poultry vaccines were mainly investigated in the context of avian influenza and ND and showed promising results such as high antigenicity and even protection from infection [44][45][46]. Most studies directly examine the protection that is conferred by a particular vaccine. For animal welfare reasons, this study focused first on determining the tolerability as well as the immune responses elicited upon vaccination.
Because MLV-based VLPs are enveloped with a membrane originating from the cells utilized for production, it was decided to generate the VLPs in a chicken cell line (LMH) to avoid immune reactions directed against heterologous host cell proteins. Expression levels of the ILTV glycoprotein variant-displaying VLPs in LMH cells was comparable to the productivity in the most frequently used human producer cell line 293T (data not shown).
The in ovo injection had no impact on hatching rate, which is on average between 85 and 95 % upon artificial incubation [47,48] and ranged between 86 and 100 % in this study. Neither in ovo administration nor i.m. injection resulted in adverse effects in chicks as rated in the vitality score. No influence on the weight at hatch was observed. Notably, the weekly weight gain was reduced in the in ovo vaccinated gG group compared to the gB group receiving no treatment prior to hatch. Unfortunately, we did not find earlier reports examining the effect of in ovo mock injections in comparison to no manipulation at all. We assumed that the observed difference was not related to the vaccine but rather a result of the injection procedure as animals in the control groups (injection of PBS and plasmid encoding ChIL-2, respectively) also revealed a delayed weight gain. Another factor that could have contributed to the delayed weight gain is the different age of the breeders at the time of egg-laying [49] as the experiments with the two groups (gG, gB) were not conducted in parallel.
Previous experiments showed that ChIL-2 was detectable in blood, thymus and liver of 20-day-old embryos 2 days after administration of ChIL-2 expression plasmids through the CAM. In the current study, expression of ChIL-2 was detected when blood was analysed on day 8 p.h. (11 days after in ovo administration of the ChIL-2 expression plasmids) or at later time points (DPH: 16,22,25,29,35). However, no differences were observed between the control group (PBS) and groups who received the ChIL-2 expression plasmids. This might be due to an onset of endogenously produced ChIL-2, which can occur between days 2 and 4 p.h. [50]. The same was observed for the expression of ChIL-18: no difference was detected between the control group (PBS) and chickens that received the expression plasmid when analysed 2 days after i.m. administration on day 14. This could also be due to an early onset of constitutive ChIL-18 expression already on ED 12 and an increase immediately upon hatching [51]. For future experiments, a detection method needs to be utilized enabling the differentiation between endogenous and recombinant interleukins.
Various studies have indicated a particular importance of the cellular immunity to protect against ILTV infection [25][26][27][28][29]. This issue was addressed in the current study by maleylation of the gB-displaying VLPs and co-administration of plasmids mediating the expression of ChIL-18. Although thymocytes in the chicken embryo respond to mitogen stimulation as early as ED 14, these cells reach their full activation capacity at an age of about 2 weeks [52]. Thus, it was decided to apply the vaccine at a stage where it was more likely to elicit a robust cellular immune response. Nevertheless, a strong cellular immune response could not be detected. The ELISPOT assay did not reveal any IFN-γ response upon stimulation with the antigen. One potential reason for this might be the time point of sampling at 21 days post-vaccination, and thus, rather late compared to other vaccination studies describing sampling between 7 and 17 days post-vaccination [53][54][55].
To monitor the cellular immune response over time, the ratio of CD4 + to CD8 + T cells was determined by FACS. This ratio is an indicator for immune competence. Upon virus infection (or vaccination), cytotoxic T lymphocytes, which are largely CD8 + , undergo massive expansion [56][57][58]. CD4 + T cells also expand, but their differentiation into subsets that possess distinct effector functions is more pronounced. The expansion phase lasts for about 2 weeks and peaks at about 1 week after antigen exposure. Thus, virus infection is usually accompanied by a decreased CD4 + /CD8 + T cell ratio. In healthy, non-vaccinated chickens the CD4 + /CD8 + T cell ratio remains relatively constant even at different ages, but it can vary between different chicken lines and housing conditions. For specific pathogen-free chickens, lower ratios (between 0.74 and 0.94) were found compared to different laying hen lines kept under regular housing conditions (Dekalb Delta hybrid: 1.2; outbred white leghorn chicken: 3.25) [59,60].
In the current study, no statistically significant difference in CD4 + /CD8 + ratio over time for control treated animals (PBS: 1.34±0.17, ChIL-18: 1.17±0.18) was observed. This is in accordance with previous studies reporting similar findings for chickens kept in regular environments. In contrast, the vaccinated chicks showed a slight trend towards an increase of CD8 + cells. This contradicts the results of an ILTV vaccination study published by Chen and colleagues, who detected an increase of the CD4 + /CD8 + ratio and furthermore associated this increase with the conferred protection [61]. In contrast, protective vaccination against ND and fowl adenovirus, respectively, were reported to be a result of a decreased CD4 + / CD8 + ratio [62,63].
To counteract the ILTV gG-induced immune evasion, this study aimed at inducing gG-specific antibodies using in ovo vaccination with gG-displaying VLPs and ChIL-2 expression constructs. To further increase the immune response, a booster vaccination was administered i.m. on day 14. On the first day of sampling (DPH 8, 11 days after vaccination), increased anti-gG antibody levels were detected. This was a promising result, as ILTV gG seems to be only weakly antigenic [64]. The success of the described approach is probably due to the high density of antigens decorated on the surface of the membrane-enveloped VLPs. The elicited anti-gG antibody levels decreased over time and reached background levels on day 22 (1 week after the boost). Surprisingly, the boost did not lead to an increased or at least constant antibody titre. The detected effect on the cellular immune response was also weaker than expected and was probably a result of the comparably low amount of antigen used. Most vaccines currently administered in ovo are either replication competent attenuated life vaccines or vector-derived vaccines. The replication incompetence of VLPs, which is an advantage regarding safety, might be a drawback regarding the induction of a robust immune response. Yet various studies have shown that a single in ovo vaccination with a replicationincompetent reagent such as outer membrane vesicles of gramnegative bacteria or antigen-antibody complexes (AACs) can induce at least an antibody response [65,66]. Nevertheless, we propose a higher dosage for the i.m. application, which should be determined according to the actual body weight at the time of injection. The average weight of a layer chicken embryo at ED 18 is 22 g, and thus the injected amount of vaccine corresponds to 14 µg g −1 body weight (bw). In contrast, the injected amount of VLP in the 14-day-old chicks was around 2-3 µg g −1 bw for VLP-gB or VLP-gG respectively. Furthermore, analysis of the cellular immune response by ELISPOT should be conducted at two time points closer to the time of vaccination, e.g. 7 and 14 days post-injection as in earlier vaccination studies [53,54].
In summary, the MLV-derived VLPs displaying ILTV glycoprotein variants were well tolerated and were able to induce a detectable immune response. Thus, this new approach could initiate the further development of ILTV vaccines and the generation of safer vaccines for poultry in general.

Funding information
The authors received no specific grant from any funding agency.