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. 2026 Feb 12;105(5):106632. doi: 10.1016/j.psj.2026.106632

A Self-amplifying mRNA vaccine for infectious laryngotracheitis virus (ILTV) induces efficient protective immunity

Huan-huan Li a,b,1, Ling Tong b,c,d,e,1, Sai-sai Chen b,c,d,e, Zhi-xiang Bi b,c,d,e, Gang Hu f, Wan-li Chen f, Jia-li Yu f, Ruo-nan Tao f, Teng Huang a,⁎, Shu-hua Xia f, Nikolaus Osterrieder g, Ji-chun Wang b,c,d,e
PMCID: PMC12926564  PMID: 41713095

Abstract

Infectious laryngotracheitis (ILT) continues to pose a significant threat to intensive poultry farming regions worldwide. Current control strategies primarily rely on live attenuated and recombinant live ILT virus (ILTV) vaccines. However, concerns persist regarding the potential reversion to virulence, recombination of vaccine viruses, and interference from maternal antibodies. mRNA vaccine platforms offer a novel and promising approach for developing next-generation ILTV vaccines. In this study, we designed a self-amplifying RNA (saRNA) vaccine incorporating the ILTV gD gene and evaluated its immunogenicity and protective efficacy in chickens. The saRNA-gD vaccine elicited robust humoral immunity, with serum neutralizing antibody titers reaching up to 1:415. All vaccinated chickens achieved seropositivity within 7 days post-vaccination (dpv). Significantly elevated levels of IL-2 cytokine were observed at 28 and 42 dpv compared to the LNP control group, suggesting the potential activation of cellular immune responses following vaccination. Animal challenge experiment revealed that the saRNA-gD vaccine effectively mitigated clinical symptoms induced by ILTV infection, conferring a protection rate of approximately 66.7%, which is comparable to that of the commercial recombinant fowl pox virus (rFPV-LT) vaccine. Quantification of viral loads in tracheal swabs and histopathological examination demonstrated that the saRNA-gD vaccine significantly reduced viral DNA copies in swabs and induced markedly milder histopathological changes. These findings indicate that the saRNA-gD vaccine induces potent immune responses and provides substantial protection against ILTV challenge, potentiating it as a highly promising vaccine candidate for the control of ILT in poultry.

Keywords: ILTV, Glycoprotein D, saRNA vaccine, Virus-neutralizing antibody, Immune protection

Introduction

Since its first report in the United States in 1925, infectious laryngotracheitis (ILT) has been detected globally (Gowthaman et al., 2020). ILT is caused by infection with ILTV, a member of the Alphaherpesvirinae subfamily (Elshafiee et al., 2022). This virus can infect various avian species, including chickens, pheasants, and peacocks (Crawshaw and Boycott, 1982), and leads to respiratory diseases, which are characterized by clinical signs such as coughing, wheezing, dyspnea, and hemoptysis (Bagust et al., 2000).

Currently, the primary strategies for clinical control and prevention of the disease rely on live attenuated vaccines and recombinant live vector vaccines (Menendez et al., 2014). However, live attenuated vaccines carry risks of latent infection and virulence reversion in vaccinated chickens, as well as the potential for vaccine virus recombination, which can lead to outbreaks of vaccine-derived infectious laryngotracheitis (VLT) (Coppo et al., 2012; Chacón et al., 2025; Dufour-Zavala, 2008; Lee et al., 2012; Oldoni et al., 2009;). Although recombinant live vector vaccines are safe for all age groups, their efficacy is often compromised by poor induction of local or cellular immunity, incomplete virus neutralization in the trachea, and high virus shedding (Johnson et al., 2010; Vagnozzi et al., 2012; Zeng et al., 2023). These limitations render conventional vaccine platforms inadequate during pandemics or rapidly expanding epidemics, underscoring the need for more effective platforms (Pardi et al., 2020).

Since the onset of the COVID-19 pandemic in 2019, mRNA-based vaccine platforms have emerged as promising candidate vaccines due to flexible antigen design and the possibility for rapid production (Blakney et al., 2019; Feldman et al., 2019). Currently, numerous veterinary mRNA vaccines are being investigated, including those protecting against foot-and-mouth disease (FMD) (Rodríguez Pulido et al., 2009), pseudorabies (PR) (Jiang et al., 2020), porcine epidemic diarrhea (PED) (Gerdts and Zakhartchouk, 2017), avian influenza (AI) (Hajam et al., 2020), Marek's disease (MD) (Fazel et al., 2024), swine influenza (SI) (Bosworth et al., 2010), and monkeypox (Tai et al., 2025). In these studies, mRNA vaccines have consistently been demonstrated to hold considerable promise.

The overall safety and immunogenicity of mRNA vaccines have been well recognized. However, their application in chickens is limited, largely due to their higher costs in comparison with conventional live attenuated vaccines, inactivated vaccines, or even subunit vaccines. Recently, self-amplifying RNA (saRNA) vaccines are emerging as a more cost-effective alternative to the prototype mRNA vaccines. Unlike conventional mRNA vaccine platforms, saRNA vaccines typically feature alphavirus-derived RNA replication systems, such as those from Venezuelan equine encephalitis virus (VEEV), Sindbis virus (SINV), or Semliki Forest virus (SFV) (Casmil et al., 2025). This design facilitates intracellular self-amplification, i.e., rapid RNA replication, and sustained in vivo expression of target antigens (Comes et al., 2023). Consequently, this tool can elicit robust protective immunity even at lower doses (Blakney et al., 2020), which reduces overall production costs and, therefore, is of particular suitability for poultry vaccine applications.

During the construction of saRNA vaccines, target antigens can be flexibly selected (Blakney et al., 2021). The surface envelope glycoproteins of ILTV play critical roles in viral entry, replication, cell-to-cell spread, and virus release (Ponnusamy et al., 2025). Among these, glycoprotein D (gD), encoded by the US6 gene, is highly conserved across the members of the alphaherpesviral subfamily, and functions as a critical ligand for virus attachment to and subsequent entry into host cells (Di Giovine et al., 2011). Furthermore, gD has been shown to elicit potent neutralizing antibodies and robust cell-mediated immunity, and has been extensively characterized as a promising candidate antigen for vaccine development (Lazear et al., 2012).

In this study, we constructed an saRNA vaccine expressing the ILTV gD gene. Vaccinated chickens developed robust immune responses and effective protection was elicited after immunization. These findings indicate its potential as a candidate vaccine for preventing and controlling ILTV, thus providing the foundation for subsequent vaccine optimization and production.

Materials and methods

Design of the saRNA vaccine

The saRNA vaccine candidate was composed of a 5′ cap structure, a 5′ untranslated region (UTR), self-amplifying elements, the open reading frame (ORF) encoding the gD protein, a 3′ UTR, and a poly (A) tail (110 bp). Briefly, the gD gene of the ILTV LJS09 strain (GenBank ID: JX458822.1) was codon-optimized based on chicken codon preference, chemically synthesized, and then cloned into the pUC57 vector (GenScript, USA) for in vitro transcription of mRNA. The resulting mRNA was enzymatically capped using CleanCap® Reagent AU, with all procedures performed according to the manufacturer's protocols (Thermo Fischer Scientific, USA).

Cells and virus

Human embryo kidney (HEK) 293T cells (ATCC® AAV-293) and primary chicken embryo kidney (CEK) cells (Guo et al., 2025) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fischer Scientific, USA) and DMEM/F-12 medium (Thermo Fischer Scientific, USA), respectively. Both media were supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (both from Sigma-Aldrich, USA). All cells were incubated at 37°C and 5% CO2. The ILTV JS1511 strain was isolated and maintained in our laboratory (unpublished data).

Production and quality control of mRNA-LNP

Lipid nanoparticles (LNPs) were synthesized using a microfluidic mixing method (iNanoE, Ignite Biotech). Lipids (ALC-0315; DSPC; cholesterol and ALC-0159) were dissolved in ethanol at a molar ratio of 46.3:9.4:42.7:1.6. The aqueous phase of mRNA was prepared by diluting the mRNA (stored in RNAse-free water) with 100 mM citrate buffer at pH 4.0. The microfluidic cartridge was then used to mix lipids and mRNA solutions at a total flow rate of 6 mL/min, in which the flow rate ratio between the aqueous phase and the organic phase was 3:1, resulting in a N/P ratio of 6. Using an Amicon®30 kDa ultrafilter for concentration and exchange, the purified and concentrated LNPs were then stored under predetermined test conditions (Ling et al., 2025). The hydrodynamic diameter, polydispersity index (PDI), and zeta potential of the LNPs were determined by dynamic light scattering (Malvern Panalytical, England).

Validation of antigen gene expression

To confirm in vitro expression of saRNA delivered by LNP, HEK293T cells were transfected with either LNP empty vector, LNP-encapsulated saRNA-gD, or left untreated. Cells were collected at 24-, 48- and 72-hours post-transfection for RNA extraction and qRT-PCR analysis to assess the self-amplifying property of saRNA-gD. Additionally, at 48 h post-transfection, cells were collected and lysed in RIPA buffer supplemented with protease inhibitor for protein extraction. The protein extract was then separated by SDS-PAGE and subjected to Western blot analysis using the chicken anti-ILTV serum (1:500 dilution, prepared in-house) and HRP-conjugated goat anti-chicken IgG secondary antibody (1:5000 dilution) (Solarbio, China) (Ling et al., 2025). The PVDF membrane was developed using a chemiluminescence method with a chemiluminescence imager (Tanon, China).

Vaccination and virus challenge

Forty-five specific pathogen-free (SPF) White Leghorn chickens were randomly divided into three groups (n=15), identified by differently colored leg bands, and housed respectively in negative-pressure isolators with the same condition. Chickens in group 1 were intramuscularly injected with 1000 ng saRNA-gD vaccine on 0 d and 21 d respectively. Chickens in group 3 were vaccinated via wing-web puncture with recombinant fowlpox virus-vectored ILTV vaccine (rFPV-LT, Guangdong Wens Dahuanong Biotechnology Co. Ltd., China) on 21 d, while animals in group 3 (empty LNP control) were intramuscularly injected with 1000 ng of LNP alone on 0 d and 21 d, respectively. Blood samples were collected from all groups on 7, 14, 21, 28, 35, and 42 dpv. On 42 d, chickens were challenged via intratracheal inoculation with 103.1/EID50 of the ILTV JS1511 strain. Clinical signs were monitored daily for 10 days after challenge in all groups. Protection was defined as the absence of any clinical signs. The clinical symptom score performed according to predefined criteria (0 = Normal, 1 = lethargy, sneezing, coughing, nasal discharge, 2 = dyspnea, gasping, 3 = death). On 3 dpc, three chickens per group were randomly selected and euthanized for the collection of tracheal and lung tissues. Laryngeal swabs were randomly collected from five chickens per group at 3, 5, 7 and 9 dpc. All surviving chickens were humanely euthanized at 10 dpc. The weight of each group of chickens was measured at different time points.

Determination of virus-neutralizing antibodies

To determine serum neutralizing antibodies in the vaccinated groups, CEK were cultured in 96-well plates. Serum samples were inactivated at 56 °C for 30 min and then subjected to serial double dilutions (1:2n). Each diluted serum sample was mixed with an equal volume of ILTV JS1511 strain virus suspension (containing 200 TCID50). The mixture was then added to the pre-prepared CEK and incubated at 37 °C for 3 to 5 days, with daily observation for cytopathic effect (CPE). The neutralizing antibody titer was calculated as the average of three measurements using the Reed-Muench method (Reed and Muench, 1938).

Determination of virus shedding in laryngeal swabs

Virue load quantification in laryngeal swabs was performed using real-time quantitative PCR (qPCR). Briefly, specific primers targeting a conserved region of the ILTV gC gene (F: 5′-GACTTGTGTCAGGATTATGCG-3′; R: 5′-CTAGCGAAGGAAGAAAGCAG-3′) were designed to amplify the target sequence. The PCR amplicon was cloned into the pUC57 vector to generate the reference plasmid. A standard curve was computed through 10-fold serial dilutions (10-1 to 10-7) of the engineered plasmid. The qPCR assay was performed in 8-tube strips using TB Green Premix Ex Taq II Fast (Takara, Japan). Each 25 μL reaction mixture contained: 12.5 μL of 2 × TB Green Premix Ex Taq II Fast, 0.4 μM each of forward and reverse primers, 1 μL of DNA template, nuclease-free ddH2O to adjust the final volume to 25 μL. The thermal cycling protocol consisted of: Initial denaturation at 95°C for 30 sec (1 cycle); Followed by 40 cycles of denaturation at 95°C for 5 sec, annealing/extension at 60°C for 10 sec. Viral copy numbers in samples were calculated from Ct values using the standard curve. Samples with quantifiable copy numbers were defined as PCR-positive. Quantitative PCR was performed on a Roche LightCycler 480 system, and the data were analyzed with the built-in LightCycler® 480 software (version 1.5; Roche Diagnostics, Switzerland).

Measurement of cytokines by ELISA

Serum concentrations of IFN-γ and IL-2 were quantified according to the manufacturer's protocols utilizing commercial chicken-specific ELISA kits (Mlbio, China). Briefly, standards and serum samples were incubated and subsequently treated with a biotinylated detection antibody, followed by HRP-conjugated streptavidin, with washing conducted between each step. The TMB substrate was added, followed by stop solution after incubation, and the absorbance was measured at 450 nm (BioTek, USA). All assays were performed using 100 μL serum samples in duplicate.

Histopathological examination

On 3 dpc, three chickens from each group were randomly selected and sacrificed. Tracheal and lung tissues were collected and fixed in 4% paraformaldehyde. Histopathological changes induced by ILTV infection were observed by hematoxylin and eosin (H&E) staining. Tissue sections were blindly examined by two independent veterinary pathologists. Histopathological lesions were evaluated using a four-grade scoring system wherein normal tissue was designated a score of 0, minimal changes received a score of 1 (characterized by inflammatory cell infiltration), mild lesions were marked as 2 (featuring swelling of respiratory epithelial cells with edema), marked lesions were assigned a score of 3 (identified by multinucleated cell formation and lymphocyte/plasma cell migration into the mucosa or submucosa), and severe pathological alterations were denoted as 4 (defined by cell disintegration and the mucosal surface being covered by a layer of basal cells or the absence of any epithelial covering). Digital images were acquired using a PANNORAMIC DESK/MIDI/250/1000 panoramic scanner (3DHISTECH, Hungary) and analyzed using CaseViewer 2.4 software (3DHISTECH, Hungary). Tissue section preparation and scoring were conducted in collaboration with Wuhan Servicebio Technology Co., Ltd.

Animal welfare and ethics statement

The SPF chickens used in the experiment were purchased from Jinan Saishi Poultry Technology Co., Ltd. All animal studies were approved by the Institutional Animal Care and Use Committee of Jiangsu Academy of Agricultural Sciences (Authorization No. IACUC-AE-2025-03-006) and were strictly conducted in accordance with guidelines provided by the Institutional Biosafety Committee. At the end of the experiment, chicks were euthanized by cervical dislocation performed by trained personnel.

Statistical analysis

All statistical analyses in this study were performed using GraphPad Prism software (version 10.1.2; GraphPad Software, San Diego, CA, USA). Statistical differences between groups (clinical symptom scores, weight gain, neutralizing antibody titers, cytokine levels, viral load in laryngeal swabs) were analyzed using two-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. Survival curves were analyzed using the Gehan-Breslow-Wilcoxon test. Statistical significance between groups is denoted as **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, and ns (not significant) P > 0.05. Differences were considered statistically significant at P < 0.05. All data are expressed as the mean ± standard deviation (SD).

Results

Formulation and characterization of the saRNA vaccine encoding ILTV gD

We designed an saRNA vaccine encoding ILTV gD. The mRNA construct incorporated optimized 5′ and 3′ untranslated regions (UTRs), with the 5′ UTR derived from the human α-globin gene and the 3′ UTR consisting of a hybrid AES/TLE5 and mtRNR1 sequence. Additionally, an alphavirus-derived replicase motif comprising four nonstructural protein (nsp) replicase motifs and a 110-nucleotide poly (A) tail were incorporated (Fig. 1a). Following synthesis of the mRNA-LNP complex, zeta potential analysis revealed a near-neutral surface charge of -2.01 mV (Fig. 1b). Particle size distribution and polydispersity index (PDI) were assessed by dynamic light scattering (DLS), demonstrating an average hydrodynamic diameter of 78.93 nm with a PDI of 0.108, which is indicative of a monodisperse solution (PDI<0.2) (Fig. 1c). In vitro transfection of HEK-293T cells confirmed the self-amplifying property of saRNA-gD (Supplementary Fig. 1), and demonstrated that the gD was expressed and properly processed (Fig. 1d). These results demonstrate the saRNA-gD vaccine was successfully constructed and could be efficiently expressed in transfected cells.

Fig. 1.

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Production and characteristics of ILTV gD-based saRNA vaccines. (a) Schematic design of the ILTV saRNA gD vaccine. The 5′ UTR is derived from human α-globin, and the 3′ UTR is a combination of AES/TLE5 and mtRNR1, with additional components including the replication motif from alphavirus and a 110 bp poly (A) tail. (b) Measurement of Zeta potential. (c) Particle size and polydispersity index (PDI) of the saRNA-LNP measured by dynamic light scattering (DLS). (d) The expression of saRNA-gD in transfected cells detected by immunoblot, with GAPDH as a loading control.

Immune response induced by saRNA-gD in chickens

To evaluate the immunogenicity of saRNA-gD, chickens were vaccinated twice with the saRNA-gD vaccine on 0 dpv and 21 dpv (Fig. 2a). After vaccination, body weights were determined, and no significant differences were observed among groups (P>0.05) (Fig. 2b). VNT results demonstrated that all chickens vaccinated with saRNA-gD reached the seropositivity threshold by 7 dpv (Supplementary Fig. 3). At 28, 35 and 42 dpv, neutralizing antibody titers in the saRNA-gD vaccinated group were significantly higher than those in the rFPV-LT vaccinated group (P<0.0001) (Fig. 2c). The neutralizing antibody titer peaked at 28 dpv (1:415). These findings indicate that the saRNA-gD vaccine can induce high levels of ILTV-specific antibodies in immunized chickens. Similar to the rFPV-LT group, chickens in the saRNA-gD group had significantly higher IL-2 levels compared to the LNP group at 28 dpv (p = 0.0003) and 42 dpv (p = 0.0014) (Fig. 2d). These results demonstrate the favorable safety profile and immunogenicity of the saRNA-gD vaccine.

Fig. 2.

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Evaluation of the immunogenicity of saRNA-gD in chickens. (a) Experimental design for vaccination and challenge. Chickens were intramuscularly immunized with 1000 ng of saRNA-gD at 0 and 21 dpv. rFPV-LT was applied via wing-web at 21 dpv. All chickens were challenged intratracheally with ILTV JS1511 at 42 dpv. (b) Body weight gain in immunized chickens across all groups was monitored. Data presented as the mean±SD (n=15 chickens per group). (c) Neutralizing antibody titers induced by saRNA-gD. Serum samples were collected at 28, 35, and 42 dpv. ILTV neutralizing antibody titers were measured by VNT (Neutralizing antibody titers > log23 were considered positive). Data presented as the mean±SD (n=15 chickens per group). (d) Expression levels of IL-2 in serum. Serum samples collected at 28 and 42 dpv were analyzed by ELISA. Data are presented as the mean±SD (n=5 chickens per treatment). (e) Expression levels of IFN-γ in serum. Serum samples collected at 28 and 42 dpv were analyzed by ELISA. Data presented as the mean±SD (n=5 chickens per group). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test: **P < 0.01, ***P < 0.001, ****P < 0.0001.

Protective efficacy of saRNA gD against ILTV challenge infection in chickens

At 42 dpv, all chickens were challenge-infected intratracheally with 103.1/EID50 of the ILTV JS1511 strain. On 3 dpc, chickens in the LNP group began to exhibit clinical signs, including depression, severe coughing, sneezing, dyspnea, and one chicken in this group died at 5 dpc (Fig. 3a, 3b). Chickens in the saRNA-gD group also started to show clinical signs at 3 dpc, but these were limited to mild coughing and sneezing. On 5 dpc, the clinical symptom scores of the saRNA-gD group were significantly lower than that of the LNP group (P<0.0001) (Fig. 3a). No statistically significant differences in body weights were observed between the groups following challenge (Fig. 3c). To further evaluate the protective efficacy of the saRNA-gD vaccine, ILTV shedding was monitored by qPCR in laryngeal swabs. The results showed that chickens in the saRNA-gD group had significantly reduced viral loads in laryngeal swabs at both 3 and 5 dpc compared to the LNP group (p < 0.05). Chickens in the rFPV-LT group reached peak viral loads of 104.5 copies/mg (mean log10 value: 3.418±0.9561) on 3 dpc, whereas animals in the saRNA-gD group reached a maximum of only 103.5 copies/mg (mean log10 value: 3.663±0.8994) (Fig. 3d). Notably, the protection rate conferred by saRNA-gD vaccination was comparable to that of the commercial rFPV-LT vaccine, with 66.7% of chickens (10/15) showing no clinical signs post-challenge (Fig. 3e). These results demonstrate that the saRNA-gD vaccine confers effective protection in chickens.

Fig. 3.

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Evaluation of the protective efficacy of saRNA-gD in chickens. (a) Clinical symptom scores (0 = Normal, 1 = lethargy, sneezing, coughing, nasal discharge, 2 = dyspnea, gasping, 3 = death). (b) Survival rate of chickens post-challenge. (c) Weight gain of chickens in each group at 42 dpv and 52 dpv. Data are presented as mean ± SD (n=15 chickens per group at 42 dpv; n=13 chickens per group at 52 dpv). (d) ILTV loads in laryngeal swabs. Laryngeal swabs were collected from 5 randomly selected chickens per group at 3, 5, 7 and 9 dpc. The viral copy numbers were quantified by qPCR. Data are presented as mean ± SD (n=5 chickens per group). (e) Vaccine protection rate. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test: *P < 0.05, **P < 0.01, ****P < 0.0001.

Histopathological lesions

On 3 dpc, three chickens per group were randomly picked for euthanasia to collect tracheal and lung tissues. In the LNP group, severe pathological changes were identified in the tracheal, including moderate thickening of the mucosal layer with substantial infiltration of lymphocytes and granulocytes, while extensive hemorrhage, diffuse lymphocyte infiltration and vascular congestion were found in the lungs. The rFPV-LT group exhibited moderate histopathological lesions, characterized by mild tracheal mucosal thickening with limited lymphocyte infiltration, along with small focal areas of capillary congestion and lymphocyte aggregation in lung tissue. In contrast, animals in the saRNA-gD vaccine group displayed only minor pathological changes, consisting of locally scattered lymphocytes in the trachea and occasional capillary congestion in the lungs (Fig. 4a). Histopathological scoring results revealed a significant difference in tracheal lesion scores between animals in the saRNA-gD group and the LNP group (P = 0.0408) (Fig. 4b), indicating that the saRNA-gD vaccine conferred effective protection against tracheal tissue damage. Although no significant difference in lung pathology scores was observed between the groups, the maximum lesion score of chickens in the saRNA-gD group was 2, compared to 6 in the LNP group (Fig. 4c). These results demonstrate that the saRNA-gD vaccine effectively reduces tracheal and pulmonary tissue damage caused by ILTV infection.

Fig. 4.

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Histopathological analysis of trachea and lung tissues following ILTV challenge. (a) Representative H&E-stained sections of trachea and lung. (b) Histopathological scores of tracheal tissues. Data are presented as mean ± SD (n=3 chickens per group). (c) Histopathological scores of lung tissues. Data are shown as mean ± SD (n=3 chickens per group). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test: *P < 0.05.

Discussion

The control of ILTV primarily relies on vaccination and biosecurity measures. Currently, only live attenuated vaccines and live vectored vaccines are commercially available for ILTV. While live attenuated vaccines confer robust protection in chickens, they have intrinsic risk of latent infection and potential to regain virulence through serial passages in vaccinated birds. Conversely, recombinant live-vectored vaccines are safer, yet their efficacy in blocking virus shedding is suboptimal, leading to incomplete protection (García, 2017). The next-generation saRNA vaccine platform holds promise as a candidate for ILTV immunization. Studies have demonstrated that saRNA and mRNA vaccines can provide comparable levels of protection against influenza virus but at significantly lower doses (Vogel et al., 2018). Moreover, a single administration of saRNA vaccine can induce durable humoral immune responses (Alwis et al., 2021). The enhanced antigen expression at lower doses improves immunogenicity and cost-effectiveness (Casmil et al., 2025). Previous studies on saRNA-based veterinary preparations have robustly demonstrated that saRNA vaccines may serve as an alternative for prophylactic strategies against ILTV and the associated disease complex.

Glycoprotein D (gD) is a ubiquitous component in nearly all alphaherpesviruses (Connolly et al., 2011). gD is essential and serves as the receptor-binding protein that mediates entry into susceptible cells (Sabir et al., 2019). During the development of second-generation ILTV live viral vector vaccines, gD has frequently been employed as a key immunogen (Zeng et al., 2023). Furthermore, the glycoprotein has been demonstrated to elicit both humoral and cell-mediated immune responses (Zhao et al., 2014). In our study, the saRNA vaccine was constructed using gD as the vaccine antigen. Zeta potential and particle size measurements demonstrated the homogeneous nature of the saRNA-LNP complexes (Fig. 1b, 1c), Western blot analyses confirmed stable expression of the antigen in vitro (Fig. 1e). Notably, the Western blot results revealed that the apparent molecular weight of the gD protein exceeded its actual molecular weight, suggesting potential glycosylation of the protein (He et al., 2022).

It is well-established that vaccine-induced immune responses play a crucial role in antiviral immunity and infection control (Fahey et al., 1983). In our study, chickens immunized with 1 μg/dose of saRNA-gD mounted neutralizing antibodies exceeding the seropositivity threshold as early as 7 after vaccination (Supplementary Fig. 3). Booster immunization induced robust humoral immunity, with peak serum neutralizing antibody titers reaching 1:415, significantly higher than levels induced by the commercial rFPV-LT vaccine (Fig. 2c). This data demonstrates the capacity of saRNA vaccines to elicit potent humoral responses. Furthermore, vaccinated chickens developed elevated IL-2 levels (Fig. 2d), suggesting the activation of T-cell responses.

The protective efficacy of ILTV vaccines was evaluated based on their ability to block or reduce the development of clinical symptoms and decrease the replication level of challenge viruses (Maekawa et al., 2021). Currently available first-generation live attenuated vaccines and second-generation live vectored vaccines against ILTV have not achieved 100% protection (Chen et al., 2020; Esaki et al., 2013; Maekawa et al., 2019; Palomino-Tapia et al., 2019). In our study, the saRNA-gD vaccine conferred a protection rate of 66.7%, which was comparable to that of the commercial vaccine rFPV-LT (Fig. 3e). Following vaccination, immunized chickens displayed normal feed and water uptake and exhibited normal behavioral patterns. Moreover, no significant difference in body weights was found compared to control animals (Fig. 2b), and injection sites showed no pathological lesions. These findings demonstrate a favorable safety of the vaccine. Notably, the saRNA-gD vaccine not only effectively alleviated clinical symptoms in challenge-infected chickens (Fig. 3a) and mitigated pathological damage in tracheal and lung tissues (Fig. 4a), but also significantly reduced virus loads in laryngeal swabs (Fig. 3d). These findings highlight the vaccine’s potential to address key challenges in ILTV vaccine development and application (Esaki et al., 2013).

Our findings also demonstrate the potential of saRNA-gD as a promising vaccine candidate. However, it is noteworthy that our data show both the commercial vaccine and the saRNA vaccine significantly induced IL-2 secretion (Fig. 2d), suggesting effective T-cell activation. Nevertheless, no significant increase in serum IFN-γ levels was observed (Fig. 2e), indicating that the Th1-type effector response or cytotoxic T-cell function was not completely elicited under these conditions. It has been established that cell-mediated immunity serves as the primary and partially protective immune response following ILTV infection (Fuchs et al., 2007; Honda et al., 1994; Shao et al., 2024). Therefore, the lack of cell-mediated immune responses may partially explain the suboptimal vaccine protection efficacy observed after viral challenge, highlighting the need for further optimization of this vaccine. Notably, this interpretation is limited by our focus on systemic immunity, therefore a comprehensive evaluation of antigen-specific cellular immune activation, using such as ELISpot or flow cytometry, is warranted for future studies.

Besides, a study of DNA vaccine against ILTV has shown that gB also induces protective immune responses, particularly in the presence of pro-inflammatory cytokine IL-18 (Chen et al., 2011). As we know, the commercial recombinant vectored vaccine rFPV-LT contains an expression cassette encoding ILTV gB. For this reason, in addition to gB, other glycoproteins (e.g., gD, gC, gE) are thought to be responsible for protective immunity against ILTV that requires the increased level of IFN-γ. Recently, a trivalent mRNA vaccine co-expressing gC-gD-gE of HSV-2 has been shown to protect against genital lesions and latent infection (Egan et al., 2020). These findings suggest that the development of effective herpesvirus vaccines should progress beyond traditional single-target strategies. A more promising strategy is to develop multivalent vaccines supplemented with immune enhancers, characterized by significantly elevated IFN-γ levels and improved cellular immunity, both of which are essential for viral clearance and the prevention of latent infection.

Finally, the saRNA-gD vaccine demonstrated favorable safety and immunogenicity in protecting against ILTV infection. Its comparatively low production cost highlights its advantage as a poultry vaccine platform. Future research should prioritize the screening of immunodominant epitopes to achieve coordinated induction of both humoral and cellular immunity, while also focusing on optimizing delivery systems to enhance mucosal immune responses and enable strong protective efficacy after a single immunization. Furthermore, exploring novel immunization routes will be essential to improve the applicability and practical utility of saRNA vaccine platform in the prevention and control of avian infectious diseases. Additionally, the development of multivalent or even combination vaccines should be explored to fully leverage the potential of this technology for use in commercial poultry.

In summary, our findings demonstrate that the saRNA-gD vaccine confers substantial immune protection against ILTV infection as evidenced by high levels of neutralizing antibodies and reduced virus replication post challenge, which in turn mitigated pathological damage and protected against clinical disease and death. Thus, our study provides evidence that the saRNA-gD vaccine may hold promise as a next-generation ILTV candidate with distinct advantages over currently used modified live virus or vectored vaccines.

Authorship contribution statement

Huan-huan Li: Conceptualization, Data curation, Methodology, Resources, Software, Validation, Writing-original draft, Writing-review & editing. Ling Tong: Conceptualization, Data curation, Methodology, Resources, Software, Validation, Writing-original draft, Writing-review & editing. Sai-sai Chen: Data curation, Formal analysis, Methodology, Resources, Software, Validation. Zhi-xiang Bi: Data curation, Formal analysis, Methodology, Resources, Software, Validation. Gang Hu: Data curation, Formal analysis, Methodology, Resources, Software, Validation. Wan-li Chen: Data curation, Formal analysis, Methodology, Resources, Software, Validation. Jia-li Yu: Data curation, Formal analysis, Methodology, Resources, Software, Validation. Ruo-nan Tao: Data curation, Formal analysis, Methodology, Resources, Software, Validation. Teng Huang: Conceptualization, Funding acquisition, Project administration, Supervision, Visualization, Writing-original draft, Writing-review & editing. Shu-hua Xia: Conceptualization, Funding acquisition, Project administration, Supervision, Visualization, Writing-original draft, Writing-review & editing. Nikolaus Osterrieder: Conceptualization, Funding acquisition, Project administration, Supervision, Visualization, Writing-original draft, Writing-review & editing.

Ji-chun Wang: Conceptualization, Funding acquisition, Project administration, Supervision, Visualization, Writing-original draft, Writing-review & editing.

CRediT authorship contribution statement

Huan-huan Li: Writing – original draft, Visualization, Validation, Software, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Ling Tong: Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Sai-sai Chen: Visualization, Supervision, Resources, Methodology, Investigation, Formal analysis, Data curation. Zhi-xiang Bi: Visualization, Supervision, Resources, Methodology, Investigation, Formal analysis, Data curation. Gang Hu: Visualization, Supervision, Resources, Methodology, Investigation, Formal analysis, Data curation. Wan-li Chen: Visualization, Supervision, Resources, Methodology, Investigation, Formal analysis, Data curation. Jia-li Yu: Visualization, Supervision, Resources, Methodology, Investigation, Formal analysis, Data curation. Ruo-nan Tao: Visualization, Supervision, Resources, Methodology, Investigation, Formal analysis, Data curation. Teng Huang: Writing – review & editing, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Shu-hua Xia: Writing – review & editing, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Nikolaus Osterrieder: Writing – review & editing, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Ji-chun Wang: Writing – review & editing, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was partially funded by Guangxi Science and Technology Major Program (AA23062050).

Footnotes

The appropriate scientific section for the paper: Immunology, Health and Disease.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2026.106632.

Contributor Information

Teng Huang, Email: thomashwang@gxu.edu.cn.

Shu-hua Xia, Email: shuhua.xia@jinfapharma.com.

Nikolaus Osterrieder, Email: Klaus.Osterrieder@tiho-hannover.de.

Ji-chun Wang, Email: 19970021@jaas.ac.cn.

Appendix. Supplementary materials

mmc1.docx (1.7MB, docx)

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