An IMS 1313-based inactivated PRV vaccine with a novel immunopotentiator induces robust protective immunity against virulent variants

Highlights

• •In mice, the IMS 1313 + c formulation induced a neutralizing antibody titer of 1:32 and 93.3% protection after two immunizations at a 28-day interval, markedly higher than control groups (1:5.6).
• •In piglets, intramuscular vaccination elicited a peak titer of 1:295.8, outperforming oil-adjuvant (1:28.2) and live vaccine (1:13.1) groups, and provided 100% clinical protection with reduced viral shedding and minimal pathological changes.
• •Intradermal administration showed slightly lower efficacy, highlighting the importance of immunization route.
• •Notably, neutralizing antibody levels did not fully correlate with protective efficacy, suggesting the involvement of additional immune mechanisms.

Abstract

Background

Pseudorabies virus (PRV) variants have emerged as major swine pathogens associated with high piglet mortality and reproductive losses in China since 2011. Conventional oil-adjuvant vaccines are effective but frequently induce adverse local reactions. Water-soluble adjuvants, such as the nano-adjuvant IMS 1313, confer improved safety and potent immunogenicity. This study aimed to develop an inactivated PRV vaccine based on IMS 1313 combined with an immunopotentiator and evaluate its immune protective efficacy.

Methods

Multiple adjuvant-immunopotentiator combinations were screened. The PRV LA1206-80 strain formulated with IMS 1313 plus immunopotentiator c exhibited the optimal comprehensive performance and was selected for subsequent evaluation. Mice were immunized to detect serum neutralizing antibody titers and survival-based protection rates. Piglets were immunized via intramuscular or intradermal routes, and immune responses, post-challenge viral shedding, as well as histopathological lesions were monitored. Oil-adjuvant inactivated vaccine and live attenuated vaccine were set as control groups. Virus neutralization assays were performed to determine neutralizing antibody responses.

Results

After two immunizations with a 28-day interval, the IMS 1313 + immunopotentiator c formulation induced a neutralizing antibody titer of 1:32 and achieved 93.3 % protection in mice, which was markedly higher than that of the control groups (1:5.6). In this preliminary exploratory piglet trial, intramuscular vaccination yielded peak neutralizing antibody titers of 1:295.8, which appeared higher than those of the oil‑adjuvant vaccine (1:28.2) and live vaccine (1:13.1). This immunization regimen achieved 100 % survival, accompanied by reduced viral shedding and minimal pathological lesions. Intradermal vaccination conferred relatively lower protective efficacy, underscoring the critical influence of immunization route.

Conclusion

IMS 1313 combined with immunopotentiator c markedly enhances the immunogenicity and safety profile of inactivated PRV vaccines in mice. Preliminary data from piglets revealed favourable protective trends upon intramuscular vaccination. This candidate represents a promising safer alternative compared with conventional oil-adjuvant vaccines and warrants further larger-scale efficacy evaluation.

1. Introduction

Pseudorabies virus (PRV) is a highly pathogenic alphaherpesvirus with pigs as its only natural host. Following infection, PRV initially replicates in the upper respiratory tract, particularly within the tonsillar epithelium (Verpoest et al., 2017). The virus subsequently spreads from infected respiratory epithelial cells into the underlying connective tissue, gaining access to regional lymph nodes and the bloodstream (Narita et al., 1994). Within these lymphoid tissues, viral amplification is pronounced, and infected leukocyte populations may contribute to systemic dissemination through lymphatic and circulatory pathways (Lamote et al., 2016). This results in a cell-associated viremia within peripheral blood mononuclear cells, which facilitates systemic spread and supports viral trafficking to multiple target organs (Lamote et al., 2016; Nauwynck and Pensaert, 1995). In pregnant sows, viremia has been considered an important prerequisite for viral dissemination to the uterus, where infection can lead to severe reproductive failure. In neonatal piglets, PRV infection is particularly devastating, with morbidity and mortality rates approaching 100%. Since 2011, novel PRV variants with enhanced pathogenicity and immune escape capacity have become endemic in China, posing new challenges for disease control (Tan et al., 2021). Although older pigs may develop partial clinical resistance, infection frequently manifests as respiratory disease accompanied by necrotizing splenitis, hepatitis, lymphadenitis, and encephalitis, collectively resulting in substantial economic losses in breeding herds. Consequently, PRV continues to represent a major threat to the swine industry, underscoring the urgent need for more effective preventive strategies.

Vaccine adjuvants play a central role in enhancing vaccine immunogenicity. These compounds, when incorporated into vaccine formulations, improve antigen uptake and presentation, activate innate immune pathways, and amplify both the magnitude and duration of adaptive immune responses (Zhao et al., 2023). Vaccination remains the cornerstone strategy for PRV control in endemic regions (Zheng et al., 2022; Zhou et al., 2023). However, most currently licensed PRV vaccines rely on oil-based adjuvant systems, which generate a depot effect that enables sustained antigen release. While such formulations are immunologically effective, they are frequently associated with local tissue reactions, including granuloma formation, sterile abscesses, and in some cases systemic inflammatory responses (Stewart-Tull, 1996; Lavelle and McEntee, 2024; Hu et al., 2021; Zhang et al., 2021). Moreover, conventional vaccines do not always provide complete protection against emerging PRV variants circulating in China. To address these limitations, aqueous adjuvant systems have attracted increasing attention as safer and more versatile alternatives. For example, Liting Hou et al. reported that an inactivated PRV vaccine formulated with ISA 206 water-in-oil-in-water adjuvant combined with the immunopotentiator c-di-GMP induced stronger CD4⁺ and CD8⁺ T-cell responses compared with both live attenuated and conventional inactivated vaccines. This formulation was able to elicit balanced innate and adaptive immune responses and achieved protection levels comparable to live vaccines (Hou et al., 2022). These findings highlight the importance of rational adjuvant–immunopotentiator selection for optimizing PRV vaccine performance.

IMS 1313 is a composite water-soluble nano-adjuvant developed by Seppic. It consists of microemulsion-based structures and immunostimulating complexes, providing broad compatibility with diverse antigen types, including bacterial, viral, parasitic, and recombinant subunit vaccines. Its physicochemical properties, including small particle size, homogeneous dispersion, and large surface area, contribute to efficient antigen adsorption and in vivo uptake. In addition, IMS 1313 exhibits strong immunostimulatory capacity and is capable of inducing both humoral and cellular immune responses. It also demonstrates favorable safety and stability profiles, and is suitable for prime–boost immunization strategies (Hussein et al., 2018; Jang et al., 2011). Compared with oil-based adjuvants, aqueous systems such as IMS 1313 offer improved syringeability, faster absorption, and reduced local reactogenicity. They are also compatible with large-scale vaccination approaches in intensive livestock production, including mass administration via drinking water or spray delivery (Gu et al., 2024). Notably, IMS 1313 has been incorporated into veterinary vaccine development programs in South Korea for swine pathogens, including PRV-related applications.

Immunopotentiators are biologically active molecules capable of enhancing both antigen-specific and non-specific immune responses, thereby accelerating immune activation and improving response magnitude. In PRV vaccine development, combining immunopotentiators with IMS 1313 may provide synergistic effects on immunogenicity and protective efficacy. Based on these considerations, the present study was designed to develop an inactivated PRV vaccine using IMS 1313 in combination with a selected immunopotentiator. Using a highly virulent PRV strain identified in our laboratory, we systematically assessed humoral immune responses and protective efficacy in both murine and piglet models. This work aims to provide a safer and more effective vaccination strategy for PRV control and to support the development of multivalent vaccine platforms applicable to intensive swine production systems.

2. Materials and methods

2.1. Virus strains, cells, and experimental animals

The PRV LA1206– 80 strain used for vaccine preparation was stored in our laboratory at the Jiangsu Academy of Agricultural Sciences, with a viral titer of 10^8.65 TCID₅₀/mL. For challenge experiments, the virulent PRV variant AH02LA (CGMCC No 10,891) was employed; this strain was originally isolated from infected pigs on a commercial farm in Anhui Province and maintained at 10^8.60 TCID₅₀/mL at the National Research Center for Veterinary Biologicals Engineering and Technology. Swine testicle (ST) cells were obtained from the same center and cultured under standard laboratory conditions. ST cells were free of mycoplasma contamination and used within 15 passages. Female ICR mice (4–6 weeks old, 20–22 g) were purchased from Yangzhou University. Eighteen piglets (21–35 days old), negative for PRV and PRRSV antigens and antibodies, were sourced from a PRV- and PRRSV-free farm. All animals were housed in controlled conditions (22–25 °C, 50–60% relative humidity, 12 h light/dark cycle) with free access to feed and water. Animals were randomly allocated into experimental groups. Clinical observation, survival recording, and histopathological scoring were performed by investigators blinded to group assignments. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Jiangsu Academy of Agricultural Sciences and conducted following institutional biosafety and animal welfare guidelines.

2.2. Main reagents and instruments

ISA 15A VG (Lot No 220,217,014,944), ISA 201 VG (Lot No 220,705,025,159), and IMS 1313 nano-adjuvant were obtained from SEPPIC (France). Immunopotentiators c, d, and e were provided by the Animal Immunotechnology Innovation Team of our institute. Immunopotentiator c is a proprietary, in‑house‑developed immune‑enhancing compound for experimental use (Wang et al., 2023). Fetal bovine serum (FBS) was purchased from Sijiqing Company (China), suspension culture medium and DMEM from Gibco (USA), trypsin (0.25%) from Beyotime Biotechnology (China), and formaldehyde from Sinopharm Chemical Reagent Co., Ltd. (China). Major instruments included a constant-temperature orbital shaker (ISH, USA), CO₂ incubator (Thermo Fisher Scientific, USA), biosafety cabinet (Suzhou Antai Air Technology Co., Ltd., China), and constant-temperature water bath (Shanghai Yiheng Scientific Instrument Co., Ltd., China).

2.3. Antigen preparation

The PRV LA1206– 80 strain was propagated in ST cells under standard conditions until cytopathic effect (CPE) reached approximately 80%; culture supernatants were then harvested, clarified by centrifugation, and titrated using the TCID₅₀ method. Viral suspensions were inactivated with 0.15% formaldehyde at 37 °C for 36 h. After inactivation, formaldehyde was neutralized with sodium thiosulfate prior to vaccine formulation. Complete viral inactivation was verified by three serial blind passages on ST cell monolayers. Each passage was cultured for 5–7 days, and no cytopathic effect was detected in any passage. The fully inactivated antigen was stored at 4 °C prior to vaccine formulation.

2.4. Verification of IMS 1313 adjuvant efficacy in mice

Seventy-five female ICR mice were randomly assigned to five groups (n = 15 per group) according to the immunization scheme in Table 1. Vaccines containing PRV LA1206– 80 antigen formulated with IMS 1313 or ISA 201, with or without immunopotentiators, were administered subcutaneously at the dorsal neck region (0.1 or 0.2 mL per mouse). A booster was given 28 days after primary immunization using the same formulation and dose. Blood samples were collected via the orbital venous plexus at 7-day intervals starting 14 days after primary immunization; serum was separated and stored at −20 °C for analysis. Serum samples were heat-inactivated at 56 °C for 30 min before detection. Neutralizing antibody titers were measured using a fixed-virus serum neutralization assay with 100 TCID₅₀ PRV per well. Fourteen days after the booster, mice were challenged subcutaneously with 0.2 mL of PRV AH02LA (10^5.50 TCID₅₀ per mouse) and monitored daily for 14 days for clinical signs and survival. Mice exhibiting severe clinical signs (e.g., paralysis, moribund status) were humanely euthanized according to animal welfare criteria. Protection rates were calculated based on survival, and dose selection was guided by preliminary optimization experiments and standard murine immunization practices for inactivated viral vaccines.

Table 1.

Experimental groups of mice.

Group	Strain	Adjuvant	Immunopotentiator	Dose (mL/mouse/time)	Challenge dose
1	LA1206–80	IMS 1313 VG	c	0.1	105.50 TCID50
2	LA1206–80	IMS 1313 VG	–	0.1	105.50 TCID50
3	LA1206–80	IMS 1313 VG	–	0.2	105.50 TCID50
4	LA1206–80	ISA 201 VG	e	0.1	105.50 TCID50
5	Challenge control	–	–	–	105.50TCID50

Footnote: IMS, immunostimulating complex; ISA, water-in-oil-in-water emulsion adjuvant; VG, veterinary grade. Immunopotentiators c and e are proprietary immune-enhancing compounds developed by the authors’ laboratory.

2.5. Verification of IMS 1313 adjuvant efficacy in piglets

Eighteen PRV-seronegative piglets aged 21–35 days were randomly assigned to six groups (n = 3 per group) as shown in Table 2. Piglets were immunized either intramuscularly (IM) or intradermally (ID) with inactivated PRV vaccines formulated with IMS 1313, with or without the addition of immunopotentiator c. live attenuated vaccine (LA2017) and oil-adjuvant inactivated (LA-A strain, 10^8.3TCID₅₀/ml before inactivation) vaccines served as positive controls, and DMEM was used as a challenge control. Inactivated‑vaccine groups received primary immunization on day 0 and a homologous booster on day 28 via the same administration route. By contrast, the live‑attenuated vaccine was delivered as a single injection on day 0. The IM dose was 2 mL per piglet, while the ID dose was 0.2 mL, reflecting differences in antigen delivery efficiency between routes. The total antigen dose was not identical for intramuscular and intradermal vaccination. This difference arises from practical constraints for intradermal injection, and should be taken into account when comparing protective outcomes across immunization routes.

Table 2.

Experimental design of IMS 1313-based vaccine evaluation in piglets.

Group	Strain	Route	Formulation	Immunopotentiator	Dose (mL/piglet)	Challenge dose
1	LA1206–80	Intramuscular (IM)	IMS 1313	–	2 mL	106.5 TCID50/piglet
2	LA1206–80	Intramuscular (IM)	IMS 1313	c	2 mL	106.5 TCID50/piglet
3	LA1206–80	Intradermal (ID)	IMS 1313	c	0.2 mL	106.5 TCID50/piglet
4	LA2017	Intramuscular (IM)	live attenuated vaccine	–	105.0 TCID50/piglet	106.5 TCID50/piglet
5	LA-A	Intramuscular (IM)	Oil-adjuvant inactivated vaccine	–	2 mL	106.5 TCID50/piglet
6	Challenge control	–	DMEM	–	–	106.5 TCID50/piglet

Footnote: IM, intramuscular injection; ID, intradermal injection; DMEM, Dulbecco’s modified Eagle’s medium; IMS, immunostimulating complex-based nano-adjuvant; LA-A, commercial oil-adjuvant inactivated PRV vaccine; LA2017, live attenuated PRV vaccine.

Challenge infection was performed using PRV strain AH02LA at 10^6.5 TCID₅₀ per piglet via intranasal inoculation. TCID₅₀ indicates 50% tissue culture infectious dose.

Blood samples were collected weekly from week 2 post-primary immunization via jugular venipuncture and stored at −20 °C until analysis. Neutralizing antibody titers were determined using a fixed-virus serum neutralization assay.

Fourteen days after the booster, all piglets were challenged intranasally with 2 mL PRV AH02LA containing 10^6.50 TCID₅₀ per animal. This challenge dose is substantially higher than the estimated infectious dose encountered under natural transmission conditions, providing a stringent test for vaccine efficacy (Ling et al., 2025). Clinical observations were recorded daily for 10 days post-challenge. Clinical observations included body temperature, respiratory signs, neurological symptoms, appetite, and general behavior, with any abnormality recorded as a clinical case. Nasal swabs were collected daily to assess viral shedding by TCID₅₀ titration, with shedding duration defined as the number of consecutive days with detectable virus. At the end of the observation period, surviving animals were humanely euthanized, and lung, tonsil, and lymph node tissues were collected for histopathological examination using hematoxylin–eosin staining. Protective efficacy was evaluated based on survival, clinical signs, and viral shedding. Due to the small sample size (n = 3 per group), this study serves only as preliminary candidate-screening data and cannot provide definitive efficacy conclusions for field application.

2.6. Statistical analysis

All quantitative data are presented as mean ± standard deviation (SD). For mouse experiments with sufficient sample size (n = 15 per group), the Shapiro‑Wilk test was used to evaluate the normality of data distribution. Normally distributed data were analyzed by one‑way analysis of variance (ANOVA) with Tukey’s post‑hoc test for multiple comparisons. Non‑normally distributed mouse data were analyzed using the Kruskal‑Wallis H test followed by Dunn’s multiple‑comparison test. Given the limited sample size of piglet experiments (n = 3 per group), reliable normality assessment was not feasible; piglet datasets were analyzed by Kruskal‑Wallis H test followed by Dunn’s multiple‑comparison test for pairwise comparisons. Survival rates and survival curves were analyzed by the Kaplan‑Meier method and compared using the log‑rank test. Statistical analyses were performed using GraphPad Prism 6.0 and SPSS 16.0. A value of P < 0.05 was considered statistically significant.

3. Results

3.1. Screening of adjuvants and immunopotentiators

The virulent PRV LA1206-80 strain, previously characterized as a highly pathogenic isolate in our laboratory, was used to evaluate different adjuvant–immunopotentiator combinations. Marked variability in both protective efficacy and safety profile was observed across formulations (Table 3). Several combinations, including Gel02 + immunopotentiator a, Gel02 + immunopotentiator b, Gel02 + immunopotentiator d, IMS 1313 + immunopotentiator c, ISA 15A + immunopotentiator b, ISA 15A + immunopotentiator d, and ISA 201 + immunopotentiator e, achieved complete protection following challenge, whereas reduced protection was recorded in IMS 1313 + immunopotentiator b (66.7%), IMS 1313 + immunopotentiator d (70%), and ISA 15A + immunopotentiator a (66.7%). In parallel, safety assessment revealed clear differences among groups, with Gel02 + immunopotentiator a showing the lowest safety performance (40%), while ISA 15A + immunopotentiator b and ISA 15A + immunopotentiator d were associated with relatively higher frequencies of injection-site residue or irritation.

Table 3.

Immune protection and safety evaluation of adjuvant–immunopotentiator combinations in mice.

Group	Adjuvant	Immunopotentiator	Deaths after immunization	Safety (%)	Deaths after challenge	Protection rate (%)
1	GEL 02	a	6/10	40	0/4	100
2	GEL 02	b	0/10	100	0/10	100
3	GEL 02	c	0/10	100	1/10	90
4	GEL 02	d	0/10	100	0/10	100
5	IMS 1313	a	2/10	80	0/8	100
6	IMS 1313	b	1/10	90	3/9	66.7
7	IMS 1313	c	0/10	100	0/10	100
8	IMS 1313	d	0/10	100	3/10	70
9	ISA 15A	a	1/10	90	3/9	66.7
10	ISA 15A	b	0/10	100	0/10	100
11	ISA 15A	c	0/10	100	1/10	90
12	ISA 15A	d	0/10	100	0/10	100
13	ISA 201	e	0/10	100	0/10	100
14	Challenge control	–	–	–	8/10 mortality	–

Footnote: Safety refers to the proportion of animals without observable adverse reactions following immunization. Protection rate = (surviving animals after challenge / total challenged animals) X 100%. ISA 201 VG is a commercial water-in-oil-in-water adjuvant. GEL 02 and ISA 15A are commercial adjuvant formulations. Immunopotentiators a–e are proprietary compounds.

Among all tested formulations, IMS 1313 combined with immunopotentiator c consistently showed the most balanced profile. As shown in Fig. 1, this group achieved complete protection while exhibiting no detectable vaccine residue or local irritation at the injection site. By contrast, several Gel02- and ISA 15A-based formulations showed either residual antigen persistence or mild local inflammatory reactions. Taken together, considering both protective efficacy and local safety, the IMS 1313 + immunopotentiator c formulation was selected for subsequent studies.

Fig. 1.

Representative macroscopic photographs showing local tissue residuals and injection-site reactions collected 7 days post-immunization with different adjuvant-immunopotentiator combinations. (a) Gel02 + immunopotentiator b; (b) Gel02 + immunopotentiator d; (c) IMS 1313 + immunopotentiator c; (d) ISA 15A + immunopotentiator b; (e) ISA 15A + immunopotentiator d; (f) ISA 201 + immunopotentiator e.

3.2. Neutralizing antibody responses in mice

Neutralizing antibody responses were assessed following primary and booster immunization in mice. As illustrated in Fig. 2, antibodies were first detected in IMS 1313-based groups after primary immunization and increased markedly following booster administration. The IMS 1313 + immunopotentiator c formulation induced significantly higher neutralizing antibody titers compared with IMS 1313 alone, indicating a clear adjuvant effect of immunopotentiator c. In addition, dose-dependent differences were observed in the absence of immunopotentiator, with the 0.2 mL IMS 1313 group eliciting stronger responses than the 0.1 mL group.

Fig. 2.

Neutralizing antibody titers in mice immunized with different vaccine formulations.

Serum neutralizing antibody titers measured in mice after primary and booster vaccination. (a) IMS 1313 + immunopotentiator c (0.1 mL group); (b) IMS 1313 (0.1 mL group); (c) IMS 1313 (0.2 mL group); (d) ISA 201 + immunopotentiator e (0.1 mL group). Data are presented as mean ± SD. Statistical comparisons were performed by one-way ANOVA with Tukey’s post-hoc test or Kruskal-Wallis H test with Dunn’s post-hoc test according to data normality. *P < 0.05; ***P < 0.001.

After booster immunization, the IMS 1313 + immunopotentiator c low-dose group reached a neutralizing antibody titer of 1:32, compared with 1:5.60 in the corresponding group without immunopotentiator (Table 4). By the second week post-booster, titers further increased to 1:44.70 in the IMS 1313 + immunopotentiator c group, surpassing those observed in the high-dose formulation. In contrast, the ISA 201 + immunopotentiator e group exhibited an early peak (1:52.98) followed by a rapid decline to 1:6.72. Overall, these results suggest that IMS 1313 combined with immunopotentiator c promotes both rapid induction and sustained maintenance of humoral immunity, with evidence of a dose-sparing effect.

Table 4.

Mouse safety and protective efficacy of IMS 1313 formulations.

Group	Strain	Adjuvant	Immunopotentiator	Dose (mL/mouse)	Safety (%)	Mortality (post-challenge)	Protection rate (%)
1	LA1206–80	IMS 1313	c	0.1	100	1/15	93.3
2	LA1206–80	IMS 1313	–	0.1	100	3/15	80
3	LA1206–80	IMS 1313	–	0.2	100	0/15	100
4	LA1206–80	ISA 201	e	0.1	100	0/15	100
5	Challenge control	–	–	–	–	14/15	–

Footnote: Safety indicates absence of visible adverse reactions after vaccination. Protection rate = (survival animals / total animals) × 100%. ISA 201 VG was used as a positive control adjuvant. TCID₅₀, 50% tissue culture infectious dose.

3.3. Protective efficacy in mice

Protective efficacy was evaluated using a virulent PRV AH02LA challenge model. As summarized in Table 4, the high-dose IMS 1313 + immunopotentiator c group achieved complete protection (100% survival), whereas the low-dose IMS 1313 + immunopotentiator c group provided 93.3% protection. In comparison, the IMS 1313 group without immunopotentiator conferred only 80% protection.

Following challenge, only mild and transient clinical signs, including ruffled fur and reduced activity, were observed in a limited number of animals in the low-dose groups. These signs resolved spontaneously within several days without progression to neurological manifestations. Importantly, no vaccine-associated adverse effects were detected in the IMS 1313 + immunopotentiator c group. Collectively, these findings confirm that immunopotentiator c markedly enhances the protective efficacy of IMS 1313-based inactivated vaccines.

3.4. Immune responses and protective efficacy in piglets

Serum neutralizing antibody responses in piglets were monitored following immunization. As shown in Fig. 3, the IMS 1313 + immunopotentiator c intramuscular group tended to elicit the strongest humoral response, reaching a peak titer of 1:295.80 after booster immunization. This response appeared higher than that observed in the oil‑adjuvant inactivated vaccine group (1:28.20) and the live vaccine group (1:13.07; Kruskal‑Wallis H test followed by Dunn’s multiple‑comparison test, *P < 0.05). Given the small sample size (n = 3 per group), these antibody differences should be interpreted as preliminary trends rather than definitive inter‑group conclusions. The intradermal IMS 1313 + immunopotentiator c group also generated detectable antibody responses, although at a lower peak level of 1:126.67. After boosting, antibody levels in intramuscular IMS 1313-based groups remained relatively stable, whereas intradermal administration resulted in a reduced magnitude of response.

Fig. 3.

Neutralizing antibody responses in piglets receiving different vaccine formulations.

Serum neutralizing antibody titers of piglets after primary and booster immunization. (a) IMS 1313 intramuscular (IM) group; (b) IMS 1313 + immunopotentiator c (IM) group; (c) IMS 1313 + immunopotentiator c intradermal (ID) group; (d) Live attenuated vaccine group; (e) Oil-adjuvant inactivated vaccine group. Data are presented as mean ± SD. Group comparisons were performed using Kruskal‑Wallis H test followed by Dunn’s multiple‑comparison test. *P < 0.05; **P < 0.01; ***P < 0.001.

Following virulent PRV challenge, all vaccinated groups achieved 100% survival, whereas the challenge control group exhibited 33.3% mortality (Table 5). Despite uniform survival, differences in clinical manifestations were observed. Piglets in the IMS 1313 + immunopotentiator c intramuscular group showed no apparent clinical abnormalities, whereas mild and transient signs, including sneezing and reduced activity, were observed in the IMS 1313-alone and intradermal IMS 1313 + immunopotentiator c groups. These signs resolved without progression to severe disease.

Table 5.

Clinical outcomes and protective efficacy of different immunization strategies in piglets.

Group	Immunization strategy	Morbidity rate	Mortality rate	Clinical protection (n/N, %)
a	IMS 1313 (intramuscular)	1/3	0/3	2/3, 66.7%
b	IMS 1313 + c (intramuscular)	0/3	0/3	3/3, 100%
c	IMS 1313 + c (intradermal)	1/3	0/3	2/3, 66.7%
d	LA-A inactivated vaccine (oil-adjuvant)	1/3	0/3	2/3, 66.7%
e	LA2017 live attenuated vaccine	0/3	0/3	3/3, 100%
f	Challenge control	3/3	1/3	0/3, 0%

Footnote: Clinical protection rate was defined as the proportion of animals surviving without overt clinical signs during the 10-day observation period after challenge infection. Due to the limited sample size (n = 3 per group), this study was designed as a preliminary exploratory evaluation rather than a statistically powered efficacy trial. Therefore, statistical comparisons between groups were not performed for Table 5. Morbidity was defined as the presence of any observable clinical signs, including fever, respiratory distress, neurological symptoms, anorexia, or reduced activity.

Consistent with clinical findings, viral-shedding patterns differed noticeably across experimental groups (Table 6). Piglets in the IMS 1313 + immunopotentiator c intramuscular group and oil-adjuvant vaccine group had shorter virus-shedding windows (2–6 days). By contrast, longer viral shedding was seen for the IMS 1313 intradermal group (3–9 days) and challenge control group (1–9 days). The live-vaccine group displayed intermediate shedding lasting 3–6 days. Examination of H&E-stained tissue sections further showed that intramuscular vaccination with IMS 1313 + immunopotentiator c largely maintained normal tissue structure in lung, tonsil and lymph node. Mild inflammatory lesions remained in animals receiving intradermal vaccination and in control piglets (Fig. 4, Fig. 5, Fig. 6). Gross pathological changes for these organs can be viewed in Figs. S1-S3.

Table 6.

Duration of viral shedding in piglets following PRV challenge.

Group	Duration of viral shedding (days)
IMS 1313 (2 mL, intramuscular)	2–8
IMS 1313 + c (2 mL, intramuscular)	2–6
IMS 1313 + c (0.2 mL, intradermal)	3–9
LA2017 live vaccine	3–6
LA-A inactivated vaccine	2–6
Challenge control	1–9

Note: Viral shedding duration was defined as the number of consecutive days during which infectious virus was detected in nasal swabs following PRV challenge. Nasal swabs were collected daily and analyzed using the TCID₅₀ assay. Shedding was considered terminated when viral detection became negative for two consecutive days. Due to the limited sample size (n = 3 per group), shedding duration is presented as group-level observed ranges rather than statistically modeled endpoints.

Fig. 4.

Histopathological changes in lung tissues after PRV AH02LA challenge.

Representative Hematoxylin-eosin (H&E)-stained micrographs, 200 × magnification, scale bar = 200 μm. (a) IMS 1313 (intramuscular) (IM) group; (b)IMS 1313 + immunopotentiator c (IM) group; (c) IMS 1313 + immunopotentiator c intradermal (ID) group; (d) Oil-adjuvant inactivated vaccine group; (e) LA2017 live vaccine; (f) Challenge control group.

Fig. 5.

Histopathological changes in tonsil tissues after PRV AH02LA challenge.

Representative Hematoxylin-eosin (H&E)-stained micrographs, 200 × magnification, scale bar = 200 μm. (a)IMS 1313 intramuscular (IM) group; (b) IMS 1313 + immunopotentiator c (IM) group; (c)IMS 1313 + immunopotentiator c intradermal (ID) group; (d) Oil-adjuvant inactivated vaccine group; (e) LA2017 live vaccine; (f) Challenge control group.

Fig. 6.

Histopathological changes in lymph node tissues after PRV AH02LA challenge.

Representative Hematoxylin-eosin (H&E)-stained micrographs, 200 × magnification, scale bar = 200 μm. (a)IMS 1313 intramuscular (IM) group; (b) IMS 1313 + immunopotentiator c (IM) group; (c) IMS 1313 + immunopotentiator c intradermal (ID) group; (d) Oil-adjuvant inactivated vaccine group; (e) LA2017 live vaccine; (f) Challenge control group.

Fig. 5.

Taken together, these preliminary exploratory data suggest that intramuscular IMS 1313 + immunopotentiator c offers favourable protective trends in piglets. Within this preliminary trial, it yielded survival outcomes comparable to the oil‑adjuvant vaccine while displaying a better safety profile.

3.5. Clinical observations in piglets

Following challenge infection with PRV AH02LA, the control group developed severe clinical signs, including hyperthermia (>41 °C), anorexia, depression, and neurological symptoms, with mortality occurring by day 7. In contrast, all vaccinated groups were protected from death. The IMS 1313 + immunopotentiator c intramuscular group showed only minimal transient signs, whereas mild clinical symptoms, including sneezing and reduced activity, were observed in the IMS 1313-alone and intradermal groups between days 2–6. These symptoms resolved spontaneously without progression to severe disease (Table 5). Overall, intramuscular administration of IMS 1313 + immunopotentiator c appeared to confer the most favorable clinical protection among tested groups.

3.6. Viral shedding assessment

Post-challenge viral shedding was further analysed based on serial nasal swab samples (Table 6). Consistent with our pathological observations, intramuscular immunization with IMS 1313 + immunopotentiator c effectively limited viral replication in the upper respiratory tract. Notably, despite measurable neutralizing antibody titers generated by the intradermal IMS 1313 + immunopotentiator c group, this route failed to restrict prolonged virus shedding. Collectively, these preliminary findings reinforce that intramuscular delivery of this candidate vaccine tends to achieve protection comparable to oil-adjuvant formulations while featuring an improved safety profile.

4. Discussion

An ideal vaccine adjuvant should exhibit multiple properties, including the ability to enhance both humoral and cellular immune responses, improve the immunogenicity of weak antigens without inducing significant toxicity, allow administration through different routes, remain effective in immunocompromised hosts, and avoid residual toxicity in food-producing animals (Stewart-Tull, 1996; Schijns and Lavelle, 2011a; Schijns and Lavelle, 2011b; Takahashi et al., 1993). Current adjuvants are generally categorized into aqueous-based and oil-emulsion systems. Oil-emulsion adjuvants are widely used due to their depot effect, which prolongs antigen retention and sustains immune stimulation, thereby enhancing both the magnitude and duration of immune responses. However, their application is limited by poor biodegradability, impaired local clearance, and risks of adverse reactions such as granuloma formation, sterile abscesses, and in severe cases, systemic toxicity or mortality (Halenova et al., 2020; Petrovsky and Aguilar, 2004). The LA-A inactivated PRV vaccine, which employs an oil-emulsion adjuvant, exemplifies this trade-off: it provides prolonged antigen stimulation yet is still associated with local tissue reactions and safety concerns (Broutin et al., 2023). Moreover, the viscous and irritant nature of oil-based adjuvants makes them poorly suited for intradermal application for practical vaccination purposes, despite the dose-sparing advantages and potent immune-inducing capacity of this route mediated by abundant dermal antigen-presenting cells (Alsakini et al., 2024; Tahara et al., 2022; Schijns et al., 2014; Li et al., 2022a). These limitations highlight the need for alternative adjuvant systems that maintain immunogenicity while improving safety profiles.

Nanoparticle-based adjuvants have emerged as a promising alternative platform. Defined by particle sizes typically below 1000 nm, nano-adjuvants can enhance immune responses by improving antigen stability, facilitating cellular uptake, and modulating antigen presentation. Their physicochemical properties, including high surface area-to-volume ratio and tunable surface chemistry, enable efficient antigen adsorption and controlled delivery, thereby improving immunogenicity and bioavailability (Saleemi et al., 2024; Shan et al., 2022). Accumulating evidence suggests that nano-adjuvants promote uptake by dendritic cells and macrophages, enhance antigen processing and presentation, and activate both Th1- and Th2-biased responses. In addition, they may contribute to the generation of long-lived memory B and T cells, which are essential for durable protection.

IMS 1313 is a water-soluble nano-adjuvant developed by Seppic, consisting of microemulsion-based structures combined with immunostimulating complexes. Its formulation enables compatibility with a broad range of antigens, including viral, bacterial, parasitic, and subunit vaccines. Compared with oil-based systems, IMS 1313 offers improved dispersibility, reduced local reactogenicity, and enhanced suitability for large-scale veterinary application. Importantly, its aqueous nature facilitates alternative delivery strategies such as mass vaccination approaches in intensive farming systems. These features have supported its application in commercial veterinary vaccines, including PRV vaccines in certain regions.

In the murine model, IMS 1313 exhibited a favorable safety profile and consistent immunogenicity across tested dose ranges. A clear dose-dependent effect was observed in groups receiving IMS 1313 without immunopotentiator, suggesting that antigen load remains a primary determinant of immune priming in inactivated vaccine systems. At the same time, the addition of immunopotentiator c significantly enhanced immune outcomes, particularly under low-antigen conditions, where it partially compensated for reduced antigen dose and improved protective efficacy. Conversely, increasing antigen dose alone also improved immune protection, indicating that both antigen quantity and adjuvant composition jointly contribute to vaccine performance. Together, these findings informed the dosing strategy used in the piglet study, ensuring balanced antigen exposure across intramuscular and intradermal routes while maintaining comparability between formulations.

In the piglet challenge model, immune outcomes were more complex and revealed a partial dissociation between humoral antibody titers and protective efficacy. It should be noted that piglet data originate from a preliminary exploratory experiment with limited sample size (n = 3 per group). Although IMS 1313 and IMS 1313+c groups induced comparable neutralizing antibody levels at certain time points, the IMS 1313+c intramuscular group tended to show favorable clinical protection and reduced viral shedding. This trend suggests that neutralizing antibody titers alone are insufficient to fully predict protective immunity against PRV infection. Cell-mediated immunity and mucosal immune responses may play complementary or even dominant roles in restricting viral dissemination within the respiratory tract. In particular, the route of administration had a marked influence on vaccine performance. Intramuscular immunization tended to produce stronger protection than intradermal delivery, likely reflecting differences in antigen uptake efficiency, local immune cell activation, and lymphatic drainage patterns.

An additional notable observation was that certain formulations, such as ISA15A+d, induced relatively high neutralizing antibody titers yet failed to confer equivalent clinical protection. This discrepancy is consistent with previous reports by Wang et al. (2014), reinforcing the concept that antibody magnitude does not necessarily correlate with functional protection during complex viral infections. Instead, qualitative aspects of immune responses, including antibody functionality, cellular immunity, and tissue-level immune surveillance, may be more critical determinants of protection.

Comparative analysis with other veterinary pathogens further underscores the context-dependent performance of adjuvants. Zhao Xudong et al. reported superior protection of ISA201 VG compared with IMS 1313 in Streptococcus suis infection (Zhao et al., 2021), whereas Pei Shixuan et al. observed better efficacy of GEL02 in Erysipelothrix rhusiopathiae vaccination (Pei et al., 2020). Similarly, Li Houwei et al. demonstrated the advantage of GEL02 in porcine deltacoronavirus vaccines (Li et al., 2022b). These differences likely reflect pathogen-specific immune requirements, host-pathogen interactions, and antigen properties, rather than intrinsic superiority of any single adjuvant system. Within the context of the present PRV vaccination study, IMS 1313 combined with immunopotentiator c showed the most balanced profile among tested formulations, showing robust protection in mice and favorable protective trends in piglets together with minimal local reactions and reduced viral shedding. Among tested formulations, IMS 1313+c administered intramuscularly showed the most consistent efficacy trend in piglets aged 21–35 days.

From a translational perspective, these findings support IMS 1313+c as a promising candidate for further PRV vaccine development oriented toward intensive pig production systems. However, several limitations should be acknowledged. First, the relatively small sample size in piglet experiments limits statistical power and may obscure subtle inter-group differences. Second, the observation period was restricted to acute post-challenge outcomes, and long-term immune memory was not evaluated. Third, the study was conducted under controlled experimental conditions, without assessment under field-relevant stressors such as co-infections, environmental variability, or maternal antibody interference. Fourth, although we speculate the involvement of cellular and mucosal immunity based on protective phenotypes, direct measurements of T-cell responses, cytokines or mucosal IgA were absent, which restricts mechanistic interpretation of the enhanced protection conferred by IMS 1313+c.

Future studies should further characterize the cellular immune responses induced by IMS 1313-based formulations, with particular attention to T-cell subsets, cytokine production, and mucosal IgA responses. At the same time, optimizing antigen dosage will be important for improving overall immunogenicity, especially under conditions that more closely reflect practical use. Efforts to enhance the effectiveness of different administration routes, including intramuscular and mucosal delivery, are also needed to increase applicability in different settings. In addition, studies on long-term protection, together with field evaluations under commercial farming conditions, will be necessary to assess real-world performance. Finally, combining IMS 1313 with other adjuvant systems, including nano- or oil-based formulations, may provide a feasible approach to further improve immune responses while maintaining safety. These directions should help support the development of next-generation PRV vaccines with more durable and broader protection.

Ethics declaration

This study was conducted in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. This study was approved by the Weifang Maternal and Child Health Hospital.

Funding

Funding statement this study was supported by the Jiangsu Natural Science Foundation (BK20181243) and Jiangsu Agriculture Science and Technology Innovation Fund [CX(24)2001].

CRediT authorship contribution statement

Ya-ting Zheng: Validation, Resources, Methodology, Investigation, Formal analysis, Data curation. Xian-zhu Jin: Validation, Methodology, Formal analysis, Data curation. Chuan-jian Zhang: Validation, Software, Resources, Methodology, Data curation. Sai-sai Chen: Validation, Investigation, Data curation. Nikolaus Osterrieder: Resources, Methodology, Investigation. Ya-mei Liu: Writing – review & editing, Supervision, Project administration, Conceptualization, Investigation, Writing – original draft. Ji-chun Wang: Supervision, Funding acquisition, Conceptualization.

Declaration of competing interest

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.

Appendix. Supplementary materials

Fig. S1. Gross lung lesions in piglets after virulent PRV AH02LA challenge.

Necropsy photographs of lung tissues collected from each experimental group post-challenge. (a) IMS 1313 intramuscular (IM) group; (b)IMS 1313 + immunopotentiator c (IM) group; (c) IMS 1313 + immunopotentiator c intradermal (ID) group; (d)Oil-adjuvant inactivated vaccine group; (e) LA2017 live vaccine; (f) Challenge control group.

Fig. S2. Gross tonsil lesions in piglets after virulent PRV AH02LA challenge.

Necropsy photographs of tonsil tissues collected from each experimental group post-challenge. (a) IMS 1313 intramuscular (IM) group; (b) IMS 1313 + immunopotentiator c (IM) group; (c) IMS 1313 + immunopotentiator c intradermal (ID) group; (d)Oil-adjuvant inactivated vaccine group; (e) LA2017 live vaccine; (f)Challenge control group.

Fig. S3. Gross lymph-node lesions in piglets after virulent PRV AH02LA challenge.

Macroscopic necropsy images of lymph node tissues across experimental groups. (a) IMS 1313 intramuscular (IM) group; (b) IMS 1313 + immunopotentiator c (IM) group; (c) IMS 1313 + immunopotentiator c intradermal (ID) group; (d)Oil-adjuvant inactivated vaccine group; (e) LA2017 live vaccine; (f) Challenge control group.

Data availability

Data will be made available on request.