Immunogenicity and safety in animals of a lyophilized live attenuated hepatitis A vaccine following stabilizer optimization

Abstract

To evaluate the immunogenicity and safety profiles of lyophilized hepatitis A live attenuated vaccine (L-A-1 attenuated strain) after stabilizer formula optimization using non-human primates and various rodent models. Immunogenicity assessment was conducted using NIH mice and rhesus monkeys comparing pre-optimization commercial vaccine with post-optimization test vaccine. Mice received single immunizations with cardiac blood collection at day 28 for ELISA-based hepatitis A antibody detection. Non-human primates were monitored for clinical status, body weight, and hepatitis A virus IgG antibody titers at baseline(D0) and post-immunization timepoints (D14, D21, D28). Safety evaluation included guinea pig systemic anaphylaxis testing, rabbit subcutaneous irritation assessment, and rat single-dose toxicity investigation using both vaccine formulations and their corresponding stabilizer components. Both mouse and non-human primate studies demonstrated equivalent immunogenicity between optimized and commercial vaccines, with 100% seroconversion rates and comparable antibody concentrations (GMCs: 390.52 vs. 388.25 mIU/ml in mice, p > 0.05). Non-human primates showed 16–256 fold antibody titer increases by day 28 with normal clinical status maintained throughout. Safety assessments revealed no systemic sensitization, local irritation, or toxicity in any animal model for both formulations and their corresponding stabilizer components. Comprehensive animal model investigations demonstrate that the optimized stabilizer formulation maintains equivalent immunogenic potency while establishing a favorable safety profile comparable to the pre-optimization formulation. These findings provide substantive preclinical evidence supporting the feasibility and benefits of the stabilizer formula optimization strategy for lyophilized hepatitis A live attenuated vaccine.

Hepatitis A virus (HAV) infection causes an acute inflammatory disease of the liver primarily transmitted through the fecal-oral route^1,2, though it can also spread via daily contact interactions and, under specific circumstances, through male-to-male sexual behavior³. The clinical manifestations of hepatitis A infection typically include fatigue, malaise, anorexia, emesis, abdominal discomfort, diarrhea, jaundice, and dark urine^4,5. In severe cases, the infection can progress to acute liver failure (ALF) and acute-on-chronic liver failure (ACLF), conditions that frequently result in mortality without liver transplantation⁶. Hepatitis A outbreaks and epidemics impose substantial medical and economic burdens on affected populations and significantly impact quality of life and public health⁷. Vaccination represents the most efficacious preventive measure against hepatitis A virus infection⁸.

Lyophilization (freeze-drying) technology involves the removal of water from frozen biological samples through sublimation and desorption processes, and is widely recognized as the most gentle approach for concentrating or drying biologically active substances⁹. This methodology constitutes one of the most extensively utilized techniques for preserving biological materials in a dehydrated state and has been successfully implemented in the preservation of proteins, liposomes, and vaccines within the pharmaceutical and biomedical sectors¹⁰. Lyophilized vaccines offer superior stability characteristics and logistical advantages in terms of transportation and storage requirements¹¹. During the lyophilization process, specialized stabilizing agents are essential to protect biologically active components from freezing-induced degradation. The selection of appropriate stabilizers directly influences product quality, as different stabilizing formulations necessitate specific lyophilization parameters.

In China, lyophilized hepatitis A live attenuated vaccine was developed in the early 21st century, with extensive post-licensure monitoring demonstrating favorable immunogenicity and safety profiles¹. Nevertheless, the stabilizers employed in the lyophilized formulation typically contain either gelatin or dextran 40. Gelatin, a common vaccine excipient, is associated with potential IgE-mediated allergic reactions^12,13. Similarly, dextran 40 carries a risk of inducing severe anaphylactic responses, including bronchospasm and fatal anaphylaxis^14,15. To enhance the safety profile of the lyophilized hepatitis A live attenuated vaccine, we undertook the optimization of the stabilizer formulation used in the production of the vaccine derived from the HAV L-A-1 attenuated strain. The original stabilizer comprised eight components: dextran 40, trehalose, sodium glutamate, arginine, vitamin C, mannitol, sorbitol, and urea. The optimized stabilizer consists of five constituents: trehalose, sodium glutamate, arginine, urea, and mannitol. This optimized formulation reduces complexity and eliminates known allergenic compounds such as dextran 40, thereby significantly improving the safety profile and supporting broader application within China’s Expanded Program on Immunization.

The present investigation was designed to comprehensively evaluate the immunogenicity and safety characteristics of the lyophilized hepatitis A live attenuated vaccine (L-A-1 attenuated strain) following stabilizer formulation optimization. This evaluation employed both non-human primate and rodent experimental models, with comparative analyses against the pre-optimization commercial vaccine to establish the feasibility and advantages of the optimized stabilizer formulation.

Materials and methods

Experimental materials

Test vaccine preparations and optimized stabilizer formulations were manufactured using the optimized lyophilization stabilizer composition; commercial lyophilized hepatitis A live attenuated vaccine and pre-optimization stabilizer were prepared using the original stabilizer formulation. All experimental materials were manufactured by Changchun Institute of Biological Products Co., Ltd. The anesthetic used was Zoletil™ 50 (tiletamine-zolazepam), purchased from Virbac Group, France.

Analytical instrumentation and immunoassay systems

Microplate spectrophotometer (model: iMarK, BIO-RAD, Japan) was employed for immunoassay. HAV-antibody qualitative detection kits were sourced from EXpress BIO (USA), and HAV-antibody quantitative detection kits were obtained from Beijing Wantai Biological Pharmacy Co., Ltd. (China).

Experimental animals

All procedures involving animals were conducted in compliance with the relevant laws and regulations of the institutional committee on the use and management of laboratory animals. All methods strictly followed the ARRIVE guidelines for reporting experimental studies involving animals. The method of euthanasia for experimental animals is inhalation of CO2 or injection of anesthetics followed by exsanguination. The number of animals, experimental design, and handling were approved by the Institutional Animal Care and Use Committee, and the IACUC approval number is detailed below.

The animal models used are as follows:

• NIH mice: SPF grade, n = 80 (40 male, 40 female), body weight 14–16 g, supplied by the Laboratory Animal Center of Changchun Institute of Biological Products Co., Ltd.(Approved institution name : Institutional Animal Care and Use Committee of Changchun Institute of Biological Products Co., Ltd., (IACUC). IACUC approval numbers: CCIBP202108-01).

• Rhesus monkeys (Macaca mulatta): Conventional grade, n = 12 (5 female, 7 male), age 1–3 years, body weight 2.65–3.75 kg, obtained from Hubei Tianqin Biotechnology Co., Ltd., Suizhou Branch. (Approved institution name : Institutional Animal Care and Use Committee of Hubei Topgene Biotechnology Research Institute Co., Ltd. (IACUC). IACUC approval numbers : IACUC(准)-YJY-2022-013).

• Guinea pigs: SPF grade, n = 48 (24 male, 24 female), body weight 276–325 g, sourced from Qingdao Kangda Biotechnology Co., Ltd. (Approved institution name: Institutional Animal Care and Use Committee of Shandong Xinbo Pharmaceutical R&D, Ltd. (IACUC). IACUC approval numbers : XB-IACUC-2021-0898).

• Japanese white rabbits: Conventional grade, n = 16 (8 male, 8 female), body weight 2.29–2.61 kg, acquired from Qingdao Kangda Biotechnology Co., Ltd. (Approved institution name: Institutional Animal Care and Use Committee of Shandong Xinbo Pharmaceutical R&D, Ltd. (IACUC). IACUC approval numbers : XB-IACUC-2021-0899).

• Sprague-Dawley rats: SPF grade, n = 50 (25 male, 25 female), body weight 195.50–260.50 g, procured from Beijing Charles River Laboratory Animal Technology Co., Ltd. (Approved institution name: Institutional Animal Care and Use Committee of Shandong Xinbo Pharmaceutical R&D, Ltd. (IACUC) (IACUC).IACUC approval numbers : XB-IACUC- 2021 − 0777).

Immunogenicity assessment protocols

Murine immunogenicity evaluation

NIH mice were randomized into eight experimental groups (n = 10 per group, gender-balanced) according to a computer-generated allocation sequence. Three groups received post-optimization test vaccine formulations; three groups received pre-optimization commercial vaccine formulations; one group received physiological saline (negative control); and one untreated group served as blank control. Immunization was administered via intraperitoneal route. On day 28 post-immunization, blood was collected via cardiac puncture, and sera were isolated for quantitative and qualitative analysis of anti-HAV antibodies.

Non-human primate immunogenicity assessment

Rhesus monkeys were stratified by body weight and randomly assigned to three experimental cohorts: negative control group immunized with sterile water for injection (n = 2; 1 female, 1 male), commercial control group immunized with pre-optimization commercial vaccine (n = 5; 2 females, 3 males), and test article group immunized with post-optimization test vaccine (n = 5; 2 females, 3 males). All administrations were performed as single intravenous injections with a standardized volume of 0.5 mL. Post-immunization monitoring included daily clinical observations, weekly body weight measurements, and serological sampling at baseline (D0) and post-immunization timepoints (D14, D21, D28) for analysis of HAV-specific IgG antibody titers.

Serological analysis methodologies

Murine serological analysis

HAV-specific antibodies in mouse sera were quantified using validated ELISA protocols. For qualitative assessment, samples with optical density (OD) value/cutoff ratio ≤ 1 were classified as positive, while those with OD value/cutoff ratio > 1 were categorized as negative. Seroconversion was defined as the transition from negative to positive antibody status following vaccination. For quantitative determination, standard curve-based linear regression analysis was applied to calculate absolute antibody concentrations from OD values.

Non-human primate serological analysis

HAV-specific IgG antibody titers in rhesus monkey sera were determined using standardized ELISA methodology. Serial dilutions of serum samples were analyzed, with the highest dilution yielding an OD value/cutoff ratio ≤ 1 designated as the endpoint titer. Seroconversion was defined as a ≥ 4-fold increase in HAV antibody titer following immunization relative to baseline (D0) values.

Statistical analyses

Statistical evaluations were conducted using SPSS 23.0 software (IBM Corp., USA). Independent samples t-tests were applied to compare antibody concentrations among groups in the immunogenicity study in mice(the data subjected to logarithmic transformation prior to analysis), a nonparametric test (Mann-Whitney U) was applied to compare antibody titers between groups in the immunogenicity study conducted in monkeys, with p < 0.05 considered statistically significant. Graphical representations were generated using GraphPad Prism 8.0 software (GraphPad Software Inc., USA). Data are presented as mean ± standard deviation unless otherwise specified.

Safety evaluation methodologies

Guinea pig systemic active anaphylaxis assessment

SPF-grade guinea pigs were randomized into six experimental groups (n = 8 per group, gender-balanced):

• Negative control group: administered 0.9% sodium chloride injection.

• Positive control group: administered 0.1% high-purity ovalbumin.

• Test group 1: administered pre-optimization vaccine.

• Test group 2: administered post-optimization vaccine.

• Test group 3: administered pre-optimization stabilizer.

• Test group 4: administered post-optimization stabilizer.

The sensitization protocol consisted of three subcutaneous injections (0.5 mL/injection) of the corresponding test article administered on alternate days. For challenge assessments conducted 14 and 21 days after the final sensitization, four animals per group (2 female, 2 male) received the corresponding test article at twice the sensitization concentration via plantar vein injection. Allergic manifestations were continuously monitored from the moment of challenge until symptom resolution, or for 30 min in the absence of observable reactions. Allergic response intensity was evaluated according to standardized scoring criteria. Statistical analyses utilized EXCEL and SPSS26 software packages, with data expressed as mean ± standard deviation.

Rabbit subcutaneous irritation evaluation

Japanese white rabbits were randomized into four experimental groups (n = 4 per group, gender-balanced):

• Test group 1: administered pre-optimization vaccine.

• Test group 2: administered post-optimization vaccine.

• Test group 3: administered pre-optimization stabilizer.

• Test group 4: administered post-optimization stabilizer.

A contralateral self-control methodology was implemented, with 0.9% sodium chloride injection and the corresponding test article administered subcutaneously (0.5 mL/site/animal, approximately 30-second injection duration) at the left and right scapular regions, respectively. Half of the animals in each group (1 male and 1 female) were euthanized at 48 h post-injection, while the remaining animals were euthanized after a 14-day recovery period. Following euthanasia, the skin and subcutaneous tissues at the injection sites were excised and macroscopically examined for irritation responses. The tissues were subsequently fixed in 10% neutral formalin and processed for routine histopathological examination using standard tissue sectioning techniques.

Rat subcutaneous Single-dose toxicity assessment

SPF-grade SD rats were randomized into five experimental groups (n = 10 per group, gender-balanced):

• Negative control group: administered 0.9% sodium chloride injection.

• Test group 1: administered pre-optimization vaccine.

• Test group 2: administered post-optimization vaccine.

• Test group 3: administered pre-optimization stabilizer.

• Test group 4: administered post-optimization stabilizer.

Test articles were administered via subcutaneous injection (1.0 mL/animal distributed across multiple injection sites, 0.5 mL/site). Post-administration monitoring included continuous clinical observations for 14 days and body weight measurements twice weekly. Terminal procedures included comprehensive gross necropsy examinations for all animals, with histopathological evaluation of organs displaying gross abnormalities. Body weight data and growth rate calculations (expressed as percentage change relative to baseline) were analyzed using EXCEL and SPSS26 software packages, with data presented as mean ± standard deviation.

Results

Immunogenicity studies

Mouse immunogenicity study

The seroconversion rate of hepatitis A virus antibodies in mouse serum from 3 batches of post-optimization test vaccines and pre-optimization commercial control vaccines was 100%. There was no statistically significant difference in antibody concentration results (t = 0.164, P = 0.870 > 0.05). The antibody seroconversion rate in the saline control group and negative control group was 0%. Detailed results are shown in Table 1.

Table 1 Mouse serum antibody seroconversion and geometric mean Concentration.

Monkey immunogenicity study

General condition observation

Within 4 weeks after administration, no abnormalities were observed in any of the animals.

Animal body weight changes

During the test period, animal body weight showed a gradual upward trend, and there was no significant difference in body weight between groups before and after administration (P > 0.05). Details are shown in Table 2.

Table 2 Effect of intravenous injection of hepatitis A live attenuated vaccine on rhesus monkey body weight (unit: kg; $\:\bar{x}$±SD).

Serum antibody results

Both the test and commercial vaccine groups demonstrated an effective and time-dependent rise in antibody levels, achieving a 100% seroconversion rate by day 28, with a greater than 4-fold (16–256×) increase in antibody titers following immunization. There was no statistically significant difference between in antibody titers results (z = 1.336, P = 0.182 > 0.05). Conversely, the negative control group showed no immune response (≤ 2-fold titer increase). Detailed results are shown in Table 3; Fig. 1.

Table 3 Serum antibody titers (1:X) in rhesus monkeys after intravenous injection of lyophilized hepatitis A live attenuated Vaccine.

Fig. 1

Changes in serum antibody titers in rhesus monkeys after intravenous injection of lyophilized hepatitis a live attenuated vaccine data are presented as geometric mean ± geometric SD. Statistical analysis was performed on log10-transformed data.

Safety evaluation studies

Guinea pig systemic active anaphylaxis test

No adverse effects on general condition or body weight were observed in any test group compared to the negative control (F = 0.008, P = 1.000 > 0.05). Furthermore, all test groups, like the negative control, showed negative allergic responses after challenge, contrasting with the extremely strong positive reaction in the positive control group.Detailed results are shown in Tables 4 and 5.

Table 4 Animal body weight statistical data (MEAN ± SD, unit: g).

Table 5 Results of systemic sensitization Evaluation.

Rabbit subcutaneous irritation test

Comprehensive macroscopic and histopathological evaluations demonstrated excellent local tolerance of the test articles. No test article-related local reactions, such as redness, swelling, congestion, exudation, degeneration, or necrosis, were observed at the administration sites in any animals from test groups 1–4. Furthermore, no drug-related histopathological changes were detected at these sites compared to the contralateral control sides. This absence of local irritation and pathological findings was consistent at both the 48-hour post-administration and 14-day recovery phase time points. All animals maintained good general condition throughout the study, with no mortality recorded.Detailed results are shown in Figs. 2 and 3.

Fig. 2

Histopathological results of skin and subcutaneous tissues at injection sites 48 h after administration.

Fig. 3

Histopathological results of skin and subcutaneous tissues at injection sites at the end of recovery period.

Rat subcutaneous Single-Dose toxicity test

No abnormalities were observed in the animals during the test period, and no animal deaths occurred. Subcutaneous injection of pre- and post-optimization lyophilized hepatitis A live attenuated vaccines and pre- and post-optimization stabilizers showed no statistically significant difference in body weight and body weight gain compared with the negative control group. At the end of the recovery period, no obvious gross pathological changes were observed in the tissues and organs of the animals, and no histopathological examination was performed. Specific data are shown in Tables 6 and 7.

Table 6 Male animal body Weight/Growth rate statistical data (MEAN ± SD, n = 5).

Table 7 Female animal body Weight/Growth rate statistical data (MEAN ± SD, n = 5).

Discussion

The global epidemiological profile of hepatitis A infection demonstrates a strong correlation with socioeconomic parameters. According to the 2019 Global Burden of Disease estimates, approximately 159 million cases of acute HAV infection occurred worldwide, resulting in 39,000 fatalities and 2.3 million disability-adjusted life years lost¹⁶. In China, the most significant hepatitis A outbreak occurred in Shanghai in 1988, affecting over 310,000 individuals and necessitating hospitalization for more than 8,000 patients¹⁷, with profound public health implications. In accordance with the World Health Organization position paper, prevention strategies for hepatitis A infection should integrate improvements in personal hygiene practices, sanitation infrastructure, water quality, and implementation of hepatitis A vaccination programs¹⁶.

Hepatitis A live attenuated vaccines offer several significant advantages including durable immunity following single-dose administration, cost-effective production methodology, and robust protective efficacy. Furthermore, this vaccine category mimics natural infection by stimulating both humoral and cell-mediated immune responses¹⁸, providing comprehensive protective immunological memory.

The hepatitis A live attenuated vaccine manufactured using the L-A-1 attenuated strain propagated in human embryonic lung diploid (2BS) cells constitutes a vaccine with independent intellectual property rights in China. Following its regulatory approval in 1992¹⁷, extensive clinical evaluation has confirmed its impressive efficacy, effectiveness, and immunological durability. The lyophilized hepatitis A live attenuated vaccine received regulatory approval in China in 2000. In 2007, hepatitis A vaccination was fully integrated into China’s National Expanded Program on Immunization, with universal free provision to all children ≥ 18 months of age, resulting in effective control of hepatitis A outbreaks. These public health interventions have contributed to a remarkable reduction in the annual incidence of hepatitis A in China from 52.6/100,000 in 1990 to 5.9/100,000 in 2007, with a further 72.1% decline documented between 2007 and 2013¹⁹.

To optimize the production process and improve product quality and safety, we simplified and optimized the stabilizer formulation used in the lyophilized hepatitis A attenuated live vaccine (L-A-1 attenuated strain), reducing the stabilizer components from 8 to 5. Experimental results demonstrated that the optimized stabilizer formulation does not adversely affect the quality of the lyophilized hepatitis A attenuated live vaccine. Additionally, the optimized formulation excludes substances that may cause allergic reactions, making it safer and more advanced. For lyophilized vaccines, stabilizer optimization, while appearing to be a minor technical improvement, may have significant implications. First, simplifying the stabilizer formulation can reduce production costs and improve economic efficiency. Reducing excipient use may decrease potential safety risks associated with certain excipients, facilitating vaccination implementation. Second, stabilizer optimization can enhance product quality and potentially extend shelf life, reducing waste and optimizing inventory management, thereby improving vaccine coverage. Third, the optimized stabilizer, due to its fewer components and greater stability, is more suitable for vaccine production and storage in developing countries, potentially increasing vaccination rates and reducing disease incidence. Therefore, the optimized stabilizer formulation may improve global vaccine accessibility.

Since the initial characterization of hepatitis A virus, non-human primate models have been instrumental in advancing our understanding of viral pathophysiology^20,21,22,23. Various species including chimpanzees, rhesus monkeys, cynomolgus monkeys, African green monkeys, marmosets, tamarins, and owl monkeys have been employed in HAV research^24,25. The phylogenetic proximity of non-human primates to humans and the similarity in HAV infection manifestations between these species have positioned non-human primate models as the gold standard for elucidating HAV replication dynamics and pathogenic mechanisms, with significant contributions to vaccine development.

Therefore, this study utilized rhesus macaques, a non-human primate model, to conduct immunogenicity studies of the lyophilized hepatitis A attenuated live vaccine, which provides a more accurate evaluation of vaccine efficacy. The immunogenicity experiments in non-human primates conducted in this study were performed according to the requirements for " tests for safety and immunogenicity in monkeys " specified in the Chinese Pharmacopoeia (2020 edition, Volume III) for lyophilized hepatitis A attenuated live vaccines. The route of administration was intravenous injection, consistent with the Chinese Pharmacopoeia requirements. Additionally, rodent models using mice can also serve as an approach for vaccine immunogenicity evaluation; therefore, this study also employed murine models to conduct parallel efficacy assessments.

In the murine immunogenicity experiments, the immunization dose was 0.5 ml per dose. Due to the small body weight of mice, intramuscular injection cannot accommodate such a large volume of liquid. The peritoneal cavity possesses a large surface area and abundant immune cells, which can similarly stimulate serum antibody production. Therefore, this experiment employed the conventional and widely used intraperitoneal injection method for mouse immunization. To ensure experimental consistency, both the pre- and post-optimization groups in the murine immunogenicity experiments and non-human primate immunogenicity studies used identical administration methods, ensuring that the immunization route, dose, and timing were consistent across all groups. This design ensured comparability of experimental results between groups. The combination of murine immunogenicity results and non-human primate experimental results provides a more comprehensive reflection of vaccine efficacy. The aforementioned experimental results demonstrated that the lyophilized hepatitis A attenuated live vaccine prepared with the optimized stabilizer exhibited good immunogenicity.

Recent studies have demonstrated that small animal models can also be utilized to evaluate hepatitis A virus (HAV), including the use of human hepatocyte-infected chimeric mice. In mice deficient in type I interferon (IFN) receptor (IFN-α/β receptor) or mitochondrial antiviral signaling (MAVS) protein expression, HAV can replicate to high titers²⁶. This provides a valuable platform for HAV mechanistic studies and serves as a critical tool for pathogenesis research, particularly demonstrating innovation in elucidating MAVS-dependent hepatocyte apoptosis. These models exhibit tremendous potential in antiviral drug screening, vaccine evaluation, and mechanistic studies. However, their clinical translation requires standardized validation, ethical compliance optimization, and international collaboration to meet global regulatory requirements. Future advances through model optimization and multidisciplinary integration are expected to promote the development of prevention and treatment strategies for HAV and related hepatitis viruses.

In compliance with regulatory requirements in China, we conducted comprehensive safety evaluation studies utilizing small animal models, including guinea pig systemic active anaphylaxis assessment, rabbit subcutaneous irritation evaluation, and rat single-dose toxicity testing. The results conclusively demonstrate comparable safety profiles between the post-optimization lyophilized hepatitis A live attenuated vaccine and the pre-optimization commercial formulation, with both meeting established safety criteria.

Conclusion

The present investigation provides comprehensive preclinical evidence supporting the optimization of the stabilizer formulation for lyophilized hepatitis A live attenuated vaccine derived from the L-A-1 attenuated strain. Immunogenicity assessment in both murine and non-human primate models demonstrates that the post-optimization formulation induces robust seroconversion with antibody titers comparable to those elicited by the pre-optimization commercial vaccine. Safety evaluation across multiple animal models confirms the absence of systemic toxicity, local irritation, or allergenic potential in the optimized formulation.

These findings collectively establish the feasibility and advantages of the stabilizer optimization strategy, which achieves equivalent or enhanced immunogenicity while potentially improving safety through elimination of allergenic components. The comprehensive preclinical dataset generated in this study provides a strong foundation for subsequent clinical evaluation of the optimized vaccine formulation.