---
title: "HRSV Pre-F protein with AlOH, AlOH+CpG, or BFA03: comparative protection in BALB/c mice"
id: "frontiers-in-immunology-4-comparison-of-the-protective-effect-of-human-respiratory-syncytial-virus-pre-f"
canonical_url: "https://medichelpline.com/clinical-feed/frontiers-in-immunology-4-comparison-of-the-protective-effect-of-human-respiratory-syncytial-virus-pre-f"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "Frontiers in Immunology"
source_url: "https://www.frontiersin.org/articles/10.3389/fimmu.2026.1904272"
published_at: "2026-07-20T00:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# HRSV Pre-F protein with AlOH, AlOH+CpG, or BFA03: comparative protection in BALB/c mice
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/frontiers-in-immunology-4-comparison-of-the-protective-effect-of-human-respiratory-syncytial-virus-pre-f
- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** Frontiers in Immunology
- **Source URL:** [Original Journal Publication](https://www.frontiersin.org/articles/10.3389/fimmu.2026.1904272)
- **Published At:** 2026-07-20T00:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- This study compared the protective efficacy of recombinant **Pre-F protein** of human respiratory syncytial virus (**HRSV**) formulated with three adjuvant strategies (AlOH, AlOH+CpG, and BFA03) in BALB/c mice. - Thirty-six female BALB/c mice were randomized into six groups (three antigen+adjuvant groups and three adjuvant-only controls); immunizations were given intramuscularly on Days 0 and 21 and mice were challenged 14 days after the second dose. - The Pre-F antigen was produced in CHO-S cells, included six stabilizing mutations and a C-terminal foldon and His tag, ran at ~65 kDa by SDS–PAGE, and showed a uniform trimer by TEM. - Humoral responses: all antigen-adjuvant groups developed higher Pre-F–specific IgG than adjuvant-only groups. Pre-F+BFA03 induced the highest IgG titre (1.9×10^5) and the highest neutralizing antibody titre (1716); Pre-F+AlOH induced the lowest IgG (4.3×10^4) and showed the lowest neutralizing activity among antigen groups. - Isotype and cellular readouts showed **AlOH** alone skewed toward a Th2 response, while adding **CpG** or using **BFA03** produced more balanced humoral and cellular responses; the Pre-F+AlOH+CpG group induced comparable neutralizing antibodies and cellular responses to Pre-F+BFA03. - After viral challenge, mice immunized with antigen groups began regaining weight by Day 3, whereas adjuvant-only mice continued to lose weight. Pre-F+BFA03 yielded the lowest lung viral load and milder pathology; Pre-F+AlOH showed the highest lung viral load (Ct 32.2) and the most severe pathology (score 2.83). - The Pre-F+AlOH+CpG group had the least lung pathological damage (score 2.16) while maintaining robust neutralizing and cellular responses. - Authors conclude that **Pre-F + AlOH+CpG** and **Pre-F + BFA03** provided similar and superior protection compared with **Pre-F + AlOH**, offering guidance for adjuvant selection in Pre-F–based HRSV vaccine development. - Ethical approval, assay methods (ELISA, PRNT, ELISPOT, RT–PCR, histopathology scoring), and basic statistical approaches (one-way ANOVA, significance at P<0.05) were reported. Data availability and specific numerical datasets beyond reported summary values were not detailed in the source.
## Clinical Analysis & Structured Key Points
About us All journals All articles Submit your research Search Login Frontiers in Immunology Sections Articles Research Topics Editorial board About journal Published in Frontiers in Immunology Viral Immunology 7 impact factor 11.3 citescore Part of a Research Topic Immune Regulation and Innovative Therapeutic Strategies in Infectious Disease Persistence and Resistance Submission open 10k views 11 articles Editor & Reviewers Edited by Hongyang Yi Reviewed by Shuai Wang Yong-Peng Sun Outline Abstract Introduction Materials and methods Results Discussion Data availability statement Ethics statement Author contributions Funding Acknowledgments Conflict of interest Generative AI statement Publisher’s note References Figures and Tables Figure 1 View in article Figure 2 View in article Figure 3 View in article Figure 4 View in article Figure 5 View in article Table 1 Animal grouping and immunizations. View in article ORIGINAL RESEARCH article Front. Immunol., 20 July 2026 Sec. Viral Immunology Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1904272 Comparison of the protective effect of human respiratory syncytial virus Pre-F protein combined with different adjuvants in BALB/c mice Mengxuan Chu 1,2† L D Liang Du 1,2† H H Hongqiao Hu 1,2† L C Lei Cao 1,2 L Z Li Zhang 1,2,3 N M Naiying Mao 1,2 Z Z Zhen Zhu 1,2 Y Z Yan Zhang 1,2 Hai Li 1,2* 1. National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Disease, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (Chinese Academy of Preventive Medicine), Beijing, China 2. National Health Commission (NHC) Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (Chinese Academy of Preventive Medicine), Beijing, China See more Article metrics View details 134 Views Abstract Introduction: Human respiratory syncytial virus (HRSV) has a high disease burden in infants and elderly individuals. In this study, the adjuvants AlOH, AlOH+CpG and BFA03 were used to compare the protective effect of HRSV prefusion protein (Pre-F) in BALB/c mice. Methods: We divided BALB/c mice into three experimental groups (Pre-F+AlOH+CpG, Pre-F+AlOH, and Pre-F+BFA03) and three adjuvant control groups (AlOH+CpG, AlOH, and BFA03). After two intramuscular immunizations, we measured serum neutralizing antibody titers and quantified the numbers of IFN-γ- and IL-4-secreting lymphocytes. After viral challenge, we monitored body weight changes, determined lung viral loads (Ct values), and scored lung pathological damage. Results: The mice in the experimental groups exhibited high titres of neutralizing antibodies and increased numbers of IFN-γ- and IL-4-secreting lymphocytes. The mice began to regain weight on the third day after challenge, but the mice in the adjuvant groups continued to lose weight. The mice immunized with Pre-F+BFA03 elicited the highest neutralizing antibody titre (1716), the lowest viral load in the lung, and milder pathological damage. The mice immunized with Pre-F+AlOH had the most severe lung pathological injury (score: 2.83) and the highest viral load in the lung (Ct value: 32.2). Compared with the BFA03 adjuvant, the AlOH adjuvant induced a Th2-biased humoral immune response in mice. The Pre-F+AlOH+CpG group had the least pathological damage in the lung (score 2.16), and the ability to induce neutralizing antibodies and cellular immune responses was comparable with that of the BFA03 adjuvant. Discussion: These findings indicate that Pre-F protein combined with AlOH+CpG or BFA03 adjuvant provided similar protection in mice and both were superior to the AlOH adjuvant, providing a reference for adjuvant selection and formulation strategies in HRSV Pre-F protein vaccine development. Introduction Human respiratory syncytial virus (HRSV) is the only virus in the genus Orthopneumovirus of the family Pneumoviridea to infect humans, and it is also known as human Orthopneumovirus (1). HRSV contains 10 genes, which can encode 11 proteins, including two nonstructural proteins (NS1 and NS2), nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), small hydrophobic protein (SH), glycoprotein (G), fusion protein (F), and M2–1 and M2–2 proteins, which can be expressed by the M2 gene and RNA-dependent RNA polymerase (L) (2). A variety of HRSV vaccines have been designed based on the structural proteins F, G, and SH. The F protein includes two structures, prefusion protein (Pre-F) and postfusion protein (Post-F), which can mediate the fusion of the viral envelope and the host cell membrane through conformational transition, and is highly conserved. The Pre-F protein conformation includes six epitopes (site I, II, III, IV, V, and Ø) and only four epitopes (site I, II, III, and IV) for Post-F, where site III is predominantly present in Pre-F and site I is predominantly present in Post-F (3). Site Ø is located at the top of the distal membrane of the Pre-F protein, and the recognition sites are aa 62–69 and aa 196-209. The neutralizing activity of antibodies induced by site Ø is much higher than that of other epitopes. Therefore, the stable site Ø is an ideal neutralizing epitope for HRSV vaccine development, and vaccine development is expected to contain a stable site Ø (4). HRSV is mainly transmitted by droplets into and adsorbed on the respiratory mucosa, causing upper respiratory tract infection, and in severe cases it can cause lower respiratory tract infection. It mainly infects infants, elderly individuals, and people with low immunity, and it is the most common viral pathogen of acute lower respiratory tract infection in infants and young children (5). Almost all infants and young children under 2 years old have been infected with HRSV, which often causes lower respiratory tract infection, usually manifested as bronchiolitis and pneumonia, while neonates younger than 3 months have more severe symptoms (6). One study estimated that in 2019 more than 45, 000 infants younger than 6 months died from acute lower respiratory infections caused by HRSV (7). Severe HRSV bronchiolitis in infancy and early childhood is a strong risk factor for the development of allergic asthma in early adolescence (8). Children and adults usually have upper respiratory tract infections, and the symptoms are usually mild (9). With increasing age, the underlying diseases of elderly individuals increase, and immunity decreases. Therefore, HRSV also has high morbidity and mortality in the elderly (10). In addition, HRSV infection can cause extrapulmonary organ diseases, including interstitial myocarditis, epilepsy, or encephalitis (11). The immune population of the HRSV vaccine includes infants, pregnant women, and elderly individuals. The specific antibody levels in these three groups are different, and the immune system maturity is different; thus, it is difficult to develop a vaccine that is suitable for all three groups at the same time. A large number of studies have shown that nonreplicating vaccines should be avoided in infants who have not been infected with HRSV (12). The elderly have been naturally infected with HRSV many times, and it is difficult for the live-attenuated vaccine to induce the ideal protective effect, and the stimulated immune response is not sufficient to prevent HRSV reinfection (13). Therefore, the HRSV vaccines used in clinical research need to have the appropriate vaccine form or adjuvant according to the different target populations to solve the problem of HRSV infection faced by different populations. At present, the vast majority of vaccines and monoclonal antibodies were designed based on the Pre-F protein. Three HRSV vaccines have been approved globally: preF3 (Arexvy) (14), preF (Abrysvo) (15), and mRNA-1345 (16). These vaccines are indicated for adults aged 60 years and older, as well as adults aged 18–59 years who are at increased risk of RSV-associated lower respiratory tract disease (17). Although no HRSV vaccine has yet been approved for infants, three RSV monoclonal antibodies for infant use have been approved globally: nirsevimab, clesrovimab, and palivizumab (18, 19). Formalin-inactivated RSV vaccines (FI-RSV) induce the body to produce a biased Th2 immune response, causing enhanced respiratory disease (ERD) (20); therefore, ideal HRSV vaccines need to induce the body to produce a biased Th1 immune response. Adjuvants can not only enhance the strength of the body’s immune response but also regulate the bias of the immune response. Aluminium adjuvants, including aluminium phosphate (Al-Phos) and aluminium hydroxide (AlOH), are the most commonly used vaccine adjuvants for human use. The antigen adsorbed by aluminium adjuvant is slowly released at the injection site and provides long-term and effective stimulation of the immune system; however, it can cause granulomas at the injection site, attract cells of the innate immune system, such as monocytes, eosinophils, and neutrophils (21), and induce the body to produce a Th2-biased immune response (22). CpG oligodeoxynucleotides (CpG), a commonly used adjuvant, are Toll-like receptor 9 (TLR9) agonists. TLR9 is mainly expressed in dendritic cells (DCs) and B cells in humans and mice and is a strong inducer of B-cell activation, plasmacytoid dendritic cell maturation, and monocyte maturation. CpG can enhance the immune activity of vaccine-induced cytotoxic T cells and induce the production of Th1-biased immune responses (23), which is helpful for solving the problems of insufficient safety and weak immunogenicity of HRSV vaccines. CpG has been successfully used as an adjuvant in the hepatitis B vaccine. The combination of CpG ODN and aluminium adjuvant has a synergistic effect to promote the balance of the Th1/Th2 immune response. The use of novel adjuvants is the secret for recombinant protein vaccines to excel. At present, a variety of novel adjuvants have been applied to viral vaccines, including AS01B adjuvant for herpes zoster vaccine, AS04 adjuvant for human papillomavirus (HPV) vaccine, Matrix M1 adjuvant for COVID-19 vaccine, and MF59 adjuvant for influenza virus vaccine. Experts of FDA agreed that the RSV PreF3 vaccine (developed by GSK) is highly effective, and it benefits from the strong humoral and cellular immune responses induced by the use of the AS01E adjuvant. BFA03 (α-tocopherol, squalene, and polysorbate 80), developed by Reco Bio, has been tested in clinical trials for COVID-19 recombinant protein vaccines. The AS01E adjuvant was not commercially available as a standalone reagent at the time of this study; therefore, we focused on comparing AlOH (a widely used aluminum adjuvant), AlOH+CpG (a TLR9 agonist combination), and BFA03 (a novel candidate adjuvant). Future studies directly comparing these formulations with AS01E would be of significant value. In this study, BALB/c mice were immunized by intramuscular injection with AlOH, AlOH+CpG or BFA03 adjuvant mixed with Pre-F (CHO cell expression). By comparing the immunogenicity of the Pre-F protein in BALB/c mice, the effects of different adjuvants on the immunogenicity of the Pre-F protein were evaluated. This study provides a theoretical basis for further research on adjuvanted HRSV vaccines. Materials and methods Viruses, BALB/c mice and Pre-F protein HEp-2 cells and the HRSV-Long strain were stored in our laboratory. Female BALB/c mice aged 6–8 weeks were purchased from Vitong Lihua Laboratory Animal Technology Co., Ltd. in Beijing. The animal experiments involved in this study were approved by the Animal Experiment Ethics Committee of the National Institute for Virus Disease Control and Prevention, Chinese Center for Disease Control and Prevention. Pre-F protein was expressed in CHO-S cells (purchased from Invitrogen). Pre-F contains six amino acid mutations (S155C, S290C, A149C, Y458C, S190F, and V207C), and a foldon sequence (GYIPEAPRDGQAYVRKDGEWVLLSTFL) and His tag were added at the C-terminus. After affinity chromatography, the purity and structure of Pre-F were verified by SDS–PAGE and transmission electron microscopy. Immunization strategies Thirty-six female BALB/c mice (6–8 weeks old) were randomly divided into 6 groups (6 mice in each group). The mice were immunized intramuscularly with 5 μg of Pre-F and adjuvant on Days 0 and 21 (Table 1). The mice were challenged 14 days after the second immunization (2×105 PFU/mouse). This challenge dose was selected based on our preliminary dose-finding experiments, which demonstrated that this dose induces detectable viral replication, significant body weight loss, and histopathological changes in BALB/c mice without causing excessive mortality, thereby allowing clear differentiation of protective efficacy among vaccine groups. The same dose was used for all groups to ensure comparability of the results. The changes in body weight were observed and recorded daily. The spleen and lungs of mice were removed aseptically for testing on the fifth day after challenge. Table 1 Group Antigen Adjuvant and dose Immunization Number Pre-F+AlOH+CpG Pre-F 25 μg CpG+50 μg AlOH 0, 21 6 Pre-F+AlOH Pre-F 50 μg AlOH 0, 21 6 Pre-F+BFA03 Pre-F 50 μL BFA03 0, 21 6 AlOH+CpG – 25 μg CpG+50 μg AlOH 0, 21 6 AlOH – 50 μg AlOH 0, 21 6 BFA03 – 50 μL BFA03 0, 21 6 Animal grouping and immunizations. IgG, IgG1 and IgG2a antibodies were detected by ELISA Specific IgG, IgG1, and IgG2a antibodies in mouse serum were detected by ELISA. The procedure was as follows: each well of the 96-well microplate was coated with 20 ng of Pre-F protein and incubated at 4 °C overnight. The next day, the microplate was blocked with PBS containing 3% BSA for 2 hours, washed, dried, and used for subsequent 4-fold dilution of the collected mouse serum (from 1:100). After incubation at 37 °C for 1 h, horseradish peroxidase (HRP)-labelled goat anti-mouse IgG (1:5000 dilution) and HRP-labelled goat anti-mouse IgG1 and IgG2a (1:50000 dilution) were added. After incubation at 37 °C for 1 h, TMB colour was observed. The cells were incubated at 37 °C for 15 min, and the absorbance (OD) was measured by a microplate reader (wavelength 450 nm) after the termination solution was added. EC50 values were calculated by 4-parameter fitting using GraphPad Prism software. Neutralizing antibody titre was detected by plaque reduction neutralization test HEp-2 cells were seeded onto 24-well plates one day in advance. A twofold ratio dilution (starting at 1:32) was performed on 60 μL of serum. The same volume of 50 PFU virus suspension was added to the diluted serum, and the mixture was mixed and neutralized in a 37 °C, 5% CO2 incubator for 2 hours. Then, the mixture was added to the cell wells, and each dilution was 2 wells. The cells were adsorbed for 1 hour. Inoculants were removed, and the overlay was added and cultured at 37 °C in a 5% CO2 incubator for 5 days. On Day 5, the overlay was removed by aspiration, the cells were fixed and stained with 0.5% crystal violet, and the number of plaques was counted. Neutralizing antibody titres were calculated using the Reed–Muench formula as the reciprocal of the serum dilution for which the number of plaques was reduced by 50%. The number of lymphocytes secreting IFN-γ and IL-4 was detected by ELISPOT Mouse spleen tissue was aseptically removed and placed into a culture dish containing 4 mL of mouse lymphocyte separation solution, and the spleen was ground with the inner core of a syringe. After grinding, the solution was transferred to a 15 mL centrifuge tube and covered with 1 mL of RPMI 1640 medium. After centrifugation at 800 × g for 25 min, the lymphocyte layer was aspirated, and 10 mL of RPMI 1640 medium was added, mixed, and centrifuged at 250 g for 5 min. Mouse spleen cells were collected and diluted to 1x106 cells/mL by adding culture medium. Each well was stimulated with 10 μL of Pre-F protein (10 μg/mL), the positive control was stimulated with 10 μL of positive stimulator (PMA+ionomycin), and the negative control was stimulated with 10 μL of culture medium. ELISPOT was used to detect the number of IFN-γ- and IL-4-secreting lymphocytes. Other procedures were performed according to the instructions of the ELISPOT kit (Daktronics Biotech, batch number: 2210002, 2210402). Cycle threshold (Ct) of HRSV nucleic acid of lung tissue The right lung tissue of mice was removed aseptically, and 2% DMEM was added for grinding. Two hundred microlitres was taken to extract nucleic acid, and the pulmonary viral load of HRSV in lung tissue was detected by real-time quantitative fluorescent PCR (RT–PCR). Pathological injury of lung tissue The left lung tissue was fixed with 4% paraformaldehyde for 24 hours and stained with haematoxylin and eosin (HE) to examine the characteristics of tissue sections, including alveolitis, bronchiolitis, and infiltration of inflammatory cells around blood vessels and in the tissue space. The lung tissues of individual mice were blindly evaluated by a pathologist at Beijing Lanrui Pu Technology Co., Ltd. The score was 0 (normal, no lesion), 1 (mild lesion, where the lesion area was less than 1/4 of the lung tissue section), 2 (moderate lesion, where the lesion area was approximately 1/4 to 2/4 of the lung tissue section), 3 (severe lesion, where the lesion area was approximately 2/4 to 3/4 of the lung tissue section), and 4 (very severe lesion, where the lesion area was more than 3/4 of the lung tissue section). Statistical analysis Excel software was used for data collation, and GraphPad Prism 9.0 software was used for statistical analysis and drawing. One-way analysis of variance was used to calculate whether a significant difference existed between groups. If P < 0.05, the difference was statistically significant and marked with *. Results Modification and analysis of the Pre-F protein Pre-F protein was expressed in CHO-S cells (purchased from Invitrogen). Pre-F contains six amino acid mutations (S155C, S290C, A149C, Y458C, S190F, and V207C), and a foldon sequence (GYIPEAPRDGQAYVRKDGEWVLLSTFL) and His tag were added at the C-terminus (Figure 1A). After affinity chromatography, the molecular weight of Pre-F was approximately 65 kDa (Figure 1B). The uniform trimer structure can be seen under transmission electron microscopy (Figure 1C). Figure 1 Modification and analysis of the Pre-F protein. (A) Schematic diagram of the Pre-F construct. The construct consists of the F2 subunit (brown), linker, and F1 subunit (light blue), followed by a C-terminal T4 fibritin trimerization motif (Foldon, dark blue), a thrombin cleavage site, and a 6× His tag (yellow). (B) SDS-PAGE analysis of the purified Pre-F protein. (C) Transmission electron microscopy (TEM) observation of the Pre-F protein. Humoral immune responses in mice To verify the humoral immune responses induced by the three adjuvants, indirect ELISA was used to detect the specific IgG antibody titres in the serum of mice. As shown in Figure 2A, the mice in the experimental groups (Pre-F+AlOH+CpG, Pre-F+AlOH, and Pre-F+BFA03) produced higher IgG antibodies than those in the adjuvant groups (AlOH+CpG, AlOH, and BFA03). The IgG antibody titre of the Pre-F+AlOH immunization group was the lowest (4.3×104) and was significantly lower than that of the Pre-F+BFA03 immunization group (P < 0.05). The IgG antibody titre of the Pre-F+BFA03 immunization group was the highest (1.9×105) but not significantly higher than that of the Pre-F+AlOH+CpG immunization group (9.1×104) (P = 0.1673). Figure 2 Humoral immune response in mice. (A) IgG antibody titres in serum on Day 35. (B) Serum neutralizing antibody titres of mice. (C) IgG1 and IgG2a titres in serum. (D) Ratio of IgG2a to IgG1 antibody. * indicates P< 0.05, ** indicates P< 0.01. Neutralizing antibodies are one of the impor
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