Human respiratory syncytial virus (HRSV) is a leading cause of lower respiratory tract infection in infants and a significant pathogen in elderly and immunocompromised populations. The fusion (F) protein, particularly the prefusion conformation (Pre-F protein) that displays the site Ø neutralizing epitope, is the major antigenic target for neutralizing antibodies and current recombinant vaccine strategies. Adjuvants influence both magnitude and bias of the immune response; aluminium hydroxide (AlOH) often induces Th2-skewed responses, while CpG oligodeoxynucleotides (TLR9 agonists) favor Th1 responses. Novel oil-in-water formulations containing α-tocopherol and squalene (here, BFA03) have been used to enhance humoral and cellular responses in other recombinant vaccines. This study directly compared three adjuvant approaches—AlOH, AlOH+CpG, and BFA03—formulated with recombinant Pre-F in BALB/c mice to inform adjuvant selection for Pre-F–based HRSV vaccines.
The recombinant Pre-F protein used in this study was expressed in CHO-S cells and incorporated six stabilizing amino acid substitutions (S155C, S290C, A149C, Y458C, S190F, V207C), a C-terminal T4 foldon trimerization motif, and a His tag. After affinity purification, the protein migrated at approximately 65 kDa by SDS–PAGE and presented as a uniform trimer by transmission electron microscopy, consistent with a stabilized prefusion conformation.
Thirty-six female BALB/c mice (6–8 weeks old) were randomized into six groups (n=6 per group): three antigen groups (Pre-F+AlOH+CpG, Pre-F+AlOH, Pre-F+BFA03) and three adjuvant-only controls (AlOH+CpG, AlOH, BFA03). Mice received intramuscular injections on Days 0 and 21 of 5 µg Pre-F with the indicated adjuvants (25 µg CpG + 50 µg AlOH; 50 µg AlOH; or 50 µL BFA03). Fourteen days after the second immunization animals were challenged intranasally with 2×105 PFU of HRSV-Long. Body weight was recorded daily, and spleen and lung tissues were collected on Day 5 post-challenge for immunological assays and viral load/histopathology assessments. The challenge dose was chosen to produce measurable replication and pathology without excessive mortality.
Pre-F–specific serum IgG was measured by ELISA and reported as EC50. All antigen-containing groups developed substantially higher IgG titres than adjuvant-only controls. The Pre-F+BFA03 group induced the highest IgG titre (1.9×105) and the Pre-F+AlOH group the lowest among antigen groups (4.3×104); the difference between Pre-F+BFA03 and Pre-F+AlOH reached statistical significance (P<0.05). Neutralizing antibody titres determined by plaque reduction neutralization assay showed the highest titre in the Pre-F+BFA03 group (1716). The Pre-F+AlOH+CpG group generated neutralizing antibody levels comparable to BFA03, while Pre-F+AlOH alone elicited the weakest neutralizing response among the antigen groups.
Cellular responses were assessed by ELISPOT for IFN-γ and IL-4–secreting splenic lymphocytes following Pre-F stimulation and by measuring IgG1 and IgG2a isotype titres. The data indicate that AlOH alone tended to promote a Th2-biased profile (higher IgG1 relative to IgG2a and IL-4), whereas the addition of CpG or use of BFA03 shifted responses toward a more balanced or Th1-favoring profile with increased IFN-γ–secreting cells. The Pre-F+AlOH+CpG group induced cellular responses comparable to Pre-F+BFA03, supporting the ability of CpG to modulate the AlOH-driven Th2 bias.
After challenge, mice in antigen-immunized groups began to regain body weight by Day 3 post-challenge, whereas adjuvant-only groups continued to lose weight. Lung viral load was assessed by RT–PCR and reported as cycle threshold (Ct) values; the Pre-F+AlOH group showed the highest pulmonary viral load among antigen groups with a Ct value of 32.2, while Pre-F+BFA03 yielded the lowest viral load. These virological findings aligned with neutralizing antibody titres and weight recovery patterns.
Left lung tissues were fixed, sectioned, and stained with hematoxylin and eosin. Blinded histopathological scoring used a 0–4 scale based on lesion area and severity (alveolitis, bronchiolitis, perivascular and interstitial inflammatory infiltration). The Pre-F+AlOH group had the most severe lung pathology among antigen groups (score 2.83). The Pre-F+AlOH+CpG group had the lowest pathology score among antigen groups (2.16), and the Pre-F+BFA03 group demonstrated milder pathological changes consistent with lower viral load and higher neutralizing titres.
Assays included ELISA for IgG/IgG1/IgG2a, plaque reduction neutralization tests (PRNT) for neutralizing antibodies, ELISPOT for IFN-γ and IL-4, RT–PCR for lung viral load, and blinded histopathology scoring. One-way analysis of variance was used to evaluate differences between groups with P<0.05 considered significant. Animal experiments were approved by the Animal Experiment Ethics Committee of the National Institute for Viral Disease Control and Prevention, Chinese CDC. Details on reagent batch numbers for ELISPOT kits and basic procedural steps were provided in the source.
In this BALB/c mouse model, recombinant Pre-F protein formulated with either AlOH+CpG or BFA03 provided superior protection compared with AlOH alone, as judged by higher neutralizing antibody titres, lower lung viral loads, earlier weight recovery, and reduced lung pathology. BFA03 delivered the highest neutralizing titres and the lowest viral load, while AlOH+CpG achieved comparable immunogenicity and the lowest pathological score among antigen groups, demonstrating that CpG can mitigate AlOH-induced Th2 bias and improve protective outcomes. These results support the use of CpG-containing combinations or oil-in-water adjuvants like BFA03 in Pre-F recombinant vaccine formulations to enhance balanced humoral and cellular immunity against HRSV.
The study was performed in BALB/c mice and may not fully predict responses in humans or other target populations (infants, pregnant women, elderly). Direct comparisons with other licensed adjuvants (for example, AS01E) were not performed; the authors note that AS01E was not available as a standalone reagent at the time of study. Numerical datasets beyond summary values reported in the article (for example, full individual animal values) were not provided in the source text. The source reports that data availability, ethics approval, funding, and author contributions are documented in the original article.