An mRNA-Based Respiratory Syncytial Virus Vaccine Elicits Strong Neutralizing Antibody Responses and Protects Rodents Without Vaccine-Associated Enhanced Respiratory Disease

Abstract

Background: Respiratory syncytial virus (RSV) causes the most common type of severe lower respiratory tract infection worldwide, and the fusion (F) protein is a target for neutralizing antibodies and vaccine development. This study aimed to investigate the immunogenicity and efficacy of an mRNA-based RSV vaccine with an F protein sequence.

Methods: We designed an mRNA construct encoding a modified RSV F protein, which was further developed into an LNP-encapsulated mRNA vaccine (LVRNA007). LVRNA007 was administered to mice and cotton rats, followed by immunogenicity analysis and viral challenge studies. Protection of rodents from the viral infection was evaluated based on the presence of the virus in the lung and pathological examination of respiratory tissues.

Results: LVRNA007 induced robust humoral and cellular immune responses in both mice and cotton rats, with neutralization antibody levels in the immunized animals maintained at high levels for over one year. Vaccination of LVRNA007 also protected the rodents from RSV challenge, judged by the much decreased virus titer and the pathological score in the lung tissue. In addition, no vaccine-enhanced disease (VED) phenomenon was observed with LVRNA007 vaccination.

Conclusions: Based on the preclinical immunogenicity and efficacy data, LVRNA007 could be a potential promising vaccine for prophylaxis of RSV infection.

Keywords: F protein; LVRNA007; RSV; mRNA vaccine; viral challenge study.

Figure 1

Design and expression of the mRNA antigens. (A) Schematic diagram of mRNA vaccine constructs (wild, DS-Cav1, or LVRNA007). SP, signal peptide. p27, p27 peptide. HRA, heptad repeat A. HRB, heptad repeat B. TM, transmembrane peptide. (B) HEK293T cells transfected with in vitro transcription (IVT) mRNA bound to antibodies against the antigenic site Φ, the trimeric site Φ, and site IV were analyzed using flow cytometry. (C) HEK293T cells were transfected with mRNA, and Western blotting was conducted to detect F0, F1, and GAPDH proteins. (D) Spherical nanoparticles were observed using a transmission electron microscope. Scale bar, 50 nm.

Figure 2

Durability of cellular and humoral immune responses after LVRNA007 administration. (A) Experimental design. Mice were immunized i.m. twice with LVRNA007 (5 μg or 15 μg, days 0 and 28) or LNP. (B–E) IFN-γ, IL-2, IL-4, and IL-10 cytokine levels in lymphocytes were detected through cytokine ELISAs on day 118 (90 days after the second vaccine dose). (F) IgG levels in serum samples were monitored for 364 days. (G) Neutralizing antibodies in serum samples were monitored for 364 days. n = 3. *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

Figure 3

LVRNA007 protected mice from RSV challenge without inducing VED. (A) Schematic depiction of the experimental strategy. Mice were immunized i.m. on days 0 and 21 with LVRNA007 (5 μg or 15 μg), FI-RSV (0.05 μg), or LNP and challenged intranasally with 5 × 10⁵ PFU of RSV A2 on day 35. The Health group was not treated. (B) Body weight was recorded and diagramed for 40 days. (C) Specific binding antibody levels in the mice were analyzed using ELISA on day 35. (D,E) Neutralizing antibody titers against RSV A2 and RSV B 18537 strains were measured in serum samples on day 35. (F) RSV titer was measured in the lung tissue. (G) Pathological score was calculated on day 40. (H) Histopathological examination of lung tissue was conducted using HE staining. Scar bar, 200 μm. The blue arrows indicate perivasculitis. The black arrows indicate peribronchiolitis. The green arrows indicate alveolitis. The cyan arrows indicate interstitial pneumonia. n = 6. *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ns, no significance.

Figure 4

Effect of LVRNA007 on binding and neutralizing antibody titers in cotton rats. (A) Schematic depiction of the experimental strategy. Cotton rats were vaccinated i.m. on days 0 and 21 with LVRNA007 (5 µg or 25 µg), FI-RSV (0.05 μg), or LNP. The RSV live virus group received 5 × 10⁵ PFU of RSV A2 intranasally on day 28. All animals were challenged intranasally with 1 × 10⁶ PFU of RSV A2 on day 56. (B) Body weights of cotton rats were monitored and diagramed for 61 days. (C) Binding antibody levels in cotton rats were determined using ELISA on day 28. (D,E) Neutralizing antibody titers in serum against RSV A2 and RSV B 18537 strains were examined on day 28. (F) Binding antibody levels in cotton rats were measured using ELISA on day 56. (G,H) Neutralizing antibody titers in serum against RSV A2 and RSV B 18537 strains were analyzed on day 56. n = 8. *, p < 0.05; ***, p < 0.001; and ns, no significance.

Figure 5

LVRNA007 protected cotton rats from RSV challenge without inducing VED. (A) RSV titer of turbinate bones in cotton rats following challenge on day 61. (B) RSV titer of lung washes in cotton rats on day 61. (C) HE staining was performed to identify histopathological changes in the lung tissue. Scar bar, 200 μm. The blue arrows indicate perivasculitis. The black arrows indicate peribronchiolitis. The green arrows indicate alveolitis. The cyan arrows indicate interstitial pneumonia. (D) Pathological score of the lung tissue was calculated on day 61. (E–I) IFN-γ, IL-2, IL-4, IL-5, and IL-13 cytokine mRNA levels in the lung tissue were examined using real-time PCR. n = 8. *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ns, no significance.