A truncated pre-F protein mRNA vaccine elicits an enhanced immune response and protection against respiratory syncytial virus

Abstract

The Food and Drug Administration (FDA) has approved vaccines designed by GSK, Pfizer and Moderna to protect high-risk populations against respiratory syncytial virus (RSV). These vaccines employ the pre-fusion F (pre-F) protein as the immunogen. In this study, we explored an mRNA vaccine based on a modified pre-F protein called LC2DM-lipid nanoparticle (LC2DM-LNP). This vaccine features a truncated version of the pre-F protein that is anchored to the cell membrane. Our experiments in young and old female mice revealed that the LC2DM-LNP vaccine elicited robust neutralizing antibody titers. Moreover, LC2DM-LNP prompted a Th1-skewed T-cell immune response in female rodent models. Female cotton rats immunized with LC2DM-LNP demonstrated strong immunity to RSV, without signs of vaccine-enhanced respiratory disease (VERD), even in cases of breakthrough infection. Importantly, when administered to pregnant female cotton rats, LC2DM-LNP ensured the transfer of pre-F-specific antibodies to the offspring and provided protection against RSV without increasing lung inflammation. Our findings suggest that LC2DM-LNP could serve as an alternative RSV vaccine candidate for high-risk groups.

Here the authors design an mRNA based RSV vaccine, expressing a truncated membrane-anchored version of the stabilized pre-F protein, and demonstrate strong humoral and Th1-skewed T-cell responses in small animal models without signs of vaccine-enhanced respiratory disease.

Introduction

Human respiratory syncytial virus (RSV), a member of the Pneumoviridae family, is a leading cause of acute lower respiratory infections in children under 5 years of age worldwide^1,2. Most children under 2 years of age experience RSV infections, among whom recurrence is common due to the relatively low natural immunity in this age group^3,4. In older adults, particularly those with a weakened immune system, RSV can have an impact similar to that of influenza A⁵. Despite its significant health burden, RSV vaccine development has faced challenges, most notably with the failure of the FI-RSV vaccine in the 1960s, which not only failed to protect against the disease but also led to vaccine-enhanced respiratory disease (VERD)^6–8. Recent breakthroughs, however, have been marked by the approval of RSV vaccines by GSK, Moderna, and Pfizer for use in older adults, with the latter also approved for pregnant women to protect their infants⁹.

The RSV F protein, a class I fusion glycoprotein responsible for viral entry into host cells, transitions between two conformations: a metastable prefusion (pre-F) and a stable postfusion (post-F) form. The pre-F conformation is an ideal vaccine target, as most neutralizing antibodies (nAbs) in human sera are directed against this form of the protein¹⁰. The failure of the FI-RSV vaccine is attributed partly to the loss of pre-F on the virion surface, thereby diminishing its protective efficacy¹¹. Advances in stabilizing pre-F proteins such as DS-Cav1¹², SC-TM¹³, and pre-F 847¹⁴ have resulted in higher nAb titers than with post-F-based vaccines in both animal studies and clinical trials^7,15–17. The three FDA-approved RSV vaccines leverage the pre-F conformation to achieve their efficacy.

Severe RSV infection in neonates and older adults is associated with the production of antibodies exhibiting low neutralization activity^18,19. Therefore, further improvement in antibody neutralization ability and quality, especially in vulnerable populations, remains a critical objective for vaccine development. One promising approach is immune focusing, which has been shown to improve antibody quality for other viruses, such as Middle East respiratory syndrome coronavirus (MERS)²⁰ and SARS-CoV-2^21,22. The RSV pre-F trimer consists of a “stalk” and a “head” region, with antigenic sites Φ and V located on the head, recognized by potent nAbs such as D25¹² and hRSV90^23,24. Site II, which is located centrally on the trimer, binds antibodies of lower potency, such as palivizumab and motavizumab (Mota)²⁵, whereas the stalk region contains antigenic sites I and IV, which are targeted by even weaker antibodies, including 4D7²⁶ and 101F^27,28. The surface of the pre-F protein, including sites Φ and V, is relatively small, suggesting the presence of several nonneutralizing epitopes that can reduce the potency of the immune response. Kwong et al. explored “head-only” immunogens to focus the antibody response onto site Φ, but these immunogens yielded lower neutralization titers than did antibodies targeting the entire DS-Cav1 trimer²⁹. Thus, while focusing on key antigenic sites may improve antibody quality, it may compromise overall immunogenicity, suggesting that further refinement is needed.

In addition to effectiveness, a significant obstacle to the development of RSV vaccines is the risk of VERD caused by a Th-2-skewed immune response³⁰. To mitigate this risk, the incorporation of Th-1-biasing adjuvants is crucial, as nonreplicating vaccines without such adjuvants may continue to pose a risk of VERD. This concern is particularly important in vulnerable populations such as infants, for whom only a limited number of Th-1-inducing adjuvants are approved for use. Furthermore, older adults, who are more susceptible to severe RSV disease, tend to exhibit Th-2-biased immune responses, with weaker RSV-specific CD8+ T-cell activity than younger adults³¹. Therefore, an ideal RSV vaccine must not only generate high titers of nAbs but also induce a robust Th1-skewed cellular immune response to provide broad and effective protection in all age groups.

Recent advances in mRNA technology offer promising solutions for designing RSV vaccines. mRNA vaccines can induce both humoral and cellular immune responses and efficiently express complex or unstable antigens³². Additionally, mRNA vaccines for SARS-CoV-2 have shown acceptable safety in various populations, including children³³, pregnant women³⁴, and older adults³⁵. Moderna’s mRNA vaccine targeting the pre-F conformation for elderly populations represents a significant milestone and highlights the potential of this approach for RSV prevention.

However, concerns regarding the safety of mRNA vaccines in pediatric populations have arisen. A recent clinical trial of Moderna’s mRNA vaccine in young children revealed an imbalance in severe RSV-associated lower respiratory tract infections between vaccinated and placebo groups in RSV-naïve infants, leading to a study pause and raising questions about the risk of VERD³⁶. It remains unclear whether these safety concerns are analogous to those associated with the FI-RSV vaccine, as Moderna’s mRNA vaccine utilizes the pre-F antigen to elicit high-affinity nAbs and induces a balanced Th1/Th2 helper T-cell response, theoretically addressing the primary hypotheses for VERD observed with FI-RSV. Given that mRNA vaccines for SARS-CoV-2 have not shown similar safety issues in children, it is more likely that the challenges stem from the intrinsic complexity of RSV itself rather than the mRNA platform. Consequently, optimizing and redesigning RSV antigens may represent a critical next step for improving the safety and efficacy of mRNA vaccines for pediatric use.

In this study, we developed a modified pre-F protein, termed LC2DM, designed to enhance immunogenicity by focusing on potent epitopes on the cell membrane. LC2DM mRNA was encapsulated in lipid nanoparticles (LNPs) to create the vaccine formulation. In vivo studies in various animal models showed that LC2DM-LNP elicited strong humoral and Th-1-skewed T-cell responses. Furthermore, cotton rats vaccinated with LC2DM-LNP demonstrated complete protection against RSV infection, without signs of VERD. Importantly, the offspring of LC2DM-LNP-immunized cotton rats also exhibited robust protection against RSV without presenting with exacerbated lung disease. These findings support the potential of the LC2DM-LNP vaccine as a promising RSV vaccine candidate, particularly for high-risk populations.

Results

Design and characterization of truncated pre-F immunogens from mRNA vaccines

“Head-only” immunogens can improve antibody quality but at the cost of slightly reduced immunogenicity²⁹. Therefore, we pursued three distinct approaches to further increase the immunogenicity of the “head-only” immunogen. First, we replaced the RSV A2 F protein signal peptide sequence with human tissue plasminogen activator (tPA) to ensure secretion^37,38 and maintained the transmembrane domain to create an immunogen that could be expressed at the immune site for long periods^39,40. Second, we investigated 13 designs generated by different truncation and connection strategies (Table S1) and identified one design, LC2, that best preserved the pre-F-specific sites (Fig. S1). Finally, to enhance the stability of the immunogen, we introduced two mutations (N67I, S215P) into LC2 and named the final construct LC2DM¹³ (Fig. 1a). We subsequently introduced the coding sequences of LC2DM into a template plasmid harboring the T7 RNA polymerase promotor, a 5′ untranslated region (UTR), a 3′ UTR, and a 3′ poly (A) tail to generate mRNA antigens. The pre-F sequence from SC-TM¹³, which contains a transmembrane domain, was also included in the template plasmid to generate a control mRNA antigen (Fig. 1a). Nucleotide-modified mRNAs were synthesized via in vitro transcription. The integrity and capping rate of the mRNAs were confirmed via microfluidic capillary electrophoresis and liquid chromatography–mass spectrometry (LC–MS). These methods revealed sharp, single peaks and capping ratios exceeded 99.7% for both mRNAs. (Fig. S2a, b). To obtain the vaccine formulation, we encapsulated the two mRNAs with LNPs (Fig. 1b). The tested mRNA-LNPs exhibited a high encapsulation efficiency (>95%) and an average diameter of 100 nm, with a narrow polydispersity index of 0.149 (Fig. S2c). The morphology of the LNPs was evaluated via transmission electron microscopy and revealed that the nanoparticles were homogeneous and had regular round shapes (Fig. S2d).

Fig. 1. Design of the truncated, membrane-anchored pre-F protein and characterization of the expressed antigens.

a Design and construction of the mRNAs expressing pre-F (mPre-F) and truncated, membrane-anchored pre-F (LC2DM). Functional domains are labeled. SP: signal peptide; F2: fusion glycoprotein F2; F1: fusion glycoprotein F1; TM: transmembrane domain; CT: cytoplasmic tail. The solid triangles indicate the furin proteolytic sites; red lines indicate point mutations. GOI: gene of interest. b The coding sequences of the two constructs were introduced into a template plasmid harboring a 5' untranslated region (UTR), a 3′ UTR, and a 3′ poly (A) tail to generate mRNA, which was finally encapsulated in a lipid nanoparticle for in vitro and in vivo experiments. c, d HeLa cells were transfected with equal molar masses of antigen mRNAs or no mRNA (negative control) using LNPs for 24 h. Mean fluorescent intensity (MFI) and percentage of cells transfected with mRNAs expressing candidate antigens bound by Mota, D25, hRSV90, or 4D7 are shown. Results are representative of three separate experiments performed under identical conditions. e HeLa cells were transfected with mRNA of sPre-F, mPre-F, or LC2DM. At 24 h, cells were fixed, permeabilized with or without Triton X-100, and stained to assess the expression and localization of the tested antigens using the epitope-specific antibodies (D25). Scale bar represents 7.5 μm. Data represents one of three independent experiments. Source data are provided as Source Data File.

The antigenic profiles, expression levels, and subcellular locations of the mRNA/LNPs were confirmed via flow cytometry, cell-based ELISA, and immunofluorescence microscopy, respectively. Equal molar masses of the mRNA of the anchored form of pre-F (mPre-F) and LC2DM were transfected into HeLa cells via LNPs, which were subsequently stained with Mota, D25, hRSV90, and 4D7 antibodies for flow cytometry analysis (Fig. 1c, d and Fig. S3a). The results demonstrated that cells transfected with either mPre-F or LC2DM mRNA exhibited significant binding to the Mota, D25, and hRSV90 antibodies, with positive rates of up to 90%, and both antigens exhibited negative binding to the post-F-specific antibody 4D7. Furthermore, compared with mPre-F mRNA-transfected cells, LC2DM-transfected cells presented with an ~1.2-fold greater mean fluorescent intensity (MFI). Cell-based ELISA was used to further investigate the membrane expression levels of mPre-F and LC2DM⁴¹. The EC50 values were calculated with gradient dilution of the mRNAs with both the transfection reagent and LNPs. When the transfection reagent was used, the EC50 values were similar for both mRNAs, whereas the EC50 value of LC2DM was ~2-fold lower than that of mPre-F when LNPs were used (Fig. S3b, c). Membrane locations were further confirmed through immunofluorescence. A secreted form of pre-F (sPre-F), which incorporates a foldon sequence at its C-terminus instead of the TM domain contained in pre-F, was constructed as a negative control. As anticipated, sPre-F-LNP remained undetectable unless it was prepermeabilized with Triton X-100, upon which it was primarily distributed in the cytoplasm. In contrast, both mPre-F-LNP and LC2DM-LNP were detectable regardless of permeabilization and were mainly localized in the cell membrane (Fig. 1e).

LC2DM-LNP induces high levels of neutralizing antibodies in young and elderly BALB/c mice

The immunogenic potency of LC2DM-LNP was compared with that of mPre-F-LNP and a Pre-F+Alum control in young adult BALB/c mice through a dose-response study ranging from 0.1 to 20 μg(Fig. 2a). Serum IgG binding to pre-F and post-F proteins after secondary immunization was assessed via ELISA. At higher doses (10 and 20 μg), LC2DM-LNP and mPre-F-LNP induced comparable levels of pre-F-binding IgG, whereas comparatively, post-F-binding IgG titers in LC2DM-LNP vaccinated mice were significantly reduced, ~13-fold less, suggesting a potential selective immune response (Fig. 2b). Neutralization assays against RSV-A2-mKate2 demonstrated a dose-dependent correlation between the vaccines and antigen-specific IgG titers. Peak nAb titers were reached at 1 μg for Pre-F+Alum and at 10 μg for both mRNA vaccines (mPre-F-LNP and LC2DM-LNP) (Fig. 2c). Remarkably, at doses of 10 and 20 μg, LC2DM-LNP achieved the highest nAb titer (~2194), significantly exceeding those of Pre-F+Alum (~351) and mPre-F-LNP (~583)(Fig. 2c). We purified serum antibodies from each vaccinated group at a 10 μg dose and quantified their half-maximal inhibitory concentration (IC50). The results revealed IC50 values of 0.42 μg/mL, 0.38 μg/mL, and 0.19 μg/mL for Pre-F+Alum, mPre-F-LNP, and LC2DM-LNP, respectively (Fig. S4), suggesting that LC2DM-LNP may enhance antibody quality in vivo.

Fig. 2. Immunogenicity of candidate vaccines in young adult and elderly mice.

a Experiment timeline. Young female adult BALB/c mice (6–8 weeks old, n = 5 mice per group in (b, c)) were intramuscularly injected with either 0.1, 1, 10, or 20 μg of Pre-F+Alum, mPre-F-LNP, and LC2DM-LNP at week 0 and 2. Serum samples were collected at week 4. b Levels of pre-F- and post-F-specific IgG in sera of vaccinated young mice determined by ELISA. c Serum neutralizing antibody (nAb) titers measured with RSV-A2-mKate2. d Experiment setup. Elderly Female BALB/c mice (18–20 months old, n = 3 mice per group in (e, f)) received 1 μg of pre-F protein with Alum or 10 μg of mRNA-LNP intramuscularly at weeks 0 and 2. Serum samples were collected and analyzed on days 14, 21, 42, 56, and 84 after priming. One animal in the Pre-F+Alum immunized group died at day 28, and one in the mPre-F-LNP immunized group died at day 70. e Levels of pre-F- and post-F-specific IgG in sera of vaccinated elderly mice determined by ELISA. f Serum neutralizing antibody (nAb) titers measured with RSV-A2-mKate2. Dotted lines in (b, c, e, f) represent the lower limit of detection. Data are presented as geometric means with geometric SD in (b, c, e, f). Statistical analysis was performed with two-way ANOVA and Tukey’s multiple comparisons test in (b, c). Asterisks indicate significance with respect to LC2DM-LNP-vaccinated animals. Source data are provided as Source Data File.

Older individuals are particularly vulnerable to RSV, so we evaluated the immunogenic potential of LC2DM-LNP in aged mice (18–20 months) to simulate the waning immunity in older adults. The mice were intramuscularly vaccinated with Pre-F+Alum, mPre-F-LNP, or LC2DM-LNP via a prime–boost regimen in which the two were spaced 14 days apart. Serum samples were collected on days 14, 21, 42, 56, and 84 after the first vaccination (Fig. 2d). LC2DM-LNP elicited a pre-F–focused response, characterized by increased pre-F-binding titers and nearly undetectable post-F binding titers (Fig. 2e). In all the immunization groups, the highest binding titers were observed 42 days post priming (Fig. 2e), and the nAb titers reached peak levels at the same time before declining (Fig. 2f). Notably, the nAb titers in the LC2DM-LNP–immunized mice after boosting were greater than those measured in the mice immunized with Pre-F+Alum or mPre-F-LNP. On day 84, all immunized mice presented with reduced nAb titers; however, compared with those on day 42, pre-F+Alum-immunized mice presented with a greater decrease in nAb titers (to undetectable levels), whereas mPre-F-LNP– and LC2DM-LNP–immunized mice still presented with some nAb titers, with LC2DM-LNP-immunized mice presenting the highest nAb titers on day 84. These findings suggest that mRNA vaccines may induce a more persistent humoral response and that the truncated, membrane-anchored pre-F–based mRNA vaccine, LC2DM-LNP, elicits a robust and sustained humoral response even in aged mice.

LC2DM-LNP induces a high-quality antibody response

Having confirmed the strong immunogenicity of LC2DM-LNP in both young and elderly mice, we next sought to evaluate the antibody quality of this vaccine. LC2DM harbors three known epitopes, namely site II, Φ, and V, with site II-targeting antibodies showing lower neutralization efficacy. Building upon the work of Swanson et al. on improving antibody quality through the removal of site II⁴², we engineered LC2DM-IIKO, a site II-deficient variant of LC2DM. We validated that this knockout was specific and did not impact the other epitopes with immunofluorescence (Fig. S5). Mice were intramuscularly vaccinated with phosphate-buffered saline (PBS), Pre-F+Alum, mPre-F-LNP, LC2DM-LNP, or LC2DM-IIKO-LNP via a prime–boost regimen in which the two injections were spaced 14 days apart. Serum samples were collected 4 weeks after the first vaccination (Fig. 3a). Postvaccination serological assessment revealed that LC2DM-LNP and LC2DM-IIKO-LNP induced comparable pre-F-binding antibodies to those of mPre-F-LNP but with diminished post-F binding (Fig. 3b). The ratio of pre-F-binding IgG to post-F binding IgG was significantly greater for these two vaccines, indicating a targeted immune response (Fig. 3c). Serological profiling for the conformation of the F protein revealed that LC2DM-LNP elicited the highest D25 and hRSV90 competitive antibody levels, whereas LC2DM-IIKO-LNP elicited the lowest for hRSV90 competitive antibody levels (Fig. 3d), suggesting a potential adverse effect of site II knockout on hRSV90 binding. Analysis of nAb titers revealed that LC2DM-LNP reached the highest nAb titers against RSV A2-mkate virus of ~3254, which was significantly greater than that of Pre-F+Alum (~336) and mPre-F-LNP (~762) (Fig. 3e). While LC2DM-IIKO-LNP presented with slightly lower nAb titers than did LC2DM-LNP (~1759), it still demonstrated higher nAb titers than the other vaccine formulations (Fig. 3e). In terms of neutralization against RSV B 18537, LC2DM-LNP still reached the highest nAb titers (~1650), significantly surpassing Pre-F+Alum (~211) and LC2DM-IIKO-LNP (~217) and achieving an ~2-fold greater nAb titers than mPre-F-LNP (~765) (Fig. 3e). Previous studies have suggested that virus-binding antibodies that are induced by vaccination but fail to provide neutralizing activity (i.e., nonneutralizing antibodies) may lead to VERD effects^43,44. Therefore, we compared the ratios of neutralizing to binding antibodies of the four tested vaccines and discovered that removing site II did not increase this ratio. Moreover, the ratios were similar among the Pre-F+Alum, mPre-F-LNP, and LC2DM-IIKO-LNP groups, whereas LC2DM-LNP elicited the highest ratio of neutralizing to binding antibodies (Fig. 3f). Consequently, the LC2DM-LNP vaccine might offer advantages in avoiding potential VERD effects.

Fig. 3. Humoral and cellular response of LC2DM-LNP in BALB/c mice.

a Experiment timeline. Young female adult BALB/c mice (6–8 weeks old, n = 5 mice per group in (b–i)) were intramuscularly injected with either 1 μg of pre-F protein with Alum or 10 μg of mRNA-LNPs at weeks 0 and 2, and serum was collected at week 4 and spleens at week 6 post-priming. b Levels of pre-F- and post-F-specific IgG in the sera of vaccinated young mice determined by ELISA. c Ratios of pre-F binding IgG titers to post-F binding IgG titers. d Serum levels of pre-F D25 competitive antibodies and pre-F hRSV90 competitive antibodies determined with competitive ELISA. e Serum neutralizing antibody (nAb) titers measured with RSV-A2-mKate2 and RSV 18537, as described in the Materials and Methods. f Ratios of nAb titers to pre-F binding Ab titers elicited by candidate vaccines. g Pre-F-specific IgG, isotype IgG1 and IgG2a serum antibodies. (h, i) Percentages of IFN-γ– and IL-4– producing CD4+ and CD8+ T cells isolated from spleens, quantified after stimulation of a peptide pool covering the entire F protein. The gating strategy is summarized in Supplementary Fig. 6. Dotted lines in (b, d, e, g) represent the lower limit of detection. Data are presented as geometric means with geometric SDs in (b, e, g) and as mean values with SDs in (c, d, f, h, i). Statistical analysis was performed with two-way ANOVA and Tukey’s multiple comparisons test in (e) and with one-way ANOVA and Tukey’s multiple comparisons test in (b–d) and (f–i). Source data are provided as Source Data File.

mRNA-LNP-based vaccines elicit Th1-biased and RSV-specific T-cell response

To evaluate the likelihood of VERD manifesting from Th2-biased immune responses, we investigated the balance between Th1- and Th2-biased immune responses in vaccinated mice. Given the negligible production of post-F-binding antibodies following LC2DM-LNP and LC2DM-IIKO-LNP administration (Fig. 3b), we conducted ELISA based on pre-F to determine the degree of Th bias. As anticipated, the IgG2a/IgG1 ratios of all the mRNA vaccines were markedly greater than those of Pre-F+Alum (Fig. 3g). Furthermore, the ratios were found to be similar among LC2DM-LNP, LC2DM-IIKO-LNP, and mPre-F-LNP, suggesting that mRNA vaccines elicit a Th1-skewed T-cell immune response.

Subsequently, we sought to assess T-cell responses via the collection of spleen cells from immunized mice 4 weeks after the second immunization, which we then stimulated with pooled peptides of RSV F. The quantity of T cells producing cytokines was measured via intracellular cytokine staining (ICS) and ELISpot assays. Following peptide stimulation, the LC2DM-LNP-, LC2DM-IIKO-LNP-, and mPre-F-LNP-immunized mice demonstrated a marked population of CD4+ and CD8+ T cells that produced IFN-γ, as demonstrated by ICS (Fig. 3h, i and Fig. S6a). LC2DM-LNP exhibited the most robust response in terms of IFN-γ production by both CD8+ and CD4+ T cells, indicating that the protein truncation did not adversely affect the T-cell response or result in a Th1-biased T-cell response (Fig. 3h, i). The Pre-F+Alum control vaccine did not induce the production of either IFN-γ or IL-4 (Fig. 3h, i). The T-cell responses were also confirmed via an ELISpot assay (Fig. S6b).

LC2DM-LNP protects cotton rats against the RSV Long challenge without leading to VERD

Researchers have previously utilized the semipermissive cotton rat as a model for RSV infection to evaluate the efficacy of potential RSV vaccines and study VERD^45,46. Here, this model was used to assess the safety and protective efficacy of the designed vaccines. Since LC2DM-IIKO-LNP produced a less than satisfactory humoral response, we investigated responses using only LC2DM-LNP. Multiple groups of cotton rats were employed; three were separately immunized intramuscularly with vaccines, including Pre-F+Alum, mPre-F-LNP, and LC2DM-LNP, and two groups received PBS injections as control. All cotton rats were treated according to a prime–boost protocol with injections spaced 3 weeks apart. Additionally, FI-RSV was included as a positive VERD vaccine control (Fig. 4a). Serum samples were collected 2 weeks after each immunization to evaluate humoral responses in the cotton rats. The results were consistent with those observed in BALB/c mice, in which all the vaccine-immunized groups, except the FI-RSV-immunized group, presented with high levels of serum antibodies against the pre-F protein (Fig. 4b). Notably, the LC2DM-LNP vaccine elicited significantly lower post-F binding titers than the Pre-F+Alum, mPre-F-LNP and FI-RSV vaccines did (Fig. 4b). As anticipated, LC2DM-LNP induced significantly higher nAb titers (~20,570) than Pre-F+Alum (~2356), mPre-F-LNP (~8633) and FI-RSV (~69) (Fig. 4c).

Fig. 4. Cotton rat immunization and RSV challenge.

a Schematic diagram of the vaccination regimen and RSV challenge protocol in cotton rats. Female cotton rats (6–8 weeks old, n = 5 cotton rats per group in (b–e)) were immunized intramuscularly with 5 μg of pre-F+Alum, 20 μg of mRNA-LNPs, PBS, PBS or FI-RSV on days 0 and 21. Serum samples were collected and analyzed on day 14 and 35. Animals were challenged with RSV Long intranasally at a titer of 1 × 10⁶ PFU on day 49, with the exception that one of the PBS vaccinated groups did not challenge the virus as a negative control. Five days after challenge, lung tissues were harvested. b Levels of pre-F- and post-F-specific IgG in the sera of vaccinated animals determined by ELISA. c Serum neutralizing antibody (nAb) titers measured with RSV-A2-mKate2. d Lung viral titers (left) and viral N-gene copies (right) determined with plaque assay and RT–qPCR. e Lung pathology following RSV challenge, alevolitis (left), and total pathology scores (right). The lower dotted lines in (b, c) and d left represent the lower limit of detection, while the upper dotted lines in (c) represents the protective threshold. Data are presented as geometric means with geometric SDs in (b, c) and as means with SDs in (d, e). Statistical analyses were performed with one-way ANOVA, Tukey’s multiple comparisons (b–d), and the Kruskal--Wallis test (nonparametric) (e). Source data are provided as Source Data File.

All vaccinated animals, except one PBS control group (unchallenged as negative control), were challenged with RSV. Samples from the right lung were collected after 5 days for viral titration and viral load quantification, while the left lung was preserved in formalin, stained with hematoxylin and eosin (H&E), and evaluated for pathological severity on a scale of 0 to 4, where 0 signifies no pathology and 4 reflects the highest severity, as outlined in previous research⁴⁷. Both the Pre-F+Alum-vaccinated cotton rats and the cotton rats vaccinated with the two mRNA/LNPs showed protection against the RSV challenge in the lungs (Fig. 4d). An examination of the lungs revealed indicators of peribronchiolitis, perivasculitis, interstitial pneumonia, and alveolitis; the last one, alveolitis, is the most pivotal markers of VERD in cotton rats vaccinated with FI-RSV⁴⁸. Compared with PBS-treated rats, FI-RSV-vaccinated cotton rats exhibited more severe VERD-related lung inflammation following RSV challenge, as evidenced by significantly elevated alveolitis and higher overall lung pathology scores (Fig. 4e and Fig. S7a). In contrast, Pre-F+Alum and both mRNA/LNP vaccines resulted in less severe lung pathology, with LC2DM-LNP producing significantly lower total pathology scores than FI-RSV (Fig. 4e and Fig. S7a). These findings suggest that the LC2DM-LNP vaccine provides effective protection against RSV in cotton rats.

To further assess whether LC2DM-LNP induces VERD during breakthrough infection, a dose–gradient experiment was conducted. Experimental cotton rats were immunized with LC2DM-LNP at doses of 20, 10, 5, 1, and 0.1 μg on days 0 and 21, and a VERD-positive FI-RSV-injected group was used as controls (Fig. 5a). Serum samples were collected 2 weeks after the second dose, and nAb titers were evaluated. All the LC2DM-LNP-immunized groups, except the 0.1 μg group, reached protective thresholds (Fig. 5b). Although the 0.1 μg LC2DM-LNP group elicited similarly low nAb titers as the FI-RSV group, only two rats in this group experienced breakthrough infections, compared to all rats in the FI-RSV group (Fig. 5c). This outcome suggests that the mRNA vaccine may provide a specific advantage in controlling viral replication via T cell responses. Lung pathology revealed the strongest inflammation in the FI-RSV group, whereas the LC2DM-LNP-immunized groups, even those with breakthrough infections, presented with alleviated inflammation and no signs of enhanced pathology (Fig. 5d and Fig. S7b). As VERD is linked to Th2-biased T-cell responses, cytokine expression was assessed in lung tissue. Specifically, mRNA in the lungs was analyzed to determine the expression of Th1- and Th2-associated cytokines, including IFN-γ, IL-4, and IL-13. As expected, the expression of IL-4 and IL-13 expression was highest in the FI-RSV group (Fig. 5e), indicating a Th2-associated VERD response. In contrast, cytokine expression in the LC2DM-LNP-immunized groups was lower than that in the PBS-treated control group (Fig. 5e). These findings indicate that LC2DM-LNP may has a favorable safety profile.

Fig. 5. Evaluation of signs of VERD associated with the candidate vaccine in cotton rats.

a Schematic diagram of the vaccination regimen and RSV challenge protocol in cotton rats. Female cotton rats (6–8 weeks old, n = 5 cotton rats per group in (b–e)) were immunized intramuscularly with 0.1, 1, 5, 10, or 20 μg of LC2DM-LNP, PBS, PBS, or FI-RSV on days 0 and 21. Serum samples were collected and analyzed on day 35. Serum nAb titers were measured with RSV-A2-mKate2. b Serum neutralizing antibody (nAb) titers measured with RSV-A2-mKate2. c All animals from (a), with the exception of one PBS-treated group as a negative control, were challenged with RSV Long via intranasal administration at a titer of 1 × 10⁶ PFU on day 49. Five days after the challenge, lung tissues were harvested. Lung viral titers was determined with plaque assays. d Lung pathology following RSV challenge for the animals from (a), alevolitis (left), and total pathology scores (right). e Cytokine gene expression after RSV challenge. mRNA was isolated from the lungs of each cotton rat, and cytokine expression levels were normalized to β-actin. The mRNA fold changes were calculated relative to unchallenged cotton rats. The lower dotted lines in (b, c) represent the lower limit of detection, while the upper dotted lines in (b) represents the protective threshold. Data are presented as geometric means with geometric SDs in (b, c) and as means with SDs in (d, e). Statistical analyses were performed with one-way ANOVA, Tukey’s multiple comparisons in (b, c, e), and the Kruskal–Wallis test (nonparametric) (d). Source data are provided as Source Data File.

Immunogenicity and protective efficacy of a single dose of LC2DM-LNP in cotton rats with prior RSV exposure

Given that most humans encounter RSV by age two, the results from RSV-naïve animal models may not fully represent human responses. To address this, we assessed the immunogenicity of a single dose of LC2DM-LNP in cotton rats previously exposed to RSV, as no previous studies have indicated additional immunogenic benefits from a second dose⁴⁹. Cotton rats were primed with intranasal administration of 2 × 10⁵ PFU of RSV Long. Four months after the initial RSV challenge, the animals were divided into groups and administered PBS, FI-RSV, Pre-F+Alum, mPre-F-LNP, or LC2DM-LNP. Two weeks postvaccination, serum samples were collected to assess the antibody responses (Fig. 6a). The results revealed that preexposing cotton rats to RSV resulted in a post-F-biased IgG response, demonstrating post-F-binding titers 33-fold higher than pre-F titers (Fig. 6b). Despite this preexisting bias, LC2DM-LNP increased pre-F-binding IgG titers 425-fold and nAb titers 59-fold in primed cotton rats, similar to those of mPre-F-LNP and Pre-F+Alum (Fig. 6b, d). Given that human RSV F-specific antibodies predominantly target the pre-F conformation⁵⁰, the enhanced immunogenicity observed with LC2DM-LNP may offer even more pronounced benefits in humans.

Fig. 6. Immunogenicity of candidate vaccines in RSV-preexposed cotton rats.

a Study timeline. Female cotton rats (6–8 weeks old, n = 5 cotton rats per group in (b–e)) were initially administered 2 × 10⁵ PFU of RSV Long intranasally and divided into five subgroups. Four months after the initial exposure, the animals were vaccinated with 5 μg of Pre-F protein with Alum, 20 μg of mRNA-LNPs, PBS, or FI-RSV. Serum samples were collected 2 weeks post-vaccination, and another 2 weeks later, the animals were challenged with 1 × 10⁶ PFU of RSV Long. b Levels of pre-F- and post-F-specific IgG in the sera of vaccinated animals, determined by ELISA. c Serum neutralizing antibody (nAb) titers measured with RSV-A2-mKate2. d Lung viral titers determined with plaque assays. e Lung pathology following RSV challenge, total pathology scores. Dotted lines represent the lower limit of detection in (b–d). Data are presented as geometric means with geometric SDs in (b, c) and as means with SDs in (d, e). Source data are provided as Source Data File.

The preexposure model established in cotton rats was also suitable for assessing vaccine protection. We attempted an RSV challenge for each immune group. Four weeks after single-dose immunization, the cotton rats were inoculated with RSV Long virus via intranasal administration of 10⁶ PFU of the virus. Five days after the challenge, the right lung was collected for viral titer detection, and the left lung was collected for pathological examination. Notably, all immunized cotton rats were completely protected against RSV infection, even the PBS-treated group, in whom no infection was observed (Fig. 6d). These results suggest that prior infection may have established robust protection in cotton rats, enabling complete control of the secondary infection. This finding is consistent with previous studies showing that preinfection with RSV results in abortive RSV infection in a rechallenge model^51,52. Furthermore, no severe pathology was observed in any of the vaccinated groups (Fig. 6e).

Pups from LC2DM-LNP–vaccinated mothers maintained high nAb titers within 4 weeks

Young children aged 2 years and younger are most vulnerable to RSV infection, and maternal immunization is a major preventive measure for newborns. Here, we developed a maternal immunization model in cotton rats to evaluate the potential utility of LC2DM-LNP as a maternal vaccine (Fig. 7a)^53–56. Female, RSV-naïve cotton rats were immunized intramuscularly with Pre-F+Alum, mPre-F-LNP, LC2DM-LNP, or two PBS controls. After 1 weeks, the females were paired with naïve males and, 1 week later, administered a booster of the same vaccine as the prime injection. Five weeks after priming, serum samples were harvested to assess the antibody response in the female cotton rats (Fig. S8a). Approximately one-third of the paired females became pregnant and gave birth at weeks 7–8 after the initial immunization, with an average of 4–6 pups per litter. The delivered pups were then divided into two groups.

Fig. 7. Maternal immunization of female cotton rats and efficacy of offspring protection.

a Study timeline. Female cotton rats were immunized intramuscularly with PBS, PBS, 5 μg of Pre-F+Alum, or 20 μg of mRNA-LNPs. After 2 weeks, the females were paired with males, 1 week later, they were boosted. Pups were delivered between weeks 7 and 8. Half of the pups were sacrificed to evaluate the antibody titers at birth, while the other half were challenged with 1 × 10⁶ PFU RSV at 4 weeks of age and sacrificed 5 days later to collect the lungs and nasal turbinates. Details on the number of animals employed are listed in Supplementary Table 2. b Levels of pre-F-- and post-F-specific IgG in the sera of neonates (0–3 days of age) and their mothers, as determined with ELISA. c Serum neutralizing antibody (nAb) titers of pups (0–3 days of age) and their mothers measured with RSV-A2-mKate2. Mothers, n = 6 cotton rats per group. Pups, Pre-F+Alum (n = 5), mPre-F-LNP (n = 4), LC2DM-LNP (n = 4) in (b, c). d Blood samples from the pups were collected on days 7, 14, 21, and 28 after birth, and levels of pre-F- and post-F-specific IgG were determined by ELISA. e Serum nAb titers measured with RSV-A2-mKate2. Pre-F+Alum (n = 5), mPre-F-LNP (n = 6), LC2DM-LNP (n = 5) at day 7. Pre-F+Alum (n = 4), mPre-F-LNP (n = 6), LC2DM-LNP (n = 4) at day 14, 21, and 28 in (d, e). f Lung viral titers (left) and viral loads (right) determined with plaque assays and RT–qPCR. g Nasal turbinate viral titers (left) and viral loads (right) determined with plaque assays and RT–qPCR. h Lung pathology following RSV challenge, total pathology scores. i Heatmap showing changes in the levels of three cytokines in lung homogenates. Unchallenged (n = 6), PBS (n = 4), Pre-F+Alum (n = 4), mPre-F-LNP (n = 6) and LC2DM-LNP (n = 4) in (f–i). Dotted lines in (b–e, f left and g left) represent the lower limit of detection, and the upper dotted line in (e) represents the protective threshold. Data are presented as geometric means with geometric SDs in (b–e) and as means with SDs in (f–h). Statistical analysis was performed with one-way ANOVA and Tukey’s multiple comparisons (b–g) and the Kruskal-Wallis test (nonparametric) (h). Source data are provided as Source Data File.

Pups in the first group were euthanized within 0–3 days of birth to obtain sufficient serum for assessing the antibody titer at birth, including both the binding titer and neutralization titer. The pre-F-binding IgG titer in neonates was similar among those whose mothers were vaccinated with Pre-F+Alum, mPre-F-LNP, and LC2DM-LNP. However, the post-F-binding IgG titer was significantly lower in the LC2DM-LNP group than in the Pre-F+Alum and mPre-F-LNP groups, suggesting that maternal immunization with LC2DM-LNP may be advantageous over immunization with the other vaccines in reducing post-F-specific nonneutralizing antibodies in neonates (Fig. 7b). Neutralization titers were also assessed in neonates, revealing that maternal immunization with LC2DM-LNP resulted in the highest nAb titers (~16,384) in pups on days 0–3, which were significantly higher than those of Pre-F+Alum (~1626) and mPre-F-LNP (~3276) (Fig. 7c). For the second group of pups, serum samples were collected at 7, 14, 21, and 28 days after birth to determine the half-life of the maternal antibodies. Antibody titers decreased steadily in all pups delivered by vaccinated mothers (Fig. 7d). Nevertheless, the nAb titers in pups delivered by LC2DM-LNP–immunized mothers were highest at all four screening timepoints, remaining at an nAb titers of  ~288 on day 28 postbirth (Fig. 7e), whereas pups delivered by Pre-F+Alum– and mPre-F-LNP–immunized mothers presented with lower titers at this time point (~41 and 102, respectively), with one pup in the Pre-F+Alum–vaccinated group presenting with undetectable titers (Fig. 7e).

Maternal immunization with LC2DM-LNP protects neonates from RSV challenges

On day 28 after birth, the pups were exposed to RSV Long (except for one PBS control group, which was left unchallenged as a negative control) and sacrificed 5 days later. Compared with those born to PBS-vaccinated mothers, those born to Pre+Alum–, Pre-F-LNP–, or LC2DM-LNP–immunized mothers presented with markedly lower viral titers and viral loads in the lungs (Fig. 7f). Notably, all four litters born to LC2DM-LNP–vaccinated female cotton rats had undetectable lung viral titers according to the plaque assay and the lowest number of lung viral N gene copies. Pups delivered by mPre-F-LNP– and LC2DM-LNP–vaccinated cotton rats with higher nAb titers showed good protection in the turbinates (Fig. 7g), with one-third of pups in the mPre-F-LNP–maternal immunization group and half of the pups in the LC2DM-LNP–maternal immunization group exhibiting undetectable viral titers. Together, these results indicate the potential of employing LC2DM-LNP as a maternal vaccine.

Low-avidity antibodies are hypothesized to be the primary cause of RSV vaccine failure and subsequent VERD development in affected children⁵⁷. To evaluate the avidity of the polyclonal antibody response, a urea-based ELISA was conducted. The results indicated that, compared with Pre-F+Alum vaccination, vaccination with the mRNA/LNPs resulted in increased antibody avidity in cotton rats (Fig. S8b). Histopathological examination of offspring lung tissues revealed that pups born to mPre-F-LNP- and LC2DM-LNP–immunized mothers presented mild pneumonia, in sharp contrast to the severe diseases observed in the PBS-vaccinated controls (Fig. 7h and Fig. S8c). Pups from mothers vaccinated with Pre-F+Alum, meanwhile, demonstrated comparable but slightly milder pneumonia than those from the PBS-vaccinated mothers (Fig. 7h). These findings parallel the enhanced protective efficacy conferred by maternal vaccination (Fig. 7e). Analysis of cytokine expression in lung tissue obtained from cotton rats revealed that although all pups from LC2DM-LNP–vaccinated mothers presented with greater cytokine expression than those from unchallenged mothers, the overall expression of cytokines was much lower than that of pups from PBS-vaccinated mothers. Notably, the expression of IFN-γ was significantly lower in pups from LC2DM-LNP–vaccinated mothers than in those from PBS-vaccinated mothers (Fig. 7i). Overall, our study provides preliminary evidence that LC2DM-LNP is an effective maternal vaccine.

Discussion

In our investigation, we expanded on the established benefits of the pre-F protein as a promising vaccine candidate^58,59, aiming to provide targeted protection to high-risk populations, such as children and elderly individuals.

We examined a new vaccine candidate, LC2DM-LNP, an mRNA construct featuring a truncated pre-F protein designed to eliminate nonneutralizing epitopes. The vaccine demonstrated effective humoral and cellular responses in female BALB/c mice, with a notably greater antibody response quality. The potential of LC2DM-LNP in high-risk populations was evaluated with an RSV-aged animal model, a preexposure cotton rat model, and a maternal cotton rat model, all of which yielded positive results.

Improving antibody quality and ensuring sufficient nAb titers are critical considerations in the design of RSV vaccines. Our development of the LC2DM immunogen aimed to elicit a high-quality and effective nAb response, featuring a significant modification that involves removing the stalk region of the pre-F protein to potentially reduce immune competition from nonneutralizing epitopes. Compared with the pre-F mRNA vaccine, the LC2DM-LNP significantly increased hRSV90 competitive antibody levels and yielded lower IC50 values. Notably, the ratio of nAb titers to binding titers was greater in animals receiving LC2DM-LNP than in those receiving other treatments, demonstrating the superior quality of the serum antibodies produced. The nAb titers of the mice and cotton rats immunized with the LC2DM-LNPs were four times greater than those of the mice immunized with the mPre-F-LNPs.

The inclusion of a transmembrane domain in LC2DM-LNP, which facilitates membrane expression, may have contributed to the observed improvement in antibody quality, thereby ensuring high nAb titers. LC2DM-LNP induced nAb titers ~8–12 times higher than those of sLC2DM-LNP (a secreted form of LC2DM with a fold-on domain) and i447-LNP²⁹ (Fig. S9). This increase was corroborated by a recent study in which a membrane-anchoring motif was added to the receptor-binding domain (RBD)⁶⁰. However, the mechanism by which this domain enhances nAb titers has not been definitively established. One hypothesis posits that secreted forms, such as sLC2DM, may expose epitopes not present in the pre-F protein, leading to a greater number of antibodies targeting these regions. Conversely, membrane expression could limit the generation of such antibodies. Another hypothesis suggests that T-cell responses may be enhanced when the antigen is presented in an anchored form⁶¹. Further investigation is necessary to elucidate the mechanisms underlying the enhancement of nAb titers via the RBD.

VERD triggered by FI-RSV represents a significant obstacle to the development of RSV vaccines. Extensive research has sought to elucidate the underlying mechanism of VERD. The prevailing consensus attributes VERD to Th2-biased helper T-cell responses^62–64 and the generation of low-affinity, nonneutralizing antibodies^11,57. The LC2DM-LNP vaccine may circumvent the risk of VERD through certain characteristics: the mRNA vaccine platform fosters balanced helper T-cell activation and promotes the maturation of antibody affinity; additionally, the elimination of the stalk region from the pre-F protein promotes the production of higher-quality antibodies.

Nevertheless, the safety issues observed in children who received the RSV mRNA vaccine developed by Moderna suggest that the previously established potential mechanism of FI-RSV based on animal models might be incomplete and thus may not accurately represent the VERD mechanism in humans³⁶. No evidence of VERD was observed in the animal experiments with the tested vaccines. However, the clinical trial in children was paused due to an imbalance in severe RSV cases. This indicates that further in-depth investigations into RSV animal models and the VERD mechanism are warranted. Notably, given the potential limitations of existing animal models, although no VERD was observed with the LC2DM - LNP vaccine in multiple animal models, these models were established in prior research and may not fully recapitulate the human immune response and VERD risk. Therefore, the assessment of safety risks remains inadequate and demands further exploration and clarification.

Elderly individuals are among the highest risk groups for RSV infection, in part due to their declining immune system. To simulate vaccination regimens in older people who tend to have a weakened immune system, we injected 18–20-month-old mice with different vaccines and compounds and found that LC2DM-LNP generated significantly greater nAb titers after two rounds of immunization and maintained high titer levels for 84 days after priming. Considering that most humans are infected with RSV by the age of two, we also evaluated the immunogenicity of LC2DM-LNP in RSV-experienced cotton rats. We discovered that prior infection with RSV led to a post-F-biased antibody response, which contrasts with the typical pre-F-biased antibody response observed following RSV infection in humans⁵⁰. It remains uncertain whether repeated RSV infections in cotton rats yield antibody responses analogous to those in humans. Nonetheless, compared with mPre-F-LNPs, a single dose of LC2DM-LNP was sufficient to increase nAb titers, demonstrating its considerable potential to increase nAb levels in RSV-seropositive elderly individuals. Owing to the robust immunogenicity of LC2DM-LNP, the proposed vaccine may also serve as a promising booster agent for individuals previously vaccinated with marketed vaccines.

RSV is a leading cause of infant hospitalization, with infants aged 6 months and younger having an elevated risk of severe RSV illness^65,66. In a phase 3 trial, nirsevimab, a monoclonal antibody, protected infants from medically treated, RSV-associated lower respiratory tract infection for 150 days^67,68. Nirsevimab was recently authorized in Europe for use in infants. Although effective, monoclonal antibodies still pose a risk of viral immune escape⁶⁹. Moreover, infants can become infected with medically attended severe RSV-associated lower respiratory tract illness even as early as 8 days of age, which highlights the need for RSV prevention from birth and throughout the early stages of life⁷⁰. Maternal antibodies can protect infants immediately from birth and during the first few months of life^71–73. Both GSK and Pfizer have developed maternal RSV vaccines, although GSK halted its late-stage clinical trial owing to safety concerns, whereas Pfizer’s phase 3 trial reported positive outcomes. Although both candidates are based on the pre-F protein without an adjuvant, the reason for the safety of GSK remains unclear. Maternal immunization with LC2DM-LNP, however, presented with distinct advantages, including generating higher and more durable nAb titers, lowering the risk of VERD by improving pre-F-binding IgG, and producing antibodies with greater avidity than Pre-F+Alum did.

Antibody administration and maternal immunization represent passive prevention strategies, whereas active prevention can confer unique benefits, including the induction of long-term immune memory. For infants, attenuated or viral vector vaccines tend to be safer options. The lower molecular weight of LC2DM makes it highly adaptable to diverse vaccine platforms, ranging from influenza virus vectors⁷⁴ to novel RNA-based vaccines, such as circular RNA⁷⁵ and self-replicating RNA vaccines. This adaptability is crucial because high-molecular-weight antigens pose a greater challenge for packaging within these platforms.

Our study has several limitations. Notably, our maternal study lacked an FI-RSV control group, which limits our ability to assess the risk of VERD effectively. Additionally, vaccinations were conducted on RSV-naïve pregnant cotton rats, a model that may not adequately reflect the various exposure histories typical in human populations. Moreover, there is growing recognition that immune responses can differ between males and females^49,76,77. While we evaluated the immunogenicity of LC2DM-LNP in both male and female cotton rats and observed comparable nAb titers between sexes (Fig. S10), this outcome contrasts with findings from other studies, possibly due to differences in vaccine types or the timing of sample collection. Additional studies are warranted to determine whether pre-F-based mRNA vaccines produce differential immunogenicity on the basis of sex⁷⁸. Furthermore, the molecular mechanisms underlying the increased immunogenicity conferred by the anchored form of the antigen require further elucidation. Finally, the specific antibody and B-cell responses induced by mPre-F-LNP and LC2DM-LNP need to be more comprehensively investigated.

In summary, we developed an RSV mRNA vaccine based on a modified version of the pre-F protein. Our findings indicate that LC2DM-LNP generates robust immunogenic responses in various animal models, including young adult and elderly mice, RSV-experienced and RSV-naïve cotton rats, and pregnant cotton rats. Additionally, LC2DM-LNP offers substantial protection against RSV infection without increasing lung inflammation. Overall, LC2DM-LNP could be a viable option for developing a vaccine for RSV-vulnerable groups.

Methods

Study design

This study investigated the immune response and protective effects of a modified pre-F mRNA vaccine in preclinical models of RSV susceptibility. To this end, various animal models were employed, with the sample sizes and ages determined on the basis of the results of prior studies. Female young adult BALB/c mice were randomly allocated into groups of five to assess the antibody and T-cell response induced by the vaccine candidate, whereas female 18–20 months-old BALB/c mice were randomly assigned to groups of three to evaluate the potential immune response of the vaccine candidate. To evaluate the protective efficacy and safety of the candidate vaccine, female cotton rats (n = 5 per group) were vaccinated and then challenged with the virus, and changes in viral titers, the viral RNA load, and histopathology in the lungs between the vaccinated and unvaccinated groups were compared. For the RSV preexposure model, 25 cotton rats were subdivided into five groups (n = 5 per group), and after 4 months, the animals were vaccinated to measure antibody titer changes. For maternal immunization, 30 young adult female cotton rats were paired with 15 male cotton rats at a ratio of 2:1 and randomly divided into five groups to evaluate the potential of the candidate vaccine as a maternal vaccine. Researchers conducting RSV neutralization assays using mouse sera and RSV virus in cotton rat tissues were blinded to the groupings. Assessments of animal pathology were also conducted under blinded conditions.

Cell lines and virus culture

HEK293T (American Type Culture Collection [ATCC]; CRL11268) and HeLa cells (ATCC; CCL2) were grown in Dulbecco’s Modified Eagle’s Medium (Gibco, Cat. No. 10569044) and Eagle’s minimum essential medium (Gibco, Cat. No. 42360099), respectively, with L-glutamine (Invitrogen, Cat. No. 25030-081), high glucose and 10% heat-inactivated fetal bovine serum (Gibco, Cat. No. 10099141) in a humidified incubator at 37 °C with 5% CO2. Both cell lines were authenticated by ATCC and were checked for Mycoplasma contamination in our laboratory.

Human RSV Long (ATCC, VR26), human RSV 18537 (ATCC VR1580), and human RSV-A2-mKate2 (pSynkRSVA2 D46F, a recombinant virus based on RSV strain A2 with insertion of a fluorescent reporter before the NS1 protein, kindly donated by Dr. Barney S. Graham, VRC, NIH) were propagated in HeLa cells.

Antibodies and proteins

Plasmids of primary antibodies for flow cytometry, cell-based ELISA, and immunostaining, including Mota and D25, were generously donated by Jason S. McLellan (Department of Molecular Biosciences, College of Natural Sciences, The University of Texas at Austin, TX, USA). 4D7 and hRSV90 were produced by subcloning the heavy and light chain variable sequences into a pTT5 vector, which was then transiently transfected into FreeStyleTM 293-F cells (Gibco). The supernatants were collected after 6 days, purified with a Protein A column (GenScript, Cat. No. L00210), and subjected to buffer exchange with 1 × phosphate buffer saline (PBS) (Gibco, Cat. No. 10010049).

Primary antibodies were used at a concentrations of 1 μg/mL for flow cytometry and immunostaining experiments and at 5 μg/mL for cell-based ELISA. Goat anti-human Alexa Fluor 647 (Thermo Fisher, Cat. No. A-21445) and donkey anti-mouse Alexa Fluor 647 (Thermo Fisher, Cat. No. A-31571) were used at a concentration of 1 μg/mL. The secondary antibodies goat anti-mouse IgG Fc-HRP (Abcam, Cat. No. ab97265) and goat anti-human IgG-HRP (Abcam, Cat. No. ab97225) were diluted 1:5000 in 5% bovine serum antigen (BSA) in PBS. The soluble post-F protein and pre-F protein SC-TM were constructed, expressed, and purified according to previous methods^12,13. In brief, RSV fusion proteins were expressed by transient transfection of plasmids encoding the SC-TM or post-F protein into Expi293F™ cells (Thermo Fisher Scientific, Cat. No. A14528) in suspension culture. 7 days after transfection, cell culture supernatants were collected, centrifuged to remove cell debris, and sterilized through filtration. The RSV F proteins were purified using a Ni Sepharose Fast Flow 6 resin (GE Healthcare, USA), followed by further concentration and purification on HiLoad 16/600 Superdex 200 pg size exclusion column (GE Healthcare, USA) according to the manufacturer’s instructions. The purified trimeric F proteins were then concentrated, aliquoted, and stored at  −80 °C until further use. The pre-fusion and post-fusion conformation identities were verified via ELISA with an RSV antibody panel (Fig. S11).

In vitro transcription (IVT) and purification of mRNA

Plasmids for IVT of the modified pre-F were designed on the basis of the RSV F sequence from RSV strain A2, and modifications were incorporated as described in the Results. The full amino acid sequence for each construct is listed in Table S1. The coding regions were codon-optimized with a GC content of ~27% and preceded by a 5′ untranslated region (5′ UTR) containing a Kozak sequence, followed by a 3′ untranslated region (3′ UTR), as used in BNT162b2⁷⁹. The sequences were inserted into a modified pUC57 vector with a T7 promotor and a poly (A) tail (A30LA70).

Modified mRNAs were produced via IVT with a T7 High Yield RNA Transcription Kit (N¹-Me-Pseudo UTP) (Vazyme, Cat. No. DD4202). A trinucleotide cap1 analog, CleanCap (TriLink, Cat. No. N-7413-10), was used to cap the in vitro-transcribed mRNAs. The capped and tailed mRNAs were then purified using lithium chloride (Thermo, Cat. No. AM9480). mRNAs were analyzed via capillary electrophoresis (Bioptic Qsep 100) and LC/MS (Agilent) and stored frozen at  −80 °C until further use.

Lipid nanoparticle formulation of the mRNA

Nucleoside-modified mRNAs were encapsulated into LNPs for animal immunization as previously described⁷². Lipids were dissolved in ethanol at molar ratios of 50:10:38.5:1.5 (ionizable lipid: DSPC: cholesterol: PEG-lipid). The formulations were dialyzed against PBS in dialysis cassettes for at least 18 h, then concentrated with Amicon ultracentrifugal filters (Millipore, Cat. No. UFC201024). The concentration and encapsulation rate of the mRNAs were measured with a Quant-it RiboGreen RNA Assay Kit (Invitrogen, Cat. No. R11490). The sizes of the mRNA-LNP particles were measured with dynamic light scattering on a NanoBrook Omni Brochure (Brookhaven Instruments).

mRNA transfection and detection of the expressed immunogens

HeLa cells or HEK293T cells were transfected with mPre-F mRNA, LC2DM mRNA, and LC2DM-IIKO mRNA via Lipofectamine MessengerMAX transfection reagent (Thermo Fisher, Cat. No. LMRNA003) according to the manufacturer’s instructions or with the corresponding LNP formulations.

For flow cytometry, HeLa cells were seeded into 6-well plates for 24 h and then transfected with equal molar masses of mRNA expressing the indicated antigens encapsulated in LNPs. After 24 h, the cells were harvested, pelleted, and washed twice with PBS. The cells were subsequently incubated with LIVE/DEAD Fixable Aqua Dead cell stain (Thermo Fisher, Cat. No. L34966) for 30 min in the dark and washed twice with stain buffer (BD, Cat. No. 566349). The cell pellets were resuspended in primary antibody and incubated for 15 min on ice in the dark, followed by two washes with stain buffer. Finally, the cells were resuspended with secondary antibody for another 15 min on ice in the dark. The stained cells were analyzed with LSRFortessa (BD Biosciences) and FlowJo (BD Biosciences).

For immunostaining, HeLa and 293T cells were seeded into 96-well plates for 24 h and then transfected with 0.1 μg of mRNAs expressing the indicated antigens. After an additional 24 h, the cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.3% Triton X-100 (Sigma, Cat. No. T8787-100ML) for 10 min. The cells were then blocked with 5% skim milk (Wondersun) for 1 h and then incubated with primary antibodies for 1 h. The cells were washed twice with PBS and then incubated with secondary antibodies for 1 h. Finally, nuclear staining was performed using 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Cat. No.D1306). Images were acquired on a Leica TCS SP8 confocal imaging systeman or an Opera Phenix with a 40 × water-immersion objective.

For cell-based ELISA, 293T cells were seeded into 96-well plates for 24 h and then transfected with the indicated mRNAs via a transfection reagent and LNPs. The mRNAs were serially diluted 2-fold from an original concentration of 5 μg/mL. After an additional 24 h, the cell supernatants were discarded, the cells were gently washed twice with PBS and then fixed with 4% paraformaldehyde for 30 min. The cultures were rinsed three times with PBS. The mixture was incubated in blocking buffer (5% skim milk) for 1 h and then incubated with Mota for another 1 h. The cells were washed five times with PBS-Tween (PBST) and then incubated with secondary antibodies. After a further 1-h incubation, the plates were washed with PBST buffer and developed with a tetramethylbenzidine (Wantai) chromogen solution (100 μL per well). The chromogen reaction was stopped after 10 min by the addition of 50 μL 2 M H₂SO₄, and the optical density at OD 450–620 was measured.

Animals, immunizations, and RSV challenge

BALB/c mice were purchased from Shanghai SLAC Laboratory Animal Co. Female cotton rats were bred and housed. Animals were maintained in specific pathogen-free (SPF) facilities under a 12-h light/dark cycle and ambient conditions, with unrestricted access to food and water. The temperature was controlled between 20 and 24 °C, and relative humidity was maintained at 45–65%. Viral infections were performed in a BSL-2 laboratory following institutional guidelines for laboratory animal care and use. All animal experiments were approved by the Institutional Animal Care and Use Committee and Laboratory Animal Management Ethics Committee of Xiamen University (ethics approval number: XMULAC20200198).

For immunogenicity evaluation, young adult female BALB/c mice (6–8 weeks old, n = 5 per group) were immunized intramuscularly with 0.1, 1, 10, or 20 μg of pre-F protein with Alum (Alhydrogel® adjuvant 2%, InvivoGen, Cat. No. vac-alu-50), mPre-F-LNP, or LC2DM-LNP in a prime–boost regimen with a 14-day interval. Aged mice (18–20 months, n = 3 per group) were immunized intramuscularly with 1 μg of Pre-F+Alum or 10 μg of each candidate mRNA vaccine. Serum samples were collected from young adult mice at week 4 via retro-orbital bleeding and from aged mice at weeks 2, 3, 6, 8, and 12 for serology. Male and female cotton rats (6–8 weeks old, n = 5 per group) were immunized intramuscularly with 5 μg and 20 μg of LC2DM-LNP with a 21-day interval to assess differences in immunogenicity between the sexes. Serum samples were collected from the cotton rats at week 5 via retro-orbital bleeding.

To evaluate the antibody quality and T-cell response, young adult female BALB/c mice (6–8 weeks old, n = 5 per group) were immunized intramuscularly with 1 μg of pre-F+Alum and 10 μg of each of the candidate mRNA vaccines in a prime–boost injection regimen with a 14-day interval between the injections. Serum samples were collected from the mice at week 4 via retro-orbital bleeding. At week 6, the spleens were collected from the mice to assess T-cell responses via ELISpot and flow cytometry. To evaluate protective efficacy, female cotton rats (6–8 weeks old, n = 5 per group) were intramuscularly administered two doses of PBS or 5 μg of the pre-F+Alum and 20 μg of each of the candidate mRNA vaccines or FI-RSV in a prime–boost injection regime with a 21-day interval between the injections. Serum samples were taken at week 5 via retro-orbital bleeding for serological analysis. At week 7, all animals except those in the PBS group in the study were intranasally inoculated with 1 × 10⁶ PFU of the RSV Long strain. Five days postchallenge, the animals were euthanized, and their lung tissues were harvested for quantification of the viral load and viral titer, as well as for lung pathology assessment. To evaluate the potential risk of VERD in the event of breakthrough infection following LC2DM-LNP vaccination, female cotton rats (6–8 weeks old, n = 5 per group) were intramuscularly administered PBS, FI-RSV, or 0.1, 1, 5, 10, or 20 μg of LC2DM-LNP in a prime–boost regimen with a 21-day interval. Serum samples were collected at week 5 via retro-orbital bleeding for serological analysis. At week 7, all the animals, except those in the PBS group, were intranasally inoculated with 1 × 10⁶ PFU of the RSV Long strain. Five days postchallenge, the animals were euthanized, and their lung tissues were harvested for quantification of viral titers and for assessment of lung pathology.

To evaluate the immune response in RSV-primed cotton rats, 25 female cotton rats (6–8 weeks old) received intranasal administration of 2 × 10⁵ PFU of RSV Long and divided into five subgroups (n = 5 per group). Four months after the initial treatment, each group of animals was intramuscularly administered PBS, FI-RSV, 5 μg of Pre-F+Alum, or 20 μg of either candidate mRNA vaccine. Serum samples were collected 2 weeks postvaccination to assess immune responses. Two weeks later, all the animals were intranasally inoculated with 1 × 10⁶ PFU of the RSV Long strain. Five days postchallenge, the animals were euthanized, and their lung tissues were harvested for viral titer quantification and lung pathology assessment.

For maternal immunization, a total of 30 young adult female cotton rats (6–8 weeks old) were intramuscularly immunized with 5 μg Pre-F+Alum and 20 μg candidate mRNA vaccines. After 2 weeks, two female cotton rats were paired with one male cotton rat (10–12 weeks old) and were given a booster injection 1 week later. Upon confirmation of pregnancy, the male was removed from the cage. Serum samples were taken at weeks 5 and 7, and the females delivered their pups between weeks 7 and 8. Half of the pups from each immunized group were sacrificed at birth to assess the transfer ratio, while the other half were screened for serum antibody levels and then challenged with 2 × 10⁶ PFU of the RSV Long strain at 4 weeks after birth to assess vaccine protective efficacy.

ELISA

Pre-F and post-F protein-specific antibody titers in the serum samples were characterized with ELISA, as previously described⁴⁷. Briefly, 96-well ELISA plates were coated with pre-F or post-F protein in PBS (100 ng/well) and incubated overnight at 4 °C. The plates were washed three times with PBST (0.05% Tween 20 in PBS) and then blocked with 5% BSA (in PBS) in PBST. The sera were serially diluted 5-fold, transferred to an antigen-coated plate, and incubated at 37 °C for 1 h. The plates were washed six times with PBST and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, IgG1, and IgG2a antibodies diluted 1:5000. After a further 1 h of incubation, the plates were washed with PBST buffer and developed with tetramethylbenzidine (Wantai) chromogen solution (100 μL per well). The chromogen reaction was stopped after 10 min with the addition of 50 μL of 2 M H₂SO₄, and the optical density at OD 450–620 was measured. Serum end-point titers were calculated as the dilutions that emitted an OD exceeding a 3 × background.

Competitive ELISA

Epitope-specific serum antibody concentrations were determined with competitive ELISA as previously described, with slight modifications^80,81. Briefly, 96-well plates were coated, blocked, and washed as described for the RSV pre-F binding ELISA. Serum samples were initially diluted 1:5, serially diluted 1:2 for a total of 11 dilutions, then added to protein-coated plates and incubated at room temperature for 30 min. An analogous antibody (unbiotinylated) was used to generate a standard curve for each assay. An equal volume of biotinylated antibody was added to the diluted serum in the plates to reach a total volume of 100 μL, and the mixture was incubated at room temperature for 1 h. The plates were then incubated with streptavidin-conjugated HRP (BioLegend, Cat. No. 405210) diluted in 5% milk in PBS for 1 h at room temperature and developed and read as described for the RSV F binding assays. The coating and biotinylated antibody concentrations for each assay were as follows: 1 μg/mL and 500 ng/mL for the pre-F D25 competitive antibody assay and 100 ng/mL and 100 ng/mL for the pre-F hRSV90 competitive antibody assay, respectively. The lower limit of quantitation was 8 mg/mL for the D25 competitive antibody assay and 4 mg/mL for the pre-F hRSV90 competitive antibody assay.

Neutralization assays

The levels of nAbs against RSV strain A2 were measured in HeLa cells with RSV-A2-mKate (pSynkRSV A2 D46F). Briefly, 10 μL of heat-inactivated serum or monoclonal antibody (mAb; 1 mg/mL) was added to 90 μL of DMEM, serially diluted 4-fold, mixed with 75 μL of RSV-A2-mKate and incubated at 37 °C for 1 h. During the incubation, 96-well microplates were seeded with HeLa cells at a density of 30,000 cells per well. The mixtures were transferred to the cell plate for incubation. The fluorescence intensity was captured with a SpectraMax Paradigm Multi-Mode Microplate Reader (Molecular Devices, LLC, San Jose, CA, USA) at 588 nm excitation and 633 nm emission after 24 h. IC50 values were computed with GraphPad Prism version 8.00 (GraphPad Software, San Diego, CA, USA).

To determine the neutralizing antibody titers against RSV B 18537, the serum samples were diluted as described above and then incubated with RSV B 18537 instead of RSV-A2-mKate. Focus-forming units (FFUs) were enumerated with HRP-conjugated Synagis. The detection procedure was conducted according to the manufacturer’s instructions. Spots were counted and analyzed with CTL-ImmunoSpot S5 (Cellular Technology Limited). NT50 values were calculated as reciprocal serum dilutions at which 50% of the virus was neutralized compared with control wells without serum.

ELISpot assay

Single-cell suspensions were obtained from mouse spleens (10⁶ cells per well) through grinding in cell strainers and seeding into the wells of ELISPOT plates precoated with anti-mouse IFN-γ/IL-4 antibody (Immunospot, Cat. No. mIFNgIL4-1M). The cells were then incubated with pooled peptides of RSV F (15-mer peptides with 11 amino acid overlaps covering the entire F protein; Sangon) and cultured at 37 °C with 5% CO2 for 20 h. The detection procedure was conducted according to the manufacturer’s instructions. Spots were counted and analyzed by using CTL-ImmunoSpot S5 (Cellular Technology Limited). The numbers of IFN-γ–secreting cells and IL-4–secreting cells were calculated by subtracting the numbers of cells in the PBS-stimulated wells from those in the F peptide pool-stimulated wells.

Flow cytometry analysis

For ICS, mouse splenocytes were stimulated with pooled F protein peptides at a concentration of 2 μg/mL in a U-bottom plate during an 18-h incubation period. Subsequently, protein transport inhibitors (BD GolgiPlug, BD Biosciences, Cat. No. 555028) were added, and the cells were incubated for an additional 5 h. The cells were then stained with Brilliant Violet 421 anti-mouse CD3 (BioLegend, Cat. No. 100336), FITC anti-mouse CD4 (BioLegend, Cat. No. 100406), and PerCP-Cy5.5 anti-mouse CD8α antibodies (BioLegend, Cat. No. 100734), and the LIVE/DEAD Fixable Aqua Dead Cell Staining Kit. Next, the cells were fixed and permeabilized with a Fixation/Permeabilization Solution Kit (BD Biosciences, Cat. No. 554722) and further stained with APC rat anti-mouse IL-4 (BioLegend, Cat. No. 562045) and PE/Cyanine7 anti-mouse IFN-γ antibodies (BioLegend, Cat. No. 505826) at 4 °C for 30 min. Finally, the samples were assessed with a BD LSRFortessa X-20 flow cytometer (BD Biosciences), and the data were analyzed with FlowJo V10.6.0.

RSV plaque assay

Titers of nasal turbinate and lung homogenates were determined with plaque assays. Briefly, virus stocks or lung homogenates were serially diluted 10-fold with DMEM. Then, 50 μL diluted samples (dilutions 1 × 10³ to 1 × 10⁷ or 1 × 10⁰ to 1 × 10²) were added to a monolayer of HeLa cells (2 × 10⁵ per well, 12-well plate) for 1 h at 25 °C, after which the cells were incubated at 37 °C with 5% CO₂. After 4 days, the plaques were stained with H&E (Sigma–Aldrich) and counted.

RSV RNA quantification

The viral RNA load in the nasal turbinate and lung tissues of challenged animals was detected via quantitative reverse-transcription polymerase chain reaction (RT–qPCR). Briefly, nasal turbinate and lung tissues were collected and homogenized with TissueLyser II (Qiagen). RNA was extracted via a Viral DNA/RNA Extraction Kit (GenMagBio, Cat. No. NA007-4-new) according to the manufacturer’s instructions. RNA quantification was performed via RT–qPCR targeting the N gene of RSV via a Bio-Rad CFX96 system. The abundance of β-actin was used as an internal reference. The viral load of the unchallenged group was normalized to 100%.

Histopathology

At 5 days postvirus challenge, lung tissue samples were extracted, fixed with 10% formalin for 48 h, embedded in paraffin, and sectioned via a microtome (Leica). Histological slides were stained with H&E and evaluated by two independent pathologists who were blinded to the animal groups with a standard scoring system. The samples were graded according to the levels of perivasculitis (inflammatory cell infiltration around small blood vessels), peribronchiolitis (inflammatory cell infiltration around bronchioles), interstitial pneumonia (inflammatory cell infiltration and thickening of alveolar walls), and alveolitis (cells within alveolar spaces). For each slide, a score was determined on the basis of its severity, with 0 indicating no pathological change, 1 indicating mild pathological change, 2 indicating moderate pathological change, 3 indicating severe pathological change, and 4 indicating very severe pathological change.

Cytokine analysis

RNA samples harvested from the lungs of challenged cotton rats were subjected to RT–qPCR to measure the levels of the cytokines IFN-γ, IL-6, and IL-1β via a Bio-Rad CFX96 system.

Statistical analysis

One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test and the Kruskal–Wallis test (nonparametric) were used for comparisons between groups, as indicated in the figure legends. All the statistical analyses were performed via Prism v9.0.2 (GraphPad Software). P < 0.05 were regarded as statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Author contributions

Conceptualization: M.L., Y.F.Y., and Z.Z.Z. Methodology: M.L. and Y.F.Y. Investigation: Y.F.Y., X.M.Z., and C.W. Funding acquisition: J.Z., Z.Z.Z., and N.S.X. Project administration: M.L., Y.F.Y., X.M.Z., C.W., X.Q.Z., L.T.Z., L.C., and X.L. Supervision: S.L.W., Z.Z.Z., J.Z., N.S.X. Writing original draft: M.L. and Y.F.Y. Review & editing: M.L., Z.Z.Z.

Peer review

Peer review information

Nature Communications thanks Dokyun Kim, Roland Zahn and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Data availability

Source data are provided with this paper.

Competing interests

Z.Z.Z., M.L., Y.F.Y., X.M.Z., C.W. C.L., J.Z. and N.S.X. are coinventors on patent NO. PCT/CN2023/102342. The other authors declare that they have no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-025-56302-1.