A Single Amino Acid Mutation Alters the Neutralization Epitopes in the Respiratory Syncytial Virus Fusion Glycoprotein

doi:10.21203/rs.3.rs-4999528/v1

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A Single Amino Acid Mutation Alters the Neutralization Epitopes in the Respiratory Syncytial Virus Fusion Glycoprotein

https://doi.org/10.21203/rs.3.rs-4999528/v1

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Respiratory syncytial virus (RSV) is the leading cause of infant hospitalization. All current available RSV therapeutics, including antibody prophylaxis and adult vaccination, target the RSV fusion glycoprotein (RSV-F). The seven neutralization sites on RSV-F are highly conserved and infrequently mutated. Here, we show that a single amino acid mutation at position 305 in RSV-F significantly alters antigenic recognition of RSV-F binding sites and reduces the susceptibility of RSV to neutralizing antibodies. In an in vitro evolution assay, we show that RSV-F L305I occurs in the majority of RSV quasi-species. Computational modeling predicted that the L305I mutation alters the epitope landscape of RSV-F, resulting in changes to neutralizing antibody sensitivity and affinity towards the RSV-F glycoprotein. Screening of published RSV-F sequences revealed that position 305 in RSV-F is a conserved leucine and isoleucine in RSV-A and RSV-B subtypes respectively. Our study suggests that select amino acids in RSV-F may act as conformational switches for RSV to evade host serum antibodies. This work has important implications in understanding RSV evolution and resistance as it suggests that mutational resistance to neutralizing antibodies can occur at sites distal to antigenic epitopes, significantly altering antibody sensitivity to viral infection. These unique antigenic landscape changes should be considered in the context of vaccine and therapeutic development in order to better understand viral mechanisms of evasion and resistance.

Biological sciences/Biochemistry

Biological sciences/Computational biology and bioinformatics

Biological sciences/Immunology

Biological sciences/Microbiology

Health sciences/Medical research

Health sciences/Pathogenesis

Respiratory Syncytial Virus

Fusion glycoprotein

MD simulation

viral evolution

Respiratory Syncytial Virus (RSV) is the leading cause of infant hospitalization and the second leading cause of infant mortality worldwide ¹. There are an estimated 33 million cases of RSV-associated acute-lower respiratory tract infections annually, over 100,000 of which lead to death ¹. Immunocompromised and elderly populations are increasingly being recognized as vulnerable high-risk groups for severe disease ². Despite the enormous disease burden presented by RSV, there are few effective antiviral therapies readily available for the treatment of active infection. Biologics in the form of prophylactic monoclonal antibodies provide a means of passive immunization and are the treatment of choice for RSV infection. Until recently, the prophylactic monoclonal antibody Palivizumab (PZMB) was the only preventative therapeutic available for high-risk infants. PZMB is administered monthly throughout the RSV season to only the highest-risk infants. PZMB is expensive, has disputed efficacy, and leaves a large population of infants and susceptible adults unprotected ³. In 2023, the FDA approved Nirsevimab for use in all infants under 8 months old entering their first RSV season, and Merck has a Phase III biologic - Clesrovimab - in development ⁴. In contrast to PZMB, Nirsevimab and Clesrovimab are more potent, and thus administration is only required once per RSV season. Two new adult vaccines created by Pfizer (Abrysvo) and GSK (Arexvy) were also approved for use in the elderly, and pregnant women ^5,6.

In light of these promising advancements, it is critical that we better understand how RSV will adapt to evade the selective pressures that are sure to be elicited by novel therapeutics. RSV is under consistent evolutionary pressure while circulating in the human population. While the primary burden of RSV is in infants, it continuously circulates in healthy adults and accounts for approximately 15–35% of all respiratory virus cases in any given season ⁷. In combination with an error-prone RNA-polymerase, the antiviral immune response mounted during the course of RSV infection places selective pressures on the virus that can lead to quasi-species displaying an array of polymorphisms. Like other RNA viruses, RSV adapts readily while circulating in the population, particularly in the highly variable gene that encodes the attachment glycoprotein, RSV-G ⁸. Genetically distinct viruses have the potential to impact viral load and pathology in humans during subsequent RSV seasons, which we have previously correlated with viral replication in tissue culture ⁹.

There are two subtypes of RSV, subtype A (RSV-A) and subtype B (RSV-B). RSV-A and RSV-B are estimated to have diverged from their most recent common ancestor approximately 338 years ago ¹⁰. In any given season, one viral subtype typically predominates over the other and cycles annually ¹¹. However, some reports show the two subtypes can co-circulate in the population, and even present as co-infections in the same patient ¹². Overall, RSV-A and RSV-B share about 90% nucleotide sequence identity, with the highest regions of sequence variability occurring in the RSV-M2-2 and RSV-G genes ¹³. RSV-A and RSV-B are primarily differentiated by high amino acid variability between their RSV-G glycoproteins ¹⁴. Notably, there are also select residues in other viral proteins, including RSV-F, that are conserved within each RSV subtype and distinguish the two. Although it is widely disputed whether genetic differences alter disease severity, genetic differences between RSV-A and RSV-B can have varying susceptibilities to serum and monoclonal antibodies. The prophylactic monoclonal antibody, Suptavumab by Regeneron Pharmaceuticals, was halted in phase III clinical trials due to two amino acid substitutions in RSV-B that rendered it ineffective ¹⁵. Therefore, it is critical that we better understand how mutations and subtype differences in RSV-F affect viral susceptibility to therapeutics in order to predict how RSV will mutate in response to antiviral pressure.

The RSV fusion glycoprotein (RSV-F) is the primary therapeutic target for the large majority of biologics and vaccines in development against RSV as it induces highly potent neutralizing antibodies ^16–19. RSV-F mediates viral entry by attachment to a primary signaling receptor and fusion protein. We have previously reported that IGF1R is a primary signaling receptor for RSV-F where the interaction of these two promotes the expression of the fusion receptor, nucleolin, to the cell surface ^20,21. This interaction triggers a dynamic conformational change of RSV-F that fuses the viral envelope and cell membrane ²². RSV-F exists in two conformational forms, a prefusion, and a post-fusion form. Prefusion RSV-F is highly antigenic and contains 7 potent neutralization sites: ∅, I, II, III, IV, V, and the newly identified VI site ^23,24. Of these, sites ∅, I, and V are lost upon fusion ²⁵. PZMB, Nirsevimab, and Clesrovimab epitopes bind to sites II, ∅, and IV respectively. The majority of RSV-F is highly conserved with the exception of the signal peptide and p27 domains where consistently high levels of sequence variability between RSV isolates are observed ^26–28. Even so, there are positions in RSV-F that appear to be more variable than others, like positions 172 and 173 at site V, and positions 206 and 209 just outside of and within antigenic site ∅ ^29,30. Additionally, mutations at positions 272 and 276 have previously been associated with PZMB resistance ³¹.

Here, we report on a key conformational change in the RSV-F glycoprotein mediated by a mutation at position 305. In an evolution assay using lab strain RSV-A2, we found that a leucine (L) to isoleucine (I) mutation at RSV-F residue 305 occurred at an evolutionary bottleneck in the presence of polyclonal antibody pressure. Importantly, position 305 in RSV-F is one of the key conserved differences between RSV-A and RSV-B subtypes, marked by amino acids L305 and I305 respectively. We found that this single amino acid difference caused a change in the RSV-F conformational landscape and reduced RSV susceptibility to neutralizing sera and monoclonal antibodies targeted at multiple sites on the RSV-F protein. In light of these findings and recent therapeutic advancements, a better understanding of RSV adaptation and evolution is critical for monitoring potential breakthrough viral isolates and future vaccine design.

Lab-adapted RSV-A2 develops a Leucine to Isoleucine mutation at position 305 under polyclonal selective pressure at bottleneck titers.

To understand how RSV mutates in response to antibody pressure, we performed an in vitro resistance and evolution assay in the presence of a neutralizing polyclonal anti-RSV antibody, a non-nucleoside polymerase inhibitor that we have previously discovered ^32,33, and a DMSO control. The virus was harvested every two days for 20 passages. Viral titer was measured by qRT-PCR and viral genomes were next-generation sequenced following significant drops in titer, which we refer to hereafter as bottlenecks (Fig. 1a and 1c). Bottlenecks were of interest to us because we sought to determine if the mutations at these positions lead to viral evasion of neutralizing antibody pressure and subsequently increased viral titer. The first bottleneck we identified under polyclonal anti-RSV antibody selective pressure was at passage 5 (Fig. 1a). Additional bottlenecks at passages 9 and 13 were observed but reliable reads could not be obtained by NGS past passage 9 and the virus was undetectable past passage 13 by RT-qPCR. Sequencing of the passage 5 viral population revealed the RSV-F L305I mutation as the only non-synonymous mutation occurring in the majority of sequencing reads (Fig. 1b, S. Table 1). Here, 42.03% of reads contained L (CTA) while 57.93% of reads contained the mutated I (ATA) (Fig. 1b, S. Table 1). An increase in the proportion of RSV-F L305I was subsequently associated with a rebound in viral titer (Fig. 1a and b). The next two most frequently occurring non-synonymous mutations were RSV-F N276S and RSV-F V152I, occurring at 36.92% and 31.82% respectively (S. Table 2). Interestingly, the RSV-F N276S has previously been associated with Palivizumab resistance, and modern RSV strains appear to be mutating away from the historic N at position 276 to an S ³⁴. To the best of our knowledge, an RSV-F V152I mutation has not been reported in the context of viral resistance. However, upon examination of published RSV-F sequences, we determined that 98.7% of sequences contain isoleucine (I) at position 152, suggesting that a V may be characteristic of the early RSV-A2 genotype and that most modern circulating RSV strains have since mutated away from this. The RSV-F L305I mutation also occurred upon the treatment with our synthesized RSV polymerase inhibitor ³³ (Fig. 1c). At 10 µM and 25µM, RSV-F L305I occurs at passage 8 (17.25%) and passage 6 (41.53%) respectively but is undetectable by passage 20 at both concentrations (data not shown).

Published and clinical RSV isolates show that amino acid position 305 is subtype specific.

Since RSV-F L305I was the only mutation to emerge in the majority of sequenced reads under selective anti-RSV pressure, we sought that it is crucial to understand its importance and relevance to viral evolution and the impact of this mutation. To determine the biological relevance of the RSV-F L305I mutation, we analyzed published RSV-F sequences from the Virus Pathogen Resource (ViPR) database and representative clinical samples from previously sequenced RSV-A and B clinical isolates from hospitalized patients in Alberta, Canada, and Nationwide Children’s Hospital in Columbus, Ohio, during the 2014 to 2016 seasons ⁹. The percent identities between clinical isolates were determined (Fig. 1d). We found that RSV-F was one of the most conserved genes among our clinical isolates, in agreement with previous reports ²⁶. RSV-F sequences shared greater than 99% identity within each individual subtype and 90.4% identity between RSV-A and RSV-B isolates (S. Table 3). Upon further analysis of our sequencing library of RSV clinical isolates ⁹, we noted that at position 305 almost all RSV-A and RSV-B sequences had a conserved L and I, respectively.

To support our modest sample size, we downloaded 3747 complete RSV-F sequences from the ViRP database and analyzed their amino acid composition (Fig. 1e and S. Table 4). As we observed in our clinical isolates, RSV-A and RSV-B subtypes differed in their amino acid composition at position 305, as others have previously noted ³⁵. In addition to position 305, there were approximately 50 other residues throughout RSV-F that appeared to be subtype-specific (Fig. 1e). It is worth noting that no other amino acid, other than L or I, was found at position 305 in any RSV isolates, suggesting that the RSV-F L305I mutation is not simply an artifact of our in vitro evolution assay and may indeed serve a biological function.

An L305I mutation in the RSV-A2 fusion glycoprotein alters the conformation of the RSV-F protomer.

The constitutional isomers, leucine and isoleucine, inherently share several biochemical properties and differ structurally only in the position of a side chain methyl group (Fig. 2a). We asked whether this structural difference could have a steric effect on nearby amino acids in the RSV-F protein. To investigate this, we introduced the L305I mutation into the cryo-EM structure of the RSV-A2 prefusion F glycoprotein using the Schrödinger Small Molecule Discovery Suite followed by a molecular dynamics simulation of both proteins. Comparison of the wild-type RSV-F L305 and mutant RSV-F I305 models revealed that an L305I mutation induces a subtle protein-wide conformational change (Fig. 2b). Superimposition of wild-type and mutant RSV-F suggests that this structural change extends to a variety of binding sites including antigenic sites ∅, II, and IV (Fig. 2b and c).

RSV-F exists on the surface of the RSV virion as a dynamic trimer that has been shown to “breathe”, a phenomenon that can alter the accessibility of specific epitopes on trimerized RSV-F ³⁶. To determine whether L305I affects trimer breathing, we evaluated the dynamics of RSV-F L305 and RSV-F I305 using Molecular Dynamics (MD) simulations (Fig. S1). Analysis of the MD trajectories revealed that the Cα of both systems each equilibrated around 200 ns of the simulation time with an average RMSD of 2.3 Å and 2.8 Å for RSV-F L305 and RSV-F I305 respectively, suggesting that the mutation has little effect on trimer breathing (Fig. S1a). Next, we investigated the flexibility of the system residues using atomic fluctuations expressed as a function of B-factor of the protein backbone atoms for both systems (Fig. S1b). The majority of protein residues were stable during the entire simulation with little flexibility observed in the region 250–305 for RSV-F I305 compared to that of the RSV-F L305 and with increased flexibility in the 420–500 region of RSV-F L305 compared to RSV-F I305 (Fig. S1b and S movie 1). The flexibility of the latter region could be attributed to the presence of a flexible loop that connects F1 and F2 protomers and mutations in this region have been previously utilized to develop a stable prefusion RSV F vaccine ³⁷; however, the stabilization of the movement in this region in the L305I mutant could be attributed to effect of the mutation at position 305 and the effect has been distally extended to affect the overall dynamics and stability of the RSV F trimer. In summary, using computational simulations, we predict that there are flexibility differences between RSV-F L305 and RSV-F I305 proteins in certain regions, which could explain the observed structural alterations between the two.

To further validate the significance of the RSV-F L305I mutation, we compared the structure of the RSV-A2 pre-fusion F protein containing an asparagine or serine at position 228. We chose this position because analysis of published NCBI data highlighted it as another subtype-specific residue in RSV-F, that is Asn (N) in RSV-A and Ser (S) in RSV-B and is found distal to the binding site ∅ (Fig. 1e). Clustering of the MD trajectories suggested that the RSV-F S228 mutant did not show a noticeable conformational shift from the wild-type RSV-F N228. Atomic alignment of the RSV-F S228 and RSV-F N228 structures had an RMSD of 2.4 Å. The RMSD of both systems showed a stable structure during the whole simulation with an average RMSD of 2.9 Å and 2.7Å for the RSV-F N228 and RSV-F S228, respectively (Fig S1c). We also analyzed the atomic fluctuation to understand the regional flexibility of the structures. Our results indicate a similar trend in region flexibility between RSV-F N228 and RSV-F S228 suggesting that this mutation causes no to minimal conformational changes (Fig S1d). Taken together, our computational modeling predicts that introducing the conservative RSV-F L305I mutation into RSV-A2 can alter the structural confirmation of the entire RSV-F glycoprotein and that this observed difference is likely not due to alterations in trimer breathing.

A conservative Leucine to Isoleucine mutation at position 305 in an RSV reverse genetics model reduces infectivity of viral particles and susceptibility to human sera.

Given that our models predict a structural difference in RSV-A2 fusion proteins containing either an L or an I at position 305, we wondered whether this single mutation would have a functional impact on RSV in vitro. To test this, we used a recombinant reverse genetics model of RSV-A2 based on the RW30 backbone (rgRSV) ^38,39. We added in an L305I mutation via Gibson assembly and the resulting plasmid was sequenced to confirm the presence of RSV-F I305. First, we looked at whether there were any changes in growth characteristics between the wild-type rgRSV-A2 L305 and mutant rgRSV-A2 I305, which we will refer to as RSV WT and RSV L305I respectively. Since RSV-B isolates have been found to grow slower than RSV-A isolates in tissue culture, we hypothesized that RSV L305I might also grow slower than RSV WT ^40,41. To test this, we created a one-step growth curve of RSV WT and RSV L305I (Fig. 2d) in which HeLa cells were infected with equivalent MOIs, and the media was collected every 5 hrs over 100 hrs. Subsequent viral particle release was measured by RT-qPCR. We found no significant difference in the number of viral transcripts released between RSV WT and RSV L305I (Fig. 2d). However, when we measured foci of infection, we found that RSV L305I resulted in significantly fewer foci than RSV WT (Fig. 2e). This suggests that although similar amounts of transcripts are created by the two viruses, the virions produced by RSV L305I appear to be significantly less infectious (Fig. 2e).

Taking into account our modeling data, we asked whether the sensitivity of RSV to human sera may be altered by the L305I mutation. We obtained polyclonal sera from otherwise healthy full-term infants that had confirmed infections with RSV-A or RSV-B. We decided to use infant sera to reduce confounding results from older patients which may be complicated by immune memory to previous RSV infections. However, our sera samples were obtained from infants ranging in age from 2 weeks − 13.3 months. Although maternal RSV antibodies have been previously reported to be mostly absent by 3 months of age, we cannot rule out whether these infants had received passive RSV antibodies from their mothers, particularly in sera samples from infants less than 3 months of age ⁴². Briefly, HeLa cells were infected at an MOI of 0.3 with RSV WT or RSV L305I incubated with a 10^− 3 dilution of each sera sample for 1 hr and analyzed by flow cytometry (Fig. 3). As was expected, different sera samples had varying levels of neutralization to RSV WT and RSV L305I (Fig. 3a). Though we found that overall RSV L305I was significantly more resistant to human sera than RSV WT (Fig. 3b). This is in line with what we observed in our in vitro evolution experiment, wherein the introduction of the L305I mutation led to a temporary rebound in viral titer (Fig. 1a).

Computational modeling of the RSV-F L305I mutation shows a structural shift in the RSV-F antigenic site II.

Currently, prophylactic monoclonal antibodies are the therapeutic of choice to prevent RSV infection in infants. PZMB and Nirsevimab have been FDA-approved for use in infants and a third antibody - Clesrovimab - is in late-phase clinical trials ⁴. These antibodies target antigenic sites II, ∅, and IV respectively, on RSV-F (Fig. 4a). We sought to investigate the impact that the L305I mutation may have in the context of these prophylactic treatments. Our in silico structural modeling shows that position 305 is located in antigenic site III, typically considered a weak antigenic site (Fig. 2b). However, we also observed that position 305 is located directly behind PZMB binding site II ²⁵ in RSV-F (Fig. 2a and 4b). Using molecular dynamics simulations at over 500 ns, we found that the L305I mutation led to a conformational change in antigenic site II (Fig. 4b). Superimposition of site II RSV-F L305 and RSV-F I305 revealed not only a movement in the PZMB binding site but also a change in the orientation of several residues in which the L305I mutation forced residue N254 to change orientation outwards by 137.4˚ (Fig. 4c). In addition, Q279 has a bond angle change of 137.2˚ from the wildtype RSV-F L305 to the mutant RSV-F I305 (Fig. 4c). In summary, it appears that the steric effects caused by the L305I mutation extend to and affect the structural confirmation of antigenic site II.

The structural change elicited by RSV-F L305I alters antibody binding affinity to site II and viral susceptibility to palivizumab.

To determine whether the predicted change in the structure of binding site II affects PZMB neutralization, we conducted an in vitro PZMB neutralization assay with RSV WT and RSV L305I. We found that RSV WT was more susceptible to palivizumab (EC₅₀ = 91.26 ng/mL) than RSV L305I (EC₅₀ = 244.1 ng/mL) (Fig. 4d). Even at a maximum concentration of 10⁵ ng/mL, PZMB was unable to fully neutralize RSV L305I, whereas RSV WT appeared to be almost fully neutralized by 10³ ng/mL of PZMB (Fig. 4d). To further validate the change in PZMB sensitivity to neutralization, we directly assessed the binding affinity of PZMB to RSV-F using surface plasmon resonance (SPR). To do this we used a recombinant pre-fusion stabilized RSV-F protein trimer called DS-Cav1¹⁸. Wildtype (DS-Cav1 L305) and mutant (DS-Cav1 I305) constructs were used to measure the kinetics of bivalent IgG PZMB binding to protein. We observed a slight, but not significant, decrease in the K_D value, for the mutant DS-Cav1 I305 (K_D = 0.10 nM) as compared with the WT DS-Cav1 L305 (K_D = 0.29 nM) (Fig. 4e and f, S. Table 6). Since K_D is inversely proportional to binding affinity, this suggests that there is no observable change to PZMB binding affinity induced by the L305I mutation.

RSV-F L305I alters the structure of RSV-F antigenic sites Ø and IV, and subsequent binding affinity of monoclonal antibodies.

Following our characterization of the effect of L305I on binding site II, we asked whether the same mutation could similarly affect more distant antigenic sites. Several therapeutic monoclonal antibodies that target various RSV-F antigenic sites are of particular interest to us, including the recently approved Nirsevimab and the phase III clinical trial antibody Clesrovimab, which target binding sites Ø and IV respectively ^4,43. To study these binding sites, we used monoclonal antibodies D25 and 101F. D25 is the parental antibody to Nirsevimab ⁴⁴ while 101F and Clesrovimab both target binding site IV, specifically the epitopes spanning residues 427–438 and 426–447 respectively ^45,46. Analysis of MD trajectories revealed that antigenic site Ø, at the apex of the RSV-F I305 monomer and trimer, was shifted by 9.1 Å for α1 and 10 Å for α5 helices when compared to RSV-F L305 (Fig. 5a), while site IV shifted by about 8 Å (Fig. 5b). Interestingly, these results suggest that the L305I mutation, which is located in antigenic site III, also affects the structural conformation of antigenic sites Ø and IV in RSV-F.

To confirm whether these structural changes have biological relevance, we evaluated the neutralizing potential of D25 and 101F to RSV WT and RSV L305I. We saw similar trends with monoclonal antibodies D25 and 101F as we did with PZMB. The EC₅₀ value for D25 was higher against RSV L305I (19.53 ng/mL) than RSV WT (6.46 ng/mL) suggesting that the L305I mutation acts as a resistance mutation (Fig. 5c). Similar to our PZMB results, RSV L305I could not be fully neutralized even at a maximum antibody concentration of 10⁴ ng/mL whereas RSV WT was fully neutralized by 10² ng/mL of antibody (Fig. 5c). Similarly, we observed that the EC₅₀ value of 101F was also higher against RSV L305I (153.7 ng/mL) compared to RSV WT (71.27 ng/mL), suggesting that the L305I mutation also makes RSV less susceptible to 101F (Fig. 5d). To validate these findings, we again compared the binding kinetics of D25 and 101F to DS-Cav1 L305 and DS-Cav1 I305 RSV-F proteins using SPR. Consistent with our D25 neutralizations, we report an increased K_D for DS-Cav1 I305 (4.40 nM) compared to DS-Cav1 L305 (0.84 nM), suggesting that the binding affinity of D25 is higher to the wildtype RSV-F than the mutant (Fig. 5e and g). SPR with 101F revealed similar results to PZMB, wherein the K_D value for DS-Cav1 L305 (0.52 nM) and DS-Cav1 I305 (0.31 nM) was about the same, suggesting that the L305I mutation does not affect the binding affinity of 101F (Fig. 5f and h). This data suggests that the increased resistance to D25 can be attributed to a change in binding affinity towards RSV-F, where D25 exhibited a weaker binding to RSV-F L305I (Fig. e and g). However, it is not clear what the source of the increased resistance to PZMB and 101F is if the L305I mutation does not alter the binding affinity of these antibodies to prefusion RSV-F (Fig. 4 and Fig. 5). Given that antigenic sites II and IV are retained during the conversion of prefusion to post-fusion RSV-F, whereas site Ø is not, it is possible that the L305I mutation also has an effect on antibody binding to post-fusion RSV-F in these cases. Overall, our results suggest that the L305I mutation can elicit significant changes across at least RSV-F antigenic sites Ø, II, and IV, and subsequently impact the effectiveness of monoclonal antibodies that target these regions.

To the best of our knowledge, this is the first in vitro analysis of longitudinal RSV-F adaptation in response to antibody pressure. Here we describe a new L305I mutation that changes the structural landscape of the RSV-F trimer and alters viral susceptibility to monoclonal antibodies, despite being located outside of the target antigenic sites. We found that the L305I mutation was the first to arise at viral bottleneck under anti-RSV polyclonal antibody pressure and is a conserved difference between RSV-A and RSV-B subtypes. We suspect that the L305I mutation was able to develop in a majority of the viral population due to the subtle differences in antibody susceptibility between the viruses so that when RSV WT load was suppressed, RSV L305I was able to grow. This would suggest that L305I could arise normally but is outcompeted in the presence of RSV-F L305 containing RSV-A viruses. We surmise that, since the emergence of RSV-F I305 in RSV-B strains, compensatory mutations may have resulted in some RSV-B isolates growing to levels that are equivalent to RSV-A isolates allowing the two subtypes to co-circulate.

Using computational modeling, we showed that L305I, located in the antigenic site III, was able to alter the conformation of the entire RSV-F trimer. Subsequently, this mutation led to a decrease in viral susceptibility to monoclonal antibodies PZMB, D25, and 101F, at the cost of viral replicative fitness in tissue culture. Interestingly, this reduced propensity for growth is analogous to the differences reported between RSV-A and RSV-B subtypes ^9,40,41. Our neutralization assays revealed that the L305I mutation also altered viral susceptibility to serum neutralization to varying degrees. It is possible that L305I, and perhaps similar mutations, may have evolved to allow RSV to escape from neutralizing serum antibodies at the cost of reduced viral fitness. Indeed, a previous paper reported on the high levels of RSV-G diversity in patients in the presence of immune pressure ⁴⁷.

We found that overall, only D25 had a significantly different K_D value between DS-Cav1 L305 and DS-Cav1 I305, while PZMB and 101F had nearly identical K_D values for both proteins. Suggesting that the resistance to D25 observed with RSV L305I can be explained by a decrease in binding affinity to RSV-F I305. Interestingly, it is possible that there are other effects resulting from the L305I mutation that are responsible for the decrease in antibody susceptibility observed with PZMB and 101F, for instance, altered binding to post-fusion RSV-F. It is also important to note that measuring EC₅₀ in tissue culture introduces additional factors compared to the highly controlled environment used to measure K_D.

With the introduction of novel RSV prophylaxis in the form of both vaccines and monoclonal antibodies, it is critical that we carefully monitor circulating RSV strains for escape mutants. Special attention should be paid to mutations located throughout RSV-F and not just in targeted antigenic sites, particularly if treatments cause a significant bottleneck and non-sterilizing reductions in viral titer in recipients. Wilkins et al. recently reported that although the Nirsevimab binding site is relatively conserved, amino acid variability is high outside of the binding site ³⁹. It is important to note that they also reported a change in PZMB susceptibility with a mutation at the Nirsevimab binding site. Interestingly, they have also reported that the RSV-A and RSV-B RSV-F proteins have less genetic diversity than other class I viral fusion proteins of influenza and beta coronaviruses ⁴⁴. However, we do not know whether this is because the RSV-F protein is structurally constrained or it undergoes more structural plasticity with fewer mutations, compared to other viruses. Other groups have also shown significant functional changes in a protein with conserved mutations. Wu et al. demonstrated that a mutation from isoleucine to leucine, in the KlenTaq1 DNA polymerase, significantly affected its temperature sensitivity ⁴⁸. Despite the mutation being located 20 Å away from the active site, this mutation had a substantial impact on the enzyme's temperature sensitivity. This suggests that even conservative alterations in the amino acid sequence, such as those in RSV-F, may be critical for maintaining the protein functionality required for viral entry.

The work described herein has novel significance with respect to antiviral and vaccine resistance and highlights the value of in silico modeling combined with functional studies and rigorous viral surveillance. This study highlights the importance of elucidating the full potential of therapeutic breakthroughs by viruses. In light of the recent therapeutic advancements, RSV will be under increasing amounts of selective pressure, particularly in the case of widespread distribution of Nirsevimab. We propose that, in addition to surveying for escape mutants in the RSV-F binding site, distal mutations should also be closely monitored, as these adaptations may lead to the emergence of viral resistance. Furthermore, it is crucial to understand that RSV persists and evolves due to imperfect immunity within the population. As highlighted by Grenfell et al., pathogens exposed to incomplete immune responses face selective pressures that drive rapid evolutionary changes ⁴⁹. This concept of imperfect immunity is particularly relevant for RSV, as it creates an environment where the virus can continuously adapt and evade neutralization. We must be vigilant in understanding the mechanisms underlying RSV evolution and adaptation to preserve current prophylactics and ensure the future development of better ones.

Cell culture

Henrietta Lack’s (HeLa) cells from American Type Culture Collection (ATCC, CCL2) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing L-glutamine, glucose, and sodium pyruvate with 10% heat-inactivated fetal bovine serum (FBS). Human airway epithelial (1HAEo-) cells (a gift from D.Gruenert, University of California, San Francisco, CA, USA) were grown in minimum essential medium (MEM) supplemented with 10% heat-inactivated FBS. Both immortalized cell lines were grown at 37℃ with 5% CO₂.

Reagents, antibodies, antisera, and DNA aptamers

The following reagents were used to stain virally infected cells via our colorimetric immunostaining assay: 0.5mg/ml X-Gal (5-Bromo-4-chloro-3-indolyl-ꞵ-D-galactopyranoside) (Fisher BioReagents™, Cat # BP1615-100) and Yellow Substrate (PBS containing 3mM potassium ferricyanide III, 3mM potassium ferrocyanide trihydrate, and 1mM magnesium chloride hexahydrate). The following commercial antibodies were used: Anti-F (RSV) D25 (human Fc) Antibody (Cambridge Bio, Cat # 01-07-0120), Anti-F (RSV) 101F (human Fc) Antibody (Cambridge Bio, Cat # 01-07-0140), Goat Anti-RSV Polyclonal Antibody (Meridian Life Science, Cat # B65860G), Rabbit Anti-Goat IgG H&L (β-galactosidase) (Abcam, Cat # ab136712), and Palivizumab (Synagis^®). Palivizumab was provided by the Stollery Children’s Hospital neonatal intensive care unit in Edmonton, Alberta, Canada. Infant Anti-RSVA and Anti-RSVB sera, and infant RSV-positive nasopharyngeal samples were kindly provided by Dr. Asuncion Mejias and Dr. Octavio Ramilo from the Nationwide Children’s Hospital in Columbus, Ohio, USA. Pre-fusion stabilized DS-Cav1 WT and L305I proteins and plasmids were kindly provided by Dr. Jason S. McLellan and Dr. Kaci Erwin and generated as previously described ²². A new batch of the RSV non-nucleoside polymerase inhibitor was synthesized according to the previously published procedure ^32,33.

DS-Cav1 protein expression and purification

High-efficiency NEB10-beta competent E.coli cells (New England Biolabs, Cat # C3019H) were transformed according to the NEB protocol using DS-Cav1 WT and L305I DS-Cav1 plasmids, provided by Dr. Jason McLellan. LB + Amp was used for all plates and cultures and all incubations were conducted at 37℃ unless otherwise specified. Single colonies were picked for starter cultures which were then used to inoculate 300mL for maxi prep recommended protocol using NucleoBond Xtra Maxi EF (Macherey Nagel, Cat # 740424). Endotoxin-free plasmid DNA was sent to Dr. Joanne Lemieux’s lab at the University of Alberta, Edmonton, Canada and her group kindly expressed the protein using the EXPi293 system (Thermo Fisher Scientific) as recommended. Briefly, EXPi293F cells were transfected as per protocol and grown for 5 days before pelleting cells and freezing down the secreted protein found in the media. We confirmed the presence of the DS-Cav1 protein via western blot. The protein was purified on an AKTAstart (Cytiva Life Science, Cat # 29022094) using a HisPur Ni-NTA chromatography cartridge (Thermo Fisher Scientific, Cat # 90099) and an isocratic elution. We concentrated the protein and exchanged the buffer using Amicon Ultra-15, 10k MWCO (MilliporeSigma, Cat # UFC901024). The final concentration was determined with the Qubit Protein Assay Kit (Invitrogen, Cat # Q33212) and stored at -80℃ in 2mM Tris, 200mM NaCl, 0.02% sodium azide.

Virus isolation, propagation, and purification

Clinical samples were tested with a NxTAG^® Respiratory Virus Panel (RVP, Luminex, Austin, TX, USA) and samples positive for RSVA or RSVB were subject to further analysis. Clinical strains of RSV were isolated from patient nasopharyngeal samples as previously described ⁹. Briefly, NP samples diluted in 1mL DMEM + 10% FBS + 1X Penicillin/Streptomycin were added to sub-confluent HeLa cells and incubated at 37℃ with 5% CO₂. After 4 hours, fresh media was added to the cells and the virus was left to propagate for 96 hours. Virus media was harvested and stored in liquid nitrogen. Recombinant lab-adapted strain RSV type-A2 expressing green fluorescent protein (GFP) (rgRSV RW30) was a gift from M.E. Peeples (Children’s Research Institute, Columbus, OH, USA). Lab strain A2 and the RSV L305 mutant were purified by sucrose density gradient purification as described previously ^9,50. Briefly, the virus was precipitated from conditioned media by stirring with 10% Polyethylene glycol (PEG)-6000 on ice for 90 min. The virus was pelleted by centrifugation at 4,300 x g at 4°C for 30 min, the pellets resuspended in NT buffer (0.15 M NaCl, 0.05 M tris, pH 7.5) and overlaid on a discontinuous sucrose gradient (35%, 45%, 60% sucrose in NT buffer) as previously described. The sucrose-purified RSV band was spun for 4 h at 217,290× g at 4℃, harvested, then aliquoted and stored in liquid nitrogen.

Construction of the RSV L305I Mutant

The rgRSV-L305I mutant was constructed by inserting a CTA to ATA mutation at amino acid position 305 into the rgRSV RW30 vector via Gibson Assembly and Cloning ⁵¹. To clone the RSV-L305I mutant, 6 primers were designed using SnapGene Software, a forward and reverse primer for each of the 3 complementary PCR fragments that were created, with one of the primers containing the CTA to ATA mutation. The online ThermoFisher Multiple Primer Analyzer was used for primer analysis. Each PCR fragment was created separately using the Q5^® High-Fidelity DNA polymerase protocol. Briefly, dNTPs (NEB, N0446S), template plasmid RSV RW30 DNA, forward and reverse primers, Q5^Ⓡ Reaction Buffer (NEB, B9027S) and Q5^Ⓡ High-Fidelity DNA polymerase (NEB, M0491S) were combined on ice. PCR reactions were carried out in a thermocycler (Biorad T100) under the following settings: 98℃ for 30s, [98℃ for 10s, 55℃ for 30s, 72℃ for 21s] for 35 cycles, 72℃ for 5 min, and set to hold at 10℃ once the reaction was complete. The sizes of PCR products were confirmed using a 1% agarose gel stained with SYBR^Ⓡ Safe DNA gel stain (ThermoFisher, #S33102). DNA was extracted according to the Qiaex II Gel Extraction Kit (Qiagen, #20051) and purified using the QIAquick PCR purification kit (Qiagen, #28104). The purified products were annealed together using the NEBuilder HiFi DNA Assembly Master Mix and accompanying protocol (NEB, #E2621L) by combining the Master Mix with the purified PCR fragments and incubating them at 50℃ for 15 min in the thermocycler. Annealed viral plasmid DNA was used to transform competent NEB10-beta E.coli (NEB, #C3019) according to the High Efficiency Transformation Protocol (NEB). Briefly, assembled plasmid and competent cells were incubated on ice for 30 min and heat shocked at 42℃ for 30s, SOC media was added and incubated on a shaker for 1 hour at room temperature. Bacteria were plated on YT + 10µg/mL tetracycline selection plates, incubated overnight at 37℃, and the following day individual colonies were selected and grown in YT broth overnight at 37℃. Bacteria were pelleted by centrifugation at 500 x g for 10 mins and plasmid DNA was isolated using the QIAprep^Ⓡ Spin Miniprep Kit (Qiagen, #27106). Extracted plasmid DNA size was confirmed on a 1% agarose gel as described above and plasmids were sent for sequencing to the Molecular Biology Service Unit (MBSU, University of Alberta, Edmonton, AB, Canada) to confirm the presence of the L305I mutation.

RSV reverse genetics

Full length rgRSV RW30 and mutant L305I RW30 cDNA were rescued into infectious virions in HeLa cells. Briefly, full length rgRSV RW30 or RSV-L305I, support plasmids (RSV N, P, L, and M2-1), and T7 RNA polymerase (a gift from Benhur Lee, Addgene plasmid #65974) were transfected into sub-confluent HeLa cells using TransIT-HeLa MONSTER (Mirus Bio, MIR 2900). After rescue, RSV was propagated in HeLa cells in T75 flasks and harvested as cell-free (clarified) RSV-conditioned DMEM with 10% FBS. RSV-conditioned media was aliquoted and stored in liquid nitrogen. The RSV-L305I mutant was used as media-virus and the rgRSV RW30 was further sucrose purified before use in experiments.

Infectious RSV quantification

RSV-infected cells were detected using a colorimetric immunostaining assay as previously described ⁵⁰. Briefly, RSV-infected monolayers were fixed and permeabilized with methanol: acetone (1:1 volume) and incubated for 10 min at RT. Cells were blocked with PBS + 10% FBS and incubated for 30 min at RT. Subsequently, cells were stained with primary goat anti-RSV (diluted 1:1000) and incubated overnight at 4°C. The following day cells were treated with a secondary rabbit anti-goat antibody conjugated to ꞵ-galactosidase (diluted 1:2000) and in the absence of light for 1 hour at RT. Cells were then stained blue with 1:1000 X-gal diluted in PBS containing 3mM potassium ferricyanide III, 3mM potassium ferrocyanide trihydrate, and 1mM magnesium chloride hexahydrate and incubated away from light at 37℃ with 5% CO₂ for 2–4 hours until blue spots were fully developed. Foci of infection stained blue and were counted under the EVOS® Fl Auto Imaging System (ThermoFisher, AMAFD1000). Viral titer was estimated in focus-forming units (FFU/mL). Note that all antibodies were diluted to appropriate concentrations in PBS + 1% FBS and that cells were washed three times with PBS following all blocking and staining treatments unless otherwise specified.

Human sera % neutralization and EC₅₀ values for monoclonal antibodies were determined via GFP fluorescence detected by flow cytometry. All human sera were diluted to 10^− 3 while monoclonal antibodies PZMB and 101F were used at starting concentrations of 1,000,000 ng/mL and D25 started at 100,000 ng/mL due to its increased potency. Monoclonal antibodies were 10-fold serially diluted 8 times and incubated with RSV WT or RSV L305I at an MOI of 0.25 (for monoclonal antibodies) or 0.3 (for human sera) for 1 hour. Sub-confluent HeLa cells grown in DMEM + 10% FBS + 1X P/S were infected with the virus antibody dilutions in 12-well (monoclonals) or 48-well (human sera) plates. 4 hours post infection fresh media was added to cells. 24 hours post-infection cells were prepared for flow cytometry by treatment with a 1:1000 dilution of Ghost Dye™ Violet 450 (Cytek, 13–0863) for 30 mins. Viral infection is indicated by GFP fluorescence and % neutralization is determined by comparison to an antibody-free control group in each experiment. Flow cytometry was conducted using the BD LSRFortessa X-20 (BD Biosciences). B530 and V450 lasers to detect GFP and live-dead stains respectively. Flow analysis was completed using FLOWJO software (BD Biosciences) and final EC₅₀ values, curves, and statistics were evaluated using GraphPad Prism.

RSV evolution experiment

Evolution experiments were done as described in our previous publication ³³. Briefly, sub-confluent HeLa cells were infected at an MOI of 0.5 with lab strain rgRSV-A2 incubated with commercial anti-RSV polyclonal goat sera, RSV polymerase inhibitor (compound 5f) at 10µM and 25µM diluted in DMSO, or 0.25% DMSO. 4 hours post-infection, fresh media containing the same antibody or drug, was added to the cells and the infection was allowed to proceed at 37℃ and 5% CO₂. After 48 hours, virus-conditioned media was clarified, and a portion was used to infect a new HeLa cell monolayer in the presence of an antibody or drug again to repeat the process. The viruses were passaged every two days for 40 days for a total of 20 passages. RNA was harvested from every passage and viral growth was measured by qRT-PCR and a foci counting assay to test infectivity.

Whole genome sequencing

Whole genome sequencing was done as described in ⁹. Briefly, viral RNA from each passage of the evolution experiment was extracted using the QIAamp® Viral RNA Mini Kit (Qiagen, 52906). Viral mRNA was isolated by poly-A pulldown with oligo d(T) 25 beads using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB, E7490L). The first strand of cDNA was prepared using the High-Capacity cDNA RT Kit (ThermoFisher, 4368814), and the second strand was synthesized with the large Klenow fragment of DNA polymerase 1 (NEB, M0210L). cDNA was purified with the MinElute™ PCR Purification Kit (Qiagen, 28004). Tagmentation and creation of the library were done using the Nextera XT DNA Library Preparation Protocol (Illumina, FC-131-1096). Index adapters used for sample identification were created by MBSU (Molecular Biology Service Unit, University of Alberta, Edmonton, AB, Canada) and libraries were cleaned up using the MagJET NGS Cleanup Kit (ThermoFisher, K2821). Sample concentration was checked using the NanoDrop ^TM 8000 Spectrophotometer (ThermoFisher, ND-8000-GL) and sample purity analyzed by the Agilent 2100 Bioanalyzer G2938C (Marshall Scientific, AG-2100C) at the MBSU. Equal amounts of DNA were combined and sent for sequencing to the MBSU for Next Generation Sequencing on the MiSeq® System (Illumina, SY-410-1003).

Surface Plasmon Resonance

Experiments were performed using the Biacore T200 Surface Plasmon Resonance System (Cytiva, 28975001). Monoclonal antibody binding affinity was determined using His-tag coupling. The kinetics protocol is as follows: NTA chip was Ni²⁺ activated according to the Series S NTA Sensor Chip protocol (Cytiva, BR100532). Prefusion-stabilized RSV-F DS-Cav1^L305 and DS-Cav1^I305 proteins (diluted to 0.1 µg/mL were injected onto the chip at 5µL/min for 60s. Antibody dilutions were run at 30µL/min for 180s, and dissociation was measured over 800s. HBS-P (pH 7.4) supplemented with 50µM EDTA was used as a running buffer and each antibody concentration was run in at least duplicate. 350mM EDTA was used for chip regeneration at 10µL/min for 60s three times. Bivalent IgG monoclonal antibodies were 3-fold serially diluted 8 times in the running buffer from a starting concentration of 100nM. An empty inactivated flow lane was used for double reference subtraction and curves were fit to a 1:1 binding model in Biacore Insight Evaluation Software (Cytiva). Final curves were created in GraphPad Prism.

Determining the % identity and similarity of RSV isolates

Amino acid identity and similarity between RSVA and B were determined using Geneious software, Biomatters, New Zealand.

Aligning multiple clinical isolates of RSV and determining aa differences in RSV-F protein

5141 RSV-F protein sequences were downloaded from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) ⁵² through the web site at http://www.viprbrc.org/. SeqKit ⁵³ was then used to filter the RSV-F sequences to include only sequences that are the correct length (574 amino acids), and are identified as RSV type-A or RSV type-B. After this filtering, 3747 sequences remained – 2313 RSV type-A (61.7%) and 1434 RSV type-B (38.3%). The RSV type-A sequences and type-B sequences were aligned separately and together using Clustal Omega ⁵⁴ with default settings and analyzed using Jalview 2.11.1.4 ⁵⁵. Amino acids with at least 65% greater conservation for both RSV type-A and type-B compared to the combined alignment were marked as being conserved by RSV type.

Protein preparation from molecular dynamics simulations.

Starting coordinates for the RSV-F protein were obtained from the Protein Data Bank for both the monomer (PDB accession: 4MMU) and the protomer (PDB accession: 5UDC). The X-ray structure of the protomer was solved with bound MEDI8897, which was removed to obtain the free protomer. Mutation at residue 305 was performed using the Schrödinger Small Molecule Discovery Suite. The Protein Preparation Wizard module in Schrödinger was used to add hydrogen atoms, minimize energy, fill missing loops, and create the appropriate protonation states of amino acid side chains. The protein structure was then subjected to three stages of energy minimization, all of which utilized the OPLS3 force field ⁵⁶.

Molecular dynamics simulations of the RSV-F

The structures of RSV-F protein were solvated in a cuboidal box of TIP3P water molecules using AMBER’s tLEaP tool ⁵⁷. The ff14SB parameters were assigned for the protein. The complexes were then neutralized and solvated in a NaCl salt concentration of 0.15 M with tLEaP using the same process described above. The simulations were performed using PMEMD in AMBER18 ⁵⁷. An initial minimization step was performed in order to relax the water and ionic positions. The whole system was then minimized and heated gradually up to 300K in 100 ps using Langevin dynamics. During the heating process, we restrained the backbone of the protein, and a time step of 0.5 fs and periodic volume conditions were employed during this phase. The time step has been set to 2 fs, and periodic pressure conditions (1atm) have been imposed and the restraints have been gradually released in four phases of 50 ps each. The production phase of the simulations at the NPT conditions was performed in triplicates for 100, 270, and 500 ns using GPU accelerated version of PMEMD (pmemd. cuda) implemented in AMBER 18 ⁵⁷.

Structural analysis of molecular dynamics simulations

The CPPTRAJ software in AMBER18 was used to compute the root mean squared deviation (RMSD) of the protein coordinates with respect to the reference X-ray structure along the MD trajectories. The coordinates of the models were also clustered using CPPTRAJ with the average-linkage clustering algorithm ⁵⁸.

Statistical analysis

Statistical analysis was done using GraphPad Prism 9 software. Unless otherwise indicated, results are expressed as mean ± s.d. Group means were compared by either two-tailed Student’s t-tests or one-way analysis of variance (ANOVA) with Tukey’s post hoc analysis, comparing each group with the appropriate control. All tests are two-tailed unless otherwise indicated. We considered a P value ≤ 0.05 to be statistically significant. Representative data from a single experiment were confirmed by 3 or more independent repeats. Independent experimental repeat details are found within the legends of each Extended Data figure.

Acknowledgments

We would like to thank the University of Alberta Faculty of Medicine and Dentistry Flow Cytometry Facility (RRID:SCR_019195) which receives financial support from the Faculty of Medicine & Dentistry and the Canada Foundation for Innovation (CFI) awards to contributing investigators. We would also like to thank Bart Hazes (University of Alberta) for his bioinformatics expertise with evolution experiments and helpful discussions. The Digital Research Alliance of Canada(alliancecan.ca) is thanked for the computational resources. This study was supported in part by the Alberta Ministry of Technology and Innovation through SPP-ARC (Striving for Pandemic Preparedness - The Alberta Research Consortium).

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary information

The online version contains supplementary material available at

Li, Y. et al. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in children younger than 5 years in 2019: a systematic analysis. Lancet 399, 2047–2064 (2022). https://doi.org/10.1016/S0140-6736(22)00478-0
Busack, B. & Shorr, A. F. Going Viral-RSV as the Neglected Adult Respiratory Virus. Pathogens 11 (2022). https://doi.org/10.3390/pathogens11111324
Caserta, M. T., O'Leary, S. T., Munoz, F. M., Ralston, S. L. & Committee On Infectious, D. Palivizumab Prophylaxis in Infants and Young Children at Increased Risk of Hospitalization for Respiratory Syncytial Virus Infection. Pediatrics 152 (2023). https://doi.org/10.1542/peds.2023-061803
Phuah, J. Y. et al. Quantification of clesrovimab, an investigational, half-life extended, anti-respiratory syncytial virus protein F human monoclonal antibody in the nasal epithelial lining fluid of healthy adults. Biomed Pharmacother 169, 115851 (2023). https://doi.org/10.1016/j.biopha.2023.115851
Kampmann, B. et al. Bivalent Prefusion F Vaccine in Pregnancy to Prevent RSV Illness in Infants. N Engl J Med 388, 1451–1464 (2023). https://doi.org/10.1056/NEJMoa2216480
Melgar, M. et al. Use of Respiratory Syncytial Virus Vaccines in Older Adults: Recommendations of the Advisory Committee on Immunization Practices - United States, 2023. MMWR Morb Mortal Wkly Rep 72, 793–801 (2023). https://doi.org/10.15585/mmwr.mm7229a4
Griffiths, C., Drews, S. J. & Marchant, D. J. Respiratory Syncytial Virus: Infection, Detection, and New Options for Prevention and Treatment. Clin Microbiol Rev 30, 277–319 (2017). https://doi.org/10.1128/CMR.00010-16
Zlateva, K. T., Lemey, P., Moes, E., Vandamme, A. M. & Van Ranst, M. Genetic variability and molecular evolution of the human respiratory syncytial virus subgroup B attachment G protein. J Virol 79, 9157–9167 (2005). https://doi.org/10.1128/JVI.79.14.9157-9167.2005
Elawar, F. et al. A Virological and Phylogenetic Analysis of the Emergence of New Clades of Respiratory Syncytial Virus. Sci Rep 7, 12232 (2017). https://doi.org/10.1038/s41598-017-12001-6
Yu, J. M., Fu, Y. H., Peng, X. L., Zheng, Y. P. & He, J. S. Genetic diversity and molecular evolution of human respiratory syncytial virus A and B. Sci Rep 11, 12941 (2021). https://doi.org/10.1038/s41598-021-92435-1
Elawar, F. et al. Pharmacological targets and emerging treatments for respiratory syncytial virus bronchiolitis. Pharmacol Ther 220, 107712 (2021). https://doi.org/10.1016/j.pharmthera.2020.107712
Utokaparch, S. et al. The relationship between respiratory viral loads and diagnosis in children presenting to a pediatric hospital emergency department. Pediatr Infect Dis J 30, e18-23 (2011). https://doi.org/10.1097/INF.0b013e3181ff2fac
Rebuffo-Scheer, C. et al. Whole genome sequencing and evolutionary analysis of human respiratory syncytial virus A and B from Milwaukee, WI 1998–2010. PLoS One 6, e25468 (2011). https://doi.org/10.1371/journal.pone.0025468
Johnson, P. R., Spriggs, M. K., Olmsted, R. A. & Collins, P. L. The G glycoprotein of human respiratory syncytial viruses of subgroups A and B: extensive sequence divergence between antigenically related proteins. Proc Natl Acad Sci U S A 84, 5625–5629 (1987).
Simoes, E. A. F. et al. Suptavumab for the Prevention of Medically Attended Respiratory Syncytial Virus Infection in Preterm Infants. Clin Infect Dis 73, e4400-e4408 (2021). https://doi.org/10.1093/cid/ciaa951
McLellan, J. S. Neutralizing epitopes on the respiratory syncytial virus fusion glycoprotein. Curr Opin Virol 11, 70–75 (2015). https://doi.org/10.1016/j.coviro.2015.03.002
McLellan, J. S. et al. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342, 592–598 (2013). https://doi.org/10.1126/science.1243283
McLellan, J. S. et al. Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science 340, 1113–1117 (2013). https://doi.org/10.1126/science.1234914
McLellan, J. S., Yang, Y., Graham, B. S. & Kwong, P. D. Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes. J Virol 85, 7788–7796 (2011). https://doi.org/10.1128/JVI.00555-11
Griffiths, C. D. et al. IGF1R is an entry receptor for respiratory syncytial virus. Nature 583, 615–619 (2020). https://doi.org/10.1038/s41586-020-2369-7
Hayes, R. S., Oraby, A. K., Camargo, C., Marchant, D. J. & Sagan, S. M. Mapping respiratory syncytial virus fusion protein interactions with the receptor IGF1R and the impact of alanine-scanning mutagenesis on viral infection. J Gen Virol 105 (2024). https://doi.org/10.1099/jgv.0.001951
McLellan, J. S., Ray, W. C. & Peeples, M. E. Structure and function of respiratory syncytial virus surface glycoproteins. Curr Top Microbiol Immunol 372, 83–104 (2013). https://doi.org/10.1007/978-3-642-38919-1_4
Bin, L. et al. Emergence of new antigenic epitopes in the glycoproteins of human respiratory syncytial virus collected from a US surveillance study, 2015-17. Sci Rep 9, 3898 (2019). https://doi.org/10.1038/s41598-019-40387-y
McCool, R. S. et al. Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein elicits antibodies targeting a membrane-proximal epitope. J Virol, e0092923 (2023). https://doi.org/10.1128/jvi.00929-23
Beeler, J. A. & van Wyke Coelingh, K. Neutralization epitopes of the F glycoprotein of respiratory syncytial virus: effect of mutation upon fusion function. J Virol 63, 2941–2950 (1989).
Mas, V., Nair, H., Campbell, H., Melero, J. A. & Williams, T. C. Antigenic and sequence variability of the human respiratory syncytial virus F glycoprotein compared to related viruses in a comprehensive dataset. Vaccine 36, 6660–6673 (2018). https://doi.org/10.1016/j.vaccine.2018.09.056
Rezende, W., Neal, H. E., Dutch, R. E. & Piedra, P. A. The RSV F p27 peptide: current knowledge, important questions. Front Microbiol 14, 1219846 (2023). https://doi.org/10.3389/fmicb.2023.1219846
Rezende, W. et al. The Efficiency of p27 Cleavage during In Vitro Respiratory Syncytial Virus (RSV) Infection Is Cell Line and RSV Subtype Dependent. J Virol 97, e0025423 (2023). https://doi.org/10.1128/jvi.00254-23
Jones, H. G. et al. Alternative conformations of a major antigenic site on RSV F. PLoS Pathog 15, e1007944 (2019). https://doi.org/10.1371/journal.ppat.1007944
Mukhamedova, M. et al. Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein induces genetically and antigenically diverse antibody responses. Immunity 54, 769–780 e766 (2021). https://doi.org/10.1016/j.immuni.2021.03.004
Zhao, X., Chen, F. P. & Sullender, W. M. Respiratory syncytial virus escape mutant derived in vitro resists palivizumab prophylaxis in cotton rats. Virology 318, 608–612 (2004). https://doi.org/10.1016/j.virol.2003.10.018
Atienza, B. J. P. et al. Dual Catalytic Synthesis of Antiviral Compounds Based on Metallocarbene-Azide Cascade Chemistry. J Org Chem (2018). https://doi.org/10.1021/acs.joc.8b00222
Oraby, A. K., Bilawchuk, L., West, F. G. & Marchant, D. J. Structure-Based Discovery of Allosteric Inhibitors Targeting a New Druggable Site in the Respiratory Syncytial Virus Polymerase. ACS Omega 9, 22213–22229 (2024). https://doi.org/10.1021/acsomega.4c01207
Hashimoto, K. & Hosoya, M. Neutralizing epitopes of RSV and palivizumab resistance in Japan. Fukushima J Med Sci 63, 127–134 (2017). https://doi.org/10.5387/fms.2017-09
Hause, A. M. et al. Sequence variability of the respiratory syncytial virus (RSV) fusion gene among contemporary and historical genotypes of RSV/A and RSV/B. PLoS One 12, e0175792 (2017). https://doi.org/10.1371/journal.pone.0175792
Gilman, M. S. A. et al. Transient opening of trimeric prefusion RSV F proteins. Nat Commun 10, 2105 (2019). https://doi.org/10.1038/s41467-019-09807-5
Krarup, A. et al. A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism. Nat Commun 6, 8143 (2015). https://doi.org/10.1038/ncomms9143
Techaarpornkul, S., Barretto, N. & Peeples, M. E. Functional analysis of recombinant respiratory syncytial virus deletion mutants lacking the small hydrophobic and/or attachment glycoprotein gene. J Virol 75, 6825–6834 (2001). https://doi.org/10.1128/JVI.75.15.6825-6834.2001
Techaarpornkul, S., Collins, P. L. & Peeples, M. E. Respiratory syncytial virus with the fusion protein as its only viral glycoprotein is less dependent on cellular glycosaminoglycans for attachment than complete virus. Virology 294, 296–304 (2002). https://doi.org/10.1006/viro.2001.1340 S0042682201913406 [pii]
Kim, Y. I. et al. Relating plaque morphology to respiratory syncytial virus subgroup, viral load, and disease severity in children. Pediatr Res 78, 380–388 (2015). https://doi.org/10.1038/pr.2015.122
Rijsbergen, L. C. et al. Human Respiratory Syncytial Virus Subgroup A and B Infections in Nasal, Bronchial, Small-Airway, and Organoid-Derived Respiratory Cultures. mSphere 6 (2021). https://doi.org/10.1128/mSphere.00237-21
Capella, C. et al. Prefusion F, Postfusion F, G Antibodies, and Disease Severity in Infants and Young Children With Acute Respiratory Syncytial Virus Infection. J Infect Dis 216, 1398–1406 (2017). https://doi.org/10.1093/infdis/jix489
Zhu, Q. et al. A highly potent extended half-life antibody as a potential RSV vaccine surrogate for all infants. Sci Transl Med 9 (2017). https://doi.org/10.1126/scitranslmed.aaj1928
Wilkins, D. et al. Nirsevimab binding-site conservation in respiratory syncytial virus fusion glycoprotein worldwide between 1956 and 2021: an analysis of observational study sequencing data. Lancet Infect Dis 23, 856–866 (2023). https://doi.org/10.1016/S1473-3099(23)00062-2
McLellan, J. S. et al. Structure of a major antigenic site on the respiratory syncytial virus fusion glycoprotein in complex with neutralizing antibody 101F. J Virol 84, 12236–12244 (2010). https://doi.org/10.1128/JVI.01579-10
Tang, A. et al. A potent broadly neutralizing human RSV antibody targets conserved site IV of the fusion glycoprotein. Nat Commun 10, 4153 (2019). https://doi.org/10.1038/s41467-019-12137-1
Grad, Y. H. et al. Within-host whole-genome deep sequencing and diversity analysis of human respiratory syncytial virus infection reveals dynamics of genomic diversity in the absence and presence of immune pressure. J Virol 88, 7286–7293 (2014). https://doi.org/10.1128/JVI.00038-14
Wu, E. Y. et al. A conservative isoleucine to leucine mutation causes major rearrangements and cold sensitivity in KlenTaq1 DNA polymerase. Biochemistry 54, 881–889 (2015). https://doi.org/10.1021/bi501198f
Grenfell, B. T. et al. Unifying the epidemiological and evolutionary dynamics of pathogens. Science 303, 327–332 (2004). https://doi.org/10.1126/science.1090727
Bilawchuk, L. M., Griffiths, C. D., Jensen, L. D., Elawar, F. & Marchant, D. J. The Susceptibilities of Respiratory Syncytial Virus to Nucleolin Receptor Blocking and Antibody Neutralization are Dependent upon the Method of Virus Purification. Viruses 9 (2017). https://doi.org/10.3390/v9080207
Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6, 343–345 (2009). https://doi.org/10.1038/nmeth.1318
Pickett, B. E. et al. ViPR: an open bioinformatics database and analysis resource for virology research. Nucleic Acids Res 40, D593-598 (2012). https://doi.org/10.1093/nar/gkr859
Shen, W., Le, S., Li, Y. & Hu, F. SeqKit: A Cross-Platform and Ultrafast Toolkit for FASTA/Q File Manipulation. PLoS One 11, e0163962 (2016). https://doi.org/10.1371/journal.pone.0163962
Madeira, F. et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res 47, W636-W641 (2019). https://doi.org/10.1093/nar/gkz268
Waterhouse, A. M., Procter, J. B., Martin, D. M., Clamp, M. & Barton, G. J. Jalview Version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009). https://doi.org/10.1093/bioinformatics/btp033
Harder, E. et al. OPLS3: A Force Field Providing Broad Coverage of Drug-like Small Molecules and Proteins. J Chem Theory Comput 12, 281–296 (2016). https://doi.org/10.1021/acs.jctc.5b00864
Case, D. A. et al. AmberTools. J Chem Inf Model 63, 6183–6191 (2023). https://doi.org/10.1021/acs.jcim.3c01153
Roe, D. R. & Cheatham, T. E., 3rd. PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data. J Chem Theory Comput 9, 3084–3095 (2013). https://doi.org/10.1021/ct400341p

No competing interests reported.

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A Single Amino Acid Mutation Alters the Neutralization Epitopes in the Respiratory Syncytial Virus Fusion Glycoprotein

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Version 1

Abstract

Figures

Introduction

Results

Discussion

Materials and Methods

Cell culture

Reagents, antibodies, antisera, and DNA aptamers

DS-Cav1 protein expression and purification

Virus isolation, propagation, and purification

Construction of the RSV L305I Mutant

RSV reverse genetics

Infectious RSV quantification

RSV evolution experiment

Whole genome sequencing

Surface Plasmon Resonance

Determining the % identity and similarity of RSV isolates

Aligning multiple clinical isolates of RSV and determining aa differences in RSV-F protein

Molecular dynamics simulations of the RSV-F

Structural analysis of molecular dynamics simulations

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1