Respiratory syncytial virus (RSV), or human respiratory syncytial virus (hRSV), is a common viral respiratory infection that triggers mild, cold-like symptoms.
However, at-risk populations, including infants, older adults, and immunocompromised individuals, are more susceptible to severe RSV infections, which can lead to bronchiolitis or pneumonia. As a result, RSV significantly impacts infant mortality, causing approximately 50,000 deaths annually in children under five.1
RSV remains a significant burden primarily due to the limited availability of prevention and treatment options. Several antibody drugs, such as palivizumab, are used to combat RSV infection; however, its use is restricted to prophylactic treatment in high-risk neonates.
Source: ACROBiosystems
| Advantages to using ACRO Recombinant Proteins |
| Conformation |
Proteins are verified to be conformation-specific with at a high-purity level. |
| Validation |
Validated Analytical methodology is performed for each batch under strict quality management systems. |
| Insightful |
Both protein conformations are available to help perform a wide array of research studies. |
This restriction is due to the low neutralizing potency of the developed RSV antibody. As a result, the treatment requires several high-dose administrations. Vaccine development has been similarly unsuccessful, with no treatments reaching market approval.
A major breakthrough for RSV research was the discovery of a conformational change of the fusion (F) protein following the establishment of infection. The RSV F protein is a trimer in two different forms: a metastable, pre-fusion form (pre-F) and stable post-fusion form (post-F), as outlined in Figure 1.
Structural research on RSV F protein has identified seven different antigenic epitopes, termed Site Ø, I, II, III, IV, V, and a quaternary dependent (QD) site, with only I, II, and IV being exposed on the post-F form.2,3 The post-F form has been shown to produce relatively weaker neutralizing antibodies that are unable to prevent infection.
Work conducted by Kwong et al. demonstrated that antibodies targeting sites exposed on both forms can neutralize late in the entry process, particularly in post-F form.4
The study discussed in this article investigates protocols and technical considerations for conformational change identification in the RSV protein, helping to ensure proteins are structurally accurate and fully evaluate vaccine products.
Figure 1. (Left) Pre- and (Right) Post-fusion forms of the RSV F protein. Protein structural arrangement occurs during the host-virus membrane fusion, signaling viral infection.5 Image Credit: ACROBiosystems
Methods and materials
Standard laboratory instrumentation and methodologies were used to analyze protein structure conformation. Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) was utilized to study conformational changes, while enzyme-linked immunosorbent assay (ELISA) was employed to confirm them.
SEC-MALS detection
Lyophilized pre-F and post-F RSV proteins were reconstituted in PBS (pH: 7.4) with 10 % trehalose as a stabilizing agent. Standard solutions were injected at 5 uL, with a constant column temperature of 25 °C during analysis. Conditions remained constant between analyses.
Table 2. SEC-MALS Experimental Conditions. Source: ACROBiosystems
| Conditions |
Description |
| Column |
TSKgel G3000SWXL 7.8 x 300 mm, 5 μm |
| Mobile Phase |
200 mM disodium phosphate (Na2HPO4) with 1% isopropyl alcohol, pH: 7.0 |
| Gradient |
Isocratic at 0.5 mL/min with a total run time of 30 min. |
| Absorption Wavelength |
280 nm |
ELISA
Direct ELISA was performed by immobilizing pre-F and post-F RSV protein to the bottom of each well. Different site-specific anti-fusion RSV antibodies were introduced and detected with peroxidase-conjugated anti-human IgG antibodies.
Table 3. Site-specific Anti-fusion RSV Glycoprotein Antibodies. Source: ACROBiosystems
| Antibody |
Site |
Description |
| D25 |
Ø |
Anti-Fusion glycoprotein F0 Antibody, Human IgG1 |
| 131-2a |
I |
Anti-RSV Fusion Protein Antibody |
| 101F |
IV |
Anti-Fusion glycoprotein F0 Antibody, Mouse IgG2a |
| AM14 |
QD |
Recombinant Human Anti-RSV Antibody (AM14) |
Table 4. ELISA Experimental Materials. Source: ACROBiosystems
| Materials |
Catalog No. |
Vendor |
| hRSV (A) Pre-Fusion Glycoprotein, His tag |
RSF-V52H7 |
ACROBiosystems |
| hRSV (A) Post-Fusion Glycoprotein, His tag |
RSF-V52H6 |
ACROBiosystems |
| Site-specific Anti-fusion glycoprotein F0 Antibody |
— |
--- |
Peroxidase AffiniPure Goat
Anti-Human IgG, Fcγ fragment specific |
109-035-098 |
Jackson
ImmunoResearch |
Costar 1 x 8 Stripwell, high binding EIA/RIA plate,
flat bottom, without lid |
42592 |
Corning |
| Bovine Serum Albumin (BSA) |
— |
Yancheng
Saibao |
| Tetramethylbenzidine (TMB) |
A600954 |
BBI Life Sciences |
Table 5. Reagents required for ELISA. Source: ACROBiosystems
| Reagents |
Description |
| Coating Buffer |
15 mmol/L Na2CO3, 35 mmol/L NaHCO3, 7.7 mmol/L NaN3, pH9.6 |
| Washing Buffer |
0.05% Tween-20 in TBS, pH7.4 |
| Blocking Buffer |
2% BSA in Washing Buffer, pH7.4 |
| Sample Dilution Buffer |
0.5% BSA in Washing Buffer, pH7.4 |
| Antibody Dilution Buffer |
0.5% BSA in Washing Buffer, pH7.4 |
| Substrate Solution |
8 μl 3% H2O2 and 100 μl 10 mg/mL TMB in 10 mL Substrate
Solution A (50 mmol/L Na2HPO4·12H2O, 25 mmol/L Citric acid, pH5.5) |
| Stop Solution |
1 mol/L sulfuric acid |
ELISA protocol
1. Coating
The plate was coated with 0.2 μg/well (2 μg/ml, 100 μl/well) HRSV (A) Fusion glycoprotein F0, His Tag (Cat. No. RSF-V52H7) at 4 °C overnight (or 16 hours). The protein was diluted with the coating buffer.
2. Washing
The wells were washed four times with washing buffer (300 μl per well). After washing, the remaining solution was removed by aspirating or decanting. The plate was inverted and left on clean paper towels, ensuring it has been dried completely. Complete removal of the washing buffer is essential.
3. Blocking
The wells were blocked with blocking buffer (300 μl per well) at 37 °C for 1.5 hours.
4. Washing
Repeat step 2.
5. Adding Sample
Anti-Fusion glycoprotein F0 Antibody, Human IgG1 (D25) (100 μl 0.152588-9.76563 ng/mL) was added to each well and incubated at 37 °C for 1 hour. The sample was then diluted in sample dilution buffer.
6. Washing
Repeat step 2 again.
7. Adding Detection Antibody
Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (Jackson, Cat. No. 109-035-098) (100 μl) was then added to each well and incubated at 37 °C for 1 hour. The antibody was diluted to 1:12000 in antibody dilution buffer (dilution buffer: 0.5% BSA in washing buffer, pH7.4).
8. Washing
Repeat step 2 one more time.
9. Adding Substrate
Substrate solution (200 μl) was then added to each well and incubated at 37 °C for 20 minutes while avoiding light exposure.
10. Termination:
Sulfuric acid (50 μl 1 mol/L) was then added to each well to stop the reaction.
11. Read and Calculate
The OD was then read at 450 nm, and the blank value was subtracted to calculate the final OD value.
Results and discussion
Preliminary confirmation analysis with SEC-MALS
Pre- and post-fusion RSV protein characterization was performed using size-exclusion chromatography connected to a MALS detector. Discussed here are the typical experimental results and key observations that may be helpful in identifying unwanted protein structural rearrangements.
Based on the SEC-MALS data, differences can be identified that may be interpreted as a structural rearrangement, as shown in Figure 2.
Firstly, there is a shift in peak retention time towards the right for the post-F form under identical experimental conditions. This underrepresentation of the actual molecular weight, excluding instrumentation errors, can be attributed to a conformational difference.
As most SEC column calibrations employ globular-shaped proteins, the non-globular, oblong shape of the post-F form produces unexpected results. The pipe-like protein shape increases the number of protein-pore interactions, eluting later than expected.
Figure 2. Chromatogram collected through SEC-MALS using standard solutions of pre-F and post-F RSV proteins. Retention time (RT) information is collected from UV detector, while molecular weight (MW) is collected from MALS. Image Credit: ACROBiosystems
Table 6. Molecular Analysis Results of Pre-F and Post-F RSV Protein. Source: ACROBiosystems
| |
Pre-fusion RSV |
Post-fusion RSV |
| Theoretical MW |
56.1 kDa |
55.2 kDa |
| SDS-PAGE |
60-65 kDa |
47-50 kDa |
| SEC-MALS (Native, Trimer) |
175-195 kDa |
148-182 kDa↓ |
| Peak Retention Time (TUV) |
13.09 min |
14.04 min↑ |
However, these analytical methods are a ‘quick & dirty’ evaluation for protein conformation. The method presented herein provides only a preliminary evaluation, utilizing non-resource-intensive methods and common laboratory instrumentation.
Protein conformation verification with ELISA
To confirm and verify protein confirmation, use of site-specific antibodies is recommended. Based on previous research, there are seven different antigenic sites that have been targeted through commercial antibodies, many of which are conserved in both pre-F and post-F forms.
By employing different anti-fusion antibodies that bind to either both or only pre-F forms, clear results verifying the protein conformation can be identified.
Table 1. RSV F protein antigenic sites, antibodies, and pre-/post-fusion binding. Source: ACROBiosystems
| Site |
Antibody |
Pre-F |
Post-F |
| Ø |
D25, 5C4, AM22 |
Binding |
No binding |
| I |
131-2a, 2F |
Binding |
Binding |
| II |
Palivizumab,
Motavizumab |
Binding |
Binding |
| III |
AM14 |
Binding |
Weak binding |
| IV |
101F, mAb19 |
Binding |
Binding |
| V |
hRSV90 |
Binding |
No binding |
Quaternary-
dependent (QD) |
AM14 |
Binding |
No binding |
Four different site-specific anti-fusion antibodies were selected (101F, 5C4, 131-2A, and AM14), of which 5C4 and AM14 were expected and verified not to bind to the post-F forms, due to the non-exposed epitopes (site Ø and QD) on the protein surface.
ELISA offers the best results by directly identifying whether site-specific antibodies have been bound or not. However, these methodologies are heavily dependent on the availability and laboratory capabilities in generating and validating these site-specific antibodies.
Figure 3. (Red) Immobilized HRSV Post-fusion glycoprotein F0, His Tag (Cat. No. RSF-V52H6) and (Blue) HRSV Pre-fusion glycoprotein F0, His Tag (Cat. No. RSF-V52H7)is coated onto each well at 1 μg/mL (100 μL/well) and exposed to (A) 101F, (B) 5C4, (C)131-2A, and (D) AM14 anti-fusion protein antibodies and detected using the relevant peroxidase-conjugated antibodies. Image Credit: ACROBiosystems
In most cases, in-depth conformation validation characterization studies is not required. However, it may be a vital troubleshooting component to ensure accurate and comprehensive assessments.
Conclusions
The identification of two structural conformations of the RSV F proteins represents a significant breakthrough in the development of an effective RSV vaccine and drug for at-risk populations.
These findings have been implemented by GSK and Pfizer to utilize the pre-F RSV protein as their immunogen for vaccine research. The availability of high purity, conformationally verified proteins is an important first step for vaccine development research combating the burden of RSV disease.
Acknowledgments
Produced from materials originally authored by Mingzhu Jia, Jie Zhang, Cynthia Qin and Spencer Chiang from ACROBiosystems.
References and further reading
- Shi, T., et al.. (2017). Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modeling study. The Lancet, [online] 390(10098), pp.946–958. https://doi.org/10.1016/S0140-6736(17)30938-8.
- McLellan, J.S., Chen, M., Leung, S., Graepel, K.W., Du, X., Yang, Y., Zhou, T., Baxa, U., Yasuda, E., Beaumont, T., Kumar, A., Modjarrad, K., Zheng, Z., Zhao, M., Xia, N., Kwong, P.D. and Graham, B.S. (2013). Structure of RSV Fusion Glycoprotein Trimer Bound to a Prefusion-Specific Neutralizing Antibody. Science, 340(6136), pp.1113–1117. https://doi.org/10.1126/science.1234914.
- Gilman, M.S.A., et al.. (2015). Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein. PLoS pathogens, [online] 11(7), p.e1005035. https://doi.org/10.1371/journal.ppat.1005035.
- Goodwin, E.C., et al.. (2018). Infants Infected with Respiratory Syncytial Virus Generate Potent Neutralizing Antibodies that Lack Somatic Hypermutation. Immunity, 48(2), pp.339-349.e5. https://doi.org/10.1016/j.immuni.2018.01.005.
About ACROBiosystems
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