Optimizing the Production of gp145, an HIV-1 Envelope Glycoprotein Vaccine Candidate and Its Encapsulation in Guanosine Microparticles
Abstract
:1. Introduction
2. Materials and Methods
2.1. CHO-K1 Cell Line Expressing gp145
2.2. Upstream and Downstream Strategies to Increase gp145 Titers
2.3. Titer Determination and Hydrophobic Footprint Analysis Using Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC)
2.4. Host Cell Protein (HCP) Determination and Binding Affinity Analysis Using Bio-Layer Interferometry
2.5. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
2.6. MALDI-TOF Mass Spectrometry
2.7. Mass Analysis
2.8. Glycoform Profiling by Imaged Capillary Isoelectric Focusing (cIEF)
2.9. Size Distribution Analysis by Dynamic Light Scattering (DLS)
2.10. Secondary Structure Analysis by Circular Dichroism
2.11. Synthesis of Supramolecular Hacky Sacks (SHS)
2.12. Fluorescent Labeling of gp145
2.13. Preparation of the gp145-FITC-SHS Complex and VRC01-Alexa Fluor 647 Complex
2.14. Flow Cytometry
2.15. Confocal Imaging
3. Results
3.1. Optimizing the Expression of gp145
3.2. Analysis of Whole gp145 by SDS PAGE and MALDI-TOF Mass Spectrometry
3.3. Glycan Analysis
3.4. Biochemical Analysis of gp145 by Capillary Isoelectric Focusing (cIEF) and RP-HPLC
3.5. Oligomeric State of gp145
3.6. Native-Like Properties of gp145
3.7. Encapsulation of gp145 for Delivery
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Code Availability
Acknowledgments
Conflicts of Interest
References
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Akamine, P.; González-Feliciano, J.A.; Almodóvar, R.; Morell, G.; Rivera, J.; Capó-Vélez, C.M.; Delgado-Vélez, M.; Prieto-Costas, L.; Madera, B.; Eichinger, D.; et al. Optimizing the Production of gp145, an HIV-1 Envelope Glycoprotein Vaccine Candidate and Its Encapsulation in Guanosine Microparticles. Vaccines 2023, 11, 975. https://doi.org/10.3390/vaccines11050975
Akamine P, González-Feliciano JA, Almodóvar R, Morell G, Rivera J, Capó-Vélez CM, Delgado-Vélez M, Prieto-Costas L, Madera B, Eichinger D, et al. Optimizing the Production of gp145, an HIV-1 Envelope Glycoprotein Vaccine Candidate and Its Encapsulation in Guanosine Microparticles. Vaccines. 2023; 11(5):975. https://doi.org/10.3390/vaccines11050975
Chicago/Turabian StyleAkamine, Pearl, José A. González-Feliciano, Ruth Almodóvar, Gloriner Morell, Javier Rivera, Coral M. Capó-Vélez, Manuel Delgado-Vélez, Luis Prieto-Costas, Bismark Madera, Daniel Eichinger, and et al. 2023. "Optimizing the Production of gp145, an HIV-1 Envelope Glycoprotein Vaccine Candidate and Its Encapsulation in Guanosine Microparticles" Vaccines 11, no. 5: 975. https://doi.org/10.3390/vaccines11050975
APA StyleAkamine, P., González-Feliciano, J. A., Almodóvar, R., Morell, G., Rivera, J., Capó-Vélez, C. M., Delgado-Vélez, M., Prieto-Costas, L., Madera, B., Eichinger, D., Pino, I., Rivera, J. H., Ortiz-Ubarri, J., Rivera, J. M., Baerga-Ortiz, A., & Lasalde-Dominicci, J. A. (2023). Optimizing the Production of gp145, an HIV-1 Envelope Glycoprotein Vaccine Candidate and Its Encapsulation in Guanosine Microparticles. Vaccines, 11(5), 975. https://doi.org/10.3390/vaccines11050975