Abstract
The display of native-like human immunodeficiency virus type 1 envelope (HIV-1 Env) trimers on liposomes has gained wide attention over the last few years. Currently, available methods have enabled the preparation of Env-liposome conjugates of unprecedented quality. However, these protocols require the Env trimer to be tagged and/or to carry a specific functional group. For this reason, we have investigated N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide/N-Hydroxysulfosuccinimide (EDC/Sulfo-NHS) chemistry for its potential to covalently conjugate tag-free, non-functionalized native-like Env trimers onto the surface of carboxyl-functionalized liposomes. The preservation of the liposome’s physical integrity and the immunogen’s conformation required a fine-tuned two-step approach based on the controlled use of β-mercaptoethanol. The display of Env trimers was strictly limited to activated liposomes of positive charge, i.e., liposomes with a positive zeta potential that carry amine-reactive Sulfo-NHS esters on their surface. In agreement with that, conjugation was found to be highly ionic strength- and pH-dependent. Overall, we have identified electrostatic pre-concentration (i.e., close proximity between negatively charged Env trimers and positively charged liposomes established through electrostatic attraction) to be crucial for conjugation reactions to proceed. The present study highlights the requirements and limitations of potentially scalable EDC/Sulfo-NHS-based approaches and represents a solid basis for further research into the controlled conjugation of tag-free, non-functionalized native-like Env trimers on the surface of liposomes, and other nanoparticles.
Highlights
The advent of native-like Env trimers has substantially contributed to the ongoing efforts to develop prophylactic vaccines against HIV-1 [1]
Excess ethylcarbodiimide hydrochloride (EDC) has to be removed before initiation of the conjugation reaction as it will otherwise induce intramolecular cross-linking of the Env trimers
The results indicate that tag-free conjugation onto cationic liposomes using EDC/Sulfo-NHS chemistry is applicable to UFO and SOSIP constructs, two representative state-of-the-art HIV-1 Env trimer constructs
Summary
The advent of native-like Env trimers has substantially contributed to the ongoing efforts to develop prophylactic vaccines against HIV-1 [1]. There is at least one approved protein-based therapeutic that comprises a His-tag (Blinatumomab), the use of conjugation techniques that rely on the use of a His-tag, might raise concerns and impede regulatory approval, especially as it has been repeatedly reported that Env-liposome conjugates prepared this way induce an anti-His immune response [11] With regard to their stability under physiological conditions, covalent Env-liposome conjugates are clearly superior [16,18]. It is a widely used zero-length crosslinker and reactions are described as proceeding with high efficiency With this in mind, we have decided to systematically investigate it for its potential to covalently couple native-like Env trimers on the surface of carboxyl-functionalised liposomes. We are confident that the present study provides a solid basis for us and others to continue working on this and similar potentially scalable, tag-free approaches for the manufacturing of Env-liposome conjugates
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