Covalent Linkage of HIV-1 Trimers to Synthetic Liposomes Elicits Improved B Cell and Antibody Responses

Abstract

We have demonstrated that a liposomal array of well-ordered trimers enhances B cell activation, germinal center formation, and the elicitation of tier-2 autologous neutralizing antibodies. Previously, we coupled well-ordered cleavageindependent NFL trimers via their C-terminal polyhistidine tails to nickel lipids integrated into the lipid bilayer. Despite favorable in vivo effects, concern remained over the potentially longer-term in vivo instability of noncovalent linkage of the trimers to the liposomes. Accordingly, we tested both cobalt coupling and covalent linkage of the trimers to the liposomes by reengineering the polyhistidine tail to include a free cysteine on each protomer of model BG505 NFL trimers to allow covalent linkage. Both cobalt and cysteine coupling resulted in a high-density array of NFL trimers that was stable in both 20% mouse serum and 100 mM EDTA, whereas the nickel-conjugated trimers were not stable under these conditions. Binding analysis and calcium flux with antiEnv-specific B cells confirmed that the trimers maintained conformational integrity following coupling. Following immunization of mice, serologic analysis demonstrated that the covalently coupled trimers elicited Env-directed antibodies in a manner statistically significantly improved compared to soluble trimers and nickel-conjugated trimers. Importantly, the covalent coupling not only enhanced gp120-directed responses compared to soluble trimers, it also completely eliminated antibodies directed to the C-terminal His tag located at the "bottom" of the spike. In contrast, soluble and noncovalent formats efficiently elicited anti-His tag antibodies. These data indicate that covalent linkage of well-ordered trimers to liposomes in highdensity array displays multiple advantages in vitro and in vivo.IMPORTANCE Enveloped viruses typically encode a surface-bound glycoprotein that mediates viral entry into host cells and is a primary target for vaccine design. Liposomes with modified lipid head groups have a unique feature of capturing and displaying antigens on their surfaces, mimicking the native pathogens. Our firstgeneration nickel-based liposomes captured HIV-1 Env glycoprotein trimers via a noncovalent linkage with improved efficacy over soluble glycoprotein in activating germinal center B cells and eliciting tier-2 autologous neutralizing antibodies. In this study, we report the development of second-generation cobalt-and maleimidebased liposomes that have improved in vitro stability over nickel-based liposomes. In particular, the maleimide liposomes captured HIV-1 Env trimers via a more stable covalent bond, resulting in enhanced germinal center B cell responses that generated higher antibody titers than the soluble trimers and liposome-bearing trimers via noncovalent linkages. We further demonstrate that covalent coupling prevents release of the trimers prior to recognition by B cells and masks a nonneutralizing determinant located at the bottom of the trimer.

Strategies for inducing effective neutralizing antibody responses against HIV-1

“…However, it was found that NiNTA-coupled trimers completely dissociate in less than one day [197]. Cobalt coupling and NiNTA-assisted covalent coupling by terminal cysteines [198] have allowed more stable immobilization of Env trimers. These liposomal nanoparticles induced increased Env-specific binding responses in mice [197,198].…”

“…Cobalt coupling and NiNTA-assisted covalent coupling by terminal cysteines [198] have allowed more stable immobilization of Env trimers. These liposomal nanoparticles induced increased Env-specific binding responses in mice [197,198]. Future studies will teach us whether similar approaches can also be used in animals that induce NAbs [199].…”

Presentation of HIV-1 Envelope Trimers on the Surface of Silica Nanoparticles

“…For instance, Env trimers have been arrayed on the surface of functionalized 150 nm liposomes via covalent and noncovalent coupling. 17 The particle-based formulations demonstrated clear advantages over the soluble trimers regarding the induction of germinal centers, T-follicular helper cells, and trimer-specific antibody responses. 11,17 However, stability concerns arose regarding liposomal carriers, which suffer from coalescence, lipid exchange, oxidation, and hydrolysis within biological media.…”

Modulating the immune system through nanotechnology

“…This new composition resulted in Th1/Th2 balanced profiles and increased titers against the antigens [180]. A similar strategy based on displaying HIV trimers on the liposomes surface in order to target B cells has been adopted by other authors, showing positive results in terms of neutralizing antibodies responses [181–183]. On the other hand, Hanson et al co-administered two liposomal formulations, one of them displaying an Env-derived peptide and encapsulating a T-helper peptide, and another one loaded with cyclic di-GMP.…”