Neutralizing Antibody Induction by HIV-1 Envelope Glycoprotein SOSIP Trimers on Iron Oxide Nanoparticles May Be Impaired by Mannose Binding Lectin

Abstract

We covalently attached human immunodeficiency virus type 1 (HIV-1) Env SOSIP trimers to iron oxide nanoparticles (IO-NPs) to create a particulate immunogen for neutralizing antibody (NAb) induction. The attached trimers, ∼20 per particle, retained native-like antigenicity, judged by reactivity with NAbs and non-NAbs. Bivalent (BG505 and B41) trimer IO-NPs were made, as were IO-NPs displaying B41 trimers carrying a PADRE T-cell helper epitope (TCHE). We immunized mice with B41 soluble or IO-NP trimers after PADRE peptide priming. After two immunizations, IO-NP presentation and the TCHE tag independently and substantially increased anti-trimer antibody responses, but titer differences waned after two further doses. Notable and unexpected findings were that autologous NAbs to the N289 glycan hole epitope were consistently induced in mice given soluble but not IO-NP trimers. Various recombinant mannose binding lectins (MBLs) and MBLs in sera of both murine and human origin bound to soluble and IO-NP trimers. MBL binding occluded the autologous NAb epitope on the B41 IO-NP trimers, which may contribute to its poor immunogenicity. The exposure of a subset of broadly active NAb epitopes was also impaired by MBL binding, which could have substantial implications for the utility of trimer-bearing nanoparticles in general and perhaps also for soluble Env proteins. IMPORTANCE Recombinant trimeric SOSIP proteins are vaccine components intended to induce neutralizing antibodies (NAbs) that prevent cells from infection by human immunodeficiency virus type 1 (HIV-1). A way to increase the strength of antibody responses to these proteins is to present them on the surface of nanoparticles (NPs). We chemically attached about 20 SOSIP trimers to NPs made of iron oxide (IO). The resulting IO-NP trimers had appropriate properties when we studied them in the laboratory but, unexpectedly, were less able to induce NAbs than nonattached trimers when used to immunize mice. We found that mannose binding lectins, proteins naturally present in the serum of mice and other animals, bound strongly to the soluble and IO-NP trimers, blocking access to antibody epitopes in a way that may impede the development of NAb responses. These findings should influence how trimer-bearing NPs of various designs are made and used.

Conjugation of Native-Like HIV-1 Envelope Trimers onto Liposomes Using EDC/Sulfo-NHS Chemistry: Requirements and Limitations

“…Also in agreement with our observations, Pejawar-Gaddy et al have reported that the inclusion of DOTAP to double-functionalised, DOPC-based interbilayer-cross-linked multilamellar vesicles (ICMVs) improved the conjugation efficiency of Env trimers from around 5% to slightly above 20% [ 17 ]. In stark contrast to our findings, Ringe et al showed that Env trimers can be coupled to the surface of negatively charged nanoparticles using EDC/Sulfo-NHS chemistry [ 32 ]. The relatively high Env trimer:nanoparticle ratio and high Env trimer bulk concentration may have contributed to the higher number of displayed Env trimers.…”

“…With regard to the oriented display, homology models and calculations/estimations of the degree of deprotonation of the Env trimer’s primary amines, suggest that not all primary amines are equally accessible and nucleophilic/reactive (unpublished observations). In concordance with this, recently published homology models of two SOSIP trimers indicate that most lysine residues at the apex of the Env trimer are shielded by glycans [ 32 ]. However, many lysine residues on the glycan-free base appear accessible.…”

“…However, many lysine residues on the glycan-free base appear accessible. Ringe et al found that tag-free, non-functionalised Env trimers coupled to iron oxide nanoparticles using EDC/Sulfo-NHS, induce reduced anti-base non-nAb responses when compared to soluble Env trimers [ 32 ]. Based on these observations, they concluded that the Env trimers must have been displayed in a (partly) oriented fashion that occludes the immunodistractive base.…”

“…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. EDC/Sulfo-NHS-based conjugation to carboxyl-functionalised nanoparticles has been described for many other proteins and peptides, but was only recently described for the first time within the context of the particulate display of native-like Env trimers [ 29 , 30 , 31 , 32 ].…”

Tailored Design of Protein Nanoparticle Scaffolds for Multivalent Presentation of Viral Glycoprotein Antigens

“…While previous studies of nanoparticle-displayed HIV-1 Env trimers have demonstrated enhanced immunogenicity39 , the effects are often modest compared to those observed for other antigens1,2,18,36 . While there may exist intrinsic peculiarities to HIV-1 Env that limit increases in Ab responses upon multivalent display40,41 , limitations associated with epitope inaccessibility caused by crowding of the trimers on nanoparticle surfaces have also been identified19 . This observation strongly motivates the exploration of antigen presentation geometry through the custom design of nanoparticle scaffolds to most effectively elicit the desired immune response.…”