Antigenicity and immunogenicity of HIV-1 gp140 with different combinations of glycan mutation and V1/V2 region or V3 crown deletion

Abstract

The carbohydrate moieties on HIV-1 envelope glycoprotein (Env) act as shields to mask conserved neutralizing epitopes, while the hyperimmunogenic variable regions are immunodominant in inducing non-neutralizing antibodies, representing the major challenge for using Env as a vaccine candidate to induce broadly neutralizing antibodies (bNAbs). In this study, we designed a series of HIV-1 gp140 constructs with the removal of N276/N463 glycans, deletion of the V1/V2 region and the V3 crown, alone or in combination. We first demonstrated that all the constructs had a comparable level of expression and were mainly expressed as trimers. Following purification of gp140s from mammalian cells, we measured their binding to bNAbs and non-NAbs in vitro and capability in inducing bNAbs in vivo. Antibody binding assay showed that removal of N276/N463 glycans together with the deletion of V1/V2 region enhanced the binding of gp140s to CD4-binding site-targeting bNAbs VRC01 and 3BNC117, and CD4-induced epitopes-targeting non-NAbs A32, 17b and F425 A1g8, whereas further deletion of V3 crown in the gp140 mutants demonstrated slightly compromised binding capability to these Abs. Immunogenicity study showed that the above mutations did not lead to the induction of a higher Env-specific IgG response via either DNA-DNA or DNA-protein prime-boost strategies in mice, while neutralization assay did not show an apparent difference between wild type and mutated gp140s. Taken together, our results indicate that removal of glycans at N276/N463 and deletion of the V1/V2 region can expose the CD4-binding site and CD4-induced epitopes, but such exposure alone appears incapable of enhancing the induction of bNAbs in mice, informing that additional modification or/and immunization strategies are needed. In addition, the strategies which we established for producing gp140 proteins and for analyzing the antigenicity and immunogenicity of gp140 provide useful means for further vaccine design and assessment.

Introduction

Vaccines are believed to be the ideal strategy to prevent infectious diseases, but an effective vaccine against HIV-1 remains elusive. As the key component in viral entry, HIV-1 envelope glycoprotein (Env) represents the primary candidate for vaccine design. However, attempts using Env-based immunogens to induce broadly neutralizing antibodies (bNAbs) against main circulating HIV-1 strains have not been successful in various models [1], [2], whereas such bNAbs have been continuously identified in HIV-1 infected individuals including elite controllers [3]. With the advances in understanding Env structure and recognition epitopes of the identified bNAbs, it is believed that Env-based immunogens, once appropriately optimized, may be able to elicit bNAbs in vivo.

Env is first synthesized as a precursor gp160, and then cleaved into noncovalently associated gp120 and gp41 on the viral membrane as heterogenous trimers. Removal of the cleavage site by mutation generates trimeric gp160 while introducing a stop codon to the end of gp41 ectodomain can yield soluble gp140 [4]. The trimeric form of Env is believed to be a better vaccine candidate than the corresponding gp120 monomer because non-neutralizing epitopes exposed in the monomer are shielded in the trimer. [5]. The surface unit gp120 can be divided into 5 conserved (C1-C5) and 5 variable (V1-V5) regions. In general, conserved regions are believed to be crucial for viral fitness and therefore remain relatively constant across strains. Conserved regions are usually shielded by glycosylation which prevents these sites from recognition by the host immune system. By contrast, variable regions are more dispensable for viral entry and can vary significantly between different strains without severe impact on viral fitness. Furthermore, variable regions are prone to be immunodominant and the immune responses diverted to these regions are usually non-neutralizing, representing one of the mechanisms that the virus evades the immune system.

Given the importance of glycosylation and variable regions on viral immune evasion, removal of glycans or/and variable regions of HIV-1 Env may represent a feasible approach to induce broadly neutralizing immune responses. We and others previously found that removal of certain specific glycans near CD4-binding site (CD4-BS) on Env not only rendered HIV-1 more sensitive to bNAbs, but also enhanced bNAb induction in animals [6], [7], [8], [9]. More importantly, a recent study reported that removal of two conserved glycans at N276 and N463 could allow Env-binding to, and activation of, B cells expressing the germline-reverted BCRs of two potent bNAbs VRC01 and NIH45-46 targeting CD4-binding site [10], [11]. In addition, genetic removal of the V1/V2 loop was shown to associate with enhanced neutralization of virus by antibodies targeting CD4-binding site and CD4-induced epitopes (CD4i) that overlap with the conserved coreceptor binding site in the bridging sheet [12]. In an HIV-1 gp140 vaccine study, a predominant anti-V3 non-neutralizing IgG response was observed, indicating that V3 may play an important role in HIV-1 immune evasion [13].

In this study, we designed, expressed and purified gp140s bearing N276/N463 glycan mutations, V1V2 and V3 deletion, alone or in combination. We compared the antigenicity and immunogenicity of the gp140s using both in vitro and in vivo assays.