Antigenicity and immunogenicity of HIV-1 gp140 with different combinations of glycan mutation and V1/V2 region or V3 crown deletion
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.
Section snippets
Cells
Cell lines 293 T and TZM-bl were obtained from the American Type Culture Collection (ATCC) and NIH AIDS Reagent Program, respectively. Cells were grown in Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and penicillin-streptomycin (both at 100 U/ml) at 37 °C with 5% CO2. The FreeStyle™ 293-F cell line, purchased from Thermo Fisher Scientific, was grown in FreeStyle™ 293 Expression Medium in a shaking incubator at 37 °C with 5% CO2.
Env constructs
Human codon-optimized CN54 (Clade CRF_07
Production of homogeneous gp140 trimers.
Based on the trimeric CRF_07 BC CN54 gp140-expressing plasmid (gp1) as previously reported [14], [15], [22], [23], [24] (Fig. 1A), we designed and constructed a series of plasmids with N276D/N463D glycan mutations, V1V2 region and V3 crown deletion, alone or in combination, named gp2, gp3, gp4, C1, C2, C3, C4, D1, D2, D3 and D4, respectively (Fig. 1A and 1B). The flow chart for construction of the above plasmids is shown in Fig. 1B. The expression of the constructs was analyzed by transient
Discussion
Although bNAbs are believed to have the potential to provide complete protection against HIV-1 infection, there are considerable challenges in inducing such antibodies by vaccination. Given that the conserved epitopes which bNAbs bind to are masked in native Env [25], a number of studies have been focusing on HIV-1 Env modification to induce bNAbs in vivo [4], [12], [26]. Previous studies have showed that removal of two conserved glycosylation sites N276 or N463, located in loop D and variable
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the National Mega-Projects against Infectious Diseases (2018ZX10301406-002 and 2018ZX10301405-003), the National Natural Science Foundation of China (81772192), and the Hotung Trust. We thank Ding Gao at the Core Facility and Technical Support, Wuhan Institute of Virology, for his technical support with Bio-layer interferometry, and Xuefang An and Fan Zhang for their assistance with mouse immunizations.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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