Design and characterization of a germ-line targeting soluble, native-like, trimeric HIV-1 Env lacking key glycans from the V1V2-loop
Graphical abstract
Introduction
The HIV-1 envelope glycoprotein (Env) is a trimer of heterodimers of gp120 and gp41 that are non-covalently associated and mediate both receptor attachment and entry into the host cell. It is also the sole antigen present on the virion surface and is the target for neutralizing antibodies. The HIV-1 Env spike is shielded from potentially neutralizing antibodies by extensive sequence variation [1], conformational masking [2] and glycan shielding [3]. Glycans also play an important role in viral assembly, infectivity, Env folding and in modulating the immune response [4]. Atomic-level structures of fully glycosylated Env trimers reveal the presence of complex oliogosaccharides which extend as prominent ridges of interlocking oligomannose to shield the underlying polypeptide surface from access to B cells and antibodies [5,6]. Antibody selection pressure also induces host-derived N-glycan masking to occlude conserved neutralizing determinants to generate escape mutants. Nevertheless, in some subjects, regardless of these viral evasion strategies, the host immune system is capable of generating neutralizing antibody (NAb) responses during natural HIV-1 infection [7]. The induction of naïve B cells, through B cell receptors (BCRs), initiate a particular antibody lineage of an unmutated germ-line antibody, which over time, leads to broadly neutralizing antibodies (bNAbs), through affinity maturation by somatic hyper mutations (SHMs) of the complementarity determining regions (CDRs). It may be essential to imitate the same naïve B cell induction followed by affinity maturation of antibodies to successfully generate bNAbs against HIV-1 by vaccination. Therefore, the designed Env-based immunogens should be capable of binding germ-line antibodies that have the potential to evolve into bNAbs.
The critical epitopes on Env, which are recognized and targeted by bNAbs are mostly conformational in nature and highly dependent on quaternary packing of the Env [8]. Many of these antibodies favourably accommodate the glycans for high affinity binding [9,10]. A HIV-1 Env subunit-based immunogen in soluble, native-like trimeric conformation, with close resemblance to the structure of the Env protein as present on viral membrane, is expected to elicit neutralizing antibodies against HIV-1 Env. However, such Envs, designed from multiple sources, did not elicit reasonable breadth and potency of bNAbs in animal immunization studies [[11], [12], [13], [14]]. This may be due to the presence of extensive glycan shielding that has evolved to protect the immunogenic patches from access to antibodies and therefore hinders priming of B cells. In spite of this bNAbs make favourable contacts with some glycans and thereby reduces entropy. On the other hand unfavorable glycans hinder access to the epitope.
An alternate approach to design Env based immunogens is to work around the glycan shield, particularly for unfavorable glycans, by creating glycan holes at potentially immunogenic locations or by introducing glycans as glycan mask [[15], [16], [17], [18]] at highly immunogenic but non-neutralizing patches in order to divert elicitation of antibodies only towards the desired site and thereby modulate desired epitope accessibility. Immunogens designed with targeted glycan deletion or glycan masking are expected to improve site-selective immunogenicity with better potential to improve site-directed priming and increase polyclonal frequency of specific antibody lineages. Subsequent boosting with wild-type Env will help in affinity maturation of the induced B-cells and produce bNAbs with reasonable titer and breadth. Immunogens designed with glycans removed from the peripheral region of CD4 binding site (CD4bs) results in increased sensitivity to neutralization against CD4bs directed antibodies such as VRC01 and also increases the affinity for germ-line antibodies directed against CD4bs [15,[19], [20], [21]]. Naturally occurring Envs from HIV-1 isolates that lack the glycosylation sites at position N130 and within the V2 region are more sensitive to V1V2 apex-directed germ-line antibodies [22]. Sequential or combinatorial immunization with glycan-altered Env along with original Env have resulted in better induction of antibodies in animal immunization studies compared to when immunized with only natural variants of Env [15,21]. These studies indicate that properly designed native-like Env trimers with altered glycans can improve antibody induction.
In the trimer context, the V1V2-loop is located at the trimer apex and forms the top layer of the Env complex. The trimer apex is structurally conserved motif and immunogenic that elicits well characterized bNAbs in naturally infected patients [23]. The apex region contains several glycans including the two conserved glycans at position N160 and N156. The N160 and N156 facilitate the binding of trimer apex directed antibodies, but the other glycans are mostly random and may occlude the epitope for apex directed antibodies as seen for other epitopes on HIV-1 Env [16,24]. A key problem is our limited understanding of the role of glycans in maintaining the trimeric structure of Env. The Env structure is highly metastable and it has been shown previously that some glycans play a critical role in maintaining the overall structure. Removal of glycans can grossly impact overall structure or may cause microscopic perturbation of local structures [25,26]. Successfully maintaining native-like trimeric conformation and immunogenicity varies with the source of the Env and the locations from which the glycans are removed or added, indicating more such designs need to be studied in order to better understand the role of glycans in maintaining trimeric conformation as well as their potential to induce neutralizing antibodies. So far only a handful of Envs from BG505, B41, 16055, 426c, CH505 and ZM106.9 strains have been conformationally stabilized with glycans removed around the CD4bs in soluble native-like trimeric form [15,21,27], but no designed Env with glycans removed from the V1V2 apex region from any strain has been reported.
In this work we selected an Indian clade C (4-2.J41) (GenBank ID: GU945316) origin Env that has been previously stabilized in the soluble, native-like, trimeric conformation by domain swap with the well characterized clade B Env BG505 (GenBank ID: DQ208458) [28] in order to design a construct with N-glycans deleted from the V1V2-loop region. A foldon stabilized version of trimeric 4-2.J41 Env when used in DNA-prime protein-boost regime induces autologous and cross-clade reactive neutralizing antibodies suggesting the potential of 4-2.J41 Env as a possible immunogen candidate [29]. We hypothesised that modifying the 4-2.J41 Env with glycans deleted from the V1V2 region will help in inducing apex directed antibodies. Here, we sought to determine how targeted deletion of N-glycans from the V1V2 apex region impacts the structural stability and functional integrity of native-like trimeric conformation of 4-2.J41 and how that influences their antigenicity towards broadly neutralizing as well as V1V2-directed germ-line antibodies.
Section snippets
Structure modelling
For structural modelling the amino acid sequence of 4-2.J41-Swapped Env was searched for best available model on SWISS-MODEL homology modelling server for closest matched Env structure available in the PDB database.The modelled structure was energy minimized using CHARMM force field through Discovery Studio 3.5 (Accelrys Inc.) [30]. Energy minimization was carried out using 1000 steps of the steepest descent algorithm followed by 1000 steps of the conjugate gradient algorithm. The minimization
Design and modelling:
In order to generate an Env construct with glycans deleted from the V1V2-loop region of 4-2.J41 Env, the three N-linked glycosylation sites were collectively removed by mutating the N residues to Q. The glycan deletion mutations were introduced in the background of DC4-2.J41.gp41(BG505) Env construct that has been stabilized by swapping of gp41 region from BG505 on 4-2.J41 Env along with the SOSIP and I201C/A433C mutations and has been described elsewhere [28]. Henceforth, the parental
Discussion
The trimeric Env as present on the viral surface has primarily five prominent epitopes that are targeted by bNAbs isolated from infected individuals. These sites include the glycan-containing apex regions of V1V2, CD4-binding site, the V3 region around the N332 glycan, the gp120-gp41 interface and the membrane-proximal external region (MPER) on gp41 [[43], [44], [45], [46]]. The V1V2 epitope region located at the trimer apex consists of variable loops 1 and 2. The V1V2-loop varies greatly in
Declaration of Competing Interest
None
Acknowledgment
We thank Prof. Rogier W. Sanders, University of Amsterdam, The Netherlands for the germ-line antibody clones as kind gift. We thank the Neutralizing Antibody Consortium and the NIH AIDS Research and Reference Reagent Program for providing monoclonal antibodies. We would also like to thank Dr. Gagandeep Kang, Executive Director, Translational Health Science and Technology Institute, Faridabad, India, Dr. Bimal K. Chakrabarti, Advanced Bioscience Laboratories Inc. Rockville, Maryland, USA, and
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