The extensive post-translational modifications of the envelope spikes of the human immunodeficiency virus (HIV) present considerable challenges and opportunities for HIV vaccine design. These oligomeric glycoproteins typically have over 30 disulfide bonds and around a 100 N-linked glycosylation sites, and are functionally dependent on protease cleavage within the secretory system. The resulting mature structure adopts a compact fold with the vast majority of its surface obscured by a protective shield of glycans which can be targeted by broadly neutralizing antibodies (bnAbs). Despite the notorious heterogeneity of glycosylation, rare B-cell lineages can evolve to utilize and cope with viral glycan diversity, and these structures therefore present promising targets for vaccine design. The latest generation of recombinant envelope spike mimetics contains re-engineered post-translational modifications to present stable antigens to guide the development of bnAbs by vaccination.

Introduction

The immense variation of the genome of HIV presents a considerable obstacle to the development of an effective, sterilizing vaccine. The failure of classical vaccination strategies has driven advances in our understanding of the molecular basis of viral infectivity and the host immune response. Lines of enquiry have included investigating correlates of protection arising from the RV144 trial such as the role of non-neutralizing antibodies [13]. Here, however, we focus on vaccination strategies aiming to develop immunogens capable of eliciting a protective broadly neutralizing antibody (bnAb) response.

Innovations towards the development of bnAb-eliciting immunogens have included the production of native-like trimers that mimic the envelope (Env) glycoprotein spikes expressed on the surface of the virion [47]. These soluble glycoproteins have revolutionized our understanding of the viral glycoprotein structure and have stimulated the design of new immunogens. Encouragingly, these mimetics bind to a growing number of bnAbs isolated from infected patients, and they are being investigated as a platform for the next generation of immunogens. Armed with the detailed structure of the envelope spike mimetics, a detailed knowledge of the intricate network of post-translational modifications has been revealed [813]. Exploiting and targeting the post-translational modifications of the viral spike has enabled the design of improved immunogens that are able to elicit protective neutralizing antibodies against a narrow but growing range of viral isolates [1416].

Engineering the envelope spike

Env is the sole viral protein expressed on the surface of the HIV virion and antibodies capable of binding the functional form of the viral spike can effectively neutralize viral infectivity [1723]. As a class I viral fusion glycoprotein, Env consists of a trimer of heterodimers comprising gp120, which consists of five constant domains interspersed with hypervariable loops (Figure 1A), responsible for receptor recognition and the transmembrane fusion glycoprotein, gp41. Env enables viral particle recognition of CD4+ T-cells, binding to the chemokine co-receptor, and ultimately fusion of the viral membrane with that of the target cell. To generate infectious viral particles, the viral spike must be extensively post-translationally modified by the host cell. These modifications include the formation of disulfide bonds (Figure 1B), extensive N-linked glycosylation (Figure 1C,D) and two proteolytic cleavage events, one after the signal peptide and the other between gp120 and gp41 (Figure 1E) [27].

Representation of the post-translational modifications of BG505 clade A envelope glycoprotein.

Figure 1.
Representation of the post-translational modifications of BG505 clade A envelope glycoprotein.

Models were generated using the cryo-EM structure of BG505 SOSIP.664 PDB ID:5ACO [10]. cryo-EM, cryo-electron microscopy. (A) 3D representation of the variable loops on gp120 and the heptad repeats of gp41. (B) Canonical disulfide bonds found in BG505 [24] with the additional stabilizing disulfide bond found in BG505 SOSIP.664 shown in orange. (C) Conservation map of the glycans of BG505 SOSIP.664. The glycans were coloured according to their conservation across 4000 Env strains, Huang et al. [25]. BG505 crystal structure with N-linked glycans modelled by Behrens et al. [26]. (D) The frequency of potential N-linked glycan sites across the Env sequence with the PNGs found in BG505 SOSIP.664 labelled on the X-axis. PNG, potential N-glycosylation site. (E) Schematic showing the locations of proteolytic cleavage for the signal peptide and the furin cleavage site.

Figure 1.
Representation of the post-translational modifications of BG505 clade A envelope glycoprotein.

Models were generated using the cryo-EM structure of BG505 SOSIP.664 PDB ID:5ACO [10]. cryo-EM, cryo-electron microscopy. (A) 3D representation of the variable loops on gp120 and the heptad repeats of gp41. (B) Canonical disulfide bonds found in BG505 [24] with the additional stabilizing disulfide bond found in BG505 SOSIP.664 shown in orange. (C) Conservation map of the glycans of BG505 SOSIP.664. The glycans were coloured according to their conservation across 4000 Env strains, Huang et al. [25]. BG505 crystal structure with N-linked glycans modelled by Behrens et al. [26]. (D) The frequency of potential N-linked glycan sites across the Env sequence with the PNGs found in BG505 SOSIP.664 labelled on the X-axis. PNG, potential N-glycosylation site. (E) Schematic showing the locations of proteolytic cleavage for the signal peptide and the furin cleavage site.

Successful attempts to recombinantly mimic the Env-based epitopes of bnAbs have involved solubilizing and stabilizing glycoprotein trimers utilizing post-translational modifications and amino acid substitutions. Crucially, native-like Env trimers have been generated by the addition of a disulfide bond between gp120 and gp41 subunits and an isoleucine-to-proline mutation in gp41 which stabilize the prefusion conformation of Env. These immunogens are termed ‘SOSIPs’ [5,28,29]. These stable soluble mimetics are not in themselves sufficient to generate a broad and protective immune response. Strategies to improve the breadth and potency of the antibody response have included the design of immunogens to stimulate the precursor lineage of potent bnAbs, for example, by the targeted elimination of key glycans and the hyperstabilization of trimers through disulfide bond engineering [2832].

Disulfides in infection and hyperstabilization

The quaternary structure of natively folded viral spikes requires the correct formation of disulfide bonds in the mammalian secretory pathway. Slow folding events allow time for the extensive network of disulfide bonds to form and to ‘shuffle’ using protein disulfide isomerase (PDI) to ensure the optimal disulfide bond configuration is achieved [3335]. The correct pairing of disulfides is an essential requirement for infectious Env, as by knocking out PDIs in vitro the resultant protein is able to bind to CD4 but not to undergo the conformational changes required for membrane fusion [36]. The incorrect formation of disulfides is also promoted when viral spike mimetics remain uncleaved by furin, this not only affects the resultant quaternary structure of the viral spike but also perturbs the glycan shield and therefore bnAb binding [24].

With the structural characterization by X-ray crystallography and mass spectrometry using the BG505 SOSIP.664 construct, it has been possible to map the locations of the network of disulfide bonds [24,37]. As well as understanding the locations of the disulfide bonds in three-dimensional space, it is also possible to utilize the biophysical analysis to design next-generation hyperstabilized constructs that employ additional disulfide bonds to minimize the intrinsic flexibility and instability of Env. Torrents de la Peña et al. [28] describe additional mutations to the original BG505 SOSIP.664 of which the most prominent is the addition of an interprotomer gp120 disulfide bond. This effectively stabilizes the trimeric Env oligomeric state and also prevents the possibility of gp120 shedding. These modifications do not affect upon previously characterized bnAb binding and do not influence other post-translational modifications such as N-linked glycosylation and therefore demonstrate the ability to configure Env post-translational modifications for superior immunogen design [28].

Glycan engineering of germline targeting immunogens

In addition to their role in protein folding, the high density of N-linked glycans is thought to be driven by immune selection whereby glycans mask underlying conserved protein epitopes. This is evidenced by their accumulation during longitudinal infection and depletion during collapse of an effective adaptive immune response in late infection [3842]. The high density of glycans means that the evolution of the glycan shield can be understood as holes being formed and being filled rather than there being a continuum of options for glycan locations [43]. This is less so in the variable loops where there is very low conservation of glycan positions (Figure 1C,D) [25]. One interesting consequence of the trimeric structure of the viral spike is the role of conserved glycans at the protomer interface. Presumably, the interfacial glycans act to obscure important surface features and may help stabilize the trimeric structure [44]. The highly conserved N-glycan sites across the surface of Env also form epitopes for a range of bnAbs and with the characterization of one such bnAb that is able to recognize the highly conserved N262 and N448 glycans the entire surface of Env forms bnAb epitopes [45,46].

The conservation of potential N-linked glycan sites across the envelope spike far exceeds that of the protein surface and presents a robust platform for immunogen design [46,47]. In addition, the high density of glycosylation and the trimeric architecture places steric restrictions on glycan processing which drives the formation of a population of under-processed oligomannose-type glycans [8,26,4851]. However, the extensive heterogeneity inherent to N-linked glycan processing means that these steric constraints alone are not entirely sufficient for native-like glycosylation [48].

The producer cell of the Env spike, either viral or recombinant, can lead to changes in glycosylation that may affect on bnAb binding. In one extreme, when the glycans of macrophage-derived HIV particles are compared with those from peripheral blood mononuclear cells (PBMCs), there was a significant shift in the composition of the population of complex-type glycans with the macrophage-derived glycans exhibiting large polylactosamine structures [52]. Importantly, the viruses exhibited different sensitivities to antibody neutralization and this may be attributed to glycosylation.

Glycan heterogeneity is an important parameter when assessing recombinant Env spike mimetics. Such heterogeneity has been shown to contribute to partial neutralization by glycan-dependent bnAbs [5355]. Encouragingly, trimeric SOSIPs exhibit native-like levels of oligomannose-type glycans. However, there will be potentially important variations in glycan structure between recombinant immunogens and their viral counterparts. For example, between PBMC and HEK293T-derived gp120, there are subtle differences such as the sialic acid linkages of complex-type glycans, with PBMCs presenting a larger population of α2,6-linkages. Such differences between viral and recombinant glycosylation may result in diminished presentation of potential glycan epitopes to the immune system when recombinant constructs are used as immunogens [56]. Despite this caveat, promising immunogens are being developed that exploit viral glycosylation.

Despite the broad neutralization and high affinities of bnAbs to the viral spike, few patients develop bnAb lineages before the genetic diversity of the viral spike overwhelms the immune system. The original goal of mimicking the natively glycosylated viral spike has produced strong autologous responses, but has not managed to elicit bnAbs in macaques [15]. The focus of immunogen design has therefore shifted and now focuses on manipulating the post-translational modifications of Env to guide the immune system to mature the rare B-cell lineages of bnAbs. This approach has been greatly stimulated by the observation that patients who have eventually developed a potent bnAb response often have virus missing key glycan sites [57]. Many investigators are now examining immunogens displaying glycan holes [2932,58,59].

One such platform is the glycan-depleted trimer, termed BG505 SOSIP.v4.1-GT1, which has been designed to trigger B cells corresponding to germline PGT121 and germline VRC01. This trimer lacks 15 glycans across the viral spike and initiates antibody responses in knock-in mice expressing the predicted germlines for these bnAbs [60]. In addition, a comparative glycan analysis revealed that the remaining oligomannose-type glycans were largely unaffected by the glycan deletions with changes in their mannose trimming localized to regions proximal to a depleted glycan [61]. The overall integrity of the mannose patch is a promising observation and broadens the possibilities of using extensive glycan holes as a design feature of new immunogens.

It is also possible to harness the microheterogeneity of conserved glycan sites to present glycan epitopes that result in the proliferation of the rare B-cell lineages of bnAbs. In trimers derived from env sequences from a prolonged infection such as the clade A BG505 sequence used for BG505 SOSIP.664, the apex sites N160 and N156 display oligomannose glycans [26,49,50]. Andrabi et al. [62] demonstrated that bnAb precursors require sialic acid to bind to the apex viral spike and that Env sequences derived from early infection appear to have sialic acid glycans at N160 and N156. Although the bnAb precursors require sialic acid, the resultant mature bnAbs are able to bind to trimers containing apex sites that are well defined as oligomannose. These observations teach us that glycan heterogeneity can evolve during the course of infection and that this feature can potentially be exploited in vaccine design.

Optimizing and bypassing furin cleavage

Another critical post-translational modification of Env is furin cleavage of the gp160 pro-protein. Furin cleavage is thought to occur in the trans-Golgi network and is an essential step in the formation of functional correctly folded trimers. Furin cleaves between the gp120 and gp41 subunits of the gp160 polypeptide, recognizing the amino acid motif Arg-X-(Arg/Lys)-Arg [63]. Negative stain electron microscopy of uncleaved trimers, which have been explored as viral spike mimetics, revealed that they do not naturally adopt the classical trimeric architecture [56,64]. The perturbation of the structural integrity resulting from a lack of furin cleavage also has implications for the post-translational modifications of candidate immunogens. Analysis of N-linked glycosylation on a global and a site-specific level on many uncleaved structures described an elevation in glycan processing, most likely resulting from aberrant trimerization [8,51]. As bnAb epitopes frequently contain N-linked glycans, the aberrant glycosylation resulting from a lack of furin cleavage will have knock-on effects on the antigenicity of those trimers. Furthermore, the destabilization of the quaternary structure ablates the binding of quaternary-specific bnAbs such as PGT151 and also reduces the affinity of other quaternary-specific bnAbs such as PGT145 [65,66].

The requirement for furin cleavage for correct assembly is an important feature in the expression of BG505 SOSIP.664 as the low levels of endogenous furin result in large populations of uncleaved trimers. Binley et al. circumvented this problem by co-transfecting plasmid containing the furin gene concomitantly with BG505 SOSIP.664. In addition, it is important to optimize the protease cleavage step as much as possible. The majority of HIV strains present a furin cleavage site consisting of a REKR motif. A panel of mutations introduced to this region demonstrated that this is not the optimal motif for furin-mediated Env glycoprotein cleavage. By replacing the REKR motif with six arginine residues, the proteolytic separation of gp120 and gp41 is greatly improved [67]. By modifying the amino acid sequence in this way, it is possible to manipulate post-translational protease cleavage to allow for a larger amount of native-like material. Impressively, efficient furin cleavage has also now been achieved in production of clinical grade BG505 SOSIP.664 using a stable CHO cell line containing the target SOSIP, possessing an optimized furin cleavage site, and also the gene encoding furin [68].

As well as using recombinant glycoproteins as immunogens, a further strategy currently under investigation to boost the neutralizing antibody titre is to use DNA-based approaches. For DNA vaccines, the env gene is administered and the trimers are expressed by the host. To utilize this approach, it is favourable to bypass the furin cleavage stage as it removes the requirement of co-transfection with furin. Although previously defined uncleaved trimers have produced large populations of misfolded trimers, several constructs now exist that are able to form native-like trimers without the need for furin cleavage. By replacing the furin cleavage site with a flexible linker, it is possible to generate native-like trimers. With additional stabilizing mutations, native flexibly linked (NFL) trimers display native-like bnAb binding and analogous glycosylation to BG505 SOSIP.664 [6,31,69]. Using in silico methods, Kong et al. [70] redesigned the HR1 loop to generate uncleaved trimers that were stabilized in the prefusion conformation (UFO) and present trimers with greater stability than the equivalent SOSIP construct. The ability to redesign fundamental post-translational modifications with little impact on the overall glycosylation and topology of the envelope spike further highlights the robustness of the glycan shield as a target for bnAb elicitation by immunization.

Perspective

As immunogen strategies continue to move towards activating precursor B cells, innovations are increasingly exploiting or bypassing post-translational modifications of the envelope glycoprotein. These advances may well stimulate developments of efficacious vaccines against a much wider range of pathogens where classical vaccine design strategies have proved ineffective.

Abbreviations

     
  • bnAb

    broadly neutralizing antibody

  •  
  • CHO

    Chinese hamster ovary

  •  
  • cryo-EM

    cryo-electron microscopy

  •  
  • Env

    envelope

  •  
  • HEK

    human embryonic kidney

  •  
  • HIV

    human immunodeficiency virus

  •  
  • NFL

    native flexibly linked

  •  
  • PBMCs

    peripheral blood mononuclear cells

  •  
  • PDI

    protein disulfide isomerase

  •  
  • PNG

    potential N-glycosylation site

  •  
  • UFO

    uncleaved prefusion-optimized

Funding

M.C. is supported by the Scripps CHAVI-ID (1UM1AI100663), the International AIDS Vaccine Initiative (IAVI) and the Bill & Melinda Gates Foundation through the Collaboration for AIDS Vaccine Discovery [OPP1084519, OPP1115782], and R.W.S. by NIH HIVRAD grant P01 AI110657. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 681137. R.W.S. is a recipient of a Vidi grant from the Netherlands Organization for Scientific Research (NWO) and a Starting Investigator Grant from the European Research Council [ERC-StG-2011-280829-SHEV]. K.J.D. was funded by the Medical Research Council (MRC) [MR/K024426/1].

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

References

References
1
Barouch,
D.H.
,
Stephenson,
K.E.
,
Borducchi,
E.N.
,
Smith,
K.
,
Stanley,
K.
,
McNally,
A.G.
et al. 
. (
2013
)
Protective efficacy of a global HIV-1 mosaic vaccine against heterologous SHIV challenges in rhesus monkeys
.
Cell
155
,
531
539
2
Chung,
A.W.
,
Kumar,
M.P.
,
Arnold,
K.B.
,
Yu,
W.H.
,
Schoen,
M.K.
,
Dunphy,
L.J.
et al. 
. (
2015
)
Dissecting polyclonal vaccine-induced humoral immunity against HIV using systems serology
.
Cell
163
,
988
998
3
Rerks-Ngarm,
S.
,
Pitisuttithum,
P.
,
Nitayaphan,
S.
,
Kaewkungwal,
J.
,
Chiu,
J.
,
Paris,
R.
et al. 
. (
2009
)
Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand
.
N. Engl. J. Med.
361
,
2209
2220
4
Chuang,
G.-Y.
,
Geng,
H.
,
Pancera,
M.
,
Xu,
K.
,
Cheng,
C.
,
Acharya,
P.
et al. 
. (
2017
)
Structure-based design of a soluble prefusion-closed HIV-1 Env trimer with reduced CD4 affinity and improved immunogenicity
.
J. Virol.
91
,
e02268-16
5
Sanders,
R.W.
,
Derking,
R.
,
Cupo,
A.
,
Julien,
J.P.
,
Yasmeen,
A.
,
de Val,
N.
, et al. 
. (
2013
)
A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies
.
PLoS Pathog.
9
,
e1003618
6
Sharma,
S.K.
,
de Val,
N.
,
Bale,
S.
,
Guenaga,
J.
,
Tran,
K.
,
Feng,
Y.
et al. 
. (
2015
)
Cleavage-independent HIV-1 Env trimers engineered as soluble native spike mimetics for vaccine design
.
Cell Rep.
11
,
539
550
7
Kulp,
D.W.
,
Steichen,
J.M.
,
Pauthner,
M.
,
Hu,
X.
,
Schiffner,
T.
,
Liguori,
A.
et al. 
. (
2017
)
Structure-based design of native-like HIV-1 envelope trimers to silence non-neutralizing epitopes and eliminate CD4 binding
.
Nat. Commun.
8
,
1655
8
Behrens,
A.-J.
,
Harvey,
D.J.
,
Milne,
E.
,
Cupo,
A.
,
Kumar,
A.
,
Zitzmann,
N.
et al. 
. (
2017
)
Molecular architecture of the cleavage-dependent mannose patch on a soluble HIV-1 envelope glycoprotein trimer
.
J. Virol.
91
,
e01894-16
9
Stewart-Jones,
G.B.E.
,
Soto,
C.
,
Lemmin,
T.
,
Chuang,
G.-Y.
,
Druz,
A.
,
Kong,
R.
et al. 
. (
2016
)
Trimeric HIV-1-Env structures define glycan shields from clades A, B, and G
.
Cell
165
,
813
826
10
Lee,
J.H.
,
de Val,
N.
,
Lyumkis,
D.
and
Ward,
A.B.
(
2015
)
Model building and refinement of a natively glycosylated HIV-1 Env protein by high-resolution cryoelectron microscopy
.
Structure
23
,
1943
1951
11
Gristick,
H.B.
,
von Boehmer,
L.
,
West
, Jr,
A.P.
,
Schamber,
M.
,
Gazumyan,
A.
,
Golijanin,
J.
et al. 
. (
2016
)
Natively glycosylated HIV-1 Env structure reveals new mode for antibody recognition of the CD4-binding site
.
Nat. Struct. Mol. Biol.
23
,
906
915
12
Lee,
J.H.
,
Ozorowski,
G.
and
Ward,
A.B.
(
2016
)
Cryo-EM structure of a native, fully glycosylated, cleaved HIV-1 envelope trimer
.
Science
351
,
1043
1048
13
Crispin,
M.
,
Ward,
A.B.
and
Wilson,
I.A.
(
2018
)
Structure and immune recognition of the HIV glycan shield
.
Annu. Rev. Biophys.
47
,
annurev-biophys-060414-034156
14
Torrents de la Peña,
A.
,
de Taeye,
S.W.
,
Sliepen,
K.
,
LaBranche,
C.C.
,
Burger,
J.A.
,
Schermer,
E.E.
et al. 
. (
2018
)
Immunogenicity in rabbits of SOSIP trimers from clades A, B and C, given individually, sequentially or in combinations
.
J. Virol.
92
,
e01957-17
15
Pauthner,
M.
,
Havenar-Daughton,
C.
,
Sok,
D.
,
Nkolola,
J.P.
,
Bastidas,
R.
,
Boopathy,
A.V.
et al. 
. (
2017
)
Elicitation of robust tier 2 neutralizing antibody responses in nonhuman primates by HIV envelope trimer immunization using optimized approaches
.
Immunity
46
,
1073
1088.e6
16
Klasse,
P.J.
,
LaBranche,
C.C.
,
Ketas,
T.J.
,
Ozorowski,
G.
,
Cupo,
A.
,
Pugach,
P.
et al. 
. (
2016
)
Sequential and simultaneous immunization of rabbits with HIV-1 envelope glycoprotein SOSIP.664 trimers from clades A, B and C
.
PLOS Pathog.
12
,
e1005864
17
Burton,
D.R.
and
Hangartner,
L.
(
2016
)
Broadly neutralizing antibodies to HIV and their role in vaccine design
.
Annu. Rev. Immunol.
34
,
635
659
18
Mascola,
J.R.
,
Stiegler,
G.
,
VanCott,
T.C.
,
Katinger,
H.
,
Carpenter,
C.B.
,
Hanson,
C.E.
et al. 
. (
2000
)
Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies
.
Nat. Med.
6
,
207
210
19
Hessell,
A.J.
,
Rakasz,
E.G.
,
Tehrani,
D.M.
,
Huber,
M.
,
Weisgrau,
K.L.
,
Landucci,
G.
et al. 
. (
2010
)
Broadly neutralizing monoclonal antibodies 2F5 and 4E10 directed against the human immunodeficiency virus type 1 gp41 membrane-proximal external region protect against mucosal challenge by Simian-Human immunodeficiency virus SHIVBa-L
.
J. Virol.
84
,
1302
1313
20
Shingai,
M.
,
Donau,
O.K.
,
Plishka,
R.J.
,
Buckler-White,
A.
,
Mascola,
J.R.
,
Nabel,
G.J.
et al. 
. (
2014
)
Passive transfer of modest titers of potent and broadly neutralizing anti-HIV monoclonal antibodies block SHIV infection in macaques
.
J. Exp. Med.
211
,
2061
2074
21
Julg,
B.
,
Liu,
P.-T.
,
Wagh,
K.
,
Fischer,
W.M.
,
Abbink,
P.
,
Mercado,
N.B.
et al. 
. (
2017
)
Protection against a mixed SHIV challenge by a broadly neutralizing antibody cocktail
.
Sci. Transl. Med.
9
,
eaao4235
22
Julg,
B.
,
Tartaglia,
L.J.
,
Keele,
B.F.
,
Wagh,
K.
,
Pegu,
A.
,
Sok,
D.
et al. 
. (
2017
)
Broadly neutralizing antibodies targeting the HIV-1 envelope V2 apex confer protection against a clade C SHIV challenge
.
Sci. Transl. Med.
9
,
eaal1321
23
Shibata,
R.
,
Igarashi,
T.
,
Haigwood,
N.
,
Buckler-White,
A.
,
Ogert,
R.
,
Ross,
W.
et al. 
(
1999
)
Neutralizing antibody directed against the HIV–1 envelope glycoprotein can completely block HIV-1/SIV chimeric virus infections of macaque monkeys
.
Nat. Med.
5
,
204
210
24
Go,
E.P.
,
Cupo,
A.
,
Ringe,
R.
,
Pugach,
P.
,
Moore,
J.P.
and
Desaire,
H.
(
2016
)
Native conformation and canonical disulfide bond formation are interlinked properties of HIV-1 Env glycoproteins
.
J. Virol.
90
,
2884
2894
25
Huang,
J.
,
Kang,
B.H.
,
Pancera,
M.
,
Lee,
J.H.
,
Tong,
T.
,
Feng,
Y.
et al. 
. (
2014
)
Broad and potent HIV-1 neutralization by a human antibody that binds the gp41–gp120 interface
.
Nature
515
,
138
142
26
Behrens,
A.-J.
,
Vasiljevic,
S.
,
Pritchard,
L.K.
,
Harvey,
D.J.
,
Andev,
R.S.
,
Krumm,
S.A.
et al. 
. (
2016
)
Composition and antigenic effects of individual glycan sites of a trimeric HIV-1 envelope glycoprotein
.
Cell Rep.
14
,
2695
2706
27
Sanders,
R.W.
and
Moore,
J.P.
(
2017
)
Native-like Env trimers as a platform for HIV-1 vaccine design
.
Immunol. Rev.
275
,
161
182
28
Torrents de la Peña
,
A.
,
Julien,
J.P.
,
de Taeye,
S.W.
,
Garces,
F.
,
Guttman,
M.
,
Ozorowski,
G.
et al. 
. (
2017
)
Improving the immunogenicity of native-like HIV-1 envelope trimers by hyperstabilization
.
Cell Rep.
20
,
1805
1817
29
Binley,
J.M.
,
Sanders,
R.W.
,
Clas,
B.
,
Schuelke,
N.
,
Master,
A.
,
Guo,
Y.
et al. 
. (
2000
)
A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure
.
J. Virol.
74
,
627
643
30
Zhou,
T.
,
Doria-Rose,
N.A.
,
Cheng,
C.
,
Stewart-Jones,
G.B.E.
,
Chuang,
G.-Y.
,
Chambers,
M.
et al. 
. (
2017
)
Quantification of the impact of the HIV-1-glycan shield on antibody elicitation
.
Cell Rep.
19
,
719
732
31
Dubrovskaya,
V.
,
Guenaga,
J.
,
de Val,
N.
,
Wilson,
R.
,
Feng,
Y.
,
Movsesyan,
A.
et al. 
. (
2017
)
Targeted N-glycan deletion at the receptor-binding site retains HIV Env NFL trimer integrity and accelerates the elicited antibody response
.
PLOS Pathog.
13
,
e1006614
32
McCoy,
L.E.
,
van Gils,
M.J.
,
Ozorowski,
G.
,
Messmer,
T.
,
Briney,
B.
,
Voss,
J.E.
et al. 
. (
2016
)
Holes in the glycan shield of the native HIV envelope are a target of trimer-elicited neutralizing antibodies
.
Cell Rep.
16
,
2327
2338
33
Stolf,
B.S.
,
Smyrnias,
I.
,
Lopes,
L.R.
,
Vendramin,
A.
,
Goto,
H.
,
Laurindo,
F.R.M.
et al. 
. (
2011
)
Protein disulfide isomerase and host-pathogen interaction
.
ScientificWorldJournal
11
,
1749
1761
34
Land,
A.
,
Zonneveld,
D.
and
Braakman,
I.
(
2003
)
Folding of HIV-1 envelope glycoprotein involves extensive isomerization of disulfide bonds and conformation-dependent leader peptide cleavage
.
FASEB J.
17
,
1058
1067
35
Go,
E.P.
,
Hua,
D.
and
Desaire,
H.
(
2014
)
Glycosylation and disulfide bond analysis of transiently and stably expressed clade C HIV-1 gp140 trimers in 293T cells identifies disulfide heterogeneity present in both proteins and differences in O-linked glycosylation
.
J. Proteome Res.
13
,
4012
4027
36
Gallina,
A.
,
Hanley,
T.M.
,
Mandel,
R.
,
Trahey,
M.
,
Broder,
C.C.
,
Viglianti,
G.A.
et al. 
. (
2002
)
Inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent HIV-1 entry
.
J. Biol. Chem.
277
,
50579
50588
37
Julien,
J.-P.
,
Cupo,
A.
,
Sok,
D.
,
Stanfield,
R.L.
,
Lyumkis,
D.
,
Deller,
M.C.
et al. 
. (
2013
)
Crystal structure of a soluble cleaved HIV-1 envelope trimer
.
Science
342
,
1477
1483
38
Wei,
X.
,
Decker,
J.M.
,
Wang,
S.
,
Hui,
H.
,
Kappes,
J.C.
,
Wu,
X.
et al. 
. (
2003
)
Antibody neutralization and escape by HIV-1
.
Nature
422
,
307
312
39
Reitter,
J.N.
,
Means,
R.E.
and
Desrosiers,
R.C.
(
1998
)
A role for carbohydrates in immune evasion in AIDS
.
Nat. Med.
4
,
679
684
40
Coss,
K.P.
,
Vasiljevic,
S.
,
Pritchard,
L.K.
,
Krumm,
S.A.
,
Glaze,
M.
,
Madzorera,
S.
et al. 
. (
2016
)
HIV-1 glycan density drives the persistence of the mannose patch within an infected individual
.
J. Virol.
90
,
11132
11144
41
Blay,
W.M.
,
Gnanakaran,
S.
,
Foley,
B.
,
Doria-Rose,
N.A.
,
Korber,
B.T.
and
Haigwood,
N.L.
(
2006
)
Consistent patterns of change during the divergence of human immunodeficiency virus type 1 envelope from that of the inoculated virus in simian/human immunodeficiency virus-infected macaques
.
J. Virol.
80
,
999
1014
42
Borggren,
M.
,
Repits,
J.
,
Sterjovski,
J.
,
Uchtenhagen,
H.
,
Churchill,
M.J.
,
Karlsson,
A.
et al. 
. (
2011
)
Increased sensitivity to broadly neutralizing antibodies of end-stage disease R5 HIV-1 correlates with evolution in Env glycosylation and charge
.
PLoS ONE
6
,
e20135
43
Moore,
P.L.
,
Gray,
E.S.
,
Wibmer,
C.K.
,
Bhiman,
J.N.
,
Nonyane,
M.
,
Sheward,
D.J.
et al. 
. (
2012
)
Evolution of an HIV glycan-dependent broadly neutralizing antibody epitope through immune escape
.
Nat. Med.
18
,
1688
1692
44
Auwerx,
J.
,
François,
K.O.
,
Covens,
K.
,
Van Laethem,
K.
and
Balzarini,
J.
(
2008
)
Glycan deletions in the HIV-1 gp120 V1/V2 domain compromise viral infectivity, sensitize the mutant virus strains to carbohydrate-binding agents and represent a specific target for therapeutic intervention
.
Virology
382
,
10
19
45
Zhou,
T.
,
Zheng,
A.
,
Baxa,
U.
,
Chuang,
G.-Y.
,
Georgiev,
I.S.
,
Kong,
R.
et al. 
. (
2018
)
A neutralizing antibody recognizing primarily N-linked glycan targets the silent face of the HIV envelope
.
Immunity
48
,
500
513.e6
46
Ward,
A.B.
and
Wilson,
I.A.
(
2017
)
The HIV-1 envelope glycoprotein structure: nailing down a moving target
.
Immunol. Rev.
275
,
21
32
47
Travers,
S.A.
(
2012
)
Conservation, compensation, and evolution of N-linked glycans in the HIV-1 group M subtypes and circulating recombinant forms
.
ISRN AIDS
2012
,
1
9
48
Behrens,
A.-J.
and
Crispin,
M.
(
2017
)
Structural principles controlling HIV envelope glycosylation
.
Curr. Opin. Struct. Biol.
44
,
125
133
49
Cao,
L.
,
Diedrich,
J.K.
,
Kulp,
D.W.
,
Pauthner,
M.
,
He,
L.
,
Park,
S.-K.R.
et al. 
. (
2017
)
Global site-specific N-glycosylation analysis of HIV envelope glycoprotein
.
Nat. Commun.
8
,
14954
50
Panico,
M.
,
Bouché,
L.
,
Binet,
D.
,
O'Connor,
M.-J.
,
Rahman,
D.
,
Pang,
P.-C.
et al. 
(
2016
)
Mapping the complete glycoproteome of virion-derived HIV-1 gp120 provides insights into broadly neutralizing antibody binding
.
Sci. Rep.
6
,
32956
51
Pritchard,
L.K.
,
Vasiljevic,
S.
,
Ozorowski,
G.
,
Seabright,
G.E.
,
Cupo,
A.
,
Ringe,
R.
et al. 
. (
2015
)
Structural constraints determine the glycosylation of HIV-1 envelope trimers
.
Cell Rep.
11
,
1604
1613
52
Willey,
R.L.
,
Shibata,
R.
,
Freed,
E.O.
,
Cho,
M.W.
and
Martin,
M.A.
(
1996
)
Differential glycosylation, virion incorporation, and sensitivity to neutralizing antibodies of human immunodeficiency virus type 1 envelope produced from infected primary T-lymphocyte and macrophage cultures
.
J. Virol.
70
,
6431
6436
PMID:
[PubMed]
53
Pritchard,
L.K.
,
Spencer,
D.I.R.
,
Royle,
L.
,
Vasiljevic,
S.
,
Krumm,
S.A.
,
Doores,
K.J.
et al. 
. (
2015
)
Glycan microheterogeneity at the PGT135 antibody recognition site on HIV-1 gp120 reveals a molecular mechanism for neutralization resistance
.
J. Virol.
89
,
6952
6959
54
McCoy,
L.E.
,
Falkowska,
E.
,
Doores,
K.J.
,
Le,
K.
,
Sok,
D.
,
van Gils,
M.J.
et al. 
. (
2015
)
Incomplete neutralization and deviation from sigmoidal neutralization curves for HIV broadly neutralizing monoclonal antibodies
.
PLoS Pathog.
11
,
e1005110
55
Katie
,
D.J.
and
Burton,
D.R.
(
2010
)
Variable loop glycan dependency of the broad and potent HIV-1-neutralizing antibodies PG9 and PG16
.
J. Virol.
84
,
10510
10521
56
Pritchard,
L.K.
,
Harvey,
D.J.
,
Bonomelli,
C.
,
Crispin,
M.
and
Doores,
K.J.
(
2015
)
Cell- and protein-directed glycosylation of native cleaved HIV-1 envelope
.
J. Virol.
89
,
8932
8944
57
Crooks,
E.T.
,
Tong,
T.
,
Chakrabarti,
B.
,
Narayan,
K.
,
Georgiev,
I.S.
,
Menis,
S.
et al. 
. (
2015
)
Vaccine-elicited tier 2 HIV-1 neutralizing antibodies bind to quaternary epitopes involving glycan-deficient patches proximal to the CD4 binding site
.
PLoS Pathog.
11
,
e1004932
58
Voss,
J.E.
,
Andrabi,
R.
,
McCoy,
L.E.
,
de Val,
N.
,
Fuller,
R.P.
,
Messmer,
T.
et al. 
. (
2017
)
Elicitation of neutralizing antibodies targeting the V2 apex of the HIV envelope trimer in a wild-type animal model
.
Cell Rep.
21
,
222
235
59
Wibmer,
C.K.
,
Gorman,
J.
,
Anthony,
C.S.
,
Mkhize,
N.N.
,
Druz,
A.
,
York,
T.
et al. 
. (
2016
)
Structure of an N276-dependent HIV-1 neutralizing antibody targeting a rare V5 glycan hole adjacent to the CD4 binding site
.
J. Virol.
90
,
10220
10235
60
Medina-Ramírez,
M.
,
Garces,
F.
,
Escolano,
A.
,
Skog,
P.
,
de Taeye,
S.W.
,
Del Moral-Sanchez,
I.
et al. 
. (
2017
)
Design and crystal structure of a native-like HIV-1 envelope trimer that engages multiple broadly neutralizing antibody precursors in vivo
.
J. Exp. Med.
214
,
2573
2590
61
Behrens,
A.-J.
,
Kumar,
A.
,
Medina-Ramirez,
M.
,
Cupo,
A.
,
Marshall,
K.
,
Cruz Portillo,
V.M.
et al. 
. (
2018
)
Integrity of glycosylation processing of a glycan-depleted trimeric HIV-1 immunogen targeting key B-cell lineages
.
J. Proteome Res.
17
,
987
999
62
Andrabi,
R.
,
Su,
C.-Y.
,
Liang,
C.-H.
,
Shivatare,
S.S.
,
Briney,
B.
,
Voss,
J.E.
et al. 
. (
2018
)
Glycans function as anchors for antibodies and help drive HIV broadly neutralizing antibody development
.
Immunity
47
,
524
537.e3
63
Molloy,
S.S.
,
Bresnahan,
P.A.
,
Leppla,
S.H.
,
Klimpel,
K.R.
and
Thomas,
G.
(
1992
)
Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen
.
J. Biol. Chem.
267
,
16396
16402
PMID:
[PubMed]
64
Ringe,
R.P.
,
Sanders,
R.W.
,
Yasmeen,
A.
,
Kim,
H.J.
,
Lee,
J.H.
,
Cupo,
A.
et al. 
. (
2013
)
Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation
.
Proc. Natl Acad. Sci. U.S.A.
110
,
18256
18261
65
Walker,
L.M.
,
Huber,
M.
,
Doores,
K.J.
,
Falkowska,
E.
,
Pejchal,
R.
,
Julien,
J.-P.
et al. 
. (
2011
)
Broad neutralization coverage of HIV by multiple highly potent antibodies
.
Nature
477
,
466
470
66
Falkowska,
E.
,
Le,
K.M.
,
Ramos,
A.
,
Doores,
K.J.
,
Lee,
J.H.
,
Blattner,
C.
et al. 
. (
2014
)
Broadly neutralizing HIV antibodies define a glycan-dependent epitope on the prefusion conformation of gp41 on cleaved envelope trimers
.
Immunity
40
,
657
668
67
Binley,
J.M.
,
Sanders,
R.W.
,
Master,
A.
,
Cayanan,
C.S.
,
Wiley,
C.L.
,
Schiffner,
L.
et al. 
. (
2002
)
Enhancing the proteolytic maturation of human immunodeficiency virus type 1 envelope glycoproteins
.
J. Virol.
76
,
2606
2616
68
Dey,
A.K.
,
Cupo,
A.
,
Ozorowski,
G.
,
Sharma,
V.K.
,
Behrens,
A.-J.
,
Go,
E.P.
et al. 
(
2018
)
cGMP production and analysis of BG505 SOSIP.664, an extensively glycosylated, trimeric HIV-1 envelope glycoprotein vaccine candidate
.
Biotechnol. Bioeng.
115
,
885
899
69
Guenaga,
J.
,
Dubrovskaya,
V.
,
de Val,
N.
,
Sharma,
S.K.
,
Carrette,
B.
,
Ward,
A.B.
et al. 
. (
2016
)
Structure-guided redesign increases the propensity of HIV Env to generate highly stable soluble trimers
.
J. Virol.
90
,
2806
2817
70
Kong,
L.
,
He,
L.
,
de Val,
N.
,
Vora,
N.
,
Morris,
C.D.
,
Azadnia,
P.
et al. 
. (
2016
)
Uncleaved prefusion-optimized gp140 trimers derived from analysis of HIV-1 envelope metastability
.
Nat. Commun.
7
,
12040