3.1. Human Immunodeficiency Virus Type-1 (HIV-1)
HIV-1 is the cause of AIDS, a disease which still causes serious global health problems [81
]. Despite enormous efforts in the vaccine development, a convincingly effective HIV vaccine is still missing. In order to evade the immune surveillance, the HIV develops numerous defense mechanisms such as frequent sequence variation and heavy glycosylation of the viral envelope glycoproteins [82
]. The trimeric HIV-1 envelope protein (Env) is responsible for host cell recognition and subsequent entry of the virus into the cytoplasm [83
]. Env is expressed as a gp160 precursor that is proteolytically cleaved into heterodimers of a surface-exposed glycoprotein, gp120, and a transmembrane glycoprotein, gp41. The outer envelope protein gp120 subunit is relevant for the adsorption of virions to chemokine and CD4 receptors on the host cell and has a highly variable surface that includes five variable loops (V1–V5) [84
]. The number of conserved N
-linked glycosylation sites of gp120 ranges from 18–33, with a median value of 25 [85
], and include complex or high-mannose type glycans [86
]. The transmembrane envelop glycoprotein gp41 possesses four conserved N
-glycans and mediates the fusion of the virus and host cell membranes [89
The Meyer group prepared gp120-based peptides and glycopeptides to investigate the impact of glycosylation of gp120 at the N197 position on binding of gp120 to CD4 by SPR and saturation transfer difference NMR spectroscopy (STD) [90
]. The peptides were assembled by microwave-assisted SPPS. Based on the amino acid sequence of gp120, linear and cyclic glycosylated and non-glycosylated peptides P1
were synthesized by combining three noncontiguous peptides according to their position in the crystal structure of the complex gp120–CD4 (Figure 11
). Additionally, two linear peptides P5
lacking the primary binding motif NMWQKVGTPL were prepared. Binding affinities of the obtained gp120 peptides towards CD4 were determined by SPR.
Peptides 1–4 bound to CD4 in the micromolar range. Preferred binding to glycosylated peptides P1 and P3 (Kd,glyc = 19 and 450 nM, respectively) over the corresponding non-glycosylated derivatives P2 and P4 (Kd,non-glyc = 83 and 540 nM, respectively) was observed. The binding affinities decreased for peptides lacking the NMWQKVGTPL binding motif. Additionally, the results indicate that the cyclic structure is another important factor that enhances the binding affinity. STD NMR analysis of the binding epitope showed that the GlcNAc moiety interacts primarily at its N-acetyl group with CD4. These findings lead to new concepts in CD4 inhibitor development.
Since the N
-glycans are assembled by the host glycosylation machinery, they are considered as ‘self’ and are therefore only weakly immunogenic. The high density of glycans generates a so called ‘glycan shield’ that usually interferes with antibody recognition. Nevertheless, some of the most potent broadly neutralizing antibodies (bNAbs) from HIV-infected patients have evolved to recognize epitopes on the Env that are formed by these glycans, including 2G12, PG9, PG16, PGT121–123, PGT125–128, and PGT135 [91
]. Current HIV-1 vaccines are not able to induce broadly neutralizing antibodies and only elicit strain-specific neutralizing antibodies. Therefore, chemical and chemoenzymatic synthesis of HIV-1 glycopeptides that mimic the bNAb epitopes but lack the other viral glycoprotein elements has become an important objective in order to characterize the epitopes and to develop new epitope-based HIV vaccines that elicit specific antibodies and thus neutralize in a broad manner.
The monoclonal HIV-1 antibody 2G12 recognizes terminal clusters of Man9
-glycans on gp120 [93
]. In order to generate glycopeptide structures in which the clustering of glycans mimics the 2G12 epitope on gp120, the Krauss group was following an original approach to generate mRNA-displayed peptide libraries of random 33-mer glycopeptides containing 3–5 high-mannose ligands (Figure 12
Here, mRNA encoding a library of random peptide sequences was cross-linked to a 3′ puromycin oligonucleotide, which forms a covalent bond to the C-terminus of the nascent peptides after ribosomal translation of the mRNAs. Thereby, methionine was substituted by the non-canonical amino acid homopropargylglycine for subsequent ‘click’-glycosylation with Man9-azide via CuAAC. The obtained mRNA-displayed glycopeptide library of ~1013 sequences was then subjected to ten rounds of selection for binding to 2G12. After each round, peptides that survived selection were amplified by PCR amplification of their cDNA, followed by transcription/translation of the PCR products. In this way, glycopeptides that bind to 2G12 in the low nanomolar to picomolar range were identified.
These candidates were then synthesized in a larger scale using fast flow peptide synthesis with subsequent glycosylation of the alkyne-functionalized peptide backbone via ‘click’ glycosylation [95
]. In fast flow peptide synthesis, all reagent solutions also including the activated amino acids are pumped through a heated reactor containing the SPPS resin, thus ensuring a continuous and fresh supply of reagents. Using this method, very short coupling times of 30 s were achieved with peptide yields comparable to microwave-assisted synthesis. The mannosylated glycopeptides were finally coupled to the carrier protein CRM197 and the obtained glycoconjugates were evaluated in binding efficacy studies to 2G12 by using enzyme-linked immunosorbent assays (ELISAs). The glycopeptide conjugates were strongly recognized by antibody 2G12. Rabbit immunogenicity studies of these conjugates to determine their ability to elicit antibody responses with 2G12-like-specificity are currently in progress.
In 2009, the potent monoclonal bNAbs PG9 and PG16 were isolated from an HIV-1-infected patient [96
]. Epitope mapping indicated that PG9 and PG16 recognize a different glycan-dependent Env epitope than 2G12 and showed preferred binding towards the trimeric gp120 and gp41 Env complex over the monomeric gp120. Crystal structures of PG9 and PG16 in complex with gp120 V1V2 showed that the antibodies interact with high mannose glycans at N160
, and an adjacent V1V2 domain consisting of four antiparallel β-strands [97
]. Whereas, both antibodies required Man5
at position 160 to neutralize HIV-1, complex type N
-glycans at position 156 or 173 play a secondary role for the binding. In terms of vaccine design, a potent gp120-based immunogen could therefore be composed of a Man5
moiety at position 160 and a complex N
-glycan at N156 or N173.
Homogenous gp120 V1V2 35mer glycopeptides carrying N
-glycans at positions 156 and 160 were synthesized and their binding to mAb PG9 was evaluated by SPR [99
]. Due to the steric demands imposed by the close proximity of the glycosylation sites, two separate peptide building blocks were assembled by SPPS and Man3
were coupled to the respective positions by aspartylation (Figure 12
b). Subsequently, both glycopeptide building blocks were joined by native chemical ligation (NCL). Binding of the glycopeptide to PG9 was investigated by SPR analysis. Significant binding affinity to the antibody was observed for both ligands with slightly better binding of Man3
= 119 nM) over Man5
= 311 nM). It was discovered, that the synthetic V1V2 peptides could form disulfide-linked dimers by spontaneous air oxidation. This finding was further investigated by chemical coupling of the synthetic glycopeptides via disulfide bond formation between the cysteines at position 157 under oxidative conditions [100
]. Circular dichroism (CD) analysis of the biophysical properties of the V1V2 glycopeptide dimers showed that the random-coiled glycopeptides adopted an ordered β-sheet conformation upon dimerization. The binding of the formed dimers to bNAbs PG9 and CH01 was evaluated by SPR. The binding affinities of both antibodies towards the Man3
glycopeptide dimers were significantly enhanced compared to the binding to the monomeric derivatives. This result indicates that adoption of the β-strand conformation of the V1V2 glycopeptides is required for binding of the antibodies. Crystal structures of PG9 and CH01 in complex with the high-mannose N
-glycan V1V2 peptides would provide additional information regarding binding preferences.
The Wang group focused on the characterization of the neutralizing epitopes of antibodies PG9 and PG16 by synthesizing V1V2 cyclic glycopeptides (aa154–177) to mimic the gp120 V1V2 epitope and apply them in antibody binding studies [101
]. Homogeneous cyclic glycopeptides corresponding to the V1/V2 domain presenting defined N
-glycans at positions N160 and N156/N173 were prepared by employing a chemoenzymatic method (Figure 13
a). Amino acid building blocks glycosylated with a GlcNAc moiety were introduced into the peptide sequence during SPPS. Then, various activated glycan oxazolines were transferred to the GlcNAc moiety by endoglycosidases via a transglycosylation reaction.
The affinities of the glycosylated cyclic peptides towards PG9/PG16 Fab were determined by SPR and ELISA experiments. It was shown that a Man5
glycan at the N160 position was essential for PG9 and PG16 recognition. Additionally, a terminal sialylated complex N
-glycan at glycosylation site N156 or N173 was reported to be important for recognition by PG9 and PG16. A disadvantage of this chemoenzymatic synthesis strategy was the difficulty to generate glycopeptides carrying two or more different glycans since the endoglycosidases usually are unable to distinguish between the GlcNAc acceptors at different glycosylation sites. Therefore, the Wang group optimized the employed method to efficiently and quickly synthesize HIV-1 V1V2 glycopeptides carrying distinct N
]. This strategy includes the introduction of two orthogonally protected GlcNAc-Asn building blocks into the peptide backbone by SPPS (Figure 13
b). After orthogonal deprotection of the GlcNAc residues, site-selective sequential extension of the glycan chains was achieved by glycosynthase-catalyzed transglycosylation reactions.
A number of the bNAbs, including V3-glycan specific antibodies, exhibit preference for the native Env trimer compared to monomeric gp120 [103
]. Therefore, multivalent V3 glycopeptides could prove to be efficient to elicit specific bNAbs. The optimized chemoenzymatic method was adopted to prepare a series of HIV-1 V3 glycopeptides [104
]. This library was employed to determine the minimal neutralizing epitopes through binding studies with the bNAbs PGT128, PGT121, and 10-1074 that bind to the N332 glycan at the base of the gp120 V3 loop [105
]. The binding experiments enabled evaluation of the glycan specificity as well as the requirement of the peptide backbone for antigen recognition by these broad neutralizing antibodies. PGT128 was shown to specifically recognize high-mannose glycans within the V3 domain. PGT121 and 10-1074 were specific for a sialylated complex N
-glycan at the N301 position and a high-mannose N
-glycan at the N332 glycosylation site, respectively. Based on these findings, a trivalent V3 glycopeptide construct that was expected to exhibited enhanced binding to bNAb 10-1074 was prepared [107
]. The afore mentioned chemoenzymatic method [101
] was used to synthesize mono- bi- and trivalent 33-mer cyclic gp120 V3 glycopeptide conjugated with Man9
-glycans at position N332 to mimic the V3 glycopeptide domains of the Env trimer. Subsequently, the V3 glycopeptides were coupled to a linear bi- or trivalent Lys(N3
)-functionalized peptide scaffold using copper(I)-catalyzed alkyne–azide 3 + 2 cycloaddition. The binding of the obtained glycopeptide conjugates to the Fab domains of PGT128 and 10-1074 antibodies was evaluated by SPR experiments. Different binding preferences of the PGT128 and 10-1074 antibodies towards the glycopeptides were observed. Whereas PGT128 showed no particular preference for mono-, bi- and trivalent V3 glycopeptides (Kd, mono
= 3.7 µM, Kd, bi
= 1.5 µM, Kd, tri
= 1.1 µM), the 10-1074 antibody showed strongly enhanced binding to the bi- and trivalent V3 glycopeptides in the nanomolar range (Kd, bi
= 0.31 µM, Kd, tri
= 0.19 µM) compared to the monomeric derivative (Kd, mono
= 3.88 µM). The SPR binding results were verified by ELISA analysis. Based on these results, a three-component vaccine construct consisting of trivalent V3 glycopeptides carrying a high-mannose Man9
glycan at the N332 site, a T helper epitope peptide derived from tetanus toxin, and the TLR ligand lipopeptide Pam3CSK4 for stimulating immune response was designed (Figure 14
]. The cyclic gp120 V3 glycopeptides carrying N
-glycans at position N332 were synthesized according to the above mentioned protocol. The T cell epitope peptide P30 bearing a C
) residue for site-specific ligation to the multivalent V3 glycopeptide scaffold via copper (I)-catalyzed alkyne-azide 3 + 2 cycloaddition, and the Pam3CSK4 lipopeptide were assembled by Fmoc-SPPS.
To evaluate the immunogenicity, the trivalent glycopeptide conjugates were incorporated into liposomes and rabbits were immunized without additional adjuvants. In comparison to the previously reported three-component monovalent glycopeptide immunogen that was able to elicit a considerable glycan-dependent antibody response [109
], the immunogenicity of the V3 glycopeptide was significantly increased due to multivalent presentation of the ligands. The binding affinities of the antisera to synthetic mono-, bi, and trivalent V3 glycopeptides [107
] immobilized on magnetic beads were determined by ELISA. While comparable binding to the mono-, bi-, and trivalent V3 glycopeptides was observed of antisera induced by the monovalent vaccine construct, the antisera induced by the trivalent vaccine construct showed up to 16-fold stronger binding to the multivalent glycopeptides. Unfortunately, neither the antisera induced by the monovalent immunogen nor the antisera induced by the trivalent immunogen showed viral neutralization activity. The authors suggested that these findings could be related to lack of somatic maturation and that further investigations are needed to elicit glycopeptide epitope-specific, broadly neutralizing antibody responses.
The Haynes group also synthesized a homogeneous cyclic V3 glycopeptide bearing Man9
glycans at N301 and N332 to target the gp120 V3-glycan epitope [110
]. The peptide was assembled using the previously introduced aspartylation/NCL protocol and rhesus macaques were immunized with monomeric Man9
-V3 glycopeptide formulated in the Toll-like receptor 4 agonist GLA-SE (glucopyranosyl lipid adjuvant-stable emulsion) adjuvant. Isolated serum antibodies were not able to neutralize Env pseudoviruses. The authors concluded that the monomeric V3 glycopeptide was not a potent immunogen and proposed that immunization with a multimerized V3 glycopeptide followed by boosts of sequential Envs may induce potent bNAbs in HIV-1 infected individuals.
A recent study focuses on the N332 to N334 mutation of the N332 high-mannose glycan on the HIV-1 gp120 V3-loop [111
]. The study shows that synthetic V3 glycopeptides bearing a N334 high-mannose Man9
glycan were recognized by bNAbs PGT128 and PGT126 but not by 10-1074. Rabbit immunization with a corresponding monovalent three-component glycopeptide immunogen elicited glycan-dependent antibodies with cross-reactivity to different HIV-1 gp120/gp140 glycoproteins carrying N332 or N334 glycosylation sites. These findings indicated that the N334 mutation represents an interesting epitope for further HIV-1 vaccine studies. High-resolution crystal structures can be used to determine differences in selectivity and affinity between closely related broad neutralizing antibodies, leading to a deeper understanding of the roles that somatic mutations might play in the enhancement of binding affinity and neutralization efficacy of bNAbs. A high-resolution crystal structure of the previously described high-mannose V3 glycopeptide carrying Man9
glycans at N322 and N301 [110
] in complex with the single chain variable fragment (scFv) of the N332-glycan recognizing bNAb DH270.6 was reported [112
]. Crystallization experiments showed that the glycopeptide was able to mimic the V3 region of a native-like HIV Env trimer forming a two-stranded β-hairpin with a bulge at the conserved 324GDIR327 motif (Figure 15
). Two major regions that participate in antibody–glycopeptide interactions were identified: (i) the Env 324
motif, which is an important binding motif for N332-glycan recognizing bNAbs, interacts with the CDRH2 and CDRH3 loops of DH270.6, and (ii) the high-mannose glycan at N332, which is in contact with the CDRH3 and CDRL2 loops of the antibody. Different mutations found in the antibody lineage influenced the binding to the antigen-glycopeptide. Based on these results, the structure of high-mannose V3 glycopeptide can be optimized leading to enhanced affinity and stability of the hairpin.
Consequently, synthetic homogeneous gp120 peptide domains carrying defined glycan structures might be able to function as minimal mimics of epitopes that are recognized by bNAbs and could be used to generate novel HIV-1 vaccines, as they may elicit similar antibody responses to target the HIV Env.