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. 2021 Oct 15;16(10):2016-2025.
doi: 10.1021/acschembio.1c00375. Epub 2021 Aug 15.

Immunogenicity Evaluation of N-Glycans Recognized by HIV Broadly Neutralizing Antibodies

Sachin S Shivatare  1 , Ting-Jen Rachel Cheng  2 , Yang-Yu Cheng  2 , Vidya S Shivatare  1 , Tsung-I Tsai  1 , Hong-Yang Chuang  2 , Chung-Yi Wu  2 , Chi-Huey Wong  1   2
Affiliations

Immunogenicity Evaluation of N-Glycans Recognized by HIV Broadly Neutralizing Antibodies

Sachin S Shivatare et al. ACS Chem Biol. .

Abstract

While the improved treatment of human immunodeficiency virus type 1 (HIV-1) infection is available, the development of an effective and safe prophylactic vaccine against HIV-1 is still an unrealized goal. Encouragingly, the discovery of broadly neutralizing antibodies (bNAbs) from HIV-1 positive patients that are capable of neutralizing a broad spectrum of HIV-1 isolates of various clades has accelerated the progress of vaccine development in the past few years. Some of these bNAbs recognize the N-glycans on the viral surface gp120 glycoprotein. We have been interested in using the glycan epitopes recognized by bNAbs for the development of vaccines to elicit bNAb-like antibodies with broadly neutralizing activities. Toward this goal, we have identified novel hybrid-type structures with subnanomolar avidity toward several bNAbs including PG16, PGT121, PGT128-3C, 2G12, VRC13, VRC-PG05, VRC26.25, VRC26.09, PGDM1400, 35O22, and 10-1074. Here, we report the immunogenicity evaluation of a novel hybrid glycan conjugated to carrier DTCRM197, a nontoxic mutant of the diphtheria toxin, for immunization in mice. Our results indicated that the IgG response was mainly against the chitobiose motif with nonspecific binding to a panel of N-glycans with reducing end GlcNAc-GlcNAc (chitobiose) printed on the glass slides. However, the IgM response was mainly toward the reducing end GlcNAc moiety. We further used the glycoconjugates of Man3GlcNAc2, Man5GlcNAc2, and Man9GlcNAc2 glycans for immunization, and a similar specificity pattern was observed. These findings suggest that the immunogenicity of chitobiose may interfere with the outcome of N-glycan-based vaccines, and modification may be necessary to increase the immunogenicity of the entire N-glycan epitope.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1:
Figure 1:
(a) Hybrid-type glycan reactive HIV-1 broadly neutralizing antibodies and location of their epitopes on HIV envelop. (b) Pictorial representation and chemical structure of hybrid type glycan I.
Figure 2:
Figure 2:
The glycan specificities of serum antibodies from mice immunized with GC-1. Female BALB/c mice were immunized with 2 μg of GC-I adjuvanted with 2 μg glycolipid C34. Mouse serum was collected two weeks after the final vaccination, and the specificities of IgG and IgM antibodies against a panel of N-linked glycans (a) were accessed after 100-fold dilution. Data of glycan binding (b) represent total intensity of five mice ± the SEM.
Figure 3:
Figure 3:
The glycan specificities of serum antibodies from mice immunized with glycoconjugates II-IV. Data represent total intensity of five mice ± the SEM.
Figure 4:
Figure 4:
The glycan specificities of serum antibodies from mice immunized with glycoconjugate V. Data represent total intensity of five mice ± the SEM.
Figure 5:
Figure 5:
Synthesis of glycoconjugate VI (a) and its immunological evaluation (b). a) (1) DTSSP, PBS, pH 7.4, rt, overnight; (2) DTT, PBS, pH 7.4, 40°C, 2 h, 70% over two steps; b) (1) PBS, pH 7.2, rt, 2 h; (2) thioethanol, PBS, pH 7.2, rt, overnight. The glycan specificities of serum antibodies from mice immunized with glycoconjugate VI. Data represent total intensity of five mice ± the SEM.
Scheme 1:
Scheme 1:
Synthesis of glycoconjugate- I Reagents and conditions: (a) p-nitrophenyl ester (homo bifunctional linker), DMF, rt, 5 h, 61%; (b) 10 mM PBS, pH 8, rt, overnight.
Scheme 2:
Scheme 2:
Synthesis of glycoconjugate II-IV Reagents and conditions: (a) p-nitrophenyl ester (homo bifunctional linker), DMF, rt, 5 h; (b) 10 mM PBS, pH 8, rt, overnight.
Scheme 3:
Scheme 3:
Synthesis of GC-V Reagents and conditions: (a) i. DTSSP, PBS buffer pH 7.4, overnight; ii. DTT, 40°C, 2hr; (b) Sulfo-EMCS, PBS buffer pH 6.4, rt, 2h; (c) Va, PBS buffer pH 7.4, rt, overnight; (d) i. VIa, PBS buffer pH 7.4, rt, overnight; ii. Mercaptoethanol, PBS buffer pH 7.4, overnight.
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