Synthesis of an Undecasaccharide Featuring an Oligomannosidic Heptasaccharide and a Bacterial Kdo-lipid A Backbone for Eliciting Neutralizing Antibodies to Mammalian Oligomannose on the HIV-1 Envelope Spike

Abstract

Lipooligosaccharides (LOS) from the bacterium Rhizobium radiobacter Rv3 are structurally related to antigenic mammalian oligomannoses on the HIV-1 envelope glycoprotein spike that are targets for broadly neutralizing antibodies. Here, we prepared a hybrid structure of viral and bacterial epitopes as part of a vaccine design strategy to elicit oligomannose-specific HIV-neutralizing antibodies using glycoconjugates based on the Rv3 LOS structure. Starting from a Kdo₂GlcNAc₂ tetrasaccharide precursor, a central orthogonally protected mannose trichloroacetimidate donor was coupled to OH-5 of the innermost Kdo residue. To assemble larger glycans, the N-acetylamino groups of the glucosamine units were converted to imides to prevent formation of unwanted imidate byproducts. Blockwise coupling of the pentasaccharide acceptor with an α-(1→2)-linked mannotriosyl trichloroacetimidate donor introduced the D1-arm fragment. Glycosylation of O-6 of the central branching mannose with an α-(1→2)-α-(1→6)-linked mannotriosyl trichloroacetimidate donor unit then furnished the undecasaccharide harboring a D3-arm extension. Global deprotection yielded the 3-aminopropyl ligand, which was activated as an isothiocyanate or adipic acid succinimidoyl ester and conjugated to CRM₁₉₇. However, representative oligomannose-specific HIV-neutralizing antibodies bound the undecasaccharide conjugates poorly. Possible reasons for this outcome are discussed herein along with paths for improvement.

Figure 1

Structure of the LOS from R. radiobacter strain Rv3 (left) and of full-sized N-linked mammalian oligomannose (Man₉GlcNAc₂) (right). Dashed lines indicate substoichiometric substitution of the LOS by a mannosyl residue at O-6 of the central branching mannose and a galactosyl residue at O-8 of the side chain Kdo.

Scheme 1. Imidate Formation and Synthesis of Pentasaccharide Acceptor 6

Scheme 2. Synthesis of Rhizobial LOS Octasaccharide Fragment 9

Scheme 3. Synthesis of Alternative Pentasaccharide Acceptor 17

Scheme 4. Synthesis of Trisaccharide Donor 21

Scheme 5. Synthesis of Undecasaccharide 24

Scheme 6. Synthesis of Neoglycoconjugates 25 and 27 and Structure of Neoglyconjugate 28

Figure 2

Broadly neutralizing antibodies PGT125, -126, -128, and -130 bind with relatively low avidity to CRM₁₉₇ conjugates of the undecasaccharide composed of an oligomannosidic heptasaccharide with a Kdo₂GlcNAc₂ backbone. All four antibodies were tested as IgGs. NIT269 and NIT273 represent the isothiocyanate-conjugated and the adipate-conjugated glycoside, respectively. NIT211, used as a comparator, is the heptamannoside conjugated via isothiocyanate to CRM₁₉₇.

Figure 3

Interaction of HIV-broadly neutralizing antibody PGT128 with a model of the undecasaccharide. (a) Overview of the modeled interaction. The crystal structure complex of PGT128 with heptamannoside NIT68A^, was used as template to create the model. The heavy and light chains of the PGT128 Fab are shown in a ribbon representation in dark gray and light gray, respectively. The modeled undecasaccharide is shown in stick representation, with the NIT68A heptamannoside in yellow, Kdo disaccharide in green, and the GlcN disaccharide in blueish purple. (b) Close-up view showing the modeled interaction of PGT128 with the undecasaccharide. The constituents of the undecasaccharide are labeled. To model the Kdo disaccharide, a crystallized Hep-Kdo₂ fragment was modeled into the PGT128 binding site by superposing the heptose residue (pink) onto the central branching mannose of the NIT68A structure. To model the lipid A disaccharide, a crystallized Kdo-GlcN₂ fragment was superposed onto the structure using the main-chain Kdo residue (Kdo I; orange) as a guide. (c) Left side view of panel b, highlighting the assumed proximity of the side chain Kdo (Kdo II) to the antibody.