Oligomannose-type glycans are among the major targets on the gp120 component of the HIV envelope protein (Env) for broadly neutralizing antibodies (bnAbs). However, attempts to elicit oligomannose-specific nAbs by immunizing with natural or synthetic oligomannose have so far not been successful, possibly due to B cell tolerance checkpoints. Here we design and synthesize oligomannose mimetics, based on the unique chemical structure of a recently identified bacterial lipooligosaccharide, to appear foreign to the immune system. One of these mimetics is bound avidly by members of a family of oligomannose-specific bnAbs and their putative common germline precursor when presented as a glycoconjugate. The crystal structure of one of the mimetics bound to a member of this bnAb family confirms the antigenic resemblance. Lastly, immunization of human-antibody transgenic animals with a lead mimetic evokes nAbs with specificities approaching those of existing bnAbs. These results provide evidence for utilizing antigenic mimicry to elicit oligomannose-specific bnAbs to HIV-1.
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.
Novel
neoglycoproteins containing oligomannosidic penta- and heptasaccharides
as structural variants of oligomannose-type N-glycans
found on human immunodeficiency virus type 1 gp120 have been prepared
using different conjugation methods. Two series of synthetic ligands
equipped with 3-aminopropyl spacer moieties and differing in the anomeric
configuration of the reducing mannose residue were activated either
as isothiocyanates or as adipic acid succinimidoyl esters and coupled
to bovine serum albumin. Coupling efficiency for adipic acid connected
neoglycoconjugates was better than for the thiourea-linked derivatives;
the latter constructs, however, exhibited higher reactivity toward
antibody 2G12, an HIV-neutralizing antibody with exquisite specificity
for oligomannose-type glycans. 2G12 binding avidities for the conjugates,
as determined by Bio-Layer Interferometry, were mostly higher for
the β-linked ligands and, as expected, increased with the numbers
of covalently linked glycans, leading to approximate KD values of 10 to 34 nM for optimized ligand-to-BSA ratios.
A similar correlation was observed by enzyme-linked immunosorbent
assays. In addition, dendrimer-type ligands presenting trimeric oligomannose
epitopes were generated by conversion of the amino-spacer group into
a terminal azide, followed by triazole formation using “click
chemistry”. The severe steric bulk of the ligands, however,
led to poor efficiency in the coupling step and no increased antibody
binding by the resulting neoglycoconjugates, indicating that the low
degree of substitution and the spatial orientation of the oligomannose
epitopes within these trimeric ligands are not conducive to multivalent
2G12 binding.
Oligomannose-type glycans on HIV-1 gp120 form a patch that is targeted by several broadly neutralizing antibodies (bnAbs) and that therefore is of interest to vaccine design. However, attempts to elicit similar oligomannose-specific bnAbs by immunizing with oligomannosidic glycoconjugates have only been modestly successful so far. A common assumption is that eliciting oligomannose-specific bnAbs is hindered by B cell tolerance, resulting from the presented oligomannosides being sensed as self molecules. Here, we present data, along with existing scientific evidence, supporting an additional, or perhaps alternate, explanation: serum mannosidase trimming of the presented oligomannosides in vivo. Mannosidase trimming lessens the likelihood of eliciting antibodies with capacity to bind fullsized oligomannose, which typifies the binding mode of existing bnAbs to the oligomannose patch. The rapidity of the observed trimming suggests the need for immunization strategies and/or synthetic glycosides that readily avoid or resist mannosidase trimming upon immunization and can overcome possible tolerance restrictions.
The
pentasaccharide fragment α-d-Man-(1 →
5)-[α-d-Kdo-(2 → 4)-]α-d-Kdo-(2
→ 6)-β-d-GlcNAc-(1 → 6)-α-d-GlcNAc equipped with a 3-aminopropyl spacer moiety was prepared
by a sequential assembly of monosaccharide building blocks. The glucosamine
disaccharide—as a backbone surrogate of the bacterial lipid
A region—was synthesized using an 1,3-oxazoline donor, which
was followed by coupling with an isopropylidene-protected Kdo-fluoride
donor to afford a protected tetrasaccharide intermediate. Eventually,
an orthogonally protected manno-configured trichloroacetimidate
donor was used to achieve the sterically demanding glycosylation of
the 5-OH group of Kdo in good yield. The resulting pentasaccharide
is suitably protected for further chain elongation at positions 3,
4, and 6 of the terminal mannose. Global deprotection afforded the
target pentasaccharide to be used for the conversion into neoglycoconjugates
and “clickable” ligands.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.