Targeted N-glycan deletion at the receptor-binding site retains HIV Env NFL trimer integrity and accelerates the elicited antibody response

Viktoriya Dubrovskaya, Javier Guenaga, Natalia de Val, Richard Wilson, Yu Feng, Arlette Movsesyan, Gunilla B. Karlsson Hedestam, Andrew B. Ward, Richard T. Wyatt

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Extensive shielding by N-glycans on the surface of the HIV envelope glycoproteins (Env) restricts B cell recognition of conserved neutralizing determinants. Elicitation of broadly neutralizing antibodies (bNAbs) in selected HIV-infected individuals reveals that Abs capable of penetrating the glycan shield can be generated by the B cell repertoire. Accordingly, we sought to determine if targeted N-glycan deletion might alter antibody responses to Env. We focused on the conserved CD4 binding site (CD4bs) since this is a known neutralizing determinant that is devoid of glycosylation to allow CD4 receptor engagement, but is ringed by surrounding N-glycans. We selectively deleted potential N-glycan sites (PNGS) proximal to the CD4bs on well-ordered clade C 16055 native flexibly linked (NFL) trimers to potentially increase recognition by naïve B cells in vivo. We generated glycan-deleted trimer variants that maintained native-like conformation and stability. Using a panel of CD4bs-directed bNAbs, we demonstrated improved accessibility of the CD4bs on the N-glycan-deleted trimer variants. We showed that pseudoviruses lacking these Env PNGSs were more sensitive to neutralization by CD4bs-specific bNAbs but remained resistant to non-neutralizing mAbs. We performed rabbit immunogenicity experiments using two approaches comparing glycan-deleted to fully glycosylated NFL trimers. The first was to delete 4 PNGS sites and then boost with fully glycosylated Env; the second was to delete 4 sites and gradually re-introduce these N-glycans in subsequent boosts. We demonstrated that the 16055 PNGS-deleted trimers more rapidly elicited serum antibodies that more potently neutralized the CD4bs-proximal-PNGS-deleted viruses in a statistically significant manner and strongly trended towards increased neutralization of fully glycosylated autologous virus. This approach elicited serum IgG capable of cross-neutralizing selected tier 2 viruses lacking N-glycans at residue N276 (natural or engineered), indicating that PNGS deletion of well-ordered trimers is a promising strategy to prime B cell responses to this conserved neutralizing determinant.

Original languageEnglish (US)
Article numbere1006614
JournalPLoS Pathogens
Volume13
Issue number9
DOIs
StatePublished - Sep 1 2017
Externally publishedYes

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Antibody Formation
Polysaccharides
Binding Sites
HIV
B-Lymphocytes
Neutralizing Antibodies
Viruses
env Gene Products
CD4 Antigens
Serum
Glycosylation
Immunoglobulin G
Rabbits

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Molecular Biology
  • Genetics
  • Virology

Cite this

Targeted N-glycan deletion at the receptor-binding site retains HIV Env NFL trimer integrity and accelerates the elicited antibody response. / Dubrovskaya, Viktoriya; Guenaga, Javier; de Val, Natalia; Wilson, Richard; Feng, Yu; Movsesyan, Arlette; Karlsson Hedestam, Gunilla B.; Ward, Andrew B.; Wyatt, Richard T.

In: PLoS Pathogens, Vol. 13, No. 9, e1006614, 01.09.2017.

Research output: Contribution to journalArticle

Dubrovskaya, V, Guenaga, J, de Val, N, Wilson, R, Feng, Y, Movsesyan, A, Karlsson Hedestam, GB, Ward, AB & Wyatt, RT 2017, 'Targeted N-glycan deletion at the receptor-binding site retains HIV Env NFL trimer integrity and accelerates the elicited antibody response', PLoS Pathogens, vol. 13, no. 9, e1006614. https://doi.org/10.1371/journal.ppat.1006614
Dubrovskaya, Viktoriya ; Guenaga, Javier ; de Val, Natalia ; Wilson, Richard ; Feng, Yu ; Movsesyan, Arlette ; Karlsson Hedestam, Gunilla B. ; Ward, Andrew B. ; Wyatt, Richard T. / Targeted N-glycan deletion at the receptor-binding site retains HIV Env NFL trimer integrity and accelerates the elicited antibody response. In: PLoS Pathogens. 2017 ; Vol. 13, No. 9.
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abstract = "Extensive shielding by N-glycans on the surface of the HIV envelope glycoproteins (Env) restricts B cell recognition of conserved neutralizing determinants. Elicitation of broadly neutralizing antibodies (bNAbs) in selected HIV-infected individuals reveals that Abs capable of penetrating the glycan shield can be generated by the B cell repertoire. Accordingly, we sought to determine if targeted N-glycan deletion might alter antibody responses to Env. We focused on the conserved CD4 binding site (CD4bs) since this is a known neutralizing determinant that is devoid of glycosylation to allow CD4 receptor engagement, but is ringed by surrounding N-glycans. We selectively deleted potential N-glycan sites (PNGS) proximal to the CD4bs on well-ordered clade C 16055 native flexibly linked (NFL) trimers to potentially increase recognition by na{\"i}ve B cells in vivo. We generated glycan-deleted trimer variants that maintained native-like conformation and stability. Using a panel of CD4bs-directed bNAbs, we demonstrated improved accessibility of the CD4bs on the N-glycan-deleted trimer variants. We showed that pseudoviruses lacking these Env PNGSs were more sensitive to neutralization by CD4bs-specific bNAbs but remained resistant to non-neutralizing mAbs. We performed rabbit immunogenicity experiments using two approaches comparing glycan-deleted to fully glycosylated NFL trimers. The first was to delete 4 PNGS sites and then boost with fully glycosylated Env; the second was to delete 4 sites and gradually re-introduce these N-glycans in subsequent boosts. We demonstrated that the 16055 PNGS-deleted trimers more rapidly elicited serum antibodies that more potently neutralized the CD4bs-proximal-PNGS-deleted viruses in a statistically significant manner and strongly trended towards increased neutralization of fully glycosylated autologous virus. This approach elicited serum IgG capable of cross-neutralizing selected tier 2 viruses lacking N-glycans at residue N276 (natural or engineered), indicating that PNGS deletion of well-ordered trimers is a promising strategy to prime B cell responses to this conserved neutralizing determinant.",
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