HIV-1 Envelope Proteins and V1/V2 Domain Scaffolds with Mannose-5 to Improve the Magnitude and Quality of Protective Antibody Responses to HIV-1

Morales, Javier F.; Morin, Trevor J.; Yu, Bin; Tatsuno, Gwen P.; O’Rourke, Sara M.; Théolis, Richard; Mesa, Kathryn A.; Berman, Phillip W.

doi:10.1074/jbc.m114.554089

Cited by 22 publications

(35 citation statements)

References 95 publications

(125 reference statements)

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“…Although the gp120 expression data presented here was derived exclusively from small-scale transient transfection experiments, we anticipate that the cell line described in this report will be useful for the development of stable MGAT1 - CHO cell lines producing vaccines based on a variety of new concepts. These include guided immunization to stimulate germline genes encoding bNAbs (79–81), Env proteins designed with features that enhance antigen processing and presentation (82) as well as glycopeptide scaffolds that enhance the immunogenicity of epitopes recognized by bN-mAbs while eliminating non-protective immunodominant epitopes (21).…”

Section: Discussionmentioning

confidence: 99%

“…Previously we reported that the same gp120s used in the RV144 trial could be modified to bind multiple bN-mAbs when expressed in a cell line (HEK 293 GnTI - ) that limited N-linked glycosylation to Man 5 , or earlier species (e.g. Man 8 , Man 9 ) (21). While in theory this cell line could be used to produce a glycan optimized gp120 vaccine, in reality this is not practical.…”

Section: Introductionmentioning

confidence: 99%

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CRISPR/Cas9 gene editing for the creation of an MGAT1 deficient CHO cell line to control HIV-1 vaccine glycosylation

Byrne

O’Rourke

Alexánder

et al. 2018

Preprint

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Over the last decade multiple broadly neutralizing monoclonal antibodies (bN-mAbs) to the HIV-1 envelope protein, gp120, have been described. Surprisingly many of these recognize epitopes consisting of both amino acid and glycan residues. Moreover, the glycans required for binding of these bN-mAbs are early intermediates in the N-linked glycosylation pathway. This type of glycosylation substantially alters the mass and net charge of HIV envelope (Env) proteins compared to molecules with the same amino acid sequence but possessing mature, complex (sialic acid containing) carbohydrates. Since cell lines suitable for biopharmaceutical production that limit N-linked glycosylation to mannose-5 (Man5) or earlier intermediates are not readily available, the production of vaccine immunogens displaying these glycan dependent epitopes has been challenging. Here we report the development of a stable suspension adapted CHO cell line that limits glycosylation to Man5 and earlier intermediates. This cell line was created using the CRISPR/Cas9 gene editing system and contains a mutation that inactivates the gene encoding Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase (MGAT1). Monomeric gp120s produced in the MGAT1- CHO cell line exhibit improved binding to prototypic glycan dependent bN-mAbs directed to the V1/V2 domain (e.g. PG9) and the V3 stem (e.g. PGT128 and 10–1074) while preserving the structure of the important glycan independent epitopes (e.g. VRC01). The ability of the MGAT1-CHO cell line to limit glycosylation to early intermediates in the N-linked glycosylation pathway, without impairing the doubling time or ability to grow at high cell densities, suggest that it will be a useful substrate for the biopharmaceutical production of HIV-1 vaccine immunogens.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9 gene editing for the creation of an MGAT1 deficient CHO cell line to control HIV-1 vaccine glycosylation

Byrne

O’Rourke

Alexánder

et al. 2018

Preprint

Self Cite

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“…These rabbit experiments are among the first to demonstrate that the immune response can be focused on the V1V2 region (54). The cross-reactivity of human V1V2 Abs has long been established although this region has the most sequence and length diversity in the HIV-1 Env (22).…”

Section: Discussionmentioning

confidence: 99%

Rationally Designed Immunogens Targeting HIV-1 gp120 V1V2 Induce Distinct Conformation-Specific Antibody Responses in Rabbits

Jiang

Totrov

et al. 2016

J Virol

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The V1V2 region of HIV-1 gp120 harbors a major vulnerable site targeted by a group of broadly neutralizing monoclonal antibodies (MAbs) such as PG9 through strand-strand recognition. However, this epitope region is structurally polymorphic as it can also form a helical conformation recognized by RV144 vaccine-induced MAb CH58. This structural polymorphism is a potential mechanism for masking the V1V2 vulnerable site. Designing immunogens that can induce conformation-specific antibody (Ab) responses may lead to vaccines targeting this vulnerable site. We designed a panel of immunogens engrafting the V1V2 domain into trimeric and pentameric scaffolds in structurally constrained conformations. We also fused V1V2 to an Fc fragment to mimic the unconstrained V1V2 conformation. We tested these V1V2-scaffold proteins for immunogenicity in rabbits and assessed the responses by enzyme-linked immunosorbent assay (ELISA) and competition assays. Our V1V2 immunogens induced distinct conformation-specific Ab responses. Abs induced by structurally unconstrained immunogens reacted preferentially with unconstrained V1V2 antigens, suggesting recognition of the helical configuration, while Abs induced by the structurally constrained immunogens reacted preferentially with constrained V1V2 antigens, suggesting recognition of the ␤-strand conformation. The Ab responses induced by the structurally constrained immunogens were more broadly reactive and had higher titers than those induced by the structurally unconstrained immunogens. Our results demonstrate that immunogens presenting the different structural conformations of the gp120 V1V2 vulnerable site can be designed and that these immunogens induce distinct Ab responses with epitope conformation specificity. Therefore, these structurally constrained V1V2 immunogens are vaccine prototypes targeting the V1V2 domain of the HIV-1 envelope. IMPORTANCEThe correlates analysis of the RV144 HIV-1 vaccine trial suggested that the presence of antibodies to the V1V2 region of HIV-1 gp120 was responsible for the modest protection observed in the trial. In addition, V1V2 harbors one of the key vulnerable sites of HIV-1 Env recognized by a family of broadly neutralizing MAbs such as PG9. Thus, V1V2 is a key target for vaccine development. However, this vulnerable site is structurally polymorphic, and designing immunogens that present different conformations is crucial for targeting this site. We show here that such immunogens can be designed and that they induced conformation-specific antibody responses in rabbits. Our immunogens are therefore prototypes of vaccine candidates targeting the V1V2 region of HIV-1 Env.

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“…38 Recent results have also illustrated that some of the bnAbs such as PG9 and PG16 require part of peptide segment of the antigen to bind to. 15,39,40 We envision that this peptide portion could be coupled to glycopolymers generated from monomer 2 (Scheme 3) by investigating end-functionalization of the polymers during the quenching stage of ROMP with an amine group, which will subsequently serve as an attachment point. 22 …”

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confidence: 99%

Synthesis of a polymerizable, bivalent glycan mimetic of the HIV envelope spike gp120

Sletten

Svec

Nguyen

2015

Tetrahedron Letters

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A synthetic study on the creation of a bivalent, ROMP capable monomer has the ability to be polymerized into the corresponding neo-glycopolymer mimetic of the surface glycans on gp120 envelope spike of the HIV virus. In our approach, we have developed a new strategy for orthogonally attaching both the terminal Manα1-2Man disaccharide unit of the D1 arm of Man9GlcNAc2 of HIV gp120 and the terminal Manα1-2 unit of its D2 arm to a bivalent scaffold to produce the corresponding polymerizable monomer. The Manα1-2 saccharide moieties were assembled using a nickel catalyst, Ni(4-F-PhCN) 4 (OTf) 2 , to activate trihaloacetimidate donors under mild and operationally simple procedure. KeywordsHIV; Glycopolymer; Carbohydrate Synthesis; Nickel Catalyzed Human immunodeficiency virus (HIV) has evolved into one of the most threatening global viruses since its first isolation in 1983. Despite extensive research efforts in more than two decades, the conception of a synthetic HIV vaccine capable of eliciting broadly-neutralizing antibodies, has thus far proven elusive. 1 A principal explanation for this phenomenon focuses on the heavily glycosylated viral envelope, comprised of oligosaccharide fragments, which may disguise the virion from immune responses if they are recognized as "self", 2 the virus also utilizes its "glycan shield" to prevent protein-specific antibodies from binding to the inner protein core. 3 The isolation of carbohydrate-specific broadly-neutralizing antibody 2G12, capable of binding to HIV gp120's surface oligosaccharides, 4,5 suggests that the carbohydrate shield of HIV could be considered as a potential target for neutralization. 6 This elucidation inspired the use of glycan antigens towards the development of potential vaccines. Along with the 2G12 antibody, several other carbohydrate-specific broadly- hien-nguyen@uiowa.edu Phone: 319-384-1887 Fax: 319-335-1270. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Supplementary dataSupplementary data associated with this article can be found in the online version, at http:// Figure 1) of gp120. The crystal structure of 2G12 in particular revealed that approximately 85% of the contact with gp120 was through this terminal disaccharide unit, 6,[8][9] and that its unusual Fab domain-swapped structure provided for additional multivalent binding sites. 6,10 This discovery suggests that a majority of the binding event may be conserved upon utilization of only a fraction of the Man 9 GlcNAc 2 epitope. Our objective is to develop a glycan mimetic of HIV gp120 that potentially binds to glycan-specific bnAbs; this synthe...

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HIV-1 Envelope Proteins and V1/V2 Domain Scaffolds with Mannose-5 to Improve the Magnitude and Quality of Protective Antibody Responses to HIV-1

Cited by 22 publications

References 95 publications

CRISPR/Cas9 gene editing for the creation of an MGAT1 deficient CHO cell line to control HIV-1 vaccine glycosylation

CRISPR/Cas9 gene editing for the creation of an MGAT1 deficient CHO cell line to control HIV-1 vaccine glycosylation

Rationally Designed Immunogens Targeting HIV-1 gp120 V1V2 Induce Distinct Conformation-Specific Antibody Responses in Rabbits

Synthesis of a polymerizable, bivalent glycan mimetic of the HIV envelope spike gp120

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