Conjugation of an scFab domain to the oligomeric HIV envelope protein for use in immune targeting

Hannah A D King; Christopher A Gonelli; Kirsteen M Tullett; Mireille H Lahoud; Damian F J Purcell; Heidi E Drummer; Pantelis Poumbourios; Rob J Center

doi:10.1371/journal.pone.0220986

Conjugation of an scFab domain to the oligomeric HIV envelope protein for use in immune targeting

PLoS One. 2019 Aug 20;14(8):e0220986. doi: 10.1371/journal.pone.0220986. eCollection 2019.

Authors

Hannah A D King^{1

2}, Christopher A Gonelli², Kirsteen M Tullett³, Mireille H Lahoud^{1

3}, Damian F J Purcell², Heidi E Drummer^{1

2

4}, Pantelis Poumbourios^{1

4}, Rob J Center^{1

2}

Affiliations

¹ Disease Elimination, Burnet Institute, Melbourne, Victoria, Australia.
² Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia.
³ Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia.
⁴ Department of Microbiology, Monash University, Melbourne, Victoria, Australia.

Abstract

A promising strategy for the enhancement of vaccine-mediated immune responses is by directly targeting protein antigens to immune cells. Targeting of antigens to the dendritic cell (DC) molecule Clec9A has been shown to enhance antibody affinity and titers for model antigens, and influenza and enterovirus antigens, and may be advantageous for immunogens that otherwise fail to elicit antibodies with sufficient titers and breadth for broad protection, such as the envelope protein (Env) of HIV. Previously employed targeting strategies often utilize receptor-specific antibodies, however it is impractical to conjugate a bivalent IgG antibody to oligomeric antigens, including HIV Env trimers. Here we designed single chain variable fragment (scFv) and single chain Fab (scFab) constructs of a Clec9A-targeting antibody, expressed as genetically fused conjugates with the soluble ectodomain of Env, gp140. This conjugation did not affect the presentation of Env neutralising antibody epitopes. The scFab moiety was shown to be more stable than scFv, and in the context of gp140 fusions, was able to mediate better binding to recombinant and cell surface-expressed Clec9A, although the level of binding to cell-surface Clec9A was lower than that of the anti-Clec9A IgG. However, binding to Clec9A on the surface of DCs was not detected. Mouse immunization experiments suggested that the Clec9A-binding activity of the scFab-gp140 conjugate was insufficient to enhance Env-specific antibody responses. This is an important first proof of principle study demonstrating the conjugation of a scFab to an oligomeric protein antigen, and that an scFab displays better antigen binding than the corresponding scFv. Future developments of this technique that increase the scFab affinity will provide a valuable means to target oligomeric proteins to cell surface antigens of interest, improving vaccine-generated immune responses.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

AIDS Vaccines / administration & dosage
AIDS Vaccines / genetics
AIDS Vaccines / immunology*
Animals
Antibodies, Neutralizing / immunology
Antibody Affinity
Antigens, Viral / genetics
Antigens, Viral / immunology
Dendritic Cells / immunology
Dendritic Cells / metabolism
Epitopes / immunology
Female
HEK293 Cells
HIV Antibodies / immunology
HIV Infections / immunology
HIV Infections / therapy*
HIV Infections / virology
Humans
Immunogenicity, Vaccine
Lectins, C-Type / immunology
Lectins, C-Type / metabolism
Mice
Proof of Concept Study
Protein Domains / genetics
Protein Domains / immunology
Receptors, Mitogen / immunology
Receptors, Mitogen / metabolism
Recombinant Fusion Proteins / administration & dosage
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / immunology*
Single-Chain Antibodies / administration & dosage
Single-Chain Antibodies / genetics
Single-Chain Antibodies / immunology*
Vaccination / methods
Vaccines, DNA / administration & dosage
Vaccines, DNA / genetics
Vaccines, DNA / immunology
env Gene Products, Human Immunodeficiency Virus / administration & dosage
env Gene Products, Human Immunodeficiency Virus / genetics
env Gene Products, Human Immunodeficiency Virus / immunology*

Substances

AIDS Vaccines
Antibodies, Neutralizing
Antigens, Viral
CLEC9a protein, human
Epitopes
HIV Antibodies
Lectins, C-Type
Receptors, Mitogen
Recombinant Fusion Proteins
Single-Chain Antibodies
Vaccines, DNA
env Gene Products, Human Immunodeficiency Virus
gp140 envelope protein, Human immunodeficiency virus 1

Grants and funding

The work was supported by an Australian Centre for HIV and Hepatitis Virology (ACH2) Research Grant, awarded to RJC, which was entitled: "Targeting HIV immunogens to immune cells to enhance vaccine efficacy”. RJC was also supported by a Project Grant from the Australian National Health and Medical Research Council (NHMRC), grant number 1146082. HADK and CAG were supported by Australian Postgraduate Awards, funded by the Australian Federal Government. MHL and KMT were supported by an NHMRC Project Grant APP1082665. DFJP was supported by the University of Melbourne. HED was supported by Project Grants from the Australian NHMRC, grant numbers 1106581 and 1146082, and a Fellowship from the NHMRC, 1041897. PP was supported by an NHMRC project grant, GNT1125822, and a grant awarded by ACH2. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.