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Journal of Virology, January 2009, p. 368-383, Vol. 83, No. 1
0022-538X/09/$08.00+0     doi:10.1128/JVI.01404-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Optimization of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins with V1/V2 Deleted, Using Virus Evolution{triangledown}

Ilja Bontjer,1 Aafke Land,2 Dirk Eggink,1 Erwin Verkade,1 Kiki Tuin,1 Chris Baldwin,1 Georgios Pollakis,1 William A. Paxton,1 Ineke Braakman,2 Ben Berkhout,1 and Rogier W. Sanders1*

Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands,1 Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands2

Received 7 July 2008/ Accepted 30 August 2008

The human immunodeficiency virus type 1 envelope glycoprotein (Env) complex is the principal focus of neutralizing antibody-based vaccines. The functional Env complex is a trimer consisting of six individual subunits: three gp120 molecules and three gp41 molecules. The individual subunits have proven unsuccessful as vaccines presumably because they do not resemble the functional Env complex. Variable domains and carbohydrates shield vulnerable neutralization epitopes on the functional Env complex. The deletion of variable loops has been shown to improve gp120's immunogenicity; however, problems have been encountered when introducing such modifications in stabilized Env trimer constructs. To address these issues, we have created a set of V1/V2 and V3 loop deletion variants in the context of complete virus to allow optimization by forced virus evolution. Compensatory second-site substitutions included the addition and/or removal of specific carbohydrates, changes in the disulfide-bonded architecture of the V1/V2 stem, the replacement of hydrophobic residues by hydrophilic and charged residues, and changes in distal parts of gp120 and gp41. These viruses displayed increased sensitivity to neutralizing antibodies, demonstrating the improved exposure of conserved domains. The results show that we can select for functionally improved Env variants with loop deletions through forced virus evolution. Selected evolved Env variants were transferred to stabilized Env trimer constructs and were shown to improve trimer expression and secretion. Based on these findings, we can make recommendations on how to delete the V1/V2 domain from recombinant Env trimers for vaccine and X-ray crystallography studies. In general, virus evolution may provide a powerful tool to optimize Env vaccine antigens.

* Corresponding author. Mailing address: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15 K3-105, 1105 AZ Amsterdam, The Netherlands. Phone: 31 20 5665279. Fax: 31 20 5669064. E-mail: r.w.sanders{at}amc.uva.nl

{triangledown} Published ahead of print on 15 October 2008.

Journal of Virology, January 2009, p. 368-383, Vol. 83, No. 1
0022-538X/09/$08.00+0     doi:10.1128/JVI.01404-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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