Single-Chain Soluble BG505.SOSIP gp140 Trimers as Structural and Antigenic Mimics of Mature Closed HIV-1 Env.
Similar to other type I fusion machines, the HIV-1 envelope glycoprotein (Env) requires proteolytic activation; specifically, cleavage of a gp160 precursor into gp120 and gp41 subunits creates an N-terminal gp41 fusion peptide and permits folding from an immature uncleaved state to a mature closed state. While the atomic-level consequences of cleavage for HIV-1 Env are still being determined, the uncleaved state is antigenically distinct from the mature closed state, and cleavage has been reported to be essential for mimicry of the mature viral spike by soluble versions of Env. Here we report the redesign of a current state-of-the-art soluble Env mimic, BG505.SOSIP, to make it cleavage independent. Specifically, we replaced the furin cleavage site between gp120 and gp41 with Gly-Ser linkers of various lengths. The resultant linked gp120-gp41 constructs, termed single-chain gp140 (sc-gp140), exhibited different levels of structural and antigenic mimicry of the parent cleaved BG505.SOSIP. When constructs were subjected to negative selection to remove subspecies recognized by poorly neutralizing antibodies, trimers of high antigenic mimicry of BG505.SOSIP could be obtained; negative-stain electron microscopy indicated these to resemble the mature closed state. Higher proportions of BG505.SOSIP-trimer mimicry were observed in sc-gp140s with linkers of 6 or more residues, with a linker length of 15 residues exhibiting especially promising traits. Overall, flexible linkages between gp120 and gp41 in BG505.SOSIP can thus substitute for cleavage, and sc-gp140s that closely mimicked the vaccine-preferred mature closed state of Env could be obtained.


OBJECTIVE
The trimeric HIV-1 envelope glycoprotein (Env) is the sole target of virus-directed neutralizing antibody responses and a primary focus of vaccine design. Soluble mimics of Env have proven challenging to obtain and have been thought to require proteolytic cleavage into two-component subunits, gp120 and gp41, to achieve structural and antigenic mimicry of mature Env spikes on virions. Here we show that replacement of the cleavage site between gp120 and gp41 in a lead soluble gp140 construct, BG505.SOSIP, with flexible linkers can result in molecules that do not require cleavage to fold efficiently into the mature closed state. Our results provide insights into the impact of cleavage on HIV-1 Env folding. In some contexts such as genetic immunization, optimized cleavage-independent soluble gp140 constructs may have utility over the parental BG505.SOSIP, as they would not require furin cleavage to achieve mimicry of mature Env spikes on virions.
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Ivelin S
IS
Georgiev
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
M Gordon
MG
Joyce
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Yongping
Y
Yang
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Mallika
M
Sastry
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Baoshan
B
Zhang
Electron Microscopy Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.
Ulrich
U
Baxa
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Rita E
RE
Chen
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Aliaksandr
A
Druz
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Christopher R
CR
Lees
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Sandeep
S
Narpala
Department of Biology, The Johns Hopkins University, Baltimore, Maryland, USA.
Arne
A
Schön
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Joseph
J
Van Galen
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Gwo-Yu
GY
Chuang
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Jason
J
Gorman
Electron Microscopy Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.
Adam
A
Harned
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Marie
M
Pancera
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Guillaume B E
GB
Stewart-Jones
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Cheng
C
Cheng
Department of Biology, The Johns Hopkins University, Baltimore, Maryland, USA.
Ernesto
E
Freire
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Adrian B
AB
McDermott
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
John R
JR
Mascola
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA pdkwong@nih.gov.
Peter D
PD
Kwong
D019943:Amino Acid Substitution; D015483:HIV Antibodies; D015488:HIV Antigens / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D015497:HIV-1 / Q000235:genetics / Q000276:immunology* / Q000502:physiology*; D006801:Humans; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D018716:Molecular Mimicry; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D054299:env Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
D015483:HIV Antibodies; D015488:HIV Antigens; D011993:Recombinant Fusion Proteins; D054299:env Gene Products, Human Immunodeficiency Virus; C097729:gp140 envelope protein, Human immunodeficiency virus 1