Characterization of VRC01, a potent and broadly neutralizing anti-HIV mAb, produced in transiently and stably transformed tobacco

Abstract

The proposed clinical trial in Africa of VRC01, a potent broadly neutralizing antibody (bNAb) capable of neutralizing 91% of known HIV-1 isolates, raises concerns about testing a treatment which will be too expensive to be accessible by the most important target population, the poor in under-developed regions such as sub-Saharan Africa. Here, we report the expression of VRC01 in plants as an economic alternative to conventional mammalian-cell-based production platforms. The heavy and light chain genes of VRC01 were cloned onto a single vector, pTRAk.2, which was transformed into Nicotiana benthamiana or Nicotiana tabacum using transient and stable expression production systems respectively. VRC01 has been successfully expressed transiently in plants with expression level of approximately 80 mg antibody/kg; stable transgenic lines expressing up to 100 mg antibody/kg were also obtained. Plant-produced VRC01 from both systems showed a largely homogeneous N-glycosylation profile with a single dominant glycoform. The binding kinetics to gp120 IIIB (approximately 1 nM), neutralization of HIV-1 BaL or a panel of 10 VRC01-sensitive HIV-1 Env pseudoviruses of VRC01 produced in transient and stable plants were also consistent with VRC01 from HEK cells.

Keywords: HIV-1 broadly neutralizing antibody; VRC01; pTRAk.2; stable transgenics; transient expression.

Figure 1

Expression profiles of VRC01 produced in plants and HEK cells. (a) Coomassie‐stained SDS‐PAGE gel showing VRC01 purified from transient plants (left panel) and stable transformants (T₀ generation; middle panel) under nonreducing (NR) and reducing (R) conditions. Commercially available human IgG1 kappa from human serum (Sigma) was used as positive control (right panel). Five micrograms of purified protein was loaded onto each lane. Full‐length IgGs on nonreducing gels, as well as the heavy and light chain on reducing gels, are indicated by black arrows. (b–e) Nonreducing (b and d) and reducing (c and e) Western blot analysis of VRC01 in 1) transient plant crude extract, 2) crude extract from stable transformants (T₀ generation) and 3) VRC01 purified from HEK cells. The heavy chain was detected using a polyclonal goat anti‐human IgG (Fc fragment) antiserum (b and c), and the light chain was detected using a goat anti‐human IgG kappa chain antibody (d and e). Full‐length IgG, as well as heavy and light chains, are indicated by black arrows.

Figure 2

Characterization of N‐glycan population of VRC01 from HEK cells, transient plants and transgenic plants. (a) Amino acid sequence of VRC01 heavy (above) and light chain (below). The highlighted portion represents the glycopeptide fragment generated after trypsin/GluC digest and subsequently analysed using LC‐ESI‐MS. Potential N‐glycosylation sites are underlined: the heavy chain has an Asn/Ser/Thr consensus sequence in the C_H2 region; the light chain has an Asn/Leu/Thr consensus sequence in the FR3 of V_L region. (b, c and d) Deconvoluted glycopeptide spectra of VRC01 derived from (b) HEK cells, (c) transiently transformed Nicotiana benthamiana plants and (d) transgenic Nicotiana tabacum plants as determined by LC‐ESI‐MS after S‐carbamidomethylation and trypsin/GluC digest. The glycopeptides analysed were EEQYNSTYR (1189.51 Da) in the heavy chain (top panel) and WGPDYNLTISNLE (1521.72 Da) in the light chain (bottom panel; glycosylation sites are underlined). Marked peaks correspond to glycans with different m/z values. Diamonds, white circles, dark squares, grey circles, triangles and stars represent N‐acetylneuraminic acid, galactose, N‐acetylglucosamine, mannose, fucose and xylose, respectively.

Figure 3

Binding kinetics of VRC01 from plant and HEK cells to HIV gp120 IIIB. (a) SPR sensorgram comparing the interaction of Protein‐A captured VRC01 from transient plants (VRC01 transient), stably transformed plants (VRC01 stable) and HEK cells (VRC01 HEK) with recombinant gp120 III. Protein A was immobilized on a CM5 chip, and VRC01 antibodies were captured to ≈100RU. gp120 were injected at a concentration of 20 μg/mL. Experimental curves are shown as grey lines and fitted curves shown in black. Sensorgram shown represents one of three similar experiments. (b) Binding kinetics: association constant (K_a), dissociation constant (K_d) and affinity (K_D) are shown for VRC01 from three sources. Each assay was performed in triplicate. Error bars denotes ± SD.

Figure 4

Neutralization of HIV‐1 virus and pseudoviruses by VRC01 produced in plants and HEK cells. (a) Neutralization curves of transient plant‐ (), stable transgenic plant ‐() and HEK cells ‐()produced VRC01 against HIV‐1BaL; tested using TZM‐bl cells. A nonspecific plant‐derived antibody was used as negative control (). Error bars denotes ± SD. Each assay was repeated three times. (b) Neutralization IC₅₀ values (μg/mL) of VRC01 produced in transient and stable transgenic plants as well as HEK cells against HIV‐1 BaL (clade B) and 10 VRC01‐sensitive HIV‐1 Env pseudoviruses from clade A, B, C and G; tested using TZM‐bl cells.