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. 2015 Apr 3;290(14):9195-208.
doi: 10.1074/jbc.M114.621185. Epub 2015 Feb 17.

Antigenicity and immunogenicity of a trimeric envelope protein from an Indian clade C HIV-1 isolate

Rangasamy Sneha Priya  1 , Menon Veena  2 , Irene Kalisz  2 , Stephen Whitney  2 , Dhopeshwarkar Priyanka  2 , Celia C LaBranche  3 , Mullapudi Sri Teja  1 , David C Montefiori  3 , Ranajit Pal  2 , Sundarasamy Mahalingam  4 , Vaniambadi S Kalyanaraman  2
Affiliations

Antigenicity and immunogenicity of a trimeric envelope protein from an Indian clade C HIV-1 isolate

Rangasamy Sneha Priya et al. J Biol Chem. .

Abstract

Human immunodeficiency virus type 1 (HIV-1) isolates from India mainly belong to clade C and are quite distinct from clade C isolates from Africa in terms of their phylogenetic makeup, serotype, and sensitivity to known human broadly neutralizing monoclonal antibodies. Because many of these properties are associated with the envelope proteins of HIV-1, it is of interest to study the envelope proteins of Indian clade C isolates as part of the ongoing efforts to develop a vaccine against HIV-1. To this end, we purified trimeric uncleaved gp145 of a CCR5 tropic Indian clade C HIV-1 (93IN101) from the conditioned medium of 293 cells. The purified protein was shown to be properly folded with stable structure by circular dichroism. Conformational integrity was further demonstrated by its high affinity binding to soluble CD4, CD4 binding site antibodies such as b12 and VRC01, quaternary epitope-specific antibody PG9, and CD4-induced epitope-specific antibody 17b. Sera from rabbits immunized with gp145 elicited high titer antibodies to various domains of gp120 and neutralized a broad spectrum of clade B and clade C HIV-1 isolates. Similar to other clade B and clade C envelope immunogens, most of the Tier 1 neutralizing activity could be absorbed with the V3-specific peptide. Subsequent boosting of these rabbits with a clade B HIV-1 Bal gp145 resulted in an expanded breadth of neutralization of HIV-1 isolates. The present study strongly supports the inclusion of envelopes from Indian isolates in a future mixture of HIV-1 vaccines.

Keywords: AIDS; Antigen; Cell Surface Protein; Human Immunodeficiency Virus (HIV); Vaccine Development.

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Figures

FIGURE 1.
FIGURE 1.
Design and purification of trimeric 93IN101 envelope immunogen. A, schematic representation of 93IN101 full-length gp160 showing the presence of the original signal peptide and intact 4E10 epitope. TM, transmembrane; CT, cytoplasmic tail. The amino acid residues are numbered according to HXB2 numbering. 93IN101 gp145 immunogen with a tissue plasminogen activator (TPA) signal peptide, abolished protease cleavage site, and cytoplasmic tail truncation is shown. A sequence alignment of 2F5-10E8 and 4E10 epitopes from HXB2 and 93IN101 is also shown. B, gel electrophoresis of purified gp120 and gp145 proteins. In native PAGE, pooled trimeric fractions of gp145 after gel filtration (lane 2) were compared with starting material (lane 3). 93IN101 gp145 (lane 2) and gp120 (lane 3) proteins were purified from stable HEK-293 cells and resolved by SDS-PAGE. Purified gp145 was analyzed under reducing conditions in SDS-PAGE after cross-linking with the indicated concentrations of ethylene glycol bis(succinimidyl succinate) (EGS). In all gels, 5 μg of protein was loaded per lane.
FIGURE 2.
FIGURE 2.
Far-UV circular dichroism spectral analysis. Purified 93IN101 gp120 and gp145 trimers were analyzed by CD at room temperature in PBS. mdeg, millidegrees.
FIGURE 3.
FIGURE 3.
Receptor and co-receptor binding site analysis using Octet RED biolayer interferometry. A, Octet sensorgrams generated by binding of serial dilutions of purified envelope gp120 and gp145 in solution to biotinylated sCD4 immobilized on streptavidin sensors. kon, rate of association; kdis, rate of dissociation. The red lines denote 1:1 global curve fits. B, exposure of co-receptor binding site antibody 17b epitope upon CD4 binding to gp120 and gp145 proteins of 93IN101. Env concentrations were 100 nm, and the Env:sCD4 molar ratio was 1:2.
FIGURE 4.
FIGURE 4.
Binding of 93IN101 gp120 and gp145 to CD4 binding site antibodies. A, binding curves generated by coating ELISA wells using gp120 or gp145 and incubating with serial dilutions of b6 (non-neutralizing antibody) or b12, VRC01, and NIH 45–46 G54W (neutralizing antibodies). B, table comparing kinetic parameters. kon is the rate of association and kdis is the rate of dissociation for immobilized gp145 and gp120 binding to various CD4 binding site antibodies in solution using Octet RED biolayer interferometry. X∧2 and R∧2 are measures of the goodness of fit.
FIGURE 5.
FIGURE 5.
Analysis of 93IN101 gp145 binding to known broadly neutralizing human monoclonal antibodies. A, ELISA binding curves generated by coating the plate with gp120 or gp145 and incubating with serial dilutions of various human monoclonal antibodies. B, summary of kinetic parameters. kon is the rate of association and kdis is the rate of dissociation calculated using Octet RED biolayer interferometry for immobilized gp145 binding to PG9, 4E10, and PG16 in solution. X∧2 and R∧2 are measures of the goodness of fit. MPER, membrane-proximal external region.
FIGURE 6.
FIGURE 6.
Protein immunization in New Zealand White rabbits. A, schematic representation of the immunization schedule showing the time of immunization and bleed. B, serial dilutions of rabbit sera were tested for reactivity by ELISA against 93IN101 gp145 protein bound on a plate. Specific IgG quantification was calculated by generating a standard curve. Env sp Ab conc, Env-specific antibody concentration.
FIGURE 7.
FIGURE 7.
Linear epitope mapping of 93IN101 gp145-immunized rabbit sera by ELISA. A, a total of 192 peptides 15 amino acids long with 11-amino acid overlapping sequence covering the entire 93IN101 gp160 protein were used. Sera from 2 weeks after the final protein immunization were tested for reactivity at a dilution of 1:50. C2507, C2508, C2509, and C2510 are rabbit identification numbers used in the study. B, titers to V1, V2, and V3 linear peptides of 93IN101 gp120. MPER, membrane-proximal external region; TM, transmembrane domain; HR, heptad repeat; IM, immuno dominant region.
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