Design of immunogens that present the crown of the HIV-1 V3 loop in a conformation competent to generate 447-52D-like antibodies

Abstract

gp120 is a subunit of the envelope glycoprotein of HIV-1. The third variable loop region of gp120 (V3 loop) contains multiple immunodominant epitopes and is also functionally important for deciding cell-tropism of the virus. 447-52D is a monoclonal antibody that recognizes the conserved tip of the V3 loop in a beta-turn conformation. This antibody has previously been shown to neutralize diverse strains of the virus. In an attempt to generate an immunogen competent to generate 447-52D-like antibodies, the known epitope of 447-52D was inserted at three different surface loop locations in the small, stable protein Escherichia coli Trx (thioredoxin). At one of the three locations (between residues 74 and 75), the insertion was tolerated, the resulting protein was stable and soluble, and bound 447-52D with an affinity similar to that of intact gp120. Upon immunization, the V3 peptide-inserted Trx scaffold was able to generate anti-V3 antibodies that could compete out 447-52D binding to gp120. Epitope mapping studies demonstrated that these anti-V3 antibodies recognized the same epitope as 447-52D. Although the 447-52D-type antibodies were estimated to be present at concentrations of 50-400 microg/ml of serum, these were not able to effect neutralization of strains like JRFL and BAL but could neutralize the sensitive MN strain. The data suggest that because of the low accessibility of the V3 loop on primary isolates such as JRFL, it will be difficult to elicit a V3-specific, 447-52D-like antibody response to effectively neutralize such isolates.

Figure 1. Structure of the V3 peptide, Trx and the sequence of the V3 loop of JRFL gp120

(A) The conformation of the V3 peptide, derived from the MN strain of HIV-1, in complex with the 447-52D antibody [31]. Only the structure of the peptide is shown for clarity. (B) Structure of Trx [43] and the positions where the V3 peptide was inserted. The loop regions used for V3 peptide insertion are indicated with arrows. (C) The amino acid sequence of the V3 loop of JRFL gp120 is shown. Residues 305–320 comprising the V3 peptide insertion described in the text are highlighted in boldface. (D) Sequence alignment of residues 305–320 of the V3 peptide from HIV-1 strains JRFL, MN and IIIB.

Figure 2. CD spectra of Trx derivatives

Spectra for NHisTrx (1), 74NHisTrxV3 (2), 83NHisTrxV3 (3), 74NHisTrxV3(307) (4) and 74NHisTrxV3(308) (5) show that there is no significant structural change in Trx after the insertion of the V3 peptide

Figure 3. Direct binding of 447-52D to TrxV3 derivatives as assayed by ELISA

TrxV3 derivatives were immobilized on the microwell surface and incubated with various concentrations of 447-52D. The binding of 74NHisTrxV3(308) to 447-52D is significantly weaker compared with other TrxV3 derivatives. OD, absorbance.

Figure 4. SPR determination of kinetic and equilibrium dissociation constants for binding of gp120 and TrxV3 derivatives to immobilized 447-52D

Surface density, 1000 RU; buffer, 10 mM phosphate (pH 7.4), 150 mM NaCl and 0.0005% Tween 20; flow rate, 30 μl/min. Sensorgram overlays are shown for the binding of gp120 (A), 74NHisTrxV3 (B), 83NHisTrxV3 (C), 74NHisTrxV3(307) (D) and 74NHisTrxV3(308) (E). The analyte concentrations are indicated along with the curves. Data points are shown as filled circles and the fits as solid lines. No fits are shown for (E) as the data could not be reliably fitted. Only every fifth data point is shown for the curves (A– D) for convenience in visualization of the data.

Figure 5. SPR-based competition of 447-52D with antibodies from sera obtained from animals immunized with 74NHisTrxV3

Sera were used at the indicated dilutions. gp120 alone (1) or pre-incubated with dilutions of 2 μl/ml of serum 1 (2), 0.5 μl/ml of serum 2 (3), 4 μl/ml of serum 3 (4) or 200 ng/ml of 447-52D (5) was passed over the 447-52D surface. The sensorgrams were obtained for several different concentrations of sera and 447-52D. However, only traces corresponding to 50% of the maximal binding in absence of antibodies are shown.

Figure 6. Competition ELISA to map the epitopes of 447-52D and V3-specific antibodies in 74NHisTrxV3-elicited immuneserum

gp120 was coated on the microwell surface and the ability of a series of overlapping peptides, spanning the V3 peptide sequence used in the present study, to compete out the binding of (A) 447-52D or (B) anti-74NHisTrxV3 serum to gp120 was monitored. Peptide sequences correspond to HIV-1 consensus subtype B envelope amino acids 297–313 (Pep1), 301–317 (Pep2), 305–321 (Pep3), 309–325 (Pep4) and 313–328 (Pep5). The region of the V3 loop present in the TrxV3 constructs consisted of amino acids 305–320 of gp120. The conserved GPG tip consists of amino acids 312–314. Amino acids are numbered according to the reference HXB2 sequence [53]. OD, absorbance.