A Directed Molecular Evolution Approach to Improved Immunogenicity of the HIV-1 Envelope Glycoprotein

A prophylactic vaccine is needed to slow the spread of HIV-1 infection. Optimization of the wild-type envelope glycoproteins to create immunogens that can elicit effective neutralizing antibodies is a high priority. Starting with ten genes encoding subtype B HIV-1 gp120 envelope glycoproteins and using in vitro homologous DNA recombination, we created chimeric gp120 variants that were screened for their ability to bind neutralizing monoclonal antibodies. Hundreds of variants were identified with novel antigenic phenotypes that exhibit considerable sequence diversity. Immunization of rabbits with these gp120 variants demonstrated that the majority can induce neutralizing antibodies to HIV-1. One novel variant, called ST-008, induced significantly improved neutralizing antibody responses when assayed against a large panel of primary HIV-1 isolates. Further study of various deletion constructs of ST-008 showed that the enhanced immunogenicity results from a combination of effective DNA priming, an enhanced V3-based response, and an improved response to the constant backbone sequences

Prime-boost immunization of rabbits with HIV-1 gp120 elicits potent neutralization activity against a primary viral isolate

<div><p>Development of a vaccine for HIV-1 requires a detailed understanding of the neutralizing antibody responses that can be experimentally elicited to difficult-to-neutralize primary isolates. Rabbits were immunized with the gp120 subunit of HIV-1 JR-CSF envelope (Env) using a DNA-prime protein-boost regimen. We analyzed five sera that showed potent autologous neutralizing activity (IC50s at ∼10³ to 10⁴ serum dilution) against pseudoviruses containing Env from the primary isolate JR-CSF but not from the related isolate JR-FL. Pseudoviruses were created by exchanging each variable and constant domain of JR-CSF gp120 with that of JR-FL or with mutations in putative N-glycosylation sites. The sera contained different neutralizing activities dependent on C3 and V5, C3 and V4, or V4 regions located on the glycan-rich outer domain of gp120. All sera showed enhanced neutralizing activity toward an Env variant that lacked a glycosylation site in V4. The JR-CSF gp120 epitopes recognized by the sera are generally distinct from those of several well characterized mAbs (targeting conserved sites on Env) or other type-specific responses (targeting V1, V2, or V3 variable regions). The activity of one serum requires specific glycans that are also important for 2G12 neutralization and this serum blocked the binding of 2G12 to gp120. Our findings show that different fine specificities can achieve potent neutralization of HIV-1, yet this strong activity does not result in improved breadth.</p> </div

Comparison of neutralization activities (IC50) of rabbit sera against JR-CSF, JR-FL, and Tier I PSVs using the PhenoSense™ assay.

<p>n.d., not determined.</p

Rabbit sera block binding of mAbs to gp120.

<p>Competition ELISA using various mAbs and sera to block binding of biotinylated (bio) 2G12 (A, C) and b12 (B, D) to immobilized JR-CSF gp120. Two normal rabbit sera are used as non-immune controls. The results shown are representative of three independent experiments. The dashed horizontal line (—) corresponds to the half-maximal signal (EC50). HIVIG: HIV immune globulin provided by J. Mascola (VRC); DEN3: a mAb to dengue virus envelope provided by D. Burton (TSRI).</p

Comparison of neutralization activities (IC50) of rabbit sera against JR-CSF and JR-FL PSVs produced in 293T cells in the presence or absence of kifunensine (kif) or in 293GnTI−/− cells using the TZM-bl assay.

<p>Comparison of neutralization activities (IC50) of rabbit sera against JR-CSF and JR-FL PSVs produced in 293T cells in the presence or absence of kifunensine (kif) or in 293GnTI−/− cells using the TZM-bl assay.</p

Rabbit sera contain neutralizing activities targeting multiple regions in JR-CSF gp120.

<p>(A) IC50 values (1/dilution) in the PhenoSense™ assay for rabbit sera and human plasma (Z23) against JR-CSF, JR-FL, and domain-swap pseudoviruses. The IC50 data are color-coded as described in the legend at the bottom to indicate the fold-change in neutralization sensitivity calculated as (IC50 JR-CSF)/(IC50 variant). (B–F) Neutralization curves for wild-type JR-CSF and those domain-swap pseudoviruses with altered sensitivity to rabbit sera (see text).</p

PNGS mutations demonstrate that similar PNGS are targeted by rabbit sera and mAbs.

<p>(A) IC50 values (µg/ml) in the PhenoSense™ assay for mAbs and CD4-IgG2 against pseudoviruses containing the indicated PNGS mutations. The color-coding scheme is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052732#pone-0052732-g003" target="_blank">Figure 3A</a>; the fold-change for pseudoviruses marked with a cross (†) is calculated relative to JR-FL. *X5 neutralization was tested in the absence of CD4. (B and C) Neutralization curves showing the activity of VRC01 against JR-CSF, JR-FL, and the indicated PNGS mutants.</p

Amino acid sequence alignment of the JR-CSF and JR-FL gp120 proteins.

<p>Amino acid differences are highlighted by grey shading. The potential N-linked glycosylation sites (PNGS) are indicated as follows: PNGS targeted by mutagenesis (yellow boxes); non-targeted PNGS common to JR-CSF and JR-FL (empty boxes); and unique PNGS present only in JR-CSF or JR-FL (red boxes). The constant and variable regions of gp120 are indicated above the aligned sequences using thick grey shading and inward pointing arrows, respectively.</p

Domain swap of C2 alters pseudovirus sensitivity to mAbs.

<p>IC50 values (µg/ml) in the PhenoSense™ assay for mAbs and CD4-IgG2 against JR-CSF, JR-FL, and domain-swap pseudoviruses. The color-coding scheme is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052732#pone-0052732-g003" target="_blank">Figure 3A</a>. * X5 neutralization was tested in the absence of CD4.</p

PNGS mutations modify the neutralizing activity of rabbit sera.

<p>IC50 values (1/dilution) in the PhenoSense™ assay for rabbit sera and human plasma (Z23) against pseudoviruses containing the indicated PNGS mutations. The color-coding scheme is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052732#pone-0052732-g003" target="_blank">Figure 3A</a>; the fold-change values for pseudoviruses marked with a cross (†) are calculated relative to JR-FL.</p