Location of mutations identified by swarm analysis that mediate resistance to neutralization in CRF01_AE viruses.

<p>(A) Ribbon diagram of the V1/V2 domain is based on the structure (PDB 3U4E) of McLellan et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119608#pone.0119608.ref020" target="_blank">20</a>]. Strands A-D of the four-stranded β-sheet structure are indicated. Locations of predicted glycosylation sites and mutations that determine sensitivity and resistance to neutralizing polyclonal or monoclonal antibodies are indicated in red. Mutations at glycosylation sites in the V1 domain of CRF01_AE viruses that determine sensitivity and resistance to neutralization are located at the junction of the A and B strands at positions 136 and 149. Also shown are the relative locations of mutations at positions 167, 180, and 197 described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119608#pone.0119608.ref015" target="_blank">15</a>], that conferred sensitivity and resistance to neutralization. The locations of glycosylation sites at positions 156 and 160 in the B strand required for the binding of the PG9 MAb [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119608#pone.0119608.ref020" target="_blank">20</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119608#pone.0119608.ref023" target="_blank">23</a>] are indicated. Disulfide bonds are shown in yellow. (B) Diagram of trimeric gp140 derived from the PDB 4NCO structure of Julien et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119608#pone.0119608.ref018" target="_blank">18</a>]. In this structure, sequences in the V1/V2 domain are colored violet and sequences from the V3 domain are colored green. Red balls indicate the location of the glycosylation sites at 136 and 149 identified by swarm analysis and the locations of the N301 and N332 glycosylation sites in the V3 domain required for the binding of the PGT121, PGT122, and PGT128 bN-MAbs. (C) Surface diagram of the gp140 trimer derived from the PDB 3J5M structure of Lyumkis et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119608#pone.0119608.ref017" target="_blank">17</a>], shows the location of the glycans at N136, N149, N301, and N332 recognized by neutralizing monoclonal and polyclonal antibodies. In panels C and D, sequences from the V1/V2 domain are shaded violet and sequences from the V3 domain are shaded green. The hypervariable connecting peptide between the A and B strands is shaded cyan. (D) Magnified view of the surface of the gp140 trimer showing the locations of glycosylation sites (red) at N136, N149, N301, and N332 that affect the binding of bNAbs. (E) Alignment of sequences from the CRF01_AE viruses described in this paper along with the HXB2 reference sequence and the sequence of the BG505 Env used for determination of the 3-dimensional structure of the gp140 trimer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119608#pone.0119608.ref017" target="_blank">17</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119608#pone.0119608.ref018" target="_blank">18</a>]. The hypervariable region is indicated in cyan. The location of PNGS in the hypervariable domain is indicated in red. Sequence number is provided with reference to the HXB2 sequence and to the numbering of BG505 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119608#pone.0119608.ref018" target="_blank">18</a>].</p

Length and number of PNGS in the V1/V2 domain of gp120 from CRF01_AE viruses.

<p>*VAX003 values represent mean titers obtained from the analysis of 563 sequences from 182 individuals. The amino acids in the V1 and V2 domains were based on the structure of Leonard et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119608#pone.0119608.ref052" target="_blank">52</a>] with numbering based on the HXB2 reference strain. The V1 domain corresponds to positions 131–156 and the V2 domain corresponds to positions 157–196. ¹ includes N130; ² includes N197.</p><p>Length and number of PNGS in the V1/V2 domain of gp120 from CRF01_AE viruses.</p

Mutational analysis to map residues responsible for differences in neutralization sensitivity and resistance in Envs from subject 113035.

<p>Amino acids from the neutralization-sensitive clone (007) were systematically inserted into the neutralization-resistant Env (045). (A) Effect of amino acid polymorphisms on neutralization by plasma from four HIV-1 infected subjects. The neutralizing antibody titer (IC₅₀) is defined as the reciprocal of the plasma dilution that produces a 50% inhibition in target cell infection. Values in bold represent significant neutralization titers that are at least three times greater than those observed against the negative control (aMLV). Panel A, open rectangle, indicates neutralization titers for the wildtype resistant (wtR) clone; black rectangle, indicates neutralization titers for the wildtype sensitive (wtS) clone; gray rectangle, indicates the single amino acid substitution that converted the neutralization-resistant Env into a neutralization-sensitive Env. NI, indicates a non-infectious mutant. Panels B and C represent graphs and statistical analysis of neutralization of 113035 mutants with the T500208 and Z23 plasma, respectively. Panels B and C, closed square (■) indicates neutralization titers of wtS clone 007; open square (□) indicates neutralization titer of wtR clone 045. Open triangles (△) indicate neutralization titers of wtR clone 045 incorporating the N150S mutation. Closed circles (●) indicate neutralization by the other mutants listed in panel A. Statistical significance was calculated using an unpaired t test.</p

Diagram of sequence differences between pairs of neutralization-sensitive and -resistant Envs of CRF01_AE viruses.

<p>The locations of sequence differences between wildtype neutralization-resistant (wtR) and wildtype neutralization-sensitive (wtS) envelope genes from subjects 107747, 113035, and 142902 are indicated by vertical lines. The location of predicted N-linked glycosylation sites in the V1 domain that alter neutralization sensitivity is marked by red circles.</p

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

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 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