In vivo emergence of HIV-1 highly sensitive to neutralizing antibodies.

BACKGROUND: The rapid and continual viral escape from neutralizing antibodies is well documented in HIV-1 infection. Here we report in vivo emergence of viruses with heightened sensitivity to neutralizing antibodies, sometimes paralleling the development of neutralization escape. METHODOLOGY/PRINCIPAL FINDINGS: Sequential viral envs were amplified from seven HIV-1 infected men monitored from seroconversion up to 5 years after infection. Env-recombinant infectious molecular clones were generated and tested for coreceptor use, macrophage tropism and neutralization sensitivity to homologous and heterologous serum, soluble CD4 and monoclonal antibodies IgG1b12, 2G12 and 17b. We found that HIV-1 evolves sensitivity to contemporaneous neutralizing antibodies during infection. Neutralization sensitive viruses grow out even when potent autologous neutralizing antibodies are present in patient serum. Increased sensitivity to neutralization was associated with susceptibility of the CD4 binding site or epitopes induced after CD4 binding, and mediated by complex envelope determinants including V3 and V4 residues. The development of neutralization sensitive viruses occurred without clinical progression, coreceptor switch or change in tropism for primary macrophages. CONCLUSIONS: We propose that an interplay of selective forces for greater virus replication efficiency without the need to resist neutralizing antibodies in a compartment protected from immune surveillance may explain the temporal course described here for the in vivo emergence of HIV-1 isolates with high sensitivity to neutralizing antibodies

A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10

Novel Envelope Determinants for CCR3 Use by Human Immunodeficiency Virus

Human immunodeficiency virus type 1 can generally use CCR3 and CCR5 for cell entry. We show that envelopes with novel phenotypes arise during “coreceptor switch”: one loses the ability to use CCR3 (R5-only phenotype), and another gains use of CXCR4 in addition to CCR5 and CCR3 (R3/R5/X4-using phenotype). The envelope determinants for CCR3 use mapped to three amino acids. One, N356 in conserved region 3, is a potential glycosylation site and has not previously been associated with coreceptor use. The other two, R440 and N448 in conserved region 4, are proximal to but distinct from residues already identified as being important for CCR5 binding

Llama Antibody Fragments with Cross-Subtype Human Immunodeficiency Virus Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120▿ †

Members of the Camelidae family produce immunoglobulins devoid of light chains. We have characterized variable domains of these heavy chain antibodies, the VHH, from llamas immunized with human immunodeficiency virus type 1 (HIV-1) envelope protein gp120 in order to identify VHH that can inhibit HIV-1 infection. To increase the chances of isolating neutralizing VHH, we employed a functional selection approach, involving panning of phage libraries expressing the VHH repertoire on recombinant gp120, followed by a competitive elution with soluble CD4. By immunizing with gp120 derived from an HIV-1 subtype B′/C primary isolate, followed by panning on gp120 from HIV-1 isolates of subtypes A, B, and C, we could select for VHH with cross-subtype neutralizing activity. Three VHH able to neutralize HIV-1 primary isolates of subtypes B and C were characterized. These bound to recombinant gp120 with affinities close to the suggested affinity ceiling for in vivo-maturated antibodies and competed with soluble CD4 for this binding, indicating that their mechanism of neutralization involves interacting with the functional envelope spike prior to binding to CD4. The most potent VHH in terms of low 50% inhibitory concentration (IC50) and IC90 values and cross-subtype reactivity was A12. These results indicate that camelid VHH can be potent HIV-1 entry inhibitors. Since VHH are stable and can be produced at a relatively low cost, they may be considered for applications such as HIV-1 microbicide development. Antienvelope VHH might also prove useful in defining neutralizing and nonneutralizing epitopes on HIV-1 envelope proteins, with implications for HIV-1 vaccine design

The gp41CHRTM antigen used for immunization.

<p><b>A</b>) Schematic representation of gp41 and of the regions present in gp41CHRTM. FP, fusion peptide; HR1, N-terminal heptad repeat; C-C loop, cysteine loop, HR2, C-terminal heptad repeat; MPER, membrane proximal external region; TM, transmembrane region. The residue numbers at the domain/region boundaries are given. <b>B</b>) Gel filtration chromatogram of recombinant gp41CHRTM, which elutes at 13.3 ml from the column, similar to the elution profile of a marker protein of 158 kDa. This indicates that gp41CHRTM is most likely trimeric and may have an elongated structure. The inset shows a Coomassie stained SDS-PAGE gel with the gp41CHRTM protein band at the left and a protein marker at the right with the marker protein sizes in kDa indicated at the right.</p

Molecular modeling of the 2H10-gp41 peptide interaction suggests that 2H10 W100 is oriented towards the membrane.

<p><b>A</b>) Molecular model of the gp41 peptide interaction with 2H10 produced by HADDOCK. Salt bridges and hydrogen bonds present in the top model and found in most of the top10 models are shown as dashed lines. <b>B</b>) Superpositioning of the Cα atoms of the gp41 peptide derived from the HADDOCK 2H10-peptide model onto the structure of a late fusion intermediate of gp41 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003202#ppat.1003202-Buzon1" target="_blank">[29]</a> shows that the CDR3 W100 is oriented towards the membrane and found in the same plane as gp41 residues W678, W680 and Y681. Note that there are no clashes between the 2H10 VHH and gp41.</p

Epitope mapping of 2H10 and binding to helical peptides.

<p><b>A</b>) The peptide Sym-903 was used for the replacement analysis with 2F5 and 2H10. Underlined cyan colored residues are recognized by the antibodies. <b>B</b>) Full substitution analysis of 2H10 epitope on gp41 MPER. The binding activity of VHH 2H10 at 2 ng/ml with a peptide is shown as a vertical line proportional to the Pepscan ELISA signal. Each group of 19 lines corresponds to the replacement set of all residues except cysteine for each amino acid position in the original 15-mer cyclic peptide: KNEQELLELDKWASL. Within each group of 19 lines the substitutions are in alphabetical order: based on the one-letter amino acid code (ADEFGHIKLMNPQRSTVWY) and the reactivity of the original nonapeptide is shown as a red line. The peptides contained an N- and C-terminal cysteine and were cyclised by addition of a T2-CLIPS <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003202#ppat.1003202-Timmerman1" target="_blank">[115]</a>. These results show that EQ at position 3 and 4 and LE at position 7 and 8 and K at position 11, cannot be replaced by another amino acid without loss of binding and are thus essential for binding 2H10. <b>C</b>) Binding of VHH 2H10 (100 ng/ml) and <b>D</b>) Mab 2F5 (1 ng/ml) to gp41-MPER peptides grafted in GCN4-like helical templates that contain a stabilizing Ile/Leu heptad repeat (top panel) and binding to peptides in which the zipper motif is mutated to destabilizing glycine residues (lower panel). Peptide sequences are all based on hybrids of HIV-1 gp41 and GCN4. Alignment is shown of peptide sequence with heptad repeat positions (top line). Residues essential for 2H10 binding according to the substitution analysis are underlined and residues essential for 2F5 binding are in green. Mutations are shown with one-letter codes and dots indicated that the residue is not changed.</p

2H10, bivalent 2H10 and mutant bi-2H10 IC50 titers against HIV-1 in TZM-bl cells.

<p>Neutralization activities of 2H10, bivalent 2H10, W100A mutant bi-2H10 and as a control 2F5 were determined against the indicated viruses. The type, clade, tier and the accession code of the envelope protein sequence are given for each virus strain. The wild-type bi-2H10s are named bi-15GS and bi-17GS and have a 15 and 17 residue linker respectively. The W100A mutant bi-2H10 is named W100A bi-15GS and has a 15 residue linker. The column labeled Tier indicates whether the virus is classified for 1,2, or 3 assessment of neutralizing antibodies <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003202#ppat.1003202-Mascola3" target="_blank">[116]</a>. The column labeled Epitope sequence, shows the sequence that aligns with the part of the HBX2 sequence to which 2H10 is directed. The underlined, boldfaced residues are essential for the binding of 2H10 and are equal to the residues at the equivalent positions in HXB2, which was used for immunization. Residues in grey are at the position of an essential residue, but deviate from the HBX2 equivalent. • indicates IC50>50 µg/ml; the empty cells represent non-performed measurements.</p

Data collection and refinement statistics.

<p>Numbers in parentheses refer to the highest resolution shell. R_merge = Σ|I _ 〈I〉|/〈ΣI〉, where I = observed intensity. R_cryst = Σ|Fo _ Fc|/Σ|Fo|, where |Fo| = observed structure factor. Amplitude and |Fc| = calculated structure factor amplitude. R_free is R_cryst for 3% of reflections excluded from the refinement.</p