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. 2008 Mar;82(5):2367-75.
doi: 10.1128/JVI.02161-07. Epub 2007 Dec 19.

4E10-resistant variants in a human immunodeficiency virus type 1 subtype C-infected individual with an anti-membrane-proximal external region-neutralizing antibody response

Elin S Gray  1 , Penny L Moore, Frederic Bibollet-Ruche, Hui Li, Julie M Decker, Tammy Meyers, George M Shaw, Lynn Morris
Affiliations

4E10-resistant variants in a human immunodeficiency virus type 1 subtype C-infected individual with an anti-membrane-proximal external region-neutralizing antibody response

Elin S Gray et al. J Virol. 2008 Mar.

Abstract

The broadly neutralizing monoclonal antibody (MAb) 4E10 recognizes a linear epitope in the C terminus of the membrane-proximal external region (MPER) of gp41. This epitope is particularly attractive for vaccine design because it is highly conserved among human immunodeficiency virus type 1 (HIV-1) strains and neutralization escape in vivo has not been observed. Multiple env genes were cloned from an HIV-1 subtype C virus isolated from a 7-year-old perinatally infected child who had anti-MPER neutralizing antibodies. One clone (TM20.13) was resistant to 4E10 neutralization as a result of an F673L substitution in the MPER. Frequency analysis showed that F673L was present in 33% of the viral variants and in all cases was linked to the presence of an intact 2F5 epitope. Two other envelope clones were sensitive to 4E10 neutralization, but TM20.5 was 10-fold less sensitive than TM20.6. Substitutions at positions 674 and 677 within the MPER rendered TM20.5 more sensitive to 4E10 but had no effect on TM20.6. Using chimeric and mutant constructs of these two variants, we further demonstrated that the lentivirus lytic peptide-2 domain in the cytoplasmic tail affected the accessibility of the 4E10 epitope, as well as virus infectivity. Collectively, these genetic changes in the face of a neutralizing antibody response to the MPER strongly suggested immune escape from antibody responses targeting this region.

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Figures

FIG. 1.
FIG. 1.
Frequency analysis of substitutions in the MPER regions of 43 gp41 molecular clones obtained from the TM20 isolate. The substitutions K665S and F673L, associated with 2F5 and 4E10 resistance, respectively, are underlined and in bold. The functional envelope clones corresponding to some of these genotypes are indicated to the left of the sequences.
FIG. 2.
FIG. 2.
Neutralization of TM20 envelope clones. The three functional clones were tested for neutralization by MAbs 4E10 and 2F5 and the entry inhibitor T-20 (enfuvirtide) (A) and by polyclonal antibodies from two broadly cross-reactive HIV-1-positive plasma specimens and autologous contemporaneous sera (B). The dotted lines indicate 50% neutralization, with only those values above the line considered positive.
FIG. 3.
FIG. 3.
Full-length amino acid sequences of the functional envelope clones TM20.5, TM20.6, and TM20.13. Variable regions, HR domains (HR1 and HR2), the MPER, the membrane-spanning domain (MSD), and LLPs (LLP-1 and LLP-2) are indicated. The mutation F673L in the MPER is underlined. The sensitivity of each of the clones to 2F5 and 4E10 neutralization is indicated, with R denoting resistance and S denoting sensitivity. TM20.6 was extremely sensitive to 4E10 neutralization and is indicated with S++.
FIG. 4.
FIG. 4.
Changes in positions 674 and 677 in the MPER affect 4E10 neutralization. TM20.6, TM20.5, and mutants TM20.6 D674N/K677N and TM20.5 N674D/N677K were tested for their sensitivity to neutralization by 4E10. Neutralization was scored as the antibody concentration required to reduce infectivity by 50% (IC50). The graph shows the means and standard deviations from three independent experiments. P values are indicated when statistically significant differences between the means were observed in a Mann-Whitney nonparametric t test analysis.
FIG. 5.
FIG. 5.
Changes in the cytoplasmic tail affect neutralization sensitivity. Schematic representations of the chimeras, constructed by exchanging gp120 or cytoplasmic tail segments of TM20.5 and TM20.6, and LLP-2 and MPER mutants are shown. All the constructs were tested for 4E10, IgG1b12, and T-20 neutralization. The mean IC50s from three independent experiments are indicated on the right. The IC50s of the chimeras and mutants were compared to those of the parental clone TM20.5 or TM20.6, and the IC50 ratio is shown in each case. Statistically significant differences between means of the parental and chimeric/mutant IC50s with P values of <0.05 and <0.01 in a Mann-Whitney t test are highlighted in light and dark gray, respectively.
FIG. 6.
FIG. 6.
Roles of gp120 and the cytoplasmic tail in infectivity and envelope incorporation. (A) JC53bl-13 cells were infected with equal amounts of p24 (10 ng) of each parental and chimeric Env-pseudotyped virus. Infectivity was determined by luciferase expression measured as relative light units (RLU). The bars are color coded according to the cytoplasmic tail carried by the construct, black for TM20.5 and white for TM20.6. Error bars indicate standard deviations. (B and C) Pelleted virions (B) and env-transfected cells (C) were lysed, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by Western blotting with anti-gp120 (D7312), anti-gp41 (7B2), or anti-p24 (D7312) antibodies. (D) Schematic representation of the gp120, gp41 ectodomain, and cytoplasmic tail encoded by the chimeric constructs. Regions derived from clone TM20.5 are shaded gray, and regions derived from TM20.6 are in white.
FIG. 7.
FIG. 7.
Anti-MPER neutralization activity present in TM20 serum. The HIV-1 MPER sequences introduced into the 7321A HIV-2 chimeric or mutant viruses used in the neutralization assay are highlighted in gray. The bold letters represent the sequence of the intact 2F5 and 4E10 epitope. The mutations N671S and T676S in C1Cm and F673L in C1CF/L are underlined. The ID50 titers are indicated on the right, with those showing activity highlighted in gray.
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