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. 2004 Jun;78(11):5651-7.
doi: 10.1128/JVI.78.11.5651-5657.2004.

Increased sensitivity to CD4 binding site-directed neutralization following in vitro propagation on primary lymphocytes of a neutralization-resistant human immunodeficiency virus IIIB strain isolated from an accidentally infected laboratory worker

Tim Beaumont  1 , Esther Quakkelaar, Ad van Nuenen, Ralph Pantophlet, Hanneke Schuitemaker
Affiliations

Increased sensitivity to CD4 binding site-directed neutralization following in vitro propagation on primary lymphocytes of a neutralization-resistant human immunodeficiency virus IIIB strain isolated from an accidentally infected laboratory worker

Tim Beaumont et al. J Virol. 2004 Jun.

Abstract

We previously described the adaptation of the neutralization-sensitive human immunodeficiency virus type 1 (HIV-1) strain IIIB to a neutralization-resistant phenotype in an accidentally infected laboratory worker. During long-term propagation of this resistant isolate, designated FF3346, on primary peripheral blood leukocytes in vitro, an HIV-1 variant appeared that had regained sensitivity to neutralization by soluble CD4 (sCD4) and the broadly neutralizing monoclonal antibody b12. When an early passage of FF3346 was subjected to limiting-dilution culture in peripheral blood mononuclear cells, eight virus variants with various degrees of neutralization resistance were isolated. Two of them, the sCD4 neutralization-resistant variant LW_H8(res) and the sCD4 neutralization-sensitive variant LW_G9(sens), were selected for further study. Interestingly, these two viruses were equally resistant to neutralization by agents that recognize domains other than the CD4 binding site. Site-directed mutagenesis revealed that the increased neutralization sensitivity of variant LW_G9(sens) resulted from only two changes, an Asn-to-Ser substitution at position 164 in the V2 loop and an Ala-to-Glu substitution at position 370 in the C3 domain of gp120. In agreement with this notion, the affinity of b12 for monomeric gp120 containing the N164S and A370E substitutions in the background of the molecular clone LW_H8(res) was higher than its affinity for the parental gp120. Surprisingly, no correlation was observed between CD4 binding affinity for monomeric gp120 and the level of neutralization resistance, suggesting that differences in sCD4 neutralization sensitivity between these viruses are only manifested in the context of the tertiary or quaternary structure of gp120 on the viral surface. The results obtained here indicate that the neutralization-sensitive strain IIIB can become neutralization resistant in vivo under selective pressure by neutralizing antibodies but that this resistance may be easily reversed in the absence of immunological pressure.

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Figures

FIG. 1.
FIG. 1.
Amino acid sequences of virus clones LW_H8res and LW_G9sens in comparison to IIIB. The viruses were isolated by limiting dilution from PBMCs infected with an isolate (FF3346) obtained 7 years after accidental infection of a laboratory worker. The predicted amino acid sequences of the envelope glycoproteins were derived from the consensus sequences of the envelope fragments ranging from V1 to V5, which were amplified and sequenced from DNA isolated from infected PBMC cultures. Variable and constant domains are indicated, and the numbering of amino acid positions is relative to HXB2; dots indicate residues identical to those in the reference sequence, and dashes indicate amino acid deletions relative to HXB2. The location of the restriction site for StuI is also depicted.
FIG. 2.
FIG. 2.
Neutralization sensitivity of LW_H8res (open circles), LW_G9sens (solid circles), and IIIB (gray diamonds). We incubated 100 TCID50 of each virus inoculum with increasing concentrations of (a) sCD4, (b) MAb b12, (c) MAb gp13, (d) MAb gp68, (e) MAb F105, (f) MAb 902, (g) pooled polyclonal serum Amshps, and (h) pooled polyclonal serum HIVIg. MAbs b12, gp13, gp68, and F105 are directed to epitopes overlapping the CD4bs of gp120, whereas MAb 902 is directed to an epitope in the V3 loop of gp120. Incubation of virus with sCD4, MAbs, or sera was performed for 2 h at 37°C before PHA-stimulated PBMCs were added. The concentration of p24 in culture supernatants was measured, and the percent viral inhibition was calculated as the mean reduction in p24 level in triplicate cultures in the presence of the neutralizing ligand compared to that in cultures with virus only. IC50s were determined by linear regression when possible. Neutralization assays were performed at least twice.
FIG. 3.
FIG. 3.
Construction of chimeric virus clones and analysis of their sensitivity to neutralization by sCD4 and MAb b12. (A) The SalI/BamHI fragment of the LW_H8res gp120 envelope was inserted into the pLAI-2 vector, which contains the molecular clone of HIV-1 strain IIIB, to yield pLAI-H8. Site-directed mutagenesis was used to engineer three different variants of pLAI-H8 with amino acid substitutions at positions 164 and 370, pLAI-H8/E370, pLAI-H8/S164, and pLAI-H8/S164/E370 (the changes of A370 to Glu and N164 to Ser in the background of pLAI-H8 are depicted). In addition, the V1/V2 fragment of strain LW_G9res was placed into pLAI-H8, to yield pLAI-H8/G9V1V2, or into pLAI-H8/E370, to yield pLAI-H8/E370/G9V1V2. (B) Neutralization sensitivity of the chimeric molecular virus clones pLAI-H8 (open squares), pLAI-H8/G9V1V2 (open circles), pLAI-H8/E370/G9V1V2 (solid circles), pLAI-H8/E370 (solid squares), pLAI-H8/S164 (open triangles), and pLAI-H8/S164/E370 (solid triangles) for sCD4 and MAb b12. Virus stocks of the chimeric molecular clones were obtained by infection of PHA-stimulated PBMCs with supernatant from transfected 293T cells. Neutralization experiments were performed as described for Fig. 1.
FIG. 4.
FIG. 4.
Binding of MAbs b12 and CD4-IgG2 to monomeric gp120 of IIIB (solid squares) and gp120 of virus derived from pLAI-H8 (solid triangles), pLAI-H8/E370 (open triangles), pLAI-H8/S164 (solid circles), and pLAI-H8/S164/E370 (open circles). gp120, standardized to equivalent amounts by using HIVIg (1 μg/ml), was captured on microtiter plate wells with an anti-C5 antibody and then probed with antibody at the concentrations indicated. Absorbance was measured at 450 nm after 30 min.
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References

    1. Beaumont, T., A. van Nuenen, S. Broersen, W. A. Blattner, V. V. Lukashov, and H. Schuitemaker. 2001. Reversal of human immunodeficiency virus type 1 IIIB to a neutralization-resistant phenotype in an accidentally infected laboratory worker with a progressive clinical course. J. Virol. 75:2246-2252. - PMC - PubMed
    1. Blaak, H., M. Brouwer, L. J. Ran, F. de Wolf, and H. Schuitemaker. 1998. In vitro replication kinetics of human immunodeficiency virus type 1 (HIV-1) variants in relation to virus load in long-term survivors of HIV-1 infection. J. Infect. Dis. 177:600-610. - PubMed
    1. Cao, Y., L. Qin, L. Zhang, J. Safrit, and D. D. Ho. 1995. Virologic and immunologic characterization of long-term survivors of human immunodeficiency virus type 1 infection. N. Engl. J. Med. 332:201-208. - PubMed
    1. Cavrois, M., C. De Noronha, and W. C. Greene. 2002. A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat. Biotechnol. 20:1151-1154. - PubMed
    1. Cecilia, D., C. Kleeberger, A. Munoz, J. V. Giorgi, and S. Zolla-Pazner. 1999. A longitudinal study of neutralizing antibodies and disease progression in HIV-1-infected subjects. J. Infect. Dis. 179:1365-1374. - PubMed

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