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. 2003 Nov;77(21):11385-97.
doi: 10.1128/jvi.77.21.11385-11397.2003.

Polyclonal immunoglobulin G from patients neutralizes human immunodeficiency virus type 1 primary isolates by binding free virions, but without interfering with an initial CD4-independent attachment of the virus to primary blood mononuclear cells

Renaud Burrer  1 , Sandrine Haessig-Einius, Anne-Marie Aubertin, Christiane Moog
Affiliations

Polyclonal immunoglobulin G from patients neutralizes human immunodeficiency virus type 1 primary isolates by binding free virions, but without interfering with an initial CD4-independent attachment of the virus to primary blood mononuclear cells

Renaud Burrer et al. J Virol. 2003 Nov.

Abstract

We investigated the relationship between human immunodeficiency virus type 1 (HIV-1) primary isolate (PI) antibody-mediated neutralization and attachment to primary blood mononuclear cells (PBMC). Incubation of PIs with immunoglobulin G (IgG) purified from infected patients did not inhibit attachment of the viruses with PBMC, but partial to complete neutralization was achieved. Neutralization of PIs already fixed on the cells was achieved by some IgG samples only and was of limited intensity compared to the former neutralization protocol. On the contrary, the binding of IgG to free virions was shown to be sufficient to reach potent neutralization, as the infectivity of IgG-PI complexes purified from the bulk of antibodies before addition to PBMC was strongly diminished compared to mock-treated controls. Monoclonal antibodies to the CDR2 domain of CD4 completely inhibited the infection of PBMC without interfering with the attachment of PIs to the cells, suggesting that, under these experimental conditions, the initial attachment of viruses to PBMC involves alternative cellular receptors. This initial interaction may also involve other components of the viral envelope than gp120, as partial depletion of the surface glycoproteins of primary viral particles that resulted in an almost complete loss of infectivity did not impair attachment to PBMC. A limited inhibition of attachment was observed when interfering with putative interactions with cellular heparan sulfate, whereas no effect was observed for cellular CD147 or nucleolin or for virion-incorporated cyclophilin A. Altogether, our results favor a mechanism of neutralization of HIV-1 PIs by polyclonal IgG where antibodies predominantly bind free virions and neutralize without interfering with the attachment to PBMC, which, in this model, is mainly CD4 independent.

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Figures

FIG. 1.
FIG. 1.
Purification of virus by gel filtration. The graph shows a representative elution profile of virus culture supernatant purification, which is intended to separate viral particles from debris. The presence of infectious PI Bx08 (grey bars) (peak value, TCID50 = 2,435 per ml) and viral proteins p24 (empty squares) (peak value, 793 ng/ml) and gp120 (black triangles) (relative units) was measured in the collected fractions. Five 1.5-ml fractions (dashed line) were pooled to obtain the purified virus sample.
FIG. 2.
FIG. 2.
Attachment of PIs to PBMC in the presence of neutralizing IgG from patients. The effect of IgG samples (diluted 1/15 compared to serum) on attachment of HIV-1 PIs Bx08, Bx17, and 11105C was determined after 2 h of incubation on ice. The attachment of the virus was measured as the p24 that remained associated to the cells after washings (empty bars). For each experiment, we added a sample treated with trypsin to confirm both the efficient removal of free virus by the washing procedure (the corresponding p24 levels should not be affected by protease treatment) and the extracellular localization of the PBMC-associated virus (cell-internalized virus becomes inaccessible to trypsin). Aliquots of each sample were cultured for 36 h at 37°C both in complete medium (black bars) and in complete medium supplemented with AZT (hatched bars) to assess neutralization and background p24, respectively. The results represent the means and standard deviations of the results of three independent experiments and are expressed as percentages of the positive controls. For these samples, the relative attachment and replication are 100% by definition, and the mean absolute p24 concentrations for attachment and replication are given above the corresponding bars.
FIG. 3.
FIG. 3.
Neutralization of PBMC-bound PIs by purified IgG from infected patients. Postattachment neutralization was determined by adding the IgG only after 2 h of adsorption of HIV-1 PIs to PBMC on ice, where free virus was removed before the addition of Abs for 2 h at 4°C (experimental condition I) (squares). In parallel, IgGs were also preincubated with the virus and present during virus-cell attachment at 4°C, before being removed together with the viral inoculum (experimental condition II) (triangles), and in experimental conditions III, the IgGs were present throughout the entire experiment (circles). Virus replication was assessed by p24 measured in the culture supernatant in the absence (solid line) or presence (dashed) of AZT as indicated in Materials and Methods. Each value corresponds to the mean of the results of two or three independent experiments.
FIG. 4.
FIG. 4.
Purification and measurement of the infectivity of IgG-virion complexes. (A) Antibodies complexed to viral particles, obtained by incubating purified virus with IgG samples at a dilution of 1/10, were separated from free IgG by gel filtration. The figure shows the representative elution profiles of 4 columns performed in parallel, with the dosage of p24 (plain lines) and IgG (dashed lines) concentrations in each fraction. (B) The fractions corresponding to the peak of p24 (arrow on panel A) were titrated on PBMC. Infectivity is expressed as the percentage of the TCID50 of each sample compared to that of the mock-treated control, with all the samples being tested in parallel with the same purified virus preparation for a given PI (i.e., up to 8 columns in parallel). Each value corresponds to the mean and standard deviation of the results of three independent experiments. Lines indicate 0% (solid), 50% (—), and 90% (.....) inhibition of infectivity. Stars denote statistically significant reductions in TCID50 (Student test, P = 0.05).
FIG. 5.
FIG. 5.
Attachment and neutralization of HIV-1 PIs in the presence of anti-CD4 MAbs, sCD4, and CD4-IgG2. Bx08 (A and B) and Bx17 (C) neutralization (diamonds) and attachment to PBMC (triangles) were measured under conditions where the PBMC were preincubated with anti-CD4 MAbs (A and C, left) or where the virus was preincubated with sCD4 or CD4-IgG2 (B and C, right). Each value corresponds to the mean and standard deviation of the results of at least two independent experiments, except for CD4-IgG2 with Bx17, which was performed only once. Cultures of samples in the presence of 10−6 M AZT (dashed line, open diamonds) give background p24 values for neutralization.
FIG. 6.
FIG. 6.
sCD4 and CD4-IgG2 induce the shedding of Bx08 gp120. Purified virus was incubated with PBS (mock-treated control, left panel) or 50-μg/ml sCD4 (central panel) or CD4-IgG2 (right panel) for 1 h at 37°C, and viral particles were further purified by parallel gel filtration columns. Dosage determinations for the viral proteins p24 (empty squares) and gp120 (black triangles) in the collected fractions were performed as indicated in Materials and Methods.
FIG. 7.
FIG. 7.
Attachment and neutralization of gp120-depleted viral particles. Bx08 culture supernatant was incubated with PBS (mock-treated control) or 50-μg/ml sCD4 for 1 h at 37°C, and viral particles were purified by gel filtration as previously described. The fraction corresponding to the first peak of p24 (Fig. 6) was used to determine the attachment to PBMC (empty bars), neutralization (black bars), and background p24 after culture in the presence of AZT (hatched bars). The p24 and gp120 concentrations were measured in the fractions that were used for the attachment experiment (bottom values) to assess the content in viral particles and the extent of shedding of the samples to be compared. The data correspond to the means and standard deviations of the results of two independent experiments.
FIG. 8.
FIG. 8.
Attachment of HIV-1 PIs in the presence of blocking reagents to putative alternative attachment interactions. The role of alternative cellular receptor candidates and virion-incorporated molecules in the initial attachment of PI Bx08 to PBMC was investigated. Experiments were performed either by preincubation of the virus with various potential attachment-blocking factors, at concentrations previously reported to efficiently inhibit attachment in some virus-cell models, before addition to the cells (A) or by preincubation of the cells with the reagents before addition of the virus (B), with controls made accordingly. Data correspond to the means and standard deviations of the results of at least two independent experiments.
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References

    1. Allaway, G. P., K. L. Davis-Bruno, G. A. Beaudry, E. B. Garcia, E. L. Wong, A. M. Ryder, K. W. Hasel, M. C. Gauduin, R. A. Koup, J. S. McDougal, et al. 1995. Expression and characterization of CD4-IgG2, a novel heterotetramer that neutralizes primary HIV type 1 isolates. AIDS Res. Hum. Retrovir. 11:533-539. - PubMed
    1. Beirnaert, E., S. De Zutter, W. Janssens, and G. G. van der Groen. 2001. Potent broad cross-neutralizing sera inhibit attachment of primary HIV-1 isolates (groups M and O) to peripheral blood mononuclear cells. Virology 281:305-314. - PubMed
    1. Binley, J. M., A. Trkola, T. Ketas, D. Schiller, B. Clas, S. Little, D. Richman, A. Hurley, M. Markowitz, and J. P. Moore. 2000. The effect of highly active antiretroviral therapy on binding and neutralizing antibody responses to human immunodeficiency virus type 1 infection. J. Infect. Dis. 182:945-949. - PubMed
    1. Blanco, J., J. Barretina, A. Gutierrez, M. Armand-Ugon, C. Cabrera, B. Clotet, and J. A. Este. 2002. Preferential attachment of HIV particles to activated and CD45RO+CD4+ T cells. AIDS Res. Hum. Retrovir. 18:27-38. - PubMed
    1. Bounou, S., N. Dumais, and M. J. Tremblay. 2001. Attachment of human immunodeficiency virus-1 (HIV-1) particles bearing host-encoded B7-2 proteins leads to nuclear factor-kappa B- and nuclear factor of activated T cells-dependent activation of HIV-1 long terminal repeat transcription. J. Biol. Chem. 276:6359-6369. - PubMed

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