1 Department of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Apartado Postal 70228, Distrito Federal, CP 04510, México
2 Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Secretaría de Salud, Distrito Federal, México
Correspondence
C. Larralde
larralde{at}servidor.unam.mx
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ABSTRACT |
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MAIN TEXT |
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Inhibition of pathogenic cellcell fusion processes by the host immune response could play a significant role in retarding disease progression. In principle, such inhibition would be attainable by HIV-1-specific neutralizing antibodies. However, antibodies detected by cell-free virus neutralization assays are not necessarily effective in inhibiting cellcell fusion. Blocking HIV-1 Env-dependent cellcell fusion often requires higher concentration of antibodies or soluble CD4 than is necessary for virus neutralization (Allaway et al., 1993; McKeating et al., 1996
; Posner et al., 1991
; Trkola et al., 1998
; Zwick et al., 2001
). Furthermore, some Env-specific antibodies that effectively inhibited infection by cell-free virus did not have an effect on cell-to-cell virus transmission (Gupta et al., 1989
; Pantaleo et al., 1995
). The amount of gp120/gp41 on the cytoplasmic membrane of infected cells (Connor & Ho, 1994
) and env-transfected cells is higher than in viral membrane. Also, close contact of the cytoplasmic membranes of cells provides a more extensive area for the establishment of a higher number of effective Envreceptor interactions potentiated by other membrane-associated molecules (Hioe et al., 1998
). As a result, cellcell fusion may differ in quantitative and qualitative requirements from viruscell fusion. Thus, although neutralizing antibodies can restrict infections by free virus particles, they could be less effective in the containment of cell-to-cell virus spread and cell death mediated by extensive fusion activity.
To our knowledge, the effect of sera from HIV-1-infected individuals on cellcell fusion and its relation to disease markers has not been explored. This is necessary to get a comprehensive understanding of the potential of the humoral immune response to influence the various ways of HIV-1 spreading and cell damage. Therefore, we determined the activity of sera from HIV-1-infected subjects on Env-mediated cellcell fusion using Jurkat cells stably transfected with a plasmid bearing the env gene from the HIV-1 syncytium-inducing virus HXBc2 (HXBc2 cells) and untransfected Jurkat cells as the CD4+ fusion partner (Cao et al., 1996). The use of cells expressing Env from a highly fusogenic prototypic clade B virus, the most prevalent clade in México and the Western world (Gudiño & Soler, 2000
; WHO, 2001
) allows a fair comparison between individuals affected by different virus variants.
An accurate quantification of fusion events was attained by a recently designed flow cytometry assay (Huerta et al., 2002), involving the labelling of fusion partner cells with the fluorescent carbocyanines DiI and DiO (Molecular Probes). Fig. 1
(a) and (b) show typical fluorescence dot plots of fusion between DiO-HXBc2 and DiI-E6 labelled Jurkat cells after 5 h of co-culture; a 1418 % of fused double fluorescent particles was consistently obtained. Dependence of the cellcell fusion on Env and CD4 is shown by the inhibition caused by an anti-CD4 mAb (clone RPA-T4; BD Pharmingen) and by three HIV-1 neutralizing mAbs (Fig. 1c
). Fusion was potently inhibited by the anti-CD4 mAb (IC50=4 µg ml1). Approximately 50 % inhibition was obtained with 52 and 64 µg ml1 of 2G12 anti-gp20 and 2F5 anti-gp41 mAbs, respectively. Less than 50 % inhibition was obtained with 50 µg ml1 of the F105 mAb. These anti-Env mAb concentrations are several to 100-fold higher than those reported to inhibit the infection of cells by HXBc2 and related strains (Posner et al., 1991
; McKeating et al., 1996
; Trkola et al., 1998
; Zwick et al., 2001
) but similar to those used by others to inhibit Env-dependent cellcell fusion (Allaway et al., 1993
).
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The effect of sera on Env-dependent cellcell fusion is in significant correlation with the number of CD4+ cells and viral load in circulation of HIV-1-infected individuals as well as with clinical progression of the disease (Fig. 3). Sera from patients with more than 200 CD4+ cells per mm3 inhibited fusion significantly above the sera from patients with the lower numbers of CD4+ lymphocytes (P=0·0054) (Fig. 3a
). Likewise, the inhibitory activity of serum is significantly higher for patients with viral loads <75 000 HIV-1 RNA copies ml1. Inhibitory activity significantly decreases when plasma viral load exceeded that of 75 000 RNA copies ml1 (P=0·0366). (Fig. 3b
). FA of sera showed significant differences between groups in distinct clinical status (Fig. 3c
). FA of sera from patients with AIDS was significantly lower than those with non-AIDS conditions (P=0·0277). Although the mean FA of HIV-1-negative sera did not differ from that of sera from patients with advanced disease, the sera from patients with more advanced infection produced the widest range of FA values. Consistent with these data, a positive correlation between FA and the CD4+ cell count (P=0·0272), and a negative correlation between FA and viral load (P=0·0362) were found significant. The three parameters considered (CD4+ T-cell count, viral load and clinical status) were available for 25 of 49 patients studied. For these patients, FA of sera was significantly inhibitory for patients with no disease progression (viral loads <75 000 RNA copies ml1, CD4+ T-cell count >200 cells per mm3 and asymptomatic or with non-AIDS related conditions) compared with that of patients with advanced disease [viral loads >75 000 RNA copies ml1, CD4+ T-cell count <200 cells per mm3 and AIDS condition (P=0·0494)].
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The comparison between clinical groups with respect to their sera activities on cell-fusion (Fig. 3c) provided an explanation for the results large variance (Fig. 3
). The sera of HIV-1-positive non-AIDS patients are significantly more inhibitory (mean=19·07, SD=20·49) than the sera of healthy persons (mean=3·65, SD=8·69) and none of them was enhancing. In AIDS cases, the mean of fusion values falls back to control levels (2·65) and 30 % of the sera enhance fusion, one of them well beyond the level of sera from healthy people, resulting in a wide distribution of FAs (SD=28·38). Enhancement of fusion was also more frequent in the higher (5 of 17, 29·4 %) than in the lower viral load group (1 of 23, 4·4 %). Thus, if there are two functional kinds of fusion-active immunoglobulins in the serum, one that inhibits cell fusion (mainly IgG) and another that enhances it (IgM), then their relative effectiveness and concentrations would determine the net degree of cell fusion along the course of the infection.
Besides the prognostic value of the quantitative cell-FA in the sera of HIV-1-infected persons, the identification of the viral or cell epitopes involved in the inhibition and enhancement of cellcell fusion may lead to immune interventions for preventing or controlling the progression of HIV-1 disease by way of interfering with Env-mediated cellcell fusion. The sensitive flow cytometry assay we have developed, may facilitate the regular exploration of the potential of HIV-1-infected patient's immune response to confront HIV-1 Env-mediated cell fusion.
Given the failure of neutralizing antibodies to control the established infection (Poignard et al., 1999) this work emphasizes that, besides neutralizing antibodies, the effect of sera on syncytium formation should be considered for a comprehensive understanding of the role of immune factors in HIV-1 spreading and cell damage. Identification of the epitopes inducing the production of fusion inhibitory antibodies could be of therapeutic and preventive value. Elucidation of the factors enhancing fusion may provide new insights in the pathogenesis of HIV-1 infection.
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ACKNOWLEDGEMENTS |
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Received 24 September 2004;
accepted 4 April 2005.
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