Department of Virology, Erasmus Medical Centre Rotterdam, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands1
Department of Internal Medicine, Erasmus Medical Centre Rotterdam, Dr Molewaterplein 40, 3015 GD Rotterdam, The Netherlands2
UMR103 CNRS BioMerieux, ENS Lyon, Lyon, France3
Bernhard Nocht Institut für Tropen Medicin, Körber Labor für AIDS Forsschung, Bernhard Nocht Straße 74, 2000 Hamburg 36, Germany4
Author for correspondence: A. D. M. E. Osterhaus. Fax +31 10 408 9485. e-mail osterhaus{at}viro.fgg.eur.nl
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Abstract |
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Introduction |
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The characteristics used to classify HIV strains in vitro include replication rate, ability to induce the formation of multinucleated cells [syncytium-inducing (SI) capacity], coreceptor requirement and ability to infect different target cells (T cell lines versus macrophages) (Asjö et al., 1986 ; Berger et al., 1998
; Cheng-Mayer et al., 1988
; Schuitemaker et al., 1992b
; Tersmette et al., 1989
). For HIV-1 it has been shown that these in vitro characteristics exhibit a high degree of correlation. For example, SI strains in general have a high replication rate, infect target cells via
and
chemokine receptors, may infect immortalized T cell lines and only infect macrophages inefficiently. NSI strains, on the other hand, have a low replication rate, require expression of
chemokine receptors, do not infect T cell lines and replicate efficiently in macrophages (Berger et al., 1998
; Schuitemaker et al., 1991
). It should, however, be noted that individual virus strains may also display intermediate biological phenotypes (Groenink et al., 1991
; Sabri et al., 1996
; Schuitemaker et al., 1992a
). Several differences in in vitro characteristics of HIV-1 and HIV-2 have become apparent. The replication rate, as defined by the time required to detect virus after initiation of standard virus culture from donor peripheral blood mononuclear cells (PBMC), is generally lower for HIV-2 than for HIV-1 (Albert et al., 1990
; van der Ende et al., 1996
). Furthermore, we and others have recently shown that clear differences exist in coreceptor requirements (Guillon et al., 1998
; McKnight et al., 1998
). The linkage between HIV-1 SI phenotype and CXCR-4 coreceptor usage was not observed for HIV-2. Furthermore, HIV-2 strains in general have a broader coreceptor usage than HIV-1. Taken together, these observations suggest that in vitro usage of the CXCR-4 coreceptor and broadening of the coreceptor usage by HIV-2 do not result in enhanced in vivo pathogenicity.
To further address this issue we studied the in vivo pathogenic potential of HIV-2 isolates and biological clones in a chimeric human-to-mouse model for in vivo HIV infection [the xeno-GvHD (graft-versus-host disease) mouse model] (Huppes et al., 1992 , 1993
; Schutten et al., 1996
). In this model high numbers of human PBMC are grafted into the peritoneal cavity of immune-deficient mice. In these mice an acute graft-versus-host reaction develops within 7 to 14 days. The human lymphocyte population that repopulates the mouse tissues is characterized by high CD4/CD8 ratios (Schutten et al., 1996
). Depletion of human CD4+ T cells from the graft results in a complete abrogation of the acute xeno-GvHD reaction. Depletion of antigen-presenting cells (APC; macrophages) from the human graft results in lower CD4/CD8 ratios and a concomitant delay in appearance of the xeno-GvHD symptoms (Huppes et al., 1993
). We therefore set out to study in this model the direct (killing of infected CD4+ T cells) and indirect (inhibition/modulation of antigen presentation) pathogenic effect of different HIV-2 strains and isolates on the development of acute xeno-GvHD symptoms, CD4/CD8 ratio and the ability of the graft to repopulate mouse tissues.
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Methods |
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HIV-2 in situ hybridization and immunohistochemistry.
Xeno-GvHD mouse tissues, including spleen, lung, bowel, heart, liver and bone, were prepared from groups of mice which had been grafted with PBMC isolated from the same individual. Tissues from two mice infected with the same HIV-2 isolate/strain were analysed pairwise. Tissues were fixed overnight with 4% formalin and subsequently embedded in paraffin. For routine immunohistochemistry, dewaxed 5 µm paraffin sections were heat-denatured with 0·01 M buffered sodium citrate (Norton et al., 1994 ). The sections were incubated with CD45 (LCA) (Dakopatts) according to the manufacturers instructions (Tenner-Racz et al., 1998
). Binding of antibodies was visualized by the alkaline phosphatase anti-alkaline phosphatase technique using New Fuchsin as red chromogen. We have previously shown that this conjugate does not stain mouse cells (Schutten et al., 1996
). After immunostaining the sections were either counter-stained with haematoxylin and mounted, or dehydrated and subjected to HIV RNA in situ hybridization. 35S-labelled, single-stranded, anti-sense HIV-2 RNA probes (Lofstrand Laboratories), which contained 1·42·7 kb fragments, collectively representing approximately 90% of the HIV-2 genome, were used as described previously (Embretson et al., 1993
; Fox et al., 1991
). Briefly, paraffin sections were either treated with Proteinase K (0·01 mg/ml) for 8 min at room temperature or heat denatured. The sections were incubated with prehybridization mixture (50% formamide, 0·5 M NaCl, 10 mM TrisHCl, pH 7·4, 1 mM EDTA, 0·02% FicollpolyvinylpyrrolidoneBSA and 7 mg of tRNA/ml) for 2 h at 37 °C and then covered with hybridization mixture (prehybridization mixture+10% dextran sulfate and 2x106 d.p.m. of probe/ml) overnight at 45 °C. The sections were washed, RNase treated (Boehringer Mannheim) for 40 min at 37 °C and further developed, counter-stained with haemalaun and mounted as previously described. As a negative control, sections were hybridized with a radiolabelled sense probe. The sections were examined with a microscope equipped with epiluminescent illumination (Axiophot; Carl Zeiss). Cells were considered positive for viral gene expression if the number of grains overlying a cell was more than six times the background.
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Results |
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Discussion |
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Both direct and indirect mechanisms have been suggested to contribute to the pronounced CD4+ T cell depletion observed in HIV-infected individuals (for review see Zinkernagel et al., 1994 ). The direct mechanisms include acute single cell lysis of infected cells (Samson et al., 1996
) and depletion of uninfected CD4+ cells by fusion with infected cells (syncytium formation) (Tersmette et al., 1993
). It has also been shown that cells of the monocyte/macrophage lineage do not function properly, which may result in dysregulation of activation and/or maturation of CD4+ T cells due to functionally disturbed antigen presentation (Meyaard et al., 1993
). This may also indirectly contribute to the decline of CD4+ T cells. Two observations suggest that this last phenomenon contributed to the HIV-2-induced CD4+ T cell depletion in our xeno-GvHD mouse model. First, no significant differences were observed in the CD4/CD8 ratio and the number of CD45+ cells in spleens and lungs of mice infected with the early, macrophage-tropic NSI isolate RH2-1 (R5) and the late non-macrophage-tropic SI isolate RH2-2 (R1,R3,R5,X4). This is despite the fact that the late SI isolate is highly cytopathic for CD4+ T cells in vitro and the early NSI isolate does not induce single cell lysis to a significant extent (unpublished observations). The fact that the percentage of infected cells was high in the RH2-1 (R5)-infected mice and low in the RH2-2 (R1,R3,R5,X4)-infected mice also suggests that these virus strains cause CD4+ T cell depletion by two different mechanisms. It seems appropriate to assume that HIV-2 RH2-2 (R1,R3,R5,X4)-infected cells are directly killed and therefore do not reach the peripheral tissues studied, whereas RH2-1 (R5)-infected cells are not killed directly, but seem rather to be functionally affected. Second, RH2-2 (R1,R3,R5,X4) and RH2-6 (X4) are both highly cytopathic for CD4+ T cells in vitro. The macrophage-tropic isolate RH2-6 (X4) proved to be far more pathogenic in vivo with respect to abrogating GvHD symptoms and inhibiting migration of T cells to peripheral tissues than the non-macrophage-tropic isolate RH2-2 (R1,R3,R5,X4).
SI capacity, replication rate and coreceptor requirements have all been shown to positively correlate with rapid disease progression in HIV-1-infected individuals. Because, for HIV-1, these factors are generally linked, it has not been possible to distinguish which factors have a causal relation with disease progression. Since SI capacity and coreceptor usage are not linked for HIV-2, we could study these factors independently. The observation that in vitro coreceptor usage of HIV-2 strains is broader than that of HIV-1 has already suggested that broadening of coreceptor usage does not necessarily lead to increased in vivo pathogenicity. Indeed, in the xeno-GvHD model, RH2-6 (X4) proved to be far more pathogenic with regard to all aspects studied than RH2-2 (R1,R3,R5,X4), suggesting that broadening of coreceptor usage does not significantly add to the in vivo pathogenicity. This is further supported by the observation that the NSI biological clone PH2-1 E6 (R1,R3,R5,X4) was less well able to inhibit migration of T cells and may therefore be regarded more pathogenic than its SI counterpart PH2-1 D5 (R5).
We have recently shown that the number of productively infected cells in lymphoid tissue from HIV-2-infected individuals is significantly lower than in HIV-1-infected individuals. Furthermore, it has been shown that the number of productively infected cells is positively correlated with the plasma viral load, which is in turn correlated with disease progression. Interestingly, we did not observe major differences in cell-associated viral loads or in pathogenicity of the HIV-2 strains tested when compared with previously studied HIV-1 strains (Schutten et al., 1996 ). It therefore seems that in the absence of a substantial humoral and cellular antiviral immune response both in vitro and in our human-to-mouse chimeric model, HIV-1 and HIV-2 may have comparable replicative and pathogenic potential. We therefore hypothesize that the lower viral load and pathogenicity observed in HIV-2-infected individuals as compared to HIV-1-infected individuals is primarily related to a difference in interaction of the virus with the specific immune response.
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Acknowledgments |
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References |
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Received 9 August 1999;
accepted 9 November 1999.