Centre for Applied Microbiology and Research (CAMR), Porton Down, Salisbury SP4 0JG, UK
Correspondence
Martin Cranage
mcranage{at}sghms.ac.uk
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ABSTRACT |
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Present address: Department of Cellular and Molecular Medicine, St George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK.
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MAIN TEXT |
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The influence of immune responses to virus-incorporated host cell antigens was dramatically illustrated when vaccination with uninfected human T-cells was shown to protect a proportion of macaques against challenge with human cell-grown SIV (Stott, 1991). Furthermore, human cell-grown SIV and HIV inactivated virus vaccines conferred potent protection against systemic autologous and heterologous virus challenge and even against mucosal challenge (Cranage et al., 1992a
) but only when the challenge virus was grown in human cells. Critically, no vaccine-induced protection was seen against SIV that had been grown in simian cells (Cranage et al., 1992b
). This xeno-immunization effect was correlated with antibody responses to human MHC class I antigens (Chan et al., 1992
; Cranage et al., 1993
). A direct demonstration of the role of xeno-MHC molecules in this vaccine effect was the finding that a proportion of macaques were protected from challenge with human cell-grown SIV following immunization with purified human MHC class I (Chan et al., 1995
), class II (Arthur et al., 1995
) or L-cells transfected with HLA-DR4 (Stott et al., 1994
). In the latter case it was shown that protective immunity could be passively transferred to
naïve
macaques.
Much less is known about the biological significance of virion-incorporated allogeneic host cell molecules, i.e. the situation arising in nature, and immune responses directed against these proteins; however, it is reasonable to assume that virion-incorporated molecules may influence virus pathogenesis including early entry events and activation of the immune system. This is evidenced by studies indicating that these molecules may maintain function after incorporation into virions (Rossio et al., 1995; Fortin et al., 1997
; Cantin et al., 1997
; Saifuddin et al., 1994
). Anti-lymphocyte antibodies have been associated with infection by HIV (Dorsett et al., 1985
; Müller et al., 1994
; Riera et al., 1992
; Daniel et al., 1989
; Ozturk et al., 1987
) and it has been suggested that immune responses to regions of MHC homology within virus-encoded proteins may be involved in HIV- and SIV-induced pathology (Habeshaw, 1994
; Kion & Hoffmann, 1991
; Grassi et al., 1991
). Antibodies to MHC molecules during infection may arise therefore through recognition of virion-acquired host proteins and/or through molecular mimicry.
In this study we have investigated the specificity of anti-cell antibody responses arising during SIV infection in comparison to those induced by vaccination with inactivated-human cell-grown virus.
Flow cytometry of concanavalin-A activated (10 µg ml-1, 72 h) human and rhesus peripheral blood mononuclear cells (PBMC) revealed that sera from SIV-infected animals (n=7), taken at various times 3 to 18 weeks after infection, bound to rhesus but not to human cells (data not shown). In contrast, sera from animals vaccinated with human cell-grown SIV or HIV-1 (n=12) bound to cells from both species although preferentially to human cells, confirming previously reported results (Langlois et al., 1992; Bergmeier et al., 1994
). Sera from neither infected nor vaccinated animals competed the binding of labelled monoclonal antibodies (W6/32 and CR3/43) against species cross-reactive monomorphic anti-MHC class I or class II antigens.
To determine directly the cell surface targets of the anti-cell antibodies and to investigate the molecular specificity of the differential antibody binding activity seen by flow cytometry, activated human and rhesus PBMC were surface labelled with biotin (Meier et al., 1992) and serum reactivity was determined by immunoprecipitation (Fig. 1
).
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In contrast to the results with human PBMC, when surface-labelled rhesus PBMC were used, sera from SIV-infected macaques precipitated a range of proteins of relative molecular mass 47 to 25 kDa (Fig. 1b, track 2), corresponding to species precipitated by sera from vaccinated animals (Fig. 1b
, track 3). In addition, sera from vaccinated animals precipitated proteins of 66, 60, 56 and 12 kDa. Sera from both infected and vaccinated macaques precipitated proteins that corresponded in migration to MHC class II and class I heavy chains (Fig. 1b
, tracks 4 and 5 respectively).
Next, to confirm the specificity of sera for MHC molecules, non-labelled lysates of mitogen-activated PBMC were used in immunoprecipitation assays. Eluted products were separated by SDS-PAGE and the presence of MHC class I and class II antigens was determined on Western blots essentially as described previously (Cranage et al., 1993). When blots of products eluted from human and rhesus PBMC were probed with anti-MHC class I antibody (HC10), sera from vaccinates were found to precipitate class I
-chain from either species, although the strongest signal was obtained with human antigen (Fig. 2
a and b, track 4). In contrast, sera from SIV-infected animals precipitated MHC class I
-chain only from rhesus PBMC lysates (Fig. 2a, b
, track 3). Similar results were obtained for
2-microglobulin and confirmed by ELISA (data not shown). The MHC class I
-chain was usually detected as a doublet of relative molecular mass 43/45 kDa in both human and rhesus preparations. Similarly, when immunoprecipitated products were probed with anti-MHC class II (CR3/43) (Fig. 2c, d
), sera from vaccinates precipitated both human and rhesus class II (track 4) but sera from SIV-infected animals precipitated only rhesus MHC class II (track 3). Interestingly, vaccinate sera precipitated only the 31 kDa class II band. Sera from SIV-infected macaques precipitated both the 33 and 31 kDa class II band, although the relative intensity of the two bands was quite variable in different experiments.
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To determine if anti-MHC responses were related to SIV-specific immune responses serial serum samples taken from seven macaques were analysed for the development of anti-rhesus MHC class I -chain antibody responses and antibody responses to SIV structural components following infection with SIV (Table 1
). Pre-inoculation sera did not react with any of the antigens tested. Anti-MHC class I responses usually occurred by 1 month after infection and in three instances were detectable prior to an anti-SIV envelope response. In one animal (K8) the anti-class I response appeared in the absence of responses to SIV Gag p27 and SIV Env gp120. This animal was definitely infected with SIV as evidenced by virus isolation from PBMC and detection of proviral DNA by PCR. Although K8 failed to make a response to gp120 at any time following infection and had a delayed response to SIV gp41, the anti-class I response peaked between 3 and 7 months post-infection. In contrast, animal 3H had no detectable anti-class I response until 3 months after infection but had seroconverted to SIV antigens 1 to 2 months after infection. The lack of correlation between the appearance of the anti-MHC class I response and the appearance of anti-SIV responses suggests that molecular mimicry, at least for MHC class I, is unlikely. The possibility of molecular mimicry cannot be excluded absolutely. It is possible that precipitating anti-MHC antibodies appear before antibodies reactive on Western blots; however, this is not supported by the results from animal 3H where anti-SIV responses precede the anti-MHC class I antibody response. If molecular mimicry does occur then the species-specific recognition of MHC implies a close evolutionary relationship between virus cross-reactive region(s) and host species perhaps conferring some selective advantage on the virus.
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Do anti-MHC antibody responses have functional significance? Recently, it has been shown that human antibodies to MHC alloantigens mediate lysis and neutralization of HIV-1 primary isolates in the presence of complement (Spear et al., 2001). Anti-MHC class I antibodies may also have a role in virus clearance through opsinization (Benkirane et al., 1994
). Although antibodies induced by alloimmunization with a B-lymphoblastoid cell line (B-LCL) failed to protect macaques against SIV infection (Polyanskaya et al., 1997
), the anti-MHC response induced was incomplete. In another study, immunization of macaques with allogeneic lymphocytes protected 50 % of animals challenged (E. J. Stott and others, personal communication). The finding that infection-related antibodies recognize virion-acquired host cell proteins needs further investigation as this may suggest novel targets for anti-envelope vaccine design.
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ACKNOWLEDGEMENTS |
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Received 19 November 2002;
accepted 28 February 2003.