In this paper, we define a mechanism of inhibition of HIV-1 infection of human PBMCs by human anti-phospholipid antibodies. Anti-phospholipid mAbs derived from human subjects can be classified into two types: those that are dependent on the presence of β2
GP1 for binding and those that are not (Zhu et al., 1999
; Iverson et al., 2002
; Ioannou et al., 2007
; Lin et al., 2007
). Although not an absolute correlation, those mAbs that require β2
GP1 for binding to phospholipids have been associated with autoimmune phenomena, including an increase in thrombogenesis and with adverse clinical outcomes (Alving, 2006
; Lin et al., 2007
), whereas those mAbs that bind directly to phospholipids and that do not require β2
GP1 for binding are not (Iverson et al., 2002
; Ioannou et al., 2006
). In addition, naturally occurring antibodies against phospholipids can be found in healthy individuals and are not associated with negative clinical consequences (Alving, 2006
). Thus, antibodies that bind to phospholipids are not always pathological, and those that possess additional beneficial properties, such as virus inhibition (Brown et al., 2007
; Soares et al., 2008
) or antitumor effects (Ran et al., 2005
), could be of interest as candidates for therapeutic monoclonal antibodies or for induction by vaccines.
The mAbs studied in this paper were generated from peripheral blood antibody libraries of healthy subjects or from blood B cells of primary and secondary anti-phospholipid antibody syndrome (APS) subjects. HIV-1–inhibiting anti-phospholipid antibodies were effective up to 48 h after HIV-1 contact with PBMC, broadly neutralized only R5 but not X4 HIV-1, and acted on peripheral blood monocytes to induce R5 chemokines. Collectively, these data explain why anti-phospholipid antibodies inhibit HIV-1 infectivity in PBMC but not in CD4+
transfected epithelial cell assays such as the TZM-bl assay (Montefiori, 2005
), and they demonstrate a novel mechanism whereby components of the adaptive immune system can activate antiviral innate immunity.
The target of the anti-phospholipid antibodies in the PBMC HIV-1 infection assay was not HIV-1 virions. Of the four mAbs that inhibited HIV-1 infectivity in PBMCs, only one, PGN632, weakly bound HIV-1 virions in SPR with a fast off rate (Fig. S3, B–D). These mAbs induced chemokines and cytokines to the point of inducing monocyte polykaryon formation in vitro (Fig. S6, A–F). R5 but not X4 chemokines were induced, with the production of MIP-1α and MIP-1β exceeding that of RANTES (), and antisera against MIP-1α and MIP-1β abrogated the ability of lipid antibodies to inhibit HIV-1 infectivity of PBMC (). Thus, virus inhibition by the anti-phospholipid antibodies is not the result of viral neutralization but rather occurs through in vitro modulation of PBMC by adaptive antibodies, resulting in inhibition of HIV-1 infectivity.
Three of the antibodies (PGN632, P1, and CL1) that inhibit HIV-1 infection bind directly to phospholipids, whereas one (IS4) requires β2
GP1 for binding. The importance of the Fab region in binding is shown by the ability of the anionic phospholipid cardiolipin to block the HIV-1 inhibition activity of CL1 and PGN632 mAbs (). For IS4, the requirement for Fab binding was shown by use of the recombinant IS4 variants with those variants that retain lipid binding showing activity in the PBMC assay. These data are consistent with direct signaling for chemokine induction through lipids or lipid-dependent signaling complexes. PS is a tenable target, as it is recognized by all four HIV-1 inhibitory mAbs, appears to be present constitutively on macrophage lineage cells (unpublished data), and is up-regulated on activated (Bevers et al., 1983
) and virally infected cells (Soares et al., 2008
). PS is enriched in lipid rafts, membrane microdomains which are known to be associated with cell signaling events. Although cardiolipin is also recognized by the four mAbs, cardiolipin is itself unlikely to be a target as it is predominantly a component of mitochondrial lipid membranes (Schlame and Hostetler, 1997
). The differing potencies of the four mAbs also suggested that the particular lipid motif bound by PGN632 and CL1 antibody Fab regions may be similar but that mAbs P1 and IS4 Fab region lipid binding motifs may differ. Thus, it is possible that the activity of certain antibodies involves binding to an unidentified lipid target in addition to PS.
Although the Fab regions of the four anti-phospholipid antibodies were clearly required for HIV-1 inhibition activity, the Fc region was also clearly required. The F(ab′)2
of mAb PGN632, the most potent anti-phospholipid mAb, did not inhibit HIV-1 infectivity in the PBMC assay and did not support ADCVI (, respectively). This finding was not a result of altered binding of the F(ab′)2
to lipids, as the F(ab′)2
for binding to lipids was identical to that of the whole IgG by SPR (). Thus, both the Fab and the Fc regions of the anti-phospholipid antibodies, together as an intact molecule, were required for mediating the HIV-1 inhibition effect of the anti-phospholipid antibodies. Triggering via the Fc receptor of IgG has been reported to stimulate chemokine release from natural killer cells (Forthal et al., 2001
), monocytes (Fernández et al., 2002
), and dendritic cells (Radstake et al., 2005
), and these data raise the possibility of synergy between two modes of triggering (e.g., lipid binding and FcR ligation; polyspecific triggering by antibody Fab plus FcR binding). Although the data in and Table S3 suggest that monocytes alone can mediate these effects, the role of other cells, such as NK cells, has not been rigorously excluded. It was notable that the inhibitory effect of anti-phospholipid antibodies was seen in only 85% of the PBMC tested. The lack of activity in PBMC that did not support the phenomenon could be the result of an inability to produce the correct chemokines, alteration in a cellular receptor, or differences in signaling pathways. Thus, it will be important to determine the mechanism of induction of chemokines and cytokines by anti-phospholipid antibodies and to determine the roles of each of the cell surface targets of this activity on multiple FcR+
It was of considerable interest that anti-phospholipid antibodies did not induce X4 chemokines but only R5 chemokines. In vitro protective effects of R5 chemokines secreted by neonatal natural killer cells have been previously described (Bernstein et al., 2004
), and antigen-specific production of R5 chemokines in vitro has been reported to correlate with decreased viral loads in HIV-1–infected subjects (Ferbas et al., 2000
). Kinter et al. (1998)
reported that R5 virus inhibition through the use of β-chemokines can be correlated with the enhancement of X4 isolate replication. This effect was most pronounced for RANTES and less substantial for MIP-1α. In contrast, in our study RANTES was minimally induced, whereas MIP-1α was increased to the greatest extent () and the use of anti-RANTES antibodies showed little effect on the antiviral activity of mAb CL1 (). In addition, Keele et al. (2008)
have recently shown that the transmitted/founder virus almost always utilizes CCR5 and not CXCR4. The anti-phospholipid antibodies studied were as potent at inhibiting the infectivity of R5 but not X4 transmitted/founder viruses as they were against chronic HIV-1 strains ( and ). It will be of interest to dissect the triggering mechanisms of CCR5 versus CXCR4 inhibition chemokines to understand how to differentially trigger both types of anti–HIV-1 innate molecules. Before the use of an intervention based on the findings of this study in human subjects, it will also be important to determine the potential for a shift from R5 to X4 infection in the presence of anti-phospholipid antibody-mediated anti–HIV-1 activity.
That the anti-phospholipid mAbs induced monocyte polykaryon formation in vitro was an indication of the stimulatory capacity of the mAbs for peripheral blood monocytes. In addition to induction of R5 chemokines, lower levels of TNF, IL-6, IL-4, and IFN-γ are induced in vitro. IFN-γ and IL-4 have been reported to induce polykaryon formation (McNally and Anderson, 1995
), and it is likely that these cytokines may be the mediators of this in vitro phenomenon. The observation of Langerin+
polykaryons in these cultures suggested that lipid antibodies may also act on dendritic cells as well as peripheral blood monocytes in culture (Fig. S6 I), although we have not studied this exhaustively.
There are prior studies of antibodies from autoimmune disease patients that have shown cross-reactivity with HIV-1 antigens (Douvas and Takehana, 1994
; Scherl et al., 2006
) and even descriptions of in vitro neutralization by such antibodies (Douvas et al., 1996
). Those antibodies have reacted with HIV-1 Env proteins and have not been targeted at host cell or virion lipids. Thus, the antibodies described in this paper represent a novel group of polyspecific antibodies worthy of further investigation to determine their potential role in protecting subjects with these antibodies from R5 HIV-1 infection. The ability of such antibodies to stimulate similar effects in vivo has yet to be determined. In separate studies, plasma was obtained from patients with autoimmune diseases and tested for anti-phospholipid antibody activity and for the presence of β-chemokines with no observed correlation between their levels (unpublished data). In another study, patients infected with HIV-1 and coinfected with syphilis were studied, and it was found that the presence of RPR-positive syphilis, and therefore the presence of anti-cardiolipin antibodies, was associated with greater HIV-1 inhibition in the PBMC assay compared with HIV-1–negative patients with syphilis infection and compared with HIV-1 patients without syphilis (unpublished data). Whether this anti–HIV-1 activity is a result of the same mechanism as that of the mAbs in this study has yet to be determined.
The development of new therapies for the prevention of HIV-1 has suffered major setbacks in efficacy trials of vaccines (Sekaly, 2008
) and microbicides (van de Wijgert and Shattock, 2007
). In HIV-1 vaccine development, recent studies have demonstrated the extremely early destruction of the immune system and early induction of immunosuppression and apoptosis (Gasper-Smith et al., 2008
; Tomaras et al., 2008
; Levesque et al., 2009
; Stacey et al., 2009
), signaling the need for a vaccine that can prime for a very early salutary immune response within days of infection. The novel mechanism outlined in this paper of anti-phospholipid antibody modulation of anti–HIV-1 chemokines accounts for the selective activity of lipid antibodies in PBMC but not epithelial-based cultures. These results suggest that a vaccine could potentially harness the adaptive immune system to trigger innate immunity for an antiviral response, in effect reversing the traditional path of protection of innate to adaptive immunity.