Role of molecular mimicry to HIV-1 peptides in HIV-1-related immunologic thrombocytopenia

Abstract

Patients with early HIV-1 infection develop an autoimmune thrombocytopenia in which antibody is directed against an immunodominant epitope of the beta3 (glycoprotein IIIa [GPIIIa]) integrin, GPIIIa49-66. This antibody induces thrombocytopenia by a novel complement-independent mechanism in which platelets are fragmented by antibody-induced generation of H2O2 derived from the interaction of platelet nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and 12-lipoxygenase. To examine whether sharing of epitope between host and parasite may be responsible for this immunodominant epitope, we screened for antibody-reactive peptides capable of inhibiting platelet lysis and oxidation in vitro, using a filamentous phage display 7-mer peptide library. Fourteen of these phage-peptide clones were identified. Five shared close sequence similarity with GPIIIa49-66, as expected. Ten were molecular mimics with close sequence similarity to HIV-1 proteins nef, gag, env, and pol. Seven were synthesized as 10-mers from their known HIV-1 sequence and found to inhibit anti-GPIIIa49-66-induced platelet oxidation/fragmentation in vitro. Three rabbit antibodies raised against these peptides induced platelet oxidation/fragmentation in vitro and thrombocytopenia in vivo when passively transferred into mice. One of the peptides shared a known epitope region with HIV-1 protein nef and was derived from a variant region of the protein. These data provide strong support for molecular mimicry in HIV-1-immunologic thrombocytopenia within polymorphic regions of HIV-1 proteins. A known epitope of nef is particularly incriminated.

Figure 1.

Effect of panned phage 7-mer peptides on inhibition of anti–GPIIIa49-66–induced platelet fragmentation. (Left) First 3 bars refer to effect of patient Ab (Pt) on platelet fragmentation compared to buffer (C) or control IgG (Ci). Phage peptides were diluted at ratios of 1:3, 1:10, 1:30, and 1:100 and then preincubated with Ab for 15 minutes prior to the addition of the combined reagents to gel-filtered platelets. First bar is buffer diluent alone. Ab-induced platelet fragmentation was about 50% inhibited with a 1:100 dilution of the 3 phage peptides. (Right) Irrelevant peptide control phage (C1 and C2) as well as inhibitory phage peptides (H20R1, PHC39, PHC34) were diluted from 1:3 to 1000, preincubated with Ab as in the left panel, and then incubated with gel-filtered platelets. Data are representative of 2 different experiments.

Figure 2.

Effect of GPIIIa49-66 and wild-type HIV-1 peptides on Ab-induced platelet fragmentation. CAPE and ESIEF refer to positive control samples in which Ab was preincubated with peptide GPIIIa49-66 (CAPESIEFPVSEARVLED) or GPIIIa52-60 (SIEFPVSE). First set of bars refer to control, patient IgG, irrelevant peptide, or positive control inhibitory peptide (CAPE) prior to incubation with gel-filtered platelets. Second set of bars refers to positive control inhibitory peptide (ESIEF). The remaining bar panels refer to preincubation with various wild-type 10-mer HIV-1 peptides. □ indicates control IgG; ▪, patient Ig; , irrelevant peptide. The horizontal line, dark gray, white, and black bars refer to molecular Ab/peptide ratios of 1:100, 1:10, 1:5, and 1:2, respectively. Numbers refer to PHC or H clones of Table 1. HR1 refers to H20R1; HR3 refers to H19R3.

Figure 3.

Effect of wild-type polymorphic HIV-1 peptides derived from their inhibitory HIV-1 peptide regions on the inhibition of Ab-induced platelet fragmentation. Peptides were preincubated with Ab for 15 minutes at 37°C prior to addition of the combined reagents to gel-filtered platelets (as in Figure 1). □ and ▪refer to preincubation with molar peptide/Ab ratios of 1:100 and 1:10, respectively. C indicates control IgG; P, patient IgG; Ph, phage peptide PHC39; HR3, HIV-1 wild-type peptide HI9R3; PHR3 and P39, reported polymorphic peptides of HI9R3 and PHC39, respectively (Table 1); Env, irrelevant conserved region of env (514-523). N=4. SEM is given.

Figure 4.

Effect of rabbit Abs raised against HIV-1 10-mer wild-type peptides or 7-mer phage peptides on platelet fragmentation and oxidation. (Top) Gel-filtered platelets were incubated with control rabbit IgG (upper left quadrant) or rabbit Ab against peptide batches 1096 (upper right quadrant), 1098 (lower left), and 1099 (lower right) at 37°C for 4 hours. The percentages refer to fragmented platelets per 10 000 events counted. (Bottom) Similar incubation with assay of Ab-induced platelet oxidation. Ct indicates control buffer; Ci, control IgG; and Pt, patient IgG. Ca and DP indicate preincubation with scavenger of reactive oxygen species and inhibitor of NADPH oxidase, catalase (60 μg/mL) and diphenylenediodonium (100 nM), respectively; 96 and 98 refer to rabbit Ab against peptide batches 1096 and 1098.

Figure 5.

Reactivity of rabbit antimolecular mimicry Ab with HIV-1 proteins. Immunoblot of control sera (lane 1); 2 positive control sera (lanes 2 and 3); 3 rabbit antimolecular mimicry Ab sera, 1096, 1098, and 1099 (lanes 4, 5, and 6, respectively); preimmune rabbit sera (lane 7); and affinity-purified HIV-1-ITP patient sera (lane 8).

Figure 6.

Effect of rabbit Ab against HIV-1 peptides on induction of thrombocytopenia in mice. Purified control IgG (Ctl), patient IgG (Pt IgG) and rabbit Ab (25 μg) against wild-type HIV-1 peptide batches 1096, 1098, and 1099 were injected intraperitoneally into Balb/c mice, and platelet counts followed for 24 hours. N=4. SEM is given.