Targeting inside-out phosphatidylserine as a therapeutic strategy for viral diseases

Abstract

There is a pressing need for antiviral agents that are effective against multiple classes of viruses. Broad specificity might be achieved by targeting phospholipids that are widely expressed on infected host cells or viral envelopes. We reasoned that events occurring during virus replication (for example, cell activation or preapoptotic changes) would trigger the exposure of normally intracellular anionic phospholipids on the outer surface of virus-infected cells. A chimeric antibody, bavituximab, was used to identify and target the exposed anionic phospholipids. Infection of cells with Pichinde virus (a model for Lassa fever virus, a potential bioterrorism agent) led to the exposure of anionic phospholipids. Bavituximab treatment cured overt disease in guinea pigs lethally infected with Pichinde virus. Direct clearance of infectious virus from the blood and antibody-dependent cellular cytotoxicity of virus-infected cells seemed to be the major antiviral mechanisms. Combination therapy with bavituximab and ribavirin was more effective than either drug alone. Bavituximab also bound to cells infected with multiple other viruses and rescued mice with lethal mouse cytomegalovirus infections. Targeting exposed anionic phospholipids with bavituximab seems to be safe and effective. Our study demonstrates that anionic phospholipids on infected host cells and virions may provide a new target for the generation of antiviral agents.

Figures

Figure 1. Bavituximab binds to Pichinde virus-infected cells and to infectious virions

(a) Flow cytometric analysis of P388D1 cells after infection at an moi of 5. Cells were stained at 48 h with bavituximab (open histograms) or control Ig (filled histograms). Live cells were gated based on their exclusion of 7-AAD. (b) Flow cytometric analysis of expression of PS and Pichinde virus antigens over time. P388D1 cells were infected with Pichinde virus at an moi of 5 and stained at the indicated time points after infection. PS expression is detected with bavituximab (closed squares) and Pichinde virus antigen expression is detected with guinea pig Pichinde-specific antiserum (closed triangles). Points, ratio of mean fluorescence intensity (MFI) for cells stained with test antibodies to MFI of cells stained with appropriate negative control antibodies. The error on the points is ± 15%. (c) Immunofluorescence staining of uninfected Vero cells (left panels) and Pichinde virus-infected cells (right panels) with bavituximab 48 h after infection. Upper panels show cells stained with bavituximab (green). Lower panels show images merged with cytoskeleton (red). Nuclei are in blue (all panels). Control Ig did not stain and is not shown. Bar, 50 μm. (d) ELISA detection of virus binding to immobilized bavituximab or control Ig. Columns, average absorbance (n = 3); bars, s.e.m. P<0.0001 (unpaired t test) (e) Depletion of infectious Pichinde by bavituximab-coated magnetic beads. Beads coated with antibodies to Pichinde virus (PV) were used as a positive control. Columns, % depletion (n = 3); bars, s.e.m. P = 0.002 (unpaired t-test).

Figure 2. Therapeutic effect against Pichinde virus in lethally infected guinea pigs displaying overt signs of disease

(a) Kaplan-Meier survival curves of guinea pigs afterlethal infection with Pichinde virus and treatment with bavituximab or control Ig. Bavituximab or control Ig (6 mg kg−1) was administered i.p. to groups of guinea pigs (n = 8), beginning after they had developed disease signs (around day 7) and three times a week thereafter. The results are representative of those in five separate experiments. Survival in the bavituximab group was significantly superior to the control Ig group (P = 0.0036, Log-rank Mantel Cox test). (b) Virus load in tissues of treated guinea pigs 14 d after infection. Columns, average PFU per gram of tissue (n = 3); bars, s.e.m. The results are representative of two separate experiments. *P = 0.0164, **P = 0.0361, ***P = 0.0436, ****P = 0.0139, *****P = 0.0992, ******P = 0.038. (c) Additive effects of bavituximab and ribavirin treatment. Kaplan-Meier survival curves are shown for Pichinde virus-infected guinea pigs treated with bavituximab and ribavirin (n = 19), bavituximab (n = 20), ribavirin (n = 18) or control Ig (n = 20). Bavituximab or control Ig (6 mg kg−1) was administered i.p. three times per week and ribavirin (8 mg kg−1) was administered i.p. daily, beginning after the guinea pigs developed disease signs. The combination was significantly more effective than bavituximab alone (P = 0.011). All treatments were significantly different from control Ig (P<0.0001). The results are representative of two separate experiments.

Figure 3. Mechanism of anti-viral effects of bavituximab

(a) Lack of Pichinde virus-specific humoral response in bavituximab-treated guinea pigs. Plasma from Pichinde virus-infected animals (n = 3) was collected 7 d after onset of treatment (14 d after infection). Antibodies (IgG and IgM) to Pichinde virus were quantified by ELISA. The titer of serum from bavituximab-treated guinea pigs was not significantly different from that of control Ig-treated guinea pigs. Points, average absorbance (n = 3); bars, s.e.m. (b) Lack of Pichinde virus antigen-specific proliferative response in splenocytes from bavituximab-treated guinea pigs. Spleens from bavituximab- or control-treated Pichinde virus-infected animals (n = 3) were removed 7 d after onset of treatment (14 d after infection). Splenocytes were stimulated with Pichinde virus antigen or mock antigen and their ability to incorporate [3H]-thymidine was determined. Bavituximab treatment did not significantly increase the stimulation index (SI). Columns, average SI (n = 3); bars, s.e.m. (c) Clearance of Pichinde virus from blood of guinea pigs treated with bavituximab. Blood samples from groups of 4 guinea pigs were harvested 1 d after treatment with bavituximab or control Ig. P = 0.0145 (unpaired t-test). Columns, average PFU per ml (n = 3); bars, s.e.m. (d) Bavituximab mediates ADCC of Pichinde virus-infected guinea pig kidney fibroblasts 48 h after infection. Specific lysis was determined by quantifying 51Cr release. Bavituximab induced specific lysis of virus infected cells, P < 0.001 (unpaired t-test). Columns, average percentages (n = 3); bars, s.e.m.

Figure 4. Broad spectrum recognition of virus infected cells and protection against cytomegalovirus infection in mice

(a) Flow cytometric analysis of virus-infected cells (right panels) and control uninfected cells (left panels). Cells were infected at an moi of 5. Cells were harvested (at 24 h for influenza A virus and VSV and at 48 h for Vaccinia virus) and stained with bavituximab (open histograms) or control Ig (filled histograms). Live intact cells were gated based on their exclusion of 7-AAD. (b) Immunofluorescence staining of mCMV-infected cells (right panels) and uninfected cells (left panels) with bavituximab. M2-10B4 cells on chamber slides were infected with mCMV at an moi of 5 and stained with bavituximab 48 h later. Upper panels show cells stained with bavituximab (green). Bavituximab is binding to externalized PS on infected cells. Lower panels show images merged with cytoskeleton (red). Nuclei are in blue (all panels). Control Ig did not stain and is not shown. Bar, 50 μm. (c) Kaplan-Meier survival curves of BALB/c mice after infection with an LD80 dose of mCMV and treatment with murine 3G4 (n = 10) or control Ig (n = 8). Treatment with 3G4 (4 mg kg−1) or control Ig was initiated 18 h after infection and administered three times a week thereafter. Survival in the 3G4 group was significantly superior to the control Ig group (P < 0.0001). The results are representative of two separate experiments.