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

Soares MM, King SW, Thorpe PE - Nat. Med. (2008)

Bottom Line: Combination therapy with bavituximab and ribavirin was more effective than either drug alone.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.

View Article: PubMed Central - PubMed

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.

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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.
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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.

Mentions: The protective effect of bavituximab does not appear to be due to direct neutralization. Bavituximab only inhibits Pichinde-virus replication by 60% in virus-yield assays in vitro, even in physiological concentrations of β2GP1 (Supplementary Fig. 3a online). Thus, PS on Pichinde virus may not be required for virus entry, in contrast to vaccinia14 where viral PS is required for infectivity14. Also, protection is not due to induction of neutralizing antibodies to Pichinde virus (Fig. 3a and Supplementary Fig. 4a online) or Pichinde virus specific T-cells (Fig. 3b and Supplementary Fig. 4b) as neither was detectable 6–7 d after onset of treatment (14 d after infection) when the treated animals begin to recover. Two mechanisms appear to explain the protective effect: 1) bavituximab causes opsonization and clearance of infectious virus from the bloodstream, leaving less virus to infect other tissues. Bavituximab treatment of viremic guinea pigs reduced infectious virus in the bloodstream by 95% within 24 h (Fig. 3c); 2) bavituximab induces antibody-dependent cellular cytotoxicity (ADCC) of virus-infected cells. Bavituximab mediates lysis of virus-infected primary guinea pig fibroblasts by primary guinea pig macrophages in vitro (Fig. 3d). Since PS exposure is an early event during virus infection, ADCC may limit virus spread.


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

Soares MM, King SW, Thorpe PE - Nat. Med. (2008)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2597367&req=5

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.
Mentions: The protective effect of bavituximab does not appear to be due to direct neutralization. Bavituximab only inhibits Pichinde-virus replication by 60% in virus-yield assays in vitro, even in physiological concentrations of β2GP1 (Supplementary Fig. 3a online). Thus, PS on Pichinde virus may not be required for virus entry, in contrast to vaccinia14 where viral PS is required for infectivity14. Also, protection is not due to induction of neutralizing antibodies to Pichinde virus (Fig. 3a and Supplementary Fig. 4a online) or Pichinde virus specific T-cells (Fig. 3b and Supplementary Fig. 4b) as neither was detectable 6–7 d after onset of treatment (14 d after infection) when the treated animals begin to recover. Two mechanisms appear to explain the protective effect: 1) bavituximab causes opsonization and clearance of infectious virus from the bloodstream, leaving less virus to infect other tissues. Bavituximab treatment of viremic guinea pigs reduced infectious virus in the bloodstream by 95% within 24 h (Fig. 3c); 2) bavituximab induces antibody-dependent cellular cytotoxicity (ADCC) of virus-infected cells. Bavituximab mediates lysis of virus-infected primary guinea pig fibroblasts by primary guinea pig macrophages in vitro (Fig. 3d). Since PS exposure is an early event during virus infection, ADCC may limit virus spread.

Bottom Line: Combination therapy with bavituximab and ribavirin was more effective than either drug alone.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.

View Article: PubMed Central - PubMed

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.

Show MeSH
Related in: MedlinePlus