Role of sulfatide in vaccinia virus infection.

BACKGROUND INFORMATION: Vaccinia virus (VACV) was used as a surrogate of variola virus (genus Orthopoxvirus), the causative agent of smallpox, to study orthopoxvirus infection. VACV infects cells via attachment and fusion of the viral membrane with the host cell membrane. Glycosphingolipids, expressed in multiple organs, are major components of lipid rafts and have been associated with the infectious route of several pathogens. RESULTS: We demonstrate that the VACV-WR (VACV Western-Reserve strain) displays no binding to Cer (ceramide) or to Gal-Cer (galactosylceramide), but binds to a natural sulfated derivative of these molecules: the Sulf (sulfatide) 3' sulfogalactosylceramide. The interaction between Sulf and VACV-WR resulted in a time-dependent inhibition of virus infection. Virus cell attachment was the crucial step inhibited by Sulf. Electron microscopy showed that SUVs (small unilamellar vesicles) enriched in Sulf bound to VACV particles. Both the A27 and L5 viral membrane proteins were shown to interact with Sulf, indicating that they could be the major viral ligands for Sulf. Soluble Sulf was successful in preventing mortality, but not morbidity, in a lethal mouse model infection with VACV-WR. CONCLUSIONS: Together the results suggest that Sulf could play a role as an alternate receptor for VACV-WR and probably other Orthopoxviruses.

ESR and NMR studies provide evidence that phosphatidyl glycerol specifically interacts with poxvirus membranes.

BACKGROUND: The lung would be the first organ targeted in case of the use of Variola virus (the causative agent of smallpox) as a bioweapon. Pulmonary surfactant composed of lipids (90%) and proteins (10%) is considered the major physiological barrier against airborne pathogens. The principle phospholipid components of lung surfactant were examined in an in vitro model to characterize their interactions with VACV, a surrogate for variola virus. One of them, Dipalmitoyl phosphatidylglycerol (DPPG), was recently shown to inhibit VACV cell infection. RESULTS: The interactions of poxvirus particles from the Western Reserve strain (VACV-WR) and the Lister strain (VACV-List) with model membranes for pulmonary surfactant phospholipids, in particular DPPG, were studied by Electron Spin Resonance (ESR) and proton Nuclear Magnetic Resonance (1H-NMR). ESR experiments showed that DPPG exhibits specific interactions with both viruses, while NMR experiments allowed us to deduce its stoichiometry and to propose a model for the mechanism of interaction at the molecular level. CONCLUSIONS: These results confirm the ability of DPPG to strongly bind to VACV and suggest that similar interactions occur with variola virus. Similar studies of the interactions between lipids and other airborne pathogens are warranted.

Postoperative Ocular Inflammation: A Single Subconjunctival Injection of XG-102 Compared to Dexamethasone Drops in a Randomized Trial.

PURPOSE: To evaluate the efficacy and safety of XG-102 (brimapitide) compared to dexamethasone eye drops in the treatment of postoperative ocular inflammation. DESIGN: Multicenter, randomized, parallel group, double-masked, noninferiority clinical trial. METHODS: Patients who underwent anterior and posterior segments combined surgery or glaucoma surgery or complex posterior segment surgery were eligible to participate. Patients were administered a single subconjunctival injection of 250 µL XG-102 90 µg (n = 47) or 900 µg (n = 48) or placebo (n = 50) at the end of ocular surgery. Subconjunctival injection for each group (XG-102 90 µg, XG-102 900 µg, or placebo) was followed by eye drops instilled 4 times per day for 21 days with placebo, placebo, or dexamethasone solution, respectively. The primary outcome measure was anterior chamber cell grades at day 28 comparing XG-102 900 µg with dexamethasone. RESULTS: The anterior cell grades for both XG-102 groups were noninferior to dexamethasone (-0.054 anterior cell grade [95% confidence interval -0.350-0.242]; P < .001 for noninferiority) for XG-102 900 µg and -0.086 anterior cell grade (95% confidence interval -0.214-0.385; P = .003 for noninferiority) for XG-102 90 µg. Rescue medication was introduced for 10 (21%), 7 (15%), and 2 (4%) patients allocated to the XG-102 90 µg, XG-102 900 µg, and dexamethasone groups, respectively. The difference between XG-102 90 µg and dexamethasone was statistically significant (P = .013). The number of patients for whom adverse events were reported and the nature of the events reported was similar between the 3 treatment groups. CONCLUSIONS: A single subconjunctival injection of XG-102 at the end of ocular surgery is noninferior to dexamethasone eye drops in the treatment of postoperative ocular inflammation.

Subconjunctival injection of XG-102, a JNK inhibitor peptide, in patients with intraocular inflammation: a safety and tolerability study.

PURPOSE: We aimed to investigate the safety, tolerability, and systemic diffusion of a single escalating dose of XG-102 (a 31-D-amino-acid peptide inhibiting JNK pathway activation), administered subconjunctivally in the treatment of post-surgery or post-trauma intraocular inflammation. METHODS: This is a dose-escalating, tolerance Phase Ib study. Twenty patients with post-surgery or post-traumatic intraocular inflammation were assigned to 1 of the 4 dose escalating (45, 90, 450, or 900 µg XG-102) groups of 5 patients each. Patients were evaluated at 24, 48 h, 8, and 28 days following the administration of XG-102, including laboratory tests, standard eye examinations, vital signs, and occurrence of adverse events. A single plasma quantification of XG-102 was performed 30 min after administration, according to previous pharmacokinetics studies performed on volunteers. RESULTS: A total of 17 non-serious adverse events, considered unrelated to the study treatment, were reported for 10 patients. The adverse event incidence was not related to the drug dose. All patients experienced a decrease in intraocular inflammation as of 24 h post-administration and this decrease was sustained up to 28 days thereafter. No patient required local injection or systemic administration of corticoids following the administration of XG-102. XG-102 was undetectable in the first 3 dose groups. In the fourth-dose group (900 µg) the XG-102 plasma levels were above the limit of detection for 3 patients and above the limit of quantification for 1 patient. CONCLUSIONS: In this first clinical trial using XG-102, administered as a single subconjunctival injection as adjunct therapy, in patients with recent post-surgery or post-trauma intraocular inflammation is safe and well tolerated. Further studies are required to evaluate its efficacy.

XG-102 administered to healthy male volunteers as a single intravenous infusion: a randomized, double-blind, placebo-controlled, dose-escalating study.

The aim of the study is to evaluate the safety, tolerability and pharmacokinetics (PK) of the JNK inhibitor XG-102 in a randomized, double blind, placebo controlled, sequential ascending dose parallel group Phase 1 Study. Three groups of male subjects received as randomly assigned ascending single XG-102 doses (10, 40, and 80 µg/kg; 6 subjects per dose) or placebo (2 subjects per dose) as an intravenous (IV) infusion over 60 min. Safety and tolerability were assessed by physical examination, vital signs, electrocardiography, eye examination, clinical laboratory tests and adverse events (AEs). PK was analyzed using noncompartmental methods. All reported AEs were mild to moderate and neither their number nor their distribution by System Organ Class suggest a dose relationship. Only headache and fatigue were considered probably or possibly study drug related. Headache frequency was similar for active and placebo, consequently this was not considered to be drug related but probably to study conditions. The other examinations did not show clinically relevant deviations or trends suggesting a XG-102 relationship. Geometric mean half-life was similar among doses, ranging from 0.36 to 0.65 h. Geometric mean XG-102 AUC0-last increased more than linearly with dose, 90% confidence intervals (CIs) did not overlap for the two highest doses. Geometric mean dose normalized C max values suggest a more than linear increase with dose but 90% CIs overlap. It may be concluded that XG-102 single IV doses of 10-80 µg/kg administered over 1 h to healthy male subjects were safe and well tolerated.

Protective effect of surfactant protein d in pulmonary vaccinia virus infection: implication of A27 viral protein.

Vaccinia virus (VACV) was used as a surrogate of variola virus (VARV) (genus Orthopoxvirus), the causative agent of smallpox, to study Orthopoxvirus infection. VARV is principally transmitted between humans by aerosol droplets. Once inhaled, VARV first infects the respiratory tract where it could encounter surfactant components, such as soluble pattern recognition receptors. Surfactant protein D (SP-D), constitutively present in the lining fluids of the respiratory tract, plays important roles in innate host defense against virus infection. We investigated the role of SP-D in VACV infection and studied the A27 viral protein involvement in the interaction with SP-D. Interaction between SP-D and VACV caused viral inhibition in a lung cell model. Interaction of SP-D with VACV was mediated by the A27 viral protein. Binding required Ca2+ and interactions were blocked in the presence of excess of SP-D saccharide ligands. A27, which lacks glycosylation, directly interacted with SP-D. The interaction between SP-D and the viral particle was also observed using electron microscopy. Infection of mice lacking SP-D (SP-D-/-) resulted in increased mortality compared to SP-D+/+ mice. Altogether, our data show that SP-D participates in host defense against the vaccinia virus infection and that the interaction occurs with the viral surface protein A27.

Lung surfactant DPPG phospholipid inhibits vaccinia virus infection.

Vaccinia virus (VACV) was used as a surrogate of Variola virus (genus Orthopoxvirus), the causative agent of smallpox, to study orthopoxvirus infection via the respiratory airway. Lung surfactant, a physiological barrier to infection encountered by the virus, is predominantly composed of phospholipids whose role during orthopoxvirus infection has not been investigated. An attenuated Lister strain, derived from the traditional smallpox vaccine and the Western Reserve (WR) strain, lethal for mice infected by the respiratory route, were examined for their ability to bind various surfactant phospholipids. Dipalmitoyl phosphatidylglycerol (DPPG) was found to interact with both VACV strains. DPPG incorporated in small unilamellar vesicle (SUV-DPPG) inhibited VACV cell infection, unlike other phospholipids tested. Both pre-incubation of virus with SUV-DPPG and pretreatment of the cell with SUV-DPPG inhibited cell infection. This specific DPPG effect was shown to be concentration and time dependent and to prevent the first step of the viral cycle, i.e. virus cell attachment. Cryo-electron microscopy highlighted the interaction between the virus and SUV-DPPG. In the presence of the phospholipid, virus particles displayed a hedgehog-like appearance due to the attachment of lipid vesicles. Mice infected intranasally with VACV-WR pre-incubated with SUV-DPPG survived a lethal infection. These data suggest that DPPG in lung surfactant could reduce the amount of orthopoxvirus particles able to infect pneumocytes at the beginning of a respiratory poxvirus infection. The knowledge acquired during this study of virus-DPPG interactions may be used to develop novel chemotherapeutic strategies for smallpox.

Phenotypic and genetic diversity of the traditional Lister smallpox vaccine.

As an initial step in the development of a second-generation smallpox vaccine derived from the Lister strain, to be prepared for a variola virus threat, diversity of the traditional vaccine was examined by characterizing a series of ten viral clones. In vitro and in vivo phenotypic studies showed that the biological behavior of the clones diverged from each other and in most cases diverged from the vaccinia virus (VACV) Lister parental population. Taken together, these results demonstrate the heterogeneity of the viral population within the smallpox vaccine and highlight the difficulty in choosing one clone which would meet the current requirements for a safe and effective vaccine candidate.