Shlomo Lustig

Prophylactic and Therapeutic Efficacy of Human Intravenous Immunoglobulin in Treating West Nile Virus Infection in Mice

West Nile virus (WNV) is a mosquito-borne disease found most commonly in Africa, West Asia, and the Middle East, where up to 40% of the human population possesses antibodies. It is an emerging disease in the United States. Humans infected with WNV develop a febrile illness that can progress to meningitis or encephalitis. In mice, WNV causes central nervous system infection, paralysis, encephalitis, and death. Currently, no specific therapy or vaccine has been approved for human use. We examined the prophylactic and therapeutic efficacy of pooled human plasma (PP) and intravenous immunoglobulin (IVIG) for treatment of WNV-infected mice. Full protection was achieved when the infected mice were treated with pooled plasma or IVIG obtained from healthy Israeli blood donors that contained WNV-specific antibodies. Similar treatments using PP or IVIG obtained from US blood donors had no protective effect. Recovery of the lethally infected mice was dependent on the dose and time of IVIG administration. These results indicate that antibodies play a major role in protection and recovery from WNV infection and that IVIG can be used as first-line therapy.

West Nile virus neuroinvasion and encephalitis induced by macrophage depletion in mice.

SummaryThe encephalitic West Nile virus and its nonneuroinvasive variant, WN-25, were used to study the effect of macrophage depletion on viral invasion of the central nervous system. The in vivo elimination of macrophages was achieved by use of liposome-encapsulated drug dichloromethylene diphosphonate. Depletion of macrophages had an exacerbating effect on the course of the viral infection, exhibited by higher and extended viremia and accelerated development of encephalitis and death. Using a low dose of West Nile virus (5 PFU/mouse), an increase in mortality (from 50% to 100%) due to macrophage depletion was demonstrated. Furthermore, the attenuated noninvasive variant WN-25 showed high and prolonged viremia in the macrophage depleted mice (≈5 log 10 PFU/ml versus 2 in control mice), that allowed the penetration of the virus into the central nervous system. The mortality rate caused by the attenuated virus in the macrophage-depleted mice was 70–75%, as compared to complete survival in the control inoculated mice. These results indicate a significant role of macrophages in the non-specific immediate defence system of the organism in case of viral infection.

Dehydroepiandrosterone protects mice from endotoxin toxicity and reduces tumor necrosis factor production.

Recent reports have demonstrated an immunomodulating activity of dehydroepiandrosterone (DHEA) different from that described for glucocorticoids. The present study was designed to test DHEAs activity in endotoxic shock and to investigate its effect on endotoxin-induced production of tumor necrosis factor (TNF). Mortality of CD-1 mice exposed to a lethal dose of lipopolysaccharide (LPS; 800 micrograms per mouse) was reduced from 95 to 24% by treatment with a single dose of DHEA, given 5 min before LPS. LPS administration resulted in high levels of TNF, a response that was significantly blocked by DHEA, both in vivo and in vitro. DHEA treatment also reduced LPS-induced increments in serum corticosterone levels, a parameter considered not to be mediated by TNF. In another experimental model, mice sensitized with D-galactosamine, followed by administration of recombinant human TNF, were subjected to 89% mortality rate, which was reduced to 55% in DHEA-treated mice. These data show that DHEA protects mice from endotoxin lethality. The protective effect is probably mediated by reduction of TNF production as well as by effecting both TNF-induced and non-TNF-induced phenomena.

West Nile virus envelope proteins: nucleotide sequence analysis of strains differing in mouse neuroinvasiveness

Several neuroinvasive and non-neuroinvasive West Nile (WN) viruses were characterized by nucleotide sequencing of their envelope (E) protein regions. Prolonged passage in mosquito cells caused loss of neuroinvasiveness and acquisition of an N-linked glycosylation site, which is utilized. Limited passage in cell culture also caused glycosylation but not attenuation, suggesting that glycosylation may not be directly responsible for attenuation and that a second mutation (L68 --> P) may also be involved. A monoclonal antibody-neutralization escape mutant with a substitution at residue 307, a site common to other flavivirus escape mutants, was also attenuated. A partially neuroinvasive revertant regained the parental E sequence, implying that determinants outside of the E region may also influence attenuation. Data suggest that the neuroinvasive determinants may be similar to those for other flaviviruses. Also, sequence comparison with the WN virus (Nigeria) strain revealed considerable divergence of the E protein at the nucleotide and amino acid levels.

Loss of active neuroinvasiveness in attenuated strains of West Nile virus : pathogenicity in immunocompetent and SCID mice

SummaryThe neuropathogenicity of West Nile virus (WNV) and two derived attenuated strains WN25 and WN25A, was studied in young adult ICR mice and in severe combined immunodeficient (SCID) mice. Similarity in serology and RNA fingerprints were found between WNV and WN25. The viral envelope proteins of the attenuates differed from WNV in their slower mobility in SDS-PAGE due probably to the presence of N-linked glycan. The three strains were lethal to ICR mice by intracerebral (IC) inoculation, but when inoculated intraperitoneally (IP), WNV caused viremia, invaded the CNS and was lethal, whereas the attenuates showed no viremia or invasion of the CNS. The attenuates elicited antibodies to comparable levels as WNV in IP-infected mice, conferring upon them immunity to IC challenge with the wild type. In IP-inoculated SCID mice the three strains exhibited similar high viremiae that lasted until death of the animals. All strains invaded the CNS and proliferated in the mouse brain to similar high titers, but differed largely in the time of invasion: WNV invaded the CNS of SCID mice (and two other mouse strains) much earlier than the attenuates, which showed large intervals in their time of invasion into individual mouse brains within the group. The data presented for SCID mice indicate that WN25 and WN25A have truly lost the neuroinvasive property, and that this property materialized by a prescribed, active process specific for WNV.

Protective effects of melatonin in mice infected with encephalitis viruses

SummaryWe examined the effect of the pineal neurohormone melatonin (MLT) on protection from viral encephalitis. The antiviral activity of MLT was evaluated in normal mice inoculated with Semliki Forest virus (SFV) and in stressed mice injected with the attenuated non-invasive West Nile virus (WN-25). Administration of MLT (s.c.) daily from 3 days before through 10 days after virus inoculation reduced viremia and significantly postponed the onset of disease and death by 7 to 10 days. Moreover, MLT injection reduced mortality of SFV (10 PFU) inoculated mice from 100% to 44%. In mice inoculated with high dose of SFV (100 PFU), MLT postponed death and reduced mortality by 20%. In all of the surviving mice anti-SFV antibodies were detected 22 days after virus inoculation. Infection of mice stressed by either isolation or dexamethasone injection with WN-25 induced mortality of 75% and 50% respectively, which was reduced by MLT administration to 31% and 25%, respectively. The efficiency of MLT in protecting from lethal viral infections warrants further investigations on its mechanisms of action.

Infection of Glioma Cells with Sindbis Virus Induces Selective Activation and Tyrosine Phosphorylation of Protein Kinase C δ IMPLICATIONS FOR SINDBIS VIRUS-INDUCED APOPTOSIS

Sindbis virus (SV) is an alpha virus used as a model for studying the role of apoptosis in virus infection. In this study, we examined the role of protein kinase C (PKC) in the apoptosis induced by SVNI, a virulent strain of SV. Infection of C6 cells with SVNI induced a selective translocation of PKCδ to the endoplasmic reticulum and its tyrosine phosphorylation. The specific PKCδ inhibitor rottlerin and a PKCδ kinase-dead mutant increased the apoptosis induced by SVNI. To examine the role of the tyrosine phosphorylation of PKCδ in the apoptosis induced by SVNI we used a PKCδ mutant in which five tyrosine residues were mutated to phenylalanine (PKCδ5). PKCδ5-overexpressing cells exhibited increased apoptosis in response to SVNI as compared with control cells and to cells overexpressing PKCδ. SVNI also increased the cleavage of caspase 3 in cells overexpressing PKCδ5 but did not induce cleavage of PKCδ or PKCδ5. Using single tyrosine mutants, we identified tyrosines 52, 64, and 155 as the phosphorylation sites associated with the apoptosis induced by SVNI. We conclude that PKCδ exerts an inhibitory effect on the apoptosis induced by SV and that phosphorylation of PKCδ on specific tyrosines is required for this function.

Protection by dehydroepiandrosterone in mice infected with viral encephalitis

SummaryDehydroepiandrosterone (DHEA) has a significant protective effect in mice infected with West Nile virus (WNV), Sindbis virus neurovirulent (SVNI) and Semliki Forest virus (SFV). Mice injected subcutaneously (SC) with a single injection of DHEA (1 g/kg) on the same day or one day pre or post infection with WNV resulted in 40–50% mortality as compared to 100% in control injected mice (p<0.05). The drug was effective following a single SC injection or serial intraperitoneal (IP) injections (5–20mg/kg) on days 0, 2, 4, and 6 following virus inoculation. Moreover, DHEA injection not only reduced viremia and death rate, but also significantly delayed the onset of the disease and mortality. The titers of antivirus antibodies in surviving mice were very high. However, DHEA had no effect on WNV growth in BHK or Vero cell cultures. In this study it was shown that DHEA protects mice against WNV, SVNI and SFV lethal infection. Though the mechanism of the protective effect of DHEA is still unknown, it seems that DHEA can modify the host resistance mechanisms rather than the virus itself.

Postexposure Immunization with Modified Vaccinia Virus Ankara or Conventional Lister Vaccine Provides Solid Protection in a Murine Model of Human Smallpox

BACKGROUND Decades after the cessation of smallpox vaccination, the potential of the deliberate release of pathogenic orthopoxviruses has forced a reconsideration of using these extremely efficient human vaccines. Scenarios of sudden biothreats have prompted demand for rapidly protective vaccination. However, the feasibility of short-term vaccination (i.e., vaccination shortly before exposure) with vaccinia virus (VACV) is uncertain. METHODS We tested the rapid protective capacity of vaccines based on VACV strain Lister (VACV-Lister) and on modified VACV Ankara (MVA) in different mouse models, comparing lethal infections with VACV strain Western Reserve (VACV-WR) or ectromelia virus (ECTV). RESULTS In contrast to VACV-WR challenge, we found extended incubation periods after ECTV challenge, allowing successful therapeutic immunization with VACV-Lister and MVA when applied 2-3 days after exposure. Rapid protection from respiratory tract ECTV infection was significantly affected by vaccine dose and was associated with occurrence of poxvirus-specific antibodies. Vaccinations in type I interferon receptor-deficient mice were protective, whereas recombination activating gene 1-deficient mice lacking mature T and B cells failed to mount immunity after short-term vaccination, confirming an essential role of adaptive immune responses. CONCLUSIONS ECTV infection in mice models the course of human smallpox. Our data provide evidence to substantiate historical data on the usefulness of postexposure vaccination with conventional VACV and the new candidate MVA to protect against fatal orthopoxvirus infections.

Clinical and Immune Responses after Revaccination of Israeli Adults with the Lister Strain of Vaccinia Virus

BACKGROUND During the winter of 2002-2003, the Israeli health authorities launched a campaign to vaccinate first responders against smallpox. METHODS In an open study, 159 healthy, preimmunized adults, 24-52 years old, who participated in the campaign were vaccinated with the Lister strain of vaccinia virus by the multipuncture technique. The safety, immunogenicity, and reactogenicity of the vaccine were assessed. RESULTS Successful vaccination rates were 61% and 56%, on the basis of clinical take and seroconversion, respectively. Adverse events among the vaccinees were minor. Seventy-nine (88%) of the 90 vaccinees with clinical take also seroconverted ( kappa =0.779). The level of preexisting antibodies inversely correlated with the rates of clinical take and seroconversion (P</=.0098). In the group of vaccinees with the lowest preexisting levels of antibodies, 89% and 86% developed clinical take or seroconverted, respectively. The time since last vaccination was significantly associated with the rates of clinical take and seroconversion (P</=0.001). CONCLUSIONS These rates of successful vaccination in previously immunized individuals are consistent with the historical experience of use of this vaccine in Israel. The rate of occurrence and the severity of local and other reactions in the vaccinees were within the expected range. Levels of preexisting antibodies and the time since last vaccination played a major role in determining success rates.