A Monovalent and Trivalent MVA-Based Vaccine Completely Protects Mice Against Lethal Venezuelan, Western, and Eastern Equine Encephalitis Virus Aerosol Challenge.

Venezuelan, eastern and western equine encephalitis viruses (EEV) can cause severe disease of the central nervous system in humans, potentially leading to permanent damage or death. Yet, no licensed vaccine for human use is available to protect against these mosquito-borne pathogens, which can be aerosolized and therefore pose a bioterror threat in addition to the risk of natural outbreaks. Using the mouse aerosol challenge model, we evaluated the immunogenicity and efficacy of EEV vaccines that are based on the modified vaccinia Ankara-Bavarian Nordic (MVA-BN®) vaccine platform: three monovalent vaccines expressing the envelope polyproteins E3-E2-6K-E1 of the respective EEV virus, a mixture of these three monovalent EEV vaccines (Triple-Mix) as a first approach to generate a multivalent vaccine, and a true multivalent alphavirus vaccine (MVA-WEV, Trivalent) encoding the polyproteins of all three EEVs in a single non-replicating MVA viral vector. BALB/c mice were vaccinated twice in a four-week interval and samples were assessed for humoral and cellular immunogenicity. Two weeks after the second immunization, animals were exposed to aerosolized EEV. The majority of vaccinated animals exhibited VEEV, WEEV, and EEEV neutralizing antibodies two weeks post-second administration, whereby the average VEEV neutralizing antibodies induced by the monovalent and Trivalent vaccine were significantly higher compared to the Triple-Mix vaccine. The same statistical difference was observed for VEEV E1 specific T cell responses. However, all vaccinated mice developed comparable interferon gamma T cell responses to the VEEV E2 peptide pools. Complete protective efficacy as evaluated by the prevention of mortality and morbidity, lack of clinical signs and viremia, was demonstrated for the respective monovalent MVA-EEV vaccines, the Triple-Mix and the Trivalent single vector vaccine not only in the homologous VEEV Trinidad Donkey challenge model, but also against heterologous VEEV INH-9813, WEEV Fleming, and EEEV V105-00210 inhalational exposures. These EEV vaccines, based on the safe MVA vector platform, therefore represent promising human vaccine candidates. The trivalent MVA-WEV construct, which encodes antigens of all three EEVs in a single vector and can potentially protect against all three encephalitic viruses, is currently being evaluated in a human Phase 1 trial.

Human-Like Neutralizing Antibodies Protect Mice from Aerosol Exposure with Western Equine Encephalitis Virus.

Western equine encephalitis virus (WEEV) causes symptoms in humans ranging from mild febrile illness to life-threatening encephalitis, and no human medical countermeasures are licensed. A previous study demonstrated that immune serum from vaccinated mice protected against lethal WEEV infection, suggesting the utility of antibodies for pre- and post-exposure treatment. Here, three neutralizing and one binding human-like monoclonal antibodies were evaluated against WEEV aerosol challenge. Dose-dependent protection was observed with two antibodies administered individually, ToR69-3A2 and ToR68-2C3. In vitro neutralization was not a critical factor for protection in this murine model, as ToR69-3A2 is a strong neutralizing antibody, and ToR68-2C3 is a non-neutralizing antibody. This result highlights the importance of both neutralizing and non-neutralizing antibodies in the protection of mice from WEEV lethality.

Novel indole-2-carboxamide compounds are potent broad-spectrum antivirals active against western equine encephalitis virus in vivo.

Neurotropic alphaviruses, including western, eastern, and Venezuelan equine encephalitis viruses, cause serious and potentially fatal central nervous system infections in humans for which no currently approved therapies exist. We previously identified a series of thieno[3,2-b]pyrrole derivatives as novel inhibitors of neurotropic alphavirus replication, using a cell-based phenotypic assay (W. Peng et al., J. Infect. Dis. 199:950-957, 2009, doi:http://dx.doi.org/10.1086/597275), and subsequently developed second- and third-generation indole-2-carboxamide derivatives with improved potency, solubility, and metabolic stability (J. A. Sindac et al., J. Med. Chem. 55:3535-3545, 2012, doi:http://dx.doi.org/10.1021/jm300214e; J. A. Sindac et al., J. Med. Chem. 56:9222-9241, 2013, http://dx.doi.org/10.1021/jm401330r). In this report, we describe the antiviral activity of the most promising third-generation lead compound, CCG205432, and closely related analogs CCG206381 and CCG209023. These compounds have half-maximal inhibitory concentrations of ∼1 µM and selectivity indices of >100 in cell-based assays using western equine encephalitis virus replicons. Furthermore, CCG205432 retains similar potency against fully infectious virus in cultured human neuronal cells. These compounds show broad inhibitory activity against a range of RNA viruses in culture, including members of the Togaviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Although their exact molecular target remains unknown, mechanism-of-action studies reveal that these novel indole-based compounds target a host factor that modulates cap-dependent translation. Finally, we demonstrate that both CCG205432 and CCG209023 dampen clinical disease severity and enhance survival of mice given a lethal western equine encephalitis virus challenge. These studies demonstrate that indole-2-carboxamide compounds are viable candidates for continued preclinical development as inhibitors of neurotropic alphaviruses and, potentially, of other RNA viruses. IMPORTANCE There are currently no approved drugs to treat infections with alphaviruses. We previously identified a novel series of compounds with activity against these potentially devastating pathogens (J. A. Sindac et al., J. Med. Chem. 55:3535-3545, 2012, doi:http://dx.doi.org/10.1021/jm300214e; W. Peng et al., J. Infect. Dis. 199:950-957, 2009, doi:http://dx.doi.org/10.1086/597275; J. A. Sindac et al., J. Med. Chem. 56:9222-9241, 2013, http://dx.doi.org/10.1021/jm401330r). We have now produced third-generation compounds with enhanced potency, and this manuscript provides detailed information on the antiviral activity of these advanced-generation compounds, including activity in an animal model. The results of this study represent a notable achievement in the continued development of this novel class of antiviral inhibitors.

A chimeric Sindbis-based vaccine protects cynomolgus macaques against a lethal aerosol challenge of eastern equine encephalitis virus.

Eastern equine encephalitis virus (EEEV) is a mosquito-borne alphavirus that causes sporadic, often fatal disease outbreaks in humans and equids, and is also a biological threat agent. Two chimeric vaccine candidates were constructed using a cDNA clone with a Sindbis virus (SINV) backbone and structural protein genes from either a North (SIN/NAEEEV) or South American (SIN/SAEEEV) strain of EEEV. The vaccine candidates were tested in a nonhuman primate (NHP) model of eastern equine encephalitis (EEE). Cynomolgus macaques were either sham-vaccinated, or vaccinated with a single dose of either SIN/NAEEEV or SIN/SAEEEV. After vaccination, animals were challenged by aerosol with a virulent North American strain of EEEV (NA EEEV). The SIN/NAEEEV vaccine provided significant protection, and most vaccinated animals survived EEEV challenge (82%) with little evidence of disease, whereas most SIN/SAEEEV-vaccinated (83%) and control (100%) animals died. Protected animals exhibited minimal changes in temperature and cardiovascular rhythm, whereas unprotected animals showed profound hyperthermia and changes in heart rate postexposure. Acute inflammation and neuronal necrosis were consistent with EEEV-induced encephalitis in unprotected animals, whereas no encephalitis-related histopathologic changes were observed in the SIN/NAEEEV-vaccinated animals. These results demonstrate that the chimeric SIN/NAEEEV vaccine candidate protects against an aerosol EEEV exposure.

Common marmosets (Callithrix jacchus) as a nonhuman primate model to assess the virulence of eastern equine encephalitis virus strains.

Eastern equine encephalitis virus (EEEV) produces the most severe human arboviral disease in North America (NA) and is a potential biological weapon. However, genetically and antigenically distinct strains from South America (SA) have seldom been associated with human disease or mortality despite serological evidence of infection. Because mice and other small rodents do not respond differently to the NA versus SA viruses like humans, we tested common marmosets (Callithrix jacchus) by using intranasal infection and monitoring for weight loss, fever, anorexia, depression, and neurologic signs. The NA EEEV-infected animals either died or were euthanized on day 4 or 5 after infection due to anorexia and neurologic signs, but the SA EEEV-infected animals remained healthy and survived. The SA EEEV-infected animals developed peak viremia titers of 2.8 to 3.1 log(10) PFU/ml on day 2 or 4 after infection, but there was no detectable viremia in the NA EEEV-infected animals. In contrast, virus was detected in the brain, liver, and muscle of the NA EEEV-infected animals at the time of euthanasia or death. Similar to the brain lesions described for human EEE, the NA EEEV-infected animals developed meningoencephalitis in the cerebral cortex with some perivascular hemorrhages. The findings of this study identify the common marmoset as a useful model of human EEE for testing antiviral drugs and vaccine candidates and highlight their potential for corroborating epidemiological evidence that some, if not all, SA EEEV strains are attenuated for humans.

Clinical and neuroradiographic manifestations of eastern equine encephalitis.

BACKGROUND: Eastern equine encephalitis occurs principally along the east and Gulf coasts of the United States. Recognition of the neuroradiographic manifestations of eastern equine encephalitis could hasten the diagnosis of the illness and speed the response to index cases. METHODS: We reviewed all cases of eastern equine encephalitis reported in the United States between 1988 and 1994. The records of 36 patients were studied, along with 57 computed tomographic (CT) scans and 23 magnetic resonance imaging (MRI) scans from 33 patients. RESULTS: The mortality rate was 36 percent, and 35 percent of the survivors were moderately or severely disabled. Neuroradiographic abnormalities were common and best visualized by MRI. Among the patients for whom MRI scans were available, the results were abnormal for all eight comatose patients as well as for all three noncomatose patients who subsequently became comatose. The CT results were abnormal in 21 of 32 patients with readable scans. The abnormal findings included focal lesions in the basal ganglia (found in 71 percent of patients on MRI, and in 56 percent on CT), thalami (found in 71 percent on MRI and in 25 percent on CT), and brain stem (found in 43 percent on MRI and in 9 percent on CT). Cortical lesions, meningeal enhancement, and periventricular white-matter changes were less common. The presence of large radiographic lesions did not predict a poor outcome, but either high cerebrospinal fluid white-cell counts or severe hyponatremia did. CONCLUSIONS: Eastern equine encephalitis produces focal radiographic signs. The characteristic early involvement of the basal ganglia and thalami distinguish this illness from herpes simplex encephalitis. MRI is a sensitive technique to identify the characteristic early radiographic manifestations of this viral encephalitis.

[The antiviral activity of inhibitors of ADP ribosylation in experimental alpha- and bunyavirus infections]. / Protivovirusnaia aktivnost' nekotorykh ingibitorov ADF-ribozilirovaniia pri éksperimental'nykh al'fa- i bun'iavirusnykh infektsiiakh.

The prophylactic and therapeutic effects of ADP-ribosylation inhibitors, 3,N-acetylaminobenzamide and 3,N-butyrylaminobenzamide, were studied in mice inoculated with an alphavirus or a bunyavirus. Both drugs were shown to have high levels of antiviral activity when given as a single subcutaneous injection in a dose of 10 mg/kg while increasing the number of injections did not increase their efficacy or might even decrease it.

Mortality of captive whooping cranes caused by eastern equine encephalitis virus.

Of 39 captive whooping cranes (Grus americana), 7 died during a 7-week period (Sept 17 through Nov 4, 1984) at the Patuxent Wildlife Research Center, Laurel, Md. Before their deaths, 4 cranes did not develop clinical signs, whereas the other 3 cranes were lethargic and ataxic, with high aspartate transaminase, gamma-glutamyl transferase, and lactic acid dehydrogenase activities, and high uric acid concentrations. Necropsies indicated that the birds had ascites, intestinal mucosal discoloration, fat depletion, hepatomegaly, splenomegaly, and visceral gout. Microscopically, extensive necrosis and inflammation were seen in many visceral organs; the CNS was not affected. Eastern equine encephalitis (EEE) virus was isolated from specimens of the livers, kidneys, lungs, brains, and intestines of 4 of the 7 birds that died, and EEE virus-neutralizing antibody was detected in 14 (44%) of the 32 surviving birds. Other infectious or toxic agents were not found. Morbidity or mortality was not detected in 240 sandhill cranes (Grus canadensis) interspersed among the whooping cranes; however, 13 of the 32 sandhill cranes evaluated had EEE virus-neutralizing antibody. Of the 41 wild birds evaluated in the area, 3 (4%) had EEE virus-neutralizing antibody. Immature Culiseta melanura (the most probable mosquito vector) were found in scattered foci 5 km from the research center.

[Effect of immunosuppressants on the enhanced sensitivity of white mice to the eastern equine encephalitis virus]. / Vliianie immunosupressantov na povyshenie chuvstvitel'nosti belykh myshei k virusu vostochnogo éntsefalomielita loshadei.

EEE virus infection could be induced in white mice whose natural resistance to this virus had been reduced by immunosuppressive drugs such as: Adreson, Cyclophosphan and hydrocortisone.

Pheasant susceptibility at different ages to Eastern encephalitis virus from various sources in Massachusetts.

Thirteen isolates of Eastern encephalitis (EE) virus recovered from various hosts in Massachusetts differed significantly in pathogenicity for pheasants, 21 days old or older, inoculated subcutaneously with graded doses of virus. Among the least pathogenic were two isolates from Culiseta melanura mosquitoes, and among the most pathogenic isolates was one of pheasant origin. Nine-day-old pheasant chicks were highly susceptible to EE virus. Mortality did not appear to be totally dose-related, for inocula larger than an infecting dose did not increase mortality substantially.

Venezuelan equine encephalitis virus: comparison of infectivity and virulence of strains V-38 and P676 in donkeys.

Two strains of Venezuelan equine encephalitis (VEE) virus were examined for the ability to replicate in, as well as to produce death among donkeys. One, a low passage strain known as strain P676 was originally isolated from mosquitos in Venezuela. The other, strain V-38 was isolated from a horse brain in 1938 and had undergone an unknown number of laboratory passages; it is used extensively for the preparation of inactivated VEE vaccine. Both strains were found to be approximately equal in their ability to infect donkeys. However, a quantity as small as 50% hamster intraperitoneal infectious units of strain V-38 resulted in fatal infection. On the other hand, as much as 631 million infectious units of strain P676 were nonfatal in one of two donkeys. It appears that strain V-38 is approximately 100 million times more virulent than strain P676 in equine species. One donkey which received strain P676 demonstrated a biphasic pattern of clinical illness and viremia, and there is suggestive evidence that another animal experienced a second and fatal clinical response 3 weeks after virus inoculation.

Pathogenicity of an attenuated, temperature-sensitive mutant of western equine encephalitis virus induced by a chemical mutagen.

To know the pathogenicity of the chemically induced, temperature-sensitive (ts) mutant of western equine encephalitis virus, designated tsNG39, the lethality for mice injected with tsNG39, virus yield, interferon production, and histological changes in the brains of these mice were examined in parallel with those of mice inoculated with the parent strain (PS). All of the mice injected intracranially with PS died within 3.5 days after injection irrespective of the inoculum size of virus, whereas the lethality of the mice inoculated with tsNG39 varied from 94.3 to 65.5% among groups of mice and this variation seemed to be correlated with the inoculum size of virus rather than with the maximum virus titer in the brain. By histological examination, two types of changes in the brain were distinguished, inflammatory and degenerative ones. Inflammatory changes were more prominent in the brains injected with tsNG39 than in those receiving PS. Degenerative changes were dominant in the brains injected with PS, but they were slight in the earlier phase of infection by tsNG39 became prominent only later. The degree of degenerative change was well correlated with both the virus titer in the mouse brain and the death pattern of mice injected with PS or tsNG39. Since degenerative changes are thought to be caused by the direct effect of injected virus, these results indicated that the factor responsible for the low virulence of tsNG39 was the slow viral growth in the brain.

Eastern equine encephalomyelitis. Histopathologic and ultrastructural changes with isolation of the virus in a human case.

Enveloped Toga virus particles were demonstrated by means of an electron microscopy in the brain tissues of a 3-year-old girl with acute encephalitis. Areas of demyelinization and necrosis throughout the white matter and brainstem were revealed by light microscopy. These viral particles were identified as eastern equine encephalomyelitis virus in postmortem isolation of the virus utilizing young mice and complement-fixation studies. To the authors' knowledge, this is the first demonstration of eastern equine encephalomyelitis virus particles in human tissues by electron microscopy.

Venezuelan equine encephalomyelitis virus (VEEV):strain differentiation and specification of virulence markers.

Fourteen strains of VEEV of diverse origin and antigenicity were classified into five virulence categories according to their efficiency of infection and nature of response in mice. Mice of ages up to 30 days represented distinct levels of responsiveness and were suitable for the differentiation of VEEV strains of highest to lowest virulence. Stepwise changes with age of the susceptibility or responsiveness of mice appeared to determined the type of response as predominantly lethal (D), dual or intermediate (DP) or protective (P).

Correlates to increased lethality of attenuated Venezuelan encephalitis virus vaccine for immunosuppressed hamsters.

Splenectomy or pretreatment of adult hamsters with cyclophosphamide (Cytoxan) increased the lethality of the TC-83 vaccine strain of Venezuelan encephalitis virus (VEE), inoculated subcutaneously, from 12% for normal hamsters to 75 and 76%, respectively. Neither splenectomy nor cyclophosphamide treatment significantly increased the lethality of Pixuna virus. Cytoxantreated (Cy) hamsters developed and maintained levels of TC-83 virus higher than normal infected controls in blood, brain, spleen, and femoral bone marrow; splenectomy had a similar but less intense effect. A severe myeloid necrosis of femoral bone marrow developed 4 to 9 days after TC-83 virus inoculation in 78% of the Cy hamsters and in 48% of the splenectomized (Sx) hamsters. In contrast, only 13% of normal TC-83-infected hamsters developed this lesion. Extensive hemorrhagic lesions in the olfactory lobes and adjacent areas of the brain also developed more frequently in Cy or Sx hamsters than in normal infected controls. Lethally infected hamsters developed and maintained a severe thrombocytopenia, which may be related to the bone marrow lesion and to the hemorrhagic manifestations of lethal VEE infections.