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Toxicity assessment of Venezuelan Equine Encephalitis virus vaccine candidate strain V3526
Abstract
DynPort Vaccine Company (DVC) LLC, a CSC Company, is under a contract with the United States Department of Defense Joint Vaccine Acquisition Program (JVAP) to develop, test and license safe and efficacious vaccines against biowarfare agents. As part of this program DVC is conducting a comprehensive toxicological assessment of the safety of V3526, a live attenuated Venezuelan Equine Encephalitis (VEE) vaccine candidate. Our review of the published pre-clinical toxicology literature, together with the data collected from DVC-sponsored investigations indicated V3526 was comparatively safer and more efficacious than TC-83, the current VEE Investigational New Drug (IND) status vaccine. Non-clinical toxicity studies on experimental systems ranging from mouse, guinea pig, equine and non-human primates (NHP) consistently revealed the V3526 vaccine candidate superior in terms of safety and related toxicological parameters such as neurovirulence when compared with the TC-83. Our experimental investigations indicated that V3526 may conform to the key requirements of a VEE virus vaccine, in that, (a) it elicits a high level of immunogenic response in mice and hamsters, both sensitive to VEE-induced pathologies, and (b) it induces a protective response in the NHP model when challenged with either virulent IA/B or IE viruses. Additional studies are underway to further confirm these findings.
... This vaccine is also used under IND status, has short-lived immunity and requires frequent boosts [14,15]. An attenuated strain, V3526, which has a deletion mutation at the furin cleavage site between the E2 and E3 glycoproteins of the Trinidad donkey strain of VEEV [16] has been extensively evaluated as a VEEV vaccine candidate17181920. V3526 has excellent immunogenic activity, but also causes febrile illness and low level neurotropism in non human primates [17,18,212223. ...
... An attenuated strain, V3526, which has a deletion mutation at the furin cleavage site between the E2 and E3 glycoproteins of the Trinidad donkey strain of VEEV [16] has been extensively evaluated as a VEEV vaccine candidate17181920. V3526 has excellent immunogenic activity, but also causes febrile illness and low level neurotropism in non human primates [17,18,212223. In phase I clinical trial, V3526 induced a robust immune response; however, a high frequency of fever and a flu-like syndrome were reported which led to the cessation of the development of V3526 as a live attenuated vaccine [24,25] .The residual virulence associated with the vaccine candidates has again focused the attention on the inactivated vaccine preparation. ...
... We also demonstrate in vitro that INA can efficiently inactivate the live attenuated vaccine strain V3526 of VEEV. Live attenuated V3526 has shown excellent protective efficacy in animal models [17,18] and robust immune response in phase I clinical trial [25]. However, its development as a live attenuated vaccine was halted due to a flu-like syndrome and fever in the recipients [24,25]. ...
We have previously shown that a hydrophobic alkylating compound, 1,5-iodonaphthyl-azide (INA) can efficiently inactivate the virulent strain of Venezuelan equine encephalitis virus (VEEV), V3000 in vitro. In this study, we have evaluated the safety of INA-inactivated V3000 and V3526 and the protective efficacy of INA-inactivated V3000. INA-inactivated V3000 and V3526 did not cause disease in suckling mice. RNA isolated from the INA-inactivated V3000 and V3526 was also not infectious. Immunization of adult mice with INA-inactivated V3000 induced an anti-VEEV antibody response and protected mice from virulent VEEV challenge. The protective efficacy of INA-inactivated V3000 increased with the use of adjuvants. Results suggest that inactivation of enveloped viruses by INA may occur by two independent mechanisms and the INA-inactivated VEEV elicit a protective antibody response in mice.
... The biohazard analysis follows the chemical and biological risk assessment components that include: (a) hazard assessment, which involves an evaluation of the intrinsic hazard characteristics attributable to the phylogenetic, biochemical, or macromolecular properties linked to hazard attributes; (b) dose-response evaluation, which in the case of a biological agent involves parameters such as minimal dose for infectivity, pathogenicity, environmental transmission, and distribution in the ecosystem populations; (c) exposure assessment such as those involved in occupational, clinical, and general environment-related activities using a set of realistic exposure scenarios; and (d) risk characterization, a formalized approach to combine the characteristics of hazard, toxicity, and exposure to derive a measure of risk associated with the biological agent. 2 2.1.1 Biohazard Analysis Based on Macromolecular Modifications: Rao et al. (2006) describes the biohazard assessment approach based on the macromolecular modification through bioengineering of wild-type strains to yield vaccine candidates as in the case of V3526, the Venezuelan Equine Encephalitis (VEE) vaccine strain. Figure 1 illustrates macromolecular construction of V3526 that results in modifying pathogenicity but retaining the immunogenic potentials. ...
... Based on Hart et al. (2000). Adapted from Rao et al. (2006) Table 1 Pathogenicity of V3526 in Mice ...
Biohazard assessment of biodefense vaccine candidates forms the basis for a facility- and activity-specific risk assessment performed to determine the biosafety levels and general safety standards required for biological product development. As part of our support to the U.S. biodefense vaccine development program, the author performed a systematic biohazard assessment of potential vaccine candidates with the primary objectives to: (a) identify and characterize hazard elements associated with the wild-type and vaccine strains; (b) provide biohazard information on the vaccine candidate to assist Phase 1 clinical trial facility sites; (c) provide a baseline agent and facility-specific risk assessment at clinical trial facilities interested in performing Phase 1 clinical trials; (d) provide comparative hazard profiles of the vaccine candidates with Material Safety Data Sheet (MSDS) for wild-type to identify and establish appropriate protective biosafety levels; and (e) support a determination of the personal protective equipment as required under the OSHA guidelines. This paper describes the biohazard analysis of two vaccine candidates, Venezuelan Equine Encephalitis Virus Strain 3526 and Francisella tularensis LVS, a viral and bacterial agent, respectively. As part of the biohazard assessment, we performed a thorough review of published literature on medical pathology, epidemiology, pre-clinical investigational studies, and environmental data on the etiologic agent subtypes and the vaccine candidates.
... While alphavirus replicon particles based on wild-type VRP 3000 or the attenuated VRP 3014 surface coats are highly successful vaccine platforms (15,17,24,25), a myriad of concerns have limited their use, including select agent/BSL3 containment, reversion, and inefficacy of the vaccine platform in vulnerable populations. To overcome these issues, we developed a platform that utilizes the VEE virus 3526 vaccine backbone, which combines the deletion of the entire furin cleavage site between E3 and E2 with a secondary site resuscitating mutation in E1 (23,26,27); this attenuated virus packages fused E3/E2 protein (PE2), has been used in humans as a vaccine candidate, and is a BSL2 pathogen (28,29). Based on this attenuated VEE virus strain, we generated a new VRP vector platform (Fig. 1A). ...
Zoonotic viruses circulate as swarms in animal reservoirs and can emerge into human populations causing epidemics that adversely affect public health. Portable, safe, and effective vaccine platforms are needed in the context of these outbreak and emergence situations. In this manuscript, we report the generation and characterization of an alphavirus replicon vaccine platform based on a non-select agent, attenuated Venezuelan equine encephalitis (VEE) virus vaccine strain 3526 (VRP 3526). Using both noroviruses and coronaviruses as model systems, we demonstrate utility of the VRP 3526 platform in generation of recombinant proteins, production of virus like particles, and in vivo efficacy as a vaccine against emergent viruses. Importantly, packaging under BSL2 conditions distinguishes VRP 3526 from previously reported alphavirus platforms and makes this approach accessible to the majority of labs around the world. In addition, improved outcomes in the vulnerable aged models as well as against heterologous challenge suggest improved efficacy compared to previous attenuated VRP approaches. Taken together, the VRP 3526 platform represents a safe and highly portable system that can be rapidly deployed under BSL2 conditions for generation of candidate vaccines against emerging microbial pathogens.
IMPORTANCE While VEE replicon particles provide a robust, established platform for antigen expression and vaccination, its utility has been limited by the requirement for high containment facilities for production and packaging. In this manuscript, we utilize an attenuated vaccine strain capable of use at lower biocontainment, but retaining the capacity of the wild type replicon particle. Importantly, the new replicon platform provides equal protection for aged mice and following heterologous challenge, which distinguishes it from other attenuated replicon platforms. Together, the new system represents a highly portable, safe system for use in the context of disease emergence.
... Figure 2 illustrates a proposed conceptual outline for risk mitigation for stem cell-derived products and therapies that takes into consideration the existing biosafety guidelines for facilities involved in cell-based products R&D, cell banking, manufacture, and testing activities, with evidence of the potential health risk associated Articles with certain sources of stem-cells used in medical therapy and product development projects. The evidencebased criteria development outlined here conceptually rests on conventional weight-of-evidence approaches to biohazard assessment of biological agents and toxins (Rao et al., 2004(Rao et al., , 2006. The proposed biohazard evidence-based risk mitigation framework takes into consideration safety and potential health hazards associated with stem cell-derived products and therapy, as well as facility operational processes likely to create occupational exposures to microbiological contamination during stem cell and feeder cell sorting and processing activities. ...
A vast spectrum of therapeutic interventions is based on stem cell technology, ranging from inert bio-materials to pluripotent, virally-modified cells. On the other hand, the scientific and regulatory community is concerned with the safety of stem cell-based products and therapies and the potential health risks associated with cell-based therapies. Scientific publications have revealed shared genetic similarities and cellular morphologic and phenotypic properties between stem cells obtained from embryonic and adult sources and cancer stem cells, thus creating considerable uncertainties about the long-term safety of stem cell-based products and therapies. Existing regulatory policy and guidelines are generally inadequate to address safety assessment and risk mitigation approaches to stem cell technology-derived products and therapies. Therefore, a need exists to develop core competencies within regulatory and standards-setting institutions to better understand the unique characteristics of stem cell-derived products and to perform safety assessments on a case-by-case basis, taking into account potential hazards and risk mitigation considerations. This article outlines an evidence-based risk mitigation framework that discusses two interrelated pathways in stem cell-based biotechnologies—stem cell-derived product development and stem cell-based therapies. The proposed conceptual framework integrates biohazard considerations for cell-based products with product and facility biosafety issues that are critical to research and product development activities. The stem cell-based clinical therapeutic pathway addresses the need for integrated Institutional Review Board and Institutional Biosafety Committee reviews as a way to develop core competencies to mitigate risks and ensure that institutional policy and safety considerations are adequately addressed in stem cell-based clinical therapy and product development activities.
... Currently live attenuated TC83 [25] which is under investigational new drug status, has limited use due to non-responders and residual virulence [26,11,13,15] and may not protect against the broad range of VEEV strains [27]. Several other passive and active immunization strategies have been tested such as genetically engineered live attenuated strain V3526 [27][28][29][30], chimeric SIN/VEE [31], recombinant baculoviruses, adenoviruses and vaccinia virus expressing VEEV structure glycoprotein [32][33][34], monoclonal antibodies [35,36] and microencapsulated VEE virus vaccine [37]. Compounds such as melatonin have also been evaluated for their efficacy in increasing the efficiency of TC-83 vaccine [38,39]. ...
... Vaccination of humans with the live attenuated vaccine followed by a booster dose of inactivated virus results is immunogenicity and is well tolerated [91]. Because of ongoing concerns about the potential use of VEE as a biowarfare weapon, impetus to develop a safe and effective VEE vaccine has intensified and several candidate vaccines are in active development [92]. Mortality in patients with VEE is typically reported as 10–25%, by contrast in symptomatic patients without encephalitis mortality is less than 1%. ...
Arboviruses continue to be a major cause of encephalitis in North America, and West Nile virus neuroinvasive disease is now the dominant cause of encephalitis. Transmission to humans of North American arboviruses occurs by infected mosquitoes or ticks. Most infections are asymptomatic or produce a flulike illness. Rapid serum or cerebrospinal fluid IgM antibody capture ELISA assays are available to diagnosis the acute infection for all North American arboviruses. Unfortunately, no antiviral drugs are approved for the treatment of arbovirus infection and current therapy is supportive.
... Against VEEV, an attenuated vaccine TC-83 and a formalin-inactivated vaccine C-84 have been used[33,70]. Currently, a promising, live-attenuated VEEV vaccine V3526 is being tested[202]. The current EEEV vaccines are inactivated products[49]that have low immunogenicity, requiring multiple inoculations and periodic boosters. ...
Sindbis virus (SINV) (genus Alphavirus, family Togaviridae) is an enveloped virus with a genome of single-stranded, positive-polarity RNA of 11.7 kilobases. SINV is widespread in Eurasia, Africa, and Australia, but clinical infection only occurs in a few geographically restricted areas, mainly in Northern Europe. In Europe, antibodies to SINV were detected from patients with fever, rash, and arthritis for the first time in the early 1980s in Finland. It became evident that the causative agent of this syndrome, named Pogosta disease, was closely related to SINV. The disease is also found in Sweden (Ockelbo disease) and in Russia (Karelian fever). Since 1974, for unknown reason, the disease has occurred as large outbreaks every seven years in Finland. This study is to a large degree based on the material collected during the 2002 Pogosta disease outbreak in Finland. We first developed SINV IgM and IgG enzyme immunoassays (EIA), based on highly purified SINV, to be used in serodiagnostics. The EIAs correlated well with the hemagglutination inhibition (HI) test, and all individuals showed neutralizing antibodies. The sensitivities of the IgM and IgG EIAs were 97.6% and 100%, and specificities 95.2% and 97.6%, respectively. E1 and E2 envelope glycoproteins of SINV were shown to be recognized by IgM and IgG in the immunoblot early in infection. We isolated SINV from five patients with acute Pogosta disease; one virus strain was recovered from whole blood, and four other strains from skin lesions. The etiology of Pogosta disease was confirmed by these first Finnish SINV strains, also representing the first human SINV isolates from Europe. Phylogenetic analysis indicated that the Finnish SINV strains clustered with the strains previously isolated from mosquitoes in Sweden and Russia, and seemed to have a common ancestor with South-African strains. Northern European SINV strains could be maintained locally in disease-endemic regions, but the phylogenetic analysis also suggests that redistribution of SINV tends to occur in a longitudinal direction, possibly with migratory birds. We searched for SINV antibodies in resident grouse (N=621), whose population crashes have previously coincided with human SINV outbreaks, and in migratory birds (N=836). SINV HI antibodies were found for the first time in birds during their spring migration to Northern Europe, from three individuals: red-backed shrike, robin, and song thrush. Of the grouse, 27.4% were seropositive in 2003, one year after a human outbreak, but only 1.4% of the grouse were seropositive in 2004. Thus, grouse might contribute to the human epidemiology of SINV. A total of 86 patients with verified SINV infection were recruited to the study in 2002. SINV RNA detection or virus isolation from blood and/or skin lesions was successful in eight patients. IgM antibodies became detectable within the first eight days of illness, and IgG within 11 days. The acute phase of Pogosta disease was characterized by arthritis, itching rash, fatigue, mild fever, headache, and muscle pain. Half of the patients reported in self-administered questionnaires joint symptoms to last > 12 months. Physical examination in 49 of these patients three years after infection revealed persistent joint manifestations. Arthritis (swelling and tenderness in physical examination) was diagnosed in 4.1% (2/49) of the patients. Tenderness in palpation or in movement of a joint was found in 14.3% of the patients in the rheumatologic examination, and additional 10.2% complained persisting arthralgia at the interview. Thus, 24.5% of the patients had joint manifestations attributable to the infection three years earlier. A positive IgM antibody response persisted in 3/49 of the patients; both two patients with arthritis were in this group. Persistent symptoms of SINV infection might have considerable public health implications in areas with high seroprevalence. The age-standardized seroprevalence of SINV (1999-2003, N=2529) in the human population in Finland was 5.2%. The seroprevalence was high in North Karelia, Kainuu, and Central Ostrobothnia. The incidence was highest in North Karelia. Seroprevalence in men (6.0%) was significantly higher than in women (4.1%), however, the average annualized incidence in the non-epidemic years was higher in women than in men, possibly indicating that infected men are more frequently asymptomatic. The seroprevalence increased with age, reaching 15.4% in persons aged 60-69 years. The incidence was highest in persons aged 50-59 years. Pogostantauti on virusinfektio, joka ilmenee ihmisellä nivel- ja ihottumaoirein. Noin 5 % suomalaisista on sairastanut infektion. Pogostantautia esiintyy syyskesällä, sillä ilmeisesti loppukesän hyttyslajit siirtävät viruksen ihmiseen. Spesifiä hoitoa tai rokotetta tautiin ei ole. Tauti löydettiin vuonna 1974 Ilomantsissa ja potilaiden vasta-ainetutkimukset osoittivat, että taudinaiheuttaja on läheistä sukua Sindbis-virukselle. Sindbis-virusta esiintyy Euraasiassa, Afrikassa ja Oseaniassa, mutta ihmisten tautitapauksia lähinnä vain Pohjois-Euroopassa, etenkin Suomessa. Pogostantauti on puhjennut satojen tai tuhansien potilaiden epidemiaksi täsmälleen seitsemän vuoden välein. Viimeisin epidemia oli syyskesällä 2002, jolloin diagnosoitiin 600 tapausta. Tämä väitöskirjatutkimus pohjautuu suurelta osin vuoden 2002 pogostantautiepidemian aikana koottuun näyte- ja potilasaineistoon. Potilaita seurattiin akuutin vaiheen jälkeen kolmen vuoden ajan. Väitöstutkimuksessa kuvattiin akuutin pogostantaudin tyyppioireiksi nivelkivut/-turvotus, kutiava ihottuma, väsymys, kuume, lihaskivut ja päänsärky. Nämä oireet alkavat usein samanaikaisesti. Tyypillisesti oireilevia niveliä ovat nilkat, polvet, sormet ja ranteet. Tutkimuksessa osoitettiin, että pogostantauti aiheuttaa osalle potilaista vuosikausia kestäviä niveloireita. Kolmen vuoden kuluttua infektiosta 4 %:lla potilaista todettiin krooninen niveltulehdus, ja kaikkiaan 14 %:lla todettiin objektiivisesti havaittavia pitkittyneitä niveloireita. Tutkimuksen mukaan Pohjois-Karjalassa noin 10 % väestöstä on sairastanut pogostantaudin, joten voidaan arvioida, että maakunnan alueella on satoja potilaita, joiden krooninen niveltulehdus johtuu aiemmin saadusta Sindbis-virusinfektiosta. Pogostantaudin mahdollisuus tulisi siis huomioida epäselvien niveltulehdusten erotusdiagnostiikassa. Tutkimuksessa kehitettiin potilasdiagnostiikkaan sopivia vasta-ainetestejä sekä kuvattiin infektion jälkeistä vasta-aineiden kinetiikkaa. Lisäksi työssä eristettiin viisi uutta Sindbis-viruskantaa suoraan akuuttien potilaiden iho- ja verinäytteistä. Kyseessä ovat ensimmäiset ihmisestä eristetyt Sindbis-viruskannat Euroopassa. Viruskantojen sukupuuanalyysi osoitti niiden olevan läheistä sukua Etelä-Afrikan kannoille. Virus saattaakin levitä maailmanlaajuisesti pohjois-eteläsuunnassa muuttolintujen välityksellä. Tutkimuksessa osoitettiin, että pienessä osassa Suomeen palaavista muuttolinnuista on Sindbis-virusvasta-aineita. Lisäksi suomalaisten metsäkanalintujen Sindbis-virusvasta-aineiden esiintyvyydessä havaittiin mahdollinen yhteys pogostantaudin vuosittaiseen esiintyvyyteen. Sekä muuttolinnuilla että vuoden ympäri Suomessa olevilla metsäkanalinnuilla saattaa siis olla rooli pogostantaudin erikoisessa seitsemän vuoden epidemiasyklissä, jonka syytä ei edelleenkään tunneta.
... V3526 is a live attenuated VEEV vaccine derived by site-directed mutagenesis from a full-length infectious VEEV isolate. It has been shown to be safe and efficacious in horses (Fine et al., 2007), is not significantly more neurovirulent than TC-83 when tested in non-human primates (Fine et al., 2008) and is able to protect mice from infection of VEEV via mosquito bite as well as i.p. challenge ( Charles et al., 1997;Rao et al., 2006). This approach to vaccine development should be conducted with caution as it can produce rapid adoption of mutations in the NSP. ...
The use of biological agents has generally been confined to military-led conflicts. However, there has been an increase in non-state-based terrorism, including the use of asymmetric warfare, such as biological agents in the past few decades. Thus, it is becoming increasingly important to consider strategies for preventing and preparing for attacks by insurgents, such as the development of pre- and post-exposure medical countermeasures. There are a wide range of prophylactics and treatments being investigated to combat the effects of biological agents. These include antibiotics (for both conventional and unconventional use), antibodies, anti-virals, immunomodulators, nucleic acids (analogues, antisense, ribozymes and DNAzymes), bacteriophage therapy and micro-encapsulation. While vaccines are commercially available for the prevention of anthrax, cholera, plague, Q fever and smallpox, there are no licensed vaccines available for use in the case of botulinum toxins, viral encephalitis, melioidosis or ricin. Antibiotics are still recommended as the mainstay treatment following exposure to anthrax, plague, Q fever and melioidosis. Anti-toxin therapy and anti-virals may be used in the case of botulinum toxins or smallpox respectively. However, supportive care is the only, or mainstay, post-exposure treatment for cholera, viral encephalitis and ricin - a recommendation that has not changed in decades. Indeed, with the difficulty that antibiotic resistance poses, the development and further evaluation of techniques and atypical pharmaceuticals are fundamental to the development of prophylaxis and post-exposure treatment options. The aim of this review is to present an update on prophylaxis and post-exposure treatment recommendations and research initiatives for biological agents in the open literature from 2007 to 2009.
... Potential sources of infected materials included animal products, infected tissues, blood, urine, vaginal discharges, aborted foetuses, contacts in abattoirs, and laboratory-acquired diseases . A comprehensive threat assessment of the Brucella species requires a systematic biological hazard assessment and environmental hazard and risk evaluation based on weighted consideration of various intrinsic biohazard and environmental factors related to transmission, infectivity and fate91011 ...
Aim:
To evaluate the current practices in laboratory disease detection, biosafety and biosecurity measures and information-sharing systems available to the public health systems in R. Macedonia and the Balkan region.
Methods:
Epidemiological studies were reviewed from the region to examine the high incidence of localized forms of human brucellosis.
Results:
There is clearly a need for the development of a South Eastern European regional network for sharing information aimed at disease surveillance, early reporting and efficient containment of human brucellosis. Given the zoonotic nature of the disease's onset and progression, public health strategies aimed at containing and eventually eliminating human brucellosis require a broader national and regional bioengagement framework that involves human and veterinary diseases detection systems, deployment of medical countermeasures, epidemiology and a supportive laboratory infrastructure, strategic stockpiles and information sharing.
Conclusions:
A systems-centric framework for a national and regional strategy to combat human brucellosis is essential, and should be comprised of: a) a disease threat and surveillance framework at the national and broader regional level, b) laboratory performance evaluation metrics, c) best practices for information-sharing and regional coordination, and d) a coordinated regional strategy to improve biosafety and biosecurity.
IntroductionViral hepatitisHighly pathogenic RNA viruses causing hemorrhagic feversHighly pathogenic RNA viruses causing encephalitisSmallpox and monkeypoxRabiesRespiratory tract infectionsHerpesviral infectionsViral gastroenteritisHuman T-lymphoma/leukemia virus 1MeaslesHuman polyomavirusesHuman enterovirusesReferences
Simian Virology is the first text to comprehensively cover all currently known simian viruses. Chapters provide an overview of nonhuman primate models of medically important viral diseases as well as natural infections of nonhuman primates with human and animal viruses. The text covers a variety of topics including primate models of medically important viral diseases such as AIDS, hypotheses on the origins of epidemic forms of HIV, and viral diseases caused by non-simian viruses in both wild and captive primates.
Precedents and Experience So Far Key Issues Titering Assays Regulatory Framework Progression from Bench to Pivotal Trials, Scale Considerations, and GMP Implementations Summaries of Individual Vector Systems in Table 1 Challenges Moving Forward References
An adenovirus-based (ad-based) vaccine delivering antigens from the Alphavirus Venezuelan equine encephalitis virus (VEEV) is a strategy that offers clinical potential. A vaccine against VEEV is desirable because of the re-emerging nature of this virus, and also the potential that it may be used as a biological weapon. This study was designed to investigate whether the co-administration of CpG oligodeoxynucleotides (ODNs) with an ad-based VEEV vaccine could enhance the protective efficacy of the vaccine. We report that the co-administration of CpG ODN was unable to increase VEEV-specific antibody responses in mice, and was unable to increase the protective efficacy of the vaccine against aerosol challenge with virulent VEEV. However, it was noted that antibody responses directed against the adenovirus vaccine vector were increased, which may be detrimental, particularly in the context of homologous boosting.
Hydrophobic alkylating compounds like 1,5-iodonaphthylazide (INA) partitions into biological membranes and accumulates selectively into the hydrophobic domain of the lipid bilayer. Upon irradiation with far UV light, INA binds selectively to transmembrane proteins in the viral envelope and renders them inactive. Such inactivation does not alter the ectodomains of the membrane proteins thus preserving the structural and conformational integrity of immunogens on the surface of the virus. In this study, we have used INA to inactivate Venezuelan equine encephalitis virus (VEEV). Treatment of VEEV with INA followed by irradiation with UV light resulted in complete inactivation of the virus. Immuno-fluorescence for VEEV and virus titration showed no virus replication in-vitro. Complete loss of infectivity was also achieved in mice infected with INA treated plus irradiated preparations of VEEV. No change in the structural integrity of VEEV particles were observed after treatment with INA plus irradiation as assessed by electron microscopy. This data suggest that such inactivation strategies can be used for developing vaccine candidates for VEEV and other enveloped viruses.
In order to reduce the toxicity of Clostridium perfringens fermentation broths used in vaccine preparation, we developed two-phase aqueous systems for removal of toxin-activating proteases. Removal of the proteases inhibits the conversion of protoxin to active toxin. In order to establish the conditions under which the phase separation occurs, binodal curves, formed by poly(ethylene glycol) (PEG) and sodium citrate, were investigated at different values of pH and PEG molar mass. A 2(4)-experimental design was used to evaluate the influence of PEG molar mass and concentration, citrate concentration and pH on protease partition coefficient, removal.factor and protease removal yield. It has been found that simultaneous increase in PEG molar mass and decrease in citrate concentration remarkably improved the removal factor, whereas the protease removal yield showed an opposite trend. The best conditions for the system under consideration (removal factor of 2.69 and yield of 116%) were obtained at pH 8.0 using PEG molar mass of 8000 g mol(-1) and concentrations of PEG and citrate of 24 and 15%, respectively.
The highly pathogenic RNA viruses that cause encephalitis include a significant number of emerging or re-emerging viruses that are also considered potential bioweapons. Many of these viruses, including members of the family Flaviviridae, the genus Alphavirus in the family Togaviridae, and the genus Henipavirus in the family Paramyxoviridae, circulate widely in their endemic areas, where they are transmitted by mosquitoes or ticks. They use a variety of vertebrate hosts, ranging from birds to bats, in their natural life cycle. As was discovered in the United States, the introduction of a mosquito-borne encephalitis virus such as West Nile virus can cause significant health and societal concerns. There are no effective therapeutics for treating diseases caused by any of these viruses and there is limited, if any, vaccine availability for most. In this review we provide a brief summary of the current status of animal models used to study highly pathogenic encephalitic RNA viruses for the development of antiviral therapeutics and vaccines.
Attenuated mutants of Venezuelan equine encephalitis virus (VEE) were isolated by selection for rapid penetration of cultured cells (R. E. Johnston and J. F. Smith, 1988, Virology 162, 437-443). Sequence analysis of these mutants identified candidate attenuating mutations at four loci in the VEE E2 glycoprotein gene: a double mutation at E2 codons 3 and 4, and single substitutions at E2 76, 120, and 209. Each candidate mutation was reproduced in an isogenic recombinant VEE strain using site-directed mutagenesis of a full-length cDNA clone of VEE. Characterization of these molecularly cloned mutant viruses showed that mutation at each of the four loci in the E2 gene was sufficient to confer both the accelerated penetration and attenuation phenotypes. Inoculation of the molecularly cloned viruses into rodent models that differ in their response to VEE suggested that individual mutations affected different aspects of VEE pathogenesis. Full-length clones containing multiple mutations were produced by combining independently attenuating mutations. Molecularly cloned viruses carrying two or three mutations were more attenuated in sensitive animal models than viruses which contained any single mutation alone. However, these highly attenuated strains still retained the ability to induce an immune response sufficient to protect against a high dose challenge with virulent VEE. These results indicate that production of a molecularly cloned live virus vaccine for VEE is feasible.
The PE2 cleavage signal in a full-length cDNA clone of the alphavirus Venezuelan equine encephalitis virus (VEE) was ablated by site-directed mutagenesis. RNA transcripts derived from the resulting plasmids programmed the production of nonviable particles upon transfection of baby hamster kidney (BHK) cells. However, the mutant RNAs also gave rise to a small proportion of viable revertants. Analysis of these biological revertants and their molecularly cloned homologs demonstrated that second-site suppressor mutations at either E2 position 243 or E1 position 253 were able to restore viability to PE2 cleavage signal mutants. The viable revertants incorporated unprocessed PE2 into particles which showed normal infectivity for BHK cells, but reduced ability to grow in C6/36 mosquito cells. A mutant carrying a lethal PE2 cleavage signal mutation in combination with a suppressor at E1 253 was either avirulent or highly attenuated in adult mice when inoculated by the subcutaneous, intracerebral, or intranasal route and conferred complete protection against both intraperitoneal and intranasal challenge with virulent VEE. These results indicate the close functional association of the E2 and E1 proteins in the alphavirus spike. They also have implications for the design of recombinant live virus vaccines for VEE, for other alphaviruses, and for other viruses that use a similar mechanism for glycoprotein maturation.
The pathogenesis of Venezuelan equine encephalitis virus (VEE) was examined in the mouse model using V3000, a virus derived from a molecular clone of the Trinidad donkey strain of VEE. These results were compared in parallel experiments with avirulent mutants of VEE derived by site-directed mutagenesis of the clone. Adult mice, inoculated subcutaneously in their left rear footpad with V3000, were followed in a time course study for 6 days in which 15 organs were tested for histopathological changes, for the presence of viral antigen by immunohistochemical staining, for the presence of viral nucleic acid by in situ hybridization analysis, and for content of viable virus. Virus was detected in the footpad inoculation site, but until 12 hr postinoculation (pi), the level of virus did not suggest early viral replication. By 4 hr pi, however, replication of V3000 was evident in the draining popliteal lymph node. At this early time point, no virus could be isolated from any other organ examined. At 12 hr, a significant serum viremia was observed, and virus was detected at a low level in a number of well vascularized organs, including spleen, heart, lung, liver, kidney, and adrenal gland. By 18 hr, high virus titers were present in serum and all the lymphoid organs examined, and these tissues appeared to be the major peripheral sites of V3000 replication. Virus in serum and peripheral organs was cleared by 3-4 days pi. In a second phase of the infection, V3000 invaded the central nervous system (CNS), replicated predominantly in neurons, and persisted in the brain until death by encephalitis. Pathologic findings as well as the results of immunocytochemical and in situ hybridization examination were generally coordinate with virus titration. A site-directed mutant of V3000, V3010, contained a mutation in the gene for the E2 glycoprotein at codon 76 (Glu to Lys) which rendered it avirulent after footpad inoculation. Detection of V3010 replication in the draining lymph node was sporadic and was sometimes delayed to as long as 3 days pi. Infrequent and/or delayed virus spread to other sites also was observed. Analogous experiments were performed with other mutants which were avirulent by the footpad inoculation route: V3014, a mutant differing from V3000 at three loci (E2 Lys 209, E1 Thr 272, and E2 Asn 239), as well as single-site mutants V3032 (E2 Lys 209) and V3034 (E1 Thr 272).(ABSTRACT TRUNCATED AT 400 WORDS)
The genetically engineered, live-attenuated Venezuelan equine encephalitis (VEE) virus vaccine candidate, V3526, was evaluated as a replacement for the TC-83 virus vaccine. Protection from lethal subcutaneous or aerosol challenge was evaluated in vaccinated mice clinically and immunohistochemically. Subcutaneous administration of V3526 induced systemic and mucosal protection more efficiently than did the TC-83 vaccine. The bronchial IgA responses induced in mice by subcutaneous administration of vaccines significantly corresponded to the ability to survive aerosol challenge with virulent virus. Furthermore, V3526 delivered by aerosol induced more complete mucosal protection than either vaccine administered subcutaneously. The ability of V3526 to induce protection in mice warrants its consideration for further testing as a potential vaccine candidate for human use.
A hazard assessment of Venezuelan equine encephalitis (VEE) virus sub-types and vaccine candidates was performed according to standard risk assessment procedures. Data from published literature demonstrates a considerable degree of safety of V3526 when compared to TC-83 vaccine, the protective measure that has been used to protect laboratory workers for over four decades. V3526 is a new recombinant vaccine candidate that is a vastly different product with a diminished hazard to public health and the general environment. A weight-of-evidence (WOE)-based scheme was employed to assign weights for relevance, quality, and adequacy of evidence in published literature on medical pathology, epidemiology, pre-clinical investigational studies, and environmental studies. The results of this assessment indicated that V3526 has a low adverse impact on public health and the general environment. Although there are currently no human infectivity or pathogenicity data for V3526, existing evidence from published experimental animal studies reveals a diminished hazard for environmental transmission and distribution. Recently, the US Centers for Disease Control and Prevention (CDC) excluded V3526 from select agent requirements set forth under the Health and Human Services (HHS) regulations in Title 42 C.F.R. Part 73 and the US Department Agriculture (USDA) regulations set forth in Title 7 C.F.R. Part 331 and Title 9 C.F.R. Part 121. This paper summarizes the background, rationale, and hazard analysis used for assessing the environmental hazard of the VEE vaccine candidate strain V3526.