Evaluation of the inactivation of Venezuelan equine encephalitis virus by several common methods.

Working with virological samples requires validated inactivation protocols for safe handling and disposal. Although many techniques exist to inactivate samples containing viruses, not all procedures have been properly validated or are compatible with subsequent assays. To aid in the development of inactivation protocols for Alphaviruses, and specifically Venezuelan equine encephalitis virus (VEEV), a variety of methods were evaluated for their ability to completely inactivate a high titer sample of the vaccine strain VEEV TC-83. The methods evaluated include reagents used in RNA extraction, fixation, treatment with a detergent, and heat inactivation. Most methods were successful at inactivating the sample; however, treatment with only Buffer AVL, SDS, and heat inactivation at 58 °C for one hour were not capable of complete inactivation of the virus in the sample. These results provide a substantial framework for identifying techniques that are safe for complete inactivation of Alphaviruses and to advise protocol implementation.

Deinococcus Mn2+-peptide complex: A novel approach to alphavirus vaccine development.

Over the last ten years, Chikungunya virus (CHIKV), an Old World alphavirus has caused numerous outbreaks in Asian and European countries and the Americas, making it an emerging pathogen of great global health importance. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, on the other hand, has been developed as a bioweapon in the past due to its ease of preparation, aerosol dispersion and high lethality in aerosolized form. Currently, there are no FDA approved vaccines against these viruses. In this study, we used a novel approach to develop inactivated vaccines for VEEV and CHIKV by applying gamma-radiation together with a synthetic Mn-decapeptide-phosphate complex (MnDpPi), based on manganous-peptide-orthophosphate antioxidants accumulated in the extremely radiation-resistant bacterium Deinococcus radiodurans. Classical gamma-irradiated vaccine development approaches are limited by immunogenicity-loss due to oxidative damage to the surface proteins at the high doses of radiation required for complete virus-inactivation. However, addition of MnDpPi during irradiation process selectively protects proteins, but not the nucleic acids, from the radiation-induced oxidative damage, as required for safe and efficacious vaccine development. Previously, this approach was used to develop a bacterial vaccine. In the present study, we show that this approach can successfully be applied to protecting mice against viral infections. Irradiation of VEEV and CHIKV in the presence of MnDpPi resulted in substantial epitope preservation even at supra-lethal doses of gamma-rays (50,000Gy). Irradiated viruses were found to be completely inactivated and safe in vivo (neonatal mice). Upon immunization, VEEV inactivated in the presence of MnDpPi resulted in drastically improved protective efficacy. Thus, the MnDpPi-based gamma-inactivation approach described here can readily be applied to developing vaccines against any pathogen of interest in a fast and cost-effective manner.

Preserving immunogenicity of lethally irradiated viral and bacterial vaccine epitopes using a radio- protective Mn2+-Peptide complex from Deinococcus.

Although pathogen inactivation by γ-radiation is an attractive approach for whole-organism vaccine development, radiation doses required to ensure sterility also destroy immunogenic protein epitopes needed to mount protective immune responses. We demonstrate the use of a reconstituted manganous peptide complex from the radiation-resistant bacterium Deinococcus radiodurans to protect protein epitopes from radiation-induced damage and uncouple it from genome damage and organism killing. The Mn(2+) complex preserved antigenic structures in aqueous preparations of bacteriophage lambda, Venezuelan equine encephalitis virus, and Staphylococcus aureus during supralethal irradiation (25-40 kGy). An irradiated vaccine elicited both antibody and Th17 responses, and induced B and T cell-dependent protection against methicillin-resistant S. aureus (MRSA) in mice. Structural integrity of viruses and bacteria are shown to be preserved at radiation doses far above those which abolish infectivity. This approach could expedite vaccine production for emerging and established pathogens for which no protective vaccines exist.

A multisystem approach for development and evaluation of inactivated vaccines for Venezuelan equine encephalitis virus (VEEV).

A multisystem approach was used to assess the efficiency of several methods for inactivation of Venezuelan equine encephalitis virus (VEEV) vaccine candidates. A combination of diverse assays (plaque, in vitro cytopathology and mouse neurovirulence) was used to verify virus inactivation, along with the use of a specific ELISA to measure retention of VEEV envelope glycoprotein epitopes in the development of several inactivated VEEV candidate vaccines derived from an attenuated strain of VEEV (V3526). Incubation of V3526 aliquots at temperatures in excess of 64 degrees C for periods >30 min inactivated the virus, but substantially reduced VEEV specific monoclonal antibody binding of the inactivated material. In contrast, V3526 treated either with formalin at concentrations of 0.1% or 0.5% (v/v) for 4 or 24 h, or irradiated with 50 kGy gamma radiation rendered the virus non-infectious while retaining significant levels of monoclonal antibody binding. Loss of infectivity of both the formalin inactivated (fV3526) and gamma irradiated (gV3526) preparations was confirmed via five successive blind passages on BHK-21 cells. Similarly, loss of neurovirulence for fV3526 and gV3526 was demonstrated via intracerebral inoculation of suckling BALB/c mice. Excellent protection against subcutaneous challenge with VEEV IA/B Trinidad donkey strain was demonstrated using a two dose immunization regimen with either fV3526 or gV3526. The combination of in vitro and in vivo assays provides a practical approach to optimize manufacturing process parameters for development of other inactivated viral vaccines.

Comparison of the immunological responses and efficacy of gamma-irradiated V3526 vaccine formulations against subcutaneous and aerosol challenge with Venezuelan equine encephalitis virus subtype IAB.

We recently developed a gamma-irradiation method to inactivate V3526, a live-attenuated Venezuelan equine encephalitis virus (VEEV) vaccine candidate. Dosage and schedule studies were conducted to evaluate the immunogenicity and efficacy of gamma-irradiated V3526 (gV3526). Subcutaneous (SC) and low dosage intramuscular (IM) administration of gV3526 were highly effective in protecting mice against a SC challenge with VEEV IA/B Trinidad Donkey strain, but not against an equivalent aerosol challenge. More robust immune responses and increased protective efficacy were noted when the IM dosage of gV3526 was increased. IM administration of gV3526 formulated with either CpG or CpG plus Alhydrogel further augmented the immune response in mice and resulted in 100% protection against aerosol challenge.

Effects of UV-irradiation upon Venezuelan equine encephalomyelitis virus.

Venezuelan equine encephalomyelitis virus labelled with [14C]aminoacids or [3H]uridine was purified and UV-irradiated. The irradiation led to the formation of uracil photodimers and the covalent linking of the nucleocapsid protein C to virion RNA. The inactivation of infectivity correlated with the formation of uracil dimers, whereas the RNA-protein links were formed at much higher doses of UV irradiation. The analysis of covalent RNA-protein complexes suggests that a fairly large fraction (at least one third) of the whole content of C protein is able to participate in the formation of UV-induced links, suggesting extensive contacts of RNA with protein with the nucleocapsid.

Photochemical inactivation of viruses with psoralens: an overview.

In the presence of longwave ultraviolet light, psoralen derivatives photoreact with the nucleic acids within intact viruses and cells. This photoreaction can leave protein antigens and other surface components relatively unmodified, while eliminating the infectivity of a wide range of infectious agents. The kinetics of inactivation differ among RNA and DNA viruses photoreacted with different derivatives of psoralen. The inactivation kinetics are nonlinear as a result of photodegradation of psoralens and the unexplained biphasic inactivation of some viruses. In spite of these complexities, the photoreaction is capable of generating broad safety margins in the disinfection of microbial products under gentle, physiologic conditions. The psoralen photoreaction provides a potential method for inactivating both known and unknown viruses in active blood products. Psoralen-inactivated viruses have already proven useful as noninfectious antigens for use in immunoassays and as successful experimental vaccines.

Effects of ultraviolet laser radiation on Venezuelan equine encephalomyelitis virus.

The effects of usual low-intensity continuous (lambda = 254 nm, I = 10 W/m2) UV radiation and high-intensity laser nanosecond (lambda = 266 nm, tau p = 10 ns, I = 10(9) W/m2) or picosecond (lambda = 266 nm, tau p = 23 ps, I = 10(12) W/m2) UV radiation on Venezuelan equine encephalomyelitis virus (a member of the Togaviridae family) were compared. The quantum yields of infectivity inactivation, pyrimidine dimer formation and RNA-protein crosslinking were determined.

[Covalent-bound aggregates of equine venezuelan encephalomyelitis virus induced by UV-irradiation]. / Kovalentno-sviazannye agregaty virusa venesuél'skogo éntsefalomielita loshadei, indutsirovannye UF-oblucheniem.

Formation of Venecuelan equine encephalomyelitis virus (VEE) aggregates induced by UV-light has been studied. The high doses of UV-irradiation induced the protein-protein cross-links resulting in formation of fast sedimenting viral structures. The latter structures are supposed to be presented by the aggregates of several virions linked by the UV-light induced RNA-protein and protein-protein covalent bonds. The lesions in the fine structure of virion envelope was registered by the electron microscopy technique.

Tetracycline-mediated photodynamic inactivation of animal viruses.

Demethylchlortetracycline (DMCT), doxycycline and, to a lesser extent, chlortetracycline were capable of mediating the in vitro photoinactivation of Venezuelan equine encephalitis (VEE) virus. Other tetracyclines tested were found to be inactive in this respect. However, no correlation between chemical structure and photosensitizing activity could be established. The photoinactivation of VEE virus by DMCT proceeds through a photodynamic mechanism as shown by the absolute requirement of O2 for the inactivation to take place. The photoinactivating effect of DMCT was also exerted upon other animal viruses tested, i.e. vesicular stomatitis virus, herpes simplex virus and poliovirus, even when, in the case of poliovirus, the capsid seems to be impermeable to the tetracycline. The fact that the two most effective photosensitizing tetracyclines for VEE virus are also the drugs more frequently associated with drug-induced phototoxicity in humans, suggests that virus photoinactivation could be used as a screening procedure for potentially phototoxic drugs developed for human application.

Photoinactivation of Venezuelan equine encephalitis virus mediated by tetracyclines.

Demethylchlortetracycline and, to a lesser extent, chlortetracycline were found to mediate the in vitro photoinactivation of Venezuelan equine encephalitis virus.

Effect of simulated solar radiation and sodium fluorescein on the recovery of Venezuelan equine encephalomyelitis virus from aerosols.

Almost 90% of the Trinidad strain of Venezuelan equine encephalomyelitis (VEE) virus survived for 1 hr after aerosolization into a dark environment at 30% relative humidity (RH), and 78% survived for 1 hr at 60% RH. After exposure to simulated solar radiation (584 mcal per cm(2) per min) 0.02% of the aerosolized virus survived for 1 hr at 30% RH and 0.006% survived for 1 hr at 60% RH. When 1.0 mg of sodium fluorescein per ml was added to suspensions prior to aerosol dissemination (to determine physical loss of aerosol), no virus was detected after 30 min at either RH upon irradiation. Sodium fluorescein also exhibited some toxicity (31% survival at 60 min) for nonirradiated aerosols of VEE virus at 60% RH; no effect was noted at 30%.