Live-attenuated CHIKV vaccine with rearranged genome replicates in vitro and induces immune response in mice.

Chikungunya fever virus (CHIKV) is a mosquito-borne alphavirus that causes wide-spread human infections and epidemics in Asia, Africa and recently, in the Americas. CHIKV is considered a priority pathogen by CEPI and WHO. Despite recent approval of a live-attenuated CHIKV vaccine, development of additional vaccines is warranted due to the worldwide outbreaks of CHIKV. Previously, we developed immunization DNA (iDNA) plasmid capable of launching live-attenuated CHIKV vaccine in vivo. Here we report the use of CHIKV iDNA plasmid to prepare a novel, live-attenuated CHIKV vaccine V5040 with rearranged RNA genome. In V5040, genomic RNA was rearranged to encode capsid gene downstream from the glycoprotein genes. Attenuated mutations derived from experimental CHIKV 181/25 vaccine were also engineered into E2 gene of V5040. The DNA copy of rearranged CHIKV genomic RNA with attenuated mutations was cloned into iDNA plasmid pMG5040 downstream from the CMV promoter. After transfection in vitro, pMG5040 launched replication of V5040 virus with rearranged genome and attenuating E2 mutations. Furthermore, V5040 virus was evaluated in experimental murine models for general safety and immunogenicity. Vaccination with V5040 virus subcutaneously resulted in elicitation of CHIKV-specific, virus-neutralizing antibodies. The results warrant further evaluation of V5040 virus with rearranged genome as a novel live-attenuated vaccine for CHIKV.

Single-Dose Immunogenic DNA Vaccines Coding for Live-Attenuated Alpha- and Flaviviruses.

Single-dose, immunogenic DNA (iDNA) vaccines coding for whole live-attenuated viruses are reviewed. This platform, sometimes called immunization DNA, has been used for vaccine development for flavi- and alphaviruses. An iDNA vaccine uses plasmid DNA to launch live-attenuated virus vaccines in vitro or in vivo. When iDNA is injected into mammalian cells in vitro or in vivo, the RNA genome of an attenuated virus is transcribed, which starts replication of a defined, live-attenuated vaccine virus in cell culture or the cells of a vaccine recipient. In the latter case, an immune response to the live virus vaccine is elicited, which protects against the pathogenic virus. Unlike other nucleic acid vaccines, such as mRNA and standard DNA vaccines, iDNA vaccines elicit protection with a single dose, thus providing major improvement to epidemic preparedness. Still, iDNA vaccines retain the advantages of other nucleic acid vaccines. In summary, the iDNA platform combines the advantages of reverse genetics and DNA immunization with the high immunogenicity of live-attenuated vaccines, resulting in enhanced safety and immunogenicity. This vaccine platform has expanded the field of genetic DNA and RNA vaccines with a novel type of immunogenic DNA vaccines that encode entire live-attenuated viruses.

Live-Attenuated CHIKV Vaccine with Rearranged Genome Replicates in vitro and Induces Immune Response in Mice.

Chikungunya fever virus (CHIKV) is a mosquito-borne alphavirus that causes wide-spread human infections and epidemics in Asia, Africa and recently, in the Americas. There is no approved vaccine and CHIKV is considered a priority pathogen by CEPI and WHO. Previously, we developed immunization DNA (iDNA) plasmid capable of launching live-attenuated CHIKV vaccine in vivo . Here we report the use of CHIKV iDNA plasmid to prepare a novel, live-attenuated CHIKV vaccine V5040 with rearranged RNA genome for improved safety. In V5040, genomic RNA was rearranged to encode capsid gene downstream from the glycoprotein genes. To secure safety profile, attenuated mutations derived from experimental CHIKV 181/25 vaccine were also engineered into E2 gene of V5040. The DNA copy of rearranged CHIKV genomic RNA with attenuated mutations was cloned into iDNA plasmid pMG5040 downstream from the CMV promoter. After transfection in vitro, pMG5040 launched replication of V5040 virus with rearranged genome and attenuating E2 mutations. Furthermore, V5040 virus was evaluated in experimental murine models for safety and immunogenicity. Vaccination with V5040 virus subcutaneously resulted in elicitation of CHIKV-specific, virus-neutralizing antibodies. The results warrant further evaluation of V5040 virus with rearranged genome as a novel live-attenuated vaccine for CHIKV.

Live-Attenuated VEEV Vaccine Delivered by iDNA Using Microneedles Is Immunogenic in Rabbits.

Effective and simple delivery of DNA vaccines remains a key to successful clinical applications. Previously, we developed a novel class of DNA vaccines, sometimes called iDNA, which encodes the whole live-attenuated vaccine viruses. Compared to a standard DNA vaccine, an iDNA vaccine required a low dose to launch a live-attenuated vaccine in vitro or in vivo. The goal of this pilot study was to investigate if iDNA vaccine encoding live-attenuated Venezuelan equine encephalitis virus (VEEV) can be efficiently delivered in vivo by a microneedle device using a single-dose vaccination with naked iDNA plasmid. For this purpose, we used pMG4020 plasmid encoding live-attenuated V4020 vaccine of VEE virus. The V4020 virus contains structural gene rearrangement, as well as attenuating mutations genetically engineered to prevent reversion mutations. The pMG4020 was administered to experimental rabbits by using a hollow microstructured transdermal system (hMTS) microneedle device. No adverse events to vaccination were noted. Animals that received pMG4020 plasmid have successfully seroconverted, with high plaque reduction neutralization test (PRNT) antibody titers, similar to those observed in animals that received V4020 virus in place of the pMG4020 iDNA plasmid. We conclude that naked iDNA vaccine can be successfully delivered in vivo by using a single-dose vaccination with a microneedle device.

An Updated View of Primary Ionization Processes in Polar Liquids.

According to picosecond radiolysis data, primary radical cations in irradiated carbonates are very rapidly deprotonated. At the same time, analysis of the radiation-induced fluorescence from carbonate solutions indicates the formation of solvent-related radical cationic species with a relatively long lifetime. We use quantum chemical methods to develop a model of carbonate ionization that reconciles these conflicting data. Using ethylene carbonate as an example and assuming that its molecules exist in solution as a collection of dimeric associates, we show that both processes are the result of the loss of an electron from such dimers. This demonstrates that the generally accepted conceptualization of a primary ionization event, based on the idea of the formation of a radical cation of an individual molecule of an irradiated substance, requires revision in the case of polar aprotic liquids that tend to form molecular associates.

Evaluation of Protective Efficacy of Influenza Virus Like Particles Prepared from H5N1 Virus of Clade 2.2.1.2 in Chickens.

Highly pathogenic Avian Influenza (HPAI) viruses continue to cause severe economic losses in poultry species worldwide. HPAI virus of subtype H5N1 was reported in Egypt in 2006, and despite vaccination efforts, the virus has become endemic. The current study aims to evaluate the efficacy of a virus-like particle (VLP) based vaccine in vivo using specific pathogen-free (SPF) chickens. The vaccine was prepared from the HPAI H5N1 virus of clade 2.2.1.2 using the baculovirus expression system. The VLPs were quantitated and characterized, including electron microscopy. In addition, the protection level of the VLPs was evaluated by using two different regimens, including one dose and two-dose vaccinated groups, which gave up to 70% and 100% protection level, respectively. The results of this study emphasize the potential usefulness of the VLPs-based vaccine as an alternative vaccine candidate for the control of AIV infection in poultry.

Influenza Virus Like Particles (VLPs): Opportunities for H7N9 Vaccine Development.

In the midst of the ongoing COVID-19 coronavirus pandemic, influenza virus remains a major threat to public health due to its potential to cause epidemics and pandemics with significant human mortality. Cases of H7N9 human infections emerged in eastern China in 2013 and immediately raised pandemic concerns as historically, pandemics were caused by the introduction of new subtypes into immunologically naïve human populations. Highly pathogenic H7N9 cases with severe disease were reported recently, indicating the continuing public health threat and the need for a prophylactic vaccine. Here we review the development of recombinant influenza virus-like particles (VLPs) as vaccines against H7N9 virus. Several approaches to vaccine development are reviewed including the expression of VLPs in mammalian, plant and insect cell expression systems. Although considerable progress has been achieved, including demonstration of safety and immunogenicity of H7N9 VLPs in the human clinical trials, the remaining challenges need to be addressed. These challenges include improvements to the manufacturing processes, as well as enhancements to immunogenicity in order to elicit protective immunity to multiple variants and subtypes of influenza virus.

Advanced Safety and Genetic Stability in Mice of a Novel DNA-Launched Venezuelan Equine Encephalitis Virus Vaccine with Rearranged Structural Genes.

The safety and genetic stability of V4020, a novel Venezuelan Equine Encephalitis Virus (VEEV) vaccine based on the investigational VEEV TC-83 strain, was evaluated in mice. V4020 was generated from infectious DNA, contains a stabilizing mutation in the E2-120 glycoprotein, and includes rearrangement of structural genes. After intracranial inoculation (IC), replication of V4020 was more attenuated than TC-83, as documented by low clinical scores, inflammation, viral load in brain, and earlier viral clearance. During the first 9 days post-inoculation (DPI), genes involved in inflammation, cytokine signaling, adaptive immune responses, and apoptosis were upregulated in both groups. However, the magnitude of upregulation was greater in TC-83 than V4020 mice, and this pattern persisted till 13 DPI, while V4020 gene expression profiles declined to mock-infected levels. In addition, genetic markers of macrophages, DCs, and microglia were strongly upregulated in TC-83 mice. During five serial passages in the brain, less severe clinical manifestations and a lower viral load were observed in V4020 mice and all animals survived. In contrast, 13.3% of mice met euthanasia criteria during the passages in TC-83 group. At 2 DPI, RNA-Seq analysis of brain tissues revealed that V4020 mice had lower rates of mutations throughout five passages. A higher synonymous mutation ratio was observed in the nsP4 (RdRP) gene of TC-83 compared to V4020 mice. At 2 DPI, both viruses induced different expression profiles of host genes involved in neuro-regeneration. Taken together, these results provide evidence for the improved safety and genetic stability of the experimental V4020 VEEV vaccine in a murine model.

Bivalent Junin & Machupo experimental vaccine based on alphavirus RNA replicon vector.

Junin (JUNV) and Machupo (MACV), two mammalian arenaviruses placed on the 2018 WHO watch list, are prevalent in South America causing Argentine and Bolivian hemorrhagic fevers (AHF and BHF), respectively. The live attenuated JUNV vaccine, Candid #1, significantly reduced the incidence of AHF. Vaccination induces neutralizing antibody (nAb) responses which effectively target GP1 (the viral attachment glycoprotein) pocket which accepts the tyrosine residue of the cellular receptor, human transferrin receptor 1 (TfR1). In spite of close genetic relationships between JUNV and MACV, variability in the GP1 receptor binding site (e.g., MACV GP1 loop 10) results in poor MACV neutralization by Candid #1-induced nAbs. Candid #1 is not recommended for vaccination of children younger than 15 years old (a growing "at risk" group), pregnant women, or other immunocompromised individuals. Candid #1's primary reliance on limited missense mutations for attenuation, genetic heterogeneity, and potential stability concerns complicate approval of this vaccine in the US. To address these issues, we applied alphavirus RNA replicon vector technology based on the human Venezuelan equine encephalitis vaccine (VEEV) TC-83 to generate replication restricted virus-like-particles vectors (VLPVs) simultaneously expressing cellular glycoprotein precursors (GPC) of both viruses, JUNV and MACV. Resulting JV&MV VLPVs were found safe and immunogenic in guinea pigs. Immunization with VLPVs induced humoral responses which correlated with complete protection against lethal disease after challenge with pathogenic strains of JUNV (Romero) and MACV (Carvallo).

Venezuelan equine encephalitis vaccine with rearranged genome resists reversion and protects non-human primates from viremia after aerosol challenge.

Live-attenuated V4020 vaccine for Venezuelan equine encephalitis virus (VEEV) containing attenuating rearrangement of the virus structural genes was evaluated in a non-human primate model for immunogenicity and protective efficacy against aerosol challenge with wild-type VEEV. The genomic RNA of V4020 vaccine virus was encoded in the pMG4020 plasmid under control of the CMV promoter and contained the capsid gene downstream from the glycoprotein genes. It also included attenuating mutations from the VEE TC83 vaccine, with E2-120Arg substitution genetically engineered to prevent reversion mutations. The population of V4020 vaccine virus derived from pMG4020-transfected Vero cells was characterized by next generation sequencing (NGS) and indicated no detectable genetic reversions. Cynomolgus macaques were vaccinated with V4020 vaccine virus. After one or two vaccinations including by intramuscular route, high levels of virus-neutralizing antibodies were confirmed with no viremia or apparent adverse reactions to vaccinations. The protective effect of vaccination was evaluated using an aerosol challenge with VEEV. After challenge, macaques had no detectable viremia, demonstrating a protective effect of vaccination with live V4020 VEEV vaccine.

Interaction of spin-correlated radical pair with a third radical: Combined effect of spin-exchange interaction and spin-selective reaction.

The reaction of electron transfer between two paramagnetic particles may be strongly dependent on the total spin state of the pair. Such dependence can be used to control electron transfer in a molecular medium via the control of the spin degrees of freedom. In this work, the spin-selective electron transfer has been studied in a three-spin system composed of a spin-correlated radical ion pair (RIP) and the nitroxide radical, TEMPONE. The RIPs were created in an n-hexane solution of tetramethylpiperidine (TMP) and para-terphenyl (p-TP) using X-rays. To monitor the spin evolution of the RIPs with a nanosecond time resolution, the method of time-resolved magnetic field effect in the RIP recombination fluorescence was applied. It was found that increasing the TEMPONE concentration increased the rate of both the radiation-induced fluorescence intensity decay and the paramagnetic relaxation of the spin-correlated RIP. For the three-spin system studied, we developed a theoretical model to calculate the singlet state population of the spin-correlated RIP that described both the spin-selective reaction and the spin-exchange interaction during an encounter between RIP partners and a third radical. It was found that the effect of the spin exchange could be neglected if the rate of the spin-selective reaction is high enough. Based on quantum chemical calculations and experiments, we found that there was a spin-selective distant electron transfer from p-TP radical anions to the TEMPONE radical. Another partner of the RIP, the radical cation formed from TMP, was only involved in the spin exchange interaction with TEMPONE radicals.

Novel DNA-launched Venezuelan equine encephalitis virus vaccine with rearranged genome.

Novel live-attenuated V4020 vaccine was prepared for Venezuelan equine encephalitis virus (VEEV), an alphavirus from the Togaviridae family. The genome of V4020 virus was rearranged, with the capsid gene expressed using a duplicate subgenomic promoter downstream from the glycoprotein genes. V4020 also included both attenuating mutations from the TC83 VEEV vaccine secured by mutagenesis to prevent reversion mutations. The full-length infectious RNA of V4020 vaccine virus was expressed from pMG4020 plasmid downstream from the CMV promoter and launched replication of live-attenuated V4020 in vitro or in vivo. BALB/c mice vaccinated with a single dose of V4020 virus or with pMG4020 plasmid had no adverse reactions to vaccinations and developed high titers of neutralizing antibodies. After challenge with the wild type VEEV, vaccinated mice survived with no morbidity, while all unvaccinated controls succumbed to lethal infection. Intracranial injections in mice showed attenuated replication of V4020 vaccine virus as compared to the TC83. We conclude that V4020 vaccine has safety advantage over TC83, while provides equivalent protection in a mouse VEEV challenge model.

Rapid Construction and Immunogenicity Testing of a Novel H5 Virus-Like Particle Prototype Vaccine Against Clade 2.3.4.4 H5N8 Highly Pathogenic Avian Influenza Virus.

From October 2016 to July 2017, 47 countries have been affected by highly pathogenic avian influenza (HPAI) viruses of the H5N8 clade 2.3.4.4 subtype, including European and African, and it has been the most severe HPAI outbreak ever in Europe. The development of effective influenza vaccines is required to combine preventive and control measures in order to avoid similar avian influenza epidemics taking place. Here we describe a novel prototype recombinant virus-like particle (VLP) vaccine based on a clade 2.3.4.4 H5 HA derived from a French duck HPAI H5N8 isolate of the 2016-2017 epidemics. Prototype vaccines with different antigen content were formulated and the immunogenicity was examined in specific-pathogen-free chickens and in ducks. Serum samples were collected at 3 and 4 weeks postvaccination, and development of the immune response was evaluated by hemagglutination inhibition test and ELISA. The VLP vaccines induced a dose-dependent and high level of antibody response in both chickens and ducks. The results of HPAI H5N8 challenge experiments in ducks are reported separately.

Construcción rápida y pruebas de inmunogenicidad de un nuevo prototipo de vacuna H5 con base en partículas similares a virus contra el clado 2.3.4.4 del virus de la influenza aviar altamente patógeno H5N8. Desde octubre del 2016 hasta julio del 2017, 47 países se han visto afectados por los virus de la influenza aviar altamente patógena del subtipo H5N8 clado 2.3.4.4, incluidos los europeos y africanos y ha sido el brote de influenza aviar de alta patogenicidad más grave en Europa. Se requiere del desarrollo de vacunas efectivas contra la influenza para combinar medidas preventivas y de control para evitar que ocurran epidemias similares de influenza aviar. En este estudio se describe un nuevo prototipo de vacuna recombinante con partículas similares a virus (VLP) basada en una hemaglutinina H5 clado 2.3.4.4 derivada de un aislamiento del virus de influenza aviar de alta patogenicidad H5N8 de patos en Francia presente en las epidemias entre los años 2016 al 2017. Se formularon prototipos de vacunas con diferente contenido de antígeno y se examinó la inmunogenicidad en pollos libres de patógenos específicos y en patos. Las muestras de suero se recolectaron a las tres y cuatro semanas después de la vacunación y el desarrollo de la respuesta inmune se evaluó mediante la prueba de inhibición de la hemaglutinación y ELISA. Las vacunas con partículas similares a virus indujeron un alto nivel de respuesta de anticuerpos dependiente de la dosis tanto en pollos como en patos. Los resultados de los experimentos de desafío con un virus de influenza aviar de alta patogenicidad H5N8 en patos se informan por separado.

Multi-clade H5N1 virus-like particles: Immunogenicity and protection against H5N1 virus and effects of beta-propiolactone.

During the past decade, H5N1 highly pathogenic avian influenza (HPAI) viruses have diversified genetically and antigenically, suggesting the need for multiple H5N1 vaccines. However, preparation of multiple vaccines from live H5N1 HPAI viruses is difficult and economically not feasible representing a challenge for pandemic preparedness. Here we evaluated a novel multi-clade recombinant H5N1 virus-like particle (VLP) design, in which H5 hemagglutinins (HA) and N1 neuraminidase (NA) derived from four distinct clades of H5N1 virus were co-localized within the VLP structure. The multi-clade H5N1 VLPs were prepared by using a recombinant baculovirus expression system and evaluated for functional hemagglutination and neuraminidase enzyme activities, particle size and morphology, as well as for the presence of baculovirus in the purified VLP preparations. To remove residual baculovirus, VLP preparations were treated with beta-propiolactone (BPL). Immunogenicity and efficacy of multi-clade H5N1 VLPs were determined in an experimental ferret H5N1 HPAI challenge model, to ascertain the effect of BPL on immunogenicity and protective efficacy against lethal challenge. Although treatment with BPL reduced immunogenicity of VLPs, all vaccinated ferrets were protected from lethal challenge with influenza A/VietNam/1203/2004 (H5N1) HPAI virus, indicating that multi-clade VLP preparations treated with BPL represent a potential approach for pandemic preparedness vaccines.

Alphavirus vector-based replicon particles expressing multivalent cross-protective Lassa virus glycoproteins.

Lassa virus (LASV) is the most prevalent rodent-borne arenavirus circulated in West Africa. With population at risk from Senegal to Nigeria, LASV causes Lassa fever and is responsible for thousands of deaths annually. High genetic diversity of LASV is one of the challenges for vaccine R&D. We developed multivalent virus-like particle vectors (VLPVs) derived from the human Venezuelan equine encephalitis TC-83 IND vaccine (VEEV) as the next generation of alphavirus-based bicistronic RNA replicon particles. The genes encoding VEEV structural proteins were replaced with LASV glycoproteins (GPC) from distantly related clades I and IV with individual 26S promoters. Bicistronic RNA replicons encoding wild-type LASV GPC (GPCwt) and C-terminally deleted, non-cleavable modified glycoprotein (ΔGPfib), were encapsidated into VLPV particles using VEEV capsid and glycoproteins provided in trans. In transduced cells, VLPVs induced simultaneous expression of LASV GPCwt and ΔGPfib from 26S alphavirus promoters. LASV ΔGPfib was predominantly expressed as trimers, accumulated in the endoplasmic reticulum, induced ER stress and apoptosis promoting antigen cross-priming. VLPV vaccines were immunogenic and protective in mice and upregulated CD11c+/CD8+ dendritic cells playing the major role in cross-presentation. Notably, VLPV vaccination resulted in induction of cross-reactive multifunctional T cell responses after stimulation of immune splenocytes with peptide cocktails derived from LASV from clades I-IV. Multivalent RNA replicon-based LASV vaccines can be applicable for first responders, international travelers visiting endemic areas, military and lab personnel.

Plasmid DNA launches live-attenuated Japanese encephalitis virus and elicits virus-neutralizing antibodies in BALB/c mice.

We describe novel plasmid DNA that encodes the full-length Japanese encephalitis virus (JEV) genomic cDNA and launches live-attenuated JEV vaccine in vitro and in vivo. The synthetic cDNA based on the sequence of JEV SA14-14-2 live-attenuated virus was placed under transcriptional control of the cytomegalovirus major immediate-early promoter. The stability and yields of the plasmid in E. coli were optimized by inserting three synthetic introns that disrupted JEV cDNA in the structural and nonstructural genes. Transfection of Vero cells with the resulting plasmid resulted in the replication of JEV vaccine virus with intron sequences removed from viral RNA. Furthermore, a single-dose vaccination of BALB/c mice with 0.5 - 5µg of plasmid resulted in successful seroconversion and elicitation of JEV virus-neutralizing serum antibodies. The results demonstrate the possibility of using DNA vaccination to launch live-attenuated JEV vaccine and support further development of DNA-launched live-attenuated vaccine for prevention of JEV infections.

Virus-like particles displaying H5, H7, H9 hemagglutinins and N1 neuraminidase elicit protective immunity to heterologous avian influenza viruses in chickens.

Avian influenza (AI) viruses circulating in wild birds pose a serious threat to public health. Human and veterinary vaccines against AI subtypes are needed. Here we prepared triple-subtype VLPs that co-localized H5, H7 and H9 antigens derived from H5N1, H7N3 and H9N2 viruses. VLPs also contained influenza N1 neuraminidase and retroviral gag protein. The H5/H7/H9/N1/gag VLPs were prepared using baculovirus expression. Biochemical, functional and antigenic characteristics were determined including hemagglutination and neuraminidase enzyme activities. VLPs were further evaluated in a chicken AI challenge model for safety, immunogenicity and protective efficacy against heterologous AI viruses including H5N2, H7N3 and H9N2 subtypes. All vaccinated birds survived challenges with H5N2 and H7N3 highly pathogenic AI (HPAI) viruses, while all controls died. Immune response was also detectable after challenge with low pathogenicity AI (LPAI) H9N2 virus suggesting that H5/H7/H9/N1/gag VLPs represent a promising approach for the development of broadly protective AI vaccine.

Mono- and quadri-subtype virus-like particles (VLPs) containing H10 subtype elicit protective immunity to H10 influenza in a ferret challenge model.

Avian-origin influenza represents a global public health concern. In 2013, the H10N8 virus caused documented human infections for the first time. Currently, there is no approved vaccine against H10 influenza. Recombinant virus-like particles (VLPs) represent a promising vaccine approach. In this study, we evaluated H10 VLPs containing hemagglutinin from H10N8 virus as an experimental vaccine in a ferret challenge model. In addition, we evaluated quadri-subtype VLPs co-localizing H5, H7, H9 and H10 subtypes. Both vaccines elicited serum antibody that reacted with the homologous H10 derived from H10N8 virus and cross-reacted with the heterologous H10N1 virus. Quadri-subtype vaccine also elicited serum antibody to the homologous H5, H7, and H9 antigens and cross-reacted with multiple clades of H5N1 virus. After heterologous challenge with the H10N1 virus, all vaccinated ferrets showed significantly reduced titers of replicating virus in the respiratory tract indicating protective effect of vaccination with either H10 VLPs or with quadri-subtype VLPs.

DNA-launched live-attenuated vaccines for biodefense applications.

A novel vaccine platform uses DNA immunization to launch live-attenuated virus vaccines in vivo. This technology has been applied for vaccine development against positive-strand RNA viruses with global public health impact including alphaviruses and flaviviruses. The DNA-launched vaccine represents the recombinant plasmid that encodes the full-length genomic RNA of live-attenuated virus downstream from a eukaryotic promoter. When administered in vivo, the genomic RNA of live-attenuated virus is transcribed. The RNA initiates limited replication of a genetically defined, live-attenuated vaccine virus in the tissues of the vaccine recipient, thereby inducing a protective immune response. This platform combines the strengths of reverse genetics, DNA immunization and the advantages of live-attenuated vaccines, resulting in a reduced chance of genetic reversions, increased safety, and improved immunization. With this vaccine technology, the field of DNA vaccines is expanded from those that express subunit antigens to include a novel type of DNA vaccines that launch live-attenuated viruses.

Next generation sequencing of DNA-launched Chikungunya vaccine virus.

Chikungunya virus (CHIKV) represents a pandemic threat with no approved vaccine available. Recently, we described a novel vaccination strategy based on iDNA® infectious clone designed to launch a live-attenuated CHIKV vaccine from plasmid DNA in vitro or in vivo. As a proof of concept, we prepared iDNA plasmid pCHIKV-7 encoding the full-length cDNA of the 181/25 vaccine. The DNA-launched CHIKV-7 virus was prepared and compared to the 181/25 virus. Illumina HiSeq2000 sequencing revealed that with the exception of the 3' untranslated region, CHIKV-7 viral RNA consistently showed a lower frequency of single-nucleotide polymorphisms than the 181/25 RNA including at the E2-12 and E2-82 residues previously identified as attenuating mutations. In the CHIKV-7, frequencies of reversions at E2-12 and E2-82 were 0.064% and 0.086%, while in the 181/25, frequencies were 0.179% and 0.133%, respectively. We conclude that the DNA-launched virus has a reduced probability of reversion mutations, thereby enhancing vaccine safety.