RNA-Based Viral Vectors
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Review RNA-based viral vectors Expert Rev. Vaccines Early online, 1–30 (2014) Mark A Mogler1 and The advent of reverse genetic approaches to manipulate the genomes of both positive (+) and Kurt I Kamrud*2 negative (-) sense RNA viruses allowed researchers to harness these genomes for basic research. Manipulation of positive sense RNA virus genomes occurred first largely because infectious RNA 1Harrisvaccines, Inc., 1102 Southern Hills Drive, Suite 101, Ames, IA 50010, could be transcribed directly from cDNA versions of the RNA genomes. Manipulation of USA negative strand RNA virus genomes rapidly followed as more sophisticated approaches to 2Synthetic Genomics Vaccines, Inc., provide RNA-dependent RNA polymerase complexes coupled with negative-strand RNA 11149 North Torrey Pines, La Jolla, CA templates were developed. These advances have driven an explosion of RNA virus vaccine vector 90237, USA *Author for correspondence: development. That is, development of approaches to exploit the basic replication and expression [email protected] strategies of RNA viruses to produce vaccine antigens that have been engineered into their genomes. This study has led to significant preclinical testing of many RNA virus vectors against a wide range of pathogens as well as cancer targets. Multiple RNA virus vectors have advanced through preclinical testing to human clinical evaluation. This review will focus on RNA virus vectors designed to express heterologous genes that are packaged into viral particles and have progressed to clinical testing. KEYWORDS: alphavirus • flavivirus • orthomyxovirus • paramyxovirus • reverse genetics • rhabdovirus • RNA virus vector This review will describe RNA virus vector nsP3 and nsP4) are translated from the genomic development and the reverse genetics approaches RNA. The nsP2 is a viral protease that processes used to support the development for five RNA the nonstructural polyprotein into its individual virus families: Togaviridae, Flaviviridae, Ortho- subunits that function to transcribe negative- For personal use only. myxoviridae, Rhabdoviridae and Paramyxoviri- sense as well as new positive-sense viral RNA. dae. The RNA vector system(s) designed for The negative-sense replicative intermediate each will be described and a summary of preclin- RNA codes for a subgenomic promoter (26S ical work that has supported advanced testing promoter) that is recognized by the nonstruc- and human clinical evaluation will also be dis- tural proteins and is used to generate a subge- cussed. Although some systems have developed nomic mRNA that represents the 3´ one-third DNA-based vectors, only RNA particle-based of the genome; the viral structural proteins are systems will be discussed in this review. Reverse translated from this subgenomic mRNA. Repli- genetics systems for the generation of viruses in cation occurs completely in the cytoplasm of the Arenaviridae family have been described, but cells and progeny viruses bud at the plasma these RNA vectors are still in very early develop- membrane as virus capsid protein encapsidates Expert Review of Vaccines Downloaded from informahealthcare.com by 174.97.229.189 on 11/08/14 ment and therefore will not be examined in this genomic RNA and associates with the envelope review. Finally, reverse genetics approaches to glycoproteins that accumulate on the membrane develop Lentivirus vectors are well established of infected cells [2,3]. but will not be covered here as they have recently Because alphaviruses are positive-sense RNA been reviewed elsewhere [1]. viruses, full-length cDNA clones of their genomes can be used to generate RNA transcripts Togaviridae that, when introduced into susceptible cells, will Genus: Alphavirus initiate a complete virus replication cycle and Alphaviruses have a single-strand, positive-sense generate infectious virus. Manipulation of alpha- RNA genome that carries a 5´ 7-mG cap and a virus genomes is possible through introduction of 3´ polyadenylated region. The alphavirus non- mutations/modifications to the cDNA of their structural proteins are coded for in the 5´ two- respective clones. Two types of alphavirus vectors thirds of the genome and the structural proteins have been developed to express heterologous are coded for in the 3´ one-third of the genome. genes: propagation-competent vectors where a The nonstructural proteins (termed nsP1, nsP2, 26S promoter region is duplicated, either informahealthcare.com 10.1586/14760584.2015.979798 Ó 2014 Informa UK Ltd ISSN 1476-0584 1 Review Mogler & Kamrud upstream or downstream of a complete structural gene region, that intravenously has been described and preliminary data have drives expression of a foreign gene or propagation-defective repli- been provided that indicate this formulation (1 Â 108 or con vectors where a foreign gene is inserted in place of the struc- 5 Â 109 particles/m2 body surface) was well tolerated and that tural protein gene region [4,5]. The replicon RNA can be packaged a transient increase (five- to tenfold) in IL-12 serum levels was into virus replicon particles (VRP) by supplying the structural pro- detected within the first 3–4 days after injection [16–18]. tein genes in trans. Replicon RNA is packaged into VRP when cells A complete summary of the clinical outcomes of this clinical are cotransfected with helper RNA which encode the full comple- trial has not yet been published. A study protocol for a ment of structural proteins. A split helper system that provides the Phase I/II clinical trial using continuous intratumoral infusion structural proteins on separate helper RNAs greatly reduces the of LSFV-IL12 in recurrent glioblastoma multiforme patients chance of an intact genome being regenerated by RNA–RNA has been described, but no data are available on the status of recombination. Thus, the VRP are defective, in that they can infect this clinical trial [18]. target cells in culture or in vivo and can express the foreign gene at high levels, but they lack critical portions of the genome (i.e., the Venezuelan equine encephalitis vectors structural protein genes) necessary to produce virus particles which Development of VEEV VRP for human clinical evaluation has could spread to other cells [4,5]. Chimeric alphavirus vectors have occurred in both an infectious disease and cancer setting [20–23]. also been developed where the structural region of one alphavirus VEEV VRP vaccines have been tested preclinically against a is exchanged for a different alphavirus structural region; the result- large range of pathogens (TABLE 1), of which only a few have pro- ing chimeric alphavirus is usually attenuated in nature [6–10]. gressed into human clinical evaluation. Some notable VEEV Alphavirus replicon vectors have been developed from a VRP preclinical testing examples include successful vaccines number of viruses within the Togaviridae family; these viruses developed against Ebola virus (EBOV), Marburg virus, Lassa consist of Salmonid alphavirus, Chikungunya virus (CHIKV), virus, Smallpox virus, equine encephalitis viruses, Clostridium Sindbis-like XJ-160, Venezuelan equine encephalitis virus botulinum neurotoxins and anthrax [24–30]; detailed descriptions (VEEV), Sindbis and Semliki forest virus (SFV), with the of these studies are not provided here in order to focus on majority of vaccine development having been conducted with those VEEV VRP vaccines that have progressed into human VEEV, Sindbis and SFV [4,11–15]. clinical evaluation. Alphavirus vectors have been developed against a large num- The initial human evaluation of a VEEV VRP vaccine was ber of pathogens. A representative list of the many alphavirus with a replicon expressing an HIV antigen carried out by vector particle-based vaccines developed and tested preclinically AlphaVax, Inc. The sequence of an HIV-1 Clade C gag gene is presented in TABLE 1. This review will focus on alphavirus vec- was identified based on a prevalent virus circulating in South For personal use only. tor particle vaccines that have reached human clinical testing or Africa, where one arm of the clinical trial would take have achieved regulatory approval/licensure to highlight how place [31,32]. This first-in-man testing of the VEEV-gag VRP far this vaccine platform has progressed. Clinical evaluation of (AVX101) vaccine demonstrated that the vaccine was safe and VRP vaccines has occurred for a number of infectious diseases well tolerated, but unlike the robust immune responses noted and cancer indications using the VEEV and SFV VRP systems. in preclinical testing of AVX101, anti-Gag immune responses in humans were limited [23]. The AVX101 vaccine expressed a Semliki forest virus vectors nonmyristoylated HIV gag gene and it is known that myristoy- To the best of our knowledge, SFV vectors have only been lation of the Gag protein is important in the production of examined clinically in a cancer setting and with the gene of Gag-derived virus-like particles (VLP) [33,34]. Furthermore, a interest being a cytokine (IL-12). Preclinical studies for the VEEV-gag VRP expressing a myristoylated version of the gag oncolytic nature of SFV supported its use in this setting [16–19]. gene was preliminarily reported to be more immunogenic in Expert Review of Vaccines Downloaded from informahealthcare.com by 174.97.229.189 on 11/08/14 A SFV replicon vector expressing murine IL-12 genes was nonhuman primates than the AVX101 VRP, suggesting that shown to have oncogenic activity when injected directly into the Gag myristoylation mutation (inhibiting