Single-round infectious particles enhance immunogenicity of a DNA vaccine against West Nile virus David C Chang 1 , Wen J Liu 1 , Itaru Anraku 2 , David C Clark 1 , Christopher C Pollitt 3 , Andreas Suhrbier 2 , Roy A Hall 1 & Alexander A Khromykh 1 DNA vaccines encoding replication-defective viruses are safer than inactivated or live attenuated viruses but may fail to stimulate an immune response sufficient for effective vaccination. We augment the protective capacity of a capsid-deleted flavivirus DNA vaccine by co-expressing the capsid protein from a separate promoter. In transfected cells, the capsid-deleted RNA transcript is replicated and translated to produce secreted virus-like particles lacking the nucleocapsid. This RNA is also packaged with the help of co-expressed capsid protein to form secreted single-round infectious particles (SRIPs) that deliver the RNA into neighboring cells. In SRIP-infected cells, the RNA is replicated again and produces additional virus-like particles, but in the absence of capsid RNA no SRIPs are formed and no further spread occurs. Compared with an otherwise identical construct that does not encode capsid, our vaccine offers better protection to mice after lethal West Nile virus infection. It also elicits virus-neutralizing antibodies in horses. This approach may enable vaccination against pathogenic flaviviruses other than West Nile virus. Flaviviruses are a large group of pathogenic, positive-strand RNA viruses that cause 450 million human infections annually. Owing primarily to efficacy and safety concerns associated with inactivated or live-attenuated vaccines 1 , there are no commercial human vaccines available for medically important flaviviruses, such as West Nile virus and dengue virus. Prototype DNA-based vaccine candidates 2 have been developed for St. Louis encephalitis virus 3 , Japanese encephalitis virus 4 , Murray Valley encephalitis virus 5 , dengue virus-2 (ref. 6), tick- borne encephalitis virus 7 and West Nile virus 8 . All use a similar strategy to express the pre-membrane (prM) and envelope (E) proteins to produce highly immunogenic secreted prM-E particles and show promise for both human and veterinary medicine. For instance, 1 mg of a DNA vaccine expressing genes encoding West Nile virus prM and E proteins generates sufficient levels of neutralizing antibody to protect horses against West Nile virus challenge by infected mosquitoes 8 . However, unlike immunization with live virus, the current DNA vaccines do not replicate and spread within the immunized host. Therefore, relatively high or multiple doses are required to induce protective immunity. Furthermore, these strategies cannot induce humoral or cell-mediated immune responses to the nonstructural viral proteins, which may be important for long-term immunity 9 . Using plasmid DNAs, encoding self-replicating RNAs allows substantial reduction of the dose of DNA vaccines 10 . Flavivirus RNAs that contain large deletions in the capsid gene are unable to produce infectious virions but retain the ability to replicate and express prM and E proteins, which are secreted as highly immuno- genic prM-E particles 11 . These can be delivered as naked RNA 11 , as plasmid DNA 12 or as pseudo-infectious virus particles 13 and were shown to be effective in mice. Here we illustrate a concept in flavivirus DNA vaccine design, which differs from other flavivirus vaccine developments and combines the advantage of replicon-based DNA technology, the ability to generate pseudo-infectious virus particles and the immunogenicity of capsid- deleted flavivirus RNAs. A naturally attenuated Australian strain of West Nile virus, Kunjin MRM61C 14 that has had an additional attenuating mutation engineered into the gene encoding NS1 ( 250 Pro to Leu), which we call WNV KUN 15 , exemplifies this vaccination strategy. We previously demonstrated that immunization with WNV KUN or with the plasmid DNA pKUN1, which encodes an infectious full-length cDNA copy of WNV KUN RNA under the control of cytomegalovirus (CMV) promoter, induced efficient immunity against the virulent New York 99 strain of West Nile virus (WNV NY99 ) 16 . As the next step in developing effective but safer West Nile virus vaccine, we generated the plasmid pKUNdC from pKUN1 DNA by deleting a large portion (codons 18–100) of the gene encoding capsid protein, while retaining the cyclization sequence. To enhance the immunogenicity of the pKUNdC vaccine, we generated a second plasmid pKUNdC/C, which is capable of simultaneously transcribing two separate RNA species from two copies of the CMV promoter configured in a back-to-back orientation (Fig. 1a). One Received 27 February; accepted 28 March; published online 20 April 2008; doi:10.1038/nbt1400 1 School of Molecular and Microbial Sciences, University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia. 2 Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia. 3 School of Veterinary Sciences, University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia. Correspondence should be addressed to A.A.K. (a.khromykh@uq.edu.au). NATURE BIOTECHNOLOGY VOLUME 26 NUMBER 5 MAY 2008 571 LETTERS