Hybrid alphavirus–rhabdovirus propagating replicon particles are versatile and potent vaccine vectors Nina F. Rose, Jean Publicover, Anasuya Chattopadhyay, and John K. Rose* Department of Pathology, Yale University School of Medicine, New Haven, CT 06510 Edited by Robert A. Lamb, Northwestern University, Evanston, IL, and approved February 20, 2008 (received for review January 11, 2008) Self-propagating, infectious, virus-like particles are generated in infected cells (12) and could be precursors involved in formation animal cell lines transfected with a Semliki Forest virus RNA of the infectious particles containing VSV G (6). replicon encoding a single viral structural protein, the vesicular Experimental SFV particle-based vaccines are normally de- stomatitis virus (VSV) glycoprotein. We show here that these rived from a complementation/packaging system in which SFV infectious particles, which we call propagating replicons, are po- replicons encoding foreign antigenic proteins are packaged into tent inducers of neutralizing antibody in animals yet are nonpatho- SFV-like particles by SFV structural proteins expressed in trans genic. Mice vaccinated with a single dose of the particles generated (5). Such a complementation system is required for alphavirus high titers of VSV-neutralizing antibody and were protected from vector systems because of the strict size limit for encapsidation a subsequent lethal challenge with VSV. Induction of antibody of viral genomic RNA. Unless the structural genes are deleted, required RNA replication. We also report that additional genes there is no space for inclusion of genes expressing foreign (including an HIV-1 envelope protein gene) expressed from the antigens. Because these complemented particles do not encode propagating replicons induced strong cellular immune responses SFV structural proteins, they replicate for only a single cycle to the corresponding proteins after a single inoculation. Our when inoculated into animals. studies reveal the potential of these particles as simple and safe The hybrid SFV/VSV propagating replicon particles that we vaccine vectors inducing strong humoral and cellular immune described infect and propagate in certain cell lines (6) with VSV responses. G as the only viral structural protein. However, the immunoge- nicity of these particles (designated SFVG particles) had not Semliki Forest virus ͉ vesicular stomatitis virus ͉ HIV-1 been tested in an animal model. Here we have examined the potential of these particles as a vaccine vector in a mouse model. NA replicons from alphaviruses including Semliki Forest virus We found that the particles induced a potent neutralizing R(SFV) have been developed and used for transient expression antibody response to VSV in mice. Mice vaccinated with these of foreign proteins in mammalian cells and also as experimental particles were protected from all weight loss and from a lethal vaccine vectors (1–4). The alphavirus genome is a capped and encephalitis caused by a high dose of wild-type VSV given polyadenylated positive-strand RNA molecule Ϸ12 kb in length. intravenously. The genomic RNA itself is an mRNA that encodes the viral We have also tested the immunogenicity of SFVG particles replicase. A subgenomic mRNA copied from the antigenomic expressing HIV-1 envelope (Env) or VSV nucleocapsid (N) RNA after replication encodes the alphavirus structural proteins. proteins behind a second SFV promoter. These vectors generate RNA transcribed from SFV cDNA can initiate viral RNA strong primary CD8 T cell responses to the foreign proteins as replication following transfection into cells (5). well as memory T cell responses that can be recalled to high Our laboratory previously tested an SFV replicon developed levels after boosting. by Liljestro¨m and Garoff (5) for expression of the vesicular Results stomatitis virus (VSV) glycoprotein (G) (6). The starting SFV Induction of Neutralizing Antibodies to VSV G Protein in Mice Inocu- RNA replicon was derived from a DNA copy of SFV from which lated with SFVG Particles Requires Vector Replication. To determine the genes for the SFV structural proteins were removed. The whether the propagating replicon particles were able to induce VSV G gene was inserted in place of genes encoding the SFV antibody responses to VSV G protein in animals and whether structural proteins. VSV G protein is the single transmembrane replication was required for such induction, we inoculated mice by glycoprotein of the prototype rhabdovirus VSV. VSV G medi- the intramuscular (i.m.) route with 6 ϫ 103 infectious units (i.u.) of ates both virus binding and membrane fusion to allow viral entry SFVG particles that were either untreated or inactivated with UV (7, 8). When this SFVG replicon RNA expressing only the SFV light to prevent RNA replication. After 1 month, serum- IMMUNOLOGY replication proteins and VSV G protein was transfected into neutralizing antibody titers to VSV were determined (Fig. 1 Left). BHK-21 cells, it initially replicated in the small fraction of These results showed 100% neutralization of VSV at serum dilu- transfected cells. Surprisingly, it also produced infectious, low tions of 1:160 for SFVG particles but no detectable neutralizing Ab density, membrane-enveloped particles lacking a nucleocapsid (Ͻ1:20) in animals given the UV-inactivated particles. These results protein that budded from the cells and infected and killed all indicate that the incoming G protein on the particles was not cells in the culture within 2–3 days. These infectious particles present in sufficient amounts to induce VSV-neutralizing antibody could be propagated (passaged) indefinitely in tissue culture, and that G protein must be synthesized in infected cells to generate and their infectivity was inactivated by a VSV-neutralizing such responses. antibody that binds the VSV G protein (6, 9). Although the precise mechanism of generation of the SFVG infectious parti- cles remains unknown, it clearly involves release of vesicles Author contributions: N.F.R., J.P., A.C., and J.K.R. designed research; N.F.R., J.P., and A.C. containing VSV G protein and SFV RNA. The replication of all performed research; N.F.R., J.P., A.C., and J.K.R. analyzed data; and N.F.R., J.P., A.C., and positive-strand RNA viruses including SFV occurs in association J.K.R. wrote the paper. with cellular membranes (10). SFV replication occurs in asso- The authors declare no conflict of interest. ciation with cytopathic vacuoles containing invaginations called This article is a PNAS Direct Submission. spherules, which are probably the sites of SFV RNA synthesis *To whom correspondence should be addressed: E-mail: [email protected]. (11–13). These spherules are also seen on the surface of SFV- © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800280105 PNAS ͉ April 15, 2008 ͉ vol. 105 ͉ no. 15 ͉ 5839–5843 Downloaded by guest on September 26, 2021 r 105 10000 t h g ei w 1000 y d 100 o b e 100 g en l al 95 SFVG imm 10 ch e- r Naïve %P 1 3 1 90 VSV neutralizing antibody tite SFVG SFVG(UV) SFVG VSV 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 6 x 103 i.u. 105 i.u. Days post challenge Fig. 2. Vaccination with SFVG particles protects mice against pathogenesis Fig. 1. Replication is required for induction of neutralizing antibody by SFVG caused by wild-type VSV. Twelve BALB/c mice were immunized with 5 ϫ 105 i.u. particles. Mice were inoculated i.m. with 6 ϫ 103 i.u. of SFVG untreated or of SFVG particles by i.m. injection. At 36 days after immunization, these mice treated with 100 mJ of UV light (UV) or with 105 i.u. of SFVG or 105 pfu of VSV were challenged with 5 ϫ 107 pfu of wild-type VSV by the i.v. route. Twelve as indicated. Pooled sera from groups of three mice were assayed for neu- nonimmunized BALB/c mice were challenged as controls. After challenge, tralizing titers to VSV on day 28 after inoculation. UV inactivation of SFVG mice were weighed daily for up to 14 days and observed for signs of patho- particles before inoculation abolished generation of anti-VSV G-neutralizing genesis. Any animal exhibiting paralysis or distress during this period was antibody. The neutralizing antibody titer to VSV in sera from mice inoculated killed. The graph shows the average weights of the mice Ϯ one standard with (105 i.u.) of SFVG was equivalent to that in sera from mice inoculated with deviation. Numbers above the x axis indicate the number of mice in the control 105 pfu of VSV (1:5,120). group that died on the corresponding day. We next determined whether the strength of the antibody SFVG Replicon Particles Are Not Pathogenic in Mice. After i.m. response to VSV G was dose-dependent. We inoculated mice injections of SFVG particles, we had not seen any signs of with 105 i.u. of SFVG particles or with 105 plaque-forming units pathogenesis in mice. To determine whether there was any (pfu) of VSV. VSV serum-neutralizing titers were determined at detectable pathogenesis caused by infection by other potentially 28 days after infection by using pooled serum from each group. more pathogenic routes, we gave the SFVG particles by both the The neutralizing antibody responses to VSV in sera from mice 5 inoculated with the high dose (105 i.u.) of SFVG was 1:5,120, i.v. and the intranasal routes (10 i.u.). We then weighed the mice 32-fold higher than that induced in mice inoculated with the daily for 2 weeks and then observed the mice for 60 days and saw lower dose (6 ϫ 103 i.u.) of SFVG. Remarkably, the neutralizing no signs of pathogenesis caused by the particles.