Alphavirus vectors as recombinant vaccines

Sammanfattning: ALPHAVIRUS VECTORS AS RECOMBINANT VACCINES Peter Berglund Doctoral dissertation from the Microbiology and TumorbiologyCenter Karolinska Institute, Sweden This thesis describes further developments of an expression system based on thealphavirus Semliki Forest virus (SFV), and its potential use as recombinant vaccine. The RNA genome of SFV contains a 3'open reading frame (ORF) encoding the structuralproteins, and a 5' ORF coding for the viral replicase. Transfection of cells withgenomic viral RNA alone is enough to induce a productive replication cycle and buddingof progeny virus particles. The viral RNA can also be produced by in vitro transcription,since the cDNA of the complete viral genome has been cloned into a transcriptionvector. The expression system is based on an expression vector where the genes encodingthe structural proteins are replaced by heterologous sequences. Transfection of cellswith expression-vector derived RNA results in high level expression of the heterologousgenes. A packaging system for production of recombinant SFV (rSFV) was previouslydeveloped using a helper vector, which encompasses the 3'0RF encoding the structuralproteins. The rSFV virions produced are able to infect cells and mediate biosynthesisof the desired protein/antigen but theoretically do not induce productive infection.However, it was found that replication-proficient virus was formed at low frequencies,probably by RNA recombination. Since the use of recombinant vaccines is associated with stringent biosafety requirements,a second helper vector was constructed. Before virus budding, the spike protein p62normally undergoes a proteolytic cleavage which is necessary for subsequent virusinfectivity. The p62 encoding sequence was modified allowing for cleavage to occuronly in vitro. Virions therefore require protease activation in vitro to become infectious.Studies in tissue culture and mice showed the absence of replicative virus, and thatrSFV could be used in vivo for vaccination studies. Immunization of mice with recombinant virus carrying the gene encoding the influenzanucleoprotein (NP) conferred prolonged humoral immune responses as well as NP-specificCD8+ cytotoxic T Iymphocytes (CTL). Repeated immunizations did not induce immunityagainst the rSFV particles to such an extent that the response induced by subsequentimmunization was inhibited. Challenge studies showed that mice immunized with bothrSFV coding for NP (rSFV NP), and influenza hemagglutinin (HA) acquired protectiveimmunity against mortality and weight loss following lethal influenza infection. Two studies were carried out in order to study if rSFV could induce immune responsesagainst lentivirus in primates. Immunization of macaques (Macaca nemestrina) withrSFV SlVmacgpl60 elicited humoral immune responses in all amimals and protectionagainst lethal acute disease following challenge with SlVmacPBjl4. In the other lentiviralvaccination study, four macaques (Macaca fascicularis) were immunized with rSFV-HlV-I-gpl60.Challenge with chimeric SIV/HIV virus gave rise to high viremia in three of fournonimmunized animals compared to one of four immunized amimals. Studies showed that the expression system could also be used for immunizationstrategies based on naked nucleic acid. Intradermal injection with RNA transcribedin vitro from expression vectors into mice resulted in antigen-specific humoral responses.A variant of the expression vector was constructed containing a promoter for eukaryoticRNA polymerase. Transfection of cells with this plasmid vector resulted in RNA replicationand heterologous protein synthesis. Mice immunized with such DNA plasmids developedstronger humoral and CTL-mediated immune responses than animals immunized with aconventional DNA vaccine. Vaccinated mice displayed significant, albeit partial,protection against mortality following influenza challenge. Taken together, thesevectors encourage further vaccination studies based on both recombinant virus aswell as naked nucleic acid. Key words: Semliki Forest virus, Alphavirus, Synthetic vaccmes, Influenza A virus,HIV-I, SIV, DNA vaccines, AIDS vaccines, Influenza vaccine ISBN 91-628-2657-3