Alphavirus vectors as recombinant vaccines

Abstract

This thesis describes further developments of an expression system based on the alphavirus 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 structural proteins, and a 5' ORF coding for the viral replicase. Transfection of cells with genomic viral RNA alone is enough to induce a productive replication cycle and budding of 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 transcription vector. The expression system is based on an expression vector where the genes encoding the structural proteins are replaced by heterologous sequences. Transfection of cells with expression-vector derived RNA results in high level expression of the heterologous genes. A packaging system for production of recombinant SFV (rSFV) was previously developed using a helper vector, which encompasses the 3'0RF encoding the structural proteins. The rSFV virions produced are able to infect cells and mediate biosynthesis of 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 p62 normally undergoes a proteolytic cleavage which is necessary for subsequent virus infectivity. The p62 encoding sequence was modified allowing for cleavage to occur only 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 that rSFV could be used in vivo for vaccination studies.

Immunization of mice with recombinant virus carrying the gene encoding the influenza nucleoprotein (NP) conferred prolonged humoral immune responses as well as NP-specific CD8+ cytotoxic T Iymphocytes (CTL). Repeated immunizations did not induce immunity against the rSFV particles to such an extent that the response induced by subsequent immunization was inhibited. Challenge studies showed that mice immunized with both rSFV coding for NP (rSFV NP), and influenza hemagglutinin (HA) acquired protective immunity against mortality and weight loss following lethal influenza infection.

Two studies were carried out in order to study if rSFV could induce immune responses against lentivirus in primates. Immunization of macaques (Macaca nemestrina) with rSFV SlVmacgpl60 elicited humoral immune responses in all animals and protection against lethal acute disease following challenge with SlVmacPBjl4. In the other lentiviral vaccination 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 four nonimmunized animals compared to one of four immunized animals.

Studies showed that the expression system could also be used for immunization strategies based on naked nucleic acid. Intradermal injection with RNA transcribed in vitro from expression vectors into mice resulted in antigen-specific humoral responses. A variant of the expression vector was constructed containing a promoter for eukaryotic RNA polymerase. Transfection of cells with this plasmid vector resulted in RNA replication and heterologous protein synthesis. Mice immunized with such DNA plasmids developed stronger humoral and CTL-mediated immune responses than animals immunized with a conventional DNA vaccine. Vaccinated mice displayed significant, albeit partial, protection against mortality following influenza challenge. Taken together, these vectors encourage further vaccination studies based on both recombinant virus as well as naked nucleic acid.