Recombinant Semliki Forest virus particles encoding the prME or NS1 proteins of louping ill virus protect mice from lethal challenge.

Abstract

Recombinant Semliki Forest virus (rSFV) vaccines encoding louping ill virus (LIV)genes prME and NS1 were examined. Cells transfected with rSFV-prME RNA showed correct processing of the precursor prME and the release into the medium of M and E proteins in particulate form, whilst rSFV-NS1-transfected cells secreted glycosylated, heat-labile NS1 dimers. Mice immunized with rSFV particles produced antibodies against prME and NS1 that were mainly of the IgG2a subtype, indicating that a T-helper 1 immune response was induced. Immunization with prME-or NS1-encoding particles induced T-cell proliferation. Mice vaccinated intraperitoneally (i.p.) with rSFVprME and/or rSFV-NS1 were significantly protected from lethal i.p. challenge by two strains of LIV, the virulent LI/31 strain, from which the commercial LIV vaccine is derived, and the less-virulent LI/I antibody-escape variant. Intranasal (i.n.) vaccination was protective for rSFV-prME only against LI/31 challenge and not against challenge with LI/I. Immunization with rSFV-NS1 was protective against i.p. and i.n. challenge with both virus strains when given i.p., but was not protective when given i.n. For unvaccinated mice infected with LIV, all animals showing clinical signs had severe degenerative and inflammatory lesions in the central nervous system. None of the rSFV-vaccinated mice that survived challenge showed central nervous system pathology, with the exception of mild leptomeningitis in a minority of LI/31-infected mice. This suggests that protection following immunization with rSFV must occur at early stages of LIV infection.

Recombinant Semliki Forest virus vaccine vectors: the route of injection determines the localization of vector RNA and subsequent T cell response

“…4 Briefly, BHK cells were co-electroporated with in vitro synthesized recombinant RNA together with two helper RNAs, one of which codes for the SFV capsid protein and the other for the SFV envelope proteins. After 24 h viral stocks were harvested, concentrated by ultracentrifugation of virus-containing medium through a 20% sucrose cushion 36 and titrated on BHK cells using indirect immunofluorescence staining. 35 Monoclonal antibodies used for ␤-galactosidase and influenza A nucleoprotein detection were purchased from Boehringer-Mannheim (Mannheim, Germany) and Virostat (Portland, OR, USA), respectively.…”

Induction of P815 tumor immunity by recombinant Semliki Forest virus expressing the P1A gene

“…29 Briefly, BHK cells transfected with rSFV mRNA encoding NP, LacZ or P1A were plated on 96-well dishes. After methanol fixation, sera obtained from immunized mice were added in blocking buffer and incubated overnight at 4°C.…”

“…The SFV system was chosen since it was earlier shown to generate strong CTL and antibody responses and to protect against challenge with Influenza virus. 23,24,[27][28][29] Our results demonstrate that vaccination of mice with recombinant SFV expressing the P815A antigen induces strong and specific CTL responses and protect mice against tumor growth. We conclude that recombinant SFV is a promising vector system which might be further optimized in order to activate immune responses against cancer.…”

A Semliki Forest virus vector engineered to express IFNα induces efficient elimination of established tumors

“…21 SFV particles were purified by ultracentrifugation as described previously. 48 Indirect immunofluorescence of infected BHK-21 cells was performed to determine the titer of recombinant virus stocks. 49 A rabbit polyclonal antiserum specific for the nsp2 subunit of SFV replicase was used as primary antibody.…”