Genetic vaccination against acute viral disease

Abstract

This thesis describes the development of recombinant vaccines based on the Semliki Forest virus (SFV) expression system. Immunisation of mice with recombinant virus particles, a layered DNA/RNA plasmid vector, and recombinant self-replicating RNA were carried out and the protective effect of these recombinant vaccines against viral challenge were examined.

The construction of a full-length infectious clone formed the basis for the SFV expression system which has previously been described. In the expression system, genes coding for the SFV structural proteins are replaced with a multiple cloning site where the gene coding for a foreign antigen can be inserted. Transfection of cells with RNA transcribed in vitro from such vectors results in transient high-level production of the antigen followed by death of the cells as a result of the induction of apoptosis. Recombinant virus particles can be made by co-transfecting cells with expression vector RNA and a defective helper RNA coding for the SFV structural proteins. Recombinant SFV (rSFV) particles lack the genes for the structural proteins and thus undergo only a single round of non-productive infection after entry into a cell.

Mice immunised with rSFV encoding influenza A virus (FLU) nucleoprotein (NP) or E.coli LacZ protein were shown to develop antigen-specific IgG and CTL responses that persisted for over one year. Humoral and cellular immune responses could be induced in mice following vaccination by peripheral and mucosal routes. Examination of IgG1/IgG2a antibody isotypes indicated that predominantly a T-helper type 1 response was induced. Importantly, this study showed that development of immune responses against the vector itself did not significantly inhibit responses following booster immunisations with rSFV. This is important for recombinant vaccine design as the use of many recombinant virus vaccines is hampered due to pre-existing immunity to the vectors.

Expression of envelope proteins, prME, and a nonstructural protein, NS1, of the tick-borne flavivirus, louping ill virus (LIV). from rSFV in cell culture was characterised. both proteins were shown to be correctly processed and secreted from transfected cells. Humoral and T-cell proliferative responses were induced in mice immunised with rSFV particles encoding LIV antigens. Mice vaccinated with rSFV` particles coding for FLU or LIV antigens were protected from challenge with FLU or from challenge with two strains of LIV. the prototype strain LI/31, and LI/l, a naturally occurring antibody escape variant.

A layered DNA/RNA vector based on the SFV replicon (pBK-SFV) was developed. A cytomegalovirus (CMV) promoter drives production, not of the antigen-encoding gene, but of the recombinant SFV RNA. In comparison to a conventional DNA vector coding for the same antigen, vaccination of mice with pBK-SFV DNA was shown to elicit stronger humoral and cellular immune responses. Mice vaccinated with pBK-SFV` encoding FLU antigens were shown to be protected against influenza A virus challenge.

In order to compare the protective efficacy of rSFV vaccines, a triple challenge model based on three strains of LIV with graded virulence was established. Results from challenge experiments showed that rSFV particles induced better protective immunity than plasmid vaccines (conventional or SFV-based) or the commercially available LIV inactivated vaccine.

Antigen-specific humoral and cellular immune responses were also elicited in mice following immunisation with naked rSFV RNA encoding FLU, LIV, or respiratory syncytial virus (RSV) antigens. Mice vaccinated with rSFV RNA were protected from challenge with FLU. LIV or RSV.

Studies showed that bone marrow derived dendritic cells (BMDDC) from mice are unable to be productively infected by rSFV particles encoding GFP (green fluorescent protein) as de novo synthesis of GFP could not be detected in these cells. However, RT-PCR analysis of rSFV` infected BMDDC showed that negative strand RNA intermediates of rSFV infection could be detected. indicating that rSFV RNA replication had been initiated in these cells. rnRNA coding for interferon-[alpha]-induced antiviral proteins and the cytokines TNF-[alpha] and IL-12 rnRNA were also upregulated in these cells. This suggests that cytokine signals required for DC maturation and activation were turned on as a result of the initiation of viral RNA replication. Uptake of antigen (GFP) by BMDDC from rSFV infected syngeneic B 16 cells was shown by confocal microscopy. Thus, induction of apoptosis in rSFV transfected cells could facilitate antigen transfer to professional antigen presenting cells for subsequent cross-priming, and might thereby explain the efficient immune responses induced following immunisation with rSFV vaccines.