J. Med. Microbiol. - Vol. 46 (1997). 129-138 ((3 1997 The Pathological Society of Great Britain and Ireland IMMUNE RESPONSE TO INFECTION Immune response to a Murray Valley encephalitis virus epitope expressed in the flagellin of an attenuated strain of Salmonella BELINDA L. WHITTLE, EVA LEE, RON C. WEIR and NARESH K. VERMA Division of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra, ACT 0200, Australia Recent developments in vaccine construction include the use of attenuated, avirulent strains of Salmonella as carriers of foreign antigens. These recombinant strains can elicit a heterologous immune response when injected into animals, demonstrating potential for their use in the construction of many vaccines. In the present study, a B-cell epitope of Murray Valley encephalitis virus (MVE) was identified and expressed in a Salmonella strain to evaluate its potential to induce a specific immune response to MVE. A synthetic oligonucleotide encoding the B-cell epitope (residues E201-224) of the envelope protein of MVE was inserted into the cloned flagellin gene of the Salmonella strain. The construct was sequenced to ensure correct orientation of the epitope. Expression of the epitope was demonstrated by Western blot analysis and immunogold electron microscopy with monoclonal antibody specific to the epitope. Electron microscopy analysis revealed multiple copies of the epitope along the flagella. The recombinant Salmonella carrying the hybrid flagellin gene elicited an immune response to the MVE epitope in a mouse model. The MVE-specific antibodies partially neutralised the virus in vitro. The significance of this system for engineering vaccines for other medically important flaviviruses is discussed. Introduction Recent developments in vaccine construction include the use of attenuated, avirulent strains of Salmonella as vectors carrying foreign antigens which elicit protective immune responses [I, 21. One important group of non- reverting Salmonella vaccine strains comprises the mutants dependent on aromatic compounds (aro- ) [3]. The aro- mutants have a deletion in one of the genes of the aromatic amino acids biosynthesis path- way resulting in strains dependent on aromatic com- pounds that are available in limited amounts in host tissues. The insertion of a tetracycline resistance transposon, Tn10, into the aroA gene of Salmonella serotype Typhimurium produced aroA strain SL326 1 [4, 51, a well-defined aro- mutant [6,7]. The aro- mutants multiply only for a few generations in host tissues, but do persist in the spleen and liver for several weeks, inducing strong humoral and cellular immune responses [7,8]. A further development has been the insertion of synthetic oligonucleotides, encoding for- eign epitopes, into a cloned Salmonella flagellin gene Received 10 April 1996; accepted 15 June 1996. Corresponding author: B. L. Whittle. [3,9]. A plasmid, pLS408, containing the H1-d flagellin gene of serotype Muenchen, has been constructed [lo]. In this plasmid, a 48-bp deletion has been made in the hypervariable segment (region IV) of the flagellin gene. The remaining EcoRV site allows the blunt end insertion of oligonucleotides. T-cell epitopes have previously been expressed in the flagellin and the immune response to the epitopes examined in mice immunised with the recombinant Salmonella strain [ll, 121. A number of epitopes of viral and eukaryotic origin have also been expressed in salmo- nella flagellin. In most cases, such recombinant Salmonella strains induced specific immune responses to the foreign epitopes [3, 131. Murray Valley encephalitis virus (MVE) belongs to the flavivirus family of small enveloped, single- stranded RNA viruses [14-161. MVE is responsible for endemic cases of encephalitis in tropical regions of Australia and Papua New Guinea, and occasional epidemics in south-eastern Australia [ 17, 181. Other flaviviruses of major public health concern are the closely related Japanese encephalitis virus (JE), yellow fever virus (YF) and dengue viruses (DEN). Currently, an attenuated vaccine for YF [19], an inactivated