Immunology 1990 70 540-546

Anti-viral immunity induced by recombinant nucleoprotein of Influenza A virus III. DELIVERY OF RECOMBINANT NUCLEOPROTEIN TO THE IMMUNE SYSTEM USING ATTENUATED SALMONELLA TYPHIMURIUM AS A LIVE CARRIER

J. P. TITE, X.-M. GAO,* C. M. HUGHES-JENKINS, M. LIPSCOMBE, D. O'CALLAGHANt G. DOUGAN & F. Y. LIEW Departments of Experimental Immunobiology and Molecular Biology, Wellcome Biotech, Beckenham, Kent

Acceptedfor publication 4 May 1990

SUMMARY A plasmid encoding the influenza nucleoprotein gene from A/NT/60/68 virus was transduced into the attenuated Salmonella typhimurium aroA- strain SL3261. The bacterial vector expressing the viral gene product was able to induce both humoral and cell-mediated immune responses to the nucleoprotein antigen. CD4+ virus-specific T cells capable of proliferation were readily induced and, in some cirumstances, class II major histocompatibility complex (MHC)-restricted cytotoxicity was detected. However, virus-specific class I MHC-restricted cytotoxic T lymphocytes (CTL) were not detected after such immunization. Mice immunized orally with the nucleoprotein-expressing bacteria mounted a strong anti-viral antibody response and spleen cells from such mice proliferated specifically to virus challenge in vitro, producing interferon-gamma (IFN-y) and interleukin-2 (IL-2). Orally immunized mice showed significant protection from challenge infection with influenza virus if the mice were also boosted intranasally before infection.

INTRODUCTION Many common pathogens against which it is desirable to vaccinate enter the human body via mucosal surfaces. These include both viral and bacterial infections of the respiratory, gastrointestinal and urinogenital tracts. There are good reasons therefore to generate protective immune responses at these surfaces to combat these infections in their early stages and prevent widespread pathogenesis. Increasing evidence is accumulating for a common mucosal immune system in which lymphocytes traffic preferentially between lymphoid tissues associated with mucosal surfaces (Mestecky, 1987). This has led to speculation that immunization of the mucosal immune system via one particular route may induce a state of immunity at anatomically distant mucosal surfaces. The oral route is clearly the most convenient and acceptable route for administration of a vaccine. This would obviate the use of needles, * Present address: Molecular Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K. t Present address: UPMTG, Institute Pasteur, 25 Rue du Dr Roux, 75015 Paris, France. Abbreviations: A/PR8, A/Puerto Rico/8/34; NP, nucleoprotein. Correspondence: Dr J. P. Tite, Dept. of Molecular Biology, Wellcome Biotech, Langley Court, Beckenham, Kent BR3 3BS, U.K.

thereby removing a psychological barrier which prevents the uptake of vaccines, especially against diseases such as influenza which are not perceived by the public as serious or lifethreatening. The use of attenuated variants as vaccine against pathogenic forms of the same organism is a well-established practice. Often, attenuation has been achieved empirically and the mechanism of attenuation ill-understood. However, recent accumulation of information on the genetic structure of pathogens has enabled the attenuation of micro-organisms in a well-defined and rational fashion. Hoiseth & Stocker (1981) described an aromatic-dependent mutant of Salmonella typhimurium (SL3261) which was capable of limited growth in vivo but was highly attenuated and able to induce protective immunity against the highly virulent wild-type organism. The attenuated organisms were still capable of penetrating the gut wall, reaching the gutassociated lymphoid tissue and initiating an immune response in these tissues (Killar & Eisenstein, 1985; Maskell et al., 1987). Advances in recombinant DNA technology has allowed this approach to be extended to the use of such attenuated organisms as carriers for heterologous antigens from other pathogens (Dougan, Hormaeche & Maskell, 1987), providing a general mechanism for the oral delivery of candidate vaccine molecules. This is especially desirable if mucosal immunity is implicated in protection from a particular pathogen. Immunity to nucleoprotein (NP) has been shown to protect mice against a subsequent lethal influenza infection (Wraith,

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Delivery of nucleoprotein by live carrier Vessey & Askonas, 1986). We have shown that immunization with recombinant derived NP, purified from S. typhimurium expressing the NP gene, generates protective immunity (Tite et al., 1990). In the experiments described in this report, we investigate the ability of attenuated aroA mutants of S. typhimurium expressing a viral gene product to induce anti-viral immunity. A plasmid containing the gene coding for the NP of Influenza A virus has been transduced into SL3261 and this construct was used to deliver NP to the immune system. This system, therefore, provides an opportunity to investigate antiviral immunity and protection after vaccination with the bacterial vector expressing the viral gene product.

MATERIALS AND METHODS Animals BALB/c and Bl0.S mice were bred in our own animal facility. Mice were used between the ages of 6 and 10 weeks. Antigens and immunizations

Purified recombinant nucleoprotein (NP) was prepared as previously described (Tite et al., 1988). Short synthetic peptides were synthesized according to the method of Houghten (1985); cleavage was performed using hydrogen fluoride. High-pressure liquid chromatography (HPLC) analysis of the peptides indicated between 80-90% purity. The production of the SL3261pNP-2 construct has been described elsewhere (Tite et al., 1988). Briefly, this construct constitutively expresses the full-length NP molecule from Influenza A/NT/60/68 virus as a fusion protein, with 12 amino acids of the cro protein plus a 20 amino acidlinker. Immunizations with bacteria were performed either intravenously, subcutaneously at the base of the tail or intragastrically, as indicated. Intranasal immunizations were performed on mice under ether anaesthetic. Viruses Viruses were grown in embryonated chicken eggs and purified by ultracentrifugation (Tite et al., 1988). The following viruses were used: A/Puerto Rico/8/34 (H 1N 1) and A/Okuda/57 (H2N2). Monoclonal antibodies The rat hybridomas YTS-1 69 (anti-CD8) (Cobbold et al., 1984) and GK1.5 (anti-CD4) (Dialynas et al., 1983) and the mouse hybridoma Y-3p (Janeway et al., 1984), specific for labs were grown as ascites in pristane primed nude mice. Immunoglobulin in ascitic fluid was partially purified by precipitation with saturated ammonium sulphate. T-cell proliferation assays Lymph node cell proliferation assays were performed as previously described (Tite et al., 1988); proliferation assays using spleen cells were performed identically after hypotonic lysis of erythrocytes. DNA synthesis was measured by incorporation of [3H]thymidine ([3H]TdR) during the last 18 hr of a 4-day culture. Generation and assay of cytotoxic T cells Cytotoxic T cells were generated in 10-ml cultures containing 2 x 107 responder lymphocytes. Medium was Clicks' extra high

amino acids (EHAA) with either 0 5% normal mouse serum or 10% fetal calf serum (FCS), as described in the figure legends. Stimulation of cultures was achieved by addition of either purified virus or virus-infected irradiated syngeneic spleen cells. Cultures were assayed for cytotoxic T lymphocytes (CTL) after 5 days using a 5"Cr-release assay, as described previously (Tite et al., 1988).

Assay of virus-specific serum antibody levels Antibody levels were measured by a solid-phase radioimmunoassay utilizing polyvinylchloride microtitre plates coated with purified A/Okuda/57 virus. Dilutions of serum were incubated on the coated plates overnight before extensive washing and application of '251-labelled affinity-purified rabbit anti-mouse immunoglobulin antibody. Titres were calculated as the dilution of serum giving 50% maximum binding of radiolabelled antibody. In vivo bacterial growth curves The growth of the aro mutants in vivo was monitored as described previously (O'Callaghan et al., 1988). Colonies in liver and spleen were measured in the presence or absence of 100 ,ug/ ml ampicillin in order to assess plasmid stability.

RESULTS

Expression of the full-length nucleoprotein gene in SL3261 The plasmid pNP-2 (Jones & Brownlee, 1985) was transduced into Salmonella typhimurium SL3261 using bacteriophage p22 and the level of NP antigen was measured. Expression of NP was evidenced by a unique band of 58,000 molecular weight (MW) in SDS-PAGE gels of SL3261 -pNP-2 compared to SL3261 (Fig. 1). Immunoblotting with a polyclonal rabbit antiNP serum confirmed this band as NP (data not shown). Expression was estimated by densitometric scanning of SDSPAGE gels at the level of 10% of total cell protein, which was found to be stable at this level throughout many experiments. Growth and stability of SL3261-pNP-2 In order to determine the in vivo stability of the transformed SL3261, mice were injected intravenously with approximately 105 bacteria, and spleens and livers assayed at various times afterwards for colony-forming units (CFU). Assays were performed in the presence and absence of ampicillin to assess plasmid stability. Figure 2 shows the growth curve obtained in such an experiment. The plasmid-containing bacterium grew less well than the untransformed SL3261, but nevertheless persisted for several weeks in both organs. There was, however, evidence of plasmid segregation such that by Day 10 after injection, approximately 90% of the colonies from mice immunized with SL3261-pNP-2 were sensitive to ampicillin. Immune responses to parenterally administered attenuated salmonella expressing the influenza NP gene In a previous series of experiments, we investigated the immune response to purified recombinant derived NP (rNP) that had been expressed in S. typhimurium (Tite et al., 1988). These experiments had revealed that whereas all mouse strains tested

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Figure 2. BALB/c mice were immunized intravenously with I05 SL3261pNP-2 or 105 SL3261. Spleens (open symbols) and livers (closed symbols) were removed at the indicates times after injection and CFU assayed on plates with or without ampicillin. SL326 1-pNP-2 immunized mice (l, *); SL3261-immunized mice (0, *); ( ) no ampicillin; (- - -) ampicillin at 100 yg/ml.

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Table 1. Serum antibody levels after parenteral immunization with SL3261-pNP-2

36,500

~

Strain

Immunogen

Route

BALB/c

SL3261 -pNP-2 SL3261 SL3261 -pNP-2 SL3261

i.v. i.v. s.c. s.c.

2-29 + 0-38