Euan Lockhart

Manipulation of immune responses to Mycobacterium bovis by vaccination with IL-2- and IL-18-secreting recombinant bacillus Calmette Guerin.

Bacillus Calmette Guerin (BCG) has been reported to show variable efficacy as a vaccine against tuberculosis. We demonstrated that the secretion of biologically active IL‐2 (rBCG/IL‐2), but not IL‐18 (rBCG/IL‐18), by BCG improves its ability to induce and maintain a strong type 1 immune response in BALB/c mice. rBCG/IL‐2 induced significantly higher Ag‐specific proliferative responses, high IFN‐γ production and serum titres of IgG2a 16 weeks after vaccination. This immune profile was correlated to an increased rate of clearance of non‐pathogenic mycobacteria (live BCG delivered intranasally). Surprisingly, however, this strong type 1 immune profile induced no greater protective immunity against aerosol challenge with virulent Mycobacterium bovis than that induced by normal BCG (nBCG). By comparison, vaccination with rBCG/IL‐18 was found to induce significantly less IFN‐γ production in splenic lymphocytes than nBCG. This impaired induction of IFN‐γ was correlated to a significantly lower protective efficacy against M. bovis challenge, as compared to nBCG. The data suggest that manipulation of the immune response to tuberculosis and tuberculosis vaccines will require a more complete understanding of the factors that are important in generating a protective immune response.

An in vivo comparison of bacillus Calmette–Guérin (BCG) and cytokine-secreting BCG vaccines

A recombinant bacillus Calmette–Guérin (BCG) vaccine has been developed, which constitutively secretes interleukin (IL)‐2. Groups of deer were immunized with either normal BCG (Pasteur 1173 P2 strain) or recombinant BCG (rBCG/IL‐2) and their immune responses were monitored over 3 months. Animals gained weight over this period and showed no signs of adverse reactions to either vaccine. Lymphocyte transformation responses did not differ significantly between the two groups. No antibody that was specific for BCG was detected in any animal. Intradermal skin‐test responses to BCG antigens showed that the rBCG/IL‐2 induced a smaller delayed‐type hypersensitivity response than the normal BCG. Cytokine transcription was determined by reverse transcription–polymerase chain reaction (RT–PCR). While IL‐2 and interferon‐γ (IFN‐γ) levels did not differ significantly between the two groups, the level of IL‐4 was found to be lower in the group given rBCG/IL‐2. This resulted in a strong interferon‐γ:IL‐4 ratio, suggesting a skewing of the immune response towards a Type 1 response. The rate at which the vaccine was eliminated from the host was the same regardless of whether BCG or rBCG was used. At autopsy (3 months after vaccination) 99·99% of the organisms had been eliminated. The small number of organisms isolated from the draining lymph node of animals given rBCG/IL‐2 were grown in antibiotic‐containing media. They were shown to still contain the shuttle plasmid and to secrete biologically active IL‐2, indicating that the plasmid was stably maintained despite the host’s immune response and in the absence of antibiotic selection.

Influenza hemagglutinin peptides fused to interferon gamma and encapsulated in liposomes protects mice against influenza infection.

The immunogenicity of a peptide vaccine may be improved by fusing antigen to a cytokine and administering this chimeric protein in a particulate delivery system. We have investigated this using a vaccine comprising an immunodominant T cell epitope and a B cell epitope from influenza haemagglutinin (HATB) fused to interferon gamma and encapsulated in liposomes (HATB/IFN-gamma/lipo). Controls comprised groups receiving HATB/IFN-gamma mixed with liposomes, HATB incorporated in liposomes or heat inactivated PR8 influenza virus (HI PR8). IFN-gamma production in mice treated with HATB/IFN-gamma/lipo was significantly higher than in mice inoculated with either HATB/IFN-gamma mixed with liposomes or HATB incorporated in liposomes but less than HI PR8. Lung viral titres were significantly lower in mice treated with HATB/IFN-gamma/lipo compared with those treated with HATB/IFN-gamma mixed with liposomes. HI PR8-treated mice recorded a nil viral titre. There was no correlation between the level of antibody production and clearance of virus from the lungs. These data suggest that particulate delivery systems may be useful adjuncts to improve immune responses to chimeric proteins and to induce protection against disease.

A prolonged immune response to antigen delivered in poly ( ∈ -caprolactone) microparticles

A single dose vaccine formulation which induces both humoral and cell‐mediated immune responses over a prolonged period would provide a potent weapon against infectious disease. We have used a water‐in‐oil‐in‐oil, solvent evaporation method for generating poly ∊‐caprolactone microparticles and tested their ability to induce an immune response against the model antigen ovalbumin. We hypothesized that the initial release of antigen from the surface of the poly ∊‐caprolactone microparticles would act as the priming dose and that the delayed release over the following months, due to diffusion from or break‐down of the microparticles, would act as a boost to the immune response. Ovalbumin encapsulated in the poly ∊‐caprolactone microparticles was able to induce both antibody and cell‐mediated immune responses. However our results suggest that the spontaneous release had little effect on the immune response. Despite this the response was maintained for at least 8 months following a single immunization. Both humoral and cell‐mediated immune responses were induced in mice. This simple method of vaccine formulation offers a cost‐efficient way to deliver antigen in a single dose to the immune system.

Bystander Help within a Polyepitope DNA Vaccine Improves Immune Responses to Influenza Antigens

A polyepitope DNA vaccine has the potential to generate protective immune responses to a range of antigens in a single construct. We investigated whether it was possible to obtain responses to individual epitopes from different antigens, directly linked in a string, and whether the response to a given epitope was enhanced by adjacent epitopes within the construct. A polyepitope plasmid was created, which included three Th epitopes (influenza haemagglutinin, moth cytochrome c and ovalbumin), a Tc epitope (ovalbumin) and two B cell epitopes (haemagglutinin and ovalbumin). Mice were immunized with DNA by using a gene gun. Responses to the polyepitope DNA vaccine were compared with those to DNA vaccine comprising only the haemagglutinin Th and B epitopes (HAThB) or with responses to the recombinant protein. These experiments showed that the polyepitope DNA vaccine induced greater antigen‐specific responses to HAThB peptide than the HAThB DNA vaccine. Antigen‐specific in vivo cytotoxic responses following polyepitope DNA vaccination were also clearly demonstrable. We conclude that a ‘naked DNA’ polyepitope vaccine generates specific responses to constituent epitopes and that adjacent irrelevant epitopes may enhance these responses.

The cloning and sequencing of cervine interleukin 10

We report the cloning and sequencing of the cervine interleukin-10 gene. Specific cDNA was amplified by PCR using primers based on the bovine sequence. This was cloned into pGEM 5Zf and several clones were sequenced. The 762 nucleotide product coded for a 179 amino acid protein which was 86% homologous with its bovine and 77% homologous with its human counterparts. There is a strongly hydrophobic signal sequence consisting of the first 20 amino acids and a potential glycosylation site at amino acids 134-136. There are three regions, comprising 34% of the protein, which show complete homology between the cervine, bovine and human sequences. The transcription of the gene was shown by Northern Blotting where a single, 1.8kb, mRNA transcript was detected 4-8 hours after activation of peripheral blood mononuclear cells with mitogen.

The Characterisation of a Cervine Immunoregulatory Cytokine, Interleukm 12

The cloning, sequencing, and production of cervine interleukin-12 is described. The cervine IL-12 p35 subunit coding sequence is 666 bp long and has highest homology to bovine p35 (94%), followed by human (79%), then murine (57%). The cDNA codes for a 221 aa long protein with predicted molecular weight of 24,902 Da. The cervine p40 subunit has a coding sequence of 984 bp and shows 96% homology to bovine, 85% homology to human, and 65% homology to murine p40 respectively. Cervine p40 cDNA codes for a 327 aa long protein with a predicted molecular weight of 37,461. Both subunits were inserted into a recombinant baculovirus that was then used to produce cervine IL-12 in Trichoplusia ni cells. Interleukin-12 was secreted into the culture medium and was biologically active as measured by proliferation of mitogen sensitised peripheral blood lymphocytes and the induction of interferon-gamma transcription in peripheral blood lymphocytes.