Glenn Buchan

A DNA prime-live vaccine boost strategy in mice can augment IFN-γ responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis

The Mycobacterium bovis bacille Calmette–Guérin (BCG) vaccine has variable efficacy for both human and bovine tuberculosis. There is a need for improved vaccines or vaccine strategies for control of these diseases. A recently developed prime‐boost strategy was investigated for vaccination against M. bovis infection in mice. BALB/c and C57BL/6 mice were primed with a DNA vaccine, expressing two mycobacterial antigens, ESAT‐6 and antigen 85 A and boosted with attenuated M. bovis strains, BCG or WAg520, a newly attenuated strain, prior to aerosol challenge. Before challenge, the antigen‐specific production of interferon‐γ (IFN‐γ) was evaluated by ELISPOT and antibody responses were measured. The prime‐boost stimulated an increase in the numbers of IFN‐γ producing cells compared with DNA or live vaccination alone, but this varied according to the attenuated vaccine strain, time of challenge and the strain of mouse used. Animals vaccinated with DNA alone generated the strongest antibody response to mycobacterial antigens, which was predominantly IgG1. BCG and WAg520 alone generally gave a 1–2 log10 reduction in bacterial load in lungs or spleen, compared to non‐vaccinated or plasmid DNA only control groups. The prime‐boost regimen was not more effective than BCG or WAg520 alone. These observations demonstrate the comparable efficacy of BCG and WAg520 in a mouse model of bovine tuberculosis. However, priming with the DNA vaccine and boosting with an attenuated M. bovis vaccine enhanced IFN‐γ immune responses compared to vaccinating with an attenuated M. bovis vaccine alone, but did not increase protection against a virulent M. bovis infection.

Interleukin‐10 does not affect phagocytosis of particulate antigen by bonemarrow‐derived dendritic cells but does impair antigen presentation

Dendritic cells (DC) are important initiators of an immune response so understanding the factors controlling antigen acquisition and presentation has important consequences for the use of these cells in vaccines and other forms of immunotherapy. We investigated the factors that influence phagocytosis by immature bone marrow‐derived DC (BMDC) and the effect of interleukin‐10 (IL‐10) on this process. Two sizes of fluorescent particles and recombinant bacillus Calmette–Guèrin expressing the green fluorescent protein (rBCG) were used as particulate antigens. The percentage of cells taking up the antigen was found to be dependent on the size and dose of the particles, and the length of exposure to them. BMDC exposed to IL‐10 at various concentrations for different periods exhibited no distinguishable change in antigen uptake. However, if BMDC treated with IL‐10 and rBCG were then exposed to a second dose of particulate antigen, uptake was increased compared with those BMDC not treated with IL‐10. The expression of major histocompatibility complex class II, CD80, CD86 and CD11c by BMDC after phagocytosing rBCG or inert beads, was inhibited when the BMDC were pretreated with IL‐10. In contrast, the expression of CD25 was increased. BMDC that had taken up BCG or purified protein derivative (PPD) were able to stimulate primed T‐cell proliferation but this was severely inhibited if the BMDC were cultured with IL‐10 before exposure to the antigen. This work suggests that although IL‐10 does not affect the phagocytic capacity of BMDC, it does inhibit maturation of the cells and consequently, T‐cell activation.

Animal models of protective immunity in tuberculosis to evaluate candidate vaccines

While the etiology of tuberculosis is well understood, the nature of the protective immune response to the causative mycobacteria has remained a mystery. There is an urgent need to define protective immunity critically, and to develop alternative animal models to evaluate the efficacy of new-generation vaccines against tuberculosis in a cost-effective way.

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.

Environmental Strains of Mycobacterium avium Interfere with Immune Responses Associated with Mycobacterium bovis BCG Vaccination

ABSTRACT Prior exposure of a vaccinee to certain species of environmental mycobacteria can prime the immune system against common mycobacterial antigens, which can in turn reduce the subsequent efficacy of live attenuated mycobacterial vaccines (such as Mycobacterium bovis BCG), in both human and livestock vaccination programs. In this study, two strains of Mycobacterium avium, both isolated from New Zealand livestock, were investigated to determine their growth characteristics and effects on the immune system in murine models. Markedly different effects on the immune system were observed; an IS901-negative strain (WAg 207) induced significant up-regulation of cell surface activation markers (major histocompatibility complex II, CD80, and CD86) on in vitro-derived dendritic cells and induced the release of proinflammatory monokines (interleukin-1β [IL-1β], IL-6, and tumor necrosis factor alpha) in dendritic cell-macrophage cocultures following direct in vitro contact of cells with bacteria. In contrast, an IS901-positive strain (WAg 206) had none of these effects. When mice were exposed to M. avium via oral infection prior to BCG parenteral immunization, both strains were shown to be capable of decreasing subsequent antigen-stimulated gamma interferon secretion by splenic lymphocytes, although this effect was more significant for strain WAg 206. Both strains also induced a mycobacterial antigen-specific serological response in M. avium-sensitized and BCG-immunized mice; this response was greater in WAg 206-sensitized mice, and there was a predominance of immunoglobulin G1 antibody. The down-regulation of IFN-γ responses and the up-regulation of antibody responses are characteristic of a switch to a type 2 immune response. The different results may be linked to the inherent growth characteristics of the two strains, since WAg 206 was shown to grow slowly in murine macrophages in vitro and to cause a persistent systemic infection following infection in vivo, while WAg 207 grew fast and did not persist in mice. The implications of these findings for BCG vaccination protocols are discussed.

Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis BCG: Anatomical sites of bacterial replication and immune activity

Lipid microencapsulation of Mycobacterium bovis bacille Calmette–Guérin (BCG) produces an oral delivery vaccine that can establish systemic cell‐mediated immune reactivity and protection against aerosol mycobacterial challenge in mice. Here, we describe the lymphatic and mucosal sites of bacterial replication, and location of Mycobacterium‐specific IFN‐γ‐secreting cell populations, following oral vaccination of BALB/c mice. Eight weeks following a single oral dose of lipid‐encapsulated BCG, viable BCG organisms were recovered from the mesenteric lymph nodes (MLN) of 11/12 mice investigated (93%). Live bacteria were also occasionally recovered from the cervical lymph nodes (17%) and Peyers patches (8%), but not from homogenates of the lungs or spleen. Strong Mycobacterium‐specific IFN‐γ production was recorded among isolated splenocytes, but not among populations of mononuclear cells derived from the MLN or lungs. Oral vaccination of mice with lipid‐encapsulated BCG thus appears to promote a state of systemic immunological reactivity more akin to that observed following parenteral rather than conventional oral vaccination, despite the fact that replicating bacilli are restricted to lymphatic tissues of the alimentary tract. Possible patterns of lymphocyte sensitization and trafficking are discussed.

Activation of an interleukin-4 mRNA-producing population of peripheral blood mononuclear cells after infection with Mycobacterium bovis or vaccination with killed, but not live, BCG.

This study examines the expression of mRNA for the Th2 cytokine, interleukin‐4 (IL‐4). Peripheral blood mononuclear cells from deer infected with Mycobacterium bovis or vaccinated with live or killed M. bovis bacillus Calmette–Guérin (BCG) were cultured with mycobacterial antigens. IL‐4 mRNA production was assayed using the polymerase chain reaction. Elevated levels of IL‐4 mRNA were detected in response to at least one antigen preparation in all animals infected with M. bovis as compared with none of the non‐infected control animals. After a primary immunization, elevated levels of IL‐4 mRNA were detected in only a proportion of vaccinated animals and this did not correlate with whether the vaccine was live BCG or killed BCG in oil. After boosting, all the animals vaccinated with killed BCG in oil exhibited elevated IL‐4 mRNA production whereas none of the animals vaccinated with live BCG showed elevated levels. The data suggest that IL‐4 is turned off during the immune response to live BCG, that boosting of low‐dose live BCG vaccine may be required to ‘imprint’ this signal and that this may be important in the development of protective immunity to tuberculosis. Killed BCG in adjuvant is not protective and as with experimental infection with virulent M. bovis it failed to switch off the IL‐4 response. IL‐4 may be useful as a diagnostic tool and as an in vitro marker of vaccine efficacy.

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.

Vaccination against tuberculosis: Is BCG more sinned against than sinner?

While extensive experimental studies of tuberculosis (Tb) have provided the foundation data for the discovery of cell‐mediated immunity, there remains much to be disclosed about the critical pathways of immunity involved in this infectious process and the factors necessary to produce protective immunity. Studies on the actiology and pathology of this disease have failed to elucidate the mechanisms of protective immunity. Although Tb research has been neglected for the past 30 years, the re‐emergence of Tb worldwide as a significant zoonotic disease has re‐focused research in this area. Scientific solutions for the control of Tb in man or domestic animals have not been found using empirical methods. Composite studies involving animal models of experimental infection will be necessary to critically evaluate vaccine efficacy and elucidate the basic immunological mechanisms involved in both disease and immunity. Available data which suggest that disease‐related hypersensitivity and immunity are dissociable highlight the prospect that immunity to infection may be induced without compromising the continued need for ongoing systems of immunodiagnosis to exclude disease. In populations with a high prevalence of disease it is likely that a combination of immunodiagnosis, chemotherapy and immunoprophylaxis will be required to eradicate the disease.