Immunogenicity and protective efficacy of mycobacterial DNA vaccines incorporating plasmid-encoded cytokines against Mycobacterium bovis.

DNA-based vaccines, alone or in combination with other sub-unit vaccination regimes, represent an alternative to live mycobacterial vaccines for protective immunization against tuberculosis. Here, we have used a murine immunization or Mycobacterium bovis aerosol challenge model to assess the immunogenicity and protective efficacy of mycobacterial DNA vaccines. Mice that received immunization with DNA constructs encoding M. bovis antigen 85A (Ag85-A) and arget(ESAT-6) produced measurable interferon-gamma (IFN-gamma) responses to CD4(+) T-cell epitope-peptide recall antigens in vitro. The magnitude of these responses was enhanced by co-delivery of a construct encoding murine cytokines (macrophage inhibitory protein (MIP)-1 alpha or interleukin(IL)-7), although they did not the match responses observed in mice that received Bacille Calmette-Guerin(BCG) immunisation. In contrast, DNA priming followed by boosting with modified vaccinia Ankara (MVA) vaccine (expressing M. tuberculosis Ag85-A) invoked higher IFN-gamma levels, with the most immunogenic regime of Ag85 or ESAT or IL-7 prime followed by MVA boost being of commensurate immunogenicity to BCG. Despite this, neither DNA alone nor DNA-prime or MVA boost regimes conferred measurable protection against aerosol challenge with virulent M. bovis. These data highlight both the promise and the shortcomings of new generation subunit tuberculosis vaccines, with particular emphasis on their potential as vaccines against M. bovis.

Strains of Mycobacterium avium differentially activate human dendritic cells.

Animal models indicate that exposure to environmental strains of mycobacteria can modulate immune responses and influence the effectiveness of live mycobacterial vaccines. Here, we describe that between the two recently reported Mycobacterium avium isolates, strain WAg 206 (but not strain WAg 207) interferes with human monocyte-derived dendritic cell (MDDC) activation. WAg 206, unlike WAg 207, did not elicit inflammatory cytokine production (TNFalpha, IL-1beta, IL-12) or costimulatory molecule expression (HLA-DR, CD83, CD80, CD86) by human MDDCs in vitro. These data highlight the potential for environmental mycobacteria to modulate immune responses in humans, and suggest a mechanism by which earlier exposure to such microbes may compromise the efficacy of live mycobacterial vaccines, as has been observed in some human BCG vaccine trials.

Environmental strains of Mycobacterium avium interfere with immune responses associated with Mycobacterium bovis BCG vaccination.

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-1beta [IL-1beta], 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-gamma 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.

Potential prophylactic value of bovine colostrum in necrotizing enterocolitis in neonates: an in vitro study on bacterial attachment, antibody levels and cytokine production.

Necrotizing enterocolitis (NEC) is an important disease of low birth-weight neonates. The immaturity of the gut mucosa may result in close contact between the host epithelium and microorganisms which are normally confined to the gut lumen. Damage of the mucosa due to endotoxin, cytokine production or other factors is believed to then occur. The aim of this study was to determine whether spray-dried bovine colostrum demonstrated potential in vitro as a prophylactic for NEC. Antiadherence was measured using a tissue culture assay and antibody levels against Enterobacteriaceae were determined by ELISA. The effect of bovine colostrum on the production of cytokines implicated in NEC was determined by a multiplex bead assay. Enterobacter cloacae, Klebsiella oxytoca, Escherichia coli, Serratia marcescens and Klebsiella pneumoniae ssp. pneumoniae were common in both NEC positive and NEC negative infants and IgA and IgG1 antibodies to these species were present in the bovine colostrum. Pretreatment with bovine colostrum produced a significant decrease (P<0.001) in attachment of bacteria to HT-29 cells. Bovine colostrum significantly increased the production of IL-8 in HT-29 cells and IL-8, IL-6 and TNF-alpha in THP-1 cells (P<0.001). The potential of bovine colostrum to increase the production of inflammatory mediators could limit its usefulness.

The use of Th1 cytokines, IL-12 and IL-23, to modulate the immune response raised to a DNA vaccine delivered by gene gun.

Unlike intramuscular injection, gene gun delivery of DNA drives a strong type 2 response. In an effort to counter this, we have genetically fused the type 1 cytokines, IL-12 and IL-23, to the hemagglutinin (HA) gene from influenza APR/8/34, and delivered these DNA constructs to Balb/c mice. Gene gun delivery of the HA gene was able to induce antibody production by all vaccinated mice. Linking of IL-12 caused almost complete suppression of immune responses whereas mice vaccinated with IL-23HA showed long-lived IgG1 antibody levels. Splenocytes from IL-23HA vaccinated mice also tended to produce more IL-5 and IFNgamma after restimulation in vitro than splenocytes from HA vaccinated mice. While codelivery of IL-23 did not change the type of immune response it may increase its longevity following vaccination.

Murine antigen-presenting cells are multifunctional in vitro biosensors for detecting the immunoactive potential of bovine milk products.

Antigen-presenting cells (APCs) are multifunctional components of the immune defense system. In this study, murine APCs were used as biosensors to detect immunologically active components of bovine milk and colostrum. By measuring changes in cell surface protein markers [major histocompatibility complex II, cluster designation (CD)40, CD86] and cytokines (tumor necrosis factor-alpha and interleukin-10) associated with APC activation, we identified a number of compounds that are immunoactive. The mouse macrophage cell line MH-S offered a simple and robust target for identification of immunoactives. The assay was shown to be adaptable for measuring immunoenhancing or immunosuppressive substances. Large-scale screening of milk extracts using this bioassay has the potential to identify substances that could be developed into nutraceuticals or pharmaceutical-grade immunotherapeutics.

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-gamma-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 Peyer's patches (8%), but not from homogenates of the lungs or spleen. Strong Mycobacterium-specific IFN-gamma 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.

A prolonged immune response to antigen delivered in poly (epsilon-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 epsilon-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 epsilon-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 epsilon-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.

IL-2-secreting recombinant bacillus Calmette Guerin can overcome a Type 2 immune response and corticosteroid-induced immunosuppression to elicit a Type 1 immune response.

The efficacy of bacillus Calmette Guerin (BCG) as a vaccine against tuberculosis is adversely affected by both genetic and environmental factors on the immune system. In this study we have demonstrated that a recombinant BCG (rBCG) secreting biologically active IL-2 has the ability to induce a T(h)1 profile in both immunocompromised and in IL-4 transgenic (Tg) mice. Dexamethasone (DXM) was administered orally to mice prior to vaccination with either rBCG or normal BCG (nBCG). Six weeks post-vaccination with rBCG, splenocytes from DXM-treated mice exhibited a strong antigen-specific proliferative response, while also secreting large amounts of IFN-gamma and low levels of IgG1. The opposite profile occurred when DXM-treated mice were vaccinated with nBCG. Splenocytes from these mice showed no significant proliferation and produced a cytokine profile associated with a T(h)2 immune response, in addition to exhibiting high levels of serum IgG1. In the IL-4 Tg model, mice vaccinated with rBCG again produced a strong T(h)1 immune response, exhibiting a high antigen-specific IFN-gamma:IL-4 ratio and a concomitantly high IgG2a:IgG1 ratio. IL-4 Tg mice vaccinated with nBCG produced the opposite profile. These findings suggest that BCG can be made more robust by incorporating immunopotentiating cytokines into the vaccine.

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-gamma 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-gamma production in splenic lymphocytes than nBCG. This impaired induction of IFN-gamma 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.