Matthew R. Lambeth

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.

LIPID-FORMULATED BCG AS AN ORAL-BAIT VACCINE FOR TUBERCULOSIS: VACCINE STABILITY, EFFICACY, AND PALATABILITY TO BRUSHTAIL POSSUMS (TRICHOSURUS VULPECULA) IN NEW ZEALAND

Bovine tuberculosis (Tb), due to infection with virulent Mycobacterium bovis, represents a threat to New Zealand agriculture due to vectorial transmission from wildlife reservoir species, principally the introduced Australian brushtail possum (Trichosurus vulpecula). An oral-delivery wildlife vaccine has been developed to immunize possums against Tb, based on formulation of the human Tb vaccine (M. bovis BCG) in edible lipid matrices. Here BCG bacilli were shown to be stable in lipid matrix formulation for over 8 mo in freezer storage, for 7 wk under room temperature conditions, and for 3–5 wk under field conditions in a forest/pasture margin habitat (when maintained in weatherproof bait-delivery sachets). Samples of the lipid matrix were flavored and offered to captive possums in a bait-preference study: a combination of 10% chocolate powder with anise oil was identified as the most effective attractant/palatability combination. In a replicated field study, 85–100% of wild possums were shown to access chocolate-flavored lipid pellets, when baits were applied to areas holding approximately 600–800 possums/km2. Finally, in a controlled vaccination/challenge study, chocolate-flavored lipid vaccine samples containing 108 BCG bacilli were fed to captive possums, which were subsequently challenged via aerosol exposure to virulent M. bovis: vaccine immunogenicity was confirmed, and protection was identified by significantly reduced postchallenge weight loss in vaccinated animals compared to nonvaccinated controls. These studies indicate that, appropriately flavored, lipid delivery matrices may form effective bait vaccines for the control of Tb in wildlife.

Lymphatic tracing and T cell responses following oral vaccination with live Mycobacterium bovis (BCG)

Oral vaccination of mice with lipid‐encapsulated Mycobacterium bovis bacille Calmette‐Guérin (BCG) expands a subset of interferon‐gamma (IFN‐γ)‐secreting T cells and mediates protection against aerosol mycobacterial challenge. We have traced the movement of the live vaccine through the regional lymphatics of mice and monitored the resultant immune response. Six hours after oral vaccination BCG was detected in low numbers systemically and in draining lymphatic tissue. However, after 48 h, BCG was predominantly associated with alimentary tract lymphatic tissues, such as the cervical and mesenteric lymph nodes and Peyers patches. Lymphocytes that produced IFN‐γ in response to PPD‐B or BCG‐pulsed dendritic cells predominated in the spleen and were almost exclusively CD4+, CD44+ and CD62L–, thus resembling an effector memory T cell population. Despite the fact that an oral route was used for immunization, splenic IFN‐γ‐secreting T cells in vaccinated mice did not express the mucosal homing antigens α4β7 integrin or αIEL (CD103). However, a proportion of BCG‐specific CD4+ T cells expressed the CD29 integrin (β1) chain, potentially involved in lung homing function. Thus, oral priming with M. bovis BCG appears to induce a subset of spleen‐resident CD4+ T cells with the potential to provide protective immunity in the lung.

Sustained protection against tuberculosis conferred to a wildlife host by single dose oral vaccination

BACKGROUND Vaccination of wildlife against bovine tuberculosis (TB) is being considered by several countries to reduce the transmission of Mycobacterium bovis infection to livestock. In New Zealand, where introduced brushtail possums (Trichosurus vulpecula) are the major wildlife hosts, we have previously shown that repeat applications of a lipid-encapsulated oral bacille Calmette-Guerin (BCG) vaccine reduce the incidence of naturally acquired TB in wild possums. Here we extend this conceptual demonstration to an operational level, assessing long-term protection against TB conferred to free-living possums by a single oral immunisation. METHODS Possums in a non-TB area were randomly allocated to receive lipid-formulated BCG vaccine or remained unvaccinated. After initial trials to assess vaccine immunogenicity and establishment of protection within the first year post-vaccination, 13 individuals of each treatment group were relocated to a biosecurity facility and challenged (at 28 months post-vaccination) by subcutaneous injection of virulent M. bovis. RESULTS Vaccine immunogenicity and short-term protection were confirmed at 2 months and 12 months post-vaccination, respectively. In the long-term assessment, vaccinated possums had significantly reduced bacterial counts in peripheral lymph nodes compared to controls, with 0.6-2.3 log(10)-fold reductions in M. bovis burdens. DISCUSSION The magnitude of protective response by possums to experimental challenge at 28 months post-vaccination is known to equate to a high degree of protection against natural infection in this species. With techniques for oral bait delivery well advanced, the longevity of protection demonstrated here shows that an operable wildlife vaccine against TB is feasible.

Immunogenicity of orally-delivered lipid-formulated BCG vaccines and protection against Mycobacterium tuberculosis infection

Lipid formulations containing BCG strains Danish 1331 or Moreau (Rio de Janeiro) were trialled as oral vaccines in rodent models. In mice, oral-delivery of either strain resulted in BCG colonisation of the alimentary tract lymphatics and induction of gamma-interferon responses. In guinea pigs, both strains provided pulmonary protection against Mycobacterium tuberculosis aerosol challenge, as shown by significantly reduced bacterial loads and lung:body weight ratios. Lipid-formulated BCG provided superior protection against M. tuberculosis over unformulated orally-delivered BCG (Moreau), and equivalent protection to sub-cutaneous BCG (Danish) immunisation. Oral-delivery of lipid-formulated BCG may offer a practical alternative to parenteral-route BCG vaccination.

Murine cytokine responses following multiple oral immunizations using lipid-formulated mycobacterial antigens.

Oral vaccination of mice with live Mycobacterium bovis BCG in lipid‐formulation induces a γ‐interferon response that can be measured systemically, and confers protection against aerosolized mycobacterial challenge. Here, we have investigated cytokine responses following the vaccination, drawing comparisons between mice that received single or multiple oral immunizations and between mice receiving formulations containing live BCG or non‐replicating mycobacterial antigens. Single oral immunization with lipid‐formulated live BCG invoked secreted and cellular IFN‐γ responses in mice 8 weeks post‐vaccination, the magnitudes of which were significantly elevated in mice receiving multiple immunizations over the 8‐week period. Single oral immunization with live BCG also invoked an interleukin‐2 response (but not TNF‐α or IL‐4), although the magnitude was not elevated by multiple immunizations. Multiple immunizations with lipid‐formulated soluble or particulate non‐replicating mycobacterial antigens failed to invoke cytokine responses, except for a low‐level IFN‐γ response in mice multiple immunized with lipid‐formulated heat‐killed BCG. These results are discussed in contrast to the known patterns of cytokine induction following parenteral‐route immunization with live or non‐replicating mycobacterial antigens and with practical reference to the development of oral‐delivery vaccines against tuberculosis.

An oral Mycobacterium bovis BCG vaccine for wildlife produced in the absence of animal-derived reagents.

ABSTRACT Cultures of Mycobacterium bovis BCG, comprising predominantly single-cell bacilli, were prepared in broth without animal-derived reagents. When formulated into a vegetable-derived lipid matrix, the vaccine was stable in vitro and was immunogenic in vivo upon feeding it to mice. This formulation could be useful for oral vaccination of wildlife against tuberculosis, where concern over transmissible prions may preclude the field use of vaccines containing animal products.

Murine immune responses to oral BCG immunization in the presence or absence of prior BCG sensitization.

Oral delivery of live Mycobacterium bovis BCG in a lipid matrix invokes cell‐mediated immune (CMI) responses in mice and consequent protection against pulmonary challenge with virulent mycobacteria. To investigate the influence of prior BCG sensitization on oral vaccine efficacy, we assessed CMI responses and BCG colonization of the alimentary tract lymphatics 5 months after oral vaccination, in both previously naive mice and in mice that had been sensitized to BCG by injection 6 months previously. CMI responses did not differ significantly between mice that received subcutaneous BCG followed by oral BCG and those that received either injected or oral BCG alone. In vivo BCG colonization was predominant in the mesenteric lymph nodes after oral vaccination; this colonizing ability was not influenced by prior BCG sensitization. From this murine model study, we conclude that although prior parenteral‐route BCG sensitization does not detrimentally affect BCG colonization after oral vaccination, there is no significant immune‐boosting effect of the oral vaccine either.