Oral vaccination with Liporale™ BCG induces effector and central memory Ag85B-specific CD4⁺ T cells in the spleen.

<p>Lymphocytes from the spleens of naïve, Liporale™-BCG vaccinated (BCG oral) or subcutaneously vaccinated (BCG s.c.) mice were stained with an Ag85B/MHCII tetramer and enriched for tetramer positive cells by magnetic bead isolation. (A) Representative flow cytometry density plots showing CD62L and CD44 expression on total CD4⁺ T cells from spleens of naïve mice, or Ag85b-specific CD4⁺ T cells from the spleens of BCG vaccinated mice. (B) Bar graphs showing the proportion of naïve (CD62L^hi, CD44^lo), T_EFF/T_EM (CD62L^lo, CD44^hi) or T_CM (CD62L^hi, CD44^hi) CD4⁺ T lymphocytes of total CD4⁺ T cells from naïve mice or Ag85B-specific CD4⁺ T cells from the spleens of BCG vaccinated mice at 4, 8 and 30 weeks post vaccination. Results are displayed as mean + SEM of n = 5 for each group: *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney test). Eight and 30 weeks results are representative of 2 independent experiments.</p

Oral vaccination with Liporale™-BCG induces CD4⁺ T cell cytokine production in the lung.

<p>Lymphocytes from the lungs of naïve, Liporale™-BCG vaccinated (BCG oral) or subcutaneous vaccinated (BCG s.c.) mice were stimulated for 6 hours <i>in vitro</i> in the presence of Brefeldin A and monensin then analyzed by flow cytometry. (A) Representative plots show CD4⁺ T cells from the lungs of naïve or BCG vaccinated mice expressing IFNγ, TNFα or IL-2. (B) Bar graphs show the percentage of CD4⁺ T cells from the lungs of naïve or BCG vaccinated mice expressing cytokines at 4, 8 or 30 weeks post immunization. Results are displayed as mean + SEM of n = 5 for each group, significance expressed relative to naïve: *p<0.05, **p<0.01, ***p<0.001 (one way ANOVA with Tukey post test). Eight and 30 weeks results are representative of 2 independent experiments.</p

Oral vaccination with Liporale™-BCG increases the number of Ag85B-specific CD4⁺ T cells in the spleen.

<p>Lymphocytes from the spleens of naïve, Liporale™-BCG vaccinated (BCG oral) or subcutaneously vaccinated (BCG s.c.) mice were stained with an Ag85B/MHCII tetramer and enriched for tetramer positive cells by magnetic bead isolation. (A) Representative flow cytometry plots show Ag85B-specific CD4⁺ T cells in the spleens of naïve or BCG vaccinated mice at 4, 8 and 30 weeks post immunization. (B) Bar graphs show the number of Ag85B-specific CD4⁺ T cells in the spleens of naïve or BCG vaccinated mice at 4, 8 and 30 weeks post vaccination. Results are displayed as mean +SEM of n = 5 for each group, significance expressed relative to naïve: *p<0.05, **p<0.01, ***p<0.001 (one way ANOVA with Tukey post test). The 8 and 30 weeks results are representative of 2 independent experiments.</p

Oral vaccination with Liporale™-BCG induces long-lived CD4⁺ effector T cells in the lung.

<p>Lymphocytes from the lungs of naïve, Liporale™-BCG vaccinated (BCG oral) or subcutaneous vaccinated (BCG s.c.) mice were analyzed by flow cytometry. (A) Representative flow cytometry plots show the gating strategy used to identify CD4⁺ T cells. (B) Bar graphs show the proportion of naïve (CD62L^hi, CD44^lo), T_EFF/T_EM (CD62L^lo, CD44^hi) or T_CM (CD62L^hi, CD44^hi) CD4⁺ T lymphocytes of total CD4⁺ T cells from the lungs of naïve or BCG vaccinated mice at 4, 8 and 30 weeks post vaccination. Results are displayed as mean + SEM of n = 5 for each group, significance expressed relative to naïve: *p<0.05, **p<0.01, ***p<0.001 (one way ANOVA with Tukey post test). Eight and 30 weeks results are representative of 2 independent experiments.</p

Field Trial of an Aerially-Distributed Tuberculosis Vaccine in a Low-Density Wildlife Population of Brushtail Possums (<i>Trichosurus vulpecula</i>)

<div><p>Oral-delivery <i>Mycobacterium bovis</i> bacillus Calmette-Guérin (BCG) vaccine in a lipid matrix has been shown to confer protection against <i>M</i>. <i>bovis</i> infection and reduce the severity of tuberculosis (TB) when fed to brushtail possums (<i>Trichosurus vulpecula</i>), the major wildlife vector of bovine TB in New Zealand. Here we demonstrate the feasibility of aerial delivery of this live vaccine in bait form to an <i>M</i>. <i>bovis</i>-infected wild possum population, and subsequently assess vaccine uptake and field efficacy. Pre-trial studies indicated a resident possum population at very low density (<0.6 possums/ha) at the field site, with a 5.1% prevalence of macroscopic TB lesions. Pilot studies indicated that flavoured lipid matrix baits in weather-proof sachets could be successfully sown aerially via helicopter and were palatable to, and likely to be consumed by, a majority of wild possums under free-choice conditions. Subsequently, sachet-held lipid baits containing live BCG vaccine were sown at 3 baits/ha over a 1360 ha area, equating to >5 baits available per possum. Blood sampling conducted two months later provided some evidence of vaccine uptake. A necropsy survey conducted one year later identified a lower prevalence of culture-confirmed <i>M</i>. <i>bovis</i> infection and/or gross TB lesions among adult possums in vaccinated areas (1.1% prevalence; 95% CI, 0–3.3%, n = 92) than in unvaccinated areas (5.6%; 0.7–10.5%, n = 89); P = 0.098. Although not statistically different, the 81% efficacy in protecting possums against natural infection calculated from these data is within the range of previous estimates of vaccine efficacy in trials where BCG vaccine was delivered manually. We conclude that, with further straightforward refinement to improve free-choice uptake, aerial delivery of oral BCG vaccine is likely to be effective in controlling TB in wild possums. We briefly discuss contexts in which this could potentially become an important complementary tool in achieving national eradication of TB from New Zealand wildlife.</p></div

Location of the Muzzle Station/Clarence Reserve study sites within the northern South Island high country region of New Zealand.

<p>Four main study blocks are depicted in the regional-scale map (left) with vaccine blocks outlined in blue and non-treatment blocks in red. The yellow lines depict the inner 700 ha areas of the two vaccine blocks over which vaccine was deployed in September 2011. The green rectangle depicts the 50 ha block on which the pilot aerial baiting and bait contact studies were conducted.</p

Overview of the flight characteristics during aerial sowing of flavoured lipid baits in sachets during the pilot trial and during the main vaccine trial (the latter containing live <i>M</i>. <i>bovis</i> BCG).

<p>Overview of the flight characteristics during aerial sowing of flavoured lipid baits in sachets during the pilot trial and during the main vaccine trial (the latter containing live <i>M</i>. <i>bovis</i> BCG).</p

Summary of results from fate-of-bait pilot trials, to determine the contact and removal rates of flavoured lipid baits from habitat on Muzzle Station/Clarence Reserve over 3–7 day periods and to identify the likely wildlife species involved.

<p>Summary of results from fate-of-bait pilot trials, to determine the contact and removal rates of flavoured lipid baits from habitat on Muzzle Station/Clarence Reserve over 3–7 day periods and to identify the likely wildlife species involved.</p

Details of possums and pigs necropsied from wildlife TB surveys conducted on Muzzle Station/Clarence Reserve between spring 2009 to autumn 2011 (final surveillance was 6 months prior to vaccine deployment).

<p>Details of possums and pigs necropsied from wildlife TB surveys conducted on Muzzle Station/Clarence Reserve between spring 2009 to autumn 2011 (final surveillance was 6 months prior to vaccine deployment).</p

Summary of LPA responses to PPD-B—stimulation among possums captured from a non-vaccinated block and two vaccine blocks, two months following aerial vaccine deployment.

<p>Diamond symbols indicate LPA responses to PPD-B in terms of a stimulation index. Left-fig, data from 61 possums, highlighting 2 animals that were excluded from subsequent analyses on the basis of likely pre-existing <i>M</i>. <i>bovis</i> infection (red symbols). Right-fig, data from 59 possums (excluding the 2 likely infected cases) re-plotted on a reduced scale graph. Yellow symbols represent responses of individual animals that were greater than the 99 percentile distribution of the control values (i.e. > control mean + 3 standard deviations, indicated by the red dashed line).</p