13 research outputs found
Survival rates and mycobacterial burden in the lung and spleen.
<p>Animals were infected with 25 colony-forming units of <i>M</i>.<i>tb</i> Erdman strain. Group coloring in the survival graph is maintained throughout the article for comparability of data. One animal in BCG/Ad(im) group died accidently and is excluded from data analysis (<b>A</b>). Overall log-rank test for trend yielded p = 0.0247, but no statistical differences between groups. Colony-forming units (CFU) of mycobacteria was measured per gram of randomly selected tissue from different lung lobs or spleen at necropsy. CFU per right lung (<b>B</b>), left lung (<b>C</b>) and spleen (<b>D</b>) were presented in logarithmic numbers. P-values above lines indicate comparison between groups. Mann-Whitney rank test is used to compare differences relative to naïve group.</p
AdHu5Ag85A Respiratory Mucosal Boost Immunization Enhances Protection against Pulmonary Tuberculosis in BCG-Primed Non-Human Primates
<div><p>Persisting high global tuberculosis (TB) morbidity and mortality and poor efficacy of BCG vaccine emphasizes an urgent need for developing effective novel boost vaccination strategies following parenteral BCG priming in humans. Most of the current lead TB vaccine candidates in the global pipeline were developed for parenteral route of immunization. Compelling evidence indicates respiratory mucosal delivery of vaccine to be the most effective way to induce robust local mucosal protective immunity against pulmonary TB. However, despite ample supporting evidence from various animal models, there has been a lack of evidence supporting the safety and protective efficacy of respiratory mucosal TB vaccination in non-human primates (NHP) and humans. By using a rhesus macaque TB model we have evaluated the safety and protective efficacy of a recombinant human serotype 5 adenovirus-based TB vaccine (AdHu5Ag85A) delivered via the respiratory mucosal route. We show that mucosal AdHu5Ag85A boost immunization was safe and well tolerated in parenteral BCG-primed rhesus macaques. A single AdHu5Ag85A mucosal boost immunization in BCG-primed rhesus macaques enhanced the antigen–specific T cell responses. Boost immunization significantly improved the survival and bacterial control following <i>M</i>.<i>tb</i> challenge. Furthermore, TB-related lung pathology and clinical outcomes were lessened in BCG-primed, mucosally boosted animals compared to control animals. Thus, for the first time we show that a single respiratory mucosal boost immunization with a novel TB vaccine enhances protection against pulmonary TB in parenteral BCG-primed NHP. Our study provides the evidence for the protective potential of AdHu5Ag85A as a respiratory mucosal boost TB vaccine for human application.</p></div
Experimental schema and plan.
<p>Depicting the timelines of vaccination (BCG priming at wk0 and AdHu5Ag85A boosting at wk14), <i>M</i>.<i>tb</i> infectious challenge (wk20) and fixed endpoint/autopsy (wk38). Scheduled times for clinical signs, blood work for acute phase protein measurement, chest X-ray and immune monitoring are indicated. Timelines during infection phase are referred relative to the 18 weeks post-challenge timepoint (indicated within parentheses).</p
Antigen-specific IFN-γ+ T cell responses post-BCG vaccination or post-BCG prime-Ad boost vaccination.
<p>Antigen-specific IFN-γ+ T cell responses in non-boosted BCG-primed animals are measured using IFN- γ ELISOPT assay at 0wk, 13wk and 17wk post BCG vaccination by stimulating fresh peripheral blood mononuclear cells (PBMC) with PPD for 24h (<b>A</b>). Scatter dotplot depicting spot forming cells/million PBMC representing the mean responses±standard error. P-values are relative to the time point before BCG vaccination (0wk). (<b>B/C/D</b>) depicts antigen-specific IFN-γ responses for each group measured using IFN-γ ELISOPT assay before vaccination (wk0) and after BCG priming (BCG) or after BCG priming and AdHu5Ag85A boosting BCG/Ad(it), BCG/Ad(aerosol), (BCG/Ad(im). Fresh PBMCs are stimulated with PPD (<b>B</b>) or rAg85A (<b>C</b>) or single pool (<b>D</b>) and scatter dotplot depicting mean of spot forming cells/million PBMC ± standard error. P-values above lines indicate comparison between groups. Wilcoxon signed rank test was used to compare measurements at different time points of same animal.</p
Characteristics of treatment groups.
<p>n.a.*: does not apply.</p><p>i.d.#: intradermal.</p><p>i.m.&: intramuscular.</p><p>i.t.: intratracheal.</p><p>CFU$:colony forming unit.</p><p>PFU@:plaque forming unit.</p><p>**one animal from BCG/Ad(im) group was removed from analysis performed post- infection due to procedure related- accidental death.</p><p>Characteristics of treatment groups.</p
Longitudinal kinetics of clinical outcomes during the course of infection.
<p>Clinical outcomes were monitored at scheduled times as indicated in the experiment plan and as described in Methods. Line graphs depict (<b>A</b>) percent of weight change relative to weight at the start of the experiment, (<b>B</b>) appetite, (<b>C</b>) change in erythrocyte sedimentation rate (ESR), and (<b>D</b>) C-reactive protein (CRP). Lines represent individual animals in each group and color assigned to individual animal in each group is maintained to display each clinical sign evaluated for comparability. Wilcoxon ranked test is used to compare clinical signs post infection relative to values before infection.</p
Clinical outcomes post-AdHu5Ag85A boost vaccination.
<p>Note: compared to naïve and BCG alone groups.</p><p>*Taken 6 weeks post AdHu5Ag85A boost immunization.</p><p>Clinical outcomes post-AdHu5Ag85A boost vaccination.</p
Evaluation of gross pathology and histopathology in M.tb-infected lungs.
<p>Individual dots represent scores for animals in each group with mean ± standard error for gross pathology (<b>A</b>). (<b>B</b>) Representative histomicrographs (x40) of lungs of animals in each group. Arrow points to granuloma with the central caseous necrosis in the lung of non-vaccinated animal (non-v), non-necrotic granuloma in the lung of BCG vaccinated animal (BCG), scattered small size granulomas lacking necrosis in the of lung of AdHu5Ag85A boosted animals (BCG/Ad(it), BCG(aerosol) and BCG(im)). (<b>C</b>) Individual dots represent scores for animals in each group with mean± standard error for histopathology. Mann-Whitney rank test is used to compare differences relative to naïve group.</p
Correlation of lung histopathology with microbiological and clinical outcomes.
<p>Bacterial burden in the lung, chest radiography (CXR) and C-reactive protein (CPR) levels in blood correlate significantly with lung histopathology. Parameters are plotted (maximum values for each of the parameters) per individual animal (color coordination consistent throughout the article) against lung histology score for right lung (<b>A</b>), left lung bacterial burden (<b>B</b>), CXR (<b>C</b>), and CRP (<b>D</b>). Spearman’s rho (Rs) correlation factors and p-values are indicated.</p
Survival rates and mycobacterial burden in the lung and spleen.
<p>Animals were infected with 25 colony-forming units of <i>M</i>.<i>tb</i> Erdman strain. Group coloring in the survival graph is maintained throughout the article for comparability of data. One animal in BCG/Ad(im) group died accidently and is excluded from data analysis (<b>A</b>). Overall log-rank test for trend yielded p = 0.0247, but no statistical differences between groups. Colony-forming units (CFU) of mycobacteria was measured per gram of randomly selected tissue from different lung lobs or spleen at necropsy. CFU per right lung (<b>B</b>), left lung (<b>C</b>) and spleen (<b>D</b>) were presented in logarithmic numbers. P-values above lines indicate comparison between groups. Mann-Whitney rank test is used to compare differences relative to naïve group.</p