Heterologous prime-boost-boost immunisation of Chinese cynomolgus macaques using DNA and recombinant poxvirus vectors expressing HIV-1 virus-like particles

Background: There is renewed interest in the development of poxvirus vector-based HIV vaccines due to the protective effect observed with repeated recombinant canarypox priming with gp120 boosting in the recent Thai placebo-controlled trial. This study sought to investigate whether a heterologous prime-boost-boost vaccine regimen in Chinese cynomolgus macaques with a DNA vaccine and recombinant poxviral vectors expressing HIV virus-like particles bearing envelopes derived from the most prevalent clades circulating in sub-Saharan Africa, focused the antibody response to shared neutralising epitopes. Methods: Three Chinese cynomolgus macaques were immunised via intramuscular injections using a regimen composed of a prime with two DNA vaccines expressing clade A Env/clade B Gag followed by boosting with recombinant fowlpox virus expressing HIV-1 clade D Gag, Env and cholera toxin B subunit followed by the final boost with recombinant modified vaccinia virus Ankara expressing HIV-1 clade C Env, Gag and human complement protein C3d. We measured the macaque serum antibody responses by ELISA, enumerated T cell responses by IFN-gamma ELISpot and assessed seroneutralisation of HIV-1 using the TZM-bl beta-galactosidase assay with primary isolates of HIV-1. Results: This study shows that large and complex synthetic DNA sequences can be successfully cloned in a single step into two poxvirus vectors: MVA and FPV and the recombinant poxviruses could be grown to high titres. The vaccine candidates showed appropriate expression of recombinant proteins with the formation of authentic HIV virus-like particles seen on transmission electron microscopy. In addition the b12 epitope was shown to be held in common by the vaccine candidates using confocal immunofluorescent microscopy. The vaccine candidates were safely administered to Chinese cynomolgus macaques which elicited modest T cell responses at the end of the study but only one out of the three macaques elicited an HIV-specific antibody response. However, the antibodies did not neutralise primary isolates of HIV-1 or the V3-sensitive isolate SF162 using the TZM-bl b-galactosidase assay. Conclusions: MVA and FP9 are ideal replication-deficient viral vectors for HIV-1 vaccines due to their excellent safety profile for use in humans. This study shows this novel prime-boost-boost regimen was poorly immunogenic in Chinese cynomolgus macaques

Unsupervised CT Lung Image Segmentation of a Mycobacterium Tuberculosis Infection Model

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis that produces pulmonary damage. Radiological imaging is the preferred technique for the assessment of TB longitudinal course. Computer-assisted identification of biomarkers eases the work of the radiologist by providing a quantitative assessment of disease. Lung segmentation is the step before biomarker extraction. In this study, we present an automatic procedure that enables robust segmentation of damaged lungs that have lesions attached to the parenchyma and are affected by respiratory movement artifacts in a Mycobacterium Tuberculosis infection model. Its main steps are the extraction of the healthy lung tissue and the airway tree followed by elimination of the fuzzy boundaries. Its performance was compared with respect to a segmentation obtained using: (1) a semi-automatic tool and (2) an approach based on fuzzy connectedness. A consensus segmentation resulting from the majority voting of three experts' annotations was considered our ground truth. The proposed approach improves the overlap indicators (Dice similarity coefficient, 94\% +/- 4\%) and the surface similarity coefficients (Hausdorff distance, 8.64 mm +/- 7.36 mm) in the majority of the most difficult-to-segment slices. Results indicate that the refined lung segmentations generated could facilitate the extraction of meaningful quantitative data on disease burden.We thank Estibaliz Gomez de Mariscal, Paula Martin Gonzalez and Mario Gonzalez Arjona for helping with the manual lung annotation. The research leading to these results received funding from the Innovative Medicines Initiative (www.imi.europa.eu) Joint Undertaking under grant agreement no. 115337, whose resources comprise funding from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies' in kind contribution. This work was partially funded by projects TEC2013-48552-C2-1-R, RTC-2015-3772-1, TEC2015-73064-EXP and TEC2016-78052-R from the Spanish Ministerio de Economia, Industria y Competitividad, TOPUS S2013/MIT-3024 project from the regional government of Madrid and by the Department of Health, UK.S

Unsupervised CT lung image segmentation of a mycobacterium tuberculosis infection model

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis that produces pulmonary damage. Radiological imaging is the preferred technique for the assessment of TB longitudinal course. Computer-assisted identification of biomarkers eases the work of the radiologist by providing a quantitative assessment of disease. Lung segmentation is the step before biomarker extraction. In this study, we present an automatic procedure that enables robust segmentation of damaged lungs that have lesions attached to the parenchyma and are affected by respiratory movement artifacts in a Mycobacterium Tuberculosis infection model. Its main steps are the extraction of the healthy lung tissue and the airway tree followed by elimination of the fuzzy boundaries. Its performance was compared with respect to a segmentation obtained using: (1) a semi-automatic tool and (2) an approach based on fuzzy connectedness. A consensus segmentation resulting from the majority voting of three experts' annotations was considered our ground truth. The proposed approach improves the overlap indicators (Dice similarity coefficient, 94% ± 4%) and the surface similarity coefficients (Hausdorff distance, 8.64 mm ± 7.36 mm) in the majority of the most difficult-to-segment slices. Results indicate that the refined lung segmentations generated could facilitate the extraction of meaningful quantitative data on disease burden.The research leading to these results received funding from the Innovative Medicines Initiative (www.imi.europa.eu) Joint Undertaking under grant agreement no. 115337, whose resources comprise funding from the European Union’s Seventh Framework Programme (FP7/2007–2013) and EFPIA companies’ in kind contribution. This work was partially funded by projects TEC2013-48552-C2-1-R, RTC-2015-3772-1, TEC2015-73064-EXP and TEC2016-78052-R from the Spanish Ministerio de Economía, Industria y Competitividad, TOPUS S2013/MIT-3024 project from the regional government of Madrid and by the Department of Health, UK

Surveillance of Daughter Micronodule Formation Is a Key Factor for Vaccine Evaluation Using Experimental Infection Models of Tuberculosis in Macaques

Tuberculosis (TB) is still a major worldwide health problem and models using non-human primates (NHP) provide the most relevant approach for vaccine testing. In this study, we analysed CT images collected from cynomolgus and rhesus macaques following exposure to ultra-low dose Mycobacterium tuberculosis (Mtb) aerosols, and monitored them for 16 weeks to evaluate the impact of prior intradermal or inhaled BCG vaccination on the progression of lung disease. All lesions found (2553) were classified according to their size and we subclassified small micronodules (<4.4 mm) as 'isolated', or as 'daughter', when they were in contact with consolidation (described as lesions ≥ 4.5 mm). Our data link the higher capacity to contain Mtb infection in cynomolgus with the reduced incidence of daughter micronodules, thus avoiding the development of consolidated lesions and their consequent enlargement and evolution to cavitation. In the case of rhesus, intradermal vaccination has a higher capacity to reduce the formation of daughter micronodules. This study supports the 'Bubble Model' defined with the C3HBe/FeJ mice and proposes a new method to evaluate outcomes in experimental models of TB in NHP based on CT images, which would fit a future machine learning approach to evaluate new vaccines

Development of a non-human primate BCG infection model for the evaluation of candidate tuberculosis vaccines.

The lack of validated immunological correlates of protection makes tuberculosis vaccine development difficult and expensive. Using intradermal bacille Calmette-Guréin (BCG) as a surrogate for aerosol Mycobacterium tuberculosis (M.tb) in a controlled human infection model could facilitate vaccine development, but such a model requires preclinical validation. Non-human primates (NHPs) may provide the best model in which to do this. Cynomolgus and rhesus macaques were infected with BCG by intradermal injection. BCG was quantified from a skin biopsy of the infection site and from draining axillary lymph nodes, by culture on solid agar and quantitative polymerase chain reaction. BCG was detected up to 28 days post-infection, with higher amounts of BCG detected in lymph nodes after high dose compared to standard dose infection. Quantifying BCG from lymph nodes of cynomolgus macaques 14 days post-high dose infection showed a significant reduction in the amount of BCG detected in the BCG-vaccinated compared to BCG-naïve animals. Demonstrating a detectable vaccine effect in the lymph nodes of cynomolgus macaques, which is similar in magnitude to that seen in an aerosol M.tb infection model, provides support for proof-of-concept of an intradermal BCG infection model and evidence to support the further evaluation of a human BCG infection model

The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays.

The current vaccine against tuberculosis, live attenuated Mycobacterium bovis BCG, has variable efficacy, but development of an effective alternative is severely hampered by the lack of an immune correlate of protection. There has been a recent resurgence of interest in functional in vitro mycobacterial growth inhibition assays (MGIAs), which provide a measure of a range of different immune mechanisms and their interactions. We identified a positive correlation between mean corpuscular haemoglobin and in vitro growth of BCG in whole blood from healthy UK human volunteers. Mycobacterial growth in peripheral blood mononuclear cells (PBMC) from both humans and macaques was increased following the experimental addition of haemoglobin (Hb) or ferric iron, and reduced following addition of the iron chelator deferoxamine (DFO). Expression of Hb genes correlated positively with mycobacterial growth in whole blood from UK/Asian adults and, to a lesser extent, in PBMC from South African infants. Taken together our data indicate an association between Hb/iron levels and BCG growth in vitro, which may in part explain differences in findings between whole blood and PBMC MGIAs and should be considered when using such assays

The Cross-Species Mycobacterial Growth Inhibition Assay (MGIA) Project, 2010-2014.

The development of a functional biomarker assay in the tuberculosis (TB) field would be widely recognized as a major advance in efforts to develop and to test novel TB vaccine candidates efficiently. We present preliminary studies using mycobacterial growth inhibition assays (MGIAs) to detect Mycobacterium bovis BCG vaccine responses across species, and we extend this work to determine whether a standardized MGIA can be applied in characterizing new TB vaccines. The comparative MGIA studies reviewed here aimed to evaluate robustness, reproducibility, and ability to reflect in vivo responses. In doing so, they have laid the foundation for the development of a MGIA that can be standardized and potentially qualified. A major challenge ahead lies in better understanding the relationships between in vivo protection, in vitro growth inhibition, and the immune mechanisms involved. The final outcome would be a MGIA that could be used with confidence in TB vaccine trials. We summarize data arising from this project, present a strategy to meet the goals of developing a functional assay for TB vaccine testing, and describe some of the challenges encountered in performing and transferring such assays