Innovation Delivered

One Size Fits All? Not in In Vivo Modeling of Tuberculosis Chemotherapeutics

 Hee-Jeong Yang¹, Decheng Wang^{2 , 3} , Xin Wen^{2 , 3} ,Danielle M. Weiner^{1 , 4} and Laura E. Via^{1 , 4 , 5 , *}

¹ Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research (DIR), National Institute of Allergy and Infectious Disease (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States,

² Medical College, China Three Gorges University, Yichang, China,

³ Institute of Infection and Inflammation, China Three Gorges University, Yichang, China,

⁴ Tuberculosis Imaging Program, DIR, NIAID, NIH, Bethesda, MD, United States,

⁵ Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa

 doi: 10.3389/fcimb.2021.613149

Abstract

Tuberculosis (TB) remains a global health problem despite almost universal efforts to provide patients with highly effective chemotherapy, in part, because many infected individuals are not diagnosed and treated, others do not complete treatment, and a small proportion harbor Mycobacterium tuberculosis (Mtb) strains that have become resistant to drugs in the standard regimen. Development and approval of new drugs for TB have accelerated in the last 10 years, but more drugs are needed due to both Mtb’s development of resistance and the desire to shorten therapy to 4 months or less. The drug development process needs predictive animal models that recapitulate the complex pathology and bacterial burden distribution of human disease. The human host response to pulmonary infection with Mtb is granulomatous inflammation usually resulting in contained lesions and limited bacterial replication. In those who develop progressive or active disease, regions of necrosis and cavitation can develop leading to lasting lung damage and possible death. This review describes the major vertebrate animal models used in evaluating compound activity against Mtb and the disease presentation that develops. Each of the models, including the zebrafish, various mice, guinea pigs, rabbits, and non-human primates provides data on number of Mtb bacteria and pathology resolution. The models where individual lesions can be dissected from the tissue or sampled can also provide data on lesion-specific bacterial loads and lesion-specific drug concentrations. With the inclusion of medical imaging, a compound’s effect on resolution of pathology within individual lesions and animals can also be determined over time. Incorporation of measurement of drug exposure and drug distribution within animals and their tissues is important for choosing the best compounds to push toward the clinic and to the development of better regimens. We review the practical aspects of each model and the advantages and limitations of each in order to promote choosing a rational combination of them for a compound’s development. 

Results from MultiScan LFER PET/CT

Tuberculosis Imaging group under the direction of Laura E. Via is using 2 MultiScan LFER PET/CT scanner for studying tuberculosis on Rabbit, Common Marmoset and Rhesus Macaque at National Institute of Allergy and Infectious Diseases.

In this substantial comprehensive review, the researchers also compared these 3 animal models in respect of PET/CT imaging:

• In longitudinal studies that combined PET/CT imaging, using FDG injected IV as a probe for metabolic activity, FDG concentrated in metabolically active regions within the Mtb HN878 lesions as they developed, but as necrosis progressed, the acellular centers of granulomas and cavities accumulated less FDG due to the lack of live cells (Figure 3A)
• NHP models are amenable to most diagnostic and therapeutic methods used in clinical studies, such as medical imaging (Figures 3B, C), serial blood sampling, and bronchoalveolar lavage (BAL) sampling for pharmacokinetic monitoring within an individual (Lewinsohn et al., 2006; Lin et al., 2013; Via et al., 2013). As serial imaging is feasible, methods to follow both individual lesion development and regression PI have been developed

Figure 3. FDG PET/CTs of a rabbit (A), marmoset (B), and rhesus macaque (C) with cavitary disease. The animals were infected for 69 to 90 days with M. tuberculosis at the time of imaging. Cavities (blue arrows) have been partially emptied of their necrotic contents and filled with air indicated by a darker central region in the lesions (lower density) surrounded by lighter walls (higher density). The scales show the range of CT Hounsfield units from higher to low density in shades of gray (+400 to −1000) on the left and FDG uptake in PET standard uptake units/body weight from high to low uptake in bright yellow to red to black (14 to 0) on the right. The width of the animal’s midsection is indicated with a bar and label to highlight the difference in size of the three animals.

Adjuvanting a subunit SARS-CoV-2 nanoparticle vaccine to induce protective immunity in non-human primates

 https://www.biorxiv.org/content/10.1101/2021.02.10.430696v1

Summary

Despite of the success of messenger RNA vaccines against SARS-CoV-2, a wider portfolio of different vaccine candidates would be needed to stop COVID-19 pandemic. In particular, vaccinating infants and the elderly could benefit from the use of subunit vaccine platforms with a demonstrable history of safety and efficacy in such populations.

Subunit vaccines include only a fragment of a pathogen which can still induce the immune system. Although, the production of these types of vaccines are safer than producing attenuated pathogen, they often require adjuvants to enhance the immune response for long term protection.

In this extensive research collaboration, the authors demonstrate the capacity of a subunit vaccine under clinical development, containing the SARS-CoV-2 Spike protein receptor binding domain displayed on a two component protein nanoparticle (RBD-NP). They assessed the immunogenicity and protective efficacy of RBD-NP vaccination using five different clinically relevant adjuvants in non-human primates.

Among the tested adjuvants, AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion, and CpG 1018 (with Alum), a Toll-like receptor 9 agonist formulated in Alum, were the most promising adjuvants.

The team concluded that the neutralizing antibody response by the RBD-NP/AS03 vaccination was durable. The study results can also help in the development of subunit vaccines to combat the ongoing pandemic.

Results from  MultiScan LFER PET/CT

The researchers in University of Pittsburgh School of Medicine evaluated inflammation in the lung tissues with no adjuvant, AS03 and CpG-Alum on pre and post challenge days using MultiScan LFER PET/CT

• Vaccinated animals showed FDG uptake, to a much lesser extent than the control animals (Fig. 3e and f)

  

  Fig 3 e, Inflammation in the lungs of two animals from each group indicated in the legend, pre-challenge (day 0) and post-challenge (day 4 or 5 after infection), measured using PET-CT scans. f, Representative PET-CT images of lungs from one animal in each group. PET signal is scaled 0 to 15 SUV.

PD-1 blockade exacerbates Mycobacterium tuberculosis infection in rhesus macaques

Keith D Kauffman, Shunsuke Sakai, Nickiana E Lora, Sivaranjani Namasivayam, Paul J Baker, Olena Kamenyeva, Taylor W Foreman, Christine E Nelson, Deivide Oliveira-de-Souza, Caian L. Vinhaes, Ziv Yaniv, Cecilia S Lindestam Arleham, Alessandro Sette, Gordon J Freeman, Rashida Moore, the NIAID/DIR Tuberculosis Imaging Program, Alan Sher, Katrin D Mayer-Barber, Bruno B Andrade, Juraj Kabat, Laura E Via, Daniel L Barber

 doi: 10.1126/sciimmunol.abf3861,  BioRxiv 

Summary

PD-1 (programmed death-1) is a coinhibitory receptor primarily expressed on activated CD4 and CD8 T cells that has been shown to limit the function of pathogen-specific T cells during chronic infection. The reactive expression of PD-L1 (PD-1 receptor ligand) on cancer cells turns off the T cells that are trying to attack the tumor. Therefore, blockade of PD-1 receptor or its ligands with monoclonal antibodies (mAbs) has become an attractive target in cancer therapy. Recognition of this pathway has led to suggestions that anti–PD-1 therapy might also boost T cell immunity in chronic infections including tuberculosis.

In this recent Science Immunology publication, Kauffman et al. examined the role of PD-1 during Mycobacterium tuberculosis (Mtb) infection of rhesus macaques. It was shown that the PD-1 blockade increased the number and functionality of Mtb-specific CD8 T cells, but not CD4 cells and was associated with increases in proinflammatory cytokines. However, animals treated with anti–PD-1 monoclonal antibody developed worse disease and higher granuloma bacterial loads compared with isotype control–treated monkeys.

These data indicate that negative regulation of immune responses is a critical aspect of host resistance to Mtb infection. Also, these findings suggest that the anti-PD-1 cancer therapy needs to be used cautiously in patients with a history of Mtb exposure.

Results from MultiScan LFER

Mediso MultiScan™ Large Field of view Extreme Resolution (LFER) PET/CT system was used to follow the course of Mycobacterium tuberculosis (Mtb) infection in rhesus macaques. The animals were imaged before infection and every 2 weeks after infection beginning at 4 weeks for a maximum of eight PET-CT scans. [18F] FDG was injected intravenously (1 mCi/kg), and after 60 minutes incubation time a 20-min PET scan was acquired.

(C) Example PET-CT image from isotype control– (top) or PD-1 (bottom)–treated animals. (D) Fold change over week 4 value of total lung standardized uptake value (SUV) in isotype control (left) and PD-1–treated (right) animals.

Comparison of SARS-CoV-2 infection in two non-human primate species: rhesus and cynomolgus macaques

Kinga P. Boszormenyi¹, Marieke A. Stammes², Zahra C. Fagrouch¹, Gwendoline Kiemenyi-Kayere¹, Henk Niphuis¹, Daniella Mortier¹, Nikki van Driel³, Ivonne Nieuwenhuis², Ella Zuiderwijk-Sick⁴, Lisette Meijer², Petra Mooij¹, Ed J. Remarque¹, Gerrit Koopman¹, Alexis C. R. Hoste⁵, Patricia Sastre⁵, Bart L. Haagmans⁶, Ronald E. Bontrop^7,8, Jan A.M. Langermans^3,9, Willy M. Bogers¹, Ernst J. Verschoor¹, and Babs E. Verstrepen¹

 ¹Department of Virology, Biomedical Primate Research Centre (BPRC), Rijswijk, The Netherlands

²Department of Parasitology, BPRC, Rijswijk, The Netherlands

³Animal Science Department, BPRC, Rijswijk, The Netherlands

⁴Alternatives unit, BPRC, Rijswijk, The Netherlands

⁵Eurofins-Inmunologia y Genetica Aplicada (Eurofins-INGENASA), Madrid, Spain

⁶Department of Viroscience, Erasmus University Medical Center, Rotterdam, The Netherlands

⁷Department of Comparative Genetics and Refinement, BPRC, Rijswijk, The Netherlands

⁸Department of Biology, Theoretical Biology and Bioinformatics, Utrecht University, Utrecht, The Netherlands

⁹Department of Population Health Sciences, Unit Animals in Science and Society, Veterinary Faculty, Utrecht University, Utrecht, The Netherlands

https://doi.org/10.1101/2020.11.05.369413

Summary

SARS-CoV-2 is a coronavirus that sparked the current COVID-19 pandemic. To stop it, effective and safe vaccines, and antiviral therapies are urgently required. To facilitate the preclinical evaluation of intervention approaches, relevant animal models need to be developed and validated. Rhesus macaques (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis) are widely used in biomedical research and serve as models for SARS-CoV-2 infection, but this is the first controlled comparative study investigating which species of them is best suited to examine specific aspects of COVID-19. This study analysed replication and symptoms for three weeks after infection. Pulmonary lesions were detected on CT images acquired with MultiScan LFER PET/CT. Elevated body temperature and decreased in physical activity was also observed. Results show that both rhesus and cynomolgus macaques represent valid models for COVID-19 prophylactic and therapeutic treatments.

Results from MultiScan LFER PET/CT

CT imaging provides a valuable tool to specifically monitor the progression of COVID-19-related lung pathology during the entire course of the study. Respiratory-gated CT scans were performed on Day0, 2, 4, 6, 8, 10, 12, 14, 16, 22 post-infection to monitor lung pathology. A semi-quantitative scoring system for chest CT evaluation was used to estimate SARS-CoV-2-induced lung disease; maximum score of 35 could be reached per timepoint.

 Scans revealed that:

• All macaques show levels of pneumonia
• Detected different types of lesions: A) ground glass opacities, B) consolidations, and C) crazy paving patterns (Figure 1)
• Around days 8 and 10 pi., lesions were manifest in all animals, and in several macaques the coverage had increased
• Cumulative CT scores increased and no difference was observed between rhesus and cynomolgus macaques

Further results showed:

• Both groups of animals, the body temperature was significantly higher during the first two weeks after infection
• Significantly lower activity in all four rhesus macaques during the first period after infection, while this difference in cynomolgus macaques was less obvious
• Antibody response became evident between day 10 and 12 pi., IgG level continued to rise for several days (development of IgM titers was barely detected)
• Certain cytokines increased in the plasma of both macaque species

Southwest National Primate Research Center in Texas Biomedical Research, San Antonio has purchased Mediso’s multiscan LFER PET/CT scanner in early 2020. Mediso USA proudly post that Professor Deepak Kaushal and his co-workers used multiscan LFER PET/CT in their recently reported comprehensive work about the course of SARS-CoV-2 infection in nonhuman primate models.

multiscan LFER PET/CT features a 20 cm axial and 15 cm transaxial field of view with a 26 cm bore opening which allows the researcher to scan non-human primates (NHP) with sub-mm PET and even 150 um CT resolution.

preliminary report title: SARS-CoV-2 infection leads to acute infection with dynamic cellular and inflammatory flux in the lung that varies across nonhuman primate species June 5, 2020. https://doi.org/10.1101/2020.06.05.136481

Summary

The authors compare SARS-CoV-2 infection in three species of experimentally infected NHPs (rhesus macaques, baboons, and marmosets). They used a wide variety of methods to describe the course of disease such as clinical parameters of viral infection, viral RNA and viral protein detection, immune response, X-ray, CXR scoring, CT scanning and pathology.

Their results show all NHPs can be infected with SARS-CoV-2 but exhibit differential progression to COVID-19. Baboons exhibit moderate to severe pathology, macaques exhibit moderate pathology and marmosets exhibit mild pathology. They also summarize that rhesus macaques and baboons develop different, quantifiable disease attributes making them immediately available essential models to test new vaccines and therapies.

Results from multiscan LFER PET/CT

The report shows the importance of state-of-the-art, non-invasive imaging – cone beam CT scanning, and the application of innovative algorithms to identify the extent of lung involvement in pneumonia in developing models of COVID-19. CT image analysis provided a quantifiable metric data which enables testing efficacy of vaccines or the impact of therapeutic intervention. Lung hyperdensity and the CT abnormality score was used as a metric parameter to follow the onset of the disease. 

• Each NHP species was infected and followed over a 3-day period to describe the early signs of infection. Cone-beam CT scans showed evidence of moderate pneumonia, which progressed over 3 days (Figure 1). CT images were analyzed using the segmentation tool in VivoQuant (Invicro, Boston,MA). Lung hyperdensity and the CT abnormality score were used as a metric parameter to follow the course of the disease.

Figure 1. a) 3D Reconstruction of ROI volume representing the location of the lesion. b-d) represent images for quantification of the lung lesion with the green area representing normal intensity lung voxels (-850 HU to -500 HU), while red areas represent hyperdense voxels (-490 HU to 500 HU). (Image courtesy of Dr. Deepak Kaushal, Texas Biomedical Research) 

• They also performed detailed imaging of macaques in a 12-day longitudinal study. Similar to the acute study. imaging revealed a significant, progressive increase in the volume of lung involved in pneumonia at 6 dpi, which normalized by 12 dpi. 

• These results suggest that pneumonia in some older macaques may persist longer than in younger animals. Although there were several smaller changes observed in older animals, old and young animals both resolved the infection.