Publications by Year: 2021


Zarkoob, Hoda, Anna Allué-Guardia, Yu-Chi Chen, Olive Jung, Andreu Garcia-Vilanova, Min Jae Song, Jun-Gyu Park, et al. (2021) 2021. “Modeling SARS-CoV-2 and Influenza Infections and Antiviral Treatments in Human Lung Epithelial Tissue Equivalents”. BioRxiv : The Preprint Server for Biology.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the third coronavirus in less than 20 years to spillover from an animal reservoir and cause severe disease in humans. High impact respiratory viruses such as pathogenic beta-coronaviruses and influenza viruses, as well as other emerging respiratory viruses, pose an ongoing global health threat to humans. There is a critical need for physiologically relevant, robust and ready to use, in vitro cellular assay platforms to rapidly model the infectivity of emerging respiratory viruses and discover and develop new antiviral treatments. Here, we validate in vitro human alveolar and tracheobronchial tissue equivalents and assess their usefulness as in vitro assay platforms in the context of live SARS-CoV-2 and influenza A virus infections. We establish the cellular complexity of two distinct tracheobronchial and alveolar epithelial air liquid interface (ALI) tissue models, describe SARS-CoV-2 and influenza virus infectivity rates and patterns in these ALI tissues, the viral-induced cytokine production as it relates to tissue-specific disease, and demonstrate the pharmacologically validity of these lung epithelium models as antiviral drug screening assay platforms.

Allué-Guardia, Anna, Rajagopalan Saranathan, John Chan, and Jordi B Torrelles. (2021) 2021. “Mycobacteriophages As Potential Therapeutic Agents Against Drug-Resistant Tuberculosis”. International Journal of Molecular Sciences 22 (2).

The current emergence of multi-, extensively-, extremely-, and total-drug resistant strains of Mycobacterium tuberculosis poses a major health, social, and economic threat, and stresses the need to develop new therapeutic strategies. The notion of phage therapy against bacteria has been around for more than a century and, although its implementation was abandoned after the introduction of drugs, it is now making a comeback and gaining renewed interest in Western medicine as an alternative to treat drug-resistant pathogens. Mycobacteriophages are genetically diverse viruses that specifically infect mycobacterial hosts, including members of the M. tuberculosis complex. This review describes general features of mycobacteriophages and their mechanisms of killing M. tuberculosis, as well as their advantages and limitations as therapeutic and prophylactic agents against drug-resistant M. tuberculosis strains. This review also discusses the role of human lung micro-environments in shaping the availability of mycobacteriophage receptors on the M. tuberculosis cell envelope surface, the risk of potential development of bacterial resistance to mycobacteriophages, and the interactions with the mammalian host immune system. Finally, it summarizes the knowledge gaps and defines key questions to be addressed regarding the clinical application of phage therapy for the treatment of drug-resistant tuberculosis.

Singh, Dhiraj Kumar, Bindu Singh, Shashank R Ganatra, Michal Gazi, Journey Cole, Rajesh Thippeshappa, Kendra J Alfson, et al. (2021) 2021. “Responses to Acute Infection With SARS-CoV-2 in the Lungs of Rhesus Macaques, Baboons and Marmosets”. Nature Microbiology 6 (1): 73-86.

Non-human primate models will expedite therapeutics and vaccines for coronavirus disease 2019 (COVID-19) to clinical trials. Here, we compare acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in young and old rhesus macaques, baboons and old marmosets. Macaques had clinical signs of viral infection, mild to moderate pneumonitis and extra-pulmonary pathologies, and both age groups recovered in two weeks. Baboons had prolonged viral RNA shedding and substantially more lung inflammation compared with macaques. Inflammation in bronchoalveolar lavage was increased in old versus young baboons. Using techniques including computed tomography imaging, immunophenotyping, and alveolar/peripheral cytokine response and immunohistochemical analyses, we delineated cellular immune responses to SARS-CoV-2 infection in macaque and baboon lungs, including innate and adaptive immune cells and a prominent type-I interferon response. Macaques developed T-cell memory phenotypes/responses and bystander cytokine production. Old macaques had lower titres of SARS-CoV-2-specific IgG antibody levels compared with young macaques. Acute respiratory distress in macaques and baboons recapitulates the progression of COVID-19 in humans, making them suitable as models to test vaccines and therapies.

Allué-Guardia, Anna, Juan I Garcia, and Jordi B Torrelles. (2021) 2021. “Evolution of Drug-Resistant Mycobacterium Tuberculosis Strains and Their Adaptation to the Human Lung Environment”. Frontiers in Microbiology 12: 612675.

In the last two decades, multi (MDR), extensively (XDR), extremely (XXDR) and total (TDR) drug-resistant Mycobacterium tuberculosis (M.tb) strains have emerged as a threat to public health worldwide, stressing the need to develop new tuberculosis (TB) prevention and treatment strategies. It is estimated that in the next 35 years, drug-resistant TB will kill around 75 million people and cost the global economy $16.7 trillion. Indeed, the COVID-19 pandemic alone may contribute with the development of 6.3 million new TB cases due to lack of resources and enforced confinement in TB endemic areas. Evolution of drug-resistant M.tb depends on numerous factors, such as bacterial fitness, strain's genetic background and its capacity to adapt to the surrounding environment, as well as host-specific and environmental factors. Whole-genome transcriptomics and genome-wide association studies in recent years have shed some insights into the complexity of M.tb drug resistance and have provided a better understanding of its underlying molecular mechanisms. In this review, we will discuss M.tb phenotypic and genotypic changes driving resistance, including changes in cell envelope components, as well as recently described intrinsic and extrinsic factors promoting resistance emergence and transmission. We will further explore how drug-resistant M.tb adapts differently than drug-susceptible strains to the lung environment at the cellular level, modulating M.tb-host interactions and disease outcome, and novel next generation sequencing (NGS) strategies to study drug-resistant TB.

Yigzaw, Wubet Birhan, Jordi B Torrelles, Shu-Hua Wang, and Belay Tessema. (2021) 2021. “Magnitude of Phenotypic and MTBDRplus Line Probe Assay First-Line Anti-Tuberculosis Drug Resistance Among Tuberculosis Patients; Northwest Ethiopia”. Infection and Drug Resistance 14: 497-505.

Background: Mycobacterium tuberculosis (Mtb) drug resistance is a key challenge in ending TB.

Objective: The study aimed to determine anti-TB drug resistance and compare the discordance between phenotypic and genotypic drug-susceptibility testing (DST).

Methods: Prospective enrollment and sputum collection from patients suspected of active pulmonary TB from May 2018 to December 2019 at the University of Gondar Hospital. Phenotypic DST study for streptomycin, isoniazid, rifampin, and ethambutol was done by MGIT 360 SIRE Kit. Genotypic resistance for isoniazid and rifampin was performed by MTBDRplus v2 line probe assay (LPA) and compared to phenotypic drug resistance.

Results: A total of 376 patients, median age 32 years, and 53.7% male were enrolled. Mtb was isolated from 126 patients. 106/126 (84%) patients were newly diagnosed with TB and 20 patients with prior TB treatment. Seventy (66.0%) were susceptible to all anti-TB drugs tested. Twenty-five (19.8%) of the isolates were resistant to isoniazid, 12 (9.5%) to rifampicin and six (5%) were multidrug resistant. Among previously treated TB patients, 4 (20.0%) and 5 (25.0%) were mono-resistant and poly-resistant, respectively. The sensitivity and specificity of LPA resistance for isoniazid were 94.4% and 100%, and for rifampin was 75.0% and 100%, respectively.

Conclusion: The frequency of mono- and poly-drug resistance among both newly diagnosed and previously treated TB patients was high to the rest of the nation. MTBDRplus showed excellent concordance for isoniazid and rifampin. We concluded that DST should be performed for all patients to improve management and decrease spread of drug-resistant Mtb strains in the community.

Singh, Dhiraj Kumar, Bindu Singh, Shashank R Ganatra, Michal Gazi, Journey Cole, Rajesh Thippeshappa, Kendra J Alfson, et al. (2021) 2021. “Author Correction: Responses to Acute Infection With SARS-CoV-2 in the Lungs of Rhesus Macaques, Baboons and Marmosets”. Nature Microbiology 6 (3): 413.
Kock, Richard, Anita L Michel, Dorothy Yeboah-Manu, Esam I Azhar, Jordi B Torrelles, Simeon I Cadmus, Lucy Brunton, et al. (2021) 2021. “Zoonotic Tuberculosis - The Changing Landscape”. International Journal of Infectious Diseases : IJID : Official Publication of the International Society for Infectious Diseases 113 Suppl 1: S68-S72.

Despite slow reductions in the annual burden of active human tuberculosis (TB) cases, zoonotic TB (zTB) remains a poorly monitored and an important unaddressed global problem. There is a higher incidence in some regions and countries, especially where close association exists between growing numbers of cattle (the major source of Mycobacterium bovis) and people, many suffering from poverty, and where dairy products are consumed unpasteurised. More attention needs to be focused on possible increased zTB incidence resulting from growth in dairy production globally and increased demand in low income countries in particular. Evidence of new zoonotic mycobacterial strains in South Asia and Africa (e.g. M. orygis), warrants urgent assessment of prevalence, potential drivers and risk in order to develop appropriate interventions. Control of M. bovis infection in cattle through detect and cull policies remain the mainstay of reducing zTB risk, whilst in certain circumstances animal vaccination is proving beneficial. New point of care diagnostics will help to detect animal infections and human cases. Given the high burden of human tuberculosis (caused by M. tuberculosis) in endemic areas, animals are affected by reverse zoonosis, including multi-drug resistant strains. This, may create drug resistant reservoirs of infection in animals. Like COVID-19, zTB is evolving in an ever-changing global landscape.

García, Juan Ignacio, Anna Allué-Guardia, Radhika P Tampi, Blanca I Restrepo, and Jordi B Torrelles. (2021) 2021. “New Developments and Insights in the Improvement of Mycobacterium Tuberculosis Vaccines and Diagnostics Within the End TB Strategy”. Current Epidemiology Reports 8 (2): 33-45.

Purpose of review: The alignment of sustainable development goals (SDGs) with the End Tuberculosis (TB) strategy provides an integrated roadmap to implement key approaches towards TB elimination. This review summarizes current social challenges for TB control, and yet, recent developments in TB diagnosis and vaccines in the context of the End TB strategy and SDGs to transform global health.

Recent findings: Advances in non-sputum based TB biomarkers and whole genome sequencing technologies could revolutionize TB diagnostics. Moreover, synergistic novel technologies such as mRNA vaccination, nanovaccines and promising TB vaccine models are key promising developments for TB prevention and control.

Summary: The End TB strategy depends on novel developments in point-of-care TB diagnostics and effective vaccines. However, despite outstanding technological developments in these fields, TB elimination will be unlikely achieved if TB social determinants are not fully addressed. Indeed, the End TB strategy and SDGs emphasize the importance of implementing sustainable universal health coverage and social protection.

Plante, Kenneth S, Varun Dwivedi, Jessica A Plante, Diana Fernandez, Divya Mirchandani, Nathen Bopp, Patricia Aguilar V, et al. (2021) 2021. “Antiviral Activity of Oleandrin and a Defined Extract of Nerium Oleander Against SARS-CoV-2”. Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie 138: 111457.

With continued expansion of the coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome 2 (SARS-CoV-2), both antiviral drugs as well as effective vaccines are desperately needed to treat patients at high risk of life-threatening disease. Here, we present in vitro evidence for significant inhibition of SARS-CoV-2 by oleandrin and a defined extract of N. oleander (designated as PBI-06150). Using Vero cells, we found that prophylactic (pre-infection) oleandrin (as either the pure compound or as the active principal ingredient in PBI-06150) administration at concentrations as low as 0.05 µg/ml exhibited potent antiviral activity against SARS-CoV-2, with an 800-fold reduction in virus production, and a 0.1 µg/ml concentration resulted in a greater than 3000-fold reduction in infectious virus production. The half maximal effective concentration (EC50) values were 11.98 ng/ml when virus output was measured at 24 h post-infection, and 7.07 ng/ml measured at 48 h post-infection. Therapeutic (post-infection) treatment up to 24 h after SARS-CoV-2 infection of Vero cells also reduced viral titers, with 0.1 µg/ml and 0.05 µg/ml concentrations causing greater than 100-fold reduction as measured at 48 h, and the 0.05 µg/ml concentration resulting in a 78-fold reduction. Concentrations of oleandrin up to 10 µg/ml were well tolerated in Vero cells. We also present in vivo evidence of the safety and efficacy of defined N. oleander extract (PBI-06150), which was administered to golden Syrian hamsters in a preparation containing as high as 130 µg/ml of oleandrin. In comparison to administration of control vehicle, PBI-06150 provided a statistically significant reduction of the viral titer in the nasal turbinates (nasal conchae). The potent prophylactic and therapeutic antiviral activities demonstrated here, together with initial evidence of its safety and efficacy in a relevant hamster model of COVID-19, support the further development of oleandrin and/or defined extracts containing this molecule for the treatment of SARS-CoV-2 and associated COVID-19 disease and potentially also for reduction of virus spread by persons diagnosed early after infection.

Silvas, Jesus A, Desarey Morales Vasquez, Jun-Gyu Park, Kevin Chiem, Anna Allué-Guardia, Andreu Garcia-Vilanova, Roy Neal Platt, et al. (2021) 2021. “Contribution of SARS-CoV-2 Accessory Proteins to Viral Pathogenicity in K18 Human ACE2 Transgenic Mice”. Journal of Virology 95 (17): e0040221.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. As of 19 May 2021, John Hopkins University's COVID-19 tracking platform reported 3.3 million deaths associated with SARS-CoV-2 infection. Currently, the World Health Organization has granted emergency use listing (EUL) to six COVID-19 vaccine candidates. However, much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse-genetics system approach to successfully engineer recombinant SARS-CoV-2 (rSARS-CoV-2) constructs; we removed individual viral ORF3a, -6, -7a, -7b, and -8 proteins from them, and we characterized the resulting recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF-deficient (ΔORF) viruses producing smaller plaques than those of the wild type (rSARS-CoV-2/WT). However, growth kinetics of ΔORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mice with the ΔORF rSARS-CoV-2s identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while ΔORF7a, ΔORF7b, and ΔORF8 rSARS-CoV-2s induced pathology comparable to that of rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse-genetics system to generate rSARS-CoV-2 constructs and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19. IMPORTANCE Despite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and the contribution of viral proteins to disease outcome remain elusive. Our study aims (i) to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins to viral pathogenesis and disease outcome and (ii) to develop a synergistic platform combining our robust reverse-genetics system to generate recombinant SARS-CoV-2 constructs with a validated rodent model of infection and disease. We demonstrate that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as a foundation for generating attenuated forms of the virus to develop live attenuated vaccines for the treatment of SARS-CoV-2.