From the Curator-in-Chief's Desk
April 14, 2020
The pandemic of SARS-CoV-2 is unprecedented at many levels. Many scientists are trying to find ways to apply their expertise to tackle the overwhelming issues from fundamental research to applications to diagnostics and treatment. The “sound bites” touting “everything you need to know about coronavirus” are everywhere, but there are few places to find actual research publications with data. Keeping up with the most relevant research publications is challenging for any investigator, but when hundreds of publications and preprints are appearing every week, the task can be overwhelming.
The registry is an effort by ASM to provide aggregated access to vetted fundamental research on SARS-CoV-2, and other relevant viruses. The registry experts will highlight relevant preprints and publications that appear every week. The goal is to ensure maximum acceleration of fundamental discoveries which are essential to optimize our chances of defeating the pandemic.
The registry is a resource for all scientists working to address the current challenge and be ready for future epidemics. For those scientists with a strong desire to contribute, but with little or no experience with viruses in general and SARS-CoV-2 in particular, this registry will provide reliable and up-to-date scientific information in selected areas, vetted by experts.
We welcome your feedback and ideas to make the Registry a useful resource for the scientific community to fight against the pandemic.
Biweekly Commentary Letters
March 26, 2021By Richard L. Hodinka, Ph.D., Professor, University of South Carolina School of Medicine Greenville, S.C.; Emeritus Professor, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pa. Dr. Hodinka is one of the curators of the Registry.
“Quantifying Absolute Neutralization Titers against SARS-CoV-2 by a Standardized Virus Neutralization Assay Allows for Cross-Cohort Comparisons of COVID-19 Sera” by Oguntuyo, K.Y., et al., published in mBio on Feb. 16, 2021.
Infection with SARS-CoV-2 elicits an immune response in the host that includes the production of neutralizing antibodies to the spike glycoprotein on the surface of the virus. Neutralizing antibodies represent a correlate of protection since they can block attachment of the virus to the ACE2 cellular receptor and prevent viral entry into the cell.
The article by Oguntuyo and colleagues describes the development and evaluation of a standardized assay to accurately quantify absolute neutralizing antibody titers against SARS-CoV-2. The assay is of high value and should provide meaningful comparative information for monitoring neutralizing antibody responses of acutely infected patients, screening convalescent-phase plasma from patients recovered from COVID-19, determining the efficacies of various vaccines and therapeutic entry inhibitors and studying naturally occuring spike mutants.
Pseudotyped viral particles bearing the SARS-CoV-2 spike glycoprotein (CoV2pp) were generated using vesicular stomatitis virus as a surrogate virus core bearing the Renilla luciferase reporter gene in place of its G glycoprotein to generate a measurable luminescent signal. A CoV2pp-based virus neutralization assay (VNA) was then developed and parameters impacting the performance of the assay were examined and standardized. The standardized VNA was evaluated by 4 independent groups using serum samples from geographically distinct and ethnically diverse COVID-19 patient cohorts. Quantitative measures of absolute 50% inhibitory concentration (absIC50), absIC80 and absIC90 were compared between laboratories. Two ultrapermissive 293T cell clones were also identified, allowing for ~150,000 infections per week in a 96-well format that can generate full neutralization curves for ~4,600 to ~6,200 samples.
Overall, this article will be invaluable to those interested in pseudotype virus production for SARS-CoV-2 and its use to quantify neutralizing antibody responses and study virus entry into host cells. Detailed and optimized protocols for producing the CoV2pp were provided. The produced CoV2pp circumvents the need for restrictive, expensive and limited biosafety level 3 (BSL 3) facilities, and the CoV2pp-based VNA represents a highly standardized, safe, reasonably simple, scalable and high-throughput system with robust metrics.
March 12, 2021
By Tom Gallagher, Ph.D., Professor of Microbiology and Immunology, Loyola University Chicago, Maywood, Ill. Dr. Gallagher is one of the curators of the registry.
“Cryo-EM structures of the SARS-CoV-2 endoribonuclease Nsp15 reveal insight into nuclease specificity and dynamics” by Pillon, M., et al., published in Nature Communications on Jan. 27, 2021.
Several coronavirus (CoV)-encoded accessory and “non-structural” proteins (nsps) operate to conceal infections from host-encoded antiviral factors and sensors that activate immune systems. CoV nsp15 is amongst these proteins facilitating virus proliferation within immunocompetent hosts. Nsp15 is an endo-uridine (endoU) specific ribonuclease that degrades polyuridine tracts from the 5’ ends of minus-strand viral RNAs, thereby quickly eliminating an RNA activator of innate immunity. Without endoU activity, CoV infections induce powerful host interferon responses, which limits their amplification in vivo. Tipping the balance towards host advantage has clinical implications. For example, live-attenuated CoV vaccines may be engineered by disabling endoU activity. Similarly, endoU-inhibiting drugs may induce interferons that quickly extinguish CoV spread and pathogenicity.
The findings reported in Pillon, M., et al. will promote nsp15-targeted antiviral drug discovery. The manuscript provides cryo-EM structures of SARS-CoV-2 nsp15 hexamers, with and without bound uridine triphosphate (UTP), in ways that further define endoU active sites and uridine substrate discrimination. The works also delineate a catalytic mechanism for endonucleolytic cleavage that brings out parallels with other endonucleases, including well-described RNase A. Importantly, the study also reveals a conformational flexibility in the hexamers that may be essential for capturing RNA substrates and mediating subsequent endonucleolytic cleavage. The findings provide frameworks for developing enzyme active-site inhibitors; i.e., uridine derivatives, as well as allosteric inhibitors that restrict the flexible “wobbling” of hexameric subunits. Finally, this study nicely illustrates how x-ray crystallography and cryo-EM techniques can be combined to reveal the conformational landscapes of complex protein machinery.
Feb. 26, 2021
By C.A.M. (Xander) de Haan, Ph.D., Associate Professor in virus-host interactions at the Faculty of Veterinary Medicine, Utrecht University, the Netherlands. Dr. de Haan is one of the curators of the registry.
“Intranasal ChAdOx1 nCoV-19/AZD1222 vaccination reduces shedding of SARS-CoV-2 D614G in rhesus macaques” by van Doremalen, N., et al., published on bioRxiv on Jan. 11, 2021.
Ideally, SARS-CoV-2 vaccines provide sterilizing immunity, meaning that they not only protect against disease, but also prevent replication in the upper respiratory tract and onward transmission. Within 1 year after the emergence of SARS-CoV-2, many vaccines targeting this virus are in clinical trials and several have already been approved. These vaccines protect against disease with different efficacies, but not necessarily prevent shedding of virus, as least as determined in non-human primates. This is probably associated with the intramuscular (IM) administration of these vaccines, which results in the induction of systemic IgG antibodies that protect the lungs, but not of mucosal IgA antibodies needed to inhibit replication in the nasal epithelium.
In a study by Neeltje van Doremalen and coworkers, intranasal (IN) and IM administration of the ChAdOx1 nCoV-19 vaccine was compared in a hamster challenge model. Both administration routes protected hamsters against disease, while IN vaccination resulted in significantly less virus shedding compared to IM vaccination. IN vaccination of rhesus macaques also protected against disease and resulted in systemic immunity comparable to that of IM-vaccinated animals. In addition, IN vaccination elicited SARS-CoV-2-specific mucosal immunity and reduced virus shedding.
Reducing virus replication and interrupting the chain of transmission is important to limit the emergence of variants, against which the currently approved vaccines may be less effective. Emergence of antigenic drift variants may even be accelerated by imperfect vaccines. Currently approved vaccines should therefore be analyzed for their ability to prevent onward transmission, and vaccines that do so should be further developed. In this respect, it is worthwhile to further investigate IN administration of adenovirus-vectored vaccines in a clinical setting.
Feb. 12, 2021
By Leo Poon, Professor, School of Public Health, The University of Hong Kong. Dr. Poon is one of the curators of the registry.
“Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice” by Cohen, A., et al., published in Science on Jan. 12, 2021.
The recent emergence of SARS-CoV-2 antigenic variants pose some serious concerns about the protective effect of COVID-19 vaccines recently rolled out in different countries. Data from preliminary studies suggest that some of these variants may partially escape neutralization by antibodies from recovered COVID-19 patients. Besides, several SARS-like betacoronaviruses (sarbecoviruses) are still circulating in animals and they continue to pose zoonotic threats to humans. Thus, there is a need to develop new strategies to control this group of pathogens.
Cohen and colleagues recently reported the use of multivalent nanoparticles containing the receptor binding domains (RBDs) of SARS-CoV-2 and other sarbecoviruses for immunization. Although the RBD of SARS-CoV-2 is a well-known neutralization target, its soluble form is not highly immunogenic. The authors found that SARS-CoV-2 RBD-specific antibody responses can be enhanced by conjugating the RBD of SARS-CoV-2 to nanoparticles. Mice treated with this nanoparticle vaccine have more robust neutralizing antibody titers than those vaccinated with the soluble form of SARS-CoV-2 RBD. In addition, serum samples from mice vaccinated with this homotypic nanoparticle vaccine can neutralize pseudoviruses of other sarbecoviruses and a boosting vaccination can further enhance such effects. Most interestingly, the authors found that mosaic nanoparticles decorated with different RBDs of sarbecoviruses (N=4/8) have even more pronounced effects in eliciting these cross-reactive responses. By contrast, convalescent plasma samples from COVD-19 patients do not have such activities.
This study from Cohen and colleagues provides a promising direction in developing pan-sarbecovirus vaccine. Further work using live virus in virus neutralization or animal studies will help to assess the potential use of this interesting approach. In particular, there is a high priority of using SARS-CoV-2 Variants of Concern (e.g. 501Y.V2 variant) for such investigations.
Jan. 22, 2021
By Jonathan D. Dinman, Ph.D., Professor of Cell Biology and Molecular Genetics, University of Maryland, College Park, Md., U.S. Dr. Dinman is one of the curators of the registry.
“SARS-CoV-2 protein ORF3a is pathogenic in Drosophila and causes phenotypes associated with COVID-19 post-viral syndrome” by Yang, S., et al., published in bioRxiv on Dec. 20, 2020.
A majority of COVID-19 patients suffer from post-recovery neurological complications including extreme fatigue and neuropsychiatric symptoms. Although a growing number of animal models, including non-human primates and hACE2 transgenic mice, have been developed to elucidate molecular mechanisms underlying the acute disease, the relatively long generation times of these mammalian systems render dissection of the bases for the long-term sequelae impractical, especially in light of the urgency attending the growing number of potential patients.
This study leveraged the power of Drosophila molecular genetics and developmental biology to address this pressing issue. Additionally, the rapid lifecycle of this model organism enables the types of longitudinal studies that would take months if not years in humans and non-human primate models. In this proof of principle work, Drosophila strains were constructed expressing the SARS-CoV-2 ORF3a protein in tissue specific manner. The authors found significant phenotypes only in flies’ expression of ORF3a in the nervous system. These included reduced lifespan, impaired motor function, abdominal swelling and partial larval lethality. Follow-up studies revealed that ORF3a expression induced apoptosis, neuroinflammation and neurotropism though immunoinflammatory pathways. The ability of ORF3a to cause lysosome deacidification suggested that this may be a source of toxic stress in neural cells. As proof of principle, transgenic flies treated with chloroquine phosphate, which blocks lysosome deacidification, increased ORF3a expressing fly lifespan, improved motor function and larval survival suggesting that chloroquine treatment may ameliorate symptoms associated with SARS-CoV-2 associated post-viral syndrome in humans. This study also validates the fly system as a platform for targeting specific proteins and pathways for drug screening.
Dec. 18, 2020
By Zhengli Shi, Ph.D., Senior Scientist, Wuhan Institute of Virology, Chinese Academy of Sciences. Dr. Shi is one of the curators of the Registry.
“Detection and Characterization of Bat Sarbecovirus Phylogenetically Related to SARS-CoV-2, Japan” by Murakami, S., et al., published in Emerging Infectious Diseases in Dec. 2020.
Coronaviruses phylogenetically related to SARS-CoV-2 were reported from 2 rhinolophus bat species in China, as well as pangolins smuggled to China from South Asian countries. This suggests that the progenitors of SARS-CoV-2 may exist in bats and other animals outside of China.
The article by Murakami, S., et. al., reported the discovery of a novel sarbecovirus (Rc-o319) in Rhinolophus cornututs, Japan, in 2 out of 4 samples collected in 2013. Rc-o319 shares 81.47% genome sequence identity with human SARS-CoV-2 and is positioned within a specific clade that includes SARS-CoV-2 and related coronaviruses from bats and pangolins. Interestingly, the Rc-o319 spike protein possesses a unique sequence variation from the known sarbecoviruses in the region corresponding to SARS-CoV and SARS-CoV-2 receptor binding domains. The authors also performed an analysis of receptor usage of this virus by using the vesicular stomatitis virus (VSV)-pseudovirus and demonstrated that Rc-o319 spike can use ACE2 of R. cornututs, but not that from human and other rhinolophus bat species. This is the first full-length genome sequence of bat sarbecovirus reported in an Asian country other than China, and highlights the necessity of much wider geographical investigations on sarbecoviruses in wildlife to find out the origins of SARS-CoV-2.
Dec. 4, 2020
By Vito Martella, Ph.D., Professor of Veterinary Medicine, Universita degli studi di Bari Aldo Moro, Bari, Italy. Dr. Martella is one of the curators of the registry.
Comparison of SARS-CoV-2 infection in two non-human primate species: rhesus and cynomolgus macaques by Böszörményi K., et al. published on bioRxiv on Nov. 5, 2020.
Animal models are essential for progress in SARS-CoV-2 research. Non-human primates (NHPs) have been employed for preclinical evaluation of vaccines and antiviral or immunomodulatory compounds to combat SARS-CoV-2. Thus far, 5 species of NHPs have been used in SARS-CoV-2 studies, including rhesus and cynomolgus macaques. Both macaque species are the 2 most widely-used NHP species in biomedical research and have been successfully used in studies of other human hypervirulent coronaviruses.
The use of different NHP species may generate variability and hamper a precise comparison of data in the study of SARS-CoV-2 infection, influencing the disease outcome considerably, as observed in other disease NHP models. In this study, the researchers made a comparative experimental infection in standardized conditions to assess which macaque species is best suited to investigate specific aspects of SARS-CoV-2 research. Rhesus and cynomolgus macaques were infected in parallel with the same virus stock, received completely identical treatment and the course of infection was followed using the same analyses and instruments. Both species showed moderate disease symptoms. Cynomolgus macaques showed elevated body temperature in the first 8 to 10 days following infection. A decrease in physical activity was only observed in the rhesus macaque. Pulmonary lesions were observed in most animals, as evidenced by computer tomography imaging. Shedding of infectious virus from the respiratory system was also documented. Immunoglobulins were detected in all animals by day 10 post-infection and the cytokine responses were highly comparable between species.
The authors conclude that both rhesus and cynomolgus macaques represent valid models for evaluation of COVID-19 vaccine and antiviral candidates in a preclinical setting.
Nov. 19, 2020
By Aaron G. Schmidt, Ph.D., Assistant Professor of Microbiology, Harvard Medical School and Ragon Institute of MGH, MIT and Harvard. Dr. Schmidt is one of the curators of the Registry.
Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants by Weisblum, Y., et al., published in eLife on Oct. 28, 2020.
In response to natural infection or vaccination, the human immune system often mounts a protective humoral response that helps prevent future infections. Viruses, however, can vary the antigenicity of their surface-exposed proteins. This serves as an effective evasion strategy to avoid host immune surveillance. Understanding how viruses might escape the mounted immune response is essential as it can directly influence the selection of antibody therapeutics as well as vaccine development.
Hatziioannou, Bieniasz and colleagues in their recent paper identify variants of the SARS-CoV-2 Spike protein that can escape neutralization by human monoclonal antibodies as well as convalescent sera. Using a chimeric vesicular stomatitis virus (VSV)/SARS-CoV-2 replication competent reporter virus, they obtained a “library” of Spike variants due to the lack of proof-reading activity of the VSV polymerase. They used this library to identify mutations that conferred resistance to three different monoclonal antibodies and convalescent sera from which the monoclonals were obtained. Key findings of their study include the observation that resistance populations were readily selected and clustered around the receptor binding domain. These mutations, however, did not impact interactions with its receptor, ACE-2. Mutations conferring escape from monoclonal antibodies remained sensitive to neutralization by the sera. Importantly, they found that cocktails of monoclonals could suppress viral escape. Lastly, through analysis of SARS-CoV-2 sequences, they also identified naturally occurring variants that are resistant to the monoclonals. Collectively, these data help further our understanding of the potential escape pathways of SARS-CoV-2 and have implications for developing therapies and vaccines.
Nov. 6, 2020
By Linda J. Saif, Distinguished University Professor, The Ohio State University, Wooster.
The effect of influenza vaccination on trained immunity: impact on COVID-19 by P. A. Debisarun, et al. medRxiv 2020.
Trained immunity is the concept that innate immune cells are trained via epigenetic and metabolic reprogramming during primary exposure to a vaccine or infection to produce enhanced secondary responses to an unrelated infection. This confers non-specific innate immune memory. Certain live vaccines, such as Bacille Calmette-Guérin (BCG), measles and oral polio, are reported to induce innate memory and cross-protection against unrelated viral infections. Currently, clinical trials are ongoing to assess if BCG vaccination may protect against COVID-19, since previous studies indicate that it can protect against certain viral respiratory tract infections in children; likewise for influenza vaccines and COVID-19.
The authors investigated the effects of an inactivated influenza vaccine ± BCG on trained immunity in vitro using peripheral blood mononuclear cells (PBMCs) from healthy volunteers. Variable and vaccine dose-related cytokine responses were observed when the primed, but not the unprimed, PBMCs were stimulated with SARS-CoV-2, suggesting varying patterns and levels of trained immunity. However, the in vitro data were limited to a few cytokines and did not include interferon responses (except Interferon (IFN)-gamma) that play key roles in innate immunity; nor were the contributing PBMC myeloid innate cell types defined. In an in vivo study, based on observation data with potential confounders, a significantly lower incidence of SARS-CoV-2 infection was reported among relatively young Dutch hospital employees (mean of 39-44 years) who received the inactivated influenza vaccine vs non-vaccines. However, there is other contradictory information on the impact of influenza vaccines on COVID-19 cases (De Wals and Divangahi, medRxiv 2020). The type of influenza vaccine (live vs. inactivated), the heterologous respiratory infection and its timing relative to vaccination, and most critically the influenza vaccinated age group and vaccine efficacy, may all influence both trained immunity and the impact of influenza vaccines.
More detailed in vitro and in vivo studies are warranted to define the impact of influenza vaccines on both trained immunity and SARS-CoV-2 infection. Relevant in vivo studies include investigations using animal models, such as ferrets susceptible to influenza and SARS-CoV-2 infections, in addition to well-designed epidemiologic studies in humans receiving influenza vaccines and exposed to SARS-CoV-2. Furthermore, as emphasized by the World Health Organization (WHO), because both positive and negative “off-target” effects of vaccines are evident (especially in children), it is critical to investigate the nonspecific effects of vaccines and trained innate immunity on subsequent infections.
Oct. 23, 2020
By Yi-Wei Tang, M.D., Ph.D., F(AAM), FIDSA, Chief Medical Officer, Danaher Diagnostic Platform and Cepheid (China), Shanghai. Dr. Tang is one of the curators of the Registry.
Saliva as a Candidate for COVID-19 Diagnostic Testing: A Meta-Analysis by Czumbel, L. M. et al. from Frontiers in Medicine.
Although the use of nasopharyngeal swab (NPS) specimens remains the “gold standard” for upper respiratory system sampling, the use of alternative specimens has been widely explored to overcome the supply shortages and to avoid clinician exposure. Saliva, which can be tracked back to 2003 in Hong Kong, when the SARS outbreak occurred, has been proposed as a promising alternative that could simplify and accelerate COVID-19 diagnosis.
The article by Czumbel and colleagues conducted a meta-analysis on the reliability and consistency of SARS-CoV-2 viral RNA detection in saliva specimens. The systematic search revealed 96 records after removal of duplicates. Twenty-six records were eventually included for analysis. Overall the sensitivity was 91% (CI 80–99%) for saliva tests and 98% (CI 89–100%) for NPS tests in previously conﬁrmed COVID-19 patients, with moderate heterogeneity among the studies. Additionally, 18 registered, ongoing clinical trials of saliva-based tests were also identified for detection of the SARS-CoV-2 virus. The authors concluded that saliva tests offer a promising alternative to NPS for COVID-19 diagnosis; however, further diagnostic accuracy studies are needed to improve their speciﬁcity and sensitivity.
Due to the nature of the metanalysis, the authors were not able to address the definition and quality control of the saliva. Instead of a simple spit, the ideal “saliva” specimen covering upper respiratory tract for SARS-CoV-2 testing should be a “hock a loggie” specimen, i.e., to cough up and spit out phlegm or saliva. To set up a standard of procedure for collecting the ideal saliva is difficult, especially for patient self collection (accessed Oct. 7, 2020). In addition, determination and standardization of saliva quality remains an unmet issue in quality assurance and quality control. Regulatory bodies need to catch up to address this deficiency. Until April 2020, the U.S. Food and Drug Administration (FDA) granted emergency use authorization (EUA) to 2 devices for use of saliva specimens for COVID-19 infection. Saliva testing on other EUA SARS-CoV-2 devices has been reported and we are looking forward to more independent, scientiﬁc analyses to establish their eﬀectiveness.
The authors acknowledged a limitation of their paper is the relatively small number of studies and small sample sizes available regarding this topic. Several relevant studies have been published after April 25, 2020 and we are looking forward to next version of metanalysis and literature review covering larger study numbers and sample sizes.
Oct. 9, 2020
By Sonia Navas-Martin, Ph.D., Associate Professor, Drexel University College of Medicine, Philadelphia. Dr. Navas-Martin is one of the curators of the Registry.
Loss of Bcl-6-Expressing T Follicular Helper Cells and Germinal Centers in COVID-19 by Kaneko, N. et al. Published in Cell on Oct. 1, 2020.
The immune response to coronaviruses remains poorly understood. Endemic human coronaviruses associated with the common cold are known to induce non-durable humoral immune responses. Similarly, there is rapid decay of anti–SARS-CoV-2 IgG in early infection in patients with mild COVID-19. Previous studies in COVID-19 autopsies have identified splenic white pulp atrophy and lymphocyte depletion in spleen and lymph nodes. Pillai, Pradera, and colleagues provide novel mechanistic insights into a paramount, unresolved question: Why humoral immunity to human coronaviruses’ infection is short lived?
This study examined post mortem thoracic lymph nodes and spleens in acute severe SARS-CoV-2 infection. Germinal centers in secondary lymphoid organs are responsible for the induction of high-affinity pathogen-specific antibodies and long-lasting B cell memory. Using quantitative multi-color immunofluorescence combined with multispectral imaging and cell-cell interaction analyses of autopsy specimens as well as analyses of peripheral blood samples in parallel cohorts with acute, severe SARS-CoV-2 infection, this study demonstrates dysregulated humoral immune induction early in COVID-19, including absence of germinal centers in the earliest stages of infection, defective Bcl6+ T follicular helper (TFH) cell generation, and aberrant lymphoid TNF (Tumor Necrosis Factor)-alpha production. Interestingly, although there is a marked reduction of germinal center B cells, activation-induced cytidine deaminase (AID)-expressing B cells are preserved, indicating that activated helper T cells are still likely to be in contact with antigen-specific B cells. Therefore, although there is robust T-cell-mediated activation of B cells, germinal centers do not form. Thus, robust activation of non-germinal center type B cell responses does not give rise to long-lived memory or high-affinity B cells. The study suggest a link between changes in the extra-follicular cytokine milieu driven by TH1 cells and the aberrant local production of TNF-a in lymphoid organs and the failure of differentiation of Bcl-6+ TFH cells. A potential hypothesis is that circulating factors in severely ill COVID-19 patients may impair GCTFH cell differentiation and thus abrogate the generation of germinal centers. Overall, these findings have implications for heard immunity and the development of efficient SARS-CoV-2 vaccines. It will be important to continue these studies in suitable SARS-CoV-2 animal models.
Sept. 17, 2020
By Dr. Catherine J. Pachuk, Chief Scientific Officer at Somahlution, Inc.
COVID-19 re-infection by a phylogenetically distinct SARS-coronavirus-2 strain confirmed by whole genome sequencing by To, K. et al. published on the Clinical Infectious Diseases on August 25, 2020.
Accounts of reinfection have been reported in individuals following apparent recovery from initial infection with SARS-CoV-2. In the absence of sufficient supporting data, it is not clear whether these reports documented reinfection or have instead described cases of prolonged viral shedding.
To et al. present the first well-documented case of SARS-CoV-2 reinfection in a patient who first tested positive in mid-March, became ill, was hospitalized and later released following two serial negative nasopharyngeal swabs. Approximately 4.5 months later, upon border screening in Hong Kong, he tested positive again, but remained asymptomatic. Whole genome sequencing on samples collected during both episodes demonstrated that the two viral genomes were phylogenetically distinct, mapping to two different GISAID clades indicating the patient was re-infected opposed to shedding virus over an extended time.
It is not known if reinfection is associated with differences in viral load compared to initial infection, however, the case for active infection during the second episode was supported by elevated CRP values and relatively high viral RNA load with gradual decline; the patient was therefore presumably still infectious. Asymptomatic reinfection or reinfection associated with milder disease is consistent with the presence of pre-existing adaptive immune responses induced following first exposure and is consistent with studies in which vaccination of Rhesus macaques conferred protection against disease but not viral infection.
The results demonstrate that reinfection is possible (at least with viruses having sufficient sequence differences) and may be associated with asymptomatic or milder disease, although the generalizability of this is not known. The results suggest that immune responses elicited by natural infection (and perhaps vaccination) may not confer “sterilizing immunity” against future infections and individuals may still become infected and transmit virus after vaccination and/or resolution of initial infection.
Sept. 4, 2020
By Dr. Ben Neuman, Professor of Biology & Chair of Biological Sciences, Texas A&M University — Texarkana
Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity by Hoffmann, M. et al., posted on BioRxiv on August 5, 2020.
Most kitchens have an “everything drawer” – a wonderful place where you can find batteries, binder clips or spare keys. The search for SARS-CoV-2 antivirals has led many groups to search the everything drawer of experimental medicine - screening libraries of approved compounds for serendipitous antiviral effects.
Among the first potential antivirals to come out of the drawer are the serine protease inhibitors nafamostat and camostat, originally developed to inhibit coagulation and treat pancreatitis. In a series of papers (Zhou Y. et al., Hoffmann M. et al., Hoffmann M. et al., culminating with the recent preprint by Hoffmann M. et al.), Stefan Pöhlmann’s lab has identified nafamostat mesylate and camostat mesylate as potent inhibitors of SARS-CoV-2 entry in cell culture. Both nafamostat and camostat prevent host transmembrane serine proteases (TMPRSS) including TMPRSS2 from cleaving the viral spike protein to free the viral fusion peptide and initiate the post-attachment steps of viral entry.
This study is the most careful and mechanistic to date on the effects of camostat. Four additional TMPRSS isoforms are identified as facilitators of SARS-CoV-2 entry. This study even tracks the conversion and potency of camostat metabolites 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) and guanidino-benzoate (GBA).
Perhaps the greatest value in a study like this is to work out a clear mechanism of action. While we wait for the results of clinical trials to find out if camostat is effective in COVID-19 patients, there is some comfort in its relative mechanistic simplicity. Even though we cannot be sure camostat will work in people, if it does, at least we will have a good idea why.
By Tom Gallagher, Ph.D., Professor, Loyola University Chicago
Aug. 14, 2020
Structural analysis of the SARS-CoV-2 methyltransferase complex involved in RNA cap creation bound to sinefungin by Krafcikova, P. et al. and Structural basis of RNA cap modification by SARS-CoV-2 by Viswanathan, T., et al., both published July 24, 2020 in Nature Communications.
Combination therapies simultaneously targeting several SARS-CoV-2 infection events can provide synergistic antiviral efficacy. Hence there are incentives for drugs disabling virus entry, polyprotein proteolysis and ribonucleic acid (RNA) replication. Additional drugs may effectively compromise the viral RNA modifications taking place as 5’ RNA caps are constructed. Coronavirus nonstructural protein 14 (nsp14) and nonstructural protein 16 (nsp16) enzymes methylate viral RNA caps, which increases RNA translation and decreases RNA recognition by the innate immune system. Therapeutics inhibiting these viral methyltransferases are promising antivirals.
Structural resolution of antiviral targets advances COVID19 therapeutic development. The articles by Krafcikova et al. and Viswanathan et al. are 2 of several recent reports on the structure of the SARS-CoV-2 nsp16. The 2 reports provide several insights. Krafcikova et al. show how nsp16 interfaces with a nonstructural protein 10 (nsp10) cofactor, revealing the challenges and the possibilities for drugs preventing nsp10-mediated methyltransferase activation. They also provide the position of a pan-methyltransferase inhibitor (sinefungin) within nsp16, along with modeled RNA substrate in a positively charged groove. This sets the stage for developing virus-specific active site inhibitors. Viswanathan et al. report RNA cap and methyl donor (SAM) locations within nsp16, and they reveal an induced fit model of RNA substrate binding. They also document a distal ligand binding site on nsp16, suggesting drug targets beyond the location of methyltransferase action. The reports provide frameworks for drug discovery, and with medicinal chemistry and clinical studies, there will be ever greater potential to bring methyltransferase inhibitors into the arsenal of therapeutics suppressing SARS-CoV-2 and related coronavirus infections.
July 31, 2020By Leo Poon, Professor, School of Public Health, The University of Hong Kong
Comprehensive mapping of immune perturbations associated with severe COVID-19 by Kuri-Cervantes, L. et al, Science Immunology 2020.
Most COVID-19 patients develop mild (40%) or moderate (40%) symptoms, whereas some can have severe (15%) or critical clinical outcomes. Such heterogeneity of disease spectrum is very different from the one of SARS. The underlying reasons accounting for this are not clear.
In order to determine immune parameters that might associate with the disease severity of COVID-19, a team from Philadelphia have conducted a comprehensive immune profiling analysis in 42 COVID-19 patients (7 mild, 28 severe and 7 recovered cases). Similar to others’ findings, several immune parameters (e.g., neutrophil-to-lymphocyte ratio and neutrophil:T cell ratio) correlate with disease severity. They also report severe COVID-19 cases tend to have reduced expression of CD16 on some innate immune cells (e.g., neutrophil, NK cells and monocytes). In addition, they report severe COVID-19 patients have increased activation of T cells and pronounced oligoclonal expansion of plasmablasts with long and divergent complementarity determining regions (CDR3) sequences. These results indicate that the immune responses of severe COVID-19 cases are different from those of mild ones. Some of these new parameters might be used as immune correlates for disease severity. Nonetheless, further systematic investigations on these immune subsets are needed to explain the pathogenesis of severe COVID-19 infection.
Several studies have demonstrated that severe COVID-19 patients have robust Immunoglobulin G (IgG) and Immunoglobulin M (IgM) responses. With the findings on B cells from the above study, it is of great interest to know the quality of antibodies produced by severe COVID-19 patients. In particular, long CDR3 sequences may relate to non-specific cross reactivity and/or immunopathology. Such analysis might provide useful information to advice clinical treatment, prognosis and vaccine development.
July 17, 2020By Michael Loeffelholz, Ph.D., D(ABMM), Senior Director, Medical Affairs, Cepheid and Adjunct Professor, Department of Pathology, University of Texas Medical Branch. Dr. Loeffelholz is one of the curators of the Registry.
“Diagnostic technology for COVID-19: comparative evaluation of antigen and serology-based SARS-CoV-2 immunoassays, and contact tracing solutions for potential use as at-home products” by Jorfi, M. et al. from medRxiv preprint server.
Testing and contact tracing are essential components of the approach to control the COVID-19 pandemic. Access to testing is crucial. At-home testing is a potential means to increase access. Manual contact tracing is labor intensive and requires a considerable amount of human resources. Personal electronic device-based contact tracing employing smartphones and wearable sensors is another potential approach. The preprint by Jorfi and colleagues describes their horizon scan, a systematic process to identify new technology with the potential for future impact, for antigen and serology diagnostics with the potential for use as at-home testing. The authors also assessed personal electronic technologies for contact tracing.
A systematic review of diagnostic tests, including literature and internet review, interviews of subject matter experts, and application of diagnostic test specification criteria such as performance characteristics, specimen types, and scalability identified over 300 candidates, including 138 serology and 44 antigen tests. The horizon scan, consisting of an algorithm to score tests identified in the systematic review, identified 24 antibody tests potentially suitable for at-home use, for further laboratory evaluation. Additionally, personal device-based electronic platforms were evaluated for potential contact tracing and the authors identified 26 potential smartphone solutions.
With increasing need for more and faster SARS-CoV-2 testing, we must consider the possibility of future at-home products. The work described in this preprint provides a framework for the identification and assessment of current diagnostic and electronic contact tracing products for their potential suitability for at-home use.
June 26, 2020By Richard L. Hodinka, Ph.D., Professor, University of South Carolina School of Medicine Greenville; Emeritus Professor, Perelman School of Medicine at the University of Pennsylvania. Dr. Hodinka is one of the curators of the Registry.
Comparative Performance of SARS-CoV-2 Detection Assays Using Seven Different Primer-Probe Sets and One Assay Kit by Nalla, A.K. et al. published in the Journal of Clinical Microbiology on May 26, 2020.
Various primer-probe sets have been developed and are being used for molecular detection of SARS-CoV-2 RNA in clinical specimens. Validation of the performance characteristics of assays using these primers and probes is needed.
The article by Nalla and colleagues is of high value and provides useful information on the comparative performance of seven different primer-probe sets and one commercial reagent kit for the detection of SARS-CoV-2 RNA when used in laboratory-designed molecular assays. Laboratories pursuing their own development, validation and implementation of in-house molecular-based assays will find this information to be helpful and it may save them much needed time and resources as they work to rapidly optimize assay performance and increase their testing capacity to meet demands.
Panels of respiratory specimens positive and negative for SARS-CoV-2 and those positive for influenza A and B, respiratory syncytial virus, parainfluenza virus, adenovirus, metapneumovirus, rhinovirus, bocavirus, and coronaviruses other that SARS-CoV-2 were examined. Nucleic acid extraction was performed on Roche MagNA Pure LC 2.0 and MagNA Pure 96 systems. Primer/probe sets against the RdRp, E, N1, N2, and N3 genes were used for SARS-CoV-2 RNA detection. RT-PCR was performed using Life Technologies AgPath-ID One Step master mix and an Applied Biosystems ABI 7500 real-time PCR system. A complete detection kit from BGI targeting the ORF1ab gene was also examined.
Overall, the authors observed variability in the sensitivities of the testing when using the different primer-probe sets and commercial reagent kit. Assays using the E-gene primer-probe set described by Corman et al. (https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045) and the N2 set developed by the Division of Viral Diseases, Centers for Disease Control and Prevention (https://www.cdc.gov/coronavirus/2019-ncov/downloads/rt-pcr-panel-primer-probes.pdf) were found to be the most sensitive. All assays tested were highly specific for SARS-CoV-2, showing no cross-reactivity with other commonly encountered respiratory viruses.
June 12, 2020By Dr. Frederick G. Hayden, Professor Emeritus, Medicine: Infectious Diseases and International Health at the University of Virginia. Dr. Hayden is one of the curators of the Registry.
Mechanism of baricitinib supports artificial intelligence-predicted testing in COVID-19 patients by Stebbing, J., et. al. published in EMBO Molecular Medicine on May 30, 2020.
The article by Stebbing and colleagues provides important new data on baricitinib, an immunomodulatory therapeutic of particular interest for COVID-19 treatment because of its documented anti-inflammatory properties and potential inhibition of coronavirus replication. The antiviral effect is postulated to be mediated by its affinity for AP2-associated protein AAK1 leading to reduced SARS-CoV-2 endocytosis. Baricitinib (trade name Olumiant, Eli Lilly and Company) is an oral inhibitor of Janus kinase (JAK)1 and JAK2 that is approved for the treatment of moderately to severely active rheumatoid arthritis in adults. Stebbing et al. show that baricitinib also inhibits numb-associated kinase (NAK) family members that includes AAK1 and that baricitinib exerts some antiviral effects against SARS-CoV-2 in in liver organoids, although at relatively high concentrations. The article also presents observational data from four baricitinib-treated COVID-19 patients.
Type I interferons trigger the Janus kinase/signal transducer and activator of transcription (Jak-Stat) signaling pathway that activates many antiviral genes, so that an inhibitor like baricitinib could facilitate virus replication. Baricitinib treatment causes dose-related decreases in interferon biomarkers in patients with interferonopathy-related auto-inflammatory disorders (Kim, 2018), and chronic therapy for rheumatoid arthritis has been associated with reactivation of latent herpes and polyomavirus infections and other serious infections (Favalli, 2020). Whether enhanced SARS-CoV-2 regulation might occur in an acute infection like COVID-19 remains to be determined.
A small, open-label observational trial of baricitinib added to lopinavir-ritonavir in hospitalized patients with moderate COVID-19 pneumonia reported that illness measures, respiratory function, and CRP elevations significantly improved both at week 1 and week 2 compared to baseline and to a historical control-group receiving lopinavir-ritonavir plus hydroxychloroquine (Cantini, 2020). A number of other clinical trials with baricitinib are in progress, including a large NIAID-sponsored randomized, placebo-controlled clinical trial (NCT04401579 ) is evaluating the safety and efficacy of a combination treatment regimen of remdesivir plus baricitinib compared to remdesivir for hospitalized COVID-19 patients.
May 29, 2020By Dr. C.A.M. de (Xander) Haan, Associate Professor, Utrecht University.
BCG-induced trained immunity: can it offer protection against COVID-19? by O’Neill, L.A. and Netea, M.G., Nature Review Immunology.
Bridging the SARS-CoV-2 vaccine gap by Bacille Calmette-Guérin (BCG) vaccination?
Development of a SARS-CoV-2 vaccine is expected to take at least 12-18 months. The authors of this comment propose that the BCG live attenuated vaccine, which was developed against tuberculosis a century ago, may be used to bridge this gap. Although currently there is no evidence that BCG protects against SARS-CoV-2 infection or disease, previous studies indicate that BCG can protect against viral respiratory tract infections in children.
This non-specific protective effect is proposed to be mediated by epigenetic changes that lead to long-term transcriptional programming of immune cells, resulting de facto in the induction of innate immune memory, termed trained immunity. Upon challenge with another pathogen, the trained immune cells then show an enhanced response, thereby promoting host defense.
Randomized controlled clinical trials are needed to provide evidence for the hypothesis that BCG vaccination may protect against COVID-19. Currently, such clinical trials are ongoing or being planned in different countries. Care should be taken that BCG vaccination to protect against COVID-19 will not cause, however, an increase of disease and deaths from tuberculosis resulting from vaccine shortages.
May 15, 2020
By Linda J Saif, Distinguished University Professor, The Ohio State University, Wooster, Ohio.
Trinity of COVID-19: Immunity, inflammation and intervention by Tay, M.Z., et al, Nat Rev Immunol 2020.
To date many approaches to therapeutic interventions for COVID-19 are empirical or based on only limited knowledge of SARS-CoV-2 infections. Based on increasing numbers of global reports, a clearer understanding of SARS-CoV-2 pathogenesis and pathophysiology is emerging to guide the rational design of targeted interventions. This is the topic of the selected review.
This review of the pathophysiology of SARS-CoV-2 infections is comprehensive and timely. The authors highlight the temporal sequence of coronavirus replication in respiratory tract cells and induction of healthy immune responses. They then characterize the chronology and potential contribution of dysfunctional immune responses to disease progression, focusing on the observed uncontrolled inflammation and cytokine storm leading to acute respiratory distress syndrome. Important and pertinent aspects of SARS and MERS coronavirus pathogenesis are compared to illuminate related data on SARS-CoV-2 infections. A table of relevant interventional clinical trials (March 2020) is included, but more updated versions are available on the WHO web site.
Significant conclusions include:
- Combined synergistic therapies are needed to inhibit both virus infection and regulate the dysfunctional immune responses.
- Studies of healthy versus dysfunctional outcomes and their chronology are critical to elucidate biomarkers of disease severity that will aid in the rational design of targeted interventions and a timeline for their application.
- Identification of biomarkers for immune correlates of protection and those related to disease severity are important for the design of safe and efficacious vaccines to circumvent immunopathology and to induce protection, respectively.
Unknowns that require additional research are how age, sex, genetics, co-morbidities, hypoxia, co-infections, immune landscape, microbiota, drug treatments, etc contribute to SARS-CoV-2 susceptibility, dysfunctional immune responses and disease severity. More information on innate immune responses is needed. A comprehensive, One Health, multi-disciplinary approach is highly relevant to answer these questions. Examples include: How do the coronavirus ancestor host species (bats) cope with SARS-like coronavirus infections to render them innocuous? How do the incidental hosts (cats, felids, ferrets, mink, non-human primates, etc) respond to SARS-CoV as a largely asymptomatic or mild infection? How do the above parameters influence the pathophysiology and severity of the disease and immune responses in humans compared with the animal disease models? Continued studies of the full spectrum of coronavirus disease (enteric, respiratory, systemic, and nervous systems) in naïve and partially immune natural host species are also warranted to delineate the impact of coronavirus infections on multiple organ systems and immune responses and to test potential targeted interventions, including vaccines.
May 1, 2020
By John J. Dennehy, Ph.D., Professor at Queens College and The Graduate Center of CUNY.
As the world struggles to overcome the COVID-19 pandemic, one fact is clear: testing and contact tracing will be a part of the solution. However, difficulties scaling up test capacity remain an issue. Test conductors are beset by logistical problems with every aspect of the workflow, from supply chains to operational coordination.
A preprint from a Yale University team suggests a way to alleviate some of these issues, while also increasing test reliability. Wyllie et al. report that tests of saliva for SARS-CoV-2 returned greater detection sensitivity and consistency throughout the course of infection than did patient-matched samples acquired from nasopharyngeal swabs. It is likely that larger sample volumes provided by saliva sampling reduce inconsistencies in sampler technique.
The results are significant for 3 reasons:
- SARS-CoV-2 test performance is already troubled by false negative results, so greater reliability is clearly indicated.
- Nasopharyngeal swabs, considered the gold standard of COVID-19 testing, have been in short supply. Dispensing with swabs will streamline test logistics.
- Nasopharyngeal swabbing is usually performed by trained medical personnel. Saliva sampling can be performed at home and submitted by post, alleviating demands on both patients and health care workers.
Further research of both COVID-19 positive and negative participants will reveal if saliva testing is ready for wider application.