Global Virus Network’s Institute of Human Virology and Italian Researchers identify a SARS-CoV-2 Viral Strain with Deletion in a Protein, Possibly Reducing Fatalities

A deletion in a protein, NSP1, which is important for reducing innate immune response may signal emergence of a less pathogenic viral strain

Baltimore, Maryland, USA, August 24, 2020: The Institute of Human Virology (IHV) at the University of Maryland School of Medicine, a Global Virus Network (GVN) Center of Excellence, in collaboration with scientists from Campus Biomedico in Rome, Italy announced today the results of studies showing the emergence of a SARS-CoV-2 viral strain with a deletion in a protein known as nsp1.  These data, accepted for publication today by the Journal of Translational Medicine, may indicate the emergence of a less pathogenic viral strain.

“Nsp1 plays a central role in hampering the anti-viral innate immune response,” said Robert C. Gallo, MD, The Homer & Martha Gudelsky Distinguished Professor, Co-founder & Director at the Institute of Human Virology, University of Maryland School of Medicine and Co-Founder and Chairman of the International Scientific Leadership Board of the Global Virus Network (GVN).  “Our data indicate that a small percentage of SARS-CoV-2 viruses is harboring a deletion in an important protein responsible for hampering the innate immune response, possibly adapting toward a decrease in pathogenicity. Scientists, including those within the Global Virus Network, will be able to expand on these data to confirm how widespread this deletion is.”

The researchers analyzed SARS-CoV-2 genome sequences from several countries and discovered a previously unknown deletion that is widespread and spans varying geographical areas. Modelling analysis of the newly identified deletion of SARS-CoV-2 nsp1 suggests that this deletion could affect the structure of the C-terminal region of the protein, important for both regulating viral replication and hampering the innate immune system response. The research indicates that the virus may become less pathogenic.

“SARS-CoV-2 seems to be undergoing profound genomic changes, but the effect of such changes on viral pathogenesis may become visible over a long period of time”, said Davide Zella, PhD, Assistant Professor of Biochemistry and Molecular Biology at the Institute of Human Virology, University of Maryland School of Medicine and member of the Global Virus Network (GVN).  “We need to confirm the spreading of this particular viral strain and research potential strains with other deletions in the nsp1 protein, both in the population of asymptomatic and pauci-symptomatic subjects, and correlate these changes in nsp1 with decreased viral pathogenicity. Also, the spreading of this deletion needs to be evaluated over time”.

“The percentage of deletions found in the cases analyzed did not seem to be geographically homogenous, possibly due to the low number of available sequences for analysis,” said Francesca Benedetti, PhD, Research Associate of Biochemistry and Molecular Biology at the Institute of Human Virology, University of Maryland School of. Medicine “The percentage was higher in Sweden with 1.89% while in certain parts of the United States was about 1%.”

“Our modeling of nsp1 protein of SARS-CoV-2 indicates that this deletion may influence potential structure in this region, thereby altering its activity and ability to interact with other proteins of the host,” says Greg Snyder, PhD, Assistant Professor of Microbiology and Immunology at the Institute of Human Virology, University of Maryland School of Medicine.

“We are pleased to work with our colleagues at the Institute of Human Virology to identify and characterize the profound alterations in the SARS-CoV-2 genomic sequences spanning the globe, and to evaluate their biological significance,” said Massimo Ciccozzi, PhD, Associate Professor of Medical Statistics, Universita’ Campus Biomedico in Rome, Italy.

# # #

About the Institute of Human Virology

Formed in 1996 as a partnership between the State of Maryland, the City of Baltimore, the University System of Maryland, and the University of Maryland Medical System, the IHV is an institute of the University of Maryland School of Medicine and is home to some of the most globally-recognized and world-renowned experts in all of virology. The IHV combines the disciplines of basic research, epidemiology, and clinical research in a concerted effort to speed the discovery of diagnostics and therapeutics for a wide variety of chronic and deadly viral and immune disorders – most notably, HIV the virus that causes AIDS. For more information, visit www.ihv.org and follow us on Twitter @IHVmaryland.

 

About the Global Virus Network (GVN)

The Global Virus Network (GVN) is essential and critical in the preparedness, defense and first research response to emerging, exiting and unidentified viruses that pose a clear and present threat to public health, working in close coordination with established national and international institutions. It is a coalition comprised of eminent human and animal virologists from 55 Centers of Excellence and 10 Affiliates in 33 countries worldwide, working collaboratively to train the next generation, advance knowledge about how to identify and diagnose pandemic viruses, mitigate and control how such viruses spread and make us sick, as well as develop drugs, vaccines and treatments to combat them. No single institution in the world has expertise in all viral areas other than the GVN, which brings together the finest medical virologists to leverage their individual expertise and coalesce global teams of specialists on the scientific challenges, issues and problems posed by pandemic viruses. The GVN is a non-profit 501(c)(3) organization. For more information, please visit www.gvn.org. Follow us on Twitter @GlobalVirusNews

 

GVN MEDIA CONTACT
Nora Samaranayake
Phone:  410-706-1966
Email:    [email protected]

 

From molecular biology to human retroviruses: in memory of GVN Italy Center Director Umberto Bertazzoni (1937-2020)

Born in Trissino (VI) in 1937, Umberto Bertazzoni graduated from the University of Pavia in Pharmacy (1960) and in Biological Sciences (1964). He was a researcher at the Joint Research Center of Ispra from 1964 to 1974, researcher at the Institute of Genetics of the CNR of Pavia from 1974 to 1994 and Head of Unit at the Directorate General for Research of the European Commission in Brussels from 1995 to 2000. He also carried out research activity in 1962 at the University of Iowa, USA; from 1971 to 1974 at the J. Monod Institute of the University of Paris VII; in 1979 and 1992 at the Naval Hospital and the NIH in Bethesda, USA. He was full professor of Molecular Biology at the University of Verona from 1990 to 2010 in the degree course of Medicine and Surgery. Prof. Bertazzoni was a Center of Excellence Director of the Global Virus Network (GVN) Italy. He authored of 110 indexed publications (h-index: 28; source. Scopus).

Prof. Umberto Bertazzoni received the “Giovanna Tosi” Prize for Cancer Research at the University of Insubria in September 2018 “For his seminal studies on human oncogenic retroviruses and for his tireless activity in foundation and support of international virology Institutions” which perfectly summarizes his long scientific life, recalls Luigi “Gino” Chieco-Bianchi (University of Padua), perhaps his closest friend, as well as collaborator and first Director of GVN Italy.

Umberto Bertazzoni in Red Square, Beijing, China during GVN’s 2015 International Meeting

Prof. Chieco-Bianchi continued, “I met Umberto in the early 1980s when, an established researcher at ISPRA (Higher Institute for Environmental Protection and Research), he dealt with the molecular mechanisms underlying the proliferation of neoplastic cells [his works on Tdt, terminal deoxynucleotidyl transferase , are among the most cited of that period – (1, 2)]. With the advent of AIDS, his skills in enzymology became invaluable for the study of reverse transcriptase, the key enzyme of retroviruses such as HIV (human immunodeficiency virus, cause of AIDS) and HTLV (human T lymphotropic virus, the first pathogenic human retrovirus, discovered in 1980, and causative agent of a relatively rare form of adult T-cell leukemia and neurological syndromes). Umberto immediately became part of the group of “human retrovirologists”, initially not very numerous – which I too had joined in after abandoning my studies on murine retroviruses – soon conquering a leading position in the study of HTLV. Those were years of intense and enthusiastic work: many young scholars, who returned from the USA and various European countries, where they had acquired new skills, were recruited together with the most valid Italian research groups in the National AIDS Project, adequately funded by the Ministry of Health and managed by the Istituto Superiore di Sanità under the intelligent and rigorous scientific direction of Giovanni Battista Rossi. Umberto participated assiduously in the periodic meetings, very animated, in which the various Operational Units of the Project reported the results achieved, thanks to which Italy in those years established itself among the leading countries for AIDS research.”

In the mid-1990s he took on an important position at the EU Research Directorate General where he was also head of the Infectious Diseases section, coordinating among other things the European projects on AIDS. The main focus of his research was the study of the characteristics of HTLV-2 (a substantially non-pathogenic virus for humans) and of its molecular and functional differences compared to HTLV-1 (absent in Italy) with the intent to clarify the absence of oncogenicity observed in infected individuals, in general drug addicts or former drug addicts. The results of these studies have been the subject of relevant publications, as evidenced by having been editor (with Vincenzo Ciminale of the University of Padua and Maria Grazia Romanelli, his close collaborator at the University of Verona) of a monographic issue of Frontiers of Microbiology dedicated to HTLV-1 (3).

Luigi “Gino” Chieco-Bianchi and and Umberto Bertazzoni

Prof. Chieco-Bianchi said, “With Umberto I also shared the adventure of the Global Virus Network (GVN), founded in Washington in 2011 by Bob Gallo of the U.S., Billy Hall of Ireland and the late Reinhardt Kurth of Germany. Bob wanted me to initially be the coordinator of the Italian Center of Excellence and so, with Umberto, Roberto Accolla, Franco and Luigi Buonaguro, Beppe Ippolito and Guido Poli, all armed with goodwill but lacking operational resources, we began to set up that involved old and young Italian virologists. After three years, due to physiological turnover, Umberto was unanimously designated to lead GVN Italy and for another three years, more effectively, he carried out his coordinating role, always animated by enthusiasm and confidence in the future.

Prof. Robert Gallo (Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, USA, Global Virus Network): “Umberto Bertazzoni was a scientific close colleague and personal friend for four decades. I always enjoyed his company and his wonderful enthusiasm for our similar scientific interests. He was early in HTLV research and some of its pioneering studies.  He was a leader in the early formation of the GVN and instrumental in its activities. He will be missed very much, but always remembered.”

Prof. Guido Poli (Vita-Salute San Raffaele University, Milan, Global Virus Network): “Immediately after his return to the University of Verona, a relationship of collaboration and friendship emerged largely mediated by his collaborator Claudio Casoli who also left us a few months ago. The intersection of our scientific interests was based on HIV-1 and HTLV-2 co-infection, mainly in drug addicts or ex-drug addicts. Keen to our scientific collaboration was the discovery in 1995-1996 of the fundamental role of CCR5-binding chemokines (and to a lesser extent CXCR4), a key receptor for HIV-1 entry into target cells. Not surprisingly, the study by Paolo Lusso and Robert Gallo of December 1995 (4) exploited the production of these chemokines by T lymphocytes immortalized by HTLV-1 or HTLV-2. We then demonstrated together with Umberto, Claudio and their collaborators that patients co-infected with HTLV-2 and HIV-1 often had the characteristics of Long-Term Non-Progressors, that is, they demonstrated a natural predisposition to control the progression of HIV-1 disease, thanks to the hyperproduction of a particularly powerful chemokine (5). Beyond his objective scientific merits, with Umberto Bertazzoni disappears one of the last ‘gentlemen scientists’ around.”

Umberto Bertazzoni and Guido Poli in Red Square, Beijing, China during GVN’s 2015 International Meeting

Prof. Davide Zella (Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, USA, Global Virus Network): “I met Umberto in 1984, at the CNR in Pavia, as a young university student. Life in his laboratory was very pleasant: I was learning and having fun. I remember the time spent discussing science, and he would have guided me towards the thesis. At the beginning he was ‘the professor,’ but he quickly became ‘Umberto,’ a friend who over time would have given me advice, at times assuming the appearance of a father figure. With the arrival of AIDS, Umberto introduced me to Robert Gallo in Florence in 1992 and from there my career began with Gallo, with whom I still work today. Umberto taught me a lot, in the laboratory and in life, and I will miss him very much.”

Franco M. Buonaguro (“Center Director”), Roberto Accolla, Luigi Buonaguro, Gino Chieco-Bianchi, Giuseppe Ippolito & Guido Poli for GVN-Italy.

 

References

  1. Bertazzoni U, Stefanini M, Noy GP, Giulotto E, Nuzzo F, Falaschi A et al. Variations of DNA polymerase-alpha and -beta during prolonged stimulation of human lymphocytes. Proc Natl Acad Sci U S A. 1976; 73 (3): 785-9
  2. Bertazzoni U, Brusamolino E, Isernia P, Scovassi AI, Torsello S, Lazzarino M et al. Prognostic significance of terminal transferase and adenosine deaminase in acute and chronic myeloid leukemia. Blood. 1982; 60 (3): 685-92
  3. Bertazzoni U, Ciminale V, Romanelli MG. Editorial: Molecular Pathology of HTLV-1. Front Microbiol. 2018; 9: 3069
  4. Cocchi F, DeVico AL, Garzino-Demo A, Arya SK, Gallo RC, Lusso P. Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8 + T cells. Science. 1995; 270 (5243): 1811-5
  5. Pilotti E, Elviri L, Vicenzi E, Bertazzoni U, Re MC, Allibardi S et al. Postgenomic up-regulation of CCL3L1 expression in HTLV-2-infected persons curtails HIV-1 replication. Blood. 2007; 109 (5): 1850-6

Hydroxychloroquine-What’s the Deal?

The absence of efficient therapeutics for COVID-19 has brought much attention to evaluation of repurposing drugs. Hydroxychloroquine (HC) is an antimalarial drug that affects endosomal function and blocks autophagosome-lysosome fusion (1). Since coronaviruses use the endolysosomal pathway to enter the cell before uncoating, HC has been shown to inhibit SARS-CoV-2 replication in cellular models. The use of HC in the treatment and/or prevention of COVID-19 has been clouded in controversy and contention. Partly, this is because it has become somewhat of a political football, with one side relentlessly touting its value and with the other side just as adamantly claiming that it has no value and is indeed harmful with various side effects. The second reason is the variety of conditions used in the reported tests. The third reason is that there seems to be a great number of risk factors or co-morbidities, and this can result in vastly different results due to differences in demographics. Results from anecdotal versus retrospective versus observational versus controlled versus randomized/blind trials have varied widely. There is also the question of whether it is necessary to include azithromycin (AZM) or other antibiotics, and whether to use zinc (Zn).  AZM, a widely used broad-spectrum antibiotic, also blocks autophagosome clearance in human cells and replication of the Zika and influenza viruses in human cells in vitro. A subset of HC advocates also thinks that inclusion of zinc is critical, and that the primary function of HC is to help Zn enter the cells.  The related drug, chloroquine, has been reported to act as a Zn ionophore(2), and Zn inhibits RNA chain elongation by SARS-CoV-1 RNA-dependent RNA polymerase in vitro(3). The authors also showed inhibition of viral infection in Vero (African green monkey kidney) cells. However, it has recently been shown that SARS-CoV-2 enters Vero cells by an alternate pathway that is not inhibited by HC. Due to the use of a different pathway for viral entry,  HC treatment in human lung cells did not significantly inhibit viral infection (4). These data suggest that if HC has a beneficial effect on treatment of COVID-19, it is either because it affects the host response to the virus or it affects a step unrelated to viral entry.

For all these reasons, studies that include randomized control groups are inherently far more reliable than observational or retrospective studies, in which data from other groups are used as a control. Thus, without randomized trials, it is difficult to draw firm conclusions. Double-blind randomized studies are the most reliable because they eliminate potential placebo effects, which can be substantial. Randomized trials are, however, more expensive and take more time to carry out, so there are far fewer of them than of observational or retrospective studies.  Sorting out all these issues is difficult, but let’s give it a try. The sources we will consider will include PubMed (comprised of peer-reviewed published studies), medRvix (preprints not yet peer-reviewed), and the internet in general (with the uncertainty that entails), including press releases and editorials, perhaps the least reliable source. We will not consider in vitro antiviral studies, as these have been performed largely with Vero cells, which as discussed above, are not useful for SARS-CoV-2 studies and HC.

Reviewing the Literature

Let’s first consider results from several randomized double-blind trials with HC. One such study looked at people with documented occupational or household exposure to individuals with confirmed COVID-19 to observe whether HC was effective prophylactically(5). Treatment with HC was for 5 days within 4 days of exposure, and both groups had somewhat more than 400 subjects. There were slightly but insignificantly fewer cases of COVID-19 in the HC arm, as judged by either a positive PCR test or development of symptoms. There was one hospitalization in each group, and no deaths occurred. HC was not associated with any serious side effects. Another randomized double-blind trial looked at patients with early COVID-19(6). Subjects were treated with HC or placebo for 5 days. There was no difference in symptom severity over 14 days between the groups. There were 4 hospitalizations and one death in the HC group compared to 8 hospitalizations and one death in the placebo group; this did not reach statistical significance. Another small randomized double blind study compared two groups (n=40) of COVID-19 patients treated with either high or lower doses of chloroquine for 10 days(7). All were taking AZM. Although there was more mortality in the high dose group, neither differed significantly from what would be expected from an untreated group of similar patients. We note that not having an internal untreated control weakens this study. Taken together, these trials strongly suggest that there is not a significant therapeutic benefit of HC, although they do not completely rule it out. In fact, the authors in these studies generally do not prove a lack of potential benefit of HC, but suggest further similar studies are needed for confirmation. The data also uniformly suggest that HC is reasonably safe.

There have also been several well controlled large randomized open-label (not blinded) trials. Among these was one testing hospital patients with COVID-19 and requiring either no supplemental O2 or <4 l/min O2. More than 500 patients were randomly assigned to 3 groups and treated for 7 days by standard of care (SOC), HC+SOC, and HC+AZM+SOC(8). Clinical status was evaluated at 15 days. There was no improvement in either group receiving HC relative to the SOC group. Elevated Q-T heart intervals and elevated liver enzymes were more prevalent in these two groups, but these were not considered serious. Another randomized open label study looked at patients with mild to moderate COVID-19 and treated them either with SOC (n=75) or SOC+HC (n=75)(9). As judged by conversion to negativity for SARS-CoV-2, judged by RT-PCR of nasal swabs, and by alleviation of symptoms, there were no differences in outcomes by day 28. Some negative events were attributed to HC, primarily diarrhea. Another randomized open label trial researched 293 patients with COVID-19 who were not hospitalized and were symptomatic for fewer than 5 days. Patients were treated either with HC for one week (n=136) or without HC (n=157). As judged by viral RNA loads, hospitalization and time to resolution of symptoms, there were no significant differences.  We note that the number of hospitalizations was too small to reach significance. Yet another randomized non-blind trial looked at patients with mild COVID-19. Patients were treated with HC for 6 days or not treated and viral RNA loads, and resolution of symptoms at day=28 were the end points. There were about 140 in each arm. No differences in outcomes were noted, and no adverse effects were reported. In contrast, another small randomized study treated patients with HC (including pneumonia) for 5 days (no AZM or Zn) or SOC (31 subjects per group) and showed a significant improvement in cough and fever resolution. The reasons for the incongruent results are unclear.

Next in degree of reliability are observational or retrospective studies, as they generally rely on statistics from patient groups that are distinct from the groups under study and may differ by genetic factors or by comorbidities. They may also differ by prior exposure to other coronaviruses or by prevalence of recent or childhood administration of vaccines such as BCG, polio or measles, all of which may affect results from SARS-CoV-2 exposure or infection because of immune memory and cross-reactivity. In general, more observational studies report a protective effect than do those that find no benefit of HC.  The quality of these studies varies widely. As with randomized studies, in general, the larger the study, the more likely the results are to be accurate. We will concentrate on several of the best and largest of these; they are reasonably representative of the multitude of observational studies.

A large (~3,700 patients) retrospective/observational study reported a benefit of HC plus AZM treatment for 5 days (n=3,119) compared to groups treated with HC alone, AZM alone or SOC (n=619), termed “others.”  Better outcomes compared to “others” were reported for mortality, hospitalization, duration of viral RNA shedding, and several other clinical parameters. It is not clear, however, as to how patients were assigned to treatment groups, and patients not receiving HC seemed sicker, judged by prevalence of cancer or hypertension. It should also be noted that there was no report of Zn usage.  A large multi-center (~2,500 patients) study treated patients with HC alone, AZM alone, HC+AZM, or neither(10) and looked at mortality rates as the primary outcome. Taking into accounts various clinical parameters from each group, the authors concluded that HC provided a hazard ratio reduction of 66% and HC+AZM a reduction of 71%. This may not account for all possible confounding factors. For example, it is not clear why the untreated patients were not given HC or whether they were sicker at admission. However, the patients not receiving HC were on average 5 years older and had a higher incidence of cancers, which seem to be serious confounding factors. It is also not totally clear how the decision was made to not administer HC. It should also be noted that steroids were administered to patients receiving HC as adjunct therapy at a far greater rate than those who did not; this is another potential substantial confounding effect. Duration of symptoms before admission were not available. Indeed, the authors caution that randomized prospective trials are needed and that their results should be interpreted with caution. There were no reported major safety issues. Again, we note there was no reported use of Zn.

Another observational study supporting a positive effect of HC looked at ~1,600 patients who were treated with one of 16 different treatments, with death or intubation as an endpoint. The only favorable treatments were HC (with a hazard ratio of 0.83) and predisone (HR=0.85). Dexamethasone treatment resulted in slightly worse outcomes. The same caveats mentioned for the previous studies are applicable to this study.

Another retrospective study with 335 subjects were apparently drawn from subjects treated by Dr. V Zelenko, who at one point claimed to have treated 1500 people with COVID-19 successfully, although this was based on symptoms rather than confirmed tests. It seems likely, based on the size of the cohort tested (335), that not all of the original patients were positive.  (For comments on this point, please read here). 141 patients were treated with HC+AZM+Zn. Oddly, public reference data on 377 patients from the same community were used as a control rather than patients in the cohort who were untreated, and no clinical or demographic data are available for this group. Although there were significantly fewer hospitalizations and deaths and no serious adverse effects in the treated group, the authors state that no conclusions can be made on efficacy or safety. This study appears to be where the idea that Zn was critical originated.

There are also observational/retrospective studies that show no effect or HC±AZM. One looked at 226 patients with mild to moderate COVID-19 who were either treated or received SOC.  No benefit was observed as judged by viral clearance, hospital stay, or duration of symptoms. It should be pointed out the treated group was relatively small (N=31). Salvarani et al. (11) looked at 4,400 people who were being treated with antimalarial drugs (HC or chloroquine) and compared them with the general population in the same geographic areas. There were no significant differences in rates of diagnoses of COVID-19 nor of positive tests for SARS-CoV-2. Geleris et al. (12) studied 1376 hospitalized COVID-19 patients, of whom 811 were treated within 48 hrs with a 5 day course of HC; the remainder were given SOC. There was no significant difference in intubation or death. As with non-randomized studies, differences in characteristics of cohorts can matter greatly. The HC-treated cohort, for example, was older and had more hypertension than the reference cohort. The Geleris study did use propensity score matching to account for these differences. The authors, however, caution that randomized trials are needed to conclude HC has no value for COVID-19.

There are many more studies than we have described, and it would not be feasible to mention them all. We have, however, tried to be as representative as possible. As we can see, there are considerable differences in outcomes reported. Looking at the data in general, it can be seen that the randomized trials have the most agreement, and most (but not all) conclude that there is no significant benefit to HC treatment with or without AZM. Concerning the observational/retrospective studies, there are more positive than negative reports. Why is this? As discussed, these are inherently less reliable than randomized trials. It may thus be easier to get positive results in an observational trial, especially if the study is small or not well controlled by cohort. Also, it is likely that positive studies in general (not simply COVID-19 and HC) are more readily publishable than negative ones. Although Zn is claimed to be critical, it should be noted that many of the positive reports do not use Zn, which would seem to negate this idea. The placebo effect may play a role.

Conclusion

Our literature review has generated somewhat contradictory findings, but strongly suggests that HC is not beneficial for COVID-19 treatment. There are positive data as well, but these come almost entirely from observational/retrospective studies, with their attendant uncertainties. However, it cannot be excluded that HC is of great benefit to an as of yet uncharacterized subset of patients. As to safety issues, there appears to be general agreement that side effects are relatively minor, and HC does not appear to be very dangerous, as would be expected from long experience with HC in connection to malaria and rheumatoid arthritis. There is far more heat than light in the public discourse on HC (internet, editorials, press releases, etc.) It will be difficult to prove benefit or safety to those whose political views inform their judgements, but we believe we have provided a balanced analysis. In addition, the U.S. Food and Drug Administration (FDA) cautions against use of hydroxychloroquine or chloroquine for COVID-19 outside of the hospital setting or a clinical trial due to risk of heart rhythm problems (13). We realize many of the studies represent something akin to battlefield medicine, are meant to save lives, and should generally be applauded, but until more blinded randomized trials are reported, it is difficult for us to ascribe value or harm to HC.

 

 

  1. KIPLIN GUY, ROBERT S. DIPAOLA, FRANK ROMANELLI, REBECCA E. DUTCH. Rapid repurposing of drugs for COVID-19. SCIENCE22 MAY 2020 : 829-830.
  2. Xue et al., Chloroquine is a zinc ionophore. PLoS One 9, e109180 (2014).
  3. J. te Velthuis et al., Zn(2+) inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture. PLoS Pathog 6, e1001176 (2010).
  4. Hoffmann et al., Chloroquine does not inhibit infection of human lung cells with SARS-CoV-2. Nature, (2020).
  5. R. Boulware et al., A Randomized Trial of Hydroxychloroquine as Postexposure Prophylaxis for Covid-19. N Engl J Med 383, 517-525 (2020).
  6. P. Skipper et al., Hydroxychloroquine in Nonhospitalized Adults With Early COVID-19: A Randomized Trial. Ann Intern Med, (2020).
  7. G. S. Borba et al., Effect of High vs Low Doses of Chloroquine Diphosphate as Adjunctive Therapy for Patients Hospitalized With Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection: A Randomized Clinical Trial. JAMA Netw Open 3, e208857 (2020).
  8. B. Cavalcanti et al., Hydroxychloroquine with or without Azithromycin in Mild-to-Moderate Covid-19. N Engl J Med, (2020).
  9. Tang et al., Hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, randomised controlled trial. BMJ 369, m1849 (2020).
  10. Arshad et al., Treatment with hydroxychloroquine, azithromycin, and combination in patients hospitalized with COVID-19. Int J Infect Dis 97, 396-403 (2020).
  11. Salvarani et al., Susceptibility to COVID-19 in patients treated with antimalarials: a population based study in Emilia-Romagna, Northern Italy. Arthritis Rheumatol, (2020).
  12. Geleris et al., Observational Study of Hydroxychloroquine in Hospitalized Patients with Covid-19. N Engl J Med 382, 2411-2418 (2020).
  13. 2020. https://www.fda.gov/drugs/drug-safety-and-availability/fda-cautions-against-use-hydroxychloroquine-or-chloroquine-covid-19-outside-hospital-setting-or

RENOWNED DOHERTY INSTITUTE IN AUSTRALIA INDEPENDENTLY VERIFIES EARLIER FINDINGS THAT AN ANTIMICROBIAL TECHNOLOGY ERADICATES SARS-COV-2 ON SURFACES FOR MORE THAN SIX WEEKS

The Findings Corroborate Research Previously Released by the Rega Medical Research Institute of KU Leuven, Belgium

Baltimore, Maryland, USA, August 13, 2020:  The Global Virus Network (GVN), a coalition comprised of the world’s preeminent human and animal virologists from 55 Centers of Excellence and 10 Affiliates in 33 countries, announced that the Peter Doherty Institute for Infection and Immunity in Melbourne, Australia released of a report on extensive tests independently confirming the Rega Medical Research Institute of KU Leuven, Belgium findings that a BIOPROTECT™ formulation by ViaClean Technologies eradicates SARS-CoV-2 (the unique coronavirus that causes COVID-19) on surfaces and provides continuous residual viricidal activity for more than six weeks.  The announcement was made today by Dr. Christian Bréchot, President of the GVN.

The Doherty and Rega Institutes both used state-of-the-art high containment virology facilities to independently conduct extensive tests on a BIOPROTECT™ formulation to study its effects on SARS-CoV-2 infectivity on various surfaces.  The standard ASTM E1053 test methodology was adapted to assess SARS-CoV-2 viricidal efficacy of microbicides on environmental surfaces.  GVN scientists at the Doherty Institute under the direction of Prof. Damian Purcell, and at the Rega Institute under the direction of Prof. Johan Neyts, definitively demonstrated that the BIOPROTECT™ formulation eliminates SARS-CoV-2 by both reducing its ability to be infectious and by destroying its genomic material.

“Our studies on numerous antiseptic agents for surfaces contaminated with SARS-CoV-2 show that the BIOPROTECT™ formulation’s long-lasting activity is far superior to conventional decontamination agents in general use,” said Prof. Damian Purcell, Head of the Molecular Virology Laboratory in the Department of Microbiology and Immunology at The Peter Doherty Institute for Infection and Immunity at The University of Melbourne.  The Doherty Institute’s report is accessible here.

The tests were conducted in both “wet” and “dry” conditions.  In the wet test, SARS-CoV-2 was coated on stainless steel disks which were then treated with a wet solution of the BIOPROTECT™ formulation. In the dry test, the BIOPROTECT™ formulation was first applied to stainless steel samples which, 46 days later, were then exposed to a high titer of SARS-CoV-2.  Proving the longevity of the BIOPROTECT™ formulation on treated surfaces, tests revealed that the presence of the BIOPROTECT™ formulation maintained the ability to inactivate SARS-CoV-2 to negligible levels.  Furthermore, test results from Rega demonstrated that the disks pretreated with the BIOPROTECT™ formulation averaged a 99.7% inactivation of the SARS-CoV-2 virus.  All tests conducted were designed to conform with the United States Environmental Protection Agency (EPA) and equivalent standards of regulatory agencies in Europe and Australia, to ensure the acceptability and credibility of the results.

“We tested BIOPROTECT™ formulation and found that it eliminated 99.7% of the SARS-CoV-2 present, 46 days after the tested material was treated with BIOPROTECT™ formulation,” said Dr. Johan Neyts, Professor of Virology at the Rega Institute for Medical Research, KU Leuven.  “This product is unique and its long-lasting ability to eliminate SARS-CoV-2 far exceeds conventional disinfectants, which makes it very helpful in the battle against COVID-19.”  The Rega Institute’s report is accessible here.

“The results of the tests conducted by the Doherty and the Rega Institutes clearly demonstrate that BIOPROTECT™ eradicates SARS-CoV-2 on surfaces and provides continuous residual antimicrobial protection for an extended period of time,” said Dr. Bréchot.  “It is clear that effective antimicrobials will be extremely important in containing the COVID-19 pandemic, given the time it will take to implement mass vaccination and fully develop novel therapies.  In this context, we are not aware of any microbicide surface treatment that continuously prohibits the growth and surface transmissibility of SARS-CoV-2 for an extended period of time.  This represents a significant breakthrough in inhibiting the spread of COVID-19 by preventing surfaces from being contaminated by the virus and stopping the spread of the virus through contact with contaminated surfaces. Identifying and exploring innovative solutions, as well as fostering and facilitating collaboration between academic and industrial partners, be it large pharmaceutical firms or small biotech companies, is one of several ways the GVN can make a consequential contribution to the fight against COVID-19.”

An official statement by Dr. Robert Gallo and Dr. Christian Bréchot on the two GVN Centers of Excellence independent verification of antimicrobial technology that eradicates SARS-CoV-2 on surfaces for more than six weeks can be found here.

 

#          #          #          #

 

About the Global Virus Network (GVN)

The Global Virus Network (GVN) is essential and critical in the preparedness, defense and first research response to emerging, exiting and unidentified viruses that pose a clear and present threat to public health, working in close coordination with established national and international institutions. It is a coalition comprised of eminent human and animal virologists from 53 Centers of Excellence and 10 Affiliates in 32 countries worldwide, working collaboratively to train the next generation, advance knowledge about how to identify and diagnose pandemic viruses, mitigate and control how such viruses spread and make us sick, as well as develop drugs, vaccines and treatments to combat them. No single institution in the world has expertise in all viral areas other than the GVN, which brings together the finest medical virologists to leverage their individual expertise and coalesce global teams of specialists on the scientific challenges, issues and problems posed by pandemic viruses. The GVN is a non-profit 501(c)(3) organization. For more information, please visit www.gvn.org. Follow us on Twitter @GlobalVirusNews

 

About the Peter Doherty Institute

Located in the heart of Melbourne’s Biomedical Precinct, the Doherty Institute is named in honor of Patron, Laureate Professor Peter Doherty, winner of the 1996 Nobel Prize in Physiology or Medicine for discovering how the immune system recognizes virus-infected cells. Under the expert guidance of Director, University of Melbourne Professor Sharon Lewin, a leader in research and clinical management of HIV and infectious diseases, the Doherty Institute has more than 700 staff who work on infection and immunity through a broad spectrum of activities. This includes discovery research; diagnosis, surveillance and investigation of infectious disease outbreaks; and the development of ways to prevent, treat and eliminate infectious diseases.

 

About the Rega Institute of Medical Research

The Rega Institute was founded in 1954 by Professor Piet De Somer and named after the 18th century philanthropist and professor Josephus Rega of Leuven. It hosts part of the Department of Microbiology and Immunology. Since its inception, the Rega Institute hosts also the Section of Medicinal Chemistry of the Department of Pharmaceutical Sciences and it is thus a true interdepartmental and interdisciplinary research institute. The Rega Institute has always been a jewel in the crown of research and innovation at KU Leuven on the basis of publications, citations and prestigious scientific prizes of its members.

GVN MEDIA CONTACT
Nora Samaranayake
Phone:  410-706-1966
Email:    [email protected]

The Role of Cytokine Storm in the Severity of COVID-19

There are two ways in which pathogens make us sick. One is by the direct effects of the pathogen itself. The other is by collateral damage from our hyperactive immune responses to the pathogen by the release of interferons (IFNs), interleukins (ILs), tumor-necrosis factors (TNF-α), chemokines, and several other mediators. This latter mechanism has appeared to play major roles in many severe cases of COVID-19. Mortality in COVID-19 patients has been linked to the presence of the so-called “cytokine storm” induced by SARS-CoV-2. Excessive production of proinflammatory cytokines leads to acute respiratory distress syndrome (ARDS) aggravation and widespread tissue damage resulting in multi-organ failure and death (1).

Cytokine storm is difficult to define. There is an excellent review of the concept found here.  It is generally thought to involve aberrant reactivity by the innate immune system, dysregulated inflammatory reactions, and over-expression of inflammatory cytokines. In particular, over-expression of IL-6 is thought to be a hallmark of the cytokine storm. In many reported cases, levels of IL-6 were significantly higher in severe cases than in mild cases. However, one study pointed out that reported levels of IL-6 in the ARDS stage of COVID-19 are one to two orders of magnitude lower than those of non-COVID-19 cases or ARDS(2). As we will further discuss in detail, drugs that target the IL-6 pathway have shown promising results in treating Covid-19 patients. Other over-expressed pro-inflammatory soluble factors include IL-2, TNF-α, and IL-1β.

What are the differences in immune system between severely and mildly ill patients?  A number of studies have attempted to determine the critical differences. The general findings are of elevated serum inflammatory cytokines and pro-inflammatory factors mostly with elevated levels of IL-6 (3-6). Correlation, however, does not necessarily indicate causation.  It is also plausible that the apparent immune hyperreactivity is a response to poorly controlled viral replication. If that were to be the case, administration of anti-inflammatory drugs could worsen rather than ameliorate disease. In general, several observational studies have concluded that administration of the IL-6 receptor targeting monoclonal antibody tocilizumab resulted in greatly improved outcomes relative to standard of care (7-8). Interestingly, one report indicated that using an IL-6 inhibitor can lead to conditionally beneficial outcomes(7), depending upon when it was administered (based upon a sole significant parameter, the patient’s %O2 requirements). Both groups benefited when comparing their death, intubation and hospital discharge rates to standard of care data. However, the benefits were more striking when treatment was initiated while O2 requirements were still below 45%. This suggests that treatment should be started before the onset of more critical disease.

In contrast, a phase 3 trial with sarilumab, another IL-6 monoclonal antibody, produced by Regeneron and used for rheumatoid arthritis, showed no beneficial effects. It is possible a difference in activities among antibodies accounts for the differences in results, but it does sound a cautionary note in concluding that IL-6 plays a major role.

A large randomized trial was carried out with another anti-inflammatory drug, dexamethasone. Against standard of care, dexamethasone treatment resulted in a strikingly lower loss of life with a 20% lower death rate in patients on oxygen. For less ill patients there was no effect on this treatment. Again, this suggests that the immune response plays a critical role in the late stage of disease. Indeed, a clear benefit was observed by a comparison study between the treatments of another anti-inflammatory drug, colchicine, and standard of care (9). These data suggest that the progress of late stage of disease results from inappropriate immune responses rather than from viral activity overcoming an increasingly active immune response. In addition, over-expression of anti-inflammatory cytokines (i.e., IL-4 and -10) have been observed in COVID-19 patients(10), although this is primarily seen in critically ill patients(11). This is further indicative of further immune dysregulation.

Aside from cytokines and other soluble mediators, what are the cellular and tissue aspects of inflammation that might be indicative of dysregulated cytokine expression? One repeated and robust observation is of an elevated ratio of neutrophils to lymphocytes. There are many reviews and meta-analyses available (12). One consequence of elevated neutrophil levels is generation of reactive oxygen species, which can induce the tissue damage typically observed in severe COVID-19 patients (13). Other markers of inflammation, such as C-reactive protein, are also commonly detected in severe COVID-19 patients. In this case, tissue damage can occur wherever neutrophil infiltration and accumulation occur. Particularly, the vascular endothelium is one of the critically affected tissues (14). Specifically, the endotheliitis observed in severe COVID-19 patients could be a prime cause in multi-organ impaired microcirculatory function, including vascular leakage followed by an increase in thrombus formation. In general, endothelial cells are activated in systemic inflammation, and exaggerated activation can lead to multi-organ failure, as occurs in sepsis(14).

Dysregulation of another component of the immune system, involving complement and coagulation, also appears to contribute to the late COVID-19 pathology. It should be noted that endothelial cells are intimately involved in regulating complement and coagulation activities. In a large retrospective observational study), a history of coagulation and complement disorders (e.g., thrombocytopenia and macular degeneration) and the presence of variants of genes associated with coagulation and complement pathways are significant morbidity and mortality risk factors to COVID-19 patients. In fact, infection with SARS-CoV-2 seems to lead to activation of these pathways.

Neutrophils also produce extracellular traps comprised of plugs of DNA with adherent toxic compounds, such as myeloperoxidase. In alveoli, this can lead to the impairer of lung function. Clinical trials are planned for intratracheal administration of aerosolized recombinant human DNAse to dissolve the DNA plugs(15), similar to what is done to treat cystic fibrosis.

Taken as a whole, the available data suggest that a cytokine storm, in the sense of overexpression of pro-inflammatory cytokines and a dysregulated and overactive immune inflammatory response, is the major contributor to the pathophysiology of the late stage of COVID-19. This may be amenable to the treatment of COVID-19 with immune modulators.

 

  1. Ragab D, Salah Eldin H, Taeimah M, Khattab R, Salem R. The COVID-19 Cytokine Storm; What We Know So Far. Front Immunol. 2020 Jun 16;11:1446. doi: 10.3389/fimmu.2020.01446. PMID: 32612617; PMCID: PMC7308649.
  2. P. Sinha, M. A. Matthay, C. S. Calfee, Is a “Cytokine Storm” Relevant to COVID-19? JAMA Intern Med, (2020).
  3. G. Chen et al., Clinical and immunological features of severe and moderate coronavirus disease 2019. J Clin Invest 130, 2620-2629 (2020).
  4. N. Chen et al., Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. Lancet, (2020).
  5. R. H. Manjili, M. Zarei, M. Habibi, M. H. Manjili, COVID-19 as an Acute Inflammatory Disease. J Immunol 205, 12-19 (2020).
  6. D. McGonagle, K. Sharif, A. O’Regan, C. Bridgewood, The Role of Cytokines including Interleukin-6 in COVID-19 induced Pneumonia and Macrophage Activation Syndrome-Like Disease. Autoimmun Rev 19, 102537 (2020).
  7. P. Sinha et al., Early administration of Interleukin-6 inhibitors for patients with severe Covid-19 disease is associated with decreased intubation, reduced mortality, and increased discharge. Int J Infect Dis, (2020).
  8. X. Xu et al., Effective treatment of severe COVID-19 patients with tocilizumab. Proc Natl Acad Sci U S A 117, 10970-10975 (2020).
  9. M. Scarsi et al., Association between treatment with colchicine and improved survival in a single-centre cohort of adult hospitalised patients with COVID-19 pneumonia and acute respiratory distress syndrome. Ann Rheum Dis, (2020).
  10. C. K. Wong et al., Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome. Clin Exp Immunol 136, 95-103 (2004).
  11. Y. Zhao et al., Detection and analysis of clinical features of patients with different COVID-19 types. J Med Virol, (2020).
  12. H. Akbari et al., The role of cytokine profile and lymphocyte subsets in the severity of coronavirus disease 2019 (COVID-19): A systematic review and meta-analysis. Life Sci, 118167 (2020).
  13. M. Laforge et al., Tissue damage from neutrophil-induced oxidative stress in COVID-19. Nat Rev Immunol, (2020).
  14. S. Pons, S. Fodil, E. Azoulay, L. Zafrani, The vascular endothelium: the cornerstone of organ dysfunction in severe SARS-CoV-2 infection. Crit Care 24, 353 (2020).
  15. J. P. Desilles et al., Efficacy and safety of aerosolized intra-tracheal dornase alfa administration in patients with SARS-CoV-2-induced acute respiratory distress syndrome (ARDS): a structured summary of a study protocol for a randomised controlled trial. Trials 21, 548 (2020).

 

 

 

View All GVN SARS-CoV-2 Perspectives