Will Neutralizing and Therapeutic Antibodies Play a Role in the Treatment of COVID-19?

Interest has increasingly been focused on the potential of virus-neutralizing monoclonal antibodies (mAbs) to treat COVID-19 by passive immunization. These antibodies generally target the viral spike protein to prevent infection by blocking ACE2 receptor binding to its receptor binding domain (RBD). There are several issues, however, that need to be addressed to determine when and how they might be used. First, the levels of neutralizing antibodies present in COVID-19 patients do not always correlate to the severity of the disease, and the mean time for seroconversion in SARS patients is known to be about 2 weeks after disease onset. It is possible that timing is critical; i.e., perhaps lack of such antibodies early in infection allow vigorous viral replication, and high levels of antibody production after the development of symptom can be too late to do much good. It may be that the T cell response also plays an important role in the course of infection. Second, although immune serum from recovered patients has been touted as a potential therapy with some successful cases, evidence of its effectiveness is at best contradictory (1).

Another consideration is the nature of the antibodies themselves. This is challenging because protective immunity against SARS-CoV-2 remains unknown. Specifically, the levels and types of antibodies required for the protection need to be defined. Neutralizing antibodies against SARS-CoV-2 appear to have two sets of targets. Some bind the RBD, while others bind the spike protein outside the RBD. Some antibodies bind to the spike protein but do not neutralize the virus (non-neutralizing antibodies). Yet another consideration is that some neutralizing antibodies can lead to antibody-dependent enhancement (we have covered this in an earlier GVN Perspectives) when they are at sub-optimal concentrations. It is obvious that great care must be taken in selecting mAbs for therapeutic development. It also seems intuitive that a mixture of mAbs targeting different epitopes would provide an extra measure of protection. In clinical use, successful treatment will depend critically upon the time of mAbs administration, doses, levels of concentrations, and duration of treatment.

The general approach to identify effective antibodies has been to identify infected patients with high titers of neutralizing antibodies, sort and recover their memory B cells, sequence the heavy and light chain of mRNAs from single sorted cells to characterize the antibody produced by each cell, synthesize their mRNAs with codon optimization for high levels of expression, clone them into expression vectors and express them in transfected cells, and then screen the resultant antibody library. This has become a standardized approach to generate recombinant neutralizing antibodies (2-8). In general, they fall into two categories. The primary group of antibodies, as might be expected, targets the receptor binding domain (RBD) of the spike protein, presumably blocking its binding to the ACE2 receptor. Others bind the N-terminal domain (NTB) of the spike protein. It is not clear how these NTB antibodies neutralize SARS-CoV-2. Presumably, this is by causing allosteric changes in the tertiary or quarternary structure of the spike trimer. Currently identified neutralizing antibodies against SARS-CoV-2 are closely related to germline sequences and do not show signs of hypermutation. This indicates that neutralizing antibodies have been derived with relatively few changes from germline sequences and is a favorable sign for successful vaccine development. It would likely be best for mAb therapeutic use to include antibodies targeting both the RBD and the NTD domains of the spike protein. It should be pointed out that most neutralization assays were done with Vero (monkey kidney) cells, which are not infected by the same pathway as human airway cells(9), although ACE2 binding is required for both pathways.

Importantly, administration of neutralizing mAbs have proven to be protective in several animal models of COVID-19, including rhesus macaques, hamsters, and mice expressing human ACE2(3, 6, 8, 10-12). It is important to note that the mAbs were administered prior to or shortly after challenge. Thus, the results apply to prophylactic or early therapeutic use, but not necessarily to general therapeutic use.

As with other antivirals, it will likely be critical to administer mAbs early in disease, or as a prophylactic. Many patients with serious disease already have high levels of neutralizing antibodies, but these do not appear to ameliorate disease severity, presumably because dysregulated immune responses are driving pathogenesis in these settings. It will also be important to determine what constitutes an effective dose as well as the biologic half-life of any protective effect.

We should mention some recently described “dark horses” for prophylactic passive immunization. These are engineered single chain single domain antibody-like proteins, called nanobodies, that are derived from camelid species (llamas, alpacas). Camelids make normal antibodies, but they also make antibodies comprised of a single heavy chain containing constant and hinge domains linked to a variable region capable of binding antigens. Interestingly, the variable domain alone is also capable of binding antigens. The variable domain is about 1/10th the size of normal antibodies; hence the name nanobody. Because of this, they are far more stable than normal antibodies, allowing them to be lyophilized, heated, and aerosolized. Their small size makes them able to penetrate tissue more readily than do normal antibodies. And they are easy to produce, which makes them scalable.

Nanobodies that are able to bind the SARS-CoV-2 spike protein (both within the RBD and outside the RBD) were identified from a yeast library of synthetic nanobody sequences. Their tiny size allowed them to be trimerized with gly-ser linkers, thus yielding a multivalent molecule with greatly increased potency.  They were then subjected to saturation mutagenesis and affinity maturation, yielding at least one nanobody with picomolar neutralization potency and sub-picomolar affinity. Similar nanobodies have been isolated from a llama immunized with stabilized SARS-CoV-2 spike protein(13). Potency was increased by creating a bivalent molecule with two linked nanobodies.

Because of their size and stability, nanobodies are easily produced and stable to aerosolization. This should make it feasible to package them in inhalers. Introduction to airway tissue would be direct to potential sites of infection. They might, thus, serve as an effective prophylactic, since it would be simple to self-administer them daily. If so, they could be a game changer, but as with other potential therapeutic possibilities, the proof will be in the results.

More than 70 antibody therapies are being developed for the treatment of COVID-19. There has been anticipation that monoclonal antibodies may provide short-term protection from SARS-CoV-2 and could serve as important components of the COVID-19 pandemic response until vaccines become available. Two Phase 3 clinical trials are currently underway in the US. In general, mass-produced antibodies are complex to manufacture and are expensive. Therefore, their availability in low- and middle-income countries can be very limited. It will be also important to have a breakthrough to produce cost-effective, large quantities of antibodies.

 

 

  1. L. Li et al., Effect of Convalescent Plasma Therapy on Time to Clinical Improvement in Patients With Severe and Life-threatening COVID-19: A Randomized Clinical Trial. JAMA, (2020).
  2. P. J. M. Brouwer et al., Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability. Science 369, 643-650 (2020).
  3. Y. Cao et al., Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients’ B Cells. Cell 182, 73-84 e16 (2020).
  4. X. Chi et al., A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. Science 369, 650-655 (2020).
  5. B. Ju et al., Human neutralizing antibodies elicited by SARS-CoV-2 infection. Nature 584, 115-119 (2020).
  6. L. Liu et al., Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike. Nature 584, 450-456 (2020).
  7. Y. Wu et al., A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2. Science 368, 1274-1278 (2020).
  8. S. J. Zost et al., Potently neutralizing and protective human antibodies against SARS-CoV-2. Nature 584, 443-449 (2020).
  9. M. Hoffmann et al., Chloroquine does not inhibit infection of human lung cells with SARS-CoV-2. Nature, (2020).
  10. A. O. Hassan et al., A SARS-CoV-2 Infection Model in Mice Demonstrates Protection by Neutralizing Antibodies. Cell 182, 744-753 e744 (2020).
  11. J. Kreye et al., A SARS-CoV-2 neutralizing antibody protects from lung pathology in a COVID-19 hamster model. bioRxiv, (2020).
  12. R. Shi et al., A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2. Nature 584, 120-124 (2020).
  13. D. Wrapp et al., Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies. Cell 181, 1004-1015 e1015 (2020).

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.

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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:    nsamaranayake@gvn.org

 

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.

 

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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:    nsamaranayake@gvn.org

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).

 

 

 

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SARS-CoV-2 Salivary Tests

The benefits of saliva sampling for frequent and massive COVID-19 testing

Saliva tests for detection of SARS-CoV-2 RNA and antigens are becoming widely available lately. What are the advantages and disadvantages of sampling saliva over the nasal swabs? Saliva sampling simply involves spitting into a collection container. Recently, the U.S. Food and Drug Administration (FDA) also authorized the first diagnostic test with the option of home-collected saliva samples by using the Spectrum Solutions LLC SDNA-1000 Saliva Collection Device. The collected samples are then sent to a lab for further processing and analysis. Therefore, saliva sampling is much simpler and less uncomfortable than nasal swab sampling. This makes taking a sample at home or point of care much easier and more practical. It does not require collection by trained and protected medical personnel wearing personal protective equipment, thus reducing a considerable risk to healthcare workers. In contrast to nasal swab sampling, this approach is not affected by global shortages of swabs and personal protective equipment. In general, saliva sampling should permit more widespread and frequent testing. It is, of course, important that the test be as reliable and sensitive as the nasal swab test, but these appear to be reasonably similar [(1-3).

The current gold standard for COVID-19 diagnosis is real-time reverse transcription polymerase chain reaction (RT-PCR) detection of SARS-CoV-2 from collected samples. Concomitant with the advent of saliva sampling, techniques to simplify the detection of viral RNA have been introduced by eliminating the need for specific equipment, thermal cyclers. This makes it far more adaptable in resource poor settings, which often don’t have the relatively expensive PCR thermal cyclers. One such technique is reverse transcription loop-mediated isothermal amplification (RT-LAMP), which has been previously used to detect other viruses, including Zika and Ebola. A typical RT-LAMP assay takes place at a constant 63°C and the presence of viral RNA generates a color change in as little as a half an hour. A recent modification of the technique that uses inhibitors of salivary RNAs has been claimed to detect a single copy of viral RNA. These methods are obviously not specific to saliva tests, but use of saliva samples should facilitate a mass testing of SARS-CoV-2.

Several new developments in testing for viral RNA combine LAMP isothermal amplification with a technique called lateral flow, in which amplified samples are applied to a strip and allowed to flow along the strip. Amplified viral cDNA is detected by application of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-CAS12 complex designed to bind to a specific viral sequence. When the CRISPR-CAS12 complex finds and binds its target, it releases a chromophore, which is visualized directly or fluorescently as a specific band on the strip. The CRISPR-CAS12 DETECTR is claimed to have a sensitivity of 95% and specificity of 100% compared to the CDC RT-PCR test (4) despite a concern of false negative results. In addition, STOP (SHERLOCK Testing in One Pot) uses similar technology. STOP has a detection limit of 100 viral RNA copies. Results from both tests are obtained in an hour or less, and analyses of the strips are obviously simple. Positive results are indicated by an exhibition of specific band on the strip.

Of course, all these tests, even though they can use point of care collection, require analyses in laboratories. A true point of care test would be one that could be used without need for laboratory involvement, similar to home pregnancy tests. Several of these kinds of tests are in development. For example, a test would involve placing a drop of saliva onto a device, the size of a quarter, and plugging it in to a smart phone. DNA aptamers on the device bind to viral proteins and then are detected by voltage generated by room temperature electron tunneling. Another potential test uses a microfluidic chip in a cartridge and isothermal amplification. Results can be read and uploaded to a smart phone.

The actual impact of these new technologies still need to be ascertained, yet, they provide a snapshot of innovative testing, not only for SARS-CoV-2, but  for other pathogens. Rapid point of care tests could ultimately be used for screening a large group of people (i.e., airline passengers, concert attendees) especially if they can be linked to smart phones. This could be an important interventional strategy in preventing transmission of the virus and in preparing for future pandemics.

 

  1. L. Azzi et al., Saliva is a reliable tool to detect SARS-CoV-2. J Infect 81, e45-e50 (2020).
  2. M. Baghizadeh Fini, Oral saliva and COVID-19. Oral Oncol 108, 104821 (2020).
  3. K. K. To et al., Consistent detection of 2019 novel coronavirus in saliva. Clin Infect Dis, (2020).
  4. J. P. Broughton et al., CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol 38, 870-874 (2020).

 

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A Statement From the Leadership of the Global Virus Network on the Passing of Renowned Chinese Virologist Yi Zeng

A top cancer researcher and leader in public service is mourned

Baltimore, Maryland, USA, July 23, 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 32 countries, collectively mourns the passing of Professor Yi Zeng, MD, Academician of the Chinese Academy of Sciences, former President of the Chinese Academy of the Preventive  Medicine and former Dean of the College of Life Science and Bioengineering at Beijing University of Technology. Prof. Zeng was best known for establishing the relationship of Epstein-Barr virus (EBV) and nasopharynx cancer, developing EBV serologic tests for nasopharynx cancer early diagnosis, and discovering the first example of co-carcinogenesis in humans, when the combination of EBV  infection and particular carcinogenic products derived from Chinese medicines and foods common to Southern China caused nasopharyngeal carcinoma.  Prof. Zeng was a founding Center Director of China’s Global Virus Network Center of Excellence and hosted GVN’s 7th International Meeting in Beijing, China in 2015.

“Prof. Yi Zeng’s loss is a tremendous one not just for China, but all of his colleagues around the world,” said Robert Gallo, MD, The Homer & Martha Gudelsky Distinguished Professor in Medicine, Co-Founder and Director, Institute of Human Virology (IHV) at the University of Maryland School of Medicine and Co-Founder and Chairman of the International Scientific Leadership Board of the Global Virus Network (GVN). “In 2012, IHV faculty unanimously voted to honor Prof. Zeng for his lifetime of leadership in virology and cancer research.  We are saddened by this immense loss and extend our deepest sympathies to his family and friends.”

“We will deeply miss Prof. Yi Zeng, whose scientific vision and commitment to the GVN have been at the heart of the cooperation with China,” said Christian Bréchot, MD, PhD, President of GVN and Professor at the University of South Florida.

“We are all saddened by the passing of Prof. Yi Zeng, the former president of the Chinese Academy of Preventive Medicine, which is the predecessor of China CDC,” said George F. Gao, DVM, DPHIL (OXON), Director General of the Chinese Center for Disease Control and Prevention (China CDC). “He was a true founder of modern Chinese disease control and prevention and public health infrastructure. He will be remembered as a great scientist, a good friend and a thoughtful mentor.”

Prof. Zeng made great achievements by pioneering, two important virology research areas in China, including, tumor virology and HIV,” said Yiming Shao, MD, the Chief Expert on AIDS, China CDC, who was Prof. Zeng’s first Doctor Degree student.  “Prof. Zeng transformed tumor virology through early diagnosis of cancer, thereby saving countless lives.  He also identified the first HIV/AIDS cases and developed initial diagnostic tools in China while educating his countrymen on AIDS prevention.”

In the early 1970s, Prof. Zeng researched the relationship of the EBV and nasopharynx cancer, established a series of EBV serologic test methods for nasopharynx cancer and increased the diagnosis rate of nasopharynx cancer at the early stage from 20-30% to 80-90%. His serological index could predict the occurrence possibility of nasopharynx cancer 5 to 10 years in advance.  He discovered carcinogens in Chinese herbal medicines and foods in areas with a high incidence of nasopharynx cancer in conjunction with EBV to cause nasopharyngeal carcinoma. Prof. Zeng was also the first to establish cell lines from nasopharynx cancers with high differentiation and low differentiation and was the first in the world to prove that the human fetal nasopharyngeal mucus tissues infected with EBV, under cooperative function of carcinogen TPA and butyric acid, could develop human nasopharynx cancer in rodents. This finding provided the first direct evidence that the EB virus could induce nasopharynx cancer and at the same time provided models for studying multiple factors of nasopharynx cancer pathogenesis and their mechanisms.  Since 1984, Prof. Zeng conducted research on HIV and AIDS and proved the introduction of HIV into China by identifying the first cases of AIDS and HIV infection and isolating the first HIV-1 virus in the country. He isolated the first Chinese HIV-1 virus in 1987 and established the rapid diagnosis method for HIV.  Prof. Zeng, with his late wife Prof. Zelin Li, also discovered Chinese herbal medicines that had a high inhibitory activity of HIV replication.

“For over five decades, Prof. Zeng was a leading virologist in China,” said Lishan Su, PhD, Professor of Immunology and Virology. Lineberger Comprehensive Cancer Center, Department of Microbiology and Immunology, School of Medicine, The University of North Carolina at Chapel Hill.  Prof. Su honored Prof. Zeng with a special lecture when he received the IHV’s 2012 Lifetime Achievement Award in Public Service. “His pioneering work in basic/clinical research on human viruses, including EBV and HIV and on public health policy, has saved millions of human lives. Prof. Zeng also played a critical role in establishing/leading the first institute of modern medical virology to train a generation of outstanding molecular virologists. He has been respected by all, will be missed and remembered in China and around the world.”

GVN is a global authority and resource for the identification and investigation, interpretation and explanation, control and suppression, of viral diseases posing threats to mankind. It enhances the international capacity for reactive, proactive and interactive activities that address mankind-threatening viruses and addresses a global need for coordinated virology training through scholarly exchange programs for recruiting and training young scientists in medical virology. The GVN also serves as a resource to governments and international organizations seeking advice about viral disease threats, prevention or response strategies, and GVN advocates for research and training on virus infections and their many disease manifestations.

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 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

 

Media Contact:
Nora Samaranayake, GVN
410-706-1966
nsamaranayake@gvn.org

Antibody Dependent Enhancement and SARS-CoV-2

When Developing a SARS-CoV-2 Vaccine, Researchers Need to Consider that Antibodies May Enhance Infection Rather than Provide Protection

There is encouraging news from recent clinical trials of SARS-CoV-2 vaccines, including several candidate vaccines that induce neutralizing antibodies with no apparent adverse effects. Their protective efficacy at preventing infections is not yet known, but will be ultimately determined by phase 3 trials. However, there are other potential concerns over vaccine outcomes, one of which is the possibility of inducing antibodies that make infection outcomes worse. One such phenomenon is called antibody dependent enhancement (ADE).

The poster child for ADE is, of course, dengue virus (DENV). Infection with one of the four common serotypes of DENV results in worse outcomes after later infection by a different serotype. Indeed, some tetravalent dengue vaccines mimic a first infection with DENV and cause worse outcomes upon later infection, even though neutralizing antibodies are elicited. It is speculated that a successful immune response to DENV requires a CD8+ T cell response. The recombinant vaccine contains only DENV envelope glycoproteins in the backbone of yellow fever attenuated 17D strain, which can be poor in inducing CD8+ T cell response. Indeed, live attenuated tetravalent DENV vaccines (National Institutes of Health ), which contain all the virion proteins, have provided enhanced protection.

How does ADE work? The most common mechanism appears to occur when a non-neutralizing or poorly neutralizing antibody binds to a virus particle. The fragment crystallizable region (Fc) of the antibody interacts with Fc receptors (FcR) expressed on certain immune cells (i.e., macrophages, B cells, Follicular dendritic cells, natural killer cells, and neutrophils) and some of the complement proteins. This facilitates viral entry into immune cells, shifting the tropism of the virus. If the virus can replicate in macrophages or other FcR-containing cell, it provides new opportunities for viral replication and spreads into neighboring cells. In addition, infection of macrophages can cause adverse immune activities. This phenomenon is often observed when antibody concentrations decrease as a result of waning immunity. In addition, an antibody may neutralize potently at high concentrations but cause enhancement of infection at sub-neutralizing concentrations.

Another way in which vaccination can result in worse disease is by enhanced respiratory disease (ERD). This was seen in children vaccinated against respiratory syncytial virus and involves non-neutralizing antibodies forming complexes that get deposited in airways, thus causing inflammation. There also appears to be priming of cell-mediated immunity towards a Th2 inflammatory type of response.

What are the reasons for thinking that ADE will or will not be a problem with SARS-CoV-2? One example of a coronavirus infection for which ADE seems to present a problem is feline infectious peritonitis virus (FIPV). Kittens inoculated with a vaccinia recombinant vaccine containing the FIPV spike protein developed high levels of non-neutralizing antibodies, but only very low levels of neutralizing antibodies. They suffered far worse infection outcome at a much higher incidence. This phenomenon was not observed when other viral proteins were used instead of spike protein; yet, it should be pointed out that FIPV is an alphacoronavirus, unlike SARS-CoV-2, a betacoronavrus.

There are some data on ADE with SARS-CoV-2-related betacoronaviruses. One study showed that a candidate vaccine containing SARS-CoV-1 spike protein elicited neutralizing antibodies in vaccinated mice. The antibodies, however, potentiated infection of B cells by an FcR-mediated mechanism.  Despite this, the vaccine provided protection to mice, so even though it elicited detectable ADE, it did not cause worse disease. A similar finding was made in hamsters.

ADE activities could be found in SARS-CoV-1-infected humans. Polyclonal antisera or of monoclonal antibodies that bind viral spike (S) protein can facilitate uptake by human monocytic cells via their Fcγ receptors (FcγRs). In the case of Middle Eastern respiratory syndrome coronavirus (MERS-CoV), Fc-mediated targeting has been observed with neutralizing antibodies that bind directly to the receptor-binding domain of S protein. For both viruses, this phenomenon is dependent on antibody concentration.

Low concentrations facilitated ADE, while high concentrations neutralized the virus. In SARS-CoV-1-infected macaques, antibodies to spike protein were associated with fatal acute lung injury, attributed to alterations in pro-inflammatory immune responses. Yan and colleagues found that a monoclonal neutralizing antibody to MERS blocked entry of a MERS-CoV pseudovirus into a typical target cell but facilitated viral entry into cells expressing FcR, such as macrophages, by a canonical viral entry pathway. The effect was attributed to the antibody loosening the spike protein trimeric structure, making it more accessible to proteolytic processing.

What about SARS-CoV-2? What of a vaccine based upon the spike protein alone? Is there a possibility that ADE may play a pathogenic role in natural infection? The reality is that there are far more questions about these possibilities than there are actual data. Epidemiological studies investigating ADE in individuals with multiple SARS-CoV-2 infections or cross-reactivity to common-cold-causing CoVs will likely take several years. One indication comes from the use of convalescent plasma. Administration into COVID-19 patients appeared to be generally safe. This does not necessarily reflect what will happen after vaccination with spike antibody protein or inactivated vaccines. Inoculation with whole inactivated virus protected macaques against subsequent challenge and showed no signs of ADE. Reducing the risk of vaccine-associated enhanced respiratory disease or ADF of replication involves induction of high-quality functional antibody responses and Th1-biased T-cell responses. If antibodies against SARS-CoV-2 with ADE potential are detected, vaccine development efforts can leverage the full suite of modern technologies around epitope mapping, protein design, adjuvant design and delivery to maximize safety. Currently, there are no data showing direct evidence of ADE for SARS-CoV-2 candidate vaccines. The answers will likely come from phase 3 trials, a number of which are underway, in recruitment, or planned. Results are most eagerly awaited.

 

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Further Insights into SARS-CoV-2 Genetic Variability: D614G

Can a single amino acid mutation in the spike protein affect the infectivity and immunogenicity of SARS-CoV-2?

Recently, a great deal of attention has become focused on a specific SARS-CoV2 mutant in which amino acid residue 614 of the spike protein is changed from aspartic acid to the less bulky and more neutral glycine. This mutant, D614G, reported by Korber et al., has become increasingly predominant after first seemingly proliferating in Europe, then spreading rapidly elsewhere. Korber et al. suggested the mutant is more infectious, based on higher viral RNA titers from patients who were infected with it and its rapid prevalence, but it does not appear to be more pathogenic, based upon the clinical pictures of the patients. Residue 614 is not in the receptor binding domain. They suggested that the mechanism of enhanced infectivity could be due to glycine not forming a hydrogen bond with the neighboring spike protein subunit, allowing the subunits to dissociate more readily and thereby facilitating virion fusion with the cell membrane.  There is a stunning virtual reality visualization of this on YouTube.  Korber et al. also reported evidence for recombination between genomes carrying both this mutation as well as other mutations, indicating recombination and simultaneous infection of cells with more than one genotype. Although suggestive, no proof was presented for an actual increase in infectivity by the mutant.

Since that report, several other studies have addressed the issue of infectivity more directly. One caveat related to many of these reports is that they use lentiviral particles pseudo typed with coronavirus envelope proteins. Viral entry is measured by a co-transduced indicator gene, such as luciferase. Although this is thought to faithfully mimic coronaviral entry, it is an inherently artificial system. The general consensus of all these studies is that the G614 variant enters cells expressing the ACE2 receptor better than the D614 variant even though the variable residue is not in the receptor binding domain, that there is no difference in clinical outcome, that infection with the G614 variant results in higher viral RNA titers in nasal swabs, and that there is not a great deal of difference in antibody neutralization (which is good news for vaccine development).

Let’s consider the individual reports separately. Ozono et al. used the lentiviral pseudovirus method to sample five different naturally occurring mutations in the spike protein, including D614G, to characterize their behavior relative to the reference genotype. Their entry characteristics varied from having a lesser to a greater ability to enter cells expressing ACE2 and the protease TMPRSS2 cells, which greatly facilitates SARS-CoV2 entry. Significantly, the D614G mutant showed the most efficient entry. Interestingly, SARS-CoV1 was much more efficient at entry than was SARS-CoV2. They performed an in silico structural analysis that suggested that the SARS-CoV1 spike trimer has a more open configuration that would result in greater accessibility to the ACE2 receptor by the receptor binding domain. They also tested COVID 19 antisera from patients infected with the D614 variant, and showed no detectable differences in neutralization between the D614 and G614 variants.

Hu et al. also used a lentiviral pseudovirus system to analyze the D614G variants. As with Korber et al., they found the G614 variant to be globally distributed. Like Ozono et al., they found about a 2.5-fold increase in entry efficiency by the G614 variant, perhaps due to more efficient protease cleavage of its spike protein. Unlike Ozono et al., they found that a minority of COVID 18 antisera failed to neutralize the G614 variant to the same extent as the D614 variant. However, it is not clear with which variants the serum donors were infected.

Wagner et al. used a more natural but messier approach. They looked at viral loads, as measured by RT-PCR, and clinical status of patients in Washington state infected with either the G614 or D614 variants. They found that patients infected with the G614 variant had higher nasal viral RNA loads, but did not have a more severe clinical picture. The age of the patients infected with G614 skewed slightly younger (~3 years). They also found that G614 became increasingly more prevalent in Washington state over time.

Lorenzo-Redondo et al. (1) reported on patients in Chicago infected with one of what they called three clades of SARS-CoV2. Clade 1, which was introduced from Washington, contains the G614 phenotype, while clades 2 and 3 have the D614 phenotype. The origin of clade 2 was ascribed to Illinois, while clade 3 was introduced from New York. Clade 1 had higher viral loads than clade 2, in agreement with Wagner et al. Interestingly, when bronchial alveolar lavage samples were tested, there was little difference in viral RNA titers between the two clades, suggesting that the increased titers were specific to upper airway tissue. This could be a factor increased spread.

It should be pointed out that all the above results are in the form of preprints. In addition, the methods used to measure entry directly are somewhat artificial. However, taken together directly from patient data, it seems that G614 may in fact be more capable of spreading, perhaps because of more facile entry into cells, perhaps due to better proteolytic processing because of a more open quarternary structure. Fortunately, this mutation does not seem to worsen the clinical outcome of infection, nor does it seem to abrogate recognition by most neutralizing antibodies.

 

References

 

  1. R. Lorenzo-Redondo et al., A Unique Clade of SARS-CoV-2 Viruses is Associated with Lower Viral Loads in Patient Upper Airways. medRxiv, (2020).
  2. Tang, Leyan & Schulkins, Allison & Chen, Chun-Nan & Deshayes, Kurt & Kenney, John. (2020). The SARS-CoV-2 Spike Protein D614G Mutation Shows Increasing Dominance and May Confer a Structural Advantage to the Furin Cleavage Domain. 10.20944/preprints202005.0407.v1.
  3. Grubaugh, N.D., Hanage, W.P., Rasmussen, A.L., Making sense of mutation: what D614G means for the COVID-19 pandemic remains unclear, Cell (2020), doi: https:// doi.org/10.1016/j.cell.2020.06.040.

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Cleveland Clinic and The University of Southern Denmark Join Global Virus Network to Combat Viral Diseases

GVN’s latest additions further bolster its 55 Centers for Excellence, expanding knowledge of viruses and treatment

Baltimore, Maryland, USA, Tuesday, July 7, 2020: The Global Virus Network (GVN), comprising foremost experts around the world in every class of virus causing disease in humans and some animals, today announced the addition of Cleveland Clinic and the University of Southern Denmark, including the Bandim Health Project in Guinea-Bissau, as its two newest Centers of Excellence. GVN is a global authority and resource for the identification and investigation, interpretation and explanation, control and suppression, of viral diseases posing threats to mankind.

“We welcome the inclusion of Cleveland Clinic and the University of Southern Denmark to our network,” said Christian Bréchot, MD, PhD, President of GVN and Professor at the University of South Florida.  “The addition of the renowned Cleveland Clinic will provide expertise and collaboration opportunities for the greater GVN on matters relating to viral-host interactions, including antiviral drug development, immune modulatory therapies and vaccine development.  The University of Southern Denmark will provide a very important contribution to novel approaches for vaccination, and also, it will further increase our outreach in Africa through the Bandim Health Project in Guinea-Bissau.”

Cleveland Clinic, headquartered in Cleveland, Ohio, USA, is a nonprofit, multispecialty academic medical center that integrates clinical and hospital care with research and education. Cleveland Clinic’s health system includes Lerner Research Institute, an integrated research institute performing investigations in basic, translational, and clinical research; Cleveland Clinic Florida Research and Innovation Center in Port Saint Lucie, Florida, which is dedicated to the discovery and advancement of innovative translational research, focuses on immuno-oncology and infectious diseases; and, the newly added Global Virus Network Center in Innate Immunity Research.  Cleveland Clinic has a 30-year history of groundbreaking advances in interferon and cytokine research. Robert Silverman, PhD, Professor at Cleveland Clinic’s Lerner Research Institute will lead this GVN Center.

“We are looking forward to collaborating with other centers in the GVN to work toward fundamental discoveries in host-virus interactions, through shared expertise in a wide range of viral infections,” said Dr. Silverman.  “Furthermore, novel antiviral strategies developed through the GVN may be implemented at Cleveland Clinic.”

The University of Southern Denmark has campuses in seven cities across Denmark and has been an established university for over 50 years. It has recently, as the first university in Denmark, made the 17 United Nations Sustainable Development Goals (SDGs) the focal point for its work as a university. The Bandim Health Project is affiliated with the Department of Clinical Research, which constitutes the university affiliation for all researchers and teachers at Odense University Hospital, Odense. The University of Southern Denmark was selected because of its long history of research into infections and vaccinations. Its key scientific contributions to the field are observations that intensity of exposure is the main determinant of severe viral infections and that vaccines have non-specific effects, affecting susceptibility toward a broad range of pathogens.  The Bandim Health Project works with population-based health research in Guinea-Bissau, one of the world’s poorest countries in West Africa. Christine Stabell Benn, MD, PhD, DMSc, Professor in Global Health at the Department of Clinical Research, University of Southern Denmark, will lead this GVN Center.

“We are honored to be part of this eminent network,” said Dr. Benn. “Vaccines and their non-specific effects may be a very important tool against emerging viral treats, allowing us to bridge the time until specific vaccines can be developed. Much more work needs to be done to understand the non-specific effects, both from an epidemiological and an immunological perspective. As a member of GVN, we will benefit greatly from interacting with the world’s leading medical virologists.”

The GVN enhances the international capacity for reactive, proactive and interactive activities that address mankind-threatening viruses and addresses a global need for coordinated virology training through scholarly exchange programs for recruiting and training young scientists in medical virology. The GVN also serves as a resource to governments and international organizations seeking advice about viral disease threats, prevention or response strategies, and GVN advocates for research and training on virus infections and their many disease manifestations.

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 nonprofit 501(c)(3) organization. For more information, please visit www.gvn.org. Follow us on Twitter @GlobalVirusNews

Media Contact:
Nora Samaranayake, GVN
410-706-1966
nsamaranayake@gvn.org