Effect of Suppressed Innate Immunity on Covid-19 Severity

One of the important determinants of severe Covid-19 appears to be an inappropriate response by the innate immune system. Specifically, this involves, on the one hand, insufficient or delayed expression and signaling by type 1 interferons (IFNs), which induce innate cell-mediated immunity. The interferon (IFN) response constitutes the major first line of defense against viruses. On the other hand, this involves an overly active inflammatory cytokine response, with its attendant tissue damage (this “cytokine storm” was explored in our previous Perspective at https://gvn.org/category/sars-cov-2/gvn-sars-cov-2-perspectives/). Let’s look at some of the lines of evidence for these contradictory events.

One way in which an aberrant immune response to SARS-CoV-2 is reflected at the cellular level is by a greatly increased ratio of neutrophils to lymphocytes(1). This is correlated with low expression of type I and III interferons and high expression of pro-inflammatory factors including IL-6 and a variety of chemokines(2) that act as attractants for neutrophils and monocyte/macrophages. These activities are the outcome of interactions between host factors that recognize pathogen associated molecular patterns (PAMPs), such as viral RNA in endosomes, and viral proteins that are antagonistic to these factors and their signaling pathways. Genetic differences in host factors can result in profound differences in host responses to pathogens (recent findings are described below). Different viruses also tend to have different or unique antagonists to host immune factors that can greatly influence the outcome of infection.

What are some of the molecular studies that point to defects in interferon activity in severe Covid-19? Recently, interest has been increasing in toll-like receptors (TLRs), especially TLR3 and TLR7, which recognize viral RNA and are important in interferon type I and inflammatory cytokine expression. TLRs play a key role in the recognition of PAMPs and trigger the activation of specific signaling pathways, thereby inducing the transcription of inflammatory and/or anti-inflammatory cytokine. One interesting report looked at two sets of two brothers(3) who, although young and otherwise healthy, had severe Covid-19 (one died). Whole exome sequencing revealed that both sets of brothers had mutations in TLR7, which serves as a sensor for viral RNA. One set had a missense mutation predicted to result in an inactive TLR7, while the other set had a frame shifting 4 nucleotide deletion, resulting in a nonsense protein. Stimulation of primary immune cells in vitro with the TLR agonist imiquimod resulted in defective expression of type I interferon-related genes normally regulated by TLR7. While the limited nature of the study does not permit a conclusion of causality, several factors make it likely that these loss of function mutations are significant. Exhibition of severe disease in young men is rare. Despite rare cases of loss of function mutations in TLR7, two different loss of function mutations in two young brother pairs with severe disease indicate its potential role in Covid-19 severity. It should be pointed out that TLR7 is located on the X chromosome, so a single mutant copy would cause loss of function. One of the mothers was heterozygous for wild type TLR7, making her a carrier. Thus, if problems with the TLR7 pathway exacerbate Covid-19, males might be likelier to have an insufficiency.

Another study analyzed the complete genomes or exomes of 659 patients with life threatening Covid-19 and compared them with those of 534 people with asymptomatic or benign infections(4). They characterized 13 genetic loci encoding factors in the TLR3-interferon regulatory factor 7 (IRF7) pathway, which also regulates type I interferon production and immunity to influenza virus. They found that 3.5% of the people with life threatening Covid-19 had loss of function variants at these loci. Moreover, when immune cells from patients with these variants were tested in vitro, they were found to be defective in type-I interferon immune activities, and further in vivo study confirmed impaired production of type I IFN during the course of SARS-CoV-2 infection. About half of these patients also had extremely low levels of serum interferon α, a type I interferon.

Yet, another study further implicates lack of appropriate interferon activity in severe Covid-19(5). In this study, 101 of 987 patients with life threatening Covid-19 had auto-antibodies against interferon α, interferon ω or both. These were not present in 663 patients with mild disease. The auto-antibodies were able to neutralize the antiviral effects of interferon in vitro (and likely in vivo). Interestingly, the auto-antibodies were about 5-fold more prevalent in men than in women.

None of these studies by themselves show a specific defect in interferon activity in a majority of cases. However, taken together, they certainly suggest that a great variety of different defects related to the antiviral activities of type 1 interferons may be surprisingly common. Probably, one of the factors explains why some people resist serious disease while, for others, it is life-threatening. Further, investigations in this area will be most interesting.

One other study identified a 3p21.31 gene cluster that conferred a risk of severe Covid-19. This region contains three chemokine receptor genes, all involved in innate immunity(6). It turns out to be a region derived from Neanderthals, and is present at variable incidence worldwide except in Africa. This provides yet another clue that the innate immune response to SARS-CoV-2 may be an important determinant of whether an infected individual will, or will not, develop critical Covid-19.

In this light, it has been proposed that some cross-protection could be afforded by administering live attenuated vaccines, such as measles-mumps-rubella, and oral polio vaccine(7) (non-specific effect of vaccination against SARS-CoV-2). The stimulation of innate immunity by these vaccines could provide temporary protection against Covid-19. If proven to be effective against Covid-19, emergency immunization with these vaccines could be used for protection against other unrelated emerging pathogens.

 

References

  1. Y. Liu et al., Neutrophil-to-lymphocyte ratio as an independent risk factor for mortality in hospitalized patients with COVID-19. J Infect 81, e6-e12 (2020).
  2. D. Blanco-Melo et al., Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19. Cell 181, 1036-1045 e1039 (2020).
  3. C. I. van der Made et al., Presence of Genetic Variants Among Young Men With Severe COVID-19. JAMA 324, 663-673 (2020).
  4. Q. Zhang et al., Inborn errors of type I IFN immunity in patients with life-threatening COVID-19. Science, (2020).
  5. P. Bastard et al., Auto-antibodies against type I IFNs in patients with life-threatening COVID-19. Science, (2020).
  6. D. Ellinghaus et al., Genomewide Association Study of Severe Covid-19 with Respiratory Failure. N Engl J Med, (2020).
  7. K. Chumakov, C. S. Benn, P. Aaby, S. Kottilil, R. Gallo, Can existing live vaccines prevent COVID-19? Science 368, 1187-1188 (2020).

Transmission Dynamics of SARS-CoV-2: Superspreaders and Superspreading Events

The concept of Superspreaders and Superspreading events has recently attracted a lot of attention. In fact it is important to understand that most of SARS-CoV-2 infected persons are in fact not contagious! Thus, transmission is really dependent on a handful of individuals we call Superspreaders who nurture Superspreading events. Let’s distinguish between Superspreaders and Superspreading events. Superspreaders are individuals who infect a high number of persons; why?  This is not clear, and this is a most important issue to clarify in the future.  We know they yield high viral load and that they are generally, but not always, young people.  But, this cannot fully explain their massive contamination impact.  Moreover, they are frequently asymptomatic, thereby significantly increasing risk of dissemination. Superspreading events, which involve at least one Superspreader, are events which favor large scale transmission, such as close contacts in indoor situations. Secondary transmissions from infected people then result in a large number of further infections, and so on. Thus, by some estimations  only 20% of infected individuals cause 80% of infections(1).  Identification of Superspreading events depends upon contact tracing.  Furthermore, DNA sequencing of viral genomes adds a great deal to a clearer understanding of these phenomena, thus, confirming the substantial role of Superspreaders in the pandemic. Let’s look at some well-characterized superspreader events to try to better understand how the majority of SARS-CoV-2 infections occur, enabling us to gain an understanding of what might be done to prevent them.

One of the early recognized superspreading events occurred in mid-March in Skagit County, Washington(2) at a 2.5 hr-long choir practice in which 61 people sang in close proximity. Probable or confirmed infections occurred in 87% of the attendees. Given the low incidence of COVID-19 at the time of this event, it is likely that all the infections originated with a single individual, and that the act of singing vigorously launched many viral laden particles into the air. People were relatively closely positioned. Thus, it is likely that superspreading occurs in an unusually favorable environment. Looking at it another way, the virus got “lucky.”  Another study, unrelated to the choir event, analyzed the sequences of 453 viral genomes collected between February and March in Washington(3).  It is possible to infer the likelihood of how many people have been infected by a single person using viral genomic epidemiology, especially given the relative genetic stability of SARS-CoV-2. A phylogenetic analysis strongly suggested that 84% of the 453 viral genomes derived from a single introduction sometime in early February.  In the choir study, it is clear how virus was transmitted. In the genomic study, it is only clear that a single infected individual somehow infected a great number of people through primary, secondary, and other less direct routes.

Another well-studied superspreader event began with a single infected individual in a meat packing plant is Postville, Iowa. In this case, viral spread could be ascertained by both contact tracing and by genomic epidemiology. Fourteen independent viral introductions were identified in the region, but the only virus to spread widely was the one from the meat packing plant. The virus from this individual passed first to numerous other workers, then to family members, then to the community in Postville (87 cases), and finally to other locations in an area of 185 square miles in Iowa, Wisconsin and Minnesota. These conclusions were supported by genomic sequences from 27 different infected individuals. Again, it appears that prolonged close contact indoors facilitated transmission and suggests that this is a critical feature of superspreading events.

Perhaps, the best characterized superspreading event, originating from an international business meeting of Biogen in Boston(4) in which more than 90 individuals became infected.  This event recently received considerable attention. The large number of infections was indicative of a possible superspreading event. A recent study looked at this event and its consequences in detail, using genomic epidemiology(5). They were able to identify and track the virus in question by a single nucleotide polymorphism (SNP), C2416T. Among 80 separate introductions from four continents into the Boston area early in the pandemic, which they inferred from phylogenetic analyses, the C2416 SNP was unique to one virus. Comparing other viral genomes from various parts of the world with C2416T, the parental origin appears to be in Europe, perhaps France, with an estimated most recent common ancestor existing about two weeks prior to the conference aroundFebruary 26-27. Of all samples collected prior to March 10, the only instances of C2416T were from people who had attended the conference, indicating that a superspreading event had indeed occurred there.

Subsequent samples (744) from infected individuals in Boston and surrounding areas were collected over a period from February to June and genomes were sequenced. Remarkably, 35% of the samples had the C2416 SNP. Since no sample prior the March 10 had this SNP, it suggests that the superspreading event at the Biogen meeting February 26-27 resulted in virus from a single individual infecting more than a third of all infected people in the Boston area. Percentages of the C2416T SNP in regions around Boston ranged from 30-46% in four adjacent counties. In addition, a second SNP, G26233T, appears to have emerged during the event, enabling further tracking. Data from this SNP shows a likely export to other states and countries, with further community spread in Virginia, Michigan and Australia. Some caution is warranted, however, since genomic sampling is not generally done on a randomized basis.

The same report looked at infection clusters at homeless shelters, nursing facilities, and a hospital to gain a better understanding of transmission dynamics. They analyzed 193 viral genomes collected from the Boston Health Care for the Homeless Program and identified 4 clusters of 20 or more highly similar genomes, including two clusters containing the C2416T SNP. They also investigated a superspreading event at a skilled nursing facility, in which 82/97 (84%) residents and 36/97 (37%) of staff were infected. In fact, 75% of viral genomes from different individuals had highly similar genomes, suggesting that they arose from a single recent introduction. This took place even though strict interventional measures were in place. Interestingly, two other clusters of three closely related genomes were detected. This represents independent introductions, but these failed to massively spread. In the case of two clusters of infection at Massachusetts General Hospital, highly similar genomes were not found, suggesting a lack of significant in-hospital spread.

What is our conclusion? First, it is now clear that a majority of transmissions result from superspreading events, facilitated by conducive conditions. These include indoor location, close contact, lengthy contact, indoor activity such as singing or talking, poor air ventilation, and lack of mitigation procedures (i.e., wearing masks and physical distancing). Tracing Superspreading events in Hong Kong also confirmed that the largest cluster (106 cases) was traced to four bars followed by a wedding (22 cases) and attendance at a temple (19 cases) (1). This study suggests that disease control efforts should focus on avoiding gathering events and mitigating their impact.  The rapid tracing and quarantine of confirmed contacts, along with the implementation of physical distancing policies including either closures or reduced capacity measures targeting high-risk social settings such as bars, weddings, religious sites and restaurants, should be efficient to prevent the occurrence of superspreading events. Overall, the issue of Superspreaders and Superspreading events illustrates the impact of molecular epidemiology for deciphering the patterns of COVID-19 dissemination. What we still clearly lack, however, is the understanding of the very early phases of the pandemics in China. This would be very useful for the whole appraisal of transmission dynamics.

  1. D. C. Adam et al., Clustering and superspreading potential of SARS-CoV-2 infections in Hong Kong. Nat Med, (2020).
  2. L. Hamner et al., High SARS-CoV-2 Attack Rate Following Exposure at a Choir Practice – Skagit County, Washington, March 2020. MMWR Morb Mortal Wkly Rep 69, 606-610 (2020).
  3. T. Bedford et al., Cryptic transmission of SARS-CoV-2 in Washington state. Science, (2020).
  4. A. Schuchat, C. C.-R. Team, Public Health Response to the Initiation and Spread of Pandemic COVID-19 in the United States, February 24-April 21, 2020. MMWR Morb Mortal Wkly Rep 69, 551-556 (2020).
  5. J. Lemieux et al., Phylogenetic analysis of SARS-CoV-2 in the Boston area highlights the role of recurrent importation and superspreading events. medRxiv, (2020).

Current Status of COVID-19 Vaccine Development

As the COVID-19 pandemic expands, interests in the progress of vaccine development are intensifying. Despite an unprecedented rate of progress, it is still uncertain when a safe, effective vaccine will be available for wide distribution to the public. Successful vaccine development goes through a series of stages, from animal studies for the evaluation of its protective immunogenicity to phase 1 (safety and antibody production), phase 2 (safety and immunogenicity by including a placebo group), and phase 3 (verification of safety, and efficacy in, a large population group) clinical trials. This is obviously a long, drawn out process, yet it is necessary to ensure the efficacy and safety of vaccines. In addition, it takes very high numbers of participants to generate meaningful and significant statistics to prove vaccine protection. This makes these trials expensive and their enrollment process lengthy, but phase 3 trials are clearly necessary, and are the most important step for its approval.

Immunogenicity studies are especially critical because there is not yet a clear understanding of what constitutes a protective immune response. Neutralizing or IgG antibody titers against the spike (S) protein do not seem to correlate inversely with disease severity, although it may be that rapid expression of such antibodies would be protective. Another issue is that neutralizing antibodies may only last a few months. However, immune memory cells may facilitate rapid production of such antibodies after infection. Even less is known about the role or relevance of T cell responses in protection. With all these caveats in mind, we will discuss nine candidate vaccines that are in the most advanced stages of development.  Most, but not all, are focused exclusively on the S protein, in large part because it is the target of neutralizing antibodies.

There are currently two vaccines in the late stage of development that depend upon injection of mRNA encoding the spike protein or portions including the receptor binding domain (RBD) of the S protein. These have an advantage of being easy to produce but have the disadvantage of needing to be stored at 4°C, requiring a cold chain supply, thus presenting difficulties for use in low-income countries. These two vaccines are produced by Moderna and Pfizer/BioNTech. Its limitations associated with the intracellular instability and inefficient delivery of mRNA have been addressed by chemically modifying the RNA and encapsulating it in lipid nanoparticles.

The Moderna vaccine (mRNA-1273) and one of the two Pfizer vaccines (BNT162b2) encode prefusion conformation of the S proteins. The other Pfizer vaccine (BNT162b1) encodes trimerized soluble S protein receptor binding domains on a peptide linker scaffold. The Moderna vaccine was protective in rhesus macaques. Human phase 1 trial results were reported in June by demonstrating its safety and immunogenicity with induction of binding and neutralizing antibodies equivalent to the levels that are seen in natural infection. The antibody levels persisted until at least day 43 post-vaccination. A phase 2 trial with 600 participants was begun in June. Phase 3 trials to determine efficacy and safety were initiated in August and will have 30,000 participants.

Pfizer decided to concentrate on BNT162b2, as it is equally immunogenic to BNT162b1 but generates fewer side effects. There do not appear to be any reports of trials with non-human primates. Three phase 1 trials showed that both vaccines elicited binding and neutralizing antibodies, but lesser side effects led to the selection of BNT162b2 for phase 3 trials (Publication 1, Publication 2). Trials began in August and, as with the Moderna trials, aim to enroll 30,000 participants.

The other nucleic acid-based vaccine, developed by Inovio (INO-4800), is comprised of DNA encoding the S protein. The DNA vaccine, unlike the mRNA vaccines, is stable at room temperature. The DNA is injected intramuscularly and then electroporated into cells by a hand-held device delivering a brief electric pulse.  The vaccine was partially protective in rhesus monkeys against a viral challenge three months after vaccination as judged by a reduction in viral titers. Inovio claims that antibody and/or T cell responses were induced after two doses of the vaccine in 94% of the 40 participants in Phase 1 trials, but they have not yet published the results. They are scheduling Phase 3 trials for September.

The Novavax vaccine candidate, NVX-CoV2373, is a full-length stabilized spike protein produced in insect cells and formulated into a lipid nanoparticle. Reports from a phase 1-2 trial showed that binding and neutralizing antibodies were elicited(1). Antibody levels were greatly increased, and T cell activities (especially Th1) were induced when NVX-CoV2373 was combined with a saponin-based adjuvant. Phase 3 trials are planned for late 2020.

There are currently three late stage vaccines that use adenoviral vectors to deliver their payloads to express the S protein. These include AstraZeneca/University of Oxford, which uses a chimpanzee adenovirus originally isolated from a chimp stool sample, Johnson and Johnson/Janssen (adeno26) and Cansino/Beijing Institute of Biotechnology (adeno5), the two latter of which are human adenoviruses. All three adenoviral vectors have been genetically modified to render them incapable of self-replication. The reasoning behind the use of a chimp adenovirus was to avoid the possibility that vaccinees previously infected by human adenoviruses would mount in an immune response against the vector, thus diminishing the efficacy of the vaccine.

The AstraZeneca vaccine, ChAdOx1 nCoV-19, was shown to partially protect rhesus macaques from viral challenge(2). Out of 6 vaccinated animals, none showed signs of pneumonia or lung pathology, while 3 of 6 controls developed interstitial pneumonia. The vaccine elicited binding and neutralizing antibodies against the S protein as well as Th1 and Th2 responses. Protection was not, however, sterilizing. Vaccinated animals had reduced viral loads in their lower respiratory tracts compared to controls, but viral loads in the nasopharynx were equivalent in both groups. A randomized phase1/2 trial with >1,000 subjects was injected with either ChAdOx1 nCoV-19 or the same vector with an unrelated antigen(3). The vaccine elicited binding and neutralizing anti-S antibodies as well as a T cell response without exhibiting serious adverse events. ChAdOx1 nCoV-19, currently in phase 3 trials, has recently been in the news because of a potential serious adverse reaction that temporarily halted the trials. A vaccinated women developed a severe spinal inflammation (transverse myelitis), which can occasionally develop following viral infections. She has since recovered, and it is not clear whether this is related to the vaccine. Trials have since resumed in Britain, but the Food and Drug Administration (FDA) has not yet approved resumption in the US. There are two ways to view this event. It could be considered to reflect the speed with which these vaccines are being developed and might be a cause for apprehension.

The Johnson and Johnson vaccine, Ad26.COV2.S, expresses a prefusion conformation of S protein (proline-stabilized S protein) in a human adeno 26 vector. In rhesus macaques, vaccinated animals developed high levels of binding and neutralizing anti-S protein antibodies and a Th1 biased T cell response(4). The authors suggested that neutralizing antibodies, but not cell-mediated immune activities, were correlative on protection. All 20 controls were infected and developed minimal disease after intratracheal and intranasal challenge. Five of six vaccinated animals were protected from detectable infection, and the sixth had a 3-4 log reduction in virus loads. Ad26.COV2.S is currently in phase 1/2 trials with 11,000 subjects that was started in June. Phase three trials are scheduled for September with 30,000 participants.

The CanSino vaccine, Ad5-S-nb2, contains a codon-optimized gene expressing the S protein. In rhesus macaques, a single dose elicited neutralizing and S protein binding antibodies and activated cell mediated immune responses after intramuscular inoculation(5). Intranasal inoculation induced antibody production but only weak cellular immunity. In an open label non-randomized trial, the vaccine was immunogenic in humans and generally well tolerated with the main adverse effect of being pain(6).  A phase 2 trial with ~600 participants confirmed immunogenicity and safety(7).

There are three vaccines, developed by Sinovac, Beijing Institute of Biological Products, and Sinopharm, that are based upon chemically inactivated whole SARS-CoV-2. These vaccines, unlike the others, contain all the viral structural proteins, and thus, might be expected to induce a wider T cell response than the other vaccines, which contain only the S protein. The Sinovac candidate, Coronavac, elicited neutralizing and binding antibodies against the S protein(8). The highest vaccine dose protected animals completely against an intratracheal challenge, and lower doses prevented severe interstitial pneumonia and resulted in greatly reduced vial loads. In a phase 1/2 trial, Sinovac claimed that 90% of the volunteers developed neutralizing antibodies and had no serious adverse effects. There was no sign of antibody-dependent enhancement within the time frame reported. Sinovac initiated phase 3 trials in Indonesia and Brazil in August and is planning another trial in Bangladesh. Another inactivated virus vaccine (BBIBP-CorV), developed by the Beijing Institute of Biological Products, induced anti-S protein binding and neutralizing antibodies in rhesus macaques and cynomolgus monkeys and protected rhesus macaques from intratracheal challenge(9). BBIBP-CorV will soon be entering human trials. Two other similarly inactivated whole vaccines, produced by Sinopharm, induced neutralizing antibodies in phase 1 trials and had no serious adverse effects(10). Phase 3 trials with this vaccine were started in July in the UAE. 

The speed of vaccine development with which this has happened is remarkable. The general take home message gleaned from an overview of these vaccines is that they induce neutralizing antibodies, stimulate T cell-mediated activity, and partially or completely protect non-human primates from infection and/or serious disease. None appear to cause an undue level of adverse events. The most pressing question is of course when one or more will be available. However, many uncertainties remain given the lack of robust clinical data. We still need to wait for finalization of phase III trials to confirm the safety and efficacy of the vaccine candidates. In particular, potential induction of antibody-dependent enhancement could be a concern. Immunogenicity of vaccine candidates are focused on the induction of neutralizing antibodies. Furthermore, they are mostly administrated by using the intramuscular route, thus limiting the induction of mucosal immunity. Intranasal immunization approach also needs to be considered. In addition, most vaccine candidates might require two doses (prime and boost vaccinations) to enhance their protective efficacy. For a global vaccination, this poses challenges financially and logistically. Therefore, we also need to consider the non-specific protective effects of live vaccines based on stimulation of innate immunity and trained innate immunity (i.e. epigenetic changes induced by live vaccines).

References

 

  1. C. Keech et al., Phase 1-2 Trial of a SARS-CoV-2 Recombinant Spike Protein Nanoparticle Vaccine. N Engl J Med, (2020).
  2. N. van Doremalen et al., ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques. Nature, (2020).
  3. P. M. Folegatti et al., Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial. Lancet 396, 467-478 (2020).
  4. N. B. Mercado et al., Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques. Nature, (2020).
  5. L. Feng et al., An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2 challenge in rhesus macaques. Nat Commun 11, 4207 (2020).
  6. F. C. Zhu et al., Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial. Lancet 395, 1845-1854 (2020).
  7. F. C. Zhu et al., Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo-controlled, phase 2 trial. Lancet 396, 479-488 (2020).
  8. Q. Gao et al., Development of an inactivated vaccine candidate for SARS-CoV-2. Science 369, 77-81 (2020).
  9. H. Wang et al., Development of an Inactivated Vaccine Candidate, BBIBP-CorV, with Potent Protection against SARS-CoV-2. Cell 182, 713-721 e719 (2020).
  10. S. Xia et al., Effect of an Inactivated Vaccine Against SARS-CoV-2 on Safety and Immunogenicity Outcomes: Interim Analysis of 2 Randomized Clinical Trials. JAMA, (2020).

 

COVID-19 Vs. Influenza: Influenza Vaccination Amid COVID-19 Pandemic

Severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2), a highly contagious virus, emerged in 2019 from Wuhan, China (1). It rapidly spread around the world causing a novel acute respiratory disease, coronavirus disease 2019 (COVID-19). The World Health Organization (WHO) declared COVID-19 a pandemic on March 11, 2020. Consequently, the current COVID-19 pandemic impacts global health and economies to unprecedented levels. As of August 17, 2020, over 21,760,000 cases have been confirmed in more than 188 countries, with over 776,580 deaths, and growing daily. The spectrum of disease with SARS-CoV-2 ranges from asymptomatic infection to severe, often fatal disease. Patients with mild disease (80%) have fever, cough, sore throat, loss of smell, headache, and body aches (2). A surge of COVID-19 patients resulted in enormous challenges for capacity and patient flow in hospitals and health care systems globally. Currently, we have limited interventional strategies in curbing COVID-19, and attention has been focused on the progress in the development of vaccines and therapeutics since the beginning of pandemic. Despite the progress, one cannot exclude that the virus would be continuously circulating as a seasonal virus even after the availability of a vaccination program.

Seasonal influenza is a major cause of morbidity, mortality, resulting in a burden on  healthcare services globally every year. According to the WHO, up to 650,000 deaths are associated with seasonal influenza respiratory infections annually. In the Northern Hemisphere, the 2020-2021 influenza season will coincide with the continued circulation of SARS-CoV-2. The nature of disease similarity between COVID-19 and influenza is cause for great concern. In addition, SARS-CoV-2 and influenza viruses have similar transmission characteristics. The two viruses are spread by contact and airborne transmission. The incubation period for influenza is short, typically 1–2 days, whereas for SARS-CoV-2, it is 4.5–5.8 days (2). The basic reproductive rate (R0, the average number of secondary transmissions from one infected person) for SARS-CoV-2 is estimated to be 2·5 (range 1·8–3·6) compared with 2·0–3·0 for the 1918 influenza pandemic, 1·5 for the 2009 influenza pandemic, and 1.3 for seasonal influenza viruses (3, 4). COVID-19 mortality risk has been highly concentrated at old ages (> 65 years old) and those, in particular, males, with underlying medical conditions (called co-morbidities), including hypertension, diabetes, cardiovascular disease, and immunocompromised states (2). Furthermore, SARS-CoV-2 can also infect younger individuals. In particular, children have shown to be susceptible to infection (5). Although most of the infections run a rather benign course, some children may develop severe primary and unique secondary inflammatory complications of infection, including multisystem inflammatory syndrome of children (6). Indeed, while children comprise 22% of the U.S. population, recent data show that 7.3% of all cases of COVID-19 in the U.S. reported to the Centers for Disease Control and Prevention (CDC) were among children (as of August 3rd, 2020). The number and rate of cases in children in the U.S. have been steadily increasing from March to July 2020, even though the incidence of SARS-CoV-2 infection in children is known to be underrated due to a lack of widespread testing. Opening schools in many locations might change a dynamic of transmission of SARS-CoV-2 and COVID-19 cases among children. Similar to COVID-19, influenza-associated excess mortality in elderly individuals related to a range of other chronic health conditions, including cardiovascular causes, diabetes, neoplasms and renal disease (2). In contrast to COVID-19, children are believed to have the highest rates of infection and complications arising from influenza, thus leading to high rates of excess outpatient visits, hospital admissions and antibiotic prescriptions (7). Infections among children can also drive influenza epidemics due to their increased susceptibility to infection and greater contribution to the spread of virus in the community.

Vaccination can be the most efficient and effective measures in controlling the current COVID-19 pandemics. Researchers are developing more than 170 vaccines against the coronavirus, and 47 vaccines are in human trials. In contrast, annual influenza vaccination is available with inactivated influenza vaccines, recombinant influenza vaccine, and live attenuated influenza vaccine. This the main public health intervention in reducing the burden of disease (8). The WHO has recognized some priority target groups for annual influenza vaccination, including pregnant women, children aged 6 months to 5 years, the elderly, subjects with specific chronic conditions, healthcare workers, and international travelers (9). However, influenza vaccination rates among children aged 6 months to 17 years remain low compared with other routinely recommended childhood vaccines. In-plan vaccination coverage during the 2016–17 season was 67.7% in infants (born 2015), 49.5% in toddlers (born 2012–2014), 35.0% in school-aged children (born 2004–2011), and 22.3% in teenagers (born 1999–2003) (10). Like vaccination coverage, vaccination opportunities decreased with age. Along with continued efforts to reduce missed opportunities, effective strategies to bring children to their doctor for annual influenza vaccination are needed, particularly for older children. Among adults, influenza vaccination coverage (≥18 years) was 45.3% in the U.S. during 2018–19 influenza season (11).

The information regarding COVID‐19 and influenza coinfection is limited. Unless screening patients with COVID‐19, the coinfection remains undiagnosed and underestimated. The severity of disease resulting from the co-infections varies by causing a more severe course with a fatal outcome or mild illness (12). Although this needs to be further evaluated, influenza immunization for high-risk groups can reduce the possibility of influenza infection and co-infection with SARS-CoV-2 and complications associated with diagnostics and antiviral treatment. A COVID-19 infection prediction model has also shown that influenza vaccines could reduce COVID-19 infection risk (13). This will also alleviate burden on the health care system by avoiding an overload of health services and hospitals associated with influenza infections (i.e., outpatient illnesses, hospitalizations, and intensive care unit admissions). Influenza vaccine is safe for elderly and children with a proven record over the past 50 years (7). Therefore, influenza vaccination can be a critical component of response to the COVID-19 pandemic. However, there has been a prediction that the COVID-19 pandemic could decrease influenza vaccination, since the pandemic resulted in a 38 percent drop in consumer spending on health care and loss of health insurance (14). In response, CDC already arranged for an additional 9.3 million doses of low-cost flu vaccine for uninsured adults, up from 500,000. The agency expanded plans to reach out to minority communities. It is uncertain how this upcoming influenza season will evolve under the current circumstance. In general, taking an influenza vaccine can be a good preventive strategy for public health.

 

Readers’ Comments are Welcome

 

References

  1. 2020. Rolling updates on coronavirus disease (COVID-19). July 31, 2020. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/events-as-they-happen.
  2. Subbarao K, Mahanty S. Respiratory Virus Infections: Understanding COVID-19. Immunity. 2020;52(6):905-909. doi:10.1016/j.immuni.2020.05.004.
  3. Petersen E, Koopmans M, Go U, Hamer DH, Petrosillo N, Castelli F, Storgaard M, Al Khalili S, Simonsen L. Comparing SARS-CoV-2 with SARS-CoV and influenza pandemics. Lancet Infect Dis. 2020 Jul 3;20(9):e238–44. doi: 10.1016/S1473-3099(20)30484-9.
  4. Biggerstaff, M., Cauchemez, S., Reed, C. et al. Estimates of the reproduction number for seasonal, pandemic, and zoonotic influenza: a systematic review of the literature. BMC Infect Dis 14, 480 (2014). https://doi.org/10.1186/1471-2334-14-480
  5. Han MS, Choi  EH, Chang  SH,  et al.  Clinical characteristics and viral RNA detection in children with coronavirus disease 2019 in the Republic of Korea.   JAMA Pediatr. Published online August 21, 2020. doi:10.1001/jamapediatrics.2020.3988.
  6. Feldstein LR, Rose  EB, Horwitz  SM,  et al; Overcoming COVID-19 Investigators and the CDC COVID-19 Response Team.  Multisystem inflammatory syndrome in U.S. children and adolescents. N Engl J Med. 2020;383(4):334-346. doi:10.1056/NEJMoa2021680.
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  12. Co-infection with COVID-19 and influenza A virus in two died patients with acute respiratory syndrome, Bojnurd S.A. Hashemi, S. Safamanesh, M. Ghafouri, M.R. Taghavi, M.S. Mohajer Zadeh Heydari, H. Namdar Ahmadabad et al. Iran. J Med Virol (2020), 10.1002/jmv.26014
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  14. Health System Tracker. 2020. How have healthcare utilization and spending changed so far during the coronavirus pandemic? https://www.healthsystemtracker.org/chart-collection/how-have-healthcare-utilization-and-spending-changed-so-far-during-the-coronavirus-pandemic/#item-start

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.

 

 

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  13. D. Wrapp et al., Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies. Cell 181, 1004-1015 e1015 (2020).

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.

 

 

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  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

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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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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