Rapid and Frequent Testing for COVID-19

The cases of new SARS-CoV-2 infections are currently increasing at an alarming rate, suggesting that winter might bring a large healthcare crisis. Diagnostics have become a critical tool in curbing COVID-19. It is important that accurate results be obtained rapidly and that collection of samples be as fast, simple, and convenient as possible.

What tests are becoming available, which promise better accuracy and greater speed and ease of use?  Thus far, testing has been based on a real-time reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2 RNA detection and serology on enzyme-linked immunosorbent assay (ELISA) for antibody tests. Yet, PCR can show false positives due to detection of fragmented RNA of virus. This has been an obstacle in determining the presence of infectious virus particles in COVID-19 patients. A recent study showed that the presence of infectious virus may be predictable by evaluating the threshold cycle (Ct value, the number of cycles of PCR amplification) (1).  Now, there are also a variety of novel tests for SARS-CoV-2 RNA detection in development that rely on CRISPR12 or 13 cleavage of SARS-CoV-2 amplified RNA, which releases a visually readable fluorescent chromophore. These rely on isothermal amplification, either LAMP(7) or recombinase polymerase amplification (RPA)(8-10), which does not require a thermal cycler. These test all have rapid turnaround times (on the order of an hour) and sensitivities that can be well comparable with other tests. One of these tests can use a cheap chemical hand warmer as the thermal source(9). Another similar CRISPR based assay can simultaneously detect and identify 169 different human viruses(11), illustrating the potential utility and efficiency of this system. These tests all have advantages of speedy detection and user-friendly application over RT-PCR.

Serological testing is also necessary. The presence of antibodies marks an ongoing or past infection, and anti-spike protein and neutralizing antibodies are considered a possible indication of immunity, although T cell activities are also contributing to protective immunity. Antibody tests can indicate past infections but are prone to be false positive in low frequency settings and have provided a difficulty with uneven results because of wide variations in test kit quality. What serological tests are likely to be most useful? As with RNA tests, this requires sensitivity, specificity, speed, and user-friendly application.

One consideration for serological tests is that there is some degree of serologic cross-reactivity of SARS-CoV-2 proteins with those of the other coronaviruses, especially SARS-CoV-1(12). It is also possible that the S2 spike domain of SARS-CoV-2  may also have some degree of cross-reactivity with that of other human coronavirus (13). Interestingly, some of these cross-reactive antibodies also appear to cross-neutralize, suggesting that there may exist conserved epitopes for the development of a broadly effective vaccine.

Most point-of care serologic assays rely in lateral flow-type assays, in which the results are read as bands on a strip and indicate the presence of IgG and/or IgM antibodies. The regulatory processes for these tests has perhaps lacked some of the usual rigor, and their accuracy tends to vary. This can be a problem in low incidence settings, where false positives are an issue. Fortunately, a recently published  study carefully compared five different test kits to ELISA and viral neutralization tests(14). As might be expected, antibodies were more readily detected when sera were collected more than 14 days after symptom onset. Using samples collected from this time period, three lateral flow tests for IgG were comparable to ELISA, but the other two kits were inferior. Prior to 14 days post symptoms, sensitivity was only on the order of 50-60% by any test. It is clear that serological tests are mostly retrospective in nature. They are useful for diagnosing past infections and viral prevalence in populations, and will be very important in evaluating vaccine durability.

In this last aspect, it would be extremely convenient to have a neutralization assay that does not involve live virus, with its attendant requirement for biosafety level 3 containment. One of the studies is based upon inhibition of binding of the receptor binding domain (RBD) of the S protein to immobilized ACE2 protein (15). The results are read colorimetrically and are near 100% sensitive and specific. An advantage of this test is that it would measure neutralization by IgA, IgM, and IgG all at the same time, increase sensitivity. This type of test will obviously be highly useful as vaccines begin to be widely deployed.

Regarding the sampling procedures, we are still using taking nasal swabs; this is a sensitive method but is prone to be false negatives due to poor sampling. Saliva is clearly far simpler to collect than nasal swabs or blood. How do tests of saliva compare with those of nasal swabs and blood?  For RNA detection by RT-PCR, tests of saliva appear to have at least comparable sensitivity to those of nasal swabs(2-4), representing a considerable simplification of testing. Potentially better is RNA detection in saliva using loop-mediated isothermal amplification (LAMP), which avoids the necessity for thermal cycler instrumentation and for RNA purification. Results appear to be equally sensitive and specific to those from RT-PCR(5). Another isothermal method that is fully automated with less sensitivity, delivers results on a dipstick in 90 minutes(6). What about antibodies in saliva? Saliva is much easier to collect. It appears that antibodies are as detectable in saliva as in serum. It’s been shown that viral antibodies persist in saliva for months(16). Another study showed persistent IgG activity against both N protein and the RBD of the S protein(17). The anti-N antibody showed 100% of sensitivity and the RBD antibody 100% specificity. Thus, a multiplexed assay should be 100% accurate.

We are heading into a period of perhaps explosive increases in rates of infection. Widespread and frequent testing with contact tracing can be a practical way to actively prevent transmission of virus, particularly, in big groups of people (i.e., school settings). The tests described above are playing a role in curbing the pandemic.

 

  1. J. Bullard et al., Predicting infectious SARS-CoV-2 from diagnostic samples. Clin Infect Dis, (2020).
  2. L. Azzi et al., Saliva is a reliable tool to detect SARS-CoV-2. J Infect 81, e45-e50 (2020).
  3. M. Baghizadeh Fini, Oral saliva and COVID-19. Oral Oncol 108, 104821 (2020).
  4. K. K. To et al., Consistent detection of 2019 novel coronavirus in saliva. Clin Infect Dis, (2020).
  5. S. Wei et al., Field-deployable, rapid diagnostic testing of saliva samples for SARS-CoV-2. medRxiv, (2020).
  6. D. A. Collier et al., Point of Care Nucleic Acid Testing for SARS-CoV-2 in Hospitalized Patients: A Clinical Validation Trial and Implementation Study. Cell Rep Med 1, 100062 (2020).
  7. Z. Ali et al., iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2. Virus Res 288, 198129 (2020).
  8. J. Arizti-Sanz et al., Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2. bioRxiv, (2020).
  9. X. Ding et al., Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay. Nat Commun 11, 4711 (2020).
  10. T. Hou et al., Development and evaluation of a rapid CRISPR-based diagnostic for COVID-19. PLoS Pathog 16, e1008705 (2020).
  11. C. M. Ackerman et al., Massively multiplexed nucleic acid detection with Cas13. Nature 582, 277-282 (2020).
  12. W. N. Chia et al., Serological differentiation between COVID-19 and SARS infections. Emerg Microbes Infect 9, 1497-1505 (2020).
  13. K. W. Ng et al., Preexisting and de novo humoral immunity to SARS-CoV-2 in humans. Science, (2020).
  14. K. Bond et al., Evaluation of Serological Tests for SARS-CoV-2: Implications for Serology Testing in a Low-Prevalence Setting. J Infect Dis 222, 1280-1288 (2020).
  15. C. W. Tan et al., A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction. Nat Biotechnol 38, 1073-1078 (2020).
  16. B. Isho et al., Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients. Sci Immunol 5, (2020).
  17. N. Pisanic et al., COVID-19 serology at population scale: SARS-CoV-2-specific antibody responses in saliva. J Clin Microbiol, (2020).

Herd Immunity – Can We Get There?

The concept of herd immunity is frequently brought up as an alternative to lockdown and intervention-based strategies for curbing the COVID-19 pandemic, this sensitive issue being much influenced by policy makers. In this context it is important to first define herd immunity. This is obtained when enough of the population has immunity to an infectious pathogen, thus resulting in prevention of its transmission. The clearest examples of achieving herd immunity are provided by successful vaccination programs (i.e., polio, smallpox and measles). The question of whether acquisition of herd immunity to SARS-CoV-2 is possible depends upon important questions. How long will it take to get such herd immunity? What will be the cost in human deaths? Will there be a successful vaccine program? And does infection provide a lasting immunity for further prevention of reinfection?

Let’s first consider the possibility of obtaining herd immunity by vaccination. Ideally, strong herd immunity can be achieved if a vaccine provides a very significant (say around 80-90%) protection (sterilizing immunity) for extended periods, and if most of the population has been vaccinated. However, it is currently uncertain that these conditions will be met. A recent survey conducted in 19 countries showed that approximately, 70% of participants were likely to take a SARS-CoV-2 vaccine(1), indicating that public acceptance will by no means be universal. Assuming such a vaccine were 70% effective, this would leave 50% of the population able to be infected. Interestingly, if a vaccine were government mandated, acceptance dropped to 50 to 60%. Obviously if we are trying to reach herd immunity by vaccination, vaccine efficacy and acceptance will be critical.

Is it possible that natural infection of a large percentage of the population would result in widespread immunity? This could in theory lead to a reduction in further viral spread to the point where it would become relatively insignificant. This depends, however, upon several factors. First, is that a large proportion of the population would need to be infected. Given an estimated infection mortality rate of 0.3 to 0.5%, this would likely result in an unacceptably high number of deaths. Second, how long would it take? Indeed we know that beside some areas such as New York or Paris for example the percentage of infected individuals has apparently remained quite low (10-15%). Third, infection would need to lead to reasonably durable immunity, and we will consider this in more detail.

As to whether infection confers immunity, this is complicated by not knowing the levels of protective immunity to SARS-CoV-2. Inducing neutralizing antibodies have been a major target for development of many vaccines based on the evaluation of immunity to other coronaviruses. Importantly, evaluation of the rate of reinfection can predict a potential acquisition of protective immunity by natural infection. Reinfection is not necessarily proven by the second positive diagnostic to SARS-CoV-2; there is a question of whether the patient has truly become reinfected or the original infecting virus has simply rebounded.

The answer seems to be that reinfection does occur but is quite rare. Indeed, there are only a handful of well documented cases showing reinfection with SARS-CoV-2. In one well characterized instance, a patient was asymptomatically reinfected 4-5 months after a first symptomatic infection(2). Viruses from the first and second infections were phylogenetically distinct, indicating that they were independent infections. Interestingly, although neutralizing and IgG antibodies were not detectable at the onset of the second infection, they rapidly and robustly appeared (3). A handful of other cases have been reported.  However, their relative rarity suggests that reinfection is the exception rather than the rule. What, then, are the potential protective mechanisms and how long do they last?

The short answer is that we don’t have exact answers yet. Much attention has focused on antibody response and durability, particularly on antibodies to the spike protein and neutralizing antibodies (NA), which are a subset of the anti-spike antibodies. Both anti-spike and neutralizing IgG (but not IgM and IgA) antibodies decrease but persist at measurable levels for at least three to six months after the resolution of the infection. Indeed, anti-spike and NA correlate reasonably well with protective immunity ((4, 5) and these two studies). and. Moreover, as mentioned above, priming by memory cells can result in rapid and robust expression of antibodies. One study showed that a better correlation between NAs and spike antibodies could be obtained by measuring NAs and both anti-receptor binding domain (RBD) and S2 domain antibodies(5). However, it is unclear what level of NAs would be protective against SARS-CoV-2 infection.

It is likely that T cell activity will be necessary for significantly durable immunity. T cell activity is more difficult to assess than antibody response, and correspondingly less is known for its role in lasting protective immunity. We should point out that some patients who have recovered from mild COVID-19 and are PCR negative for SARS-CoV-2, show T cell activity against viral peptides even though they were antibody negative(6), suggesting the involvement of cell-mediated immunity. A strong T cell cytotoxic response is evident during disease, and in the convalescent phase, a memory T cell response is common even in the absence of antibodies(7). This suggests that immune responses will be durable, although this is not yet known for SARS-CoV-2.

Many immunologists are still evaluating a lasting immunity to SARS-CoV-2.  No matter what, infecting a substantial majority of the population to reach herd immunity would likely result in a massive death toll. For example, in the U.S., probably one to two million people would die. In addition, we need to consider serious medical and financial consequences for ill individuals with COVID-19. Further, many people who have recovered from the virus report lingering health effects. Earlier in the pandemic, Sweden was pursuing a herd immunity strategy by essentially relaxed containment; yet that strategy has led in June and July 2020 to the highest rate of death in Northern Europe.

Overall containing the pandemics will not be the result of a single intervention; it will combine efficient prevention measures, novel preventive and curative treatments as well as sufficiently effective and safe vaccine. In any case, waiting for spontaneous herd immunity does not appear as a reasonable option for the population.

  1. J. V. Lazarus et al., A global survey of potential acceptance of a COVID-19 vaccine. Nature Medicine, (2020).
  2. K. K. To et al., COVID-19 re-infection by a phylogenetically distinct SARS-coronavirus-2 strain confirmed by whole genome sequencing. Clin Infect Dis, (2020).
  3. K. K. To et al., Serum antibody profile of a patient with COVID-19 reinfection. Clin Infect Dis, (2020).
  4. A. S. Iyer et al., Persistence and decay of human antibody responses to the receptor binding domain of SARS-CoV-2 spike protein in COVID-19 patients. Sci Immunol 5, (2020).
  5. T. J. Ripperger et al., Detection, prevalence, and duration of humoral responses to SARS-CoV-2 under conditions of limited population exposure. medRxiv, (2020).
  6. S. Schwarzkopf et al., Cellular Immunity in COVID-19 Convalescents with PCR-Confirmed Infection but with Undetectable SARS-CoV-2-Specific IgG. Emerg Infect Dis 27, (2020).
  7. T. Sekine et al., Robust T Cell Immunity in Convalescent Individuals with Asymptomatic or Mild COVID-19. Cell, (2020).
  8. N. Le Bert et al., SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature 584, 457-462 (2020).

How Long Is A SARS-CoV-2 Infected Person Contagious?

As the COVID-19 pandemic rages on, the global community has become accustomed to comprehensive, interventional strategies such as wearing a mask, social distancing, hand hygiene, and surface cleaning and disinfection. It is well known that transmission of SARS-CoV-2 occurs through direct, indirect, or close contact with infected people through infectious secretions, such as saliva and respiratory secretions or their respiratory droplets. In addition, the scientific community has confirmed that the airborne transmission of SARS-CoV-2 viruses in aerosols (smaller than 100 μm) can remain suspended in air for many seconds to hours and are highly concentrated near an infected person, thus infecting people most easily in close proximity (1). Furthermore, aerosols containing infectious virus can also travel more than 2 m and accumulate in poorly ventilated indoor air, leading to superspreading events. World Health Organization and Centers for Disease Control and Prevention have also acknowledged this under certain circumstances, such as enclosed spaces, prolonged exposure to respiratory particles (i.e., shouting, singing, and exercising), and inadequate ventilation or air (2, 3). Thus, effective control strategies and standardized guidance to the public are integral in mitigating COVID-19. In particular, understanding the duration of infectiousness in infected persons with SARS-CoV-2 is critical for developing evidence-based public health policies on isolation, contact tracing and returning to work. In general, the levels of viral RNA were determined by using quantitative reverse transcription-polymerase chain reaction. However, detection of viral RNA does not necessarily indicate that a person is infectious and able to transmit the virus to another person. Although it is critical to determine the levels of infectious virus particles in infected COVID-19 patients, requirement of Biosafety Level-3 laboratory for the virus titration has hindered this approach. In this perspective section, we will discuss currently available scientific data regarding the levels of infectious virus particles in asymptomatic individuals, in mild and severe COVID-19 patients and in children and young adults.

Asymptomatic and presymptomatic individuals represent a source of potentially transmissible virus (4). Asymptomatic infections have no specific incubation period due to no clinical signs. However, the viral loads detected in asymptomatic populations have been reported in several studies to be similar to those in symptomatic patients. In a nursing facility, quantitative SARS-CoV-2 viral loads detected in residents were similarly high in the four symptom groups (residents with typical symptoms, those with atypical symptoms, those who were presymptomatic, and those who remained asymptomatic). Notably, 17 of 24 specimens (71%) from presymptomatic persons had viable virus by culture 1 to 6 days before the development of symptoms. In a surveillance study, asymptomatic cases of samples were collected through swabbing of contacts or facility/family/household testing in the context of outbreak investigations. Despite the uncertainty of their date of exposure or start of infection, cultivable virus was isolated from samples collected from asymptomatic individuals (41% of tested samples).

Detection of infectious SARS-CoV-2 from upper respiratory tract of mild-to-moderate COVID-19 patients showed that infectious virus can persist for more than a week after symptom onset, declining over time (5). At 10 days after symptom onset, probability of culturing virus declines to 6%. This is in line with current WHO guidance on release from isolation. Similarly, shedding of the virus in mild COVID-19 patients was determined by measuring the levels of transcribed subgenomic mRNA and isolation of infectious viruses (6). Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.1 × 108 RNA copies per throat swab on day 4. In addition, infectious viruses were successfully isolated from these samples, confirming active virus replication in the upper respiratory tract. No viruses were isolated after day 7 onset. These findings suggest efficient transmission of SARS-CoV-2, through active pharyngeal viral shedding at a time at which symptoms are still mild and typical of infections of the upper respiratory tract. In a surveillance study in Manitoba, Canada, the presence of infectious viruses was determined by evaluating samples from day of symptom onset (day 0) up to 21 days after symptom onset (7). Within this range of samples, positive cultures were observed up to day 8 after symptom onset with the probability of obtaining peak titers on day 3. Similarly, in Hong Kong, virus was isolated from the samples of mild patients collected within the first 8 days of illness with median viral RNA load of 7.54 log10 genome copies/mL (8). From severe COVID patients, prolonged duration of cultivable virus was detected for up to 20 days after symptom onset, suggesting that prolonged excretion of infectious virus is associated to the severity of the disease (9).

Severity in most children is limited, and children do not seem to be major drivers of transmission. However, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects children of all ages. In South Korea, large-scale testing, aggressive contact tracing and testing, and isolation/direct observation of asymptomatic or mildly symptomatic children have identified the presence of asymptomatic (20 of 91 [22%]), presymptomatic (18 of 91 [20%]), and symptomatic children (53 of 91 [58%]) (10). Presymptomatic children remained symptom free for a median (range) of 2.5 (1-25) days before exhibiting any symptoms. A minority of children (6 [7%]) were identified as infected; this highlights the concept that infected children may be more likely to go unnoticed either with or without symptoms and continue on with their usual activities, which may contribute to viral circulation in the community. In a separate study with 12 symptomatic children, infectious viruses were detected at a median of 2 days after symptom onset (11). Median viral RNA load at diagnosis was 3.0 × 106 copies/mL (mean 4.4 × 108 ranging from 6.9 × 103 to 4.4 × 108 copies/mL. A limitation of this study is the small number of children assessed. However, viral load at diagnosis is comparable to that of adults and symptomatic children of all ages shed infectious virus in early acute illness, a prerequisite for further transmission. Considering the relatively low frequency of infected children, even in severely affected areas, biological or other unknown factors could lead to the lower transmission in this population. Large serologic investigations and systematic surveillance for acute respiratory diseases and asymptomatic presentations are still needed to assess the role of children in this pandemic.

Although the spectrum of COVID-19 ranges from asymptomatic to severe infections, most patients experience mild disease (80%). Scientific data indicate that infectious virus in mild patients can persist for a week after symptom onset. Furthermore, infectious virus can be isolated from asymptomatic individuals. This clearly enforces the importance of wearing a mask, quarantine, and contact tracing to mitigate the transmission of SARS-CoV-2. Recent studies support that wearing masks can save lives not only by cutting down the chances of both transmitting and catching the coronavirus (12) but also by reducing the severity of infection in contracted individuals (13). It is well demonstrated that self-quarantine of close contacts exposed to COVID-19 prevents transmission to others. Contact tracing must be conducted for close contacts (any individual within 6 feet of an infected person for at least 15 minutes) of laboratory-confirmed or probable COVID-19 patients. However, relaxed social distancing and opposition of wearing masks are hindering the mitigation of SARS-CoV-2, resulting in continuous increase of COVID-19 cases. Even when the vaccine will be available, we would not immediately stop social distancing, wearing masks, and other interventional measures until reaching efficient levels of viral mitigation. The message is clear that a simple practice of wearing masks can protect ourselves and save other lives from circulating SARS-CoV-2.

 

References

  1. PRATHER, K.A., MARR, L.C., SCHOOLEY, R.T. MCDIARMID, M.A., WILSON, M.E., MILTON, D.K. 2020. Airborne transmission of SARS-CoV-2. DOI: 10.1126/science.abf0521
  2. 2020. Transmission of SARS-CoV-2: implications for infection prevention precautions. https://www.who.int/news-room/commentaries/detail/transmission-of-sars-cov-2-implications-for-infection-prevention-precautions
  3. 2020. Scientific Brief: SARS-CoV-2 and Potential Airborne Transmission. https://www.cdc.gov/coronavirus/2019-ncov/more/scientific-brief-sars-cov-2.html
  4. Arons MM, Hatfield KM, Reddy SC, et al. 2020. Presymptomatic SARS-CoV-2 infections and transmission in a skilled nursing facility. N Engl J Med 382:2081-2090.
  5. Singanayagam Anika, Patel Monika , Charlett Andre , Lopez Bernal Jamie , Saliba Vanessa , Ellis Joanna , Ladhani Shamez , Zambon Maria , Gopal Robin. 2020. Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19, England, January to May 2020. Euro Surveill. 25.
  6. Wölfel R, Corman  VM, Guggemos  W, Seilmaier  M, Zange  S, Müller  MA, et al. 2020. Virological assessment of hospitalized patients with COVID-2019. Nature. 581:465–469.
  7. Bullard J, Dust K, Funk D, Strong JE, Alexander D, Garnett L, et al. 2020. Predicting infectious SARS-CoV-2 from diagnostic samples. Clin Infect Dis. ciaa638.
  8. Perera RAPM, Tso E, Tsang OTY, Tsang DNC, Fung K, Leung YWY, et al. SARS-CoV-2 virus culture and subgenomic RNA for respiratory specimens from patients with mild coronavirus disease. Emerg Infect Dis. 2020;26(11).
  9. van Kampen JJA, van de Vijver DAMC, Fraaij PLA, Haagmans BL, Lamers MM, Okba N, et al. Shedding of infectious virus in hospitalized patients with coronavirus disease-2019 (COVID-19): duration and key determinants. medRxiv. 2020.06.08.20125310.
  10. 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. doi:10.1001/jamapediatrics.2020.3988.
  11. L’Huillier AG, Torriani G, Pigny F, Kaiser L, Eckerle I. Culture-Competent SARS-CoV-2 in Nasopharynx of Symptomatic Neonates, Children, and Adolescents. Emerg Infect Dis. 2020 Oct;26(10):2494-2497. doi: 10.3201/eid2610.202403. Epub 2020 Jun 30. PMID: 32603290; PMCID: PMC7510703.
  12. Leffler, C. T. et al.Preprint at medRxiv https://doi.org/10.1101/2020.05.22.20109231 (2020).
  13. Gandhi, M., Beyrer, C. & Goosby, E. Gen. Intern. Med. https://doi.org/10.1007/s11606-020-06067-8 (2020).

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.
  7. Sullivan SG, Price OH, Regan AK. Burden, effectiveness and safety of influenza vaccines in elderly, paediatric and pregnant populations. Ther Adv Vaccines Immunother. 2019 Feb 7;7:2515135519826481. doi: 10.1177/2515135519826481. PMID: 30793097; PMCID: PMC6376509.
  8. Grohskopf LA, Alyanak E, Broder KR, et al. Prevention and Control of Seasonal Influenza with Vaccines: Recommendations of the Advisory Committee on Immunization Practices – United States, 2020-21 Influenza Season. MMWR Recomm Rep. 2020;69(8):1-24. Published 2020 Aug 21. doi:10.15585/mmwr.rr6908a1.
  9. World Health Organization (WHO). Vaccines against influenza WHO position paper – November 2012. Wkly. Epidemiol. Rec. 2012, 87, 461–476.
  10. Fangjun Zhou, Megan C. Lindley, Variability in influenza vaccination opportunities and coverage among privately insured children, Vaccine, 2020, ISSN 0264-410X, https://doi.org/10.1016/j.vaccine.2020.07.061.
  11. 2019. Flu vaccination coverage, United States, 2018–19 influenza season. https://www.cdc.gov/flu/fluvaxview/coverage-1819estimates.htm#:~:text=Flu%20vaccination%20coverage%20among%20adults,than%20the%202016%E2%80%9317%20season.
  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
  13. Jehi L, Ji X, Milinovich A, Erzurum S, Rubin B, Gordon S, Young J, Kattan MW. Individualizing risk prediction for positive COVID-19 testing: results from 11,672 patients. Chest. 2020 Jun 10:S0012-3692(20)31654-8. doi: 10.1016/j.chest.2020.05.580.
  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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  9. M. Hoffmann et al., Chloroquine does not inhibit infection of human lung cells with SARS-CoV-2. Nature, (2020).
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  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).

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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  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).
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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.
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  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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