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. These latter mechanisms appear 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 in this article.  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 shows 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 include 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.

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

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

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

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

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

 

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

 

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SARS-CoV-2 Salivary Tests

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

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

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

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

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

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

 

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

 

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Antibody Dependent Enhancement and SARS-CoV-2

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

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

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

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

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

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

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

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

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

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

 

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

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

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

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

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

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

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

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

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

 

References

 

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

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On the Surface, Using a Good Disinfection Strategy Matters

One approach to COVID-19 that has been gaining traction, is the idea of decontaminating surfaces to inactivate viruses that have been deposited upon them, either by aerosols, droplets, or contaminated touch. Infection from surfaces is a major concern for public entities and private businesses such as schools, public transportation, airlines, hotels, hospitals and doctors’ offices, restaurants, gyms and cruise ships. The technical issues are not trivial. Treatments of surfaces by disinfectants must be highly effective on their antiviral activity.  Additionally, they should not be too expensive; otherwise, their economic benefits in terms of reduced labor costs, lower infection rates and increased customer traffic would fail to offset costs. Safety is of significance with several approaches that have been developed, particularly in the context of SARS-CoV-2, an enveloped virus which is relatively easy to inactivate. Ideal virucides would also be effective against the more difficult, non-enveloped viruses such as norovirus, and against bacteria as well.

One general approach uses quarternary amine (QA) compounds. These contain a central nitrogen bound covalently to four hydrocarbon groups. They, thus, have both a strong positive charge and hydrophobic properties. As such, they disrupt lipid membranes through surfactant activity, attract the negatively charged surfaces of microbes and can cause protein aggregation (1). Two of the GVN’s Centers of Excellence (the Peter Doherty Institute for Infection and Immunity in Melbourne, Australia and the Rega Medical Research Institute of KU Leuven, Belgium) tested one such formulation, BioProtect, developed by ViaClean Technologies, which contains a mixture of five different QAs. When sprayed onto a surface, the QAs are covalently attached to the surface and self-assemble into nanoparticle spikes, which disinfect the surface by the typical QA activities. The covalent attachment gives the preparation durable activity that is claimed to last for at least three months. Results from both the Doherty Institute and the Rega Institute with this agent demonstrated SARS-CoV-2 inactivation of 99.7% within 10 minutes, 46 days after application, and led to degradation of the viral RNA. Another approach by Exilva uses a formulation of QAs with microfibrillated cellulose as a spray onto surfaces. The microfibrillated cellulose is not water soluble and is claimed to provide a durable coating.

A different approach covalently attaches a titanium dioxide nanoparticle photocatalyst to surfaces. The catalyst acts on water and oxygen in the presence of light to generate a variety of reactive oxygen species that attack bacteria and viruses. The product, called ACT CleanCoat, is currently being used by some cruise line operators, hotels and food processors to defend against SARS-CoV-2. Durability is claimed to be up to a year. Tests by the European Chemical Union have shown it to be effective against both enveloped and non-enveloped viruses, including MERS and murine norovirus, as judged by EN 14476 standards. This approach does, however, require that the surfaces be exposed to light.

Surface transmission of SARS-CoV-2 is certainly not a negligible concern, and such approaches could also be quite useful against other microbes, such as norovirus and bacteria. The use of advanced and durable methods for surface decontamination seems to be increasingly useful in different industries, including: academia, medical institutions, transportation, hospitality, travel, food processing and health and wellness centers.

It is clear, however, that airborne transmission, particularly in enclosed spaces, is a major route of transmission and needs to be dealt with. Masks are at least partially effective in providing protection from airborne transmission; although, they are not completely effective depending on the mask types. So how might such a problem be approached?

As evidenced by superspreading events at a choir in Washington State and a call center in South Korea, transmission powered by breath and carried in the form of droplets and aerosols are major contributors. Control of air circulation, therefore, and the decontamination from viral, airborne particles will be necessary. Heavier droplets would probably not be captured by air recirculation, and would drop to surfaces, but the approaches mentioned above for surface decontamination would rectify this. One of the ways to remove viruses and other microbes is by recirculating air through a high-efficiency particle (HEPA) filter. In fact, this is routine on commercial airliners, and is why disease transmission rarely occurs in that setting. 

Another mechanism to inactivate coronavirus is far ultraviolet UVC light (207 to 222 nm), as shown by results on the cold-related human coronaviruses alpha HCoV-229E and beta HCoV-OC43. Their biologic similarity to SARS-CoV-2 suggests that this approach should be applicable and the wavelengths used are not harmful to people. Areas that the light reaches directly could be disinfected with a limitation. Directing airflow to the closest proximity to a UVC source would be needed to maximize the virucidal effect. While surfaces that receive UVC light directly would be decontaminated, it seems that the greatest virus protection in indoor spaces would result from a combination of long-term surface decontamination combined with airflow control and UVC light/HEPA filtration.

 

  1. G. A. Knauf et al., Exploring the Antimicrobial Action of Quaternary Amines against Acinetobacter baumannii. mBio 9, (2018).

 

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So Where Did the Virus Come From, Anyway?

The question of where viruses involved in spillover zoonoses originate is always important and fascinating. Identifying their origin is critical in understanding how zoonotic epidemics originate, and in preparing preventive strategies for future epidemic. Fascinating, because identification, like solving a difficult whodunit, involves a series of forensics including those from epidemiology, molecular genetics, phylogenetics, and the study of reservoir and intermediate hosts (i.e., wild and domestic animals). Finding the immediate animal precursor virus is normally difficult; indeed, coronaviruses compound the difficulty because they have a predilection for recombination (1), constantly creating novel viruses, and making it necessary to identify more than one ancestral virus; unless, one has the rare good fortune of finding the exact parental recombinant. Of course, after the ancestral virus(es) has been identified, there remains the question of where and when did it first get into the human leading to the secondary transmission.

In this time of inexpensive and rapid genomic sequencing, the entire genome of the virus causing COVID-19 was rapidly obtained and published, identified as a beta coronavirus (2), and designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It was distantly related to SARS-CoV-1, basically refuting the idea that it had simply mutated from SARS-CoV1. A much more closely related virus (>96% over its entire genome) was sequenced from horseshoe bats living in a cave in Yunnan province, called CoV RaTG13b (3). Large stretches of the CoV RaTG13 were virtually identical to SARS-CoV-2; although, other stretches appeared to be sufficiently distinct as to rule out a direct ancestral relationship. Some attention has also been focused on pangolins, an endangered species that also carry coronaviruses, and that is imported illegally into China in great numbers for food and traditional medicine. One coronavirus isolated from a Malayan pangolin confiscated in Guangdong province was >90% identical to that of SARS-CoV-2 over its whole genome, but was clearly not a direct precursor (4).

Recently a phylogenetic analysis has provided a reasonably convincing explanation for how SARS-CoV-2 was originally formed. The study shows that the virus is likely a triple recombinant, containing genetic elements of two bat coronaviruses and a pangolin coronavirus. Likely recombinational break points are before and after the angiotensin I Converting Enzyme 2 (ACE2) receptor binding motif in the spike protein of SARS-CoV-2. The receptor binding motif of SARS-CoV-2 is virtually identical to that of an isolate from pangolins. The upstream portion of the ORF1A region appears to have been acquired from a second bat coronavirus. Interestingly, the authors present data that SARS-CoV-1 is also the product of multiple recombination events involving different bat coronaviruses. Another study has analyzed in-depth evolutionary history of bat coronaviruses, and their ability to jump species to better understand how and where zoonoses are most likely to occur. Importantly, the SARS-CoV-2 spike protein displays a unique feature, namely the presence of a furin cleavage site insertion (PRRA) at the junction of two subunits of the S protein. Neither the bat beta coronaviruses, nor the pangolin beta coronaviruses sampled thus far have polybasic cleavage sites.  No animal coronavirus has been identified that is sufficiently similar to have served as the direct progenitor of SARS-CoV-2; although, the diversity of coronaviruses in bats and other species is massively under sampled (6).

All of the above has bearing on the evolution of virus. No clear answer for the origin of the virus has been concluded prior to human infection. At first, it seemed most likely to have emerged from the wet market in Hunan, but follow-up analyses of the market and the animals present have not yielded proof. This quickly gave rise to conspiracy theories that the virus was generated in a virology lab in Wuhan.  The possibility that the virus could be man-made was fairly convincingly refuted by Anderson et al., (6) and is not supported by academic data. What then was the direct source of the virus, and when did the virus first enter humans?

Given the prevalence of recombination among bat coronavirus and the myriad of possibilities for the introduction of SARS-CoV-2, it is probable that sample collection from diverse hosts and genomic sequencing analysis need to be continue to identify the origin of SARS-CoV-2. However, global spread of these viruses and their potential for cross-species transmission makes it clear that it is possible to have waves of COVID-19 outbreaks. The important thing is how well we be prepared for the next one.

  1. B. Hu et al., Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus. PLoS Pathog 13, e1006698 (2017).
  2. F. Wu et al., A new coronavirus associated with human respiratory disease in China. Nature 579, 265-269 (2020).
  3. P. Zhou et al., A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273 (2020).
  4. T. Zhang, Q. Wu, Z. Zhang, Probable Pangolin Origin of SARS-CoV-2 Associated with the COVID-19 Outbreak. Curr Biol 30, 1346-1351 e1342 (2020).
  5. V. D. Menachery et al., A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Nat Med 21, 1508-1513 (2015).
  6. K. G. Andersen, A. Rambaut, W. I. Lipkin, E. C. Holmes, R. F. Garry, The proximal origin of SARS-CoV-2. Nat Med 26, 450-452 (2020).
  7. N. Wang et al., Serological Evidence of Bat SARS-Related Coronavirus Infection in Humans, China. Virol Sin 33, 104-107 (2018).

 

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What’s Going to Happen When Summer Gets Here?

How seasonality and climate affect SARS-CoV-2

There has been much speculation over the effects of climate on the spread of SARS-CoV-2. This is obviously a difficult question, as there are many variables to tease apart, including: population density, socioeconomic status, geography, and cultural norms. Viral -transmission seems to occur more easily indoors than outdoors. Another question which has a bearing on the effect of climate is whether infection is acquired from surfaces to any degree; although the virus is known to be spread via droplets or aerosol. A key question is the extent to which seasonal and geographic climate variations are relevant in the pandemic phase of SARA-CoV-2 infection.

What are the data from the real world, as distinguished from the laboratory? It was observed by Sajadi et al. early in the pandemic that infection spread correlated with latitude, humidity and temperature (1). Specifically, the computational analysis based on climate data suggested that spread was hampered by higher temperatures and tropical climates, and that SARS-CoV2 could be seasonal. Other studies also support that cold, dry conditions increase the transmission of the virus (2-3). In Mexico, a local scale study showed that greater spread of SARS-CoV-2 correlated with a moderate climate versus tropical or dry climates (4). However, the spread of viruses in locales such as Singapore suggest that the situation is likely more complicated. As far as is possible, we’ll try to separate out individual variables such as sunlight, temperature, and humidity.

A broad consensus exists that there is at most a weak correlation with temperature; (although some of the reports are conflicting), and that higher temperatures will not suffice to greatly influence virus transmission. Scafetta reported that in selected areas in Italy, the United States, and China, transmission seemed to be most efficient at temperatures between 4 °C and 12 °C, and relative humidity between 60% and 80% https://www.mdpi.com/1660-4601/17/10/3493/htm; although differences in age of the respective populations were a possible confounding factor.  Huang et al. (5) reported that globally the optimal conditions for transmission appeared to be temperatures between 4 °C and 12 °C, and relative humidity between 60% and 80%. Prata et al. reported an opposite correlation with a negative linear relationship between temperatures and daily cases of COVID-19 in from 16.8 °C to 27.4 °C.  Bashir et al. (6) presented essentially similar findings for New York. Taking all of the reports into account, there appears to be a weak but consistent effect of higher temperatures on reducing viral transmission. However, the mechanism is unclear and is muddled by other variables such as population density and changes in behavior. In contrast, Auler et al. (-7) reported that in Brazil, higher temperatures and average humidity favored transmission.

What about humidity? Juni et al. (8) looked worldwide at infection rates and found no correlation with temperature, but did find an inverse correlation with relative humidity that was quite weak compared with correlations with other factors such as school closures, restrictions on social gatherings and social distancing. Ward et al. (9) showed an inverse correlation of viral spread in Australia with morning humidity, but not with afternoon humidity or temperature. As with temperature, the relationship with humidity is likely to be complex and associated with various behavioral factors.

Finally, what about sunlight intensity? Again, there are many variables. A significant one mentioned earlier, is the extent to which transmission is from droplets, as opposed to from surfaces. If, as is seeming more likely, most transmission is from droplets, inactivation of virus on surfaces by sunlight will probably not factor into transmission rates to any great extent.

So what will summer in temperate climates bring? One recent study suggests that while climate may play a role in modulating detailed aspects of the size and timescales of a pandemic outbreak within a particular location, population immunity is a much more fundamental driver of pandemic invasion dynamics (10). In terms of the SARS-CoV-2 pandemic, summertime temperatures will not effectively limit the spread the infection. A more detailed understanding of climate drivers, as well as immunity length will be crucial for understanding the implications of control measures. As observed with other infectious diseases, achieving herd immunity to COVID-19 will be crucial to protect the public.

The GVN believes that we will have to wait and see to separate for proper multifactorial analysis. However, to the extent that people will spend more time outdoors in the summer, due to sports, gardening, outdoor seating at restaurants and other venues, and other outdoor activities, and to a reduction in indoor gatherings where much of the infections appear to take place, and to the recent trend in the slowing of new infections, our thoughts are that we will see further and substantial reductions in the summer. What happens in fall and winter is, at this point, totally unclear. Hopefully, by then we will have better antivirals and perhaps be well on the road to a vaccine.

 

  1. M. Sajadi, P. Habibzadeh, A. Vintzileos, S. Shokouhi, F. Miralles-Wilhelm, A. Amoroso, Temperature, humidity and latitude analysis to predict potential spread and seasonality for COVID-19. 5 March 2020; .doi:10.2139/ssrn.3550308
  2. Francesca Benedetti, Robert Gallo, Davide Zella, et al: Increase correlation between average monthly high temperature and COVID-19 related death rates in different geographical areas. 20 May 2020; doi: 1021203/rs.3.rs-290239/v1
  3. Q. Bukhari, Y. Jameel, Will coronavirus pandemic diminish by summer? 17 March 2020; .doi:10.2139/ssrn.3556998
  4. J. Wang, K. Tang, K. Feng, W. Lv, High temperature and high humidity reduce the transmission of COVID-19. 9 March 2020; .doi:10.2139/ssrn.3551767
  5. F. Mendez-Arriaga, The temperature and regional climate effects on communitarian COVID-19 contagion in Mexico throughout phase 1. Sci Total Environ 735, 139560 (2020).
  6. Z. Huang et al., Optimal temperature zone for the dispersal of COVID-19. Sci Total Environ, 139487 (2020).
  7. M. F. Bashir et al., Correlation between climate indicators and COVID-19 pandemic in New York, USA. Sci Total Environ 728, 138835 (2020).
  8. A. C. Auler, F. A. M. Cassaro, V. O. da Silva, L. F. Pires, Evidence that high temperatures and intermediate relative humidity might favor the spread of COVID-19 in tropical climate: A case study for the most affected Brazilian cities. Sci Total Environ 729, 139090 (2020).
  9. P. Juni et al., Impact of climate and public health interventions on the COVID-19 pandemic: A prospective cohort study. CMAJ, (2020).
  10. M. P. Ward, S. Xiao, Z. Zhang, The Role of Climate During the COVID-19 epidemic in New South Wales, Australia. Transbound Emerg Dis, (2020).
  11. Baker RE, Yang W, Vecchi GA, Metcalf CJE, Grenfell BT. Susceptible supply limits the role of climate in the early SARS-CoV-2 pandemic. 2020 May 18. Science. 2020; eabc2535. doi:10.1126/science.abc2535

 

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New Insights into the SARS-CoV-2 Vaccine

There has been some very interesting news in the last several days that relate to vaccine development. The first of these is a report of results on the Phase 1 trials of the Moderna mRNA vaccine.  Most importantly, the vaccine appeared to be safe; no major adverse events were reported. Vaccines developed binding antibodies to the spike protein of SARS-CoV-2 at levels seen in immunized people, and of those eight who were tested, all had neutralizing antibodies to the virus. This is hopeful; neutralizing antibodies are generally a critical part of a successful immune response. However, researchers measured virus-recognizing antibodies in 25 participants, and detected levels similar to or higher than those found in the blood of people who have recovered from COVID-19. In general, most people who have recovered from COVID-19 without hospitalization do not produce high levels of neutralizing antibodies. Therefore, it is uncertain whether the responses are enough to protect people from infection. In addition, the data of this study has not been published yet. The report also begs the question, what about T cell responses? These will likely be important for long term immunity. he Oxford vaccine, an adenoviral vectored vaccine expressing the spike protein of SARS-CoV-2, wsa also addressed in the previous vaccine post.  In rhesus monkey challenge, a significantly reduced viral load in bronchoalveolar lavage fluid and respiratory tract tissue of vaccinated animals challenged with SARS-CoV-2 compared with control animals. Importantly, vaccinated rhesus macaques were protected from pneumonia without evidence of immune-enhanced disease. However, the scientists found similar levels of viral titers in animals’ noses between unvaccinated and vaccinated groups, probably due to a high dose of challenge virus used in this study.  This has raised a concern whether the vaccination can provide the protection against transmission. The outcome of ongoing clinical trial will provide a better answer whether the vaccine can protect humans from COVID-19.  Eli Lilly is proceeding with a passive immunization trial using monoclonal neutralizing antibodies to SASR-CoV2. It will be interesting to see the results.

Two recent reports (in Cell and MedRxiv) represent an important beginning to answering this question (or questions). In addition to B cells in producing antibodies, T cell can also play a role in preventing infection. Helper T cells activate B cells to secrete antibodies and macrophages to destroy ingested microbes. Cytotoxic T cells kill infected target cells. These two studies  showed, using peptide pools covering predicted epitopes derived from the viral genome, that most convalescent patients had CD4+ and CD8+ responses to a wide variety of viral proteins, including the spike protein. Other common responses include those against the matrix and nucleoprotein as well as nonstructural proteins, such as nsp3 and 4 and ORFs 3a and 8. This suggests that vaccine inducing an immune response to not only the S protein but also these proteins could enhance its protective efficacy. Interestingly, these reports also showed that 40-60% of people who had never been exposed to SARS-CoV-2 also had T cell responses to the virus, and the authors speculated that this could be due to T cell cross-reactivity with the common seasonal cold coronaviruses. This raises some interesting questions. Could previous exposure to cold coronaviruses afford some protection against SARS-CoV2? Do these activities differ in people with severe disease? Is it predictive; that is, could it be a factor in whether infection results in mild or severe disease? How might the presence or absence of these T cell reactivities affect vaccine responses? In any event, it is likely that elicitation of T cell responses will need to be part of a successful vaccine response.

The flip side of T cell responses is that they could be harmful. A cytokine storm has been invoked by many to account for aspects of the end stage disease. If an overactive immune reaction is important, it seems intuitive it should affect younger people disproportionately, as happened in the Spanish flu pandemic. On the other hand, perhaps people with weaker immune systems don’t mount an effective response until late in the disease and generate an inappropriate inflammatory response as a result. A recent report investigated this possibility. The authors looked at moderate, severe and critical patients. They reported a depletion of CD4+ and CD8+ T cells with a CD11a++ memory/effector phenotype, and survival was correlated with recovery of these cells. Conversely, more severe disease was correlated with an increase in S protein-reactive cells producing inflammatory cytokines. Unsurprisingly, with SARS-CoV-2 the immune system appears to be a two-edged sword.

 

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Why Do Genes and Mutations Matter in SARS-CoV-2?

Why should we concern ourselves with genetics and mutations in SARS-CoV-2? There are many reasons why these are of interest, including the rate at which mutations are occurring. One reason is that mutations can result in immune evasion. Changes can occur in recognition sites (epitopes) for antibodies and for cytotoxic T cells, and these could, in theory, make vaccines less effective. In fact, our immune response tends to select for exactly these kinds of mutational changes in the virus. Another reason for our interest is that mutational signatures allow us to identify chains of transmission and origins of clusters of related mutants. Comparisons of genome sequences between current and previously circulating strains  may allow us to trace the current pandemic back to a common ancestral virus and, perhaps, identify its natural source, making it easier to anticipate and better deal with the next “spillover” event. Mutations may allow a virus to become more or less virulent or transmissible. Knowledge of the sequence of viral genes can lead to the prediction of the 3D structure of viral proteins, such as the RNA-dependent RNA polymerase and viral proteases, and inform the development of effective antiviral drugs. Sequence analysis will also allow us to characterize drug resistant mutants and develop alternative drugs. Let’s consider some of these points in a little more detail.

First, though, RNA viruses generally have a high mutation rate. Some mutations have no effect on the eventual protein sequence and are called silent or synonymous. Others alter the amino acid sequence of the protein and can result in a neutral effect or an increase or decrease in protein function. These are called non-synonymous, and most have no effect on protein function. Their RNA-dependent RNA polymerases have a high intrinsic error rate. SARS-CoV-2 seems to have a mutation rate much lower than many RNA viruses. This is because it has a proofreading nuclease that removes misincorporated bases. This is both good and bad news. It’s good because it makes the virus less likely to generate escape mutants from the immune response or from antiviral drugs. It’s potentially bad news because it could cause difficulties for drugs that target the viral polymerase by causing misincorporation of modified nucleotides, such as remdesivir. Fortunately in spite of this, remdesivir causes chain termination of RNA synthesis in an in vitro SARS-CoV-2 RNA replication system three bases downstream of its site of incorporation (1), so perhaps it won’t be a problem.

Is the virus mutating to become more infectious or pathogenic? Early in the epidemic a claim was made that there were two genetically distinguishable forms of SARS-CoV-2, called the S and L forms, in which the S form was ancestral to the L form and that the L form was spreading more aggressively, based on a relative frequency of 30% for the S form and 70% for the L. This has been debunked as being reflective of limited sampling, skewing the results, as well as founder effects, in which a single infected traveler seeds a new community with a single viral genotype. Later analyses showed the S form again becoming more predominant. More recently, similar observations have been made using a larger number of samples and focusing on the gene encoding the spike protein. They suggested that a mutation in the spike protein is becoming more dominant, thus, increasing a genotype variation, but here the same criticism would apply. Interestingly, they also present evidence for recombinant forms of SARS-CoV-2, suggesting some people become infected with more than one genotype. Nothing in the data reports differences in pathogenicity among different genotypes.

What about the origins of the virus? This is unclear and may remain so. Not only does the high number of coronavirus species make finding the exact ancestor problematic, but the proclivity of coronaviruses to recombine with each other adds considerable difficulty. The possibility that it was genetically engineered seems unlikely (2), among other reasons, because it is not optimally engineered. Two possibilities remain: that it directly infected humans or that it first infected an intermediate species, was adapted, and then infected humans, perhaps in the Wuhan wet market. The closest known related bat virus is more than 96% identical by nucleotide sequence to SARS-CoV-2. While this sounds quite close, its spike protein diverges in the receptor binding domain, suggesting that it may not bind efficiently to the human receptor, ACE2.. The Wuhan Institute of Virology scientists had a continuing study in which they collected bats and their blood and feces from caves in Yunnan Province to identify and catalog coronaviruses in response to SARS-1, and, in anticipation of future pandemics. Could one of them have become infected by inadvertently inhaling bat guano? It is a short high-speed train ride to Wuhan. Or could local villagers have acquired the virus? Bat guano is used in traditional Chinese medicine and is sometimes put in the eyes. You can buy it on Amazon. The virus may have been rare in humans until recently. Usually, however, transmission occurs through an intermediate host; civet cats with SARS-1 and camels with MERS. A fascinating book on the subject is Spillover, by David Quammen. Although pangolins have been suggested as an intermediate host, there is no convincing evidence so far. Perhaps in this case, humans are serving as the intermediate hosts. The definitive answer will require further sequencing efforts or it may never be answered conclusively.  Genetic studies of the virus have shown worldwide geographic routes of transmission that have occurred; see https://nextstrain.org/ncov/global.

Genomic sequences that have specific mutations can be thought of as having a convenient barcode that allows chains of transmissions to be traced. This can be highly valuable to epidemiologists. For example, a man who had traveled from China and arrived in Washington state in January tested positive for SARS-CoV-2. His virus had three distinct mutations that identified Wuhan as its origin. More than a month later, a high school student tested positive. The virus had the same mutations plus some new mutations. This led to the conclusion that the virus had been circulating in the general population during that time interval. Infections on the Grand Princess had the same mutational signature. Similar studies showed that the epidemic in New York had its origins primarily in travelers from Europe. The epidemic in Europe was mostly seeded by viruses from China. California virus samples also showed similarity to viruses from China but were distinct from the Washington state cluster, indicating a separate introduction, probably a bit earlier than those in Washington.

Another useful aspect of genetic studies is that they provide tools to understand potential targets for the immune system and by doing so, facilitate the development of vaccines. As an example, a study looked at B and T cell epitopes identified for SARS-CoV1 infections, then researchers looked for genetically similar regions in SARS-CoV-2 to identify possible immune targets (3). The authors further noted that these parts of the genome, which encode structural proteins of the virus, show very little variation among a large number of isolates, suggesting they may represent regions where the virus does not have much mutational latitude. Another method uses nucleotide sequences to determine the epitope , then synthesizes an overlapping set of peptides representing subsets of the epitope. These synthesized peptides are then be tested against various antisera, including natural immune sera, to identify regions of the protein that are immune targets. The viral genes can be cloned and expressed as protective antigens or directly synthesized proteins can be used for vaccine development…. Nucleotide sequence analysis, along with recombinant DNA techniques, have become extremely efficient, powerful and inexpensive, and are one of the main reasons that our understanding of emerging viruses comes as rapidly as it now does.

  1. C. J. Gordon et al., Remdesivir is a direct-acting antiviral that inhibits RNA-dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency. J Biol Chem, (2020).
  2. K. G. Andersen, A. Rambaut, W. I. Lipkin, E. C. Holmes, R. F. Garry, The proximal origin of SARS-CoV-2. Nat Med 26, 450-452 (2020).
  3. S. F. Ahmed, A. A. Quadeer, M. R. McKay, Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies. Viruses 12, (2020).

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So Will and When Will We Have a Vaccine?

Just to cut to the chase, we just do not know; but we may hope for one year and a half to two years, maybe less. And that will be great, but we have to distinguish between getting early evidence of efficacy and getting a vaccine ready for mass vaccination. But let’s consider background material that suggests it is likely we will indeed have a vaccine.

First, it is helpful to consider different mechanisms of immunity. There are two general types of immunity, innate and adaptive. The innate system is quick and dirty and recognizes general patterns widely shared by pathogens, such as double stranded RNA or bacterial lipopolysaccharides, but not normally present. It is mediated through the activity of cytokines and involves inflammation. The other mechanism, the adaptive immune system, is slower to respond but more precise and is mediated through antibodies and cytotoxic T cells.

Second, there are several kinds of immunization. Transfusion of immune serum or of purified antibodies, called passive immunization, can protect temporarily against infection and disease. This is currently being tried in patients.  Another type of immunization may induce a general innate immune response. Administration of some vaccines appear to induce a temporary immunity against viruses other than their targets, probably through stimulation of innate immunity. In theory, they should be able to protect against a relatively broad array of targets, which might include SARS-CoV2. This is likely the basis for the activities of BCG, which is used as immunotherapy against bladder cancer, for example. Phase 3 trials are currently enrolling subjects in Australia (BRACE) and the Netherlands (BCG-CORONA). More recently, Dr. Konstantin Chumakov (Food and Drug Administration, Office of Vaccines Research and Review) and Dr. Robert Gallo (Institute of Human Virology, University of Maryland School of Medicine, Baltimore) of the Global Virus Network have suggested repurposing polio vaccine for use against SARS-CoV2 through its induction of innate immunity. Finally, targeted vaccines can stimulate both innate and adaptive immunity. Targeted vaccines are the most commonly used vaccines, as they are the most specific and can provide durable protection.

Is a targeted vaccine for SARS-CoV2 possible? Several lines of evidence suggest the answer is yes. Worry number one is whether immune responses to the virus will be protective. Sera from infected rhesus monkeys and infected people can neutralize the virus, indicating it is possible to generate an effective response given an appropriate stimulus.  A second concern is that the virus will mutate rapidly and escape the immunity induced by vaccines. However, available sequence data suggest the overall genetic variability might be low. The virus has an RNA proofreading mechanism that reduces its mutation rate. A hopeful finding is that immune sera from people infected with the SARS1 virus (which is only 79% related at the whole genome level) neutralizes the SARS2 virus, suggesting elicited immunity will be broadly reactive. Similarly, a monoclonal antibody has been reported that neutralizes both SARS-CoV1 and SARS-CoV2.

There are many antibody-based passive immunization approaches with human and animal serum and recovered antibodies in pre-clinical development. There are also currently more than 200 targeted vaccines in widely various stages of development, including at least seven phase 1 or 2 clinical trials, and there are currently at least eight different approaches to developing a vaccine. One approach is simply to inject one or more of the viral proteins. Two other approaches involve injection and cellular take-up of either RNA or DNA encoding one or more viral proteins. Two other approaches involve vaccination with a heterologous viral vector derived from adenovirus, lentivirus, measles, or influenza virus, which has been engineered to express SARS-CoV2 proteins. These viral vectors can be either replication incompetent or able to replicate to various extents. Another type of vaccine vehicle is a virus-like particle (VLP), made artificially from viral proteins but lacking a viral genome. The SARS-CoV2 protein of interest is attached to the outer surface of the VLP. The other two vaccine approaches use whole SARS-CoV2 virus, either in killed form or attenuated so that its ability to replicate is severely compromised.

Obviously, testing for safety and efficacy is critical. Animal testing provides a way to obtain preliminary safety and efficacy data from candidate vaccines relatively quickly, because animals can be challenged with live virus. Drawbacks are that they may not accurately mimic the pathogenesis in humans and their immune responses may differ from that of humans. A study from China reported that vaccination with inactivated virus protected 8 rhesus macaques from viral challenge, but the numbers are small. The vaccinated macaques also developed antibodies able to neutralize different strains of the virus. They observed no harmful effects, including enhancement, in which a vaccine induces an immune response that makes infection more severe.

The gold standard in vaccine efficacy testing is obviously controlled clinical trials in humans. There are two ways to do this. One is with a large cohort of people from a group that has a high attack rate from the virus. Obvious, the more susceptible the test population, the fewer the numbers and shorter the time period required for a definitive answer. The most straightforward way, however, is to challenge vaccinated and unvaccinated unpaid volunteers with virus. This has the advantage that far fewer numbers are needed than in trials using an unchallenged general population, and results are obtained more quickly. However, such an approach is obviously fraught with ethical concerns.

As mentioned above, there are at least seven phase 1 or 2 trials of targeted vaccines now underway, several of which hope to have a vaccine by fall. An RNA-based vaccine by Moderna (clinical trials ID NCT04283461) has been reviewed by the FDA for phase 2 testing, and it is hoped to start phase 3 trials this summer. A Chinese whole killed virus vaccine candidate by Sinopharm (Chinese Clinical Trial Registry IdentifierChiCTR2000031809) has reached phase 2 status. The Oxford Vaccine Group is conducting a phase 1/2 trial with a vaccine (clinical trials ID NCT04324606) based on a chimp adenoviral vaccine vector that reportedly has shown protection in rhesus macaques.  A vaccine developed by Pfizer and BioNTech, called BNT162, is based on modified RNA (clinical trial ID NCT04368728). A phase 1/2 trial is planned in Germany and the United States. Inovio’s vaccine, called INO4800, is a DNA-based vaccine. They are currently planning a phase 1 trial in the United States and a phase 1/2 trial in South Korea (clinical trials ID NCT04336410). CanSino Biologics has an adenovius 5-based vaccine candidate that the company announced has passed phase 1 trials and are initiating a phase 2 trial (clinical trials ID NCT04313127). Sinovac has initiated phase 1 trials with a vaccine candidate based on inactivated virus plus adjuvant (clinical trials ID NCT04352608).

With the large number of approaches, the relative genetic stability of the virus, and hopeful results from animal trials, it seems likely a vaccine will be successfully developed. The big question, of course, is how soon. The best case seems to be by fall of 2020. Pressure to speed up the schedule may spur further considerations of the ethics of human volunteer challenge studies. The development of better antiviral therapies may influence considerations of these types of trials, as it would make them somewhat safer.

 

Read the June 2, 2020 update here

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