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).

 

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).

The Global Virus Network’s Position Regarding the Risk of COVID-19 Transmission by Asymptomatic Infected Subjects

BALTIMORE, MD, June 12, 2020: According to the Global Virus Network (GVN), a coalition comprised of the world’s preeminent human and animal virologists from 53 Centers of Excellence and 10 Affiliates in 32 countries, whether or not asymptomatic COVID-19 infected subjects can transmit the virus has tremendous impact on the global public health strategy against SARS-CoV-2 viral infection. In this context, the recent statement on that topic offered by the World Health Organization (WHO) and the discussions around this statement clearly provide an excellent opportunity to discuss this major issue.

We understand that there is confusion between asymptomatic and pre-symptomatic cases. Pre-symptomatic transmission has been defined as the transmission of SARS-CoV-2 from an infected person to a secondary patient before the source patient has developed symptoms. Published studies have shown substantial transmission of SARS-CoV-2 before symptom onset; indeed, importantly, shedding of the virus may begin 2 to 3 days before the appearance of the first symptoms (1), and this is in contrast to what had been observed with SARS-CoV-1. Although virus shedding has not been quantitated, specimens (71%) from pre-symptomatic persons had viable virus by culture 1 to 6 days before the development of symptoms in a nursing facility (2). After symptom onset, viral loads decreased monotonically. The proportion of pre-symptomatic transmission ranged from 46% to 55% (1). In general, as expected, the reported proportion of pre-symptomatic transmission is higher in those geographical areas where diagnostic tests for SARS-CoV-2 have been widely used. Pre-symptomatic transmission occurs through generation of respiratory droplets, as well as, possibly, aerosol or through indirect transmission (3-5). In contrast to the pre-symptomatic transmission, it has been a challenge to quantify the contributions of asymptomatic individuals to the transmission of SARS-CoV-2 (3). Lack of quantitative analysis of viral shedding also hindered in evaluating the role of asymptomatic individuals in viral transmission. However, clinical studies showed that asymptomatic individuals had substantial viral shedding for potential transmission (2, 6). For example, a recent study clearly pointed to viral transmission by asymptomatic individuals (6). Certainly, comprehensive studies are required for quantitative analysis of viral shedding, viremia, and innate immune responses in asymptomatic and pre-symptomatic individuals.

Unfortunately, the current COVID-19 pandemic is expanding because it is difficult to trace mild or pre-symptomatic infections. We have learned a very important lesson from the initial response to the outbreaks of SARS-CoV-2. For example, most countries worldwide had delayed state and local responses, thus allowing SARS-CoV-2 to spread rapidly (7). A critical problem was testing: symptom-based screening alone failed to detect a high proportion of infectious cases and was not enough to control transmission in this setting. Consequently, many countries have been shut down to prevent a rapidly expanding epidemic because only isolating people who are sick might not be enough to contain the epidemic. In contrary, several countries, such as Australia, South Korea, Germany, Singapore, and Taiwan, managed to contain the virus early and have worked hard to keep it suppressed with efficient testing and contact tracing system.

Until vaccines are widely available, available infection prevention approaches are case isolation, contact tracing and quarantine, physical distancing, decontamination, and hygiene measures. To implement the right measures at the right time, it is of crucial importance to understand the routes and timings of transmission. It is certain that the existence of pre-symptomatic transmission would present difficult challenges to contact tracing. More inclusive criteria for contact tracing to capture potential transmission events 2 to 3 days before symptom onset should be urgently considered for effective control of the outbreak as has been done in Hong Kong and mainland China since late February (1). Although WHO recently clarified its position on COVID-19 asymptomatic transmission, all these scientific findings emphasize that efficient control of asymptomatic and pre-symptomatic transmission is a critical intervention strategy for COVID-19. To support this, a massive testing with contact tracing system should be provided for the detection of viral shedding in asymptomatic individuals.

Reference

  1. He X, Lau EHY, Wu P, et al. Temporal dynamics in viral shedding and transmissibility of COVID-19. Nat Med 26, 672–675. 2020.
  2. Arons MM, Hatfield KM, Reddy SC, et al. Presymptomatic SARS-CoV-2 infections and transmission in a skilled nursing facility. NEJM 2020 382:2081-2090.
  3. Gandhi M, Yokoe DS, Havlir DV. Asymptomatic transmission, the Achilles’ Heel of current strategies to control Covid-19; NEJM 2020 382:2158-2160.
  4. Asadi S, Wexler AS, Cappa CD, Barreda S, Bouvier NM, Ristenpart WD. Aerosol emission and superemission during human speech increase with voice loudness. Sci Rep 2019 9:2348.
  5. Hijnen D, Marzano AV, Eyerich K, GeurtsvanKessel C, Giménez-Arnau AM, Joly P, et al. SARS-CoV-2 transmission from presymptomatic meeting attendee, Germany. Emerg Infect Dis. 2020 Jul. DOI: 10.3201/eid2608.201235.
  6. Chau NVV, Lam VT, et al. The natural history and transmission potential of asymptomatic SARS-CoV-2 infection, Clinical Infectious Diseases, https://doi.org/10.1093/cid/ciaa711.
  7. Schneider EC. Failing the test — the tragic data gap undermining the U.S. pandemic response. NEJM. 2020. DOI: 10.1056/NEJMp2014836.

Global Virus Network Suggests Oral Polio Vaccine May Provide Temporary Protection Against COVID-19

World-renowned scientists publish strong argument for the live attenuated vaccine in Journal Science

BALTIMORE, MD, June 11, 2020: The Global Virus Network (GVN), a coalition comprised of the world’s preeminent human and animal virologists from 53 Centers of Excellence and 10 Affiliates in 32 countries, published a viewpoint in Science today that the stimulation of innate immunity by live attenuated vaccines in general, and oral poliovirus vaccine (OPV) in particular, could provide temporary protection against coronavirus disease 2019 (COVID-19).

“We know specific interventions such as vaccines against a novel virus that can cause pandemic will take years to prove they work, are safe, durable, inexpensive and readily available for the world,” says Dr. Robert Gallo, The Homer & Martha Gudelsky Distinguished Professor in Medicine, Co-Founder & Director of the Institute of Human Virology at the University of Maryland School of Medicine and Co-Founder & Chairman of the International Scientific Leadership Board of the Global Virus Network,  “Clearly, these vaccines need to go forward.  However, until there are proven efficacy, safety and global availability of the classical vaccines for SARS-CoV-2, we believe our strategy relying on simple, safe, oral, inexpensive, live vaccines will have a broad benefit against COVID-19. This can also likely be used in future pandemics, particularly of respiratory viruses, by inducing innate immunity, which is immediate and not as limiting as a specific vaccine.”

OPV is a live attenuated vaccine that was safely used in the United States from 1963-2000 and is still being used in more than 140 countries.  Large-scale clinical studies of OPV for nonspecific prevention of diseases were carried out in the 1960s and 1970s. These involved more than 60,000 individuals and showed that OPV was effective against influenza virus infection, reducing morbidity 3.8-fold on average. OPV vaccination also had a therapeutic effect on genital herpes simplex virus infections, accelerating healing. OPV not only demonstrated positive effects against viral infections, but also oncolytic properties, both by directly destroying tumor cells and by activating cellular immunity toward tumors. More recent studies confirm these broad protective effects of OPV.

“Repeated immunization has an additive effect on stimulation of non-specific protection despite antibodies induced by the first vaccination,” says Dr. Konstantin Chumakov, Associate Director for Research for the U.S. Food and Drug Administration’s (FDA) Office of Vaccines Research and Review and a GVN Center Director.  “Further, recent reports indicate that COVID-19 may result in suppressed innate immune responses, and thus, their stimulation by OPV immunization might increase resistance to SARS-CoV-2 as well as a broad spectrum of other pathogens.”

“The GVN serves as a catalyst to bring together the world’s foremost virologists,” says Dr. Christian Bréchot, President of the GVN, and a Professor at the University of South Florida.  “We are pleased to bring this idea to fruition, and we look forward to working with varying nations to initiate clinical trials.”

In addition to Dr. Robert Gallo and Dr. Konstantin Chumakov, the authors of the viewpoint in Science include Dr. Christine Benn of OPEN and the Danish Institute for Advanced Study, University of Southern Denmark, Odense, Denmark and Dr. Peter Aaby of the Bandim Health Project, Bissau, Guinea-Bissau, who are both renowned experts in clinical vaccine research, and Dr. Shyam Kottilil professor of medicine and director of the Clinical Care and Research Division of the Institute of Human Virology at the University of Maryland School of Medicine, a GVN Center of Excellence, Baltimore, Maryland, USA.  Dr. Kottilil, as the colleague of Dr. Gallo and Dr. Chumakov, will be the chief clinician operating the clinical trials studying OPV against SARS-CoV-2 infection.

“Pandemics are unpredictable and have devastating impact on human lives,” says Dr. Kottilil.  “Our strategy allows a rapid, simple, low-cost, global approach to curtail the present and future pandemics.”

“Studies in low-income countries have shown that OPV is associated with strong reductions in child mortality even if there was no circulating polio virus,” say Dr. Christine Benn and Dr. Peter Aaby. “In Denmark we found that OPV-vaccinated children had lower risk of getting hospitalized for respiratory infections. We think that OPV may have the same beneficial non-specific effect among adults. We will soon be starting a randomized trial including 3,400 adults above 50 years of age in Guinea-Bissau to assess whether OPV can reduce the risk of COVID-19 and other infections.”

“OPV has a strong safety record, the existence of more than one serotype that could be used sequentially to prolong protection against SARS-CoV-2, a low cost, ease of administration and much availability,” says Dr. Gallo.  “This is not complicated, the science is there to support the idea, and we need to act fast.”

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

 

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

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

 

TWO CENTERS OF EXCELLENCE OF THE GLOBAL VIRUS NETWORK INDEPENDENTLY VERIFY AN ANTIMICROBIAL TECHNOLOGY THAT ERADICATES SARS-CoV-2 ON SURFACES FOR MORE THAN SIX WEEKS

Global Virus Network Also Advances The Study Of The Oral Polio Vaccine As A Preventive Measure Against SARS-CoV-2

Baltimore, Maryland, USA, June 4, 2020:  The Global Virus Network (GVN), a coalition comprised of the world’s preeminent human and animal virologists from 53 Centers of Excellence and 10 Affiliates in 32 countries, announced that two of its Centers of Excellence, the Peter Doherty Institute for Infection and Immunity in Melbourne, Australia and the Rega Medical Research Institute of KU Leuven, Belgium, demonstrated that a BIOPROTECT™  formulation by ViaClean Technologies eradicates SARS-CoV-2 (the unique coronavirus that causes COVID-19) on surfaces and provides continuous residual viricidal activity for more than six weeks.  The announcement was made today by Dr. Christian Bréchot, President of the GVN.

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

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

Figure. When applied to a surface or incorporated into a material, BIOPROTECT™ forms a covalent bond with the substrate and creates a microbiostatic antimicrobial protective layer, making it unreceptive to microorganisms. The coating forms a nano-bed shield of spikes (self-assembling monolayer), each of which carry a positive charge that attracts the negatively charged microorganism. Once attracted, the molecular spikes pierce the cell and rupture its cell membrane, causing the microorganism to die. Image from ViaClean Technologies.

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

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

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

 

GVN Also Advances The Concept Of The Oral Polio Vaccine As A Preventive Measure Against SARS-CoV-2

The GVN has also advanced a concept developed by Dr. Robert Gallo, The Homer & Martha Gudelsky Distinguished Professor in Medicine, Co-Founder & Director of the Institute of Human Virology at the University of Maryland School of Medicine and Co-Founder & Chairman of the International Scientific Leadership Board of the Global Virus Network, and by Dr. Konstantin Chumakov, Associate Director for Vaccines at the Food & Drug Administration (FDA) and a GVN Center Director, to use the existing and proven safe Oral Polio Vaccine (OPV) as a preventive measure against SARS-CoV-2.  Non-specific protective effects of OPV have been demonstrated several times against a broad set of different virus outbreaks in the 1960’s and 70’s.  More recent studies confirmed these observations and revealed that other live vaccines produce pronounced non-specific protective effects, whereas inactivated vaccines do not.  Data from randomized clinical studies showed that OPV immunization campaigns reduced all-cause mortality despite the complete absence of poliovirus circulation.  The emerging body of evidence suggests that besides inducing specific humoral and cellular immune responses, OPV may activate multiple branches of the immune system, including training innate immunity and thus increasing resistance to a broad spectrum of pathogens, including SARS-CoV-2.  The Institute of Human Virology at the University of Maryland School of Medicine, a GVN Center of Excellence, submitted a proposal to the National Institutes of Health (NIH) for an 11,000-person clinical trial to demonstrate and establish the efficacy of OPV against SARS-CoV-2.

“The GVN is playing a very meaningful role in the battle against SARS-CoV-2 by coalescing the world’s foremost virologists and COVID-19 specialists to collaboratively share their expertise, findings and research, and by bringing together academia and industry to collaborate on the development and advancement of novel technologies, therapeutics and vaccine candidates for COVID-19,” said Dr. Gallo.  “I am pleased the GVN was able to identify laboratories to independently verify the efficacy of BIOPROTECT™, bring the potential benefit of OPV to the forefront of the scientific community and spearhead OPV clinical studies in China, Iran, Russia and the United States.”

 

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About the Global Virus Network (GVN)

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

About the Peter Doherty Institute

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

About the Rega Institute of Medical Research

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

 

GVN MEDIA CONTACT

Nora Samaranayake
Phone:  410-706-1966
Email:    nsamaranayake@gvn.org

 

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.

 

Read more GVN SARS-CoV-2 Perspectives

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).

 

Read more GVN SARS-CoV-2 Perspectives

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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Testing for SARS-COV-2

Testing, Testing, Testing…

The uncertainties about SARS-CoV-2 include critical questions such as what the death rate is for infected people, and what the infection rate is for the general public. This makes testing for infection of critical importance. There are numerous ways to establish the presence of infection. One is recovery of live virus, which in case of SARS-CoV-2 requires culturing the virus in biosafety level 3 containment. Although this would seem to be a gold standard, BSL3 facilities are not widely available, so this type of test is not routinely done. Another is to test for viral proteins, known as antigen testing. This seems to be amenable to high throughput testing, but reliable diagnostic tests have not yet become available. A third is to detect and measure viral RNA, generally done by PCR amplification following reverse transcription of viral RNA. It can be performed relatively readily in high quantity, but it is only an indirect indication of live virus. Even badly fragmented RNA can register as a positive, and positive results simply indicate that virus is being shed from somewhere within a patient, or has been recently. Thus, although recovered patients and asymptomatic people can be viral RNA positive, it is unclear as to what extent they can actually transmit virus without confirming live virus by isolation in a BSL3 facility. Fourth, serologic tests for antibodies to the virus can be performed on a massive scale, but depend critically on what the false positive and false negative rates are for a given test, and these can vary considerably among different test kits. The presence of antibodies means a person has been or is infected but may not still be infected at the time of the test.  Fifth, PCR tests similar to viral RNA tests can detect spliced or subgenomic RNA. In infected cells, full length viral RNA is spliced into a variety of smaller RNAs in order to encode many of the viral proteins. Thus, the presence of spliced subgenomic RNA indicates the presence of currently infected cells somewhere within the test subject. This process is somewhat labor intensive.

  • Of than these types of tests, the two that are amenable to mass testing and currently being used are viral RNA analysis and serologic tests, which are being widely and increasingly employed. To date, by far most testing that has been reported is for viral RNA. RNA is usually quantified based upon how many cycles of polymerase chain reaction are required to generate a detectable signal. These types of tests are highly specific (defined as the rate of false positives), but may lack sensitivity (defined as the rate of false negatives. This is not because of an intrinsic lack of sensitivity, but is because detection depends on the site from which the tested sample is taken. While throat and nasal swabs give reasonable sensitivity, bronchial alveolar lavage would be more sensitive, but collection is probably too onerous to be practicable. Saliva has been suggested as being conveniently obtained, but provides somewhat less sensitivity than nasopharyngeal swabs.(1) Wolfel et al.(2) performed RNA analysis of a number of tissues from moderately sick infected individuals. Swabs, saliva and stool contained viral RNA, although no live virus could be obtained from stool samples. The swabs and saliva contained live virus. Live virus could not be obtained after 8 days, although viral RNA remained positive. Urine and serum samples were never positive for viral RNA. Thus, detection of RNA in a sample is not tantamount to detection of infectious virus in the sample, or even in the tested individual.

Serologic tests for antibodies are becoming widely available and have the potential to make widespread testing much faster. As mentioned above, the presence of antibodies can indicate either that a person is infected or has been infected, and so does not necessarily indicate the presence of live virus. Following infection, IgM is the first antibody class produced, and so is more of an indicator of recent infection than is IgG, which is produced later. IgG tends to persist longer than IgM, and more sensitive to detect than past infections. Some antibody tests detect both classes; others only one. Samples collected include drawn blood or finger pricks. Widespread use of these tests has been hampered somewhat by the large number of tests of varying quality that are available; it is currently somewhat of a Wild West situation.

The specificity and sensitivity of these tests, defined respectively as the rate of false positives and false negatives, is critical for their usefulness, depending on whether they’re used for mass screening or diagnostic purposes. For diagnostic testing, sensitivity and specificity are obviously both important. For mass testing, specificity is absolutely critical.  Let’s consider using a test that is 95% specific for a population of 100 people that is 5% infected. The test will detect the 5 infected people. In addition, ~5 uninfected people will test positive, leading to the conclusion that 10% of the population is infected, even though the true infection rate is only half that. For reference, the test made by Abbot, which will be used in mass testing, has been reported as 100% sensitive, 99.5% specific. The other test that is likely to be used in mass testing is one by Roche. As of this writing, the accuracy and sensitivity data are not available, but will presumably be similarly high. Consideration of specificity should be taken into account when evaluating reports of unexpectedly high seroprevalence such as the study from Santa Clara County. The article can be accessed here, along with a number of critiques. Mass testing in the future should lend a lot more clarity to the true situation. It would be of interest to follow up positive serologic tests with confirmatory tests for RNA.

 

  1. E. Williams, K. Bond, B. Zhang, M. Putland, D. A. Williamson, Saliva as a non-invasive specimen for detection of SARS-CoV-2. J Clin Microbiol, (2020).
  2. R. Wolfel et al., Virological assessment of hospitalized patients with COVID-2019. Nature, (2020).

 

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