Researchers at Karolinska Institutet in Sweden have identified a small neutralizing antibody, a so-called nanobody, that has the capacity to block SARS-CoV-2 from entering human cells.

The researchers believe this nanobody has the potential to be developed as an antiviral treatment against COVID-19. The results are published in the journal Nature Communications.

"We hope our findings can contribute to the amelioration of the COVID-19 pandemic by encouraging further examination of this nanobody as a therapeutic candidate against this viral infection."

Gerald McInerney, Study Corresponding Author and Associate Professor of Virology, Department of Microbiology, Tumor and Cell Biology, Karolinska Institute

The search for effective nanobodies--which are fragments of antibodies that occur naturally in camelids and can be adapted for humans--began in February when an alpaca was injected with the new coronavirus' spike protein, which is used to enter our cells.

After 60 days, blood samples from the alpaca showed a strong immune response against the spike protein.

Next, the researchers cloned, enriched, and analysed nanobody sequences from the alpaca's B cells, a type of white blood cell, to determine which nanobodies were best suited for further evaluation.

They identified one, Ty1 (named after the alpaca Tyson), that efficiently neutralizes the virus by attaching itself to the part of the spike protein that binds to the receptor ACE2, which is used by SARS-CoV-2 to infect cells. This blocks the virus from slipping into the cells and thus prevents infection.

"Using cryo-electron microscopy, we were able to see how the nanobody binds to the viral spike at an epitope which overlaps with the cellular receptor ACE2-binding site, providing a structural understanding for the potent neutralisation activity."

Leo Hanke, Study First Author and Postdoc in the McInerney Group

Nanobodies offer several advantages over conventional antibodies as candidates for specific therapies. They span less than one-tenth the size of conventional antibodies and are typically easier to produce cost-effectively at scale.

Critically, they can be adapted for humans with current protocols and have a proven record of inhibiting viral respiratory infections.

"Our results show that Ty1 can bind potently to the SARS-CoV-2 spike protein and neutralize the virus, with no detectable off-target activity" says Ben Murrell, assistant professor in the Department of Microbiology, Tumor and Cell Biology and co-senior author of the publication. "We are now embarking on preclinical animal studies to investigate the neutralizing activity and therapeutic potential of Ty1 in vivo".

This project is the first arising from the CoroNAb consortium, which is coordinated by Karolinska Institutet, and funded by the European Union's Horizon 2020 research and innovation programme. Additional funding for this project was obtained from the Swedish Research Council and KI Development Office.

The sequence of Ty1 is available in the scientific article and will also be posted on the NCBI GenBank sequence database under the accession code MT784731.

As the COVID-19 pandemic progresses, it is crucial to track the viral changes in order to link the clinical and epidemiological features of the disease with these mutations. A new study published on the preprint server bioRxiv* in September 2020 reports on a common severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutation, D614G, and its effects on the virus.

Early in the course of the pandemic, a new mutation emerged, namely, D614G, in the spike protein, superseding most local strains wherever it appeared. The current study is based on incorporating this mutation into a wildtype SARS-CoV-2 strain in order to understand how it affects virus-host interactions.

The outcomes they measured included viral replication on human lung epithelial cells and primary human airway tissues, viral fitness in hamster upper airway, and neutralization susceptibility. This study should contribute to understanding the role played by this mutation in the transmission of the virus, the effectiveness of various vaccines, and of convalescent antibody-rich plasma.

Spike Mutations and Their Importance

Even though most COVID-19 cases develop mild illness only, the pandemic has claimed almost 900,000 lives worldwide in the last eight months. Notably, severe COVID-19 is linked to dysregulated immune response, with excessive and systemic inflammation. However, viral factors such as virulence could also play a part in the evolution of the illness.

Researchers have discovered mutations that encode amino acids of a different sort in the SARS-CoV-2 spike protein sequence. This protein is key for viral entry into the host cell via the angiotensin-converting enzyme 2 (ACE2). Such mutations may cause alterations in the host range, pathogenesis and tissue tropism.

In the earlier SARS outbreak of 2002, a single mutation in the spike protein allowed the infection to spill over from the original reservoir to the intermediate civet host and to humans.

The D614G Mutation

In the current pandemic, the D614G mutation has become dominant after March, to cover ~75% of all published viral sequences by June 2020. There were three accompanying mutations as well.

This mutation set has become the predominant one both worldwide and also within distinct localities after introduction, showing that it may have conferred a fitness advantage and not just due to the founder effect or genetic drift. One especial advantage appears to be increased infectious potential, supported by the finding that this mutant is associated with higher viral loads in the nasopharynx in COVID-19 patients.

Pseudoviruses Show Improved Infectivity of Mutant Strain

Transmissibility is a key factor in viral fitness, but direct measurements were required to confirm the role of the mutation in improved fitness. The researchers first described the phenotype of the D614G mutation in pseudoviruses. They found higher viral titers in many different types of cell cultures when infected by the G614 variant. This indicated that perhaps the variant could cause higher viral entry into cells and replication in the airway.

To confirm this, they used wildtype virus with the spike mutation to infect a cell culture, primary human airway 3D tissue, and experimental hamsters. They also used neutralization tests for serum samples and monoclonal antibodies (mAbs), based on reporter SARS-CoV-2 viruses carrying either D614 or G614, labeled with mNeonGreen.

Higher Replication and Infectivity of Mutant Strain

The researchers observed that the D614G substitution in the spike protein in human lung epithelial cells caused more significant viral replication and infectivity by comparing the two variants, first in Vero cell culture. They found similar-looking plaques, but with a higher infectious titer with the G614 virus after 12 hours; later, both displayed similar titers. The same was seen for extracellular viral RNA loads. This showed that the mutation did not affect these two parameters on Vero cells.

The next step was to repeat the experiment on human lung epithelial Calu3 cells. They found that the G614 virus had somewhat higher infectious viral titers up to a maximum of 2.4 times higher over the next 48 hours. Still, the viral RNA load was lower or equivalent over the same period with this variant. This indicates that the D614G mutation makes the virus in the human lung cell line more infectious.

How did this happen? To know more, the researchers then looked at how the spike protein was being processed in the two variants. They found that full-length spike protein obtained from the virions grown in human lung epithelial cells was almost fully processed to the cleavage form in both variants. However, those obtained from Vero cells showed markedly lower cleavage efficiencies by 20% and 30% less for the D614 and G614, respectively.

This suggests the influence of the cell culture on the cleavage efficiency, but no differential effect was observed between the two viruses.

Mutation Improves Fitness in Hamster Upper Airway

The D614G mutation was then tested in the golden Syrian hamster model, where it was found that either variant showed similar pathological features, including weight loss, but without other visible symptoms. The infectious viral titers from the upper and lower respiratory tract were similar for both viruses on the second day, but the differences were more apparent in the upper than the lower airway. The most significant difference between the titer of infectious virus for the two variants was seen on day 4 after infection, in the upper airway, particularly in the nasal epithelium.

Even though the infectious viral titer was higher for G614 than for D614 in hamsters, the viral RNA load was almost identical. Thus, the ratio of RNA to PFU for G614 remained lower across the range of airway tissues.

The researchers' comment, “If the lower viral RNA/PFU ratio of the G614 virus could be extrapolated to COVID-19 patients, the modest differences in cycle threshold (Ct) values of RT-qPCR in patients’ nasopharyngeal swabs would translate to ≥10-fold infectious G614 virus, underscoring the potential for enhanced transmission and spread.” In fact, it has been observed that if the same patient has two different strains, one in throat swabs and the other in sputum, the former alone is the cause of spread.

On the seventh day after infection, no infectious virus could be detected. However, viral RNA copies were still abundantly found in nasal wash samples, indicating that viral RNA persists after the infectious virions are cleared. This explains positive RT-PCR findings in some COVID-19 patients, even when the infectious virus is undetectable. Moreover, it reflects the clinical finding that disease severity is not correlated with the dominant mutation.

Direct fitness comparison was carried out using a competition experiment. This allows the elimination of host-host variations while using internal controls, with more precise quantification of the ratio of the virus strains than comes from finding the titers individually. Using intranasal inoculation, they infected hamsters with 10⁴ PFU each for each virus. To compensate for the Vero cells of origin, they were also given viral RNA to levels equivalent to the above. They found that in all airway tissues, the G614 virus is superior to D614G614/D614, indicating its replication advantage.

Viral Replication Boosted

The researchers then evaluated the replication of D614 and G614 viruses in a 3D primary airway tissue model. They found that titers of infectious virus were much higher for the latter, at 2-8 times, but not the viral RNA loads. This confirms the effect of the substitution on replication by increasing viral replication in the human upper airway tissue.

Competition experiments to compare replication fitness showed that even when the initial infectious ratio of D614 and G614 went from 1:1 to 9:1, the latter still increased to fivefold the infectious viral titer of the former within five days. The G614 strain can thus outperform the other at great speed to achieve the numerical advantage, even when it is first present as the minor variant in a mixed virus population.

Susceptibility to Neutralization

The neutralization testing of a set of sera collected from hamsters that had been infected with the D614 strain showed that in all cases, the neutralization titer required for G614 was 1.4- to 2.3 times higher than for D614, showing that this mutation may increase the susceptibility to neutralization. Thus, any vaccine effective against the former will not lose its efficacy against the latter.

The researchers then looked at 11 anti-RBD mAbs against the two viruses. They found that only one showed double the potency against G614 compared to D614, but the other 10 showed similar neutralization efficacy to both. The mutation may reduce the neutralization efficacy depending on the conformation of the spike protein, based on which epitope is presented to the mAb.

Conclusion

The researcher sum up their findings as demonstrating that “spike substitution D614G enhances viral replication in the upper respiratory tract and increases neutralization susceptibility.” This could help develop better vaccines and antibodies to contain the pandemic in the future. (written By Dr. Liji Thomas, MD)

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

As a disease surveillance tool for COVID-19, the Ateneo Center for Computing Competency and Research (ACCCRe) of ADMU collaborated with the University of the Philippines Manila - National Telehealth Center (UP-NTHC) and the Department of Health-Epidemiology Bureau to develop the technology which is now publicly accessible at https://fassster.ehealth.ph/covid19 and uses localized indices from Philippine health records.

The current landscape of COVID-19 vaccine initiatives in the country is still dynamic - as discussions are still ongoing with potential partners. Here are some of the frequently asked questions (FAQs) on COVID-19 vaccine initiatives, as addressed by the office of DOST Assistant Secretary for International Cooperation. The contents of these FAQs will be updated regularly.


1. What is Coronavirus-2019 (COVID-19)? 

It is an infectious disease caused by a newly discovered coronavirus –SARS-CoV-2. The disease is transmitted to humans through respiratory droplets or discharges from sneeze and cough, and saliva. Since its first recorded case in China, COVID- 19 had widely spread in the different parts of the world. With the continuous increase in the number of infected people, countries have taken numerous measures and/or strategies to control the spread of the disease – physical distancing, the mandatory wearing of face masks, and increased detection of cases with early isolation and quarantine among others. Also, the global scientific community has been working tirelessly searching for the vaccines to provide immunity against this virus.

2. How do vaccines work? 

Vaccines work by introducing inactivated, weakened, or killed copies of the whole or part of the disease-causing pathogen, for instance, SARS-CoV-2, to our bodies just enough to stimulate our immune system to naturally produce cells or specific antibodies that will fight the virus. Once our immune system has successfully eliminated these foreign elements inside our bodies, it will remember the disease it caused and the antibodies it previously produced to fight it. If we are then exposed to the real SARS- CoV-2 in the future, our immune system already knows how to destroy the virus before it can even make us seriously sick.

3. What if there is no vaccine?

If there is no vaccine, transmission of the virus may slow down if enough people get infected and the country approaches herd immunity and gain at least a temporary respite from major outbreaks Some experts have estimated reaching that point would require as much as 60 percent to 70 percent of the population getting infected. However, the death rate at 60 70 infections in the population will also increase.

4. Will there be a vaccine for COVID 19?

The SARS CoV 2 is a novel coronavirus, and vaccines against it have yet to be developed But experts think there will be a COVID 19 vaccine. In fact, there are more than 160 ongoing SARS CoV 2 vaccine developments being conducted across the globe by many research institutions, private and state-owned pharmaceutical companies, and universities. To date, these vaccine candidates are in various trial stages to ensure their efficacy and safety for human use. As of 20 August 2020 the World Health Organization ( records 30 candidate vaccines that are in various clinical trial stages while 139 are in the pre-clinical trial stage.

Health experts are accelerating research to study the origins of the virus and how it is spreading. The virus has been differentiated from SARS and MERS, but its contagiousness and virulence are still being studied.

5. What are the stages of vaccine development?

Vaccines undergo various clinical trial stages to ensure their safety and efficacy These stages include:

• Pre-clinical - Vaccines are tested in animals such as mice and monkeys to see if it produces an immune response
• Phase 1 - Vaccines are given to a small number of people 10 100 people) to test its safety
• Phase 2 - Vaccine are given to hundreds of people 100 to 1 000 people) to test its efficacy, determine the right dosage, and ensure that the desired effects are achieved
• Phase 3 Vaccine are tested in a larger group of people to confirm its efficacy and safety when compared to other treatments

6. How are clinical trials done?

In a clinical trial, a volunteer is usually assigned a specific study group Volunteers in one study group may receive the COVID 19 vaccine while others may receive a placebo or a comparator vaccine in order to assess its efficacy. The trial is usually a double-blind test where participants, physicians, and research staff do not know which volunteer receives a placebo and the active treatment. This will eliminate bias so that physicians and research staff will provide the same level of medical attention and care to all participants. The vaccine recipients are monitored for side effects at various time points during the trial, and tested for their immune responses to the vaccine components.

7. Who can participate in the clinical trials?

All potential recipients of the vaccines under the clinical trial will first be screened for certain inclusion and exclusion criteria. These criteria include physical examination, general state of health, and ability to follow instructions among others. Laboratory tests will also be performed such as baseline RT PCR for SARS CoV 2 viral RNA, IgM and IgG tests, clinical chemistry examinations to detect abnormalities or disease conditions that may not be detected on physical examination alone.

8. What will participants get for joining in the clinical trial?

Participants of clinical trials in the Philippines will be given excellent healthcare services and closely monitored by the attending physicians They will also receive a minimal allowance to reimburse meals and transportation associated with participation in the clinical trial.

9. Are there risks associated with participation in the clinical trials?

Vaccination of investigational drugs or vaccines may have side effects including pain, redness, itchiness, or swelling at the injection site, which may last a few hours Other side effects may also include fever, feeling of weakness or fatigue, headache, dizziness, diarrhea, and nausea. However, during the trial, the attending physician will determine if the side effects are causally related to the vaccines. Participants are also given diary cards and are expected to report to the vaccine trial monitors any side effects or development of COVID 19 signs and symptoms during the clinical trial period. Generally, the benefits of taking the vaccine outweigh the risks associated with its side effects.

10. Are all vaccines the same? Will they work for everyone?

There are several platforms being used for the development of COVID 19 vaccine, each one having their own advantages and disadvantages Some are tried and proven and have worked for other viral illnesses Some can be produced much faster, but it is unclear if they will all be as effective Hence, parallel development of multiple types of vaccines is a good thing.

Also, a COVID 19 vaccine should have at least 50 efficacy ratings which refer to the percentage reduction of disease in a vaccinated group of people compared to an unvaccinated group. The efficacy depends on the types of the vaccine and the population inoculated This means that different types of vaccines and different populations may produce different outcomes.

11. What are the initiatives of PH government to provide vaccines for Filipinos?

The Philippines does not currently have the capacity to produce and manufacture its own vaccines Hence, the government has been in close collaboration with several countries and international organizations that are engaged in the development and manufacturing of possible COVID 19 vaccine. So far, the Philippines has had talks with seven foreign vaccine R&D institutes and manufacturers who are ahead in the race for COVID 19 vaccine Also, the government has signified its intent to participate in both the WHO Solidarity Trial on Vaccines and the GAVI COVAX Facility.

12. What are the criteria in choosing possible PH bilateral partners for COVID 19 vaccine?

The Philippines is looking into possible partnerships with bilateral partners who ( have COVID 19 vaccine development in the advance stage (i e vaccine candidate has finished Phase II Clinical Trial or currently on Phase III Clinical Trials), and ( willing to locally manufacture their vaccines in the Philippines.

13. What is WHO Solidarity Trial for Vaccine?

The World Health Organization leads the Solidarity Trial for vaccines which aims to harness global cooperation to develop and evaluate vaccine candidates as quickly as possible (identify vaccine candidates and their progress; (define the desired characteristics of safe and effective vaccines to combat the pandemic and ( coordinate the clinical trials across the world giving the best chance of safe and effective vaccines for all.

14. Can other vaccine developers outside the WHO Solidarity Vaccine Trial still conduct clinical trial in the Philippines?

Yes. Other vaccine developers can conduct independent clinical trials in the Philippines given that they will be able to fund their own trials and register their application with the FDA Independent trials would require a larger sample size based on the size requirements of Phase III clinical trial and will be mainly overseen by the vaccine developers/manufacturers and the local. Contract Research Organization and/or medical team they will engage in.

15. What is COVAX Facility?

The COVAX Facility, co-led by Gavi, the Coalition for Epidemic Preparedness Innovations (CEPI), and the World Health Organization (WHO), is a platform that aims to accelerate the development and manufacture of COVID 19 vaccines and to ensure that every country in the world is able to access the successful vaccines.

16. How will the COVAX Facility ensure equitable access to COVID 19 vaccines?

The COVAX Facility works by incentivizing manufacturers to accelerate the development and manufacture of possible vaccine candidates This allows the facility to create the largest and most diverse portfolio of COVID 19 vaccines As soon as it becomes available, COVAX will deliver doses for at least 20 of each country’s population, prioritizing the most vulnerable and at risk.

17. Will there be financing support available for the Philippines?

Yes. The COVAX facility provides opportunities for low income and lower-middle-income countries to access subsidies through the Gavi COVAX Advanced Marketing Commitment (AMC). The Philippines, as a lower-middle-income country, was recently announced as one of the countries eligible to access the COVAX facility.

Overall, the facility will require at least US 2 Billion of seed funds to fund the volume guarantees and deliver the vaccines to Low-Income Countries (LICs)and Lower Middle-Income Countries (LMICs).

18. What are the criteria for choosing a successful vaccine?

The vaccine technical evaluators use the WHO internationally accepted standards for vaccines for pandemic or outbreak response use or for long term use The WHO Target Product Profiles are used as reference standards, as well as other WHO Technical standards and FDA requirements.

A matrix is prepared to give weights on compliance of the vaccine candidates with respect to the following vaccine characteristics indication for use, contraindication, target population, safety/ reactogenicity, measures of efficacy, dose regimen, the durability of protection, route of administration, product stability and storage, co-administration with other vaccines, presentation, WHO Emergency Use Listing registration and pre-qualification, and accessibility. All pre-clinical and clinical data are rigorously evaluated.

19. What are the government’s measures to ensure the vaccine’s safety?

Aside from the Solidarity Trials, overseas clinical trials, and prequalification from the WHO, the Philippine government has established additional measures to ensure vaccines safety and efficacy The sub-Technical Working Group on Vaccine Development has created the DOST Vaccine Expert Panel, a group of technical experts and scientists tasked to identify, evaluate, and recommend possible vaccine candidates for the Philippines. The DOH Health Technology Assessment Unit as well as different medical and specialty societies have also suggested to the IATF safety nets to ensure vaccine safety when already in use through pharmacovigilance and surveillance.

20. When will the vaccine be available?

We are anticipating that the first vaccine supply will come from the GAVI COVAX Facility in the second quarter of 2021 But a lot of factors may affect this timeline that may result in advances or delays in the release of the vaccine.

There are other sources of vaccines aside from the COVAX Facility that PH is also exploring Vaccines may also be obtained through bilateral engagements with governments and foreign vaccine developers. However, there is no definite timeline yet for the release of these vaccines as it will depend on the results of the vaccine clinical trials.

21. Who will have the first access to the vaccines?

Once the vaccines become available, the government may prioritize vulnerable or high-risk groups such as healthcare workers to receive the first doses of the vaccine Also, the indigent sector of the population (senior citizens, most vulnerable citizens, poorest of the poor, etc and those communities and areas with high-risk factors, may also be provided access to the initial doses.

Succeeding supplies of the vaccine will be provided to the general population after the priority population has been given with the vaccine. 

22. Will the immunity lasts for a lifetime?

Immunity induced from administering a vaccine fades over time and the protection differs with each kind of disease and their causative agent Since SARS CoV 2 is a novel coronavirus, any long term immunity may only be determined once vaccines become available and data on their efficacy of becoming available after about 6 months from when the phase III trials.

23. Does the Philippines intend to produce its own COVID 19 vaccines?

The Philippines currently does not have the capacity to produce a COVID 19 vaccine for its population But the government is working with foreign vaccine developers who would be willing to invest and manufacture their vaccines in the country Currently, local pharmaceutical companies have expressed willingness to establish a fill and finish facility in the Philippines With this, the country will only import bulk antigens from foreign vaccine developers and fill them in ampoules and vials These facilities are intended to be modular so that it can be used and repurposed for other vaccines in the future.

24. What is the Philippines’ long-term goals to ensure the vaccine self-sufficiency?

The DOST has initiated two long term action plans to achieve vaccine self-sufficiency One is the establishment of a Virology Institute of the Philippines ( that is currently a pending bill in the legislature, together with the reinstitution of the DOST Pharma Center The sub-Technical Working Group on Vaccine Development suggested to expand the VIP to include not only viruses but other pathogens as well These institutions will serve as research arms of the government to build its capacity to produce its vaccines and drugs especially for future pandemic situations The second long term action plan is to establish a vaccine manufacturing facility through the Vaccine Self Reliance Project of the Research Institute for Tropical that is currently in the pipeline.

25. What is the manufacturing process for a vaccine?

The tools that are needed for manufacturing a vaccine vary considerably depending on its type But in many cases, vaccine development requires a bioreactor a giant tank that allows the organisms to grow that will be the source of vaccines

In addition to the bioreactor, there are other things to take into consideration, such as medical-grade glass, a sterile vial, or syringe which are generally limited and may eventually become a bottleneck if the goal is to produce billions of doses of the vaccine.

26. Are there other ways to protect us against the novel coronavirus?

A vaccine will be our best protection against the novel coronavirus. However, while the vaccine development is still underway, we have to continue instituting measures to limit the spread of the virus such as physical distancing, early detection and isolation of cases, use of face masks and other PPEs We should sustain our efforts to:

• Improve testing availability and turnaround times to help detect outbreaks
• Hire and train more contact tracers, creating more tools to assist them
• Produce high-quality N95 masks for daily public use
• Find effective treatments that speed up recovery and increase survivability

Source: http://www.pchrd.dost.gov.ph/index.php/news/6594-frequently-asked-questions-on-covid-19-vaccines-2 (Written by: Christine Jane Gonzalez)