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COVID-19

COVID-19 Antibody Testing: Answers from Dr. Robyn Heister

Key points:

  • COVID-19 appears to follow a typical seroconversion and immunoglobulin class (isotype) switching time course, IgM to IgG, which is important for antibody testing.
  • Based on what is known today, immunity may develop after infection by SARS-CoV-2, which could last at least for several months.
  • The clinical severity of COVID-19 may correlate positively with antibody titer two weeks after symptom onset.
  • The future of COVID-19 is uncertain but antibody testing is likely to play a strong part.

Questions answered:

  • ‘What kind of a virus is SARS-CoV-2 and how does it get into our body?’
  • How does our immune system respond to SARS-CoV-2 infection?
  • How quickly does one develop SARS-CoV-2 specific antibodies, and what is the proportion of patients presenting seroconversion?
  • Does primary infection with SARS CoV-2 result in immunity? 
  • If so, how long does the immunity last?
  • Why is antibody testing important? 
  • If I test positive am I no longer a threat to others?
  • How accurate are antibody tests?
  • What is the difference between antibody tests and viral RNA detection (swab) tests?


What kind of a virus is SARS-CoV-2 and how does it get into our body?

The virus underlying COVID-19, the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2), is a betacoronavirus, which is part of a family of viruses that are crown-shaped and tend to cause respiratory infections. From the currently available evidence, SARS-CoV-2 is mainly spread via respiratory droplets or contaminated surfaces. It ultimately enters our bodies through inhalation of virus-carrying droplets or when we touch our eyes, mouth, or nose with virus-contaminated hands.


Four proteins are mainly responsible for the structure of the virus: the membrane, envelope, nucleocapsid, and spike (S) proteins. The S proteins, which give the virus its crown-like feature, are involved in helping the virus enter cells via the ACE-2 receptor, mainly in the alveoli of the lungs. S proteins are also important in the body’s ability to develop immunity to the virus. Antibodies to the S protein specifically can block its ability to enter human cells and vaccines in development are therefore mainly targeting this part of the virus (1)


How does our immune system respond to infections?

Our immune response

When we are exposed to a new pathogen (disease-causing organism), our immune system responds in two waves. The first response, almost immediate, comes from our ‘innate’ immune system, which includes barriers like our skin but also immune cells like neutrophils. This response is quick but generic in that it doesn’t matter what the pathogen is, the innate immune system will respond in a similar way to fight it. The second wave is the ‘adaptive’ or ‘acquired’ immune response, occurring after a number of days. This is driven by T and B cells, and it is where antibodies come in. While this response is slower, it is tailored to the specific pathogen, with specially designed antibodies, and is more effective at launching a coordinated fight against the infection.


The role of antibodies

Antibodies released by B cells represent the humoral part of the adaptive immune response. When the body is exposed to a new pathogen that it has never seen before, such as SARS-CoV-2 in this case, after its initial innate response, the adaptive response includes the release of antibodies to launch a stronger attack. IgM antibodies respond fastest and are the first type to become detectable in the blood. Over days to weeks, IgG antibodies are also released, with the B cells switching from producing IgM to producing IgG. IgG has two roles: to continue and augment the fight against the infection but also to develop a ‘memory’ for the pathogen, so that in future it can respond more quickly and efficiently to repeat exposure. Therefore, when a person is subsequently exposed to the same pathogen, IgG levels rise earlier with a quicker and stronger antibody response.


Credit: Youtube

In the image, ‘primary response’ refers to first exposure to a pathogen and ‘secondary response’ refers to second or subsequent exposures to the same pathogen.


Seroconversion and class switching

This antibody development process is called seroconversion and when antibodies are detectable in the blood, that is referred to as being seropositive.(2) The switching of antibody production by B cells, for example from IgM to IgG, is called class, or isotype, switching.


How does our immune system respond to SARS-CoV-2 infection?

As SARS-CoV-2 is a new virus, we cannot assume that our bodies will respond in the same way as for other similar pathogens but we are learning more about it every day. There are two important questions that need to be answered regarding how our immune system responds to SARS-CoV-2.


  1. What is the proportion of patients who develop SARS-CoV-2 specific antibodies i.e. seroconvert, and how quickly does someone develop these antibodies after exposure?
  2. Are these antibodies effective at preventing future infection by SARS-CoV-2? And how long does this last?


A recent study has explored how our immune system does respond to this particular virus and found that it may follow a typical timeline for seroconversion and class switching (. This new information is a breakthrough in terms of understanding how best to harness and implement antibody testing for COVID-19.


How quickly does one develop SARS-CoV-2 specific antibodies, and what is the proportion of patients who develop these antibodies i.e. seroconvert?

This recent paper on the SARS-CoV-2 antibody response aimed to answer this particular question, and the authors studied 173 patients with confirmed COVID-19 over time to measure their antibody responses after symptom onset for up to 40 days (although this time frame varied between patients) using the ELISA lab technique. (3) They reported that it takes 11 days after symptom onset for 50% of those who are exposed to this pathogen to ‘seroconvert’ or develop detectable total antibody levels. For IgM and IgG sub-types, specifically, it took 12 and 14 days, respectively. Overall, 93.1% of patients seroconverted, as measured using total antibody level. That meant that 12 patients did not seroconvert; the authors speculated that this may have been due to their tests being drawn too soon after symptom onset. By 39 days after symptom onset, 100% of patients with samples taken had seroconverted (Figure 1, below). Overall, what this tells us is that a majority of people may have measurable antibodies to SARS-CoV-2 over two weeks after symptom onset. 


Average Seroconversion Timing in COVID-19 Patients


  • Overall, the seroconversion of Ab was significantly quicker than that of IgM (p = 0.012) and IgG (p<0.001), that possibly attributed to the double-antigen sandwich form of the assay used which usually show much higher sensitivity than capture assay (IgM) and indirect assay (IgG).


Does primary infection with SARS CoV-2 result in immunity? If so, how long does the immunity last?

At this time, SARS-CoV-2 has not been around for long enough to have a robust answer to this question for humans. There is one study that aimed to find clues using apes (4). The authors reported that, in four monkeys, recent exposure to SARS-CoV-2 resulted in protection against re-exposure. We do not yet know for how long this effect may last. An ongoing German study has released preliminary results to the media that are not yet peer-reviewed and officially published. In Heidelberg, the German epicentre of COVID-19, they have found that infection rates have reached 15%, with 14% showing evidence of immunity. Overall death rates may be lower than anticipated, at 0.4%. This suggests that immunity may be sustained after initial infection. Longer studies in SARS-CoV-1, a previous SARS coronavirus, found that healthcare workers who had been infected had sustained IgG antibody levels for years after infection (6). An ongoing study in healthcare workers is also aiming to understand what proportion have been exposed to SARS-CoV-2 and seroconverted (, while another is validating the use of at-home antibody testing in this high risk group (7).


Why is antibody testing important?

Antibody testing is vital for several reasons. Firstly, it will have a central role in determining the extent of the current pandemic. Reports suggest that anywhere from 20% to 50% of people infected may be asymptomatic (8,9,10) and many more only mildly symptomatic to the point that they do not get tested, which means that the reported rates of infection under-represent the true rates. However, if antibody testing was done at a large scale, we could discover what proportion of the population has already been exposed to SARS-CoV-2. This epidemiologic information would be valuable in informing policy decisions and even determining how to ease the restrictions currently in place regarding social interaction, working from home, and travel. Secondly, knowing whether healthcare professionals have been exposed already and recovered could have implications for how workers are deployed at the frontline. Thirdly, antibodies from people who have been exposed already can be taken from blood samples and repurposed into potentially life-saving treatment called convalescent plasma, which can be given to others with severe active infection who are unable to mount an adequate immune response. A final point on antibody testing is that it may be useful for doctors in determining who is at risk for a more severe form of COVID-19. The aforementioned study on seroconversion in COVID-19 found that there was a positive correlation between total antibody levels and developing critical illness (3), meaning that those who had a stronger antibody response tended to have more severe COVID-19.


If I test positive am I no longer a threat to others?

Given that we have not yet determined how effective our antibody response is at preventing future SARS-CoV-2 infection, we cannot say for sure that previous exposure renders someone completely immune. If you are not immune, you can still contract the virus yourself and pass it on to others. However, initial results are promising, and many are of the opinion that we will develop at least a temporary immunity to COVID-19 after initial infection.


How accurate are antibody tests?

Antibody tests specific for SARS-CoV-2 are being developed as fast as possible. In samples tested between 15 and 39 days after symptom onset, antibody tests can be very accurate (3). With time, as we learn more about the virus and its features, tests will become more consistent and accurate. Accuracy is determined by two factors: sensitivity and specificity. 


Sensitivity in this case refers to the ability of a test to detect SARS-CoV-2 when it is, in fact, present. If test sensitivity is low, it means that a negative test does not necessarily out-rule the presence of the virus. Thus, it is vital that diagnostic tests have high sensitivity. 


Specificity refers to the ability of the test to accurately diagnose the specific virus of interest. Low specificity means that a positive result may not necessarily be due to SARS-CoV-2 infection, but could be caused by the presence of antibodies to another coronavirus or another virus family. In the case of antibody testing, this would give someone a false sense of security in believing that they had already been exposed to SARS-CoV-2 and may therefore be immune.


Available tests for SARS-CoV-2 antibodies have shown variable sensitivity and specificity (11, 12, 13) These vary for different reasons. Firstly, antibody tests can differ in how they try to detect SARS-CoV-2 antibodies. Recall the structure of the virus discussed earlier and its different proteins - some tests aim to use S protein antibodies to detect virus exposure, while others use antibodies to other aspects of the virus like the Nucleocapsid protein. Secondly, different laboratory techniques can be used. ELISA seems to be more accurate than the quicker lateral immunochromatography technique  Finally, how the test is deployed plays an important role. Remembering that it takes a number of weeks for antibody levels to rise to detectable levels means that testing at the appropriate time following exposure will minimise false negative results, thus increasing sensitivity.


What is the difference between antibody tests and viral RNA detection PCR (swab) tests?

There are several differences to be aware of between antibody tests and RNA detection tests. We have discussed antibody tests and their application in detail already: they allow you to determine whether someone has previously been exposed to the virus. It is important to understand what antibody tests can not do. They can’t reliably diagnose an infection in the early stages. That makes sense when you think about how antibodies respond to a new infection. However, RT-PCR tests of nasal swabs or sputum samples are the current gold standard for diagnosing the presence of SARS-CoV-2. They are used in testing centers and hospitals currently and are the test results that are widely reported to keep track of global rates of SARS-CoV-2 infection. This technique involves looking for viral RNA in a collected sample but the standard and widely used process is much slower than antibody testing. A number of weeks after you have recovered from an infection, there will be no detectable viral RNA left in your samples and your PCR test will be negative. It won’t show whether you were previously exposed to the virus.



The future of COVID-19: what could happen?

At this point, it is unclear exactly how the rest of 2020 will unfold. There are two overarching factors to consider: firstly, how will the virus behave? And secondly, how should and will governments respond? Scientists are working tirelessly to better understand SARS-CoV-2 and to make recommendations regarding the most effective and least disruptive public health response. There are many possible scenarios. Most begin with the current ‘lockdown’ period extending anywhere from at least 1.5 to 3 months. After this, the potential scenarios diverge. Three main scenarios are outlined in table 1 below. 


The most likely of these at this point is the ‘long game’ outcome. Different strategic options have been implemented in other countries and the outcomes of these will inform decision-makers as to what may be most effective and most acceptable. These range widely. It could involve cycling between periods of restrictive lockdown and periods of less restrictive physical distancing measures depending on infection trends and hospital and critical care capacity until a safe and effective vaccine is developed. Another possibility is implementing testing at an unprecedented scale in order to identify all those infected (or already immune), which would help prevent further transmission and allow restrictions to be lifted - ongoing, regular testing would be performed to detect and isolate new cases early until a vaccine is developed.


The ‘containment’ option involves maintaining current restrictions until transmission rates are under control nationally and globally and then easing restrictions given the low risk of new cases coming from other countries. This would require significant collaboration within the US and between all countries globally. This is unlikely to occur based on how the pandemic and public health responses have played out to date.


The ‘surge’ outcome represents a decision not to pursue ‘flattening of the curve’ but to try and protect the most vulnerable and ‘raise the line’ (increase healthcare capacity). Many believe this would be the most devastating outcome from a public health perspective, possibly culminating in millions of deaths and collapse of the healthcare system, because transmission rates would be high and the surge in cases would overwhelm hospitals.


Table 1: If you look at three potential future scenarios & the possible applicability of testing, it might look like this:



Sources

Sources:

1) NCBI "SARS-CoV-2 Vaccines: Status Report," 2020 Apr 6

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136867/

2) World Health Organization, "Guidelines on clinical evaluation of vaccines: regulatory expectations," 2016

https://www.who.int/biologicals/expert_committee/Clinical_change

3) Oxford Academic, "Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019," 2020 Mar 28

https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa344/5812996

4) BioRxIV / CSH, "Reinfection could not occur in SARS-CoV-2 infected rhesus macaques," 2020

https://www.biorxiv.org/content/10.1101/2020.03.13.990226v2

5) MedRxIV / CSH / BMJ / Yale - "Long-Term Persistence of IgG Antibodies in SARS-CoV Infected Healthcare Workers", 2020

https://www.medrxiv.org/content/10.1101/2020.02.12.20021386v1

6) ClinicalTrials.gov, "COVID-19 Seroconversion Among Front Line Medical and Paramedical Staff in Emergency, Intensive Care Units and Infectious Disease Departments During the 2020 Epidemic (SEROCOV)," 2020

https://www.clinicaltrials.gov/ct2/show/NCT04304690

7) ClinicalTrials.gov, "Rapid SARS-CoV-2 IgG Antibody Testing in High Risk Healthcare Workers (COVID-Antibody)," 2020

https://clinicaltrials.gov/ct2/show/NCT04334876

8) International Journal of Infectious Diseases, "Estimation of the asymptomatic ratio of novel coronavirus infections," 2020

https://www.ijidonline.com/article/S1201-9712(20)30139-9/pdf

9) National Library of Medicine, "Estimating the Asymptomatic Proportion of Coronavirus Disease 2019 (COVID-19) Cases on Board the Diamond Princess Cruise Ship, Yokohama, Japan" 2020

https://pubmed.ncbi.nlm.nih.gov/32183930/

10) Government of Iceland, "Large scale testing of general population in Iceland underway," 2020

https://www.government.is/news/article/2020/03/15/Large-scale-testing-of-general-population-in-Iceland-underway/

11) CDC.gov, "Severe Acute Respiratory Syndrome Coronavirus 2−Specific Antibody Responses in Coronavirus Disease 2019 Patients," 2020

https://wwwnc.cdc.gov/eid/article/26/7/20-0841_article

12) Stadlbauer, et al, "A detailed protocol for a serological assay to detect SARS-CoV-2 seroconversion in humans: antigen production and test setup," 2020

https://labs.icahn.mssm.edu/krammerlab/wp-content/uploads/sites/68/2020/03/Stadlbauer-and-Amanat-COVID19-antigen-expression-and-serology-protocol-3_22_20.pdf

13) Journal of Medical Virology, Wiley "Development and clinical application of a rapid IgM‐IgG combined antibody test for SARS‐CoV‐2 infection diagnosis" 2020

https://onlinelibrary.wiley.com/doi/epdf/10.1002/jmv.25727

14) MedRxIV "A serological assay to detect SARS-CoV-2 seroconversion in humans" 2020

https://www.medrxiv.org/content/10.1101/2020.03.17.20037713v1.full.pdf

Primary Reference Study:

https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa344/5812996 


Other relevant studies / commentaries: 

1) BMJ - April 7th.

2) Norwegian Public Health rapid review.

3) Testing different SARS-CoV-2 antibody assays

4) Large ongoing study on French healthcare providers to ascertain seroconversion rate.

5) A serological assay to detect SARS-CoV-2 seroconversion in humans - April 16.

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