By Pushpa Larsen, ND

If you’ve been alive and breathing on this planet in the last year and half, you don’t need me to tell you what devastation has been wrought by the novel coronavirus known as SARS-CoV-2.  Pretty much everyone knows someone who has been infected with COVID-19.  If you’re lucky, none of your friends or family have died from this disease, but everyone has been impacted.  Pandemic fatigue set in months ago, and I don’t know anyone who isn’t looking forward to walking down the street without a mask, or being able to watch live music and sporting events, or attend weddings again – or just have friends over for dinner.  Mask-wearing and social distancing (where they are practiced) have definitely helped in mitigating the spread of the disease.  The availability of the vaccine is expected to reduce risk of transmission even further, but some key questions concerning the vaccination programs remain:

1. Given the widely varying efficacy between different vaccines, will I be protected from infection post-vaccination?
2. Presuming I am protected, how long will protection persist after vaccination?
3. To what degree do vaccines inhibit transmission, as opposed to just reducing serious disease?
4. Are there physiological factors that might give me less or no protection despite vaccination, such as obesity, diabetes, old age, or being immunocompromised?

The journal Nature recently published an article which listed five reasons why achieving that holy grail, herd immunity, might be impossible.¹ Among those reasons are that immunity – whether acquired from infection or by vaccination – may wane within a few months, and that immunity acquired in either way may not confer protection against new variants. One chart on the Centers for Disease Control website showed the prevalence of 17 different variants of the virus with an 18^th category for “other.”  “Other” turns out to include “>200 additional lineages that are each circulating at <1% of viruses.”²

The measures taken to try to stop the spread of COVID-19 – masking, social distancing, remote work when possible, varying degrees of shutdown of business and gathering places – have had a fair degree of success where they have been most consistently practiced, such as western Washington State and San Francisco.  This success has come with a cost, however, and not just economically. Mental health professionals have seen dramatic increases in anxiety and depression, from 11% of US adults in 2019 to 42% in 2020.³ Suicide ideation is also increased, with 10.7% of respondents reporting “serious consideration of suicide in the previous 30 days” in 2020 compared to 4.3% in 2018.⁴   Domestic violence towards intimate partners and children has also increased.⁵ The mental health impacts of the pandemic are primarily a function of social isolation and fear of the disease.

The National Institutes of Health suggests that widespread testing is “the key to getting back to normal.”⁶ They are referring to diagnostic testing, such as PCR and antigen tests, but COVID antibody tests also have an important role to play in allowing us to move toward a full re-engagement with society with confidence.  The ability to assess your patient’s likely immunity by monitoring antibodies to SARS-CoV-2 may be a useful tool to help decrease anxiety and increase confidence for a “back to normal” lifestyle.  The lack of such antibodies is also important information, especially as more people are vaccinated with the assumption that they are protected, which may not be the case.

This article presents a novel SARS-CoV-2 neutralizing antibody test that measures the functional potential of a patient’s antibodies to block the virus from propagating in the event of future exposure. Essentially, these specialized antibodies represent the frontline “special forces” defense that are tailor-made to attack and bind to the virus so it cannot enter the host cells and propagate infection.

SARS-CoV-2 Antibody Basics

SARS-Corona Virus 2 (SARS-CoV-2) infection starts when the virus binds to Angiotensin-Converting Enzyme 2 (ACE2) receptors on the surface of cells.  ACE2 receptors are found abundantly in the epithelial cells of lung alveoli and enterocytes in the small intestine, as well as in arterial and venous epithelial cells and in arterial smooth muscle cells.⁷ Binding of the virus to the ACE2 receptor allows penetration of the viral RNA into the cell and replication of the virus. This virus-cell interaction and subsequent viral infection leads to the activation of an individual’s immune response, which generates a population of antibodies against the virus. Vaccination also stimulates the production of COVID antibodies.

Not all antibodies produced against the SARS-CoV-2 virus are effective at preventing infection in the event of subsequent exposure. This may be why some patients have contracted COVID-19 more than once, and why vaccinations are not 100% effective.⁸ Individuals also seem to have a variable ability to produce antibodies in response to either infection or vaccination.⁹  Antibody production may vary from minimal to robust. Antibodies induced by infection will bind to a wide variety of viral proteins and regions.  Current vaccinations target the viral spike protein, so antibodies thus produced will bind primarily to that protein.⁸

Antibodies that protect against a viral infection are known as Neutralizing Antibodies, or NAbs.  SARS-CoV-2 NAbs bind primarily to a specific region of the viral spike protein known as the Receptor Binding Domain, or S-RBD.  This is the part of the spike protein that interacts directly with ACE2 receptors.  Not all antibodies that bind to the S-RBD will effectively neutralize the virus.  A COVID serology test that can distinguish between NAbs and other COVID binding antibodies can provide information on the functional immunity of an individual should there be future exposure to the virus.

The presence of NAbs from prior SARS-CoV-2 infection has been shown to be significantly associated with protection against reinfection. In a small retrospective analysis, the first of its kind in humans, preexisting neutralizing antibodies to SARS-CoV-2 were identified in three individuals on a fishery vessel which subsequently experienced a >85% attack rate at sea among its 122 crewmembers. None of the three crewmembers showed evidence of bona fide viral infection or experienced any symptoms during the viral outbreak, demonstrating the protective effect of the neutralizing antibodies.¹⁰  

Being able to measure the neutralizing potential of an individual’s COVID antibodies could be useful in determining how soon they can ease precautions or whether they are at risk if they return to work in a high exposure setting, etc.  For example, one Washington doctor reported that she had 20 patients who had contracted COVID-19 more than once.  Of those, five were health care workers who worked in a COVID ICU. (personal communication). These patients might have benefitted from testing that assessed the capacity of their COVID antibodies to neutralize the virus.

Does Everyone Make Neutralizing Antibodies?

Most people will make antibodies, including NAbs, to SARS-CoV-2 if they have been previously infected with the virus or if they have been vaccinated.  However, it is estimated that up to 12% of individuals do not make neutralizing antibodies with an additional 27% seroreverting after 180 days post infection.⁹ It is important to remember that the vaccine trials performed to date have been observational in nature.  That is, participants received either the vaccine or a placebo and then were followed to assess COVID-19 infection rates over a period of weeks or months.  This was not a live virus challenge and presumably, no one was actually trying to catch the virus.  Participants continued to practice masking and social distancing as they had before. Under these circumstances the efficacy of currently available vaccines globally ranges between 50% to 95%.⁸ The confidence and sense of security generated by vaccination becoming more widespread, as well as fatigue with the restrictions of the past year, will likely lead to a decrease in restrictions imposed and precautions taken.  This “return to normal” is certainly what most people are looking forward to.  For those individuals who have not made NAbs, a relaxing of precautions could have dire consequences. 

How Long Do Antibodies Last?

NAbs may provide sustained immunity to COVID, but there is evidence that a decrease in the neutralizing activity may occur more quickly than previously expected.  One study of 26 health care workers with mild cases of COVID-19 demonstrated a significant decline in neutralization capacity between 21 days after symptom onset and three months post onset.¹¹  While some studies confirm this decline in antibodies and/or neutralizing function, others suggest that neutralizing antibody response may be quite robust for a period of months.^12-16  As with all research, there are multiple ways to approach the question and there is no clear answer at this time.

For this reason, as we begin to rely on vaccination (or past infection) to protect us, monitoring neutralizing antibodies can be a useful tool to confirm whether that protection indeed exists and persists.  Identifying those individuals who are not protected or whose protection is waning will allow for appropriate safety measures to be taken in a more targeted manner.

What Is the cPass™ COVID Antibody Test?

The cPass™ Neutralization Antibody test provides accurate and rapid detection of total neutralizing antibodies (NAbs) to SARS-CoV-2 in response to previous infection or vaccination. There are a number of tests that detect COVID antibodies, and even a few that specifically identify antibodies to the spike protein.  cPass™ specifically detects those antibodies that block the receptor binding domain of the spike protein from interacting with ACE II receptors on the surface of cells.  Identifying neutralizing antibodies permits the assessment of a productive immune response assuring the presence of these important functional antibodies in the frontline immune response and possible protection to COVID-19.

What Is the cPass™ Methodology? (the technical stuff)

The cPass™ methodology is a surrogate virus neutralization test (sVNT) that utilizes a competition/inhibition ELISA, also known as a blocking ELISA. In this technique, ACE2 receptor protein reference material is coated in a multi-well plate. A patient sample is initially mixed with the detection antigen (SARS-CoV-2 RBD conjugated with Horse Radish Peroxidase, or RBD-HRP) and this mixture is then added to the ACE2-coated multi-well plate.  (See figure 1)

NAbs from the patient sample, if present, compete with and therefore block RBD-HRP from binding to the ACE2 receptor protein. After incubation, the RDB-HRP solution is washed out, leaving only that RBD-HRP that was not bound by the patient’s NAbs and so could bind to the ACE2 receptor protein coating the wells. A colorimetric reagent is then added to the plate wells, which causes a color change when interacting with the Horse Radish Peroxidase. The higher the concentration of NAbs present, the less RBD-HRP will have bound to the ACE2 receptor protein, and the weaker the output signal.  The strength of the signal correlates inversely with the amount of NAb present in the patient’s sample. So a weak output signal is observed with higher NAbs, and a strong output signal indicates a lack of NAbs.¹⁷

A key advantage to this method is that the RBD is in solution rather than coated to the plate as in most commercial serology tests. This increases the specificity of the assay as it allows the immunoglobulins present in the patient’s sample to interact with a more native conformation of RBD and its associated antigenic sites. A second advantage is that it is isotype-independent, allowing for the detection of any immune response antibody that binds to RBD and blocks its interaction with ACE2 (i.e.: not limited to IgG or IgM). This in effect confers a higher sensitivity as it detects all classes of NAbs compared to other commercial immunoassays that are isotype-specific.¹⁸

Traditionally, the presence of neutralizing antibodies has been detected by conventional virus neutralization tests (cVNT). cVNT is considered the gold standard because it uses live virus mixed with the patient’s serum which is then inoculated into cell cultures.  The process takes several days and must be performed in a high level safety lab facility (biosafety level 3). Such a laboratory setting makes this type of assay expensive as well as time-consuming and so is not widely used.  cPass™ is a surrogate virus neutralization test (sVNT) that can be done in a typical commercial laboratory.  The cPass™ assay takes less than 1.5 hours to run for 92 samples providing an accurate measurement of neutralizing antibodies in serum samples without the need of live virus and biosafety containment.¹⁹  cPass™ results have been shown to correlate well with cVNT.^18-20 The test has also been shown to have minimal cross-reactivity to other human CoV infections with the exception of SARS-CoV-1.¹⁹

Using cPass™ to Measure Longevity of NAbs

A critical question surrounding vaccination relates to the duration of protection. Thus, measuring the titer of neutralizing antibodies post infection or vaccination will permit individuals to track their levels over time. A semi-quantitative protocol has been written for cPass to assess titer levels of protective NAbs. The instructions for use and associated validation data have been recently submitted to the FDA to amend the current qualitative emergency use authorization for cPass.  FDA approval of the amended EUA is anticipated and may have been granted by the time of publication of this article.  The protocol is currently being used in research and permits the measurement of titer levels of NAbs in research samples.

With this ability to measure actual titers of NAbs, cPass™ can be used to evaluate the relative robustness of a patient’s antibody response.  It is important to note that at this time there is not sufficient research to determine what level of NAbs actually confers protection.  The cPass™ technology holds great promise for helping answer this question.   The ability to monitor titers also provides a mechanism for understanding the longevity of NAbs and the point at which an individual is conceivably no longer protected.  This can be important in determining the need for additional precautions or booster vaccinations, especially in times of resurgence.  If herd immunity is indeed not attainable, it is expected that we will see continue to see future surges of COVID-19.¹ This is illustrated by the dramatic surge in cases in Michigan this past March, in which cases soared from about 1,000 per day to over 5,600 per day in just over four weeks.²¹

Does cPass™ Find NAbs to SARS-CoV-2 Variants?

The cPass test has been designed to bind antibodies against the receptor binding domain of the original wild-type SARS CoV-2 strain.   This makes it ideal to assess the presence of protective neutralizing antibodies post-vaccination since all the current vaccines illicit antibodies to the wild type strain. With the emergence of dominant variant strains of SARS CoV-2, concerns have been raised around vaccine efficacy. Although there are some solid data that demonstrate a significant decrease in the neutralizing capacity of vaccine-elicited antibodies to the South African strain, the authors of some studies state that the levels are still high enough to offer adequate protection.^22-24 Hence, a strong NAb response to vaccination suggests adequate protection from the current SARS CoV-2 variants. Since cPass precisely measures these NAbs, it represents an ideal solution to assure post-vaccination immunity.

How Does cPass™ Compare with Other COVID-19 Antibody Tests?

Other commercially available COVID antibody tests, such as traditional ELISAs, measure total binding antibodies (BAbs) and are unable to distinguish NAbs from binding antibodies which do not possess neutralizing function. These COVID-19 antibody tests evaluate the ability of a patient to generate antibodies that bind the virus, such as IgG, IgM, or total antibody, but they do not tell whether the patient’s antibodies can block the virus from binding to the ACE2 receptor.  This is the essential step that breaks the life cycle of the virus.  cPass™ is not measuring IgG or IgM or total antibodies.  It is measuring the interaction between purified spike protein (S-RBD) and purified ACE2 receptor protein that is inhibited by the NAbs present in the patient serum.  The cPass™ test is, therefore, a better measure of potential resilience to re-infection.²⁵ In one study comparing cPass™ NAb methodology with BAb kits using RBD coated plates, the RBD coated plate method had an accuracy of 87.2%, a positive predictive value (PPV) of 44.87%, and negative predictive value (NPV) of 99.44%.  This is in contrast to the cPass™ methodology, which had an accuracy of 98.67%, a PPV of 100% and an NPV of 98.54%.  In other side-by-side testing, cPass™ demonstrated equal or greater sensitivity and specificity to traditional ELISA tests, including those from Roche, Abbot, and Beckman.^18,25

Most of the COVID antibody tests available are qualitative, meaning that they give a YES/NO result – either antibodies were detected, or no antibodies were detected.  The cPass™, as noted above, is currently available as a semi-quantitative assay for research use and approval for clinical use is anticipated.   Although there are a few tests that provide semi-quantitative results for total COVID antibodies, or COVID IgG and IgM antibodies, once the amended EUA has been authorized, the cPass™ technology will be the only commercially available test that can provide titers of neutralizing antibodies. 

What Do the cPass™ Test Results Mean?

It is important to remember this test is not diagnostic for a current COVID infection.   A diagnostic test for SARS-CoV-2 should be performed if acute infection is suspected. Diagnostic tests check genetic material from the virus itself, usually by polymerase chain reaction (PCR), to diagnose active infections through nasal swab, throat swab, saliva, etc. 

A positive cPass™ test result means the person has developed neutralizing antibodies to SARS-CoV-2, indicating potential immunity against the virus. Antibodies may be detectable in the blood as soon as several days after initial infection or vaccination. When present, they are usually an indication of a past infection, but a recently infected patient who is still contagious might also have positive antibodies.  If there is any question that a patient might have a current infection, a PCR test, as mentioned above, is the appropriate test.  With a confirmed negative PCR test, a positive cPass™ test shows viral clearance.

A negative cPass™ test result means that SARS-CoV-2 specific neutralizing antibodies were not present in the specimen above the limit of detection.  Patients tested early after infection or vaccination may not yet have detectable antibodies.  It can take one-to-three weeks after infection/vaccination to generate antibodies. Because not all infected patients will develop neutralizing antibodies to SARS-CoV-2, a negative result should not be used to rule out past infection.

Is the cPass™ Test FDA Cleared?

As with all other SARS-CoV-2 antibody tests, the cPass™ has not been FDA cleared or approved. cPass™ has been authorized by FDA under an Emergency Use Authorization (EUA) for testing samples by authorized complex CLIA laboratories for the presence of total neutralizing antibodies against SARS-CoV-2 and reporting the results back to the patients.

Applications of cPass™

cPass™ testing has promising applications both clinically and in research.  The most obvious is for testing individuals who are post-recovery from an active infection or post-vaccination. For those that have been infected with COVID-19 in the past and are recovered more than two weeks, the test can assess the immune system’s response to the infection. This could be an important tool in identifying those individuals who have not made neutralizing antibodies and are therefore at risk for reinfection.  Those individuals can then take greater precautions than they might if they assumed that they were immune.  Likewise, being able to identify those individuals who have robust neutralizing antibodies may allow a speedier return to normal.  A test about two weeks after vaccination is complete can similarly identify those who have had an inadequate response to vaccination. 

Repeat testing may help us better understand the longevity of immunity whether from previous infection or vaccination, as well as signal when an individual’s immunity is beginning to wane.  Early in the pandemic we saw that a significant percentage of people who test positive for COVID-19 never developed symptoms.  Many people never were tested if they didn’t have symptoms, even if they had been in close proximity with someone who tested positive for COVID.  cPass™ may be useful in identifying whether those individuals not previously tested have been exposed and developed antibodies.  This could also have a bearing on when we can confidently and safely relax restrictions. 

There are many potential research applications of cPass™.  A few that come to mind are the following:

• Evaluating differences in the effectiveness of the various vaccines to generate neutralizing antibodies (if there are any);
• Teasing out the role that neutralizing antibodies, or the lack thereof, might play in the long-lasting symptoms that some COVID patients experience;
• Evaluating whether certain therapeutics – natural or pharmaceutical – make a difference in the strength or effectiveness of the immune response.  For example, some studies have shown that low vitamin D levels put people at greater risk for severe COVID, while higher levels appear to decrease that risk.^26-28 On the other hand, vitamin D supplementation after someone has already contracted COVID may not make much of a difference.²⁹ Theeffect of vitamin D might have nothing to do with neutralizing antibodies, of course, but there may be therapeutics that could improve antibody response.

We are all tired of the pandemic.  Part of that exhaustion is the very high degree of uncertainty that we’ve been living with for the past year and half.  The cPass™ technology is one tool that can be used to provide greater understanding and certainty for our patients and ourselves.

Dr. Pushpa Larsen graduated from Bastyr University in naturopathic medicine, naturopathic midwifery, and spirituality, health and medicine. She worked as a research clinician for the Bastyr University Research Institute and as affiliate clinical faculty for Bastyr University, training students in her clinic. She practiced in West Seattle for 10 years before joining Meridian Valley Lab as a consulting physician eleven years ago. She is currently the manager of consulting and education and a member of the development team. Dr. Larsen has consulted with hundreds of doctors every year on the use and interpretation of 24-hour urine hormone profiles, blood viscosity tests, and other tests offered by Meridian Valley Lab. She is working with Dr. Jonathan Wright on the development of an educational program for health care practitioners in bioidentical hormone replacement therapy following Dr. Wright’s principle of “copy nature.” plarsen@meridianvalleylab.com

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