COVID-19 Antibodies: New York University Scientist Discover That Up To 75 Percent Of Recovered COVID-19 Patients Have Low Neutralizing Antibody Titers

COVID-19 Antibodies: A new study by researchers from New York University reports that up to about 75% of recovered COVID-19 patients have low neutralizing antibody titers. The study findings also showed that high titers of multiple antibody isotypes against both the SARS-CoV-2 spike receptor-binding domain and nucleoprotein are needed for effective and better neutralization.


 
The study findings has implications that current conventional convalescent plasma protocols might be not be effective after all since the right type of antibodies isotypes and in the right proportions are needed to achieve effective neutralization.
 
The study findings were published on a preprint server and are pending peer review. https://www.biorxiv.org/content/10.1101/2020.08.15.252353v1
 
A key question has always been to understand whether the immune response to this virus produces durable protection against reinfection. This will also determine the usefulness of a vaccine.
 
Current antibody testing to distinguish the infected from the non-infected in a community only provides a partial seroprevalence perspective to the question of whether an infection has occurred or not. Such test does not help to understand whether the detected antibodies are neutralizing and capable of preventing reinfection.
 
The study team from NYU used convalescent serum obtained from 101 healthcare workers who had tested positive for the virus. They analyzed the different classes of antibody as well as the neutralizing potency of the serum on both the wildtype virus and a lentiviral pseudovirus.
 
The pseudotyped viruses were used as they are not pathogenic, unlike the native virus, and are therefore used more frequently in research. However they do have differences relative to the wildtype virus, as shown by the variation in their neutralization by convalescent serum.
 
The study team looked at the separate Immunoglobulin G (IgG), Immunoglobulin M (IgM), and Immunoglobulin A (IgA) isotypes to the SARS-CoV-2 receptor-binding domain (RBD) on its spike glycoprotein, as well as to the nucleoprotein.
 
The minimum inhibitory concentration or MIC which is minimum dilution at which convalescent serum provided complete protection against cell infection by the virus, was calculated for each neutralization assay using the virus.
 
In addition when the pseudovirus was used, the researchers employed a luciferase assay to calculate the IC50 and the IC90.
 
The research shows that neutralization assays using these two systems are positively correlated. The researchers also found three categories of antibody response:
 
1. Most individuals, about 75%, had a low neutralizing antibody capacity, as measured by MIC and IC90, against the wildtype virus and the pseudovirus.
 
2. About 20% had intermediate neutralization capacity against both.
 
3. Only about 6% had high neutralizing capacity against the wildtype virus, but this was zero when pseudotyped virus neutralization was considered. Thus, there was a significant difference in the proportion of high neutralizers found using a pseudotyped virus.
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The study team  said that based on this, pseudovirus neutralization is an efficient option to the natural virus for such testing, but warn, “Authentic SARS-CoV-2 virus may be better able to detect potent neutralizing serums, which has implications for the selection of donors for passive immunization therapy.”
 
The resarch used samples from individuals at different times from symptom onset, which may account for the difference in neutralizing capacity. However, no timeline could be observed concerning this group. That is to say, low, high, and intermediate neutralization capacity was found at various time points between 32 and 57 days.
 
However one significant finding is that at this timeline, “serum SARS-CoV-2 neutralizing antibody capacity is low in most recovered individuals.”
The study team also analyzed the isotypes against two different viral antigens and the range of antibody isotypes.
 
The team found high IgG, IgM, and IgA titers against both antigens, corresponding to the neutralization capacity. This is important to know since many a time, antibodies are overproduced in response to one or a few viral proteins, and if so, using an antigen other than these may result in a falsely low antibody titer.
 
The research showed a uniform antibody response to both spike and N proteins, with the strongest correlation being with IgG, followed by IgA and IgM. However, most patients who developed anti-RBD IgA were negative for anti-N IgA, unlike IgG and IgM agreement for the RBD and N proteins. This would mean that the test used to detect IgA to the N protein, as compared to the RBD, would be negative in many cases.
 
However study failed to show any relationship between the time of symptom onset and the titer of each isotype.
 
Interestingly, IgM was detected at 50 days from the date of infection.
 
The study team inferred, “The antibody isotype response to SARS-CoV-2 may be antigen-specific, with IgA skewed towards the RBD.”
 
The study team found that neutralization capacity could be predicted by the antibody titers obtained from ELISA assays. Comparing each ELISA antibody titer with virus neutralization capacity, the anti-RBD antibody titer for all isotypes bore a strong correlation to the ability to neutralize the wildtype virus. Anti-N IgG also showed significant correlation but not other isotypes.
 
It was observed however that the pseudovirus neutralization capacity correlated best with the IgG titer rather than the other isotypes. For both types of viral particles, the correlation with neutralizing capacity was weaker for anti-N IgA and IgM compared to anti-N IgG.
 
The study team postulates that this may be due to misfolding of the folding of the RBD in the purified form, in the wildtype virus, and in the pseudovirus, causing the antibody isotypes to recognize them differently. IgM antibodies are generally thought to signify recent infection, with declining titers over time as antibody production switches to IgG and IgA. Instead, this may indicate that all the isotypes have to be evaluated to understand the true timeline of the immune response in this infection.
 
The team said, “These findings suggest that ELISA methods based on the RBD may benefit from detection of additional isotypes, rendering them better suited as predictors of sera neutralization.”
 
The study team  then compared the titers for each isotype and each antigen, finding that the highest neutralizing sera had higher antibody isotype titers against both antigens. Within each serum sample, the isotypes targeting the RBD agreed better than those against the N protein.
 
The tea found that mounting a robust antibody response, consisting of diverse isotypes, leads to efficient neutralization for all isotypes.
 
The study team classified the patients into 21 different clusters depending on the combination of antibody isotypes, from those who were positive for all six to being negative for all. Of the 6 individuals with high neutralizing titers, four were positive for all isotypes against both antigens, while two had a combination common to medium and low neutralizers as well. In fact, one cluster which is positive for all except anti-RBD IgM has low neutralizing capacity.
 
Significantly, this may means that certain combinations are needed for the highest neutralization to occur.
 
Most individuals were positive for IgG and IgA against the RBD, and for IgG against N protein, suggesting that IgG alone may not suffice for good neutralization.
 
The team said “It remains to be elucidated how these clusters are generated and why certain clusters elicit potent SARS-CoV-2 neutralization, and others do not. Since we use a targeted approach focusing on only two antigens and specific epitopes, it is possible that there are other anti-SARS-CoV-2 antibodies that bind viral particles and impact neutralization.”
 
As disease severity is well known to correlate with antibody production, in high neutralizers, the multiple isotypes and high neutralizing titer could be driven by severe disease.
 
In stark contrast, three patients did not seroconvert, as shown in some earlier studies too. Perhaps they really did not produce neutralizing antibodies, or the response declined to undetectable levels by the time of this study.
 
Proper follow-up studies over longer periods are needed to show how antibody titer is related to protection against reinfection.
 
A critical and significant caveat is that laboratory neutralization assays show just that but do not necessarily convey the protective capacity of antibodies in the clinical setting.
 
The study team stresses that detailed studies monitoring initial and possible reinfections along with the antibody response are needed for the important understanding of immunity to the SARS-CoV-2 coronavirus.
 
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