New study on COVID-19 patients

Just as I said it is too early to have any studies on CoronaVirus a new study got published. Please note it has not been peer reviewed yet, so it is not 100% official as of yet. As a personal note I am not a big fan of ELISA (Enzyme-linked immunosorbent assay) technique in testing for various reason, however I do understand the necessity of it. We have far better approaches to testing emerging that have been discovered by so many innovative labs, unfortunately due to complicated reasons, money being one, these methods hasn’t been inherited as a mainstream approach. Getting sidetracked here. Maybe one day I will write an article to explain the various reasons down the road.

Overview

As you read this study please consider it was done on a small sample and it is a very quick turnaround study. I am sure more in-depth studies are on the horizon. Most peer reviewed medical publications (remember this one hasn’t been peer reviewed yet) use a similar layout in displaying the information. I am sure there is a term for this structure/layout. Below I attached both screenshots and the direct link to the pdf, however I enclosed the abstract and the results in thus quick article for ease of access. If you read the study you will first find the listed authors, the abstract and the introduction. After the abstract and the introduction the credentials and other factors about this study is listed. This information is necessary to prove the credentials and the validity of the study. It is always good to review these things, but not always necessary to read in depth as a novice reader. I always look into what their sample base is to get an idea on where the samples were obtained from, what type of samples are being used. It is good to know overall what methods are used to test. Here in this study ELISA was used as the assay technique. This is expected considering ELISA and it’s accurate sensitivity is what this study is testing for. PRNT 50 was used as the measurement. This is also expected since PRNT 50 is considered to be the gold standard for detecting and measuring antibodies.

I’ve listed some terminologies in case you are new to reading medical studies. I’ve captured the most simplistic definitions for you to make it easier to understand. At the bottom I’ve listed my resources in case you are interested in reading further on the the topics. As a novice reader you might find it difficult to understand at first glance, this is normal and it is the very reason why I wrote this to help you get familiar with the concept especially since the findings of this study is very short. As you get more familiar with the jargon you will find it becomes more readable overall and keep in mind nobody expects you to understand everything since you are not in the medical field. You might ask why it is necessary to read medical studies. One it’s the most accurate information. First hand knowledge is better than some watered down version that’s filled with inaccuracies. Two educating yourself as a patient or concerned citizen is powerful both from education point of view and putting anxieties at ease point of view.

Terminology

ELISA:

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. … In an ELISA, an antigen must be immobilized on a solid surface and then complexed with an antibody that is linked to an enzyme. *1

PRNT 50:

The plaque reduction neutralization test (PRNT) is used to quantify the titer of neutralizing antibody for a virus. The concentration of serum to reduce the number of plaques by 50% compared to the serum free virus gives the measure of how much antibody is present or how effective it is. This measurement is denoted as the PRNT50 value. Currently it is considered to be the “gold standard” for detecting and measuring antibodies that can neutralise the viruses that cause many diseases. *2

Antibody:

Antibodies, also known as immunoglobulins, are Y-shaped proteins that are produced by the immune system to help stop intruders from harming the body. When an intruder enters the body, the immune system springs into action. These invaders, which are called antigens, can be viruses, bacteria, or other chemicals. *3, 4, 5

1. Immunoglobulin A (IgA), which is found in high concentrations in the mucous membranes, particularly those lining the respiratory passages and gastrointestinal tract, as well as in saliva and tears.

2. Immunoglobulin G (IgG), the most abundant type of antibody, is found in all body fluids and protects against bacterial and viral infections.

3. Immunoglobulin M (IgM), which is found mainly in the blood and lymph fluid, is the first antibody to be made by the body to fight a new infection.

4. Immunoglobulin E (IgE), which is associated mainly with allergic reactions (when the immune system overreacts to environmental antigens such as pollen or pet dander). It is found in the lungs, skin, and mucous membranes.

5. Immunoglobulin D (IgD), which exists in small amounts in the blood, is the least understood antibody. *4

SARS-CoV-2:

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), previously known as 2019 Novel Coronavirus (2019-nCoV), is a positive-sense, single-stranded RNA virus that causes the potentially lethal COVID-19 respiratory tract infection. This new virus belongs to the genus Betacoronavirus, which also includes SARS-CoV and MERS-CoV. *6

SARS-CoV-2 specific antibody responses in COVID-19 patients

Link to the study: https://www.medrxiv.org/content/10.1101/2020.03.18.20038059v1.full.pdf

Written By

NISREEN M.A. OKBA17 (n.okba@erasmusmc.nl), Marcel A Muller2 (marcel.mueller@charite.de), Wentao Li3 (w.li@uu.nl), Chunyan Wang3 (c.wang1@uu.nl), Corine H. GeurtsvanKessel1, Victor M. Corman2 (victor.corman@charite.de), Mart M. Lamers1, Reina S. Sikkema1, Erwin de Bruin1, Felicity D. Chandler1, Yazdan Yazdanpanah4, Quentin Le Hingrat4, Diane Descamps4, Nadhira Houhou-Fidouh5, Chantal B. E. M. Reusken1, Berend-Jan Bosch3, Christian Drosten6, Marion P.G. Koopmans1 and Bart L. Haagmans1 (b.haagmans@erasmusmc.nl)

• 1 Erasmus Medical Center;

• 2 Charite Universitatsmedizin;

• 3 Utrecht University;

• 4 Universite de Paris;

• 5 Hopital Bichat-Claude Bernard;

• 6 Charite-Universitatsmedizin

• ↵* Corresponding author; email: n.okba@erasmusmc.nl

Abstract

A new coronavirus, SARS-CoV-2, has recently emerged to cause a human pandemic. Whereas molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. Validated serologic assays are important for contact tracing, identifying the viral reservoir and epidemiological studies. Here, we developed serological assays for the detection of SARS-CoV-2 neutralizing, spike- and nucleocapsid-specific antibodies. Using serum samples from patients with PCR-confirmed infections of SARS-CoV-2, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial ELISAs. We demonstrate that most PCR-confirmed SARS-CoV-2 infected individuals seroconverted, as revealed by sensitive and specific in-house ELISAs. We found that commercial S1 IgG or IgA ELISAs were of lower specificity while sensitivity varied between the two, with IgA showing higher sensitivity. Overall, the validated assays described here can be instrumental for the detection of SARS-CoV-2-specific antibodies for diagnostic, seroepidemiological and vaccine evaluation studies.

Results

We evaluated SARS-CoV-2 specific antibody responses in severe and mild cases using serum samples collected at different times post-disease onset from three French PCR-confirmed CoVID-19 patients. We tested sera for SARS-CoV-2 specific antibodies using different ELISAs. Following infections, all three patients seroconverted between days 13 and 21 post onset of disease (Figure 1), and antibodies were elicited against the SARS-CoV-2 S and S1 subunit including the N-terminal (S1A) domain and the receptor binding domain (RBD). Since the N protein of SARS-CoV-2 is 90% similar to that of SARS-CoV (Table 2), we used SARS-CoV N as an antigen to test for SARS-CoV-2 N-directed antibodies in an ELISA format. We found that, following infection, antibodies were elicited against the N protein and when tested in a PRNT assay these sera were able to neutralize SARS-CoV-2. We observed cross-reactivity with the SARS-CoV S and S1 proteins, and to a lower extent with MERS-CoV S protein, but not with the MERS-CoV S1 protein (Figure 1 G-H). This was evident from analyzing the degree of similarity of the different CoV S protein domains to their corresponding SARS-CoV-2 proteins (Table 2), where SARS-CoV showed high similarities in all different S domains. The analysis showed that the spike S2 subunit is more conserved among CoVs and thus plays a role in the cross-reactivity seen when the whole S was used as an antigen. Thus, S1 is a more specific than S as an antigen for SARS-CoV-2 serological diagnosis.

We further assessed the specificity of the S1 assay using cohorts A-E (Table 1) comprising of sera from healthy blood donors (A), PCR-confirmed acute respiratory non-CoV infections (B), acute to convalescent PCR-confirmed alpha- and beta-HCoV infections (C), and PCR-confirmed MERS-CoV (D) and SARS-CoV (E) infections. None of the sera from specificity cohorts A-D were reactive in our in-house S1 ELISA at the set cut-off indicating 100% specificity, whereas sera from SARS-CoV patients cross-reacted (Figure 2A). Additionally, the specificity of S1 as an antigen for SARS-CoV-2 serology was further supported by the fact that 87-100 % of the cohort A-C sera included in this study were seropositive for the endemic HCoVs (HCoV-HKU1, HCoV-OC43, HCoV-NL63, and -229E) as determined by the S1 protein microarray (Figure 2B). Nonetheless, all were seronegative for SARS-CoV and MERS-CoV. Using the same cohort, we also validated the specificity of the anti-nucleocapsid and anti-RBD IgG ELISAs for detecting SARS-CoV-2 specific antibodies. At the set cut-off, except of SARS-CoV patient sera, none of the control sera tested positive for anti-RBD nor anti- nucleocapsid antibodies (Figure 2 C,D), whereas we detected seroconversion among the three COVID-19 patients. These validated ELISAs for different antigens can be useful for epidemiological studies as well as for evaluation of vaccine-induced immune responses.

Next, we validated the sensitivity and specificity of 2 commercial ELISA kits for detecting S1-specific IgG and IgA antibodies using the same cohort (Table1, Figure 3). All three COVID-19 patients had reactive antibodies by both the IgG (6/10 serum samples) and IgA (7/10 serum samples) ELISAs (Figure 3). While SARS-CoV patient’s sera were reactive as noted earlier, we also detected reactivity of serum samples from the validation cohorts A-D; 10/203 for IgA and 7/203 for IgG ELISAs. Two HCoV-OC43 (a β-CoV) patients’ sera were reactive in both IgG and IgA ELISA kits. We confirmed the cross-reactivity of the two sera by testing twelve serum samples from both patients collected at different time points, pre- and post- OC43 infection. While all pre-infection sera were negative, all post-infection sera were reactive in both IgG and IgA based ELISAs. We have earlier reported cross-reactivity of these sera in a MERS-CoV S1 IgG ELISA kit (6). Further validation was also done in a different laboratory using 31 serum samples collected from 9 COVID-19 German patients (Wölfel et al submitted manuscript) at different time points (3-23 days post disease onset) as well as a specificity cohort comprising of 18 serum samples from HCoV (4x HCoV-229E, 3x HCoV-HKU1, 4x HCoV-NL63, 7x HCoV-OC43) as well as MERS-CoV (n=3) and SARS-CoV (n=3) infected persons collected 4-56 days post disease onset (Figure 4). All 9 COVID-19 patients were previously confirmed to seroconvert at days 6-15 post onset of disease using recombinant immunofluorescence test and PRNT. 8/9 seroconverted patients showed reactivity above the implemented cut-off values in the IgG and IgA ELISA. One patient (Figure 4, black line) maintained slightly below the cut-off which might be explained by an overall reduced antibody response of this patient (PRNT90=10). Overall, the IgA-based ELISA kit was more sensitive but less specific than the IgG based ELISA kit.

Finally, we compared the performance of the different ELISAs for the detection of antibodies among PCR-confirmed COVID-19 patients to that of PRNT, as the gold standard for CoV serology (Table 3). PRNT50 correlated strongly with the different ELISAs, with the commercial IgA showing the strongest correlation followed by the S and N ELISAs indicating their capacity to detect SARS-CoV-2 specific antibodies. However, a larger patient cohort is needed to assess the sensitivity of these platforms.