A new set-up of vanishing antibodies: A biennial follow-up of five different clients' humoral responses against SARS-CoV-2 after systemic vaccination in an oncology hospital in Poland.

Abstract

The common severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has infected approximately 600 million people across 228 countries worldwide, leaving behind natural immunity.1 Acute COVID-19 infection can cause a cytokine storm that leads to acute respiratory failure (ARDS) and death,2 while severe inflammatory disease can result in chronic polymyositis syndrome in children (PIMS).3 Almost half (44.8%) of symptomatic infections in children and adults manifest as varied symptoms related to chronic infection, in the form of long-COVID/post-COVID clinical syndrome, cognitive and physical deficits, pulmonary fibrosis, myocarditis, or neurological deficits.4, 5 The findings so far show that to acquire antiviral immunity to SARS-CoV-2, one does not need to come into contact with the virus and become infected.6 Being around immunized people can provide a cellular and humoral response. To what extent is it active and provides self-immunity? How to check if we have acquired the appropriate antivirus response, or only had contact with it? When should a booster be given? The situation applies to everyone, including the healthy, those sick with a viral disease in any stage, those with impaired immunity, and young people. The data we collected show that it is necessary to measure the concentration of IgG-specific antibodies after the onset of SARS-CoV-2 and to measure the concentration of post-vaccine IgG (anti-S, anti-RBD, and S-RBD).6 IgM and IgG antibody levels (SARS-CoV-2-specific) may indicate early- or late-phase infection.7 Administration of antibody response acquisition during the early phase has only demonstrated therapeutic implications for symptomatic COVID-19 cases,8 and vaccination of people with a history of SARS-CoV-2 without knowledge of their humoral response is considered safe.9 In our hospital, most medical staff and patients underwent vaccinations without knowing that they had acquired a humoral response beforehand.10 The decline in humoral immunity over time following coronavirus infection, including SARS-CoV-2, is typical in mild cases.11-16 Then, do all infections require antibody monitoring? Barrière et al.17 found that after administering a second dose of the vaccine (complete vaccination) in cancer patients, the individual humoral anti-S antibody response level could be determined after three to four weeks. It has been proven that the level of S-RBD antibodies is correlated with protection against symptomatic SARS-CoV-2 infection,18, 19 and not asymptomatic infection.20 Thus, after the third dose and subsequent boosters, does the level of only anti-S antibodies determine immunity? In a selected population of healthy people, the level of the humoral response can last up to 13 months.19 During our study, the Delta strain of SARS-CoV-2 accounted for over 95% of infections in Poland. At that time, rapid neutralization by current vaccines of the new Delta virus20, 21 was reported after complete inoculation with BNT162b2. We asked whether, during the next wave of infections in Europe with the different Omicron virus types, the administration of a booster in healthy people would stimulate the immune system again,22, 23 or if it would only cause an increase in the S-RBD in response to the vaccine? The description of five different courses of SARS-CoV-2 infection until the administration of subsequent doses of the Comirnaty vaccine shows that a specific immune response expires approximately 1 year after the onset of the disease and is not induced after subsequent artificial immunization.10 We call this effect vanishing antibodies. Another illness with a new variant of the SARS-CoV-2 virus can stimulate a new response to a different level.24-26 We call this a new set-up. In other data, currently unpublished, article focused on a year-long case observation of the COVID-19 humoral response, we saw that an early double infection (without or after vaccination), rather than a third booster, raises antibodies to a higher “protective” IgG level. Before vaccines were introduced in 2021, the natural immunity in the SARS-CoV-2 pandemic was investigated by analyzing antibody concentrations. Antibody measurements were analyzed routinely for oncology patients and hospital staff for medical purposes during the COVID-19 pandemic. Retrospectively, we decided to follow five previously selected people for approximately 24 months—with most measurements taken monthly (timeline). (Supplementary Table). The complete characteristics of the antibody collection in five subjects (Case #1,2,3,4,5) in months (samples timeline). Event: SARS—SARS-CoV-2 infection time (positive PCR or antigen test); Com1, Com2, Com3, Com4—vaccination time (Comirnaty). Three patients (#2, #4, #5) and two hospital staff persons (#1, #3) were chosen. Case #2 was chosen because of their rapid response to SARS-CoV-2 infection (hyper-responder with SARS-CoV-2 IgG > 2000 AU/mL in less than 14 days), providing an excellent case of early seroconversion relevant to acute viral infection. The other two persons were selected as the controls, one being COVID-19 convalescent (#3) and one lacking a SARS-CoV-2 history (#1). The other two patients passed COVID-19 twice with mild (#4) and severe (#5) respiratory problems. Case #3 stayed at the hospital and was cured against COVID-19 using remdesivir (Veklury) for five days. Case #1 patient was chosen because of a lack of a history of COVID-19, but analyses of his seroconversion proved past SARS-CoV-2 infection (IgG cut-off > 0.2 AU/mL). Case #4 stayed and cured COVID-19 at home. Case #5 remained at the hospital because of bilateral pneumonia and was given steroids and early plasma antibodies. All five were vaccinated with BNT162b2 messenger RNA (mRNA) (Comirnaty) according to the manufacturer's schedules and took booster vaccines at different times (gray arrows in figures; Com1, Com2, Com3, Com4). Chest CTs for Cases #2, #3, and #5 showed no post-COVID changes 3 months after treatment. Our study materials were blood specimens taken through venipuncture sampling. The concentration of antibodies was evaluated 4 hours after blood collection. If an immediate assessment was impossible, the serum was collected and stored at –80°C. Due to the fact that most of the data (2021) are in AU/mL, the results of measurements in BAU/mL in 2022 were converted into AU/mL. The antibody concentrations were detected by the chemiluminescent immunoassay CLIA (MAGLUMI; Snibe Diagnostic). Per the manufacturer's protocol, results greater or equal to 1.0 AU/mL of SARS-CoV-2 IgG, IgM, and S-RBD were considered reactive and positive. The maximum limits of the antibody measurements were assumed: 2000 AU/mL of S-RBD (initially, it was 100 AU/mL), with the upper limit of the SARS-CoV-2 antibody measurements being 2000 AU/mL. According to our earlier observations in this population, the cut-off value for SARS-CoV-2 IgG should be >0.2 AU/mL for a positive test result. Antibody tests were ordered by a hospital doctor at different times, from vaccination to illness. The results are presented in a table depending on the time of the test (timeline). The patients provided their written consent for performing humoral immunity tests and for participating in this study. The Bioethics Commission of the Medical University provided consent for our research. SARS-CoV-2 IgM with IgG; SARS-CoV-2 IgG with S-RBD IgG; (A‘) or (B‘) and (B“) is a part of the diagram (A) or (B), respectively, to explain the dynamic changes of humoral response; (C) All three antibody concentrations are time-related in the logarithmic chart. Figures 1-5 presents the humoral response of each person after infection and vaccination in time. The legend in the figures shows arrows indicating systemic vaccinations (Com1, Com2, Com3, and Com4) and the symbol of the virus pointing to SARS infections. Figure 1. Case #1: (A) All IgG and IgM antibodies (measurements 1–24). A retrospective case study showed that the person had had SARS-CoV-2 infection in October 2020 without clinical symptoms, so we present (A‘) an additional diagram with only 1–20 measurements separately to point out this phenomenon. There is a typical seroconversion, seen as an increase in IgM and IgG, assumed as positive results (cut-offs: IgM > 1.0 AU/mL and IgG > 0.2 AU/mL). The results should be considered clinically asymptomatic of previous SARS-CoV-2 infection. As hospital staff, he was routinely vaccinated in January 2021 with two doses of BNT162b2 (Comirnaty) on a schedule over 28 days (see arrows). (B) Postvaccination effects are seen as S-RBD gains and triple antibody resynthesis in SARS-CoV-2 infection and vaccination ([A‘] The three peaks of IgM and IgG mean three seroconversions in a row). (C) Logarithmic representation of the three antibody concentrations. Notable here is a conversion to higher SARS-CoV-2 IgG concentrations than baseline before SARS-CoV-2 infection at the beginning of the diagram. A booster was given 8 months after the second dose of Comirnaty (see arrow) due to another wave of illness (autumn) for medical personnel (only increased S-RBD). On the contrary, at that time, the second vaccination did not work—(B) only increased S-RBD after the booster, but together with COVID-19 (raised S-RBD with SARS-CoV-2 IgG). This phenomenon is called enhanced humoral response without classical seroconversion (fast release IgG-specific antibodies). (C) After a few months, he passed SARS-CoV-2 asymptomatically, which provided a new set-up of humoral response (in the red frame). Case #2: (A) Perspective case analysis; the person, had symptomatic COVID-19 infection in March 2021, with a sore throat and muscles, a fever, and the chills. (A) Antibody collection for assays was routinely scheduled (considered a hyper-responder) as a hospital-monitored oncology patient, vaccinated in July 2021 with two doses of BNT162b2 (Comirnaty) on a schedule over 28 days (see arrows). A booster was given after half a year delay, as an auto-immunologic person. (C) A logarithmic representation of the three antibody concentrations. (B) Postvaccination effects, shown as an increase in S-RBD extended to 6 months and no resynthesis of antibodies in the vaccine prompt. (B, C) No fluctuations in the IgM and IgG concentrations. The conversion to higher IgG concentrations before SARS-CoV-2 infection and the exceptional smooth/stable course of the immune antibody curves are noteworthy. (C) A new set-up of antibodies can be seen in the red frame. Case #3: (A) Retrospective case analysis. The person had symptomatic COVID-19 infection in December 2020, including a sore throat, a cough, a fever, and dyspnea. Routinely, antibodies were collected for assay; (A‘) during inpatient treatment, remdesivir (Veklury) was administered for 5 days while being monitored by medical personnel in the hospital, and vaccinations occurred in January and in May 2021 with two doses of BNT162b2 (Comirnaty) because this patient underwent an urgent spine operation in March. A booster was given 5 months after the second dose of Comirnaty because of lost antibodies—SARS-CoV-2 IgG of 0.26 AU/mL and S-RBD IgG of 32.40 AU/mL (see arrows). (B) Here, we can see the postvaccination effects, shown as an increase in both S-RBD and SARS-CoV-2 IgG extended to over 3 months, as well as a decrease in IgG antibodies in the prompt for SARS-CoV-2 (IgG = 0.22 AU/mL and IgM = 0.05 AU/mL) and vaccination (S-RBD IgG = 63.56 AU/mL) after 5 months. Case #4: (A) Retrospective case analysis. Delayed humoral response. She past twice SARS-CoV-2 with mild symptoms (COVID-19). She is an out-patient at the breast unit. In Figure 4 (A) and (A’), we see a crossing-line dynamic of all antibodies with little correlation with vaccination (Com3; timeline 24). Figure 4. (B), (B‘), and (C) present delayed effects in time SARS-CoV-2 infection (ending “old” SARS-CoV-2 IgG and replacing them with “new”). Now all three antibodies are in response. Building a new humoral strategy: seroconversion (IgM), humoral response (S-RBD), immunity (SARS-CoV-2 IgG). That is also an example of a new set-up raise of IgG-specific antibodies at the end of observation. That person is still a riddle. That is why she did not yet decide on vaccination (Com4). Case #5: Figure 5 (A) Retrospective case analysis (measurement 2−39). The selected patient underwent COVID-19 with pneumonia and was administered steroids for respiratory failure and convalescent plasma with antibodies. Another “reinfection” of SARS-CoV-2 may have been due to the long-standing genetic material of the virus (RT-PCR positive) before urological surgery (no clinical correlation). Figure 5 (A), (A’), (B), and (C) are the only evidence of correlation with vaccinations (Com1 and Com2). As you can see, stable levels of the three classes of antibodies are maintained in cancer patients after surgery and prostate cancer hormone therapy (Com3 and Com4 vaccinations). This is evidence that COVID-19 in some immunosuppressed patients is well tolerated. Due to the significant decrease in immunity, the antibodies reduction, and the patient's clinical condition, a third dose of the vaccine was used (Figure 3A',B). After a few months, he passed into mild symptomatic COVID-19, cured after five days by molnupiravir (Lagevrio), which provided a new set-up of humoral responses (Figure 3C in the red frame). A logarithmic representation of all three antibody concentrations can be seen in Figures 1C, 2C, 3C, 4C, and 5C. The effect of the new increasing level of IgG antibodies was obtained. Conversion to higher IgG concentrations was noticeable (after disease and after complete vaccination) initially after infection with SARS-CoV-2 and a symmetrical course of S-RBD IgG curves of immune antibodies in the time immediately after the third dose of Comirnaty. There was a difference in antibody surge after COVID-19 getting sick with the boosted vaccine (third dose) between a symptomatic patient (Figure 3B) and SARS-CoV-2 asymptomatic patient (Figure 1B). The symptom convalescent patient had a higher IgG-specific level. Following autoimmunity (Case #2), the antibody response after disease and inoculation was stable. Significantly, it was strengthened by administering the first mRNA vaccine, respectively, after 3 months. The results indicate that the active autoimmune disease stabilized the level of specific IgG after systemic mRNA vaccination for at least half a year. Looking at all cases of vaccinated convalescents, we believe a booster dose should be given later than 28 days after SARS-CoV-2 infection. Then, we can observe fluctuations in antibodies, suggesting a change in response to the subsequent immunization, depending on the patient's condition. We expected a longer duration of neutralizing antibodies after vaccination. The level of postvaccine IgG antibodies decreased when vaccination was performed within 3 months after disease onset, depending on the patient's condition. We can change this by appropriately tracking S-RBD IgG and SARS-CoV-2 IgG antibodies 1 month after the disease.17 According to observations, raising IgG-specific antibodies will better occur by administering the vaccine's second (next) dose 3 months after the first. The administered booster dose (third dose) only increased the concentration of S-RBD antibodies. Despite the decrease in specific IgG, their level was higher than before disease and vaccination. Declining IgG-specific antibodies were observed. S-RBD IgG levels are not correlated with SARS-CoV-2 IgG levels in vaccinated convalescents,10 but they are related to symptomatic ones.16 Surprisingly, there was no apparent spike in IgG antibody levels after the third dose of the vaccine after 6 months, despite a persistently elevated level. Based on the collected data, we suggest that monitoring S-RBD antibodies is sensitive, but not equivalent to a specific humoral response for SARS-CoV-2 IgG. The observations of the five presented cases show that the measurement of S-RBD correlates with specific IgG only in the period from 1 to 3 months following disease and inoculation, but not for the booster doses given in the 6 months after the initiation of vaccination. Many clinical data indicate that vaccination with a third dose of Comirnaty is useful in immunocompromised people,10, 11 the chronically ill,12 or people undergoing oncological treatment.13, 14 An analysis of specific antibody concentration charts at a cut-off for SARS-CoV-2 of >0.2 AU/mL was used,10 allowing us to observe asymptomatic COVID-19 and the natural course of the disease.15 Patterns of antibody seroconversion in response to SARS-CoV-2 infection and the BNT162b2 mRNA vaccine were apparent in the five separate cases. In the case of Case #3, it turned out to be effective. Currently, a wave of infection with the SARS-CoV-2 virus strain Omicron is going through Europe and the USA, as the BNT162b2 vaccine is effective against this strain. The ideal past seroconversion is when IgG antibodies are formed after stimulation and are maintained for longer. For Case #1, Comirnaty vaccination needed to be postponed one more month after getting sick; for Case #2, double vaccination did not show/change anything (excess antibodies); for Case #3, in terms of the vaccination, the third dose significantly improved the initial level of antibodies, indicating that it is safe and advisable, and similarly to Case #1, the first dose of the Comirnaty vaccine was taken too early. Analyzing a new set-up in these five cases shows that in Case #2, subsequent infection was not needed to reach a stable level of IgG antibodies. To sum up, administration of the vaccine too early in the case of people with excess antibodies (e.g., Case #2) does not produce the expected effect, as too early an administration (Cases #1 and #3) does not allow to hit the serological window after falling ill. Following readministration (Case #3) after 5 months from the administration of the first dose, based on individual indications for vaccination, the level of antibodies responds correctly. The analysis of these five cases of antibody changes over time during the COVID-19 pandemic provides students training material and allows them to observe and control the effects of treatment—including when to administer the third or subsequent dose of the vaccine at the appropriate time for the natural course of the disease.25, 26 Persistently elevated levels of specific IgG after vaccination, even at low levels, after COVID-19 infection suggests that SARS-CoV-2 IgG-specific antibodies help in monitoring humoral immunity for a long time.26 Crucially, however, they provide a new humoral response re-build up, observed twice following infection. Moreover, we have to add that in the five cases, we primarily saw good SARS-CoV-2 response seroconversion (IgM and IgG) after mild infection and some different responses after vaccination. A lack of a humoral-specific response for complete BNT162b2 vaccination and a booster (Case #2) does not mean seroconversion (S-RBD response). The mechanism of poor, late seroconversion is supposed to be involved in a substantial primary SARS-CoV-2 response (early responder). Moreover, a poor humoral response (seroconversion of IgG and IgM) was observed for the second and subsequent booster vaccinations in healthy SARS-CoV-2 asymptomatic cases (Case #1 and #3), not only for immunocompromised people.27, 28 A preprint has previously been published.29 Piotr Kosiorek, Samuel Stróż, and Anna Stasiak-Barmuta analyzed the data and drafted the manuscript. Robert Milewski, Dorota Elżbieta Kazberuk, Anna Hryniewicz, and Klaudia Bartoszewicz participated in data analysis and extensively reviewed the manuscript. Piotr Kosiorek, Samuel Stróż, Magdalena J. Borkowska, and Anna Stasiak-Barmuta contributed to the clinical and laboratory data acquisition and reviewed the manuscript. All authors read and agreed to the published version of the manuscript. The authors thank all of the healthcare workers involved in the examinations at the Maria Sklodowska-Curie Bialystok Oncology Center in Bialystok, Poland. All of the individuals included in this work consented to the acknowledgments. This research did not receive specific funding but was performed as part of the work of the authors at Maria Sklodowska-Curie Bialystok Oncology Centre, Poland. The authors declare no conflict of interest. The Bioethics Commission of the Medical University approved this research. Approval code: APK.002.267.2021; approval date: 29 April 2021. Written informed consent was obtained from the patient(s) to publish this paper. The lead author (Piotr Kosiorek) affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained. 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