Recovery Of Innate Immune Cells And Persisting Alterations in Adaptive Immunity in The Peripheral Blood Of Convalescent Plasma Donors At Eight Months Post SARS-CoV-2 Infection Part 1

Abstract:

Persisting alterations and unique immune signatures have been previously detected in the peripheral blood of convalescent plasma (CP) donors approximately two months after the initial SARS-CoV-2 infection. This article presents the results of the sequential analysis of 47 CP donors at a median time of eight months (range 7.5–8.5 months) post-infection, as assessed by flow cytometry. 

With the outbreak of the new crown pneumonia epidemic, more and more people have begun to pay attention to the impact of convalescent plasma on immunity. Convalescent plasma is currently an effective means of treating new coronary pneumonia. It can improve the immunity of patients, enhance the body's resistance, and help patients recover as soon as possible.

Convalescent plasma is a substance extracted from the plasma of recovered patients with new coronary pneumonia, which contains a large number of antibodies and immune cells, which can help the immune system recognize and attack the virus, thus playing an antiviral role. Therefore, convalescent plasma can improve immunity to a certain extent and enhance the body's resistance.

At present, many hospitals have begun to use convalescent plasma to treat patients with new coronary pneumonia, and have achieved certain curative effects. Studies have shown that convalescent plasma therapy can greatly shorten the hospitalization time of patients, alleviate the condition and reduce the mortality rate. Therefore, we should actively support and participate in the treatment of convalescent plasma to help more patients recover as soon as possible.

In addition, in daily life, we also need to pay attention to strengthening our immunity to prevent the occurrence of diseases such as new coronary pneumonia. For example, pay attention to a balanced diet and eat more nutritious foods such as vegetables, fruits, and fish; strengthen exercise to enhance physical fitness and immunity; pay attention to hygiene, wash hands frequently, and reduce the risk of infection.

In short, convalescent plasma does have a positive impact on immunity, which can enhance the body's resistance to a certain extent and help patients recover as soon as possible. We should actively support and participate in the treatment of convalescent plasma, and pay attention to health care in daily life so that our bodies will be healthier and full of vitality! So in summary, we need to improve our immunity. Cistanche can significantly improve immunity because the polysaccharides in the meat can regulate the immune response of the human immune system, improve the stress ability of immune cells, and strengthen immune cells' bactericidal effect.

Click cistanche deserticola supplement

Interestingly, our results show a significant variation in the relevant immune subset composition among CP donors. Regarding innate immunity, both non-classical monocytes, and CD11b- granulocytes had fully recovered at eight months post COVID-19 infection. Intermediate monocytes and natural killer (NK) cells had already been restored at the two-month evaluation and remained stable. Regarding adaptive immunity, the COVID-19-related skewed Th1 and Th2 cell polarization remained at the same levels as in two months. However, low levels of total B cells were detected even after eight months of infection. 

A persisting reduction of CD8+ Tregs and changes in the NKT cell compartment were also remarkable. CP donors present with a unique immune landscape at eight months post-COVID-19 infection, which is characterized by the notable restoration of the components of innate immunity along with a persisting imprint of SARS-CoV-2 in cells of the adaptive immunity.

Keywords:

SARS-CoV-2; COVID-19; convalescence plasma donors; immune profiling; immune restoration; 8 months.

1. Introduction

Coronavirus disease 2019 (COVID-19) is caused by the potentially fatal and highly contagious airborne transmission of severe acute respiratory syndrome coronavirus 2 (SARSCoV-2). The severity of COVID-19 largely varies among infected people, ranging from cases entirely asymptomatic [1], to milder conditions experiencing fever, cough, and tiredness, and to more serious cases suffering from pneumonia leading to acute respiratory distress syndrome and unrestrained multi-organ failure, which are often fatal [2]. 

Symptomatic cases of COVID-19 are characterized by a systemic hyper-inflammatory immune response, and there is now ample evidence that variations in the severity of the symptoms following SARS-CoV-2 infection are directly related to the preceding host’s immune status [3–5]. Factors related to more dysfunctional immunocompromised immune profiles, and consequently, higher risk of mortality, including increased age and certain underlying chronic medical conditions, like type 2 diabetes, cardiomyopathy, obesity, chronic obstructive pulmonary disease, and chronic kidney disease [6,7].
Extensive research on COVID-19 has revealed important information on the immune cascades and particular changes in various immune subsets accompanying SARS-CoV-2 infection [3,4,8,9]. However, there is still limited information regarding the alterations that may persist in the host’s immune system over time after the complete resolution of the infection. 

Recently, it has been shown that distinct components of immunological memory to SARS-CoV-2 may have different kinetics over time [9,10]. For instance, anti-SARS-CoV-2 memory and cytotoxic T cells seem to decline more rapidly compared to B cell immunity, whereas both memory B cells and immunoglobulin (Ig)G levels against the spike protein of SARS-CoV-2 persist for at least six months post COVID-19 onset [10]. 

In a different cohort, Breton et al. showed that central memory CD4+ and CD8+ T cells decreased, but antigen-specific T cells persisted at six months after infection [11]. As for innate immunity cells, their profile seems to be age-dependent, and alterations in monocytes and neutrophils were observed up to ca. two months post-infection [12]. 

We have also recently reported particular persisting alterations and unique immune signatures in the peripheral blood (PB) of convalescent plasma (CP) donors at approximately two months after initial SARS-CoV-2 infection. These immune signatures correlated both with the ability of patients to mount a protective humoral response, i.e., to produce sufficient and detectable anti-SARS-CoV-2 IgG and/or IgA antibodies, but also with the severity of the experienced symptoms, with previously hospitalized CP donors being less immunocompetent than CP donors who experienced milder symptoms [13].
Following the same approach and to explore long-term imprints on the immune profile of COVID-19-recovered CP donors, we present here the results of the sequential analysis of 47 CP donors at a median time of eight months (range 7.5–8.5 months) post-infection.

2. Materials and Methods

2.1. Selection of Participants

The present report includes sequential testing of CP donors who participated in a phase 2 study (NCT04408209) and provided paired PB samples to analyze their immune profile at two and eight months, post-infection with SARS-CoV-2. All participants had been infected by SARS-CoV-2 and were diagnosed after positive nasopharyngeal and/or oropharyngeal swabs with RT-qPCR, as previously described [14]. 

Before the donation of CP, they had two negative PCR tests with an interval of at least seven days. The second negative test was performed 1–7 days before plasmapheresis. They also developed detectable anti-SARS-CoV-2 IgG antibodies against the spike domain 1 (S1) protein of SARS-CoV-2 when examined with a sensitive commercially available, and Food and Drug Administration-approved Enzyme-linked immunosorbent assay (Euroimmun Medizinische Labordiagnostika AG, Lubeck, Germany), described in detail previously [14]. 

Inclusion criteria also included at least 14 days after a complete recovery from SARS-CoV-2 infection (no symptoms, complete resolution of organ dysfunction which was caused by SARS-CoV-2), male donors without transfusion history, and females without a history of transfusion or pregnancy, a normal complete blood count, negative serological tests for hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), venereal disease research laboratory (VDRL), human T cell lymphotropic virus (HTLV)-1 and negative for HIV, HBV, HCV with nucleic acid amplification testing, and donors fulfilled all the general criteria for blood donation in terms of age, general condition, hemoglobin levels, and vital signs.

Ten healthy individuals with similar ages (median 47 years, range—45 to 73 years) and gender (6 male/4 female) were also evaluated and used as normal controls. These subjects had no known health problems and had a negative PCR test for SARS-CoV-2 from nasopharyngeal swabs at the time of blood sampling.

All participants were informed of this study and provided signed informed consent according to the declaration of Helsinki and the local ethics committee. 

All study procedures were carried out by the declaration of Helsinki (18th World Medical Association Assembly), its subsequent amendments, the Greek regulation guidelines, as well as the Good Clinical Practice (GCP) guidelines defined by the International Conference of Harmonization. The protocol was approved by the Ethics Committee of “Alexandra” General Hospital, Athens, Greece (Ref No 245/16 April 2020).

2.2. Flow Cytometry Analysis

PB samples were collected in EDTA-coated blood collection tubes (BD Vacutainer, BD Biosciences, San Jose, CA, USA; #367841) and were processed within an hour post-collection following the Stain-Lyse-no-Wash protocol (BD Biosciences). In specific, for each PB sample, 100 µL of total anticoagulated blood was transferred to a 12 × 75 mm round-bottom polystyrene FACS tube and stained with a panel consisting of appropriate fluorophore-conjugated anti-human antibodies against the surface markers CD3-PerCP, CD4-BV510, CD8-PE, CD14-BV605, CD16-APC, CD25-PECF594, CD45-APCH7, CD56- APCR700, CD183-BV421, CD194-BV650, CD196-BB515 and CD11b-BV786 (all from BD Biosciences), at room temperature (RT) for 20 min protected from light. Further, 450 µL of 1× FACS Lysing Solution was added, and cells were incubated for another 10 min at RT protected from light. All samples were analyzed on a 3-laser BD FACSCelesta (BD Biosciences), and 100,000 events were acquired per sample.

The 12-color panel referred to here consisted of our reference panel for the phenotypic analysis of CP donors at two months post-infection and allowed for the identification and analysis of 24 distinct immune populations, including B cells, CD3+ T cells, natural killer (NK) cells, monocytes, granulocytes, and their subsets following the gating strategy previously described [13]. 

To be consistent with our previous batch analyses, we followed a regular and extensive set-up performance. The cytometer set-up was performed using unstained control cells for the configuration of the appropriate PMT voltages and with BD CompBeads Set anti-mouse Igκ (BD Biosciences) for the inter-channel fluorescence spillover compensation. Moreover, a CS&T daily performance was applied before each acquisition to verify the cytometer's stable condition. 

Finally, an additional standardization step was applied daily using rainbow beads (Spherotech Inc, Lake Forest, IL, USA) to check for laser stability through the control of PMT fluctuations to detect the targeted fluorescent intensities of the beads.

2.3. Statistical Analysis

Data were analyzed using GraphPad Prism 8.0.2 software (San Diego, CA, USA). Results are expressed as means ± standard deviation (SD). For statistical analysis, a Student’s t-test was used to compare controls and CP donors eight months post SARSCoV-2 infection, whereas changes in CP donors at two and eight months post-infection were compared using paired Student’s t-test. p-values < 0.05 were considered statistically significant.

3. Results

3.1. Characteristics of CP Donors

The present analysis included 47 CP donors with paired PB samples collected at a median of two and eight (range 7.5–8.5) months post-COVID-19 infection. The clinical characteristics of the participants are presented in Table 1.

3.2. The Immune Profiling at Eight Months Post-SARS-CoV-2 Infection Does Not Correlate With the Clinical Characteristics of CP Donors

Consistent with our previous analysis at two months post SARS-CoV-2 infection [13], at eight months, we observed a significant variation of the relevant immune subset composition among CP donors. This variation exceeded the demographic and clinical characteristics of our cohort, as no significant differences could be associated with sex, age, the number of experienced symptoms, or the values of serum anti-SARS-CoV-2-specific antibodies against S1 viral antigen. 

Of note, no significant differences were obtained in the immune profiling of CP donors who were previously hospitalized and those who had milder symptoms and did not meet the criteria for admission in intensive care units (ICUs) (data not shown). Therefore, we did not further subdivide CP donors into more categories in our subsequent analysis of the dynamics of immune profiling over time (Table 2).

3.3. Innate Immunity Recovery at Eight Months Post COVID-19

We further assessed to evaluate the dynamics of particular immune profiles by comparing the relevant compositions of the various immune subsets over time. The analysis of the major immune populations belonging to the innate and the adaptive arms of immunity and their subpopulations are presented in Figures 1 and 2. In general, our results show that by eight months post-infection, the majority of innate immune cell subsets recovered to levels similar to those of the control group, contrarily to cells of the adaptive immunity, in which specific lymphocyte subsets showed evidence of persistent long-term activation.

Regarding monocyte subsets, we have previously reported a 7-fold increase of intermediate monocytes and a 2.2-fold increase of the non-classical compartment (at the expense of classical monocytes) during the active COVID-19 disease [11]. By two months post-infection, the levels of intermediate and classical monocytes showed evidence of recovery and had practically fully restored by eight months post-infection. The percentages of non-classical monocytes, which remained increased at two months post-infection (mean percentage—1.16% vs. 0.27% in the control group, p = 0.01), also fully recovered by eight months and reached the levels of the control group.

Similar observations were recorded for granulocytes. Although their total percentage at eight months was slightly reduced compared to controls, at eight months post-infection, the prevalence of the CD11b+ and CD11b- subsets have significantly decreased and increased, respectively, resulting in a CD11b+/CD11b- granulocyte ratio similar to that of normal healthy donors, as compared with the deregulated granulocyte expansion at the two-month evaluation [13].

As for NK cells, our analyses did not show any significant fluctuation between two and eight months, post-infection. The percentages of total NK cells and their subsets were almost restored at two months post-active disease resolution, and the same profile was also seen at eight months, with percentages remaining at near-normal levels.

For more information:1950477648nn@gmail.com