Collection of COVID-19 convalescent plasma in institute for transfusion medicine of republic of North Macedonia and evaluation of anti-SARS-CoV-2 antibody concentration in different donors subgroups

Background: Plasma collected from patients that have recovered from an infectious disease has been transfused over many decades for prophylaxis and treatment of various infectious diseases. Taking into consideration the expansion of COVID-19 pandemics, we started the COVID-19 convalescent plasma (CCP) programme. Aims: The aim of our study is to show our experience with collecting the CCP and to evaluate the SARS-CoV-2 antibody concentration in different convalescent plasma donors' subgroups. Methods: This is a prospective study performed in the Institute for Transfusion Medicine of Republic of North Macedonia since 30 April 2020 till July 2021. Antibody testing was performed at the Institute for Immunobiology and Human Genetics in Skopje using CLIA method with Snibe Maglumi SARS-CoV-2 S-RBD IgG (quantitative) with IgG cut-off larger than 5 AU/ml. All potential donor were tested for: negative RTPCR for SARSCoV-2 before donation, anti-SARS-CoV-2 antibodies, anti- HLA antibodies (where applicable), blood count, blood group, TTI and biochemistry. Preferred method for plasma collection was plasmapheresis which was performed with Terumo BCT Trima Accel and donation of whole blood, depending on the donor preference and venous access. All donors signed inform consent for donation and inclusion in the study. Results: There were 1476 potential CCP donors, but only 700(47.9%) donors fulfilled all the criteria and we obtained 793 units of CCP;639 (80.6%) units from whole blood donors and 154 (19.4%) CCP units from 61 plasmapheresis donors, 485 (69.3%) males and 215 (30.7%) females. Mean age of the donors was 40 years (range 18-63). Mean value of SARS-CoV-2 S-RBD IgG concentration was 31.05 AU/ml, (range from 5.1 AU/ml to >100 AU/ml), mean value of SARS-CoV-2 S-RBD IgG in men was 37.6 AU/ml and 28.9 AU/ml in women (p < 0.05). Distribution of CCP donors according to the ABO blood group was: 301 blood group A (43%) with median value of SARS-CoV-2 S-RBD IgG = 27.15 AU/ml, 220 blood group O (31.4%) median value of SARS-CoV-2 S-RBD IgG = 32.1 AU/ml, 116 blood group B (16.6%) median value of SARS-CoV-2 S-RBD IgG = 35.9 AU/ml and 63 donors had blood group AB (9%) median value of SARS-CoV-2 S-RBD IgG = 26.45 AU/ml. There were 69 donors that were previously hospitalized with mean value of SARS-CoV-2 S-RBD IgG = 48.6 AU/ml, and 629 that were treated at home with mean value of SARS-CoV-2 SRBD IgG = 29.1 AU/ml (p < 0.05), of which 578 had symptoms with mean value of SARSCoV- 2 S-RBD IgG = 29.1 AU/ml and 51 were asymptomatic with mean value of SARS-CoV-2 S-RBD IgG = 29.3 AU/ml. The CCP donors had the following distribution according to the age: 125 donors in the 18-29 age group with median value of SARS-CoV-2 S-RBD IgG = 23.0 AU/ml, 200 donors in the 30-39 age group with mean value of SARSCoV-2 S-RBD IgG = 28.2 AU/ml, 217 donors in the 40-49 age group with mean value of SARS-CoV-2 SRBD IgG = 32.9 AU/ml and 156 donors in the 50-63 age group mean value of SARS-CoV-2 S-RBD IgG = 38.3 AU/ml (p < 0.05). Summary/Conclusions: The collection procedures are safe and effective and collected CCP units were with high concentration and quality. The concentration of SARS-CoV-2 S-RBD IgG in CCP obtained from previously hospitalized patients was significantly larger than in ones that were treated at home. The concentration of SARS-CoV-2 S-RBD IgG was higher in men, in advanced age group and in donors with blood group B. The further studies are needed to clarify the impact of different variables on antibodies concentration/ titre in donors.

Reasons for deferral of COVID-19 convalescent plasma donors

Background: Taking into consideration historic success of convalescent plasma in prophylaxis and treatment of various infectious diseases over the century and expansion of COVID-19 pandemics, we started the COVID-19 convalescent plasma program in our country. Aims: The aim of our study was to show the reasons for deferral of the COVID-19 convalescent plasma (CCP) donors. Methods: This is a prospective study organized in the Institute for Transfusion Medicine of Republic of North Macedonia since 30 April 2020 till July 2021. CCP donors eligible for donation were donors without a history of blood transfusion, female donors who have never been pregnant, or who have been tested and found negative for anti- HLA antibodies, age 18-65, in good health that fulfil all other criteria for regular blood donors. All potential donor were tested for: negative RT-PCR for SARSCoV-2 before donation, anti-SARS-CoV-2 antibodies, anti-HLA antibodies (where applicable), blood count, blood group, TTI and biochemistry. Antibody testing was performed at the Institute for Immunobiology and Human Genetics in Skopje using CLIA method with Snibe Maglumi SARS-CoV-2 S-RBD IgG with IgG cut-off for CCP donation larger than 5 AU/ml (cut-off for regular positive result was larger than 1 AU/ml). All donors signed inform consent for donation and inclusion in the study. Results: There were 1462 potential CCP donors, of which 762(52.1%) did not fulfil criteria for CCP donation, 424(55.6%) women and 338 (44.4%) men. There were 454(59.6%) potential CCP donors that did not have enough anti-SARS-CoV-2 S-RBD IgG antibodies (Ab), of which 223(49.1%) had Ab concentration less than 1 AU/ml (29.3% of all deferred donors) and 231(50.9%) had Ab concentration between 1 and 5 AU/ml (30.3% of all deferred donors). In this subgroup of deferred donors, there were 227 women (50%) and 227 men (50%), with mean age 37.2 ± 10.1 (range 18-63). There were statistical significant correlation between the gender and the Ab concentration less than 1 AU/ml and from 1 to 5 AU/ml (Pearson Chi-square: 3.88667, df = 1, p = 0.048671). Multiple regression analysis showed that gender is independently connected with the Ab concentration, OR = 1.4495 (95%CI 1.0016-2.0976), i.e. male gender increases the chance for increased Ab concentration for one and a half times. According to the age group, the majority of deferred CCP donors, because of low Ab concentration, are in the age group from 30 till 39(39.6%), followed by age group from 40 till 49(23.8%), and least deferred donors were in 50 and above age group (13.2%). The most of these deferred donors were treated at home, 451(99.3%) and had symptoms 240(52.9%). Distribution according to blood group in CCP donors deferred due to low Ab concentration was: blood group A- 182(40.1%), blood group O-153(33.7%), blood group B-69(15.1%) and blood group AB-42(9.3%). In the whole investigated group, 65 (8.5%) potential donors were deferred because of low haemoglobin level (less than 12.5 g/dl for women and less than 13.5 g/dl for men), 26 (3.4%) were deferred because of positive TTI markers and 102 (13.4%) women of the total number of deferred donors were deferred because of presence of HLA antibodies (i.e. 24% of investigated women). Summary/Conclusions: There is large percentage of deferred CCP donors mostly because of low Ab concentration, presence of HLA antibodies and low haemoglobin level, but starting of COVID-19 convalescent plasma program was a big success for our institution and our country and helped a lot of patients.

Is COVID-19-associated cytokine storm distinct from non-COVID-19 secondary hemophagocytic lymphohistiocytosis?

Cytokine storm is an umbrella term that describes an inflammatory syndrome characterized by elevated levels of circulating cytokines and hyperactivation of innate and/or adaptive immune cells. One type of cytokine storm is hemophagocytic lymphohistiocytosis (HLH), which can be either primary or secondary. Severe COVID-19-associated pneumonia and acute respiratory distress syndrome (ARDS) can also lead to cytokine storm/cytokine release syndrome (CS/CRS) and, more rarely, meet criteria for the diagnosis of secondary HLH. Here, we review the immunobiology of primary and secondary HLH and examine whether COVID-19-associated CS/CRS can be discriminated from non-COVID-19 secondary HLH. Finally, we review differences in immunobiology between these different entities, which may inform both clinical diagnosis and treatment of patients.

A third dose of SARS-CoV-2 vaccine increases neutralizing antibodies against variants of concern in solid organ transplant recipients.

Vaccine-induced SARS-CoV-2 antibody responses are attenuated in solid organ transplant recipients (SOTRs) and breakthrough infections are more common. Additional SARS-CoV-2 vaccine doses increase anti-spike IgG in some SOTRs, but it is uncertain whether neutralization of variants of concern (VOCs) is enhanced. We tested 47 SOTRs for clinical and research anti-spike IgG, pseudoneutralization (ACE2 blocking), and live-virus neutralization (nAb) against VOCs before and after a third SARS-CoV-2 vaccine dose (70% mRNA, 30% Ad26.COV2.S) with comparison to 15 healthy controls after two mRNA vaccine doses. We used correlation analysis to compare anti-spike IgG assays and focused on thresholds associated with neutralization. A third SARS-CoV-2 vaccine dose increased median total anti-spike (1.6-fold), pseudoneutralization against VOCs (2.5-fold vs. Delta), and neutralizing antibodies (1.4-fold against Delta). However, neutralization activity was significantly lower than healthy controls (p < .001); 32% of SOTRs had zero detectable nAb against Delta after third vaccination compared to 100% for controls. Correlation with nAb was seen at anti-spike IgG >4 Log10 (AU/ml) on the Euroimmun ELISA and >4 Log10 (AU/ml) on the MSD research assay. These findings highlight benefits of a third vaccine dose for some SOTRs and the need for alternative strategies to improve protection in a significant subset of this population.

ALVR109, an off-the-shelf partially HLA matched SARS-CoV-2-specific T cell therapy, to treat refractory severe COVID-19 pneumonia in a heart transplant patient: Case report.

An unvaccinated adult male heart transplant recipient patient with recalcitrant COVID-19 due to SARS-CoV-2 delta variant with rising nasopharyngeal quantitative viral load was successfully treated with ALVR109, an off-the-shelf SARS-CoV-2-specific T cell therapy. Background immunosuppression included 0.1 mg/kg prednisone, tacrolimus, and mycophenolate mofetil 1 gm twice daily for historical antibody-mediated rejection. Prior therapies included remdesivir, corticosteroids, and tocilizumab, with requirement for high-flow nasal oxygen. Lack of clinical improvement and acutely rising nasopharyngeal viral RNA more than 3 weeks into illness prompted the request of ALVR109 through an emergency IND. The day following the first ALVR109 infusion, the patient's nasopharyngeal SARS-CoV-2 RNA declined from 7.43 to 5.02 log10 RNA copies/ml. On post-infusion day 4, the patient transitioned to low-flow oxygen. Two subsequent infusions of ALVR109 were administered 10 and 26 days after the first; nasopharyngeal SARS-CoV-2 RNA became undetectable on Day 11, and he was discharged the following day on low-flow oxygen 5 weeks after the initial diagnosis of COVID-19. The clinical and virologic improvements observed in this patient following administration of ALVR109 suggest a potential benefit that warrants further exploration in clinical trials.

Negative immune responses to two-dose mRNA COVID-19 vaccines in renal allograft recipients assessed with simple antibody and interferon gamma release assay cellular monitoring.

Studies are urgently needed to characterize immunogenicity, efficacy, and safety of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines in kidney transplant (KT) recipients, excluded from major clinical trials. Complex ELISPOT and other cellular response techniques have been applied, but simpler tools are needed. An easy-to-use real-world monitoring of SARS-CoV-2 IgG antibodies against the Spike protein and QuantiFERON® SARS-CoV-2 IFNγ release assay (IGRA) were performed at baseline and 28 days after the second dose in KT recipients and controls (dialysis patients and healthy ones). All healthy controls and >95% dialysis controls became positive for anti-S IgG antibodies, while only 63.3% of KT patients seroconverted with a very low antibody level. A positive IGRA was documented in 96.9% of controls, 89.3% peritoneal dialysis, 77.6% hemodialysis, 61.3% of KT patients transplanted more than 1 year ago and only 36% of those transplanted within the previous 12 months. Overall, 100% of healthy controls, 95.4% of dialysis patients and 78.8% KT recipients developed any immune response (humoral and/or cellular) against SARS-CoV-2. KT patients showed low rates of immune responses to mRNA Coronavirus infectious disease 2019 vaccines, especially those with recent transplantations. Simple humoral and cellular monitoring is advisable, so that repeated doses may be scheduled according to the results.

An interactive single cell web portal identifies gene and cell networks in COVID-19 host responses.

Numerous studies have provided single-cell transcriptome profiles of host responses to SARS-CoV-2 infection. Critically lacking however is a data mine that allows users to compare and explore cell profiles to gain insights and develop new hypotheses. To accomplish this, we harmonized datasets from COVID-19 and other control condition blood, bronchoalveolar lavage, and tissue samples, and derived a compendium of gene signature modules per cell type, subtype, clinical condition, and compartment. We demonstrate approaches to interacting with, exploring, and functional evaluating these modules via a new interactive web portal ToppCell (http://toppcell.cchmc.org/). As examples, we develop three hypotheses: (1) alternatively-differentiated monocyte-derived macrophages form a multicelllar signaling cascade that drives T cell recruitment and activation; (2) COVID-19-generated platelet subtypes exhibit dramatically altered potential to adhere, coagulate, and thrombose; and (3) extrafollicular B maturation is driven by a multilineage cell activation network that expresses an ensemble of genes strongly associated with risk for developing post-viral autoimmunity.

Positive flow cytometry crossmatch with discrepant antibody testing results following COVID-19 vaccination.

The impact of COVID-19 vaccination on the alloimmunity of transplant candidates is unknown. We report a case of positive B cell flow cytometry crossmatch in a patient waiting for second kidney transplantation, 37 days after receiving the COVID-19 vaccine. The preliminary crossmatch, using sample collected before COVID-19 vaccination, was negative. The antibodies to mismatched donor HLA-DR7 were detected only with multi-antigen beads but not with single-antigen beads, excluding possible prozone effects in solid-phase antibody assays. The crossmatches were positive with HLA-DR7-positive surrogates (n = 2) while negative with HLA-DR7-negative surrogates (n = 3), which confirms the HLA-DR7 alloreactivity. The antigen configurations on B lymphocytes are similar to that on the multi-antigen beads while distinct from the single-antigen beads. HLA-DR7 was the repeating mismatched antigen with the failing first kidney allograft. The newly emerged antibody to HLA-DR7 probably is the consequence of bystander activation of memory response by the COVID-19 vaccination. This case highlights the importance of verifying allo-sensitization history and utilizing multiple assays, including cell-based crossmatch and solid-phase assays with multi-antigens. COVID-19 immunization may deserve special attention when assessing the immunological risk before and after organ transplantation.

Microarray patches enable the development of skin-targeted vaccines against COVID-19.

The COVID-19 pandemic is a serious threat to global health and the global economy. The ongoing race to develop a safe and efficacious vaccine to prevent infection by SARS-CoV-2, the causative agent for COVID-19, highlights the importance of vaccination to combat infectious pathogens. The highly accessible cutaneous microenvironment is an ideal target for vaccination since the skin harbors a high density of antigen-presenting cells and immune accessory cells with broad innate immune functions. Microarray patches (MAPs) are an attractive intracutaneous biocargo delivery system that enables safe, reproducible, and controlled administration of vaccine components (antigens, with or without adjuvants) to defined skin microenvironments. This review describes the structure of the SARS-CoV-2 virus and relevant antigenic targets for vaccination, summarizes key concepts of skin immunobiology in the context of prophylactic immunization, and presents an overview of MAP-mediated cutaneous vaccine delivery. Concluding remarks on MAP-based skin immunization are provided to contribute to the rational development of safe and effective MAP-delivered vaccines against emerging infectious diseases, including COVID-19.

Alternative strategies of posttransplant influenza vaccination in adult solid organ transplant recipients.

Solid organ transplant (SOT) recipients are at increased risk of influenza disease and associated complications. The mainstay of prevention is the annual standard-dose influenza vaccine, as studies showed decreased influenza-related morbidity and mortality in vaccinated SOT recipients compared to those unvaccinated. Nonetheless, the immune response in this high-risk population is suboptimal compared to healthy individuals. Over the past two decades, several vaccination strategies have been investigated to overcome this inadequate immune response in SOT recipients. Howbeit, the best vaccination strategy and optimal timing of influenza vaccination remain unclear. This review will provide a detailed summary of studies of various influenza vaccination strategies in adult SOT recipients, discussing immunogenicity results, and addressing their limitations and knowledge gaps.

T cell and antibody responses to SARS-CoV-2: Experience from a French transplantation and hemodialysis center during the COVID-19 pandemic.

Immunosuppressed organ-transplanted patients are considered at risk for severe forms of COVID-19. Moreover, exaggerated innate and adaptive immune responses might be involved in severe progression of the disease. However, no data on the immune response to SARS-CoV-2 in transplanted patients are currently available. Here, we report the first assessment of antibody and T cell responses to SARS-CoV-2 in 11 kidney-transplanted patients recovered from RT-PCR-confirmed (n = 5) or initially suspected (n = 6) COVID-19. After reduction of immunosuppressive therapy, RT-PCR-confirmed COVID-19 transplant patients were able to mount vigorous antiviral T cell and antibody responses, as efficiently as two nontherapeutically immunosuppressed COVID-19 patients on hemodialysis. By contrast, six RT-PCR-negative patients displayed no antibody response. Among them, three showed very low numbers of SARS-CoV-2-reactive T cells, whereas no T cell response was detected in the other three, potentially ruling out COVID-19 diagnosis. Low levels of T cell reactivity to SARS-CoV-2 were also detected in seronegative healthy controls without known exposure to the virus. These results suggest that during COVID-19, monitoring both T cell and serological immunity might be helpful for the differential diagnosis of COVID-19 but are also needed to evaluate a potential role of antiviral T cells in the development of severe forms of the disease.

Evidence of potent humoral immune activity in COVID-19-infected kidney transplant recipients.

Whether kidney transplant recipients are capable of mounting an effective anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) adaptive immune response despite chronic immunosuppression is unknown and has important implications for therapy. Herein, we analyzed peripheral blood cell surface and intracellular cytokine phenotyping by flow cytometry along with serum antibody testing in 18 kidney transplant recipients with active coronavirus disease 2019 (COVID-19) infection and 36 matched, transplanted controls without COVID-19. We observed significantly fewer total lymphocytes and fewer circulating memory CD4+ and CD8+ T cells in the COVID-19 subjects. We also showed fewer anergic and senescent CD8+ T cells in COVID-19 individuals, but no differences in exhausted CD8+ T cells, nor in any of these CD4+ T cell subsets between groups. We also observed greater frequencies of activated B cells in the COVID-19 patients. Sixteen of 18 COVID-19 subjects tested for anti-SARS-CoV-2 serum antibodies showed positive immunoglobulin M or immunoglobulin G titers. Additional analyses showed no significant correlation among immune phenotypes and degrees of COVID-19 disease severity. Our findings indicate that immunosuppressed kidney transplant recipients admitted to the hospital with acute COVID-19 infection can mount SARS-CoV-2-reactive adaptive immune responses. The findings raise the possibility that empiric reductions in immunosuppressive therapy for all kidney transplant recipients with active COVID-19 may not be required.