Here, we present the case of a 74-year-old man in complete remission of a chronic lymphoid leukemia (CLL) after 6 cycles of rituximab and bendamustin (last therapy administrated in December 2019). On April 1st, he presented to our emergency unit with asthenia, loss of weight, dry cough and diarrhea since a month. He was otherwise healthy with well-controlled arterial hypertension and type 2 diabetes mellitus. SARS-CoV–2 RNA was detected (7x106 copies/ml) from a nasopharyngeal swab (defined as day 1). The patient presented a moderate neutropenia, but severe T (114 cell/mm3) and B (1 cell/mm3) lymphopenia with reduced total immunoglobulin (Ig) G and IgM levels (Supp. Fig. 1), while inflammatory markers (C-reactive protein and ferritin) were elevated (Fig. 1A). Chest computed tomography (CT) revealed bilateral multifocal subpleural and peribronchial ground-glass opacities typical of COVID–19 pneumonia18 (Fig. 1B, Supp. Fig. 2). The clinical condition gradually deteriorated with sub-febrile episodes, persisting dry cough and diarrhea, and progressive weight loss and cognitive dysfunction (Fig. 1C, D; Suppl. Information). Inflammatory parameters and blood cell counts remained abnormal (Fig. 1A, E), in line with the repeated SARS-CoV–2 positive nasopharyngeal swabs at high copy numbers (Suppl. Fig. 1). Complementary investigations excluded other diagnoses, while persisting SARS-CoV–2 infection was confirmed (Suppl. Information, Suppl. Fig. 1). No specific antiviral agents were introduced given the mild symptoms of COVID–19 (e.g. absence of hypoxemia).
In summary, the patient presented a long-lasting SARS-CoV–2 infection likely related to his severe immunosuppressive status. Consequently, we hypothesized that convalescent plasma could be beneficial in this particular case, by providing virus-specific neutralizing antibodies as well as a potential anti-inflammatory effect. The first cycle of ABO-compatible plasma transfusion (two units on two consecutive days) was given on days 72 and 73 after diagnosis of SARS-CoV–2 infection, followed by three additional cycles, administered 10 to 15 days apart (Suppl. Fig. 3). Plasma units from three convalescent donors were selected, each with relatively high IgG antibody titers against the S1 (spike)-protein using ELISA (Supp. Fig. 3). Within the first eight days after the start of plasma transfusions, the patient improved clinically, biologically and radiologically (Fig. 1). We observed a rapid normalization of the C-reactive protein, while absolute platelet counts and hemoglobin levels showed a more gradual return-to-normal. Follow-up chest CT scan confirmed a significant improvement in pulmonary infiltrates (Fig. 1B, Supp. Fig. 2). Absolute lymphocyte counts also substantially improved, and largely consisted of increased levels of memory-effector CD4 and CD8 T cells and of NK cells (Fig. 2A, Supp. Fig. 4). Only a moderate rise was observed for total B cell counts (mostly unswitched memory B cells at day 121), while total IgM and IgG remained globally stable (Fig. 2B, Supp. Fig. 1 and 4). These data indicate clear improvements of inflammation, pneumonia and blood cell counts, already after the 1st cycle of convalescent plasma transfusion.
Most patients with COVID–19 develop SARS-CoV–2 IgM and IgG antibody responses within 19 days after symptom onset19. To investigate whether such antibodies were transferred to our patient following successive cycles of plasma transfusion, we monitored the anti-SARS-CoV–2 S protein-specific IgG, IgA and IgM antibody levels by Luminex. There was a remarkable heterogeneity among the three plasma donors, with plasma/donor 3 exhibiting highest levels of specific antibodies (Fig. 2C, Supp. Fig. 3). Whereas no anti-S IgG response was detected in the patient’s serum before the start of plasma transfusion, the antibody titers increased progressively, up to 30-fold over the baseline reference after the 4th cycle (Fig. 2D, Supp. Fig. 3). A similar trend, albeit at much lower levels, was found for anti-S IgA antibodies. Instead, anti-S IgM antibodies revealed two peaks following serial plasma transfusions (Fig. 2D), again related to the level of specific-IgM antibodies of each plasma donor, with plasma/donor 3 showing highest titers (Fig. 2C). We also observed a boost of SARS-CoV–2 neutralizing activity after each cycle of plasma administration, in line with the higher antibody activity found in plasma/donor 3 when compared to the two others (Fig. 2E). Importantly, passive transfer of SARS-CoV–2 neutralizing antibodies inversely correlated to the gradual decline observed in viral loads in nasopharyngeal swabs, becoming undetectable for both E and RdRP genes by day 111 of diagnosis (Fig. 2F). To assess whether shedding of infectious SARS-CoV–2 still occurred after the start of plasma transfusions, we measured the presence of cultivable SARS-CoV–2 at different time-points. While infectious virus could be isolated from nasopharyngeal swabs prior and following the 1st cycle of convalescent plasma transfusion, this was no longer the case upon the 2nd cycle (Fig. 2F). Finally, the patient received one dose of intravenous immunoglobulin (IVIg; 0.4 g/kg) on day 114, providing additional passive immune protection against common pathogens, before being discharged to home. He was then followed by weekly outpatient care and considered as cured after two weeks of consecutive negative swabs (on day 127), but remaining potentially vulnerable to SARS-CoV–2 re-infection.
Collectively, our study offers novel evidence for a clear benefit of convalescent plasma in this particular case of COVID–19 disease, with the resolution of clinical, inflammatory and radiological parameters (Fig. 1). Improvement of inflammation, pneumonia and blood cell counts preceded viral clearance. Importantly, this patient showed a temporal association between detection of SARS-CoV–2 neutralizing antibodies and virus clearance following successive cycles of plasma transfusion (Fig. 2). Thus, our data provide insight into at least two distinct modes of action of plasma components. The first one is related to its proposed anti-inflammatory activity, similar to IVIg, widely used at high-dose for the treatment of several autoimmune diseases. In this line, convalescent plasma therapy may help in modulating the immune response via F(ab’)2-dependent mechanisms including blockade of cell-cell interactions (via cell-surface receptors) and neutralization of cytokines, complement and autoantibodies (by anti-idiotypic antibodies)12,20. In addition, convalescent plasma activity might involve Fc-dependent pathways (e.g. modulation of Fcreceptors on innate immune effector cells and B cells)12,20. However, unlike IVIg requiring the repetitive infusion of large amounts of Ig to achieve anti-inflammatory activity, this effect was readily observed after the 1st cycle of convalescent plasma transfusion. Of note, the total IgG found within convalescent plasma was 10 to 20-fold less concentrated as compared to IVIg, largely composed of monomeric IgG (Suppl. Fig. 3). Consequently, the immunomodulatory mechanisms by which immune plasma contributes to control COVID–19 pathogenesis may differ from those of the IVIg immunotherapy21 and deserves further studies.
Our observations support a second mechanism of action by SARS-CoV–2-specific IgG and neutralizing antibodies present in convalescent immune plasma (Fig. 2), which may mediate direct virus neutralization or other antibody-mediated pathways (e.g. complement activation, antibody-dependent cellular cytotoxicity). Passive antibody therapy has the great advantage to confer immediate immunity to vulnerable individuals22. Interestingly, our data further suggest a relatively rapid effect of convalescent plasma with disappearance of cultivable SARS-CoV–2 readily after the second cycle of transfusion, despite intermediate levels of neutralizing antibodies and high RNA-positivity by qRT-PCR (Fig. 2). This is in line with recent findings showing that neutralizing antibodies derived from COVID–19 patients may be effective, even at concentrations of 9 ng/ml or less23. Strikingly, successive cycles of plasma transfusion (every 10–15 days) led to the cumulative increase in anti-S IgG titers, associated with a boost of neutralizing antibodies after each administration. Anti-S-specific IgM titers also followed a kinetic pattern related to the antibody levels of each transfused donor-plasma unit, indicating that a de novo endogenous antibody response was improbable at this stage. Finally, it is certainly possible that CD4 and CD8 T cells played critical roles24 in the recovery of this patient who was primarily B cell deficient. This view is supported by reported cases of agammaglobulinemia patients who only presented mild COVID–19 disease25. In summary, convalescent immune plasma therapy revealed stepwise anti-inflammatory and anti-SARS-CoV–2 effects, resulting in full clinical recovery from infection. It remains to be seen how this compares to future monoclonal anti-SARS-CoV–2 antibodies and other novel COVID–19 therapies.