The lack of either IRF9, or STAT2, has surprisingly little effect on human natural killer cell development and function.

Analysis of genetically defined immunodeficient patients allows study of the effect of the absence of specific proteins on human immune function in real-world conditions. Here we have addressed the importance of type I interferon signalling for human NK cell development by studying the phenotype and function of circulating NK cells isolated from patients suffering primary immunodeficiency disease due to mutation of either the human interferon regulatory factor 9 (IRF9) or the signal transducer and activator of transcription 2 (STAT2) genes. IRF9, together with phosphorylated STAT1 and STAT2, form a heterotrimer called interferon stimulated gene factor 3 (ISGF3) which promotes the expression of hundreds of IFN-stimulated genes that mediate antiviral function triggered by exposure to type I interferons. IRF9- and STAT2-deficient patients are unable to respond efficiently to stimulation by type I interferons and so our experiments provide insights into the importance of type I interferon signalling and the consequences of its impairment on human NK cell biology. Surprisingly, the NK cells of these patients display essentially normal phenotype and function.

Elevated levels of cell-free NKG2D-ligands modulate NKG2D surface expression and compromise NK cell function in severe COVID-19 disease.

Introduction: It is now clear that coronavirus disease 19 (COVID-19) severity is associated with a dysregulated immune response, but the relative contributions of different immune cells is still not fully understood. SARS CoV-2 infection triggers marked changes in NK cell populations, but there are contradictory reports as to whether these effector lymphocytes play a protective or pathogenic role in immunity to SARS-CoV-2. Methods: To address this question we have analysed differences in the phenotype and function of NK cells in SARS-CoV-2 infected individuals who developed either very mild, or life-threatening COVID-19 disease. Results: Although NK cells from patients with severe disease appeared more activated and the frequency of adaptive NK cells was increased, they were less potent mediators of ADCC than NK cells from patients with mild disease. Further analysis of peripheral blood NK cells in these patients revealed that a population of NK cells that had lost expression of the activating receptor NKG2D were a feature of patients with severe disease and this correlated with elevated levels of cell free NKG2D ligands, especially ULBP2 and ULBP3 in the plasma of critically ill patients. In vitro, culture in NKG2DL containing patient sera reduced the ADCC function of healthy donor NK cells and this could be blocked by NKG2DL-specific antibodies. Discussion: These observations of reduced NK function in severe disease are consistent with the hypothesis that defects in immune surveillance by NK cells permit higher levels of viral replication, rather than that aberrant NK cell function contributes to immune system dysregulation and immunopathogenicity.

Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection.

Background: Helicobacter pylori (H. pylori) uses various strategies that attenuate mucosal immunity to ensure its persistence in the stomach. We recently found evidence that H. pylori might modulate the natural killer group 2, member 2 (NKG2D) system. The NKG2D receptor and its ligands are a major activation system of natural killer and cytotoxic T cells, which are important for mucosal immunity and tumor immunosurveillance. The NKG2D system allows recognition and elimination of infected and transformed cells, however viruses and cancers often subvert its activation. Here we aimed to identify a potential evasion of the NKG2D system in H. pylori infection. Methods: We analyzed expression of NKG2D system genes in gastric tissues of H. pylori gastritis and gastric cancer patients, and performed cell-culture based infection experiments using H. pylori isogenic mutants and epithelial and NK cell lines. Results: In biopsies of H. pylori gastritis patients, NKG2D receptor expression was reduced while NKG2D ligands accumulated in the lamina propria, suggesting NKG2D evasion. In vitro, H. pylori induced the transcription and proteolytic shedding of NKG2D ligands in stomach epithelial cells, and these effects were associated with specific H. pylori virulence factors. The H. pylori-driven release of soluble NKG2D ligands reduced the immunogenic visibility of infected cells and attenuated the cytotoxic activity of effector immune cells, specifically the anti-tumor activity of NK cells. Conclusion: H. pylori manipulates the NKG2D system. This so far unrecognized strategy of immune evasion by H. pylori could potentially facilitate chronic bacterial persistence and might also promote stomach cancer development by allowing transformed cells to escape immune recognition and grow unimpeded to overt malignancy.

SARS-CoV-2 membrane protein-specific antibodies from critically ill SARS-CoV-2-infected individuals interact with Fc receptor-expressing cells but do not neutralize the virus.

The membrane (M) glycoprotein of SARS-CoV-2 is one of the key viral proteins regulating virion assembly and morphogenesis. Immunologically, the M protein is a major source of peptide antigens driving T cell responses, and most individuals who have been infected with SARS-CoV-2 make antibodies to the N-terminal, surface-exposed peptide of the M protein. We now report that although the M protein is abundant in the viral particle, antibodies to the surface-exposed N-terminal epitope of M do not appear to neutralize the virus. M protein-specific antibodies do, however, activate antibody-dependent cell-mediated cytotoxicity and cytokine secretion by primary human natural killer cells. Interestingly, while patients with severe or mild disease make comparable levels of M antigen-binding antibodies, M-specific antibodies from the serum of critically ill patients are significantly more potent activators of antibody-dependent cell-mediated cytotoxicity than antibodies found in individuals with mild or asymptomatic infection.

Antibody-dependent NK-cell and neutralizing antibody responses against the Spike protein of Wuhan-Hu-1 and Omicron BA.1 SARS-CoV-2 variants in vaccinated experienced and vaccinated naïve individuals.

Antibodies triggering Fc-mediated NK cell activity may contribute to protection against disease caused by SARS-CoV-2 infection in humans. However, how these Fc-mediated humoral responses compare between individuals displaying hybrid immunity (Vac-ex) and those fully vaccinated with no history of SARS-CoV-2 infection (Vac-n) and whether they correlate with neutralizing antibody (NtAb) responses remains largely undetermined. In this retrospective study serum samples from 50 individuals (median age, 44.5 years; range, 11-85; 25 males), 25 Vac-ex and 25 Vac-n were studied. A flow-cytometry-based antibody-mediated NK-cell activation assay was used to quantitate effector NK-cells stimulated to express LAMP1 (lysosomal associated membrane protein 1), MIP1 (Macrophage inflammatory protein 1), and interferon-γ (IFNγ); NK cells isolated from two donors (D1 and D2) were used. NtAb levels targeting the Spike protein of Wuhan-Hu-1 and Omicron BA.1 SARS-CoV-2 variants were quantitated using a SARS-CoV-2 S pseudotyped neutralization assay. Regardless of the SARS-CoV-2 variant S antigen used in the NK-cell activation assay, the frequency of NK cells stimulated to express LAMP-1, MIP1ß, and IFNγ was higher in Vac-ex compared with Vac-n (p values ranging from 0.07 to 0.006) for D1; this was only seen for BA.1 when NK cells from D2 were employed. The frequency of functional NK cells activated by antibody binding to either Wuhan-Hu-1 or Omicron BA.1 S protein was not significantly different for both VAC-ex and VAC-n. In contrast, NtAb titers against BA.1 were around 10-fold lower than that against Wuhan-Hu-1. Vac-ex displayed higher NtAb titers against both (sub)variants than Vac-n. NK-cell responses correlated poorly with NtAb titers (ρ ≤ 0.30). The data demonstrate higher cross-reactivity across variants of concern for antibodies triggering Fc-mediated NK cell than for NtAb. Moreover, Vac-Ex seemed to display more robust functional antibody responses as compared with Vac-n.

BCG-activation of leukocytes is sufficient for the generation of donor-independent innate anti-tumor NK and γδ T-cells that can be further expanded in vitro.

Bacillus Calmette-Guérin (BCG), the nonpathogenic Mycobacterium bovis strain used as tuberculosis vaccine, has been successfully used as treatment for non-muscle invasive bladder cancer for decades, and suggested to potentiate cellular and humoral immune responses. However, the exact mechanism of action is not fully understood. We previously described that BCG mainly activated anti-tumor cytotoxic NK cells with upregulation of CD56 and a CD16+ phenotype. Now, we show that stimulation of human peripheral blood mononuclear cells with iBCG, a preparation based on BCG-Moreau, expands oligoclonal γδ T-cells, with a cytotoxic phenotype, together with anti-tumor CD56high CD16+ NK cells. We have used scRNA-seq, flow cytometry, and functional assays to characterize these BCG-activated γδ T-cells in detail. They had a high IFNγ secretion signature with expression of CD27+ and formed conjugates with bladder cancer cells. BCG-activated γδ T-cells proliferated strongly in response to minimal doses of cytokines and had anti-tumor functions, although not fully based on degranulation. BCG was sufficient to stimulate proliferation of γδ T-cells when cultured with other PBMC; however, BCG alone did not stimulate expansion of purified γδ T-cells. The characterization of these non-donor restricted lymphocyte populations, which can be expanded in vitro, could provide a new approach to prepare cell-based immunotherapy tools.

CyTOF analysis identifies unusual immune cells in urine of BCG-treated bladder cancer patients.

High grade non-muscle-invasive bladder tumours are treated with transurethral resection followed by recurrent intravesical instillations of Bacillus Calmette Guérin (BCG). Although most bladder cancer patients respond well to BCG, there is no clinical parameter predictive of treatment response, and when treatment fails, the prognosis is very poor. Further, a high percentage of NMIBC patients treated with BCG suffer unwanted effects that force them to stop treatment. Thus, early identification of patients in which BCG treatment will fail is really important. Here, to identify early stage non-invasive biomarkers of non-responder patients and patients at risk of abandoning the treatment, we longitudinally analysed the phenotype of cells released into the urine of bladder cancer patients 3-7 days after BCG instillations. Mass cytometry (CyTOF) analyses revealed a large proportion of granulocytes and monocytes, mostly expressing activation markers. A novel population of CD15+CD66b+CD14+CD16+ cells was highly abundant in several samples; expression of these markers was confirmed using flow cytometry and qPCR. A stronger inflammatory response was associated with increased cell numbers in the urine; this was not due to hematuria because the cell proportions were distinct from those in the blood. This pilot study represents the first CyTOF analysis of cells recruited to urine during BCG treatment, allowing identification of informative markers associated with treatment response for sub-selection of markers to confirm using conventional techniques. Further studies should jointly evaluate cells and soluble factors in urine in larger cohorts of patients to characterise the arms of the immune response activated in responders and to identify patients at risk of complications from BCG treatment.

Natural killer (NK) cell-derived extracellular-vesicle shuttled microRNAs control T cell responses.

Natural killer (NK) cells recognize and kill target cells undergoing different types of stress. NK cells are also capable of modulating immune responses. In particular, they regulate T cell functions. Small RNA next-generation sequencing of resting and activated human NK cells and their secreted extracellular vesicles (EVs) led to the identification of a specific repertoire of NK-EV-associated microRNAs and their post-transcriptional modifications signature. Several microRNAs of NK-EVs, namely miR-10b-5p, miR-92a-3p, and miR-155-5p, specifically target molecules involved in Th1 responses. NK-EVs promote the downregulation of GATA3 mRNA in CD4+ T cells and subsequent TBX21 de-repression that leads to Th1 polarization and IFN-γ and IL-2 production. NK-EVs also have an effect on monocyte and moDCs (monocyte-derived dendritic cells) function, driving their activation and increased presentation and costimulatory functions. Nanoparticle-delivered NK-EV microRNAs partially recapitulate NK-EV effects in mice. Our results provide new insights on the immunomodulatory roles of NK-EVs that may help to improve their use as immunotherapeutic tools.

Cross-reactive cellular, but not humoral, immunity is detected between OC43 and SARS-CoV-2 NPs in people not infected with SARS-CoV-2: Possible role of cTFH cells.

Multiple questions about SARS-CoV-2 humoral and cellular immunity remain unanswered. One key question is whether preexisting memory T or B cells, specific for related coronaviruses in SARS-CoV-2-unexposed individuals, can recognize and suppress COVID-19, but this issue remains unclear. Here, we demonstrate that antibody responses to SARS-CoV-2 antigens are restricted to serum samples from COVID-19 convalescent individuals. In contrast, cross-reactive T cell proliferation and IFN-γ production responses were detected in PBMCs of around 30% of donor samples collected prepandemic, although we found that these prepandemic T cell responses only elicited weak cTFH activation upon stimulation with either HCoV-OC43 or SARS-CoV-2 NP protein. Overall, these observations confirm that T cell cross-reactive with SARS-CoV-2 antigens are present in unexposed people, but suggest that the T cell response to HCoV-OC43 could be deficient in some important aspects, like TFH expansion, that might compromise the generation of cross-reactive TFH cells and antibodies. Understanding these differences in cellular responses may be of critical importance to advance in our knowledge of immunity against SARS-CoV-2.

Aberrant inflammatory responses to type I interferon in STAT2 or IRF9 deficiency.

BACKGROUND: Inflammatory phenomena such as hyperinflammation or hemophagocytic lymphohistiocytosis are a frequent yet paradoxical accompaniment to virus susceptibility in patients with impairment of type I interferon (IFN-I) signaling caused by deficiency of signal transducer and activator of transcription 2 (STAT2) or IFN regulatory factor 9 (IRF9). OBJECTIVE: We hypothesized that altered and/or prolonged IFN-I signaling contributes to inflammatory complications in these patients. METHODS: We explored the signaling kinetics and residual transcriptional responses of IFN-stimulated primary cells from individuals with complete loss of one of STAT1, STAT2, or IRF9 as well as gene-edited induced pluripotent stem cell-derived macrophages. RESULTS: Deficiency of any IFN-stimulated gene factor 3 component suppressed but did not abrogate IFN-I receptor signaling, which was abnormally prolonged, in keeping with insufficient induction of negative regulators such as ubiquitin-specific peptidase 18 (USP18). In cells lacking either STAT2 or IRF9, this late transcriptional response to IFN-α2b mimicked the effect of IFN-γ. CONCLUSION: Our data suggest a model wherein the failure of negative feedback of IFN-I signaling in STAT2 and IRF9 deficiency leads to immune dysregulation. Aberrant IFN-α receptor signaling in STAT2- and IRF9-deficient cells switches the transcriptional output to a prolonged, IFN-γ-like response and likely contributes to clinically overt inflammation in these individuals.

Malaria hospitalisation in East Africa: age, phenotype and transmission intensity.

BACKGROUND: Understanding the age patterns of disease is necessary to target interventions to maximise cost-effective impact. New malaria chemoprevention and vaccine initiatives target young children attending routine immunisation services. Here we explore the relationships between age and severity of malaria hospitalisation versus malaria transmission intensity. METHODS: Clinical data from 21 surveillance hospitals in East Africa were reviewed. Malaria admissions aged 1 month to 14 years from discrete administrative areas since 2006 were identified. Each site-time period was matched to a model estimated community-based age-corrected parasite prevalence to provide predictions of prevalence in childhood (PfPR2-10). Admission with all-cause malaria, severe malaria anaemia (SMA), respiratory distress (RD) and cerebral malaria (CM) were analysed as means and predicted probabilities from Bayesian generalised mixed models. RESULTS: 52,684 malaria admissions aged 1 month to 14 years were described at 21 hospitals from 49 site-time locations where PfPR2-10 varied from < 1 to 48.7%. Twelve site-time periods were described as low transmission (PfPR2-10 < 5%), five low-moderate transmission (PfPR2-10 5-9%), 20 moderate transmission (PfPR2-10 10-29%) and 12 high transmission (PfPR2-10 ≥ 30%). The majority of malaria admissions were below 5 years of age (69-85%) and rare among children aged 10-14 years (0.7-5.4%) across all transmission settings. The mean age of all-cause malaria hospitalisation was 49.5 months (95% CI 45.1, 55.4) under low transmission compared with 34.1 months (95% CI 30.4, 38.3) at high transmission, with similar trends for each severe malaria phenotype. CM presented among older children at a mean of 48.7 months compared with 39.0 months and 33.7 months for SMA and RD, respectively. In moderate and high transmission settings, 34% and 42% of the children were aged between 2 and 23 months and so within the age range targeted by chemoprevention or vaccines. CONCLUSIONS: Targeting chemoprevention or vaccination programmes to areas where community-based parasite prevalence is ≥10% is likely to match the age ranges covered by interventions (e.g. intermittent presumptive treatment in infancy to children aged 2-23 months and current vaccine age eligibility and duration of efficacy) and the age ranges of highest disease burden.

Single-reaction multi-antigen serological test for comprehensive evaluation of SARS-CoV-2 patients by flow cytometry.

Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen-based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS-CoV-2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine-like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory-confirmed patients no SARS-CoV-2-specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS-CoV-2 virus might provide insight into the wide array of clinical presentations of COVID-19.

Malaria infection and severe disease risks in Africa.

The relationship between community prevalence of Plasmodium falciparum and the burden of severe, life-threatening disease remains poorly defined. To examine the three most common severe malaria phenotypes from catchment populations across East Africa, we assembled a dataset of 6506 hospital admissions for malaria in children aged 3 months to 9 years from 2006 to 2020. Admissions were paired with data from community parasite infection surveys. A Bayesian procedure was used to calibrate uncertainties in exposure (parasite prevalence) and outcomes (severe malaria phenotypes). Each 25% increase in prevalence conferred a doubling of severe malaria admission rates. Severe malaria remains a burden predominantly among young children (3 to 59 months) across a wide range of community prevalence typical of East Africa. This study offers a quantitative framework for linking malaria parasite prevalence and severe disease outcomes in children.

FCGR Genetic Variation in Two Populations From Ecuador Highlands-Extensive Copy-Number Variation, Distinctive Distribution of Functional Polymorphisms, and a Novel, Locally Common, Chimeric FCGR3B/A (CD16B/A) Gene.

Fcγ receptors (FcγR), cell-surface glycoproteins that bind antigen-IgG complexes, control both humoral and cellular immune responses. The FCGR locus on chromosome 1q23.3 comprises five homologous genes encoding low-affinity FcγRII and FcγRIII, and displays functionally relevant polymorphism that impacts on human health. Recurrent events of non-allelic homologous recombination across the FCGR locus result in copy-number variation of ~82.5 kbp-long fragments known as copy-number regions (CNR). Here, we characterize a recently described deletion that we name CNR5, which results in loss of FCGR3A, FCGR3B, and FCGR2C, and generation of a recombinant FCGR3B/A gene. We show that the CNR5 recombination spot lies at the beginning of the third FCGR3 intron. Although the FCGR3B/A-encoded hybrid protein CD16B/A reaches the plasma membrane in transfected cells, its possible natural expression, predictably restricted to neutrophils, could not be demonstrated in resting or interferon γ-stimulated cells. As the CNR5-deletion was originally described in an Ecuadorian family from Llano Grande (an indigenous community in North-Eastern Quito), we characterized the FCGR genetic variation in two populations from the highlands of Ecuador. Our results reveal that CNR5-deletion is relatively frequent in Llano Grande (5 carriers out of 36 donors). Furthermore, we found a high frequency of two strong-phagocytosis variants: the FCGR3B-NA1 haplotype and the CNR1 duplication, which translates into an increased FCGR3B and FCGR2C copy-number. CNR1 duplication was particularly increased in Llano Grande, 77.8% of the studied sample carrying at least one such duplication. In contrast, an extended haplotype CD16A-176V - CD32C-ORF+2B.2 - CD32B-2B.4 including strong activating and inhibitory FcγR variants was absent in Llano Grande and found at a low frequency (8.6%) in Ecuador highlands. This particular distribution of FCGR polymorphism, possibly a result of selective pressures, further confirms the importance of a comprehensive, joint analysis of all genetic variations in the locus and warrants additional studies on their putative clinical impact. In conclusion, our study confirms important ethnic variation at the FCGR locus; it shows a distinctive FCGR polymorphism distribution in Ecuador highlands; provides a molecular characterization of a novel CNR5-deletion associated with CD16A and CD16B deficiency; and confirms its presence in that population.

Rutin Is a Low Micromolar Inhibitor of SARS-CoV-2 Main Protease 3CLpro: Implications for Drug Design of Quercetin Analogs.

The pandemic, due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has stimulated the search for antivirals to tackle COVID-19 infection. Molecules with known pharmacokinetics and already approved for human use have been demonstrated or predicted to be suitable to be used either directly or as a base for a scaffold-based drug design. Among these substances, quercetin is known to be a potent in vitro inhibitor of 3CLpro, the SARS-CoV-2 main protease. However, its low in vivo bioavailability calls for modifications to its molecular structure. In this work, this issue is addressed by using rutin, a natural flavonoid that is the most common glycosylated conjugate of quercetin, as a model. Combining experimental (spectroscopy and calorimetry) and simulation techniques (docking and molecular dynamics simulations), we demonstrate that the sugar adduct does not hamper rutin binding to 3CLpro, and the conjugated compound preserves a high potency (inhibition constant in the low micromolar range, Ki = 11 µM). Although showing a disruption of the pseudo-symmetry in the chemical structure, a larger steric volume and molecular weight, and a higher solubility compared to quercetin, rutin is able to associate in the active site of 3CLpro, interacting with the catalytic dyad (His41/Cys145). The overall results have implications in the drug-design of quercetin analogs, and possibly other antivirals, to target the catalytic site of the SARS-CoV-2 3CLpro.

A multiplex antigen microarray for simultaneous IgG and IgM detection against SARS-CoV-2 reveals higher seroprevalence than reported.

The surge of SARS-CoV-2 has challenged health systems worldwide and efficient tests to detect viral particles, as well as antibodies generated against them, are needed. Specificity, sensitivity, promptness or scalability are the main parameters to estimate the final performance, but rarely all of them match in a single test. We have developed SCOVAM, a protein microarray with several viral antigens (spike, nucleocapsid, main protease Nsp5) as capturing probes in a fluorescence immunoassay for COVID-19 serological testing. SCOVAM depicts IgG and IgM antibody responses against each of these proteins of 22 individuals in a single microscope slide. It detects specific IgM (0.094 µg ml-1 ) and IgG (~0.017 µg ml-1 ) and is scalable and cost-effective. We validated SCOVAM by comparing with a widely used chemiluminescent commercial serological test (n = 742). SCOVAM showed twice the sensitivity and allowed following seroconversion in a single assay. By analysing the prevalence 4 months later in a subset of 76 positive sera, we still detected 93.42% of positives, almost doubling the detection of the commercial assay. The higher sensitivity of SCOVAM is especially relevant to screen sera for convalescent plasma-based treatments, high-throughput antibody response monitoring after vaccination or evaluation of vaccine efficiency.

CD4+ T Cell Immune Specificity Changes After Vaccination in Healthy And COVID-19 Convalescent Subjects.

The immune response promoted by SARS-CoV-2 vaccination is relevant to develop novel vaccines and optimized prevention strategies. We analyzed the adaptive immunity in healthy donors (HD) and convalescent individuals (CD), before and after administering BNT162b2 vaccine. Our results revealed specific changes in CD4+ T cell reactivity profile in vaccinated HD and CD, with an increase in S1 and S2 positive individuals, proportionally higher for S2. On the contrary, NCAP reactivity observed in HD and CD patients was no longer detectable after vaccination. Despite the substantial antibody response in CD, MPro-derived peptides did not elicit CD4+ lymphocyte activation in our assay in either condition. HD presented an increment in anti-S and anti-RBD IgG after first dose vaccination, which increased after the second vaccination. Conversely, anti-S and anti-RBD IgG and IgA titers increased in already positive CD after first dose administration, remaining stable after second dose inoculation. Interestingly, we found a strong significant correlation between S1-induced CD4+ response and anti-S IgA pre-vaccination, which was lost after vaccine administration.

Deregulated cellular circuits driving immunoglobulins and complement consumption associate with the severity of COVID-19 patients.

SARS-CoV-2 infection causes an abrupt response by the host immune system, which is largely responsible for the outcome of COVID-19. We investigated whether the specific immune responses in the peripheral blood of 276 patients were associated with the severity and progression of COVID-19. At admission, dramatic lymphopenia of T, B, and NK cells is associated with severity. Conversely, the proportion of B cells, plasmablasts, circulating follicular helper T cells (cTfh) and CD56- CD16+ NK-cells increased. Regarding humoral immunity, levels of IgM, IgA, and IgG were unaffected, but when degrees of severity were considered, IgG was lower in severe patients. Compared to healthy donors, complement C3 and C4 protein levels were higher in mild and moderate, but not in severe patients, while the activation peptide of C5 (C5a) increased from the admission in every patient, regardless of their severity. Moreover, total IgG, the IgG1 and IgG3 isotypes, and C4 decreased from day 0 to day 10 in patients who were hospitalized for more than two weeks, but not in patients who were discharged earlier. Our study provides important clues to understand the immune response observed in COVID-19 patients, associating severity with an imbalanced humoral response, and identifying new targets for therapeutic intervention.