Pre-existing Immunocompromising Conditions and Outcomes of Acute COVID-19 Patients Admitted for Pediatric Intensive Care.

BACKGROUND: We aimed to determine if pre-existing immunocompromising conditions (ICCs) were associated with the presentation or outcome of patients with acute coronavirus disease 2019 (COVID-19) admitted for pediatric intensive care. METHODS: 55 hospitals in 30 U.S. states reported cases through the Overcoming COVID-19 public health surveillance registry. Patients <21 years admitted March 12, 2020-December 30, 2021 to the pediatric intensive care unit (PICU) or high acuity unit for acute COVID-19 were included. RESULTS: Of 1,274 patients, 105 (8.2%) had an ICC including 33 (31.4%) hematologic malignancies, 24 (22.9%) primary immunodeficiencies and disorders of hematopoietic cells, 19 (18.1%) nonmalignant organ failure with solid organ transplantation, 16 (15.2%) solid tumors and 13 (12.4%) autoimmune disorders. Patients with ICCs were older, had more underlying renal conditions, and had lower white blood cell and platelet counts than those without ICCs, but had similar clinical disease severity upon admission. In-hospital mortality from COVID-19 was higher (11.4% vs. 4.6%, p = 0.005) and hospitalization was longer (p = 0.01) in patients with ICCs. New major morbidities upon discharge were not different between those with and without ICC (10.5% vs 13.9%, p = 0.40). In patients with ICC, bacterial co-infection was more common in those with life-threatening COVID-19. CONCLUSIONS: In this national case series of patients <21 years of age with acute COVID-19 admitted for intensive care, existence of a prior ICCs were associated with worse clinical outcomes. Reassuringly, most patients with ICCs hospitalized in the PICU for severe acute COVID-19 survived and were discharged home without new severe morbidities.

NFKB2 haploinsufficiency identified via screening for IFN-α2 autoantibodies in children and adolescents hospitalized with SARS-CoV-2-related complications.

BACKGROUND: Autoantibodies against type I IFNs occur in approximately 10% of adults with life-threatening coronavirus disease 2019 (COVID-19). The frequency of anti-IFN autoantibodies in children with severe sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is unknown. OBJECTIVE: We quantified anti-type I IFN autoantibodies in a multicenter cohort of children with severe COVID-19, multisystem inflammatory syndrome in children (MIS-C), and mild SARS-CoV-2 infections. METHODS: Circulating anti-IFN-α2 antibodies were measured by a radioligand binding assay. Whole-exome sequencing, RNA sequencing, and functional studies of peripheral blood mononuclear cells were used to study any patients with levels of anti-IFN-α2 autoantibodies exceeding the assay's positive control. RESULTS: Among 168 patients with severe COVID-19, 199 with MIS-C, and 45 with mild SARS-CoV-2 infections, only 1 had high levels of anti-IFN-α2 antibodies. Anti-IFN-α2 autoantibodies were not detected in patients treated with intravenous immunoglobulin before sample collection. Whole-exome sequencing identified a missense variant in the ankyrin domain of NFKB2, encoding the p100 subunit of nuclear factor kappa-light-chain enhancer of activated B cells, aka NF-κB, essential for noncanonical NF-κB signaling. The patient's peripheral blood mononuclear cells exhibited impaired cleavage of p100 characteristic of NFKB2 haploinsufficiency, an inborn error of immunity with a high prevalence of autoimmunity. CONCLUSIONS: High levels of anti-IFN-α2 autoantibodies in children and adolescents with MIS-C, severe COVID-19, and mild SARS-CoV-2 infections are rare but can occur in patients with inborn errors of immunity.

Life-Threatening Complications of Influenza vs Coronavirus Disease 2019 (COVID-19) in US Children.

BACKGROUND: Clinical differences between critical illness from influenza infection vs coronavirus disease 2019 (COVID-19) have not been well characterized in pediatric patients. METHODS: We compared demographics, clinical characteristics, and outcomes of US children (aged 8 months to 17 years) admitted to the intensive care or high-acuity unit with influenza or COVID-19. Using mixed-effects models, we assessed the odds of death or requiring life support for influenza vs COVID-19 after adjustment for age, sex, race and Hispanic origin, and underlying conditions including obesity. RESULTS: Children with influenza (n = 179) were younger than those with COVID-19 (n = 381; median, 5.2 years vs 13.8 years), less likely to be non-Hispanic Black (14.5% vs 27.6%) or Hispanic (24.0% vs 36.2%), and less likely to have ≥1 underlying condition (66.4% vs 78.5%) or be obese (21.4% vs 42.2%), and a shorter hospital stay (median, 5 days vs 7 days). They were similarly likely to require invasive mechanical ventilation (both 30.2%), vasopressor support (19.6% and 19.9%), or extracorporeal membrane oxygenation (2.2% and 2.9%). Four children with influenza (2.2%) and 11 children with COVID-19 (2.9%) died. The odds of death or requiring life support in children with influenza vs COVID-19 were similar (adjusted odds ratio, 1.30; 95% confidence interval, .78-2.15; P = .32). CONCLUSIONS: Despite differences in demographics and clinical characteristics of children with influenza or COVID-19, the frequency of life-threatening complications was similar. Our findings highlight the importance of implementing prevention measures to reduce transmission and disease severity of influenza and COVID-19.

Host Respiratory Transcriptome Signature Associated with Poor Outcome in Children with Influenza-Staphylococcus aureus Pneumonia.

Respiratory coinfection of influenza with Staphylococcus aureus often causes severe disease; methicillin-resistant S. aureus (MRSA) coinfection is frequently fatal. Understanding disease pathogenesis may inform therapies. We aimed to identify host and pathogen transcriptomic (messenger RNA) signatures from the respiratory compartment of pediatric patients critically ill with influenza-S. aureus coinfection (ISAC), signatures that predict worse outcomes. Messenger RNA extracted from endotracheal aspirate samples was evaluated for S. aureus and host transcriptomic biosignatures. Influenza-MRSA outcomes were worse, but of 190 S. aureus virulence-associated genes, 6 were differentially expressed between MRSA-coinfected versus methicillin-susceptible S. aureus-coinfected patients, and none discriminated outcome. Host gene expression in patients with ISAC was compared with that in patients with influenza infection alone. Patients with poor clinical outcomes (death or prolonged multiorgan dysfunction) had relatively reduced expression of interferons and down-regulation of interferon γ-induced immune cell chemoattractants CXCL10 and CXCL11. In ISAC, airway host but not pathogen gene expression profiles predicted worse clinical outcomes.

Process intensification for production of Streptococcus pneumoniae whole-cell vaccine.

The available pneumococcal conjugate vaccines provide protection against only those serotypes that are included in the vaccine, which leads to a selective pressure and serotype replacement in the population. An alternative low-cost, safe and serotype-independent vaccine was developed based on a nonencapsulated pneumococcus strain. This study evaluates process intensification to improve biomass production and shows for the first time the use of perfusion-batch with cell recycling for bacterial vaccine production. Batch, fed-batch, and perfusion-batch were performed at 10 L scale using a complex animal component-free culture medium. Cells were harvested at the highest optical density, concentrated and washed using microfiltration or centrifugation to compare cell separation methods. Higher biomass was achieved using perfusion-batch, which removes lactate while retaining cells. The biomass produced in perfusion-batch would represent at least a fourfold greater number of doses per cultivation than in the previously described batch process. Each strategy yielded similar vaccines in terms of quality as evaluated by western blot and animal immunization assays, indicating that so far, perfusion-batch is the best strategy for the intensification of pneumococcal whole-cell vaccine production, as it can be integrated to the cell separation process keeping the same vaccine quality.

Vancomycin Monotherapy May Be Insufficient to Treat Methicillin-resistant Staphylococcus aureus Coinfection in Children With Influenza-related Critical Illness.

Background: Coinfection with influenza virus and methicillin-resistant Staphylococcus aureus (MRSA) causes life-threatening necrotizing pneumonia in children. Sporadic incidence precludes evaluation of antimicrobial efficacy. We assessed the clinical characteristics and outcomes of critically ill children with influenza-MRSA pneumonia and evaluated antibiotic use. Methods: We enrolled children (<18 years) with influenza infection and respiratory failure across 34 pediatric intensive care units 11/2008-5/2016. We compared baseline characteristics, clinical courses, and therapies in children with MRSA coinfection, non-MRSA bacterial coinfection, and no bacterial coinfection. Results: We enrolled 170 children (127 influenza A, 43 influenza B). Children with influenza-MRSA pneumonia (N = 30, 87% previously healthy) were older than those with non-MRSA (N = 61) or no (N = 79) bacterial coinfections. Influenza-MRSA was associated with increased leukopenia, acute lung injury, vasopressor use, extracorporeal life support, and mortality than either group (P ≤ .0001). Influenza-related mortality was 40% with MRSA compared to 4.3% without (relative risk [RR], 9.3; 95% confidence interval [CI], 3.8-22.9). Of 29/30 children with MRSA who received vancomycin within the first 24 hours of hospitalization, mortality was 12.5% (N = 2/16) if treatment also included a second anti-MRSA antibiotic compared to 69.2% (N = 9/13) with vancomycin monotherapy (RR, 5.5; 95% CI, 1.4, 21.3; P = .003). Vancomycin dosing did not influence initial trough levels; 78% were <10 µg/mL. Conclusions: Influenza-MRSA coinfection is associated with high fatality in critically ill children. These data support early addition of a second anti-MRSA antibiotic to vancomycin in suspected severe cases.

Evaluation of the Role of stat3 in Antibody and TH17-Mediated Responses to Pneumococcal Immunization and Infection by Use of a Mouse Model of Autosomal Dominant Hyper-IgE Syndrome.

Loss-of-function mutations in the signal transducer and activator of transcription 3 gene (stat3) result in autosomal dominant hyper-IgE syndrome (AD-HIES), a condition in which patients have recurrent debilitating infections, including frequent pneumococcal and staphylococcal pneumonias. stat3 mutations cause defective adaptive TH17 cellular responses, an immune mechanism believed to be critical for clearance of pneumococcal colonization and diminished antibody responses. Here we wished to evaluate the role of stat3 in the clearance of pneumococcal carriage and immunity using mice with a stat3 mutation recapitulating AD-HIES. We show here that naive AD-HIES mice have prolonged nasal carriage of pneumococcus compared to WT mice. Mutant and wild-type mice were then immunized with a pneumococcal whole-cell vaccine (WCV) that provides TH17-mediated protection against pneumococcal colonization and antibody-mediated protection against pneumonia and sepsis. WCV-immunized AD-HIES mice made significantly less pneumococcus-specific interleukin-17A (IL-17A) and antibody than WT mice. The WCV-elicited protection against colonization was abrogated in AD-HIES mice, but immunization with WCV still protected AD-HIES mice against aspiration pneumonia/sepsis. Taken together, our results suggest that impaired clearance of nasopharyngeal carriage due to poor adaptive IL-17A responses may contribute to the increased rates of pneumococcal respiratory infection in AD-HIES patients.

Acute lymphoblastic leukemia in a patient with MonoMAC syndrome/GATA2 haploinsufficiency.

Patients with GATA2 haploinsufficiency have a significant predisposition to developing cytopenias, unique infectious manifestations, and myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). We report a unique case of a patient who presented with B-cell acute lymphoblastic leukemia (B-ALL) and was subsequently diagnosed with monocytopenia and mycobacterium avium complex (MonoMAC) syndrome/GATA2 haploinsufficiency. The development of MDS/AML in patients with GATA2 haploinsufficiency is well described, however, the development of ALL has not been reported in the literature. ALL may be associated with GATA2 haploinsufficiency. Clinicians should be attuned to the features of the MonoMAC syndrome in patients with ALL that would prompt additional testing and alter treatment.

Toll-like receptor 2-dependent protection against pneumococcal carriage by immunization with lipidated pneumococcal proteins.

Infections with Streptococcus pneumoniae cause substantial morbidity and mortality, particularly in children in developing nations. Polysaccharide-conjugate vaccines provide protection against both invasive disease and colonization, but their use in developing countries is limited by restricted serotype coverage and expense of manufacture. Using proteomic screens, we recently identified several antigens that protected mice from pneumococcal colonization in a CD4(+) T cell- and interleukin-17A (IL-17A)-dependent manner. Since several of these proteins are lipidated, we hypothesized that their immunogenicity and impact on colonization are in part due to activation of Toll-like receptor 2 (TLR2), a receptor for lipoproteins. Here we show that lipidated versions of the antigens elicited significantly higher activation of both human embryonic kidney cells engineered to express TLR2 (HEK-TLR2) and wild-type (WT) murine macrophages than nonlipidated mutant antigens. Lipoprotein-stimulated secretion of proinflammatory cytokines was ∼10× to ∼100× lower in murine TLR2-deficient macrophages than in WT macrophages. Subcutaneous immunization of C57BL/6 mice with protein subunit vaccines containing one or two of these lipoproteins or protein fusion constructs bearing N-terminal lipid adducts elicited a robust IL-17A response and a significant reduction in colonization compared with immunization with alum alone. In contrast, immunization of Tlr2(-/-) mice elicited no detectable IL-17A response and no protection against pneumococcal colonization. These experiments suggest that the lipid moieties enhance the immunogenicity and protective efficacy of pneumococcal TH17 antigens through activation of TLR2. Thus, triggering TLR2 with an antigen-specific protein subunit formulation is a possible strategy for the development of a serotype-independent pneumococcal vaccine that would reduce pneumococcal carriage.

B cell-intrinsic deficiency of the Wiskott-Aldrich syndrome protein (WASp) causes severe abnormalities of the peripheral B-cell compartment in mice.

Wiskott Aldrich syndrome (WAS) is caused by mutations in the WAS gene that encodes for a protein (WASp) involved in cytoskeleton organization in hematopoietic cells. Several distinctive abnormalities of T, B, and natural killer lymphocytes; dendritic cells; and phagocytes have been found in WASp-deficient patients and mice; however, the in vivo consequence of WASp deficiency within individual blood cell lineages has not been definitively evaluated. By conditional gene deletion we have generated mice with selective deficiency of WASp in the B-cell lineage (B/WcKO mice). We show that this is sufficient to cause a severe reduction of marginal zone B cells and inability to respond to type II T-independent Ags, thereby recapitulating phenotypic features of complete WASp deficiency. In addition, B/WcKO mice showed prominent signs of B-cell dysregulation, as indicated by an increase in serum IgM levels, expansion of germinal center B cells and plasma cells, and elevated autoantibody production. These findings are accompanied by hyperproliferation of WASp-deficient follicular and germinal center B cells in heterozygous B/WcKO mice in vivo and excessive differentiation of WASp-deficient B cells into class-switched plasmablasts in vitro, suggesting that WASp-dependent B cell-intrinsic mechanisms critically contribute to WAS-associated autoimmunity.

Community-Onset Bacterial Coinfection in Children Critically Ill With Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Background: Community-onset bacterial coinfection in adults hospitalized with coronavirus disease 2019 (COVID-19) is reportedly uncommon, though empiric antibiotic use has been high. However, data regarding empiric antibiotic use and bacterial coinfection in children with critical illness from COVID-19 are scarce. Methods: We evaluated children and adolescents aged <19 years admitted to a pediatric intensive care or high-acuity unit for COVID-19 between March and December 2020. Based on qualifying microbiology results from the first 3 days of admission, we adjudicated whether patients had community-onset bacterial coinfection. We compared demographic and clinical characteristics of those who did and did not (1) receive antibiotics and (2) have bacterial coinfection early in admission. Using Poisson regression models, we assessed factors associated with these outcomes. Results: Of the 532 patients, 63.3% received empiric antibiotics, but only 7.1% had bacterial coinfection, and only 3.0% had respiratory bacterial coinfection. In multivariable analyses, empiric antibiotics were more likely to be prescribed for immunocompromised patients (adjusted relative risk [aRR], 1.34 [95% confidence interval {CI}, 1.01-1.79]), those requiring any respiratory support except mechanical ventilation (aRR, 1.41 [95% CI, 1.05-1.90]), or those requiring invasive mechanical ventilation (aRR, 1.83 [95% CI, 1.36-2.47]) (compared with no respiratory support). The presence of a pulmonary comorbidity other than asthma (aRR, 2.31 [95% CI, 1.15-4.62]) was associated with bacterial coinfection. Conclusions: Community-onset bacterial coinfection in children with critical COVID-19 is infrequent, but empiric antibiotics are commonly prescribed. These findings inform antimicrobial use and support rapid de-escalation when evaluation shows coinfection is unlikely.

Cross-reactive immunity against the SARS-CoV-2 Omicron variant is low in pediatric patients with prior COVID-19 or MIS-C.

Neutralization capacity of antibodies against Omicron after a prior SARS-CoV-2 infection in children and adolescents is not well studied. Therefore, we evaluated virus-neutralizing capacity against SARS-CoV-2 Alpha, Beta, Gamma, Delta and Omicron variants by age-stratified analyses (<5, 5-11, 12-21 years) in 177 pediatric patients hospitalized with severe acute COVID-19, acute MIS-C, and in convalescent samples of outpatients with mild COVID-19 during 2020 and early 2021. Across all patients, less than 10% show neutralizing antibody titers against Omicron. Children <5 years of age hospitalized with severe acute COVID-19 have lower neutralizing antibodies to SARS-CoV-2 variants compared with patients >5 years of age. As expected, convalescent pediatric COVID-19 and MIS-C cohorts demonstrate higher neutralization titers than hospitalized acute COVID-19 patients. Overall, children and adolescents show some loss of cross-neutralization against all variants, with the most pronounced loss against Omicron. In contrast to SARS-CoV-2 infection, children vaccinated twice demonstrated higher titers against Alpha, Beta, Gamma, Delta and Omicron. These findings can influence transmission, re-infection and the clinical disease outcome from emerging SARS-CoV-2 variants and supports the need for vaccination in children.

IL-17A and complement contribute to killing of pneumococci following immunization with a pneumococcal whole cell vaccine.

The pneumococcal whole cell vaccine (PWCV) has been investigated as an alternative to polysaccharide-based vaccines currently in use. It is a non-encapsulated killed vaccine preparation that induces non-capsular antibodies protecting mice against invasive pneumococcal disease (IPD) and reducing nasopharyngeal (NP) carriage via IL-17A activation of mouse phagocytes. Here, we show that PWCV induces antibody and IL-17A production to protect mice against challenge in a fatal aspiration-sepsis model after only one dose. We observed protection even with a boiled preparation, attesting to the stability and robustness of the vaccine. PWCV antibodies were shown to bind to different encapsulated strains, but complement deposition on the pneumococcal surface was observed only on serotype 3 strains; using flow cytometer methodology, variations in PWCV quality, as in the boiled vaccine, were detected. Moreover, anti-PWCV induces phagocytosis of different pneumococcal serotypes by murine peritoneal cells in the presence of complement or IL-17A. These findings suggest that complement and IL-17A may participate in the process of phagocytosis induced by PWCV antibodies. IL-17A can stimulate phagocytic cells to kill pneumococcus and this is enhanced in the presence of PWCV antibodies bound to the bacterial cell surface. Our results provide further support for the PWCV as a broad-range vaccine against all existing serotypes, potentially providing protection for humans against NP colonization and IPD. Additionally, we suggest complement deposition assay as a tool to detect subtle differences between PWCV lots.

Rationale and prospects for novel pneumococcal vaccines.

Streptococcus pneumoniae remains one of the most frequent bacterial causes of morbidity and mortality worldwide. National immunization programs implementing pneumococcal polysaccharide conjugate vaccines (PCVs) have successfully reduced rates of vaccine-type invasive disease and colonization both via direct effects in immunized children and, in some settings, indirect effects in unimmunized individuals. Limitations of the current PCV approach include the emergence of non-vaccine serotypes contributing to carriage and invasive disease in high-PCV coverage settings and the high cost of goods of PCVs which limits their accessibility in developing countries where the burden of disease remains highest. Furthermore, the distribution of serotypes causing disease varies geographically and includes more serotypes than are currently covered in a single PCV formulation. Researchers have long been exploring the potential of genetically conserved non-capsular pneumococcal antigens as vaccine candidates that might overcome such limitations. To better evaluate the rationale of such approaches, an understanding of the mechanisms of immunity to the various phases of pneumococcal infection is of paramount importance. Herein we will review the evolving understanding of both vaccine-induced and naturally acquired immunity to pneumococcal colonization and infection and discuss how this informs current approaches using serotype-independent pneumococcal vaccine candidates. We will then review the alternative vaccine candidates that have been or are currently under evaluation in clinical trials.

Identification of protective pneumococcal T(H)17 antigens from the soluble fraction of a killed whole cell vaccine.

Mucosal or parenteral immunization with a killed unencapsulated pneumococcal whole cell antigen (WCA) with an adjuvant protects mice from colonization by a T(H)17 CD4+ cell-mediated mechanism. Using preparative SDS gels, we separated the soluble proteins that compose the WCA in order to identify fractions that were immunogenic and protective. We screened these fractions for their ability to stimulate IL-17A secretion from splenocytes obtained from mice immunized with WCA and adjuvant. We identified 12 proteins within the stimulatory fractions by mass spectrometry; these proteins were then cloned, recombinantly expressed and purified using an Escherichia coli expression system. The ability of these proteins to induce IL-17A secretion was then evaluated by stimulation of mouse splenocytes. Of the four most stimulatory proteins, three were protective in a mouse pneumococcal serotype 6B colonization model. This work thus describes a method for identifying immunogenic proteins from the soluble fraction of pneumococcus and shows that several of the proteins identified protect mice from colonization when used as mucosal vaccines. We propose that, by providing protection against pneumococcal colonization, one or more of these proteins may serve as components of a multivalent pneumococcal vaccine.

Broad antibody and T cell reactivity induced by a pneumococcal whole-cell vaccine.

Injecting mice with killed cells of non-capsulated strain RM200 adsorbed on Al(OH)3 (pneumococcal whole-cell vaccine; WCV) reduces nasopharyngeal colonization by capsular serotype 6B and prevents fatal aspiration pneumonia by serotype 3 or serotype 5 strains. To further examine the potential for omni-strain immunity, we here examined a panel of clinical isolates and a library of capsule-switch variants in the TIGR4 background. IgG binding to these bacteria in sera of rabbits injected with WCV or Al(OH)3 alone was assayed by ELISA without and with adsorption with cell-wall polysaccharide, a species-common antigen. The examined strains were 23 primary isolates including at least 10 different MLS types and 13 serotypes; 15 of these strains were invasive isolates, subsequently mouse-passed. Additionally, to investigate the effect of capsulation, TIGR4 strain constructs with the capsulation genes of 20 different serotypes were evaluated. In ELISA all strains showed a large difference in IgG binding due to the immunization, of which most of the antibody typically was not CWPS-adsorbed and presumably directed to exposed protein antigens. Increased binding of IgG in the WCV-immunized serum to the 20 isogenic capsule-switch strains was shown also by flow cytometry. Further, all these 20 strains elicited IL-17A in T cells of WCV-vaccinated mice, a cytokine known to accelerate pneumococcal clearance. Thus WCV induced both humoral and T(H)17 cell-mediated immunity against all tested strains.

Next generation pneumococcal vaccines.

Currently licensed pneumococcal vaccines are based on the generation of antibodies to the pneumococcal polysaccharide, of which there are more than 90 different types. While these vaccines are highly effective against the serotypes included, their high cost and limited serotype coverage limit their usefulness worldwide, particularly in low resource areas. Thus alternative or adjunctive options are being actively pursued. This review will present these various approaches, including variations of the polysaccharide-protein conjugate strategy, protein-based strategies, and whole cell pneumococcal vaccines. The immunological basis for these different approaches is discussed as well.

T(H)17-based vaccine design for prevention of Streptococcus pneumoniae colonization.

Streptococcus pneumoniae is a leading cause of mortality in young children. While successful conjugate polysaccharide vaccines exist, a less expensive serotype-independent protein-based pneumococcal vaccine offers a major advancement for preventing life-threatening pneumococcal infections, particularly in developing nations. IL-17A-secreting CD4+ T cells (T(H)17) mediate resistance to mucosal colonization by multiple pathogens including S. pneumoniae. Screening an expression library containing >96% of predicted pneumococcal proteins, we identified antigens recognized by T(H)17 cells from mice immune to pneumococcal colonization. The identified antigens also elicited IL-17A secretion from colonized mouse splenocytes and human PBMCs suggesting that similar responses are primed during natural exposure. Immunization of two mouse strains with identified antigens provided protection from pneumococcal colonization that was significantly diminished in animals treated with blocking CD4 or IL-17A antibodies. This work demonstrates the potential of proteomic screening approaches to identify specific antigens for the design of subunit vaccines against mucosal pathogens via harnessing T(H)17-mediated immunity.