A Third COVID-19 Vaccine Dose in Kidney Transplant Recipients Induces Antibody Response to Vaccine and Omicron Variants but Shows Limited Ig Subclass Switching.

Solid organ transplant recipients (SOTRs) suffer more frequent and more severe infections due to their compromised immune responses resulting from immunosuppressive treatments designed to prevent organ rejection. Pharmacological immunosuppression can adversely affect immune responses to vaccination. A cohort of kidney transplant recipients (KTRs) received their third dose of ancestral, monovalent COVID-19 vaccine in the context of a clinical trial and antibody responses to the vaccine strain, as well as to Omicron variants BA.1 and BA.5 were investigated and compared with healthy controls. Total IgG and live virus neutralizing antibody titers were reduced in KTRs compared to controls for all variants. KTRs displayed altered IgG subclass switching, with significantly lower IgG3 antibodies. Responses in KTRs were also very heterogeneous, with some individuals showing strong responses but a significant number showing no Omicron-specific neutralizing antibodies. Taken together, immune responses after COVID-19 vaccination in KTRs were not only lower than healthy controls but highly variable, indicating that simply increasing the number of vaccine doses alone may not be sufficient to provide greater protection in this population. Importance: This study addresses the challenges faced by kidney transplant recipients (KTRs) in mounting effective immune responses against COVID-19. By evaluating the antibody responses to a third dose of monovalent mRNA COVID-19 vaccine and its effectiveness against Omicron subvariants (BA.1 and BA.5), this study reveals significant reductions in both binding and neutralizing antibodies in KTRs compared to healthy controls. The research highlights altered IgG subclass switching and heterogeneous responses within the KTR population. Reduced recognition of variants, coupled with differences in IgG subclasses, decreases both the quality and quantity of protective antibodies after vaccination in KTRs. These findings underscore the need for tailored vaccination strategies for immunosuppressed populations such as KTRs. Alternative formulations and doses of COVID-19 vaccines should be considered for people with severely compromised immune systems, as more frequent vaccinations may not significantly improve the response, especially regarding neutralizing antibodies.

Bystander monocytic cells drive infection-independent NLRP3 inflammasome response to SARS-CoV-2.

The pathogenesis of COVID-19 is associated with a hyperinflammatory immune response. Monocytes and macrophages play a central role in this hyperinflammatory response to SARS-CoV-2. NLRP3 inflammasome activation has been observed in monocytes of patients with COVID-19, but the mechanism and consequences of inflammasome activation require further investigation. In this study, we inoculated a macrophage-like THP-1 cell line, primary differentiated human nasal epithelial cell (hNEC) cultures, and primary monocytes with SARS-CoV-2. We found that the activation of the NLRP3 inflammasome in macrophages does not rely on viral replication, receptor-mediated entry, or actin-dependent entry. SARS-CoV-2 productively infected hNEC cultures without triggering the production of inflammasome cytokines IL-18 and IL-1ß. Importantly, these cytokines did not inhibit viral replication in hNEC cultures. SARS-CoV-2 inoculation of primary monocytes led to inflammasome activation and induced a macrophage phenotype in these cells. Monocytic cells from bronchoalveolar lavage (BAL) fluid, but not from peripheral blood, of patients with COVID-19, showed evidence of inflammasome activation, expressed the proinflammatory marker CD11b, and displayed oxidative burst. These findings highlight the central role of activated macrophages, as a result of direct viral sensing, in COVID-19 and support the inhibition of IL-1ß and IL-18 as potential therapeutic strategies to reduce immunopathology without increasing viral replication. IMPORTANCE: Inflammasome activation is associated with severe COVID-19. The impact of inflammasome activation on viral replication and mechanistic details of this activation are not clarified. This study advances our understanding of the role of inflammasome activation in macrophages by identifying TLR2, NLRP3, ASC, and caspase-1 as dependent factors in this activation. Further, it highlights that SARS-CoV-2 inflammasome activation is not a feature of nasal epithelial cells but rather activation of bystander macrophages in the airway. Finally, we demonstrate that two pro inflammatory cytokines produced by inflammasome activation, IL-18 and IL-1ß, do not restrict viral replication and are potential targets to ameliorate pathological inflammation in severe COVID-19.

Building a successful transplant research center: Blueprints and barriers.

A successful multidisciplinary research center depends on the quality of the science being conducted and the quality of the center's design, culture, infrastructure, and institutional support. In this perspective, we describe our experience building and maintaining a multidisciplinary transplant research center with a large focus on transplant infectious diseases. We identify principles that we believe contributed to our success including: taking inventory, defining culture, creating a multidisciplinary shared leadership model, establishing expertise in a multiple method approach, investing in operations and management, building and sharing resources, and securing institutional support. We share our experience putting these principles into practice and highlight potential roadblocks.

COVID-19 Diarrhea is Inflammatory, Caused by Direct Viral Effects Plus Major Role of Virus-induced Cytokines.

BACKGROUND & AIMS: Diarrhea occurs in up to 50% of cases of COVID-19. Nonetheless, the pathophysiologic mechanism(s) have not been determined. METHODS: This was examined using normal human enteroid monolayers exposed apically to live SARS-CoV-2 or non-replicating virus-like particles (VLPs) bearing the 4 SARS-CoV-2 structural proteins or irradiated virus, all of which bound and entered enterocytes. RESULTS: Live virus and VLPs incrieased secretion of multiple cytokines and reduced mRNAs of ACE2, NHE3, and DRA. Interleukin (IL)-6 plus IL-8 alone reduced NHE3 mRNA and protein and DRA mRNA and protein. Neither VLPs nor IL-6 plus IL-8 alone altered Cl- secretion, but together they caused Cl- secretion, which was Ca2+-dependent, CFTR-independent, blocked partially by a specific TMEM16A inhibitor, and entirely by a general TMEM16 family inhibitor. VLPs and irradiated virus, but not IL-6 plus IL-8, produced Ca2+ waves that began within minutes of VLP exposure, lasted for at least 60 minutes, and were prevented by pretreatment with apyrase, a P2Y1 receptor antagonist, and general TMEM16 family inhibitor but not by the specific TMEM16A inhibitor. CONCLUSIONS: The pathophysiology of COVID-19 diarrhea appears to be a unique example of a calcium-dependent inflammatory diarrhea that is caused by direct viral effects plus the virus-induced intestinal epithelial cytokine secretion.

Minimizing risk while maximizing opportunity: The infectious disease organ offer process survey.

BACKGROUND: The purpose of this study was to understand how transplant infectious disease (TID) physicians assess a potential donor with known or suspected infection and describe posttransplant management. METHODS: We designed a survey of 10 organ offer scenarios and asked questions pertaining to organ acceptability for transplantation and management posttransplant. The survey was distributed to TID clinicians via transplant society listservs and email. Responses were recorded in REDCap, and descriptive statistics were employed. RESULTS: One hundred thirteen infectious disease physicians responded to the survey, of whom 85 completed all cases. Respondents were generally in agreement regarding organ acceptability, although some divergence was seen when evaluating lungs from donors with influenza, tuberculosis, or multidrug-resistant Acinetobacter infection. Posttransplant management showed more variation. Areas of optimization were identified: (1) Further understanding of where risk-mitigation strategies within the donor offer process may improve donor acceptability and therefore organ utilization; (2) importance of recipient considerations in assessing degree of infectious risk; and (3) gaps in evidenced-based data regarding optimal posttransplant management of recipients. CONCLUSION: Evaluation of donor offers by TID clinicians is a complex process. Although the survey does not itself serve to make recommendations regarding best practices, it highlights areas where generation of data to inform acceptance and management practices may allow for improved organ utilization and recipient management.

Rapid Wane and Recovery of XBB Sublineage Neutralization After Sequential Omicron-based Vaccination in Solid Organ Transplant Recipients.

Durability of variant neutralization in solid organ transplant recipients following Omicron-containing boosters is unknown. We report wane in XBB.1.5 neutralization by 3 months following a first bivalent booster, improved by a second booster; hybrid immunity improved peak, and duration of neutralization. Boosting at 3 to 6 months appears necessary to maintain neutralization.

COVID-19 vaccination induces distinct T-cell responses in pediatric solid organ transplant recipients and immunocompetent children.

Immune responses to COVID-19 vaccination are attenuated in adult solid organ transplant recipients (SOTRs) and additional vaccine doses are recommended for this population. However, whether COVID-19 mRNA vaccine responses are limited in pediatric SOTRs (pSOTRs) compared to immunocompetent children is unknown. Due to SARS-CoV-2 evolution and mutations that evade neutralizing antibodies, T cells may provide important defense in SOTRs who mount poor humoral responses. Therefore, we assessed anti-SARS-CoV-2 IgG titers, surrogate neutralization, and spike (S)-specific T-cell responses to COVID-19 mRNA vaccines in pSOTRs and their healthy siblings (pHCs) before and after the bivalent vaccine dose. Despite immunosuppression, pSOTRs demonstrated humoral responses to both ancestral strain and Omicron subvariants following the primary ancestral strain monovalent mRNA COVID-19 series and multiple booster doses. These responses were not significantly different from those observed in pHCs and significantly higher six months after vaccination than responses in adult SOTRs two weeks post-vaccination. However, pSOTRs mounted limited S-specific CD8+ T-cell responses and qualitatively distinct CD4+ T-cell responses, primarily producing IL-2 and TNF with less IFN-γ production compared to pHCs. Bivalent vaccination enhanced humoral responses in some pSOTRs but did not shift the CD4+ T-cell responses toward increased IFN-γ production. Our findings indicate that S-specific CD4+ T cells in pSOTRs have distinct qualities with unknown protective capacity, yet vaccination produces cross-reactive antibodies not significantly different from responses in pHCs. Given altered T-cell responses, additional vaccine doses in pSOTRs to maintain high titer cross-reactive antibodies may be important in ensuring protection against SARS-CoV-2.

Detectable plasma severe acute respiratory syndrome coronavirus 2 spike antigen is associated with poor antibody response following third messenger RNA vaccination in kidney transplant recipients.

BACKGROUND: Kidney transplant recipients (KTRs) generate lower antibody responses to messenger RNA (mRNA)-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination, yet precise mechanisms for this poor response remain uncertain. One potential contributor is suboptimal spike antigen (sAg) translation and expression owing to transplant immunosuppression, which might lead to insufficient exposure to develop humoral and/or cellular immune responses. METHODS: Within a single-arm clinical trial, 65 KTRs underwent ultrasensitive plasma sAg testing before, and 3 and 14 days after, the third mRNA vaccine doses. Anti-SARS-CoV-2 spike antibodies (anti-receptor binding domain [anti-RBD]) were serially measured at 14 and 30 days post-vaccination. Associations between sAg detection and clinical factors were assessed. Day 30 anti-RBD titer was compared among those with versus without sAg expression using Wilcoxon rank sum testing. RESULTS: Overall, 16 (25%) KTRs were sAg positive (sAg+) after vaccination, peaking at day 3. Clinical and laboratory factors were broadly similar in sAg(+) versus sAg(-) KTRs. sAg(+) status was significantly negatively associated with day 30 anti-RBD response, with median (interquartile range) 10.8 (<0.4-338.3) U/mL if sAg(+) versus 709 (10.5-2309.5) U/mL if sAg(-) (i.e., 66-fold lower; p = .01). CONCLUSION: Inadequate plasma sAg does not likely drive poor antibody responses in KTRs, rather sAg detection implies insufficient immune response to rapidly clear vaccine antigen from blood. Other downstream mechanisms such as sAg trafficking and presentation should be explored.

Emergency Myelopoiesis Distinguishes Multisystem Inflammatory Syndrome in Children From Pediatric Severe Coronavirus Disease 2019.

BACKGROUND: Multisystem inflammatory syndrome in children (MIS-C) is a hyperinflammatory condition caused by recent infection with severe acute respiratory syndrome coronavirus 2, but the underlying immunological mechanisms driving this distinct syndrome are unknown. METHODS: We utilized high-dimensional flow cytometry, cell-free (cf) DNA, and cytokine and chemokine profiling to identify mechanisms of critical illness distinguishing MIS-C from severe acute coronavirus disease 2019 (SAC). RESULTS: Compared to SAC, MIS-C patients demonstrated profound innate immune cell death and features of emergency myelopoiesis (EM), an understudied phenomenon observed in severe inflammation. EM signatures were characterized by fewer mature myeloid cells in the periphery and decreased expression of HLA-DR and CD86 on antigen-presenting cells. Interleukin 27 (IL-27), a cytokine known to drive hematopoietic stem cells toward EM, was increased in MIS-C, and correlated with immature cell signatures in MIS-C. Upon recovery, EM signatures decreased and IL-27 plasma levels returned to normal levels. Despite profound lymphopenia, we report a lack of cfDNA released by adaptive immune cells and increased CCR7 expression on T cells indicative of egress out of peripheral blood. CONCLUSIONS: Immune cell signatures of EM combined with elevated innate immune cell-derived cfDNA levels distinguish MIS-C from SAC in children and provide mechanistic insight into dysregulated immunity contributing toward MIS-C, offering potential diagnostic and therapeutic targets.

Application of machine learning models to identify serological predictors of COVID-19 severity and outcomes.

Critically ill people with COVID-19 have greater antibody titers than those with mild to moderate illness, but their association with recovery or death from COVID-19 has not been characterized. In 178 COVID-19 patients, 73 non-hospitalized and 105 hospitalized patients, mucosal swabs and plasma samples were collected at hospital enrollment and up to 3 months post-enrollment (MPE) to measure virus RNA, cytokines/chemokines, binding antibodies, ACE2 binding inhibition, and Fc effector antibody responses against SARS-CoV-2. The association of demographic variables and >20 serological antibody measures with intubation or death due to COVID-19 was determined using machine learning algorithms. Predictive models revealed that IgG binding and ACE2 binding inhibition responses at 1 MPE were positively and C1q complement activity at enrollment was negatively associated with an increased probability of intubation or death from COVID-19 within 3 MPE. Serological antibody measures were more predictive than demographic variables of intubation or death among COVID-19 patients.

Epitope Mapping of SARS-CoV-2 Spike Antibodies in Vaccinated Kidney Transplant Recipients Reveals Poor Spike Coverage Compared to Healthy Controls.

Kidney transplant recipients (KTRs) develop decreased antibody titers to SARS-CoV-2 vaccination compared to healthy controls (HCs), but whether KTRs generate antibodies against key epitopes associated with neutralization is unknown. Plasma from 78 KTRs from a clinical trial of third doses of SARS-CoV-2 vaccines and 12 HCs underwent phage display immunoprecipitation and sequencing (PhIP-Seq) to map antibody responses against SARS-CoV-2. KTRs had lower antibody reactivity to SARS-CoV-2 than HCs, but KTRs and HCs recognized similar epitopes associated with neutralization. Thus, epitope gaps in antibody breadth of KTRs are unlikely responsible for decreased efficacy of SARS-CoV-2 vaccines in this immunosuppressed population.

Cell-free DNA reveals distinct pathology of multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) is a rare but life-threatening hyperinflammatory condition induced by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes pediatric COVID-19 (pCOVID-19). The relationship of the systemic tissue injury to the pathophysiology of MIS-C is poorly defined. We leveraged the high sensitivity of epigenomics analyses of plasma cell-free DNA (cfDNA) and plasma cytokine measurements to identify the spectrum of tissue injury and glean mechanistic insights. Compared with pediatric healthy controls (pHCs) and patients with pCOVID-19, patients with MIS-C had higher levels of cfDNA primarily derived from innate immune cells, megakaryocyte-erythroid precursor cells, and nonhematopoietic tissues such as hepatocytes, cardiac myocytes, and kidney cells. Nonhematopoietic tissue cfDNA levels demonstrated significant interindividual variability, consistent with the heterogenous clinical presentation of MIS-C. In contrast, adaptive immune cell-derived cfDNA levels were comparable in MIS-C and pCOVID-19 patients. Indeed, the cfDNA of innate immune cells in patients with MIS-C correlated with the levels of innate immune inflammatory cytokines and nonhematopoietic tissue-derived cfDNA, suggesting a primarily innate immunity-mediated response to account for the multisystem pathology. These data provide insight into the pathogenesis of MIS-C and support the value of cfDNA as a sensitive biomarker to map tissue injury in MIS-C and likely other multiorgan inflammatory conditions.

Heterologous versus homologous boosting elicits qualitatively distinct, BA.5-cross-reactive T cells in transplant recipients.

BackgroundThe SARS-CoV-2 Omicron BA.5 subvariant escapes vaccination-induced neutralizing antibodies because of mutations in the spike (S) protein. Solid organ transplant recipients (SOTRs) develop high COVID-19 morbidity and poor Omicron variant recognition after COVID-19 vaccination. T cell responses may provide a second line of defense. Therefore, understanding which vaccine regimens induce robust, conserved T cell responses is critical.MethodsWe evaluated anti-S IgG titers, subvariant pseudo-neutralization, and S-specific CD4+ and CD8+ T cell responses from SOTRs in a national, prospective, observational trial (n = 75). Participants were selected if they received 3 doses of mRNA (homologous boosting) or 2 doses of mRNA followed by Ad26.COV2.S (heterologous boosting).ResultsHomologous boosting with 3 mRNA doses induced the highest anti-S IgG titers. However, antibodies induced by both vaccine regimens demonstrated lower pseudo-neutralization against BA.5 compared with the ancestral strain. In contrast, vaccine-induced S-specific T cells maintained cross-reactivity against BA.5 compared with ancestral recognition. Homologous boosting induced higher frequencies of activated polyfunctional CD4+ T cell responses, with polyfunctional IL-21+ peripheral T follicular helper cells increased in mRNA-1273 compared with BNT162b2. IL-21+ cells correlated with antibody titers. Heterologous boosting with Ad26.COV2.S did not increase CD8+ responses compared to homologous boosting.ConclusionBoosting with the ancestral strain can induce cross-reactive T cell responses against emerging variants in SOTRs, but alternative vaccine strategies are required to induce robust CD8+ T cell responses.FundingBen-Dov Family; NIH National Institute of Allergy and Infectious Diseases (NIAID) K24AI144954, NIAID K08AI156021, NIAID K23AI157893, NIAID U01AI138897, National Institute of Diabetes and Digestive and Kidney Diseases T32DK007713, and National Cancer Institute 1U54CA260492; Johns Hopkins Vice Dean of Research Support for COVID-19 Research in Immunopathogenesis; and Emory COVID-19 research repository.

Persistent SARS-CoV-2-specific immune defects in kidney transplant recipients following third mRNA vaccine dose.

Kidney transplant recipients (KTRs) show poorer response to SARS-CoV-2 mRNA vaccination, yet response patterns and mechanistic drivers following third doses are ill-defined. We administered third monovalent mRNA vaccines to n = 81 KTRs with negative or low-titer anti-receptor binding domain (RBD) antibody (n = 39 anti-RBDNEG; n = 42 anti-RBDLO), compared with healthy controls (HCs, n = 19), measuring anti-RBD, Omicron neutralization, spike-specific CD8+%, and SARS-CoV-2-reactive T cell receptor (TCR) repertoires. By day 30, 44% anti-RBDNEG remained seronegative; 5% KTRs developed BA.5 neutralization (vs 68% HCs, P < .001). Day 30 spike-specific CD8+% was negative in 91% KTRs (vs 20% HCs; P = .07), without correlation to anti-RBD (rs = 0.17). Day 30 SARS-CoV-2-reactive TCR repertoires were detected in 52% KTRs vs 74% HCs (P = .11). Spike-specific CD4+ TCR expansion was similar between KTRs and HCs, yet KTR CD8+ TCR depth was 7.6-fold lower (P = .001). Global negative response was seen in 7% KTRs, associated with high-dose MMF (P = .037); 44% showed global positive response. Of the KTRs, 16% experienced breakthrough infections, with 2 hospitalizations; prebreakthrough variant neutralization was poor. Absent neutralizing and CD8+ responses in KTRs indicate vulnerability to COVID-19 despite 3-dose mRNA vaccination. Lack of neutralization despite CD4+ expansion suggests B cell dysfunction and/or ineffective T cell help. Development of more effective KTR vaccine strategies is critical. (NCT04969263).

Neutralizing activity and 3-month durability of tixagevimab and cilgavimab prophylaxis against Omicron sublineages in transplant recipients.

Neutralizing antibody (nAb) responses are attenuated in solid organ transplant recipients (SOTRs) despite severe acute respiratory syndrome-coronavirus-2 vaccination. Preexposure prophylaxis (PrEP) with the antibody combination tixagevimab and cilgavimab (T+C) might augment immunoprotection, yet in vitro activity and durability against Omicron sublineages BA.4/5 in fully vaccinated SOTRs have not been delineated. Vaccinated SOTRs, who received 300 + 300 mg T+C (ie, full dose), within a prospective observational cohort submitted pre and postinjection samples between January 31, 2022, and July 6, 2022. The peak live virus nAb was measured against Omicron sublineages (BA.1, BA.2, BA.2.12.1, and BA.4), and surrogate neutralization (percent inhibition of angiotensin-converting enzyme 2 receptor binding to full length spike, validated vs live virus) was measured out to 3 months against sublineages, including BA.4/5. With live virus testing, the proportion of SOTRs with any nAb increased against BA.2 (47%-100%; P < .01), BA.2.12.1 (27%-80%; P < .01), and BA.4 (27%-93%; P < .01), but not against BA.1 (40%-33%; P = .6). The proportion of SOTRs with surrogate neutralizing inhibition against BA.5, however, fell to 15% by 3 months. Two participants developed mild severe acute respiratory syndrome-coronavirus-2 infection during follow-up. The majority of fully vaccinated SOTRs receiving T+C PrEP achieved BA.4/5 neutralization, yet nAb activity commonly waned by 3 months postinjection. It is critical to assess the optimal dose and interval of T+C PrEP to maximize protection in a changing variant climate.