NK Cell-Monocyte Cross-talk Underlies NK Cell Activation in Severe COVID-19.

NK cells in the peripheral blood of severe COVID-19 patients exhibit a unique profile characterized by activation and dysfunction. Previous studies have identified soluble factors, including type I IFN and TGF-ß, that underlie this dysregulation. However, the role of cell-cell interactions in modulating NK cell function during COVID-19 remains unclear. To address this question, we combined cell-cell communication analysis on existing single-cell RNA sequencing data with in vitro primary cell coculture experiments to dissect the mechanisms underlying NK cell dysfunction in COVID-19. We found that NK cells are predicted to interact most strongly with monocytes and that this occurs via both soluble factors and direct interactions. To validate these findings, we performed in vitro cocultures in which NK cells from healthy human donors were incubated with monocytes from COVID-19+ or healthy donors. Coculture of healthy NK cells with monocytes from COVID-19 patients recapitulated aspects of the NK cell phenotype observed in severe COVID-19, including decreased expression of NKG2D, increased expression of activation markers, and increased proliferation. When these experiments were performed in a Transwell setting, we found that only CD56bright CD16- NK cells were activated in the presence of severe COVID-19 patient monocytes. O-link analysis of supernatants from Transwell cocultures revealed that cultures containing severe COVID-19 patient monocytes had significantly elevated levels of proinflammatory cytokines and chemokines, as well as TGF-ß. Collectively, these results demonstrate that interactions between NK cells and monocytes in the peripheral blood of COVID-19 patients contribute to NK cell activation and dysfunction in severe COVID-19.

Vascular and Non-HLA autoantibody profiles in hospitalized patients with COVID-19.

Introduction: Severe COVID-19 illness is characterized by an overwhelming immune hyperactivation. Autoantibodies against vascular, tissue, and cytokine antigens have been detected across the spectrum of COVID-19. How these autoantibodies correlate with COVID-19 severity is not fully defined. Methods: We performed an exploratory study to investigate the expression of vascular and non-HLA autoantibodies in 110 hospitalized patients with COVID-19 ranging from moderate to critically ill. Relationships between autoantibodies and COVID- 19 severity and clinical risk factors were examined using logistic regression analysis. Results: There were no absolute differences in levels of expression of autoantibodies against angiotensin II receptor type 1 (AT1R) or endothelial cell proteins between COVID-19 severity groups. AT1R autoantibody expression also did not differ by age, sex, or diabetes status. Using a multiplex panel of 60 non- HLA autoantigens we did identify seven autoantibodies that differed by COVID-19 severity including myosin (myosin; p=0.02), SHC-transforming protein 3 (shc3; p=0.07), peroxisome proliferator-activated receptor gamma coactivator 1-beta (perc; p=0.05), glial-cell derived neurotrophic factor (gdnf; p=0.07), enolase 1 (eno1; p=0.08), latrophilin-1 (lphn1; p=0.08), and collagen VI (coll6; p=0.05) with greater breadth and higher expression levels seen in less severe COVID-19. Discussion: Overall, we found that patients hospitalized with COVID-19 demonstrate evidence of auto-reactive antibodies targeting endothelial cells, angiotensin II receptors, and numerous structural proteins including collagens. Phenotypic severity did not correlate with specific autoantibodies. This exploratory study underscores the importance of better understanding of the role of autoimmunity in COVID-19 disease and sequelae.

Assessment of antibody levels to SARS-CoV-2 in patients with idiopathic inflammatory myopathies receiving treatment with intravenous immunoglobulin.

Antibodies to Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) have been reported in pooled healthy donor plasma and intravenous immunoglobulin products (IVIG). It is not known whether administration of IVIG increases circulating anti-SARS-CoV-2 antibodies (COVID ab) in IVIG recipients. COVID ab against the receptor binding domain of the spike protein were analyzed using a chemiluminescent microparticle immunoassay in patients with idiopathic inflammatory myopathies (IIM) both receiving and not receiving IVIG (IVIG and non-IVIG group, respectively). No significant differences in COVID ab levels were noted between IVIG and non-IVIG groups (417 [67-1342] AU/mL in IVIG vs 5086 [43-40,442] AU/mL in non-IVIG, p = 0.11). In linear regression models including all post-vaccination patient samples, higher number of vaccine doses was strongly associated with higher COVID ab levels (2.85 [1.21, 4.48] log AU/mL, regression coefficient [Formula: see text] [95% CI], p = 0.001), while use of RTX was associated with lower ab levels (2.73 [- 4.53, - 0.93] log AU/mL, [Formula: see text][95%CI], p = 0.004). In the IVIG group, higher total monthly doses of IVIG were associated with slightly higher COVID ab levels (0.02 [0.002-0.05] log AU/mL, p = 0.04). While patients on IVIG did not have higher COVID ab levels compared to the non-IVIG group, higher monthly doses of IVIG were associated with higher circulating levels of COVID ab in patients receiving IVIG, particularly in patients concomitantly receiving RTX. Our findings suggest that IIM patients, especially those at increased risk of COVID infection and worse COVID outcomes due to RTX therapy may have protective benefits when on concurrent IVIG treatment.

Persistent memory despite rapid contraction of circulating T Cell responses to SARS-CoV-2 mRNA vaccination.

Introduction: While antibodies raised by SARS-CoV-2 mRNA vaccines have had compromised efficacy to prevent breakthrough infections due to both limited durability and spike sequence variation, the vaccines have remained highly protective against severe illness. This protection is mediated through cellular immunity, particularly CD8+ T cells, and lasts at least a few months. Although several studies have documented rapidly waning levels of vaccine-elicited antibodies, the kinetics of T cell responses have not been well defined. Methods: Interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) were utilized to assess cellular immune responses (in isolated CD8+ T cells or whole peripheral blood mononuclear cells, PBMCs) to pooled peptides spanning spike. ELISA was performed to quantitate serum antibodies against the spike receptor binding domain (RBD). Results: In two persons receiving primary vaccination, tightly serially evaluated frequencies of anti-spike CD8+ T cells using ELISpot assays revealed strikingly short-lived responses, peaking after about 10 days and becoming undetectable by about 20 days after each dose. This pattern was also observed in cross-sectional analyses of persons after the first and second doses during primary vaccination with mRNA vaccines. In contrast, cross-sectional analysis of COVID-19-recovered persons using the same assay showed persisting responses in most persons through 45 days after symptom onset. Cross-sectional analysis using IFN-γ ICS of PBMCs from persons 13 to 235 days after mRNA vaccination also demonstrated undetectable CD8+ T cells against spike soon after vaccination, and extended the observation to include CD4+ T cells. However, ICS analyses of the same PBMCs after culturing with the mRNA-1273 vaccine in vitro showed CD4+ and CD8+ T cell responses that were readily detectable in most persons out to 235 days after vaccination. Discussion: Overall, we find that detection of spike-targeted responses from mRNA vaccines using typical IFN-γ assays is remarkably transient, which may be a function of the mRNA vaccine platform and an intrinsic property of the spike protein as an immune target. However, robust memory, as demonstrated by capacity for rapid expansion of T cells responding to spike, is maintained at least several months after vaccination. This is consistent with the clinical observation of vaccine protection from severe illness lasting months. The level of such memory responsiveness required for clinical protection remains to be defined.

Hybrid Immunity Shifts the Fc-Effector Quality of SARS-CoV-2 mRNA Vaccine-Induced Immunity.

Despite the robust immunogenicity of SARS-CoV-2 mRNA vaccines, emerging data have revealed enhanced neutralizing antibody and T cell cross-reactivity among individuals that previously experienced COVID-19, pointing to a hybrid immune advantage with infection-associated immune priming. Beyond neutralizing antibodies and T cell immunity, mounting data point to a potential role for additional antibody effector functions, including opsinophagocytic activity, in the resolution of symptomatic COVID-19. Whether hybrid immunity modifies the Fc-effector profile of the mRNA vaccine-induced immune response remains incompletely understood. Thus, here we profiled the SARS-CoV-2 specific humoral immune response in a group of individuals with and without prior COVID-19. As expected, hybrid Spike-specific antibody titers were enhanced following the primary dose of the mRNA vaccine but were similar to those achieved by naive vaccinees after the second mRNA vaccine dose. Conversely, Spike-specific vaccine-induced Fc-receptor binding antibody levels were higher after the primary immunization in individuals with prior COVID-19 and remained higher following the second dose compared to those in naive individuals, suggestive of a selective improvement in the quality, rather than the quantity, of the hybrid humoral immune response. Thus, while the magnitude of antibody titers alone may suggest that any two antigen exposures-either hybrid immunity or two doses of vaccine alone-represent a comparable prime/boost immunologic education, we find that hybrid immunity offers a qualitatively improved antibody response able to better leverage Fc-effector functions against conserved regions of the virus. IMPORTANCE Recent data indicates improved immunity to SARS-CoV-2 in individuals who experience a combination of two mRNA vaccine doses and infection, "hybrid immunity," compared to individuals who receive vaccination or experience infection alone. While previous infection accelerates the vaccine-induced immune response following the first dose of mRNA vaccination, subsequent doses demonstrate negligible increases in antibody titers or T cell immunity. Here, using systems serology, we observed a unique antibody profile induced by hybrid immunity, marked by the unique induction of robust Fc-recruiting antibodies directed at the conserved region of the viral Spike antigen, the S2-domain, induced at lower levels in individuals who only received mRNA vaccination. Thus, hybrid immunity clearly redirects vaccine-induced immunodominance, resulting in the induction of a robust functional humoral immune response to the most highly conserved region of the SARS-CoV-2 Spike antigen, which may be key to protection against existing and emerging variants of concern. Thus, next-generation vaccines able to mimic hybrid immunity and drive a balanced response to conserved regions of the Spike antigen may confer enhanced protection against disease.

Dominant CD8+ T Cell Nucleocapsid Targeting in SARS-CoV-2 Infection and Broad Spike Targeting From Vaccination.

CD8+ T cells have key protective roles in many viral infections. While an overall Th1-biased cellular immune response against SARS-CoV-2 has been demonstrated, most reports of anti-SARS-CoV-2 cellular immunity have evaluated bulk T cells using pools of predicted epitopes, without clear delineation of the CD8+ subset and its magnitude and targeting. In recently infected persons (mean 29.8 days after COVID-19 symptom onset), we confirm a Th1 bias (and a novel IL-4-producing population of unclear significance) by flow cytometry, which does not correlate to antibody responses against the receptor binding domain. Evaluating isolated CD8+ T cells in more detail by IFN-γ ELISpot assays, responses against spike, nucleocapsid, matrix, and envelope proteins average 396, 901, 296, and 0 spot-forming cells (SFC) per million, targeting 1.4, 1.5, 0.59, and 0.0 epitope regions respectively. Nucleocapsid targeting is dominant in terms of magnitude, breadth, and density of targeting. The magnitude of responses drops rapidly post-infection; nucleocapsid targeting is most sustained, and vaccination selectively boosts spike targeting. In SARS-CoV-2-naïve persons, evaluation of the anti-spike CD8+ T cell response soon after vaccination (mean 11.3 days) yields anti-spike CD8+ T cell responses averaging 2,463 SFC/million against 4.2 epitope regions, and targeting mirrors that seen in infected persons. These findings provide greater clarity on CD8+ T cell anti-SARS-CoV-2 targeting, breadth, and persistence, suggesting that nucleocapsid inclusion in vaccines could broaden coverage and durability.

Longitudinal Assessment of Coronavirus Disease 2019 Vaccine Acceptance and Uptake Among Frontline Medical Workers in Los Angeles, California.

BACKGROUND: Sentiments of vaccine hesitancy and distrust in public health institutions have complicated the government-led coronavirus disease 2019 (COVID-19) vaccine control strategy in the United States. As the first to receive the vaccine, COVID-19 vaccine attitudes among frontline workers are consequential for COVID-19 control and public opinion of the vaccine. METHODS: In this study, we used a repeated cross-sectional survey administered at 3 time points between 24 September 2020 and 6 February 2021 to a cohort of employees of the University of California, Los Angeles Health and the Los Angeles County Fire Department. The primary outcome of interest was COVID-19 vaccination intent and vaccine uptake. RESULTS: Confidence in COVID-19 vaccines and vaccine uptake rose significantly over time. At survey 1, confidence in vaccine protection was 46.4% among healthcare workers (HCWs) and 34.6% among first responders (FRs); by survey 3, this had risen to 90.0% and 75.7%, respectively. At survey 1, about one-third of participants intended to receive a vaccine as soon as possible. By survey 3, 96.0% of HCWs and 87.5% of FRs had received a COVID-19 vaccine. CONCLUSIONS: Attitudes toward vaccine uptake increased over the study period, likely a result of increased public confidence in COVID-19 vaccines, targeted communications, a COVID-19 winter surge in Los Angeles County, and ease of access from employer-sponsored vaccine distribution.

Humoral responses to SARS-CoV-2 mRNA vaccines: Role of past infection.

Two mRNA vaccines (BNT162b2 and mRNA-1273) against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) are globally authorized as a two-dose regimen. Understanding the magnitude and duration of protective immune responses is vital to curbing the pandemic. We enrolled 461 high-risk health services workers at the University of California, Los Angeles (UCLA) and first responders in the Los Angeles County Fire Department (LACoFD) to assess the humoral responses in previously infected (PI) and infection naïve (NPI) individuals to mRNA-based vaccines (BNT162b2/Pfizer- BioNTech or mRNA-1273/Moderna). A chemiluminescent microparticle immunoassay was used to detect antibodies against SARS-CoV-2 Spike in vaccinees prior to (n = 21) and following each vaccine dose (n = 246 following dose 1 and n = 315 following dose 2), and at days 31-60 (n = 110) and 61-90 (n = 190) following completion of the 2-dose series. Both vaccines induced robust antibody responses in all immunocompetent individuals. Previously infected individuals achieved higher median peak titers (p = 0.002) and had a slower rate of decay (p = 0.047) than infection-naïve individuals. mRNA-1273 vaccinated infection-naïve individuals demonstrated modestly higher titers following each dose (p = 0.005 and p = 0.029, respectively) and slower rates of antibody decay (p = 0.003) than those who received BNT162b2. A subset of previously infected individuals (25%) required both doses in order to reach peak antibody titers. The biologic significance of the differences between previously infected individuals and between the mRNA-1273 and BNT162b2 vaccines remains uncertain, but may have important implications for booster strategies.

Primary, Recall, and Decay Kinetics of SARS-CoV-2 Vaccine Antibody Responses.

Studies of two SARS-CoV-2 mRNA vaccines suggested that they yield ∼95% protection from symptomatic infection at least short-term, but important clinical questions remain. It is unclear how vaccine-induced antibody levels quantitatively compare to the wide spectrum induced by natural SARS-CoV-2 infection. Vaccine response kinetics and magnitudes in persons with prior COVID-19 compared to virus-naïve persons are not well-defined. The relative stability of vaccine-induced versus infection-induced antibody levels is unclear. We addressed these issues with longitudinal assessments of vaccinees with and without prior SARS-CoV-2 infection using quantitative enzyme-linked immunosorbent assay (ELISA) of anti-RBD antibodies. SARS-CoV-2-naïve individuals achieved levels similar to mild natural infection after the first vaccination; a second dose generated levels approaching severe natural infection. In persons with prior COVID-19, one dose boosted levels to the high end of severe natural infection even in those who never had robust responses from infection, increasing no further after the second dose. Antiviral neutralizing assessments using a spike-pseudovirus assay revealed that virus-naïve vaccinees did not develop physiologic neutralizing potency until the second dose, while previously infected persons exhibited maximal neutralization after one dose. Finally, antibodies from vaccination waned similarly to natural infection, resulting in an average of ∼90% loss within 90 days. In summary, our findings suggest that two doses are important for quantity and quality of humoral immunity in SARS-CoV-2-naïve persons, while a single dose has maximal effects in those with past infection. Antibodies from vaccination wane with kinetics very similar to that seen after mild natural infection; booster vaccinations will likely be required.

A cell-penetrating ester of the neural metabolite lanthionine ketimine stimulates autophagy through the mTORC1 pathway: Evidence for a mechanism of action with pharmacological implications for neurodegenerative pathologies.

Autophagy is a fundamental cellular recycling process vulnerable to compromise in neurodegeneration. We now report that a cell-penetrating neurotrophic and neuroprotective derivative of the central nervous system (CNS) metabolite, lanthionine ketimine (LK), stimulates autophagy in RG2 glioma and SH-SY5Y neuroblastoma cells at concentrations within or below pharmacological levels reported in previous mouse studies. Autophagy stimulation was evidenced by increased lipidation of microtubule-associated protein 1 light chain 3 (LC3) both in the absence and presence of bafilomycin-A1 which discriminates between effects on autophagic flux versus blockage of autophagy clearance. LKE treatment caused changes in protein level or phosphorylation state of multiple autophagy pathway proteins including mTOR; p70S6 kinase; unc-51-like-kinase-1 (ULK1); beclin-1 and LC3 in a manner essentially identical to effects observed after rapamycin treatment. The LKE site of action was near mTOR because neither LKE nor the mTOR inhibitor rapamycin affected tuberous sclerosis complex (TSC) phosphorylation status upstream from mTOR. Confocal immunofluorescence imaging revealed that LKE specifically decreased mTOR (but not TSC2) colocalization with LAMP2(+) lysosomes in RG2 cells, a necessary event for mTORC1-mediated autophagy suppression, whereas rapamycin had no effect. Suppression of the LK-binding adaptor protein CRMP2 (collapsin response mediator protein-2) by means of shRNA resulted in diminished autophagy flux, suggesting that the LKE action on mTOR localization may occur through a novel mechanism involving CRMP2-mediated intracellular trafficking. These findings clarify the mechanism-of-action for LKE in preclinical models of CNS disease, while suggesting possible roles for natural lanthionine metabolites in regulating CNS autophagy.

Predicting the potential public health impact of disease-modifying HIV vaccines in South Africa: the problem of subtypes.

Current HIV vaccines in development appear unlikely to prevent infection, but could provide benefits by increasing survival; such vaccines are described as disease-modifying vaccines. We review the current status of vaccines and modeling vaccines. We also predict the impact that disease-modifying vaccines could have in South Africa, where multiple subtypes are co-circulating. We model transmissibility/fitness differences among subtypes. We used uncertainty analyses to model vaccines with four characteristics: (i) take, (ii) duration of immunity, (iii) reduction in transmissibility/fitness, and (iv) increase in survival. We reconstructed, and forecasted, the South African epidemic from 1940 to 2140 (assuming no vaccination). We predict that: (i) incidence will peak in 2014, decline, and stabilize, (ii) prevalence will continue to rise, and (iii) the AIDS death rate curve will peak in 2022. Our predictions show that (over the next 135 years) the epidemic in South Africa will switch from a predominantly Subtype C epidemic to an epidemic driven by other subtypes. We predict that the epidemic could remain unchanged, even with mass vaccination with a vaccine that is equally effective against all co-circulating subtypes. However, if the non-C subtypes are less (or equally) transmissible as Subtype C then disease-modifying vaccines could result in eradication. Thus, in countries where multiple-subtypes are co-circulating it is critical to realize that small biological differences among subtypes will have dramatic consequences for the effectiveness of HIV vaccination campaigns. A slight difference in fitness will determine whether a disease-modifying vaccine has almost no impact on the epidemic or can achieve eradication.

Potential role for CD63 in CCR5-mediated human immunodeficiency virus type 1 infection of macrophages.

Macrophages and CD4(+) lymphocytes are the principal target cells for human immunodeficiency virus type 1 (HIV-1) infection, but the molecular details of infection may differ between these cell types. During studies to identify cellular molecules that could be involved in macrophage infection, we observed inhibition of HIV-1 infection of macrophages by monoclonal antibody (MAb) to the tetraspan transmembrane glycoprotein CD63. Pretreatment of primary macrophages with anti-CD63 MAb, but not MAbs to other macrophage cell surface tetraspanins (CD9, CD81, and CD82), was shown to inhibit infection by several R5 and dualtropic strains, but not by X4 isolates. The block to productive infection was postfusion, as assessed by macrophage cell-cell fusion assays, but was prior to reverse transcription, as determined by quantitative PCR assay for new viral DNA formation. The inhibitory effects of anti-CD63 in primary macrophages could not be explained by changes in the levels of CD4, CCR5, or beta-chemokines. Infections of peripheral blood lymphocytes and certain cell lines were unaffected by treatment with anti-CD63, suggesting that the role of CD63 in HIV-1 infection may be specific for macrophages.