Identification of natural killer markers associated with fatal outcome in COVID-19 patients.

Introduction: Increasing evidence has shown that coronavirus disease 19 (COVID-19) severity is driven by a dysregulated immunological response. Previous studies have demonstrated that natural killer (NK) cell dysfunction underpins severe illness in COVID-19 patients, but have lacked an in-depth analysis of NK cell markers as a driver of death in the most critically ill patients. Methods: We enrolled 50 non-vaccinated hospitalized patients infected with the initial virus or the alpha variant of SARS-CoV-2 with moderate or severe illness, to evaluate phenotypic and functional features of NK cells. Results: Here, we show that, consistent with previous studies, evolution NK cells from COVID-19 patients are more activated, with the decreased activation of natural cytotoxicity receptors and impaired cytotoxicity and IFN-γ production, in association with disease regardless of the SARS-CoV-2 strain. Fatality was observed in 6 of 17 patients with severe disease; NK cells from all of these patients displayed a peculiar phenotype of an activated memory-like phenotype associated with massive TNF-α production. Discussion: These data suggest that fatal COVID-19 infection is driven by an uncoordinated inflammatory response in part mediated by a specific subset of activated NK cells.

Plasticity of natural killer cells in pregnant patients infected with SARS-CoV-2 and their neonates during childbirth.

The COVID-19 pandemic has occurred due to infection caused by the SARS-CoV-2 coronavirus, which impacts gestation and pregnancy. In SARS-CoV-2 infection, only very rare cases of vertical transmission have been reported, suggesting that fetal immune imprinting due to a maternal infection is probably a result of changes in maternal immunity. Natural killer (NK) cells are the leading maternal immune cells that act as a natural defense system to fight infections. They also play a pivotal role in the establishment and maintenance of pregnancy. While peripheral NK cells display specific features in patients infected with SARS-CoV-2 in the general population, information remains elusive in pregnant mothers and neonates. In the present study, we analyzed the characteristics of NK cells isolated from both neonatal umbilical cord blood and maternal peripheral blood close to the time of delivery. Phenotype and functions were compared in 18 healthy pregnant women and 34 COVID-19 patients during pregnancy within an ongoing infection (PCR+; N = 15) or after recovery (IgG+PCR-; N = 19). The frequency of NK cells from infected women and their neonates was correlated with the production of inflammatory cytokines in the serum. The expression of NKG2A and NKp30, as well as degranulation of NK cells in pregnant women with ongoing infection, were both negatively correlated to estradiol level. Furthermore, NK cells from the neonates born to infected women were significantly decreased and also correlated to estradiol level. This study highlights the relationship between NK cells, inflammation, and estradiol in patients with ongoing infection, providing new insights into the impact of maternal SARS-CoV-2 infection on the neonate.

CAR-NK Cells: A Chimeric Hope or a Promising Therapy?

Immunotherapy with chimeric antigen receptor-engineered T cells (CAR-T) has revolutionized the treatment landscape of relapsed/refractory B-cell malignancies. Nonetheless, the use of autologous T cells has certain limitations, including the variable quality and quantity of collected effector T cells, extended time of cell processing, limited number of available CAR cells, toxicities, and a high cost. Thanks to their powerful cytotoxic capabilities, with proven antitumor effects in both haploidentical hematopoietic stem cell transplantation and adoptive cell therapy against solid tumors and hematological malignancies, Natural Killer cells could be a promising alternative. Different sources of NK cells can be used, including cellular lines, cord blood, peripheral blood, and induced pluripotent stem cells. Their biggest advantage is the possibility of using them in an allogeneic context without major toxic side effects. However, the majority of the reports on CAR-NK cells concern preclinical or early clinical trials. Indeed, NK cells might be more difficult to engineer, and the optimization and standardization of expansion and transfection protocols need to be defined. Furthermore, their short persistence after infusion is also a major setback. However, with recent advances in manufacturing engineered CAR-NK cells exploiting their cytolytic capacities, antibody-dependent cellular cytotoxicity (ADCC), and cytokine production, "off-the-shelf" allogeneic CAR-NK cells can provide a great potential in cancer treatments.

Dichotomy in Neutralizing Antibody Induction to Peptide-Conjugated Vaccine in Squalene Emulsion Contrast With Aluminum Hydroxide Formulation.

W614A-3S peptide is a modified 3S motif of the HIV-gp41 (mutation W614A). We previously detected the presence of natural neutralizing antibodies directed against W614A-3S peptide (NAbs) in long-term non-progressor HIV+ patients. Here, we compared the efficacy of W614A-3S peptide formulated in either squalene emulsion (SQE) or in aluminum hydroxide (Alum) in inducing broadly-NAbs (bNAbs). Rabbit and mouse models were used to screen the induction of bNAbs following 4 immunizations. SQE was more efficient than Alum formulation in inducing W614A-3S-specific bNAbs with up to 67%-93% of HIV strains neutralized. We then analyzed the quality of peptide-specific murine B cells by single-cell gene expression by quantitative reverse transcription-PCR and single-cell V(D)J sequencing. We found more frequent germinal center B cells in SQE than in Alum, albeit with a different gene expression profile. The V(D)J sequencing of W614A-3S-specific BCR showed significant differences in BCR sequences and validates the dichotomy between adjuvant formulations. All sixteen BCR sequences which were cloned were specific of peptide. Adjuvant formulations of W614A-3S-peptide-conjugated immunogen impact the quantity and quality of B cell immune responses at both the gene expression level and BCR sequence.

NK Cell Responses in Zika Virus Infection Are Biased towards Cytokine-Mediated Effector Functions.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged as a global concern because of its impact on human health. ZIKV infection during pregnancy can cause microcephaly and other severe brain defects in the developing fetus and there have been reports of the occurrence of Guillain-Barré syndrome in areas affected by ZIKV. NK cells are activated during acute viral infections and their activity contributes to a first line of defense because of their ability to rapidly recognize and kill virus-infected cells. To provide insight into NK cell function during ZIKV infection, we have profiled, using mass cytometry, the NK cell receptor-ligand repertoire in a cohort of acute ZIKV-infected female patients. Freshly isolated NK cells from these patients contained distinct, activated, and terminally differentiated, subsets expressing higher levels of CD57, NKG2C, and KIR3DL1 as compared with those from healthy donors. Moreover, KIR3DL1+ NK cells from these patients produced high levels of IFN-γ and TNF-α, in the absence of direct cytotoxicity, in response to in vitro stimulation with autologous, ZIKV-infected, monocyte-derived dendritic cells. In ZIKV-infected patients, overproduction of IFN-γ correlated with STAT-5 activation (r = 0.6643; p = 0.0085) and was mediated following the recognition of MHC class 1-related chain A and chain B molecules expressed by ZIKV-infected monocyte-derived dendritic cells, in synergy with IL-12 production by the latter cells. Together, these findings suggest that NK cells contribute to the generation of an efficacious adaptive anti-ZIKV immune response that could potentially affect the outcome of the disease and/or the development of persistent symptoms.

Natural Killer Cells in Post-Transplant Lymphoproliferative Disorders.

Post-transplant lymphoproliferative disorders (PTLDs) are life-threatening complications arising after solid organ or hematopoietic stem cell transplantations. Although the majority of these lymphoproliferations are of B cell origin, and are frequently associated with primary Epstein-Barr virus (EBV) infection or reactivation in the post-transplant period, rare cases of T cell and natural killer (NK) cell-originated PTLDs have also been described. A general assumption is that PTLDs result from the impairment of anti-viral and anti-tumoral immunosurveillance due to the long-term use of immunosuppressants in transplant recipients. T cell impairment is known to play a critical role in the immune-pathogenesis of post-transplant EBV-linked complications, while the role of NK cells has been less investigated, and is probably different between EBV-positive and EBV-negative PTLDs. As a part of the innate immune response, NK cells are critical for protecting hosts during the early response to virus-induced tumors. The complexity of their function is modulated by a myriad of activating and inhibitory receptors expressed on cell surfaces. This review outlines our current understanding of NK cells in the pathogenesis of PTLD, and discusses their potential implications for current PTLD therapies and novel NK cell-based therapies for the containment of these disorders.

Distinct immunopathological mechanisms of EBV-positive and EBV-negative posttransplant lymphoproliferative disorders.

EBV-positive and EBV-negative posttransplant lymphoproliferative disorders (PTLDs) arise in different immunovirological contexts and might have distinct pathophysiologies. To examine this hypothesis, we conducted a multicentric prospective study with 56 EBV-positive and 39 EBV-negative PTLD patients of the K-VIROGREF cohort, recruited at PTLD diagnosis and before treatment (2013-2019), and compared them to PTLD-free Transplant Controls (TC, n = 21). We measured absolute lymphocyte counts (n = 108), analyzed NK- and T cell phenotypes (n = 49 and 94), and performed EBV-specific functional assays (n = 16 and 42) by multiparameter flow cytometry and ELISpot-IFNγ assays (n = 50). EBV-negative PTLD patients, NK cells overexpressed Tim-3; the 2-year progression-free survival (PFS) was poorer in patients with a CD4 lymphopenia (CD4+ <300 cells/mm3 , p <  .001). EBV-positive PTLD patients presented a profound NK-cell lymphopenia (median = 60 cells/mm3 ) and a high proportion of NK cells expressing PD-1 (vs. TC, p = .029) and apoptosis markers (vs. TC, p < .001). EBV-specific T cells of EBV-positive PTLD patients circulated in low proportions, showed immune exhaustion (p = .013 vs. TC) and poorly recognized the N-terminal portion of EBNA-3A viral protein. Altogether, this broad comparison of EBV-positive and EBV-negative PTLDs highlight distinct patterns of immunopathological mechanisms between these two diseases and provide new clues for immunotherapeutic strategies and PTLD prognosis.

Human ACE2 peptide-mimics block SARS-CoV-2 pulmonary cells infection.

In light of the recent accumulated knowledge on SARS-CoV-2 and its mode of human cells invasion, the binding of viral spike glycoprotein to human Angiotensin Converting Enzyme 2 (hACE2) receptor plays a central role in cell entry. We designed a series of peptides mimicking the N-terminal helix of hACE2 protein which contains most of the contacting residues at the binding site, exhibiting a high helical folding propensity in aqueous solution. Our best peptide-mimics are able to block SARS-CoV-2 human pulmonary cell infection with an inhibitory concentration (IC50) in the nanomolar range upon binding to the virus spike protein with high affinity. These first-in-class blocking peptide mimics represent powerful tools that might be used in prophylactic and therapeutic approaches to fight the coronavirus disease 2019 (COVID-19).

Clinical and laboratory findings of acute Zika virus infection in patients from Salvador during the first Brazilian epidemic

Abstract Several major epidemics of Zika fever, caused by the ZIKA virus (ZIKV), have emerged in Brazil since early 2015, eventually spreading to other countries on the South American continent. The present study describes the clinical manifestations and laboratory findings of patients with confirmed acute ZIKV infection during the first epidemic that occurred in Salvador, Brazil. All included patients were seen at the emergency room of a private tertiary hospital located in Salvador, Brazil from 2015 through 2017. Patients were considered eligible if signs of systemic viral febrile disease were present. All individuals were tested for ZIKV and Chikungunya infection using PCR, while rapid test was used to detect Dengue virus antibodies or, alternatively, the NS1 antigen. A diagnosis of acute ZIKV infection was confirmed in 78/434 (18%) individuals with systemic viral febrile illness. Positivity was mainly observed in blood, followed by saliva and urine. Coinfection with Chikungunya and/or Dengue virus was detected in 5% of the ZIKV-infected patients. The most frequent clinical findings were myalgia, arthralgia and low-grade fever. Laboratory analysis demonstrated normal levels of hematocrit, platelets and liver enzymes. In summary, in acute settings where molecular testing remains unavailable, clinicians face difficulties to confirm the diagnosis of ZIKV infection, as they rely only on clinical examinations and conventional laboratory tests.

Skewed T cell responses to Epstein-Barr virus in long-term asymptomatic kidney transplant recipients.

Kidney transplant recipients (KTRs) abnormally replicate the Epstein Barr Virus (EBV). To better understand how long-term immunosuppression impacts the immune control of this EBV re-emergence, we systematically compared 10 clinically stable KTRs to 30 healthy controls (HCs). The EBV-specific T cell responses were determined in both groups by multiparameter flow cytometry with intra cellular cytokine staining (KTRs n = 10; HCs n = 15) and ELISpot-IFNγ assays (KTRs n = 7; HCs n = 7). The T/B/NK cell counts (KTRs n = 10; HCs n = 30) and the NK/T cell differentiation and activation phenotypes (KTRs n = 10; HCs n = 15/30) were also measured. We show that in KTRs, the Th1 effector CD4+ T cell responses against latent EBV proteins are weak (2/7 responders). Conversely, the frequencies total EBV-specific CD8+T cells are conserved in KTRs (n = 10) and span a wider range of EBNA-3A peptides (5/7responders) than in HCs (5/7responders). Those modifications of the EBV-specific T cell response were associated with a profound CD4+ T cell lymphopenia in KTRs compared to HCs, involving the naïve CD4+ T cell subset, and a persistent activation of highly-differentiated senescent CD8+ T cells. The proportion of total NK / CD8+ T cells expressing PD-1 was also increased in KTRs. Noteworthy, PD-1 expression on CD8+ T cells normalized with time after transplantation. In conclusion, we show modifications of the EBV-specific cellular immunity in long term transplant recipients. This may be the result of both persistent EBV antigenic stimulation and profound immunosuppression induced by anti-rejection treatments. These findings provide new insights into the immunopathology of EBV infection after renal transplantation.

NK Cells in the Human Lungs.

The lung offers one of the largest exchange surfaces of the individual with the elements of the environment. As a place of important interactions between self and non-self, the lung is richly endowed in various immune cells. As such, lung natural killer (NK) cells play major effector and immunoregulatory roles to ensure self-integrity. A better understanding of their abilities in health and diseases has been made possible over the past decade thanks to tremendous discoveries in humans and animals. By precisely distinguishing the different NK cell subsets and dissecting the ontogeny and differentiation of NK cells, both blood and tissue-resident NK populations now appear to be much more pleiotropic than previously thought. In light of these recent findings in healthy individuals, this review describes the different lung NK cell populations quantitatively, qualitatively, phenotypically, and functionally. Their identification, immunological diversity, and adaptive capacities are also addressed. For each of these elements, the impact of the mutual interactions of lung NK cells with environmental and microenvironmental factors are questioned in terms of functionality, competence, and adaptive capacities. As pulmonary diseases are major causes of morbidity and mortality worldwide, special attention is also given to the involvement of lung NK cells in various diseases, including infectious, inflammatory, autoimmune, and neoplastic lung diseases. In addition to providing a comprehensive overview of lung NK cell biology, this review also provides insight into the potential of NK cell immunotherapy and the development of targeted biologics.

Functional capacity of natural killer cells in HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients.

BACKGROUND: Natural killer (NK) cells are part of the innate immune system and provide surveillance against viruses and cancers. The ability of NK cells to kill virus-infected cells depends on the balance between the effects of inhibitory and activating NK cell receptors. This study aimed to investigate the phenotypic profile and the functional capacity of NK cells in the context of HTLV-1 infection. METHODS: This cross-sectional study sequentially recruited HTLV-1 infected individuals with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and asymptomatic HTLV-1 (AS) from the Integrated and Multidisciplinary HTLV Center in Salvador, Brazil. Blood samples from healthy blood donors served as controls. NK cell surface receptors (NKG2D, KIR2DL2/KIR2DL3, NKp30, NKG2A, NKp46, TIM-3 and PD-1), intracellular cytolytic (Granzyme B, perforin) and functional markers (CD107a for degranulation, IFN-γ) were assayed by flow cytometry in the presence or absence of standard K562 target cells. In addition, cytotoxicity assays were performed in the presence or absence of anti-NKp30. RESULTS: The frequency of NKp30+ NK cells was significantly decreased in HAM/TSP patients [58%, Interquartile Range (IQR) 30-61] compared to controls (73%, IQR 54-79, p = 0.04). The production of cytolytic (perforin, granzyme B) and functional markers (CD107a and IFN-γ) was higher in unstimulated NK cells from HAM/TSP and AS patients compared to controls. By contrast, stimulation with K562 target cells did not alter the frequency of CD107a+ NK cells in HAM/TSP subjects compared to the other groups. Blockage of the NKp30 receptor was shown to decrease cytotoxic activity (CD107a) and IFN-γ expression only in asymptomatic HTLV-1-infected individuals. CONCLUSIONS: NK cells from individuals with a diagnosis of HAM/TSP present decreased expression of the activating receptor NKp30, in addition to elevated degranulation activity that remained unaffected after blocking the NKp30 receptor.

Reverse Immunology Approach to Define a New HIV-gp41-Neutralizing Epitope.

The design of immunogens susceptible to elicit potent and broadly neutralizing antibodies against the human immunodeficiency virus type 1 (HIV-1) remains a veritable challenge in the course of vaccine development. Viral envelope proteins adopt different conformational states during the entry process, allowing the presentation of transient neutralizing epitopes. We focused on the highly conserved 3S motif of gp41, which is exposed to the surface envelope in its trimeric prefusion state. Vaccination with a W614A-modified 3S peptide induces in animals neutralizing anti-HIV-1 antibodies among which we selected clone F8. We used F8 as bait to select for W614A-3S phage-peptide mimics. Binding and molecular docking studies revealed that F8 interacts similarly with W614A-3S and a Mim_F8-1 mimotope, despite their lack of sequence homology, suggesting structural mimicry. Finally, vaccination of mice with the purified Mim_F8-1 phage elicited HIV-1-neutralizing antibodies that bound to the cognate W614A-3S motif. Collectively, our findings provide new insights into the molecular design of immunogens to elicit antibodies with neutralizing properties.

HLA-C-restricted viral epitopes are associated with an escape mechanism from KIR2DL2+ NK cells in Lassa virus infection.

BACKGROUND: Lassa virus (LASV) is the etiologic agent of an acute hemorrhagic fever endemic in West Africa. Natural killer (NK) cells control viral infections in part through the interaction between killer cell immunoglobulin-like receptors (KIRs) and their ligands. LASV infection is associated with defective immune responses, including inhibition of NK cell activity in the presence of MHC-class 1+-infected target cells. METHODS: We compared individual KIR and HLA-class 1 genotypes of 68 healthy volunteers to 51 patients infected with LASV in Sierra Leone, including 37 survivors and 14 fatalities. Next, potential HLA-C1, HLA-C2, and HLA-Bw4 binding epitopes were in silico screened among LASV nucleoprotein (NP) and envelope glycoprotein (GP). Selected 10-mer peptides were then tested in peptide-HLA stabilization, KIR binding and polyfunction assays. FINDINGS: LASV-infected patients were similar to healthy controls, except for the inhibitory KIR2DL2 gene. We found a specific increase in the HLA-C1:KIR2DL2 interaction in fatalities (10/11) as compared to survivors (12/19) and controls (19/29). We also identified that strong of NP and GP viral epitopes was only observed with HLA-C molecules, and associated with strong inhibition of degranulation in the presence of KIR2DL+ NK cells. This inhibitory effect significantly increased in the presence of the vGP420 variant, detected in 28.1% of LASV sequences. INTERPRETATION: Our finding suggests that presentation of specific LASV epitopes by HLA-C alleles to the inhibitory KIR2DL2 receptor on NK cells could potentially prevent the killing of infected cells and provides insights into the mechanisms by which LASV can escape NK-cell-mediated immune pressure.

Molecularly Imprinted Polymer Nanoparticles as Potential Synthetic Antibodies for Immunoprotection against HIV.

We describe the preparation and characterization of synthetic antibodies based on molecularly imprinted polymer nanoparticles (MIP-NPs) for the recognition and binding of the highly conserved and specific peptide motif SWSNKS (3S), an epitope of the envelope glycoprotein 41 (gp41) of human immunodeficiency virus type 1 (HIV-1). This motif is implicated in the decline of CD4+ T cells and leads to the deterioration of the immune system during HIV infection. Therefore, the development of MIP-NPs that can target and block the 3S peptide to prevent subsequent cascade interactions directed toward the killing of CD4+ T cells is of prime importance. Because most antibodies recognize their protein antigen via a conformational or structured epitope (as opposed to a linear epitope commonly used for molecular imprinting), we employed protein molecular modeling to design our template epitope so that it mimics the three-dimensional structure fold of 3S in gp41. The resulting template peptide corresponds to a cyclic structure composed of CGSWSNKSC, with the 3S motif well orientated for imprinting. MIP-NPs with a size of 65 nm were obtained by solid-phase synthesis and were water-soluble. They were prepared by a judicious combination of multiple functional monomers affording hydrogen bonding, ionic, π-π, and hydrophobic interactions, conferring high affinity and selectivity toward both the cyclic peptide and the whole gp41 protein. These results suggest that our MIPs could potentially be used for blocking the function of the 3S motif on the virus.

The Hard Way towards an Antibody-Based HIV-1 Env Vaccine: Lessons from Other Viruses.

Although effective antibody-based vaccines have been developed against multiple viruses, such approaches have so far failed for the human immunodeficiency virus type 1 (HIV-1). Despite the success of anti-retroviral therapy (ART) that has turned HIV-1 infection into a chronic disease and has reduced the number of new infections worldwide, a vaccine against HIV-1 is still urgently needed. We discuss here the major reasons for the failure of "classical" vaccine approaches, which are mostly due to the biological properties of the virus itself. HIV-1 has developed multiple mechanisms of immune escape, which also account for vaccine failure. So far, no vaccine candidate has been able to induce broadly neutralizing antibodies (bnAbs) against primary patient viruses from different clades. However, such antibodies were identified in a subset of patients during chronic infection and were shown to protect from infection in animal models and to reduce viremia in first clinical trials. Their detailed characterization has guided structure-based reverse vaccinology approaches to design better HIV-1 envelope (Env) immunogens. Furthermore, conserved Env epitopes have been identified, which are promising candidates in view of clinical applications. Together with new vector-based technologies, considerable progress has been achieved in recent years towards the development of an effective antibody-based HIV-1 vaccine.

Optimization of the EC26-2A4 Epitope in the gp41 Membrane Proximal External Region Targeted by Neutralizing Antibodies from an Elite Controller.

The analysis of patient derived HIV neutralizing antibodies (nAbs) and their target epitopes in the viral envelope (Env) protein provides important basic information for vaccine design. In this study we optimized an epitope, EC26-2A4, that is targeted by neutralizing antibodies from an elite controller (EC26) and localizes in the membrane-proximal external region from the gp41 transmembrane protein. Due to its overlap with the epitope of the first generation broadly neutralizing monoclonal Ab (mAb) 2F5 associated with autoreactivity, we first defined the minimal core epitope reacting with antibodies from EC26 plasma, but not with mAb 2F5. The optimized minimal epitope, EC26-2A4ΔM, was able to induce neutralizing antibodies in vaccinated mice. We further analyzed the frequency of antibodies against the EC26-2A4ΔM peptide in HIV-positive patient sera from a treated cohort and an untreated long-term nonprogressor (LTNP) cohort. Interestingly, 27% of the LTNP sera reacted with the peptide, whereas only 9% showed reactivity in the treated cohort. Although there was no association between the presence of antibodies against the EC26-2A4ΔM epitope and viral load or CD4 count in these patients, the CD4 nadir in the treated cohort was higher in patients positive for EC26-2A4ΔM antibodies, in particular in patients having such antibodies at an early and a late timepoint after infection.