Soluble SARS-CoV-2 RBD and human ACE2 peptidase domain produced in Drosophila S2 cells show functions evoking virus-cell interface.

The interaction between the receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 and the peptidase domain of the human angiotensin-converting enzyme 2 (ACE2) allows the first specific contact at the virus-cell interface making it the main target of neutralizing antibodies. Here, we show a unique and cost-effective protocol using Drosophila S2 cells to produce both RBD and soluble human ACE2 peptidase domain (shACE2) as thermostable proteins, purified via Strep-tag with yields >40 mg L-1 in a laboratory scale. Furthermore, we demonstrate its binding with KD values in the lower nanomolar range (independently of Strep-tag removal) and its capability to be blocked by serum antibodies in a competition ELISA with Strep-Tactin-HRP as a proof-of-concept. In addition, we assess the capacity of RBD to bind native dimeric ACE2 overexpressed in human cells and its antigen properties with specific serum antibodies. Finally, for completeness, we analyzed RBD microheterogeneity associated with glycosylation and negative charges, with negligible effect on binding either with antibodies or shACE2. Our system represents an accessible and reliable tool for designing in-house surrogate virus neutralization tests (sVNTs), enabling the rapid characterization of neutralizing humoral responses elicited against vaccines or infection, especially in the absence of facilities to conduct virus neutralization tests. Moreover, our biophysical and biochemical characterization of RBD and shACE2 produced in S2 cells lays the groundwork for adapting to different variants of concern (VOCs) to study humoral responses elicited against different VOCs and vaccine formulations.

What have we learned from a case of convalescent plasma treatment in a two-time kidney transplant recipient COVID-19 patient? A case report from the perspective of viral load evolution and immune response.

Coronavirus disease 2019 (COVID-19), an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, can have a wide range of clinical manifestations, ranging from asymptomatic disease to potentially life-threatening complications. Convalescent plasma therapy has been proposed as an effective alternative for the treatment of severe cases. The aim of this study was to follow a two-time renal transplant patient with severe COVID-19 treated with convalescent plasma over time from an immunologic and virologic perspective. A 42-year-old female patient, who was a two-time kidney transplant recipient, was hospitalized with COVID-19. Due to worsening respiratory symptoms, she was admitted to the intensive care unit, where she received two doses of convalescent plasma. We analyzed the dynamics of viral load in nasopharyngeal swab, saliva, and tracheal aspirate samples, before and after convalescent plasma transfusion. The levels of pro-inflammatory cytokines and antibody titers were also measured in serum samples. A significant decrease in viral load was observed after treatment in the saliva and nasopharyngeal swab samples, and a slight decrease was observed in tracheal aspirate samples. In addition, we found evidence of an increase in antibody titers after transfusion, accompanied by a decrease in the levels of several cytokines responsible for cytokine storm.

Low switched memory B cells are associated with no humoral response after SARS-CoV-2 vaccine boosters in kidney transplant recipients.

Introduction: The humoral response after SARS-CoV-2 vaccination and boosters in kidney transplant recipients (KTRs) is heterogeneous and depends on immunosuppression status. There is no validated immune measurement associated with serological response in clinical practice. Multicolor flow cytometric immunophenotyping could be useful for measuring immune response. This study aimed to study B- and T-cell compartments through Standardized EuroFlow PID Orientation after SARS-CoV-2 vaccination and their association with IgG SARS-CoV-2 seropositivity status after two doses or boosters. Methods: We conducted a multicenter prospective study to evaluate humoral response after SARS-CoV-2 vaccination in KTRs. Heterologous regimen: two doses of inactivated SARS-CoV-2 and two boosters of BNT162b2 mRNA (n=75). Homologous vaccination: two doses of BNT162b2 mRNA and one BNT162b2 mRNA booster (n=13). Booster doses were administrated to KTRs without taking into account their IgG SARS-CoV-2 seropositivity status. Peripheral blood samples were collected 30 days after the second dose and after the last heterologous or homologous booster. A standardized EuroFlow PID Orientation Tube (PIDOT) and a supervised automated analysis were used for immune monitoring cellular subsets after boosters. Results: A total of 88 KTRs were included and divided into three groups according to the time of the first detected IgG SARS-CoV-2 seropositivity: non-responders (NRs, n=23), booster responders (BRs, n=41), and two-dose responders (2DRs, n=24). The NR group was more frequent on mycophenolate than the responder groups (NRs, 96%; BRs, 80%; 2DRs, 42%; p=0.000). Switched memory B cells in the 2DR group were higher than those in the BR and NR groups (medians of 30, 17, and 10 cells/ul, respectively; p=0.017). Additionally, the absolute count of central memory/terminal memory CD8 T cells was higher in the 2DR group than in the BR and NR groups. (166, 98, and 93 cells/ul, respectively; p=0.041). The rest of the T-cell populations studied did not show a statistical difference. Conclusion: switched memory B cells and memory CD8 T-cell populations in peripheral blood were associated with the magnitude of the humoral response after SARS-CoV-2 vaccination. Boosters increased IgG anti-SARS-CoV-2 levels, CM/TM CD8 T cells, and switched MBCs in patients with seropositivity after two doses. Interestingly, no seropositivity after boosters was associated with the use of mycophenolate and a lower number of switched MBCs and CM/TM CD8 T cells in peripheral blood.

Humoral immune response characterization of heterologous prime-boost vaccination with CoronaVac and BNT162b2.

BACKGROUND: Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has proven to be a successful strategy for prevent severe infections. CoronaVac and BNT162b2 are the most used vaccines worldwide, but their use in heterologous vaccination schedules is still subjected to evaluation. METHODS: Fifty healthy individuals who received heterologous prime-boost vaccination with CoronaVac and BNT162b2 were enrolled in a post-vaccination serological follow-up longitudinal prospective study. We evaluated specific serum anti-receptor binding domain (RBD) IgG antibody levels, and their capacity to block RBD-ACE2 interaction with a surrogate neutralization assay. In 20 participants, we assessed antibody binding kinetics by surface plasmon resonance, and Fc-mediated functions by ADCC and ADCP reporter assays. RESULTS: Our baseline seronegative cohort, displayed seroconversion after two doses of CoronaVac and an important decrease in serum anti-RBD IgG antibodies levels 80 days post-second dose. These levels increased significantly early after the third dose with BNT162b2, but 73 days after the booster we found a new fall. Immunoglobulin functionalities showed a similar behavior. CONCLUSIONS: The heterologous prime-boost vaccination with CoronaVac and BNT162b2 generated an impressive increase in serum anti-RBD specific antibody levels followed by a drop. Nevertheless, these titers remained well above those found in individuals only vaccinated with CoronaVac in the same elapsed time. Serum IgG levels showed high correlation with antibody binding analysis, their capacity to block RBD-ACE2 interaction, and Fc-effectors mechanisms. Our work sheds light on the humoral immune response to heterologous vaccination with CoronaVac and BNT162b2, to define a post-vaccination correlate of protection against SARS-CoV-2 infection and to discuss the scheduling of future vaccine boosters in general population.

Kinetics of Bovine leukemia virus aspartic protease reveals its dimerization and conformational change.

The retropepsin (PR) of the Bovine leukemia virus (BLV) plays, as in other retroviruses, a crucial role in the transition from the non-infective viral particle to the infective virion by processing the polyprotein Gag. PR is expressed as an immature precursor associated with Gag, after an occasional -1 ribosomal frameshifting event. Self-hydrolysis of PR at specific N- and C-terminal sites releases the monomer that dimerizes giving rise to the active protease. We designed a strategy to express BLV PR in E. coli as a fusion protein with maltose binding protein, with a six-histidine tag at its N-terminal end, and bearing a tobacco etch virus protease hydrolysis site. This allowed us to obtain soluble and mature recombinant PR in relatively good yields, with exactly the same amino acid composition as the native protein. As PR presents relative promiscuity for the hydrolysis sites we designed four fluorogenic peptide substrates based on Förster resonance energy transfer (FRET) in order to characterize the activity of the recombinant enzyme. These substrates opened the way to perform kinetic studies, allowing us to characterize the dimer-monomer equilibrium. Furthermore, we obtained kinetic evidence for the existence of a conformational change that enables the interaction with the substrate. These results constitute a starting point for the elucidation of the kinetic properties of BLV-PR, and may be relevant not only to improve the chemical warfare against this virus but also to better understand other viral PRs.

Comparison of antibody response to SARS-CoV-2 after two doses of inactivated virus and BNT162b2 mRNA vaccines in kidney transplant.

BACKGROUND: Antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after mRNA or adenoviral vector-based vaccines is weak in kidney transplant (KT) patients. However, few studies have focused on humoral response after inactivated virus-based vaccines in KT. Here, we compare antibody response following vaccination with inactivated virus (CoronaVac®) and BNT162b2 mRNA. METHODS: A national multicentre cross-sectional study was conducted. The study group was composed of patients from all KT centres in Uruguay, vaccinated between 1 and 31 May 2021 (CoronaVac®, n = 245 and BNT162b2, n = 39). The control group was constituted of 82 healthy individuals. Participants had no prior confirmed coronavirus disease 2019 (COVID-19) test. Blood samples were collected between 30 and 40 days after the second dose. Serum-specific immunoglobulin G (IgG) antibodies against the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein were determined using the COVID-19 IgG QUANT ELISA Kit. RESULTS: Only 29% of KT recipients showed seroconversion (36.5% BNT162b2, 27.8% inactivated virus, P = 0.248) in comparison with 100% in healthy control with either vaccine. Antibody levels against RBD were higher with BNT162b mRNA than with inactivated virus [median (interquartile range) 173 (73-554) and 29 (11-70) binding antibody units (BAU)/mL, P < 0.034] in KT and 10 times lower than healthy control [inactivated virus: 308 (209-335) and BNT162b2: 2638 (2608-3808) BAU/mL, P < 0.034]. In multivariate analysis, variables associated with negative humoral response were age, triple immunosuppression, estimated glomerular filtration rate and time post-KT. CONCLUSION: Seroconversion was low in KT patients after vaccination with both platforms. Antibody levels against SARS-CoV-2 were lower with inactivated virus than BNT162b mRNA. These findings support the need for strategies to improve immunogenicity in KT recipients after two doses of either vaccine.

PD-1/PD-L1 blockade abrogates a dysfunctional innate-adaptive immune axis in critical ß-coronavirus disease.

Severe COVID-19 is associated with hyperinflammation and weak T cell responses against SARS-CoV-2. However, the links between those processes remain partially characterized. Moreover, whether and how therapeutically manipulating T cells may benefit patients are unknown. Our genetic and pharmacological evidence demonstrates that the ion channel TMEM176B inhibited inflammasome activation triggered by SARS-CoV-2 and SARS-CoV-2-related murine ß-coronavirus. Tmem176b-/- mice infected with murine ß-coronavirus developed inflammasome-dependent T cell dysfunction and critical disease, which was controlled by modulating dysfunctional T cells with PD-1 blockers. In critical COVID-19, inflammasome activation correlated with dysfunctional T cells and low monocytic TMEM176B expression, whereas PD-L1 blockade rescued T cell functionality. Here, we mechanistically link T cell dysfunction and inflammation, supporting a cancer immunotherapy to reinforce T cell immunity in critical ß-coronavirus disease.

CpG-ODN Shapes Alum Adjuvant Activity Signaling via MyD88 and IL-10.

Aluminum-containing adjuvants usually referred as Alum are considered as T helper type-2 (Th2) adjuvants, while agonists of toll-like receptors (TLRs) are viewed as adjuvants that favor Th1/Th17 immunity. Alum has been used in numerous vaccine formulations; however, its undesired pro-Th2 adjuvant activity constitutes a caveat for Alum-based vaccines. Combining Alum with TLR-dependent, pro-Th1/Th17 adjuvants might dampen the pro-Th2 activity and improve the effectiveness of vaccine formulations. Here, using the ovalbumin (OVA) model of allergic lung inflammation, we found that sensitization with the synthetic TLR9 agonist, which is composed of oligodeoxynucleotides containing CpG motifs adsorbed to Alum, inhibited the development of OVA-induced lung allergic Th2 responses without shifting toward a Th1 pattern. The conversion of T cell immunity from the polarized allergic Th2 response to a non-polarized form by sensitization with OVA/Alum/CpG was dependent on MyD88 signaling in myeloid cells. Notably, sensitization of IL-10-deficient mice with OVA/Alum/CpG resulted in the development of neutrophilic lung inflammation associated with IFNγ production. However, in IL-10/IL-12-deficient mice, it resulted in neutrophilic inflammation dominated by IL-17 production. We conclude that OVA/Alum/CpG sensitization signaling via MyD88 and IL-10 molecules results in non-polarized immunity. Conversely, OVA/Alum/CpG sensitization in presence of MyD88 but absence of IL-10 or IL-10/IL-12 molecules results, respectively, in neutrophilic inflammation associated with IFNγ or IL-17 production. Our work provides novel OVA models of lung inflammation and suggests that Alum/CpG-based formulations might be of potential use in anti-allergic or anti-infectious processes.