B cell analyses after SARS-CoV-2 mRNA third vaccination reveals a hybrid immunity like antibody response.

The continuous evolution of SARS-CoV-2 generated highly mutated variants able to escape natural and vaccine-induced primary immunity. The administration of a third mRNA vaccine dose induces a secondary response with increased protection. Here we investigate the longitudinal evolution of the neutralizing antibody response in four donors after three mRNA doses at single-cell level. We sorted 4100 spike protein specific memory B cells identifying 350 neutralizing antibodies. The third dose increases the antibody neutralization potency and breadth against all SARS-CoV-2 variants as observed with hybrid immunity. However, the B cell repertoire generating this response is different. The increases of neutralizing antibody responses is largely due to the expansion of B cell germlines poorly represented after two doses, and the reduction of germlines predominant after primary immunization. Our data show that different immunization regimens induce specific molecular signatures which should be considered while designing new vaccines and immunization strategies.

p66Shc Deficiency in Chronic Lymphocytic Leukemia Promotes Chemokine Receptor Expression Through the ROS-Dependent Inhibition of NF-κB.

The microenvironment of lymphoid organs is central to the pathogenesis of chronic lymphocytic leukemia (CLL). Within it, tumor cells find a favourable niche to escape immunosurveillance and acquire pro-survival signals. We have previously reported that a CLL-associated defect in the expression of the pro-apoptotic and pro-oxidant adaptor p66Shc leads to enhanced homing to and accumulation of leukemic cells in the lymphoid microenvironment. The p66Shc deficiency-related impairment in intracellular reactive oxygen species (ROS) production in CLL cells is causally associated to the enhanced expression of the chemokine receptors CCR2, CXCR3 and CCR7, that promote leukemic cell homing to both lymphoid and non-lymphoid organs, suggesting the implication of a ROS-modulated transcription factor(s). Here we show that the activity of the ROS-responsive p65 subunit of the transcription factor NF-κB was hampered in the CLL-derived cell line MEC-1 expressing a NF-κB-luciferase reporter following treatment with H2O2. Similar results were obtained when intracellular ROS were generated by expression of p66Shc, but not of a ROS-defective mutant, in MEC-1 cells. NF-κB activation was associated with increased expression of the chemokine receptors CCR2, CXCR3 and CCR7. Reconstitution of p66Shc in CLL cells normalized intracellular ROS and hampered NF-κB activation, which led to a decrease in the expression of these homing receptors. Our data provide direct evidence that the p66Shc-deficiency-related ROS depletion in CLL cells concurs to NF-κB hyperactivation and homing receptor overexpression, providing a mechanistic basis for the enhanced ability of these cells to accumulate in the pro-survival lymphoid niche.

Anatomy of Omicron BA.1 and BA.2 neutralizing antibodies in COVID-19 mRNA vaccinees.

SARS-CoV-2 vaccines, administered to billions of people worldwide, mitigate the effects of the COVID-19 pandemic, however little is known about the molecular basis of antibody cross-protection to emerging variants, such as Omicron BA.1, its sublineage BA.2, and other coronaviruses. To answer this question, 276 neutralizing monoclonal antibodies (nAbs), previously isolated from seronegative and seropositive donors vaccinated with BNT162b2 mRNA vaccine, were tested for neutralization against the Omicron BA.1 and BA.2 variants, and SARS-CoV-1 virus. Only 14.2, 19.9 and 4.0% of tested antibodies neutralize BA.1, BA.2, and SARS-CoV-1 respectively. These nAbs recognize mainly the SARS-CoV-2 receptor binding domain (RBD) and target Class 3 and Class 4 epitope regions on the SARS-CoV-2 spike protein. Interestingly, around 50% of BA.2 nAbs did not neutralize BA.1 and among these, several targeted the NTD. Cross-protective antibodies derive from a variety of germlines, the most frequents of which were the IGHV1-58;IGHJ3-1, IGHV2-5;IGHJ4-1 and IGHV1-69;IGHV4-1. Only 15.6, 20.3 and 7.8% of predominant gene-derived nAbs elicited against the original Wuhan virus cross-neutralize Omicron BA.1, BA.2 and SARS-CoV-1 respectively. Our data provide evidence, at molecular level, of the presence of cross-neutralizing antibodies induced by vaccination and map conserved epitopes on the S protein that can inform vaccine design.

Structural insights of a highly potent pan-neutralizing SARS-CoV-2 human monoclonal antibody.

As the coronavirus disease 2019 (COVID-19) pandemic continues, there is a strong need for highly potent monoclonal antibodies (mAbs) that are resistant against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoCs). Here, we evaluate the potency of the previously described mAb J08 against these variants using cell-based assays and delve into the molecular details of the binding interaction using cryoelectron microscopy (cryo-EM) and X-ray crystallography. We show that mAb J08 has low nanomolar affinity against most VoCs and binds high on the receptor binding domain (RBD) ridge, away from many VoC mutations. These findings further validate the phase II/III human clinical trial underway using mAb J08 as a monoclonal therapy.

Hybrid immunity improves B cells and antibodies against SARS-CoV-2 variants.

The emergence of SARS-CoV-2 variants is jeopardizing the effectiveness of current vaccines and limiting the application of monoclonal antibody-based therapy for COVID-19 (refs. 1,2). Here we analysed the memory B cells of five naive and five convalescent people vaccinated with the BNT162b2 mRNA vaccine to investigate the nature of the B cell and antibody response at the single-cell level. Almost 6,000 cells were sorted, over 3,000 cells produced monoclonal antibodies against the spike protein and more than 400 cells neutralized the original SARS-CoV-2 virus first identified in Wuhan, China. The B.1.351 (Beta) and B.1.1.248 (Gamma) variants escaped almost 70% of these antibodies, while a much smaller portion was impacted by the B.1.1.7 (Alpha) and B.1.617.2 (Delta) variants. The overall loss of neutralization was always significantly higher in the antibodies from naive people. In part, this was due to the IGHV2-5;IGHJ4-1 germline, which was found only in people who were convalescent and generated potent and broadly neutralizing antibodies. Our data suggest that people who are seropositive following infection or primary vaccination will produce antibodies with increased potency and breadth and will be able to better control emerging SARS-CoV-2 variants.

SARS-CoV-2 escape from a highly neutralizing COVID-19 convalescent plasma.

To investigate the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the immune population, we coincupi bated the authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for seven passages, but, after 45 d, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed, at day 80, by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom, South Africa, Brazil, and Japan of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed.

Extremely potent human monoclonal antibodies from COVID-19 convalescent patients.

Human monoclonal antibodies are safe, preventive, and therapeutic tools that can be rapidly developed to help restore the massive health and economic disruption caused by the coronavirus disease 2019 (COVID-19) pandemic. By single-cell sorting 4,277 SARS-CoV-2 spike protein-specific memory B cells from 14 COVID-19 survivors, 453 neutralizing antibodies were identified. The most potent neutralizing antibodies recognized the spike protein receptor-binding domain, followed in potency by antibodies that recognize the S1 domain, the spike protein trimer, and the S2 subunit. Only 1.4% of them neutralized the authentic virus with a potency of 1-10 ng/mL. The most potent monoclonal antibody, engineered to reduce the risk of antibody-dependent enhancement and prolong half-life, neutralized the authentic wild-type virus and emerging variants containing D614G, E484K, and N501Y substitutions. Prophylactic and therapeutic efficacy in the hamster model was observed at 0.25 and 4 mg/kg respectively in absence of Fc functions.

Enhanced IL-9 secretion by p66Shc-deficient CLL cells modulates the chemokine landscape of the stromal microenvironment.

The stromal microenvironment is central to chronic lymphocytic leukemia (CLL) pathogenesis. How leukemic cells condition the stroma to enhance its chemoattractant properties remains elusive. Here, we show that mouse and human CLL cells promote the contact-independent stromal expression of homing chemokines. This function was strongly enhanced in leukemic cells from Eµ-TCL1 mice lacking the pro-oxidant p66Shc adaptor, which develop an aggressive disease with organ infiltration. We identified interleukin-9 (IL-9) as the soluble factor, negatively modulated by p66Shc, that is responsible for the chemokine-elevating activity of leukemic cells on stromal cells. IL-9 blockade in Eµ-TCL1/p66Shc-/- mice resulted in a decrease in the nodal expression of homing chemokines, which correlated with decreased leukemic cell invasiveness. IL-9 levels were found to correlate inversely with residual p66Shc in p66Shc-deficient human CLL cells (n = 52 patients). p66Shc reconstitution in CLL cells normalized IL-9 expression and neutralized their chemokine-elevating activity. Notably, high IL-9 expression in CLL cells directly correlates with lymphadenopathy, liver infiltration, disease severity, and overall survival, emerging as an independent predictor of disease outcome. Our results demonstrate that IL-9 modulates the chemokine landscape in the stroma and that p66Shc, by regulating IL-9 expression, fine tunes the ability of leukemic cells to shape the microenvironment, thereby contributing to CLL pathogenesis.

Optimization of Organotypic Cultures of Mouse Spleen for Staining and Functional Assays.

By preserving cell viability and three-dimensional localization, organotypic culture stands out among the newest frontiers of cell culture. It has been successfully employed for the study of diseases among which neoplasias, where tumoral cells take advantage of the surrounding stroma to promote their own proliferation and survival. Organotypic culture acquires major importance in the context of the immune system, whose cells cross-talk in a complex and dynamic fashion to elicit productive responses. However, organotypic culture has been as yet poorly developed for and applied to primary and secondary lymphoid organs. Here we describe in detail the development of a protocol suitable for the efficient cutting of mouse spleen, which overcomes technical difficulties related to the peculiar organ texture, and for optimized organotypic culture of spleen slices. Moreover, we used microscopy, immunofluorescence, flow cytometry, and qRT-PCR to demonstrate that the majority of cells residing in spleen slices remain alive and maintain their original location in the organ architecture for several days after cutting. The development of this protocol represents a significant technical improvement in the study of the lymphoid microenvironment in both physiological and pathological conditions involving the immune system.

SARS-CoV-2 escape in vitro from a highly neutralizing COVID-19 convalescent plasma.

To investigate the evolution of SARS-CoV-2 in the immune population, we co-incubated authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for 7 passages, but after 45 days, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed at day 80 by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom and South Africa of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed. ONE SENTENCE SUMMARY: Three mutations allowed SARS-CoV-2 to evade the polyclonal antibody response of a highly neutralizing COVID-19 convalescent plasma.

p66Shc deficiency in the Eµ-TCL1 mouse model of chronic lymphocytic leukemia enhances leukemogenesis by altering the chemokine receptor landscape.

The Shc family adaptor p66Shc acts as a negative regulator of proliferative and survival signals triggered by the B-cell receptor and, by enhancing the production of reactive oxygen species, promotes oxidative stress-dependent apoptosis. Additionally, p66Shc controls the expression and function of chemokine receptors that regulate lymphocyte traffic. Chronic lymphocytic leukemia cells have a p66Shc expression defect which contributes to their extended survival and correlates with poor prognosis. We analyzed the impact of p66Shc ablation on disease severity and progression in the Eµ-TCL1 mouse model of chronic lymphocytic leukemia. We showed that Eµ-TCL1/p66Shc-/- mice developed an aggressive disease that had an earlier onset, occurred at a higher incidence and led to earlier death compared to that in Eµ-TCL1 mice. Eµ-TCL1/p66Shc-/- mice displayed substantial leukemic cell accumulation in both nodal and extranodal sites. The target organ selectivity correlated with upregulation of chemokine receptors whose ligands are expressed therein. This also applied to chronic lymphocytic leukemia cells, where chemokine receptor expression and extent of organ infiltration were found to correlate inversely with these cells' level of p66Shc expression. p66Shc expression declined with disease progression in Eµ-TCL1 mice and could be restored by treatment with the Bruton tyrosine kinase inhibitor ibrutinib. Our results highlight p66Shc deficiency as an important factor in the progression and severity of chronic lymphocytic leukemia and underscore p66Shc expression as a relevant therapeutic target.

p66Shc deficiency enhances CXCR4 and CCR7 recycling in CLL B cells by facilitating their dephosphorylation-dependent release from ß-arrestin at early endosomes.

Neoplastic cell traffic abnormalities are central to the pathogenesis of chronic lymphocytic leukemia (CLL). Enhanced CXC chemokine receptor-4 (CXCR4) and chemokine receptor-7 (CCR7) recycling contributes to the elevated surface levels of these receptors on CLL cells. Here we have addressed the role of p66Shc, a member of the Shc family of protein adaptors the expression of which is defective in CLL cells, in CXCR4/CCR7 recycling. p66Shc reconstitution in CLL cells reduced CXCR4/CCR7 recycling, lowering their surface levels and attenuating B-cell chemotaxis, due to their accumulation in Rab5+ endosomes as serine-phosphoproteins bound to ß-arrestin. This results from the ability of p66Shc to inhibit Ca2+ and PP2B-dependent CXCR4/CCR7 dephosphorylation and ß-arrestin release. We also show that ibrutinib, a Btk inhibitor that promotes leukemic cell mobilization from lymphoid organs, reverses the CXCR4/CCR7 recycling abnormalities in CLL cells by increasing p66Shc expression. These results, identifying p66Shc as a regulator of CXCR4/CCR7 recycling in B cells, underscore the relevance of its deficiency to CLL pathogenesis and provide new clues to the mechanisms underlying the therapeutic effects of ibrutinib.