Altered somatic hypermutation patterns in COVID-19 patients classifies disease severity.

Introduction: The success of the human body in fighting SARS-CoV2 infection relies on lymphocytes and their antigen receptors. Identifying and characterizing clinically relevant receptors is of utmost importance. Methods: We report here the application of a machine learning approach, utilizing B cell receptor repertoire sequencing data from severely and mildly infected individuals with SARS-CoV2 compared with uninfected controls. Results: In contrast to previous studies, our approach successfully stratifies non-infected from infected individuals, as well as disease level of severity. The features that drive this classification are based on somatic hypermutation patterns, and point to alterations in the somatic hypermutation process in COVID-19 patients. Discussion: These features may be used to build and adapt therapeutic strategies to COVID-19, in particular to quantitatively assess potential diagnostic and therapeutic antibodies. These results constitute a proof of concept for future epidemiological challenges.

In silico and in vitro evaluation of imatinib as an inhibitor for SARS-CoV-2.

The rapid geographic expansion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the infectious agent of Coronavirus Disease 2019 (COVID-19) pandemic, poses an immediate need for potent drugs. Enveloped viruses infect the host cell by cellular membrane fusion, a crucial mechanism required for virus replication. The SARS-CoV-2 spike glycoprotein, due to its primary interaction with the human angiotensin-converting enzyme 2 (ACE2) cell-surface receptor, is considered a potential target for drug development. In this study, around 5,800 molecules were virtually screened using molecular docking. Five molecules were selected for in vitro experiments from those that reported docking scores lower than -6 kcal/mol. Imatinib, a Bcr-Abl tyrosine kinase inhibitor, showed maximum antiviral activity in Vero cells. We further investigated the interaction of imatinib, a compound under clinical trials for the treatment of COVID-19, with SARS-CoV-2 RBD, using in silico methods. Molecular dynamics simulations verified that imatinib interacts with RBD residues that are critical for ACE2 binding. This study also provides significant molecular insights on potential repurposable small-molecule drugs and chemical scaffolds for the development of novel drugs targeting the SARS-CoV-2 spike RBD.Communicated by Ramaswamy H. Sarma.

B-Cell Receptor Features and Database Establishment in Recovered COVID-19 Patients by Combining 5'-RACE with PacBio Sequencing.

BACKGROUND: Antibodies induced by viral infection can not only prevent subsequent virus infection, but can also mediate pathological injury following infection. Therefore, understanding the B-cell receptor (BCR) repertoire of either specific neutralizing or pathological antibodies from patients convalescing from Coronavirus disease 2019 (COVID-19) infection is of benefit for the preparation of therapeutic or preventive antibodies, and may provide insight into the mechanisms of COVID-19 pathological injury. METHODS: In this study, we used a molecular approach of combining 5' Rapid Amplification of cDNA Ends (5'-RACE) with PacBio sequencing to analyze the BCR repertoire of all 5 IgH and 2 IgL genes in B-cells harvested from 35 convalescent patients after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. RESULTS: We observed numerous BCR clonotypes within most COVID-19 patients, but not in healthy controls, which validates the association of the disease with a prototypical immune response. In addition, many clonotypes were found to be frequently shared between different patients or different classes of antibodies. CONCLUSIONS: These convergent clonotypes provide a resource to identify potential therapeutic/prophylactic antibodies, or identify antibodies associated with pathological effects following infection with SARS-CoV-2.

A SARS-CoV-2 neutralizing antibody discovery by single cell sequencing and molecular modeling.

Although vaccines have been developed, mutations of SARS-CoV-2, especially the dominant B.1.617.2 (delta) and B.1.529 (omicron) strains with more than 30 mutations on their spike protein, have caused a significant decline in prophylaxis, calling for the need for drug improvement. Antibodies are drugs preferentially used in infectious diseases and are easy to get from immunized organisms. The current study combined molecular modeling and single memory B cell sequencing to assess candidate sequences before experiments, providing a strategy for the fabrication of SARS-CoV-2 neutralizing antibodies. A total of 128 sequences were obtained after sequencing 196 memory B cells, and 42 sequences were left after merging extremely similar ones and discarding incomplete ones, followed by homology modeling of the antibody variable region. Thirteen candidate sequences were expressed, of which three were tested positive for receptor binding domain recognition but only one was confirmed as having broad neutralization against several SARS-CoV-2 variants. The current study successfully obtained a SARS-CoV-2 antibody with broad neutralizing abilities and provided a strategy for antibody development in emerging infectious diseases using single memory B cell BCR sequencing and computer assistance in antibody fabrication.

COVID-19 in patients with chronic myeloid leukaemia on tyrosine kinase inhibitor therapy: a Honduran observational study.

Introduction: In the earliest cases of COVID-19, a higher percentage of severe and fatal cases was observed in patients with cancer, including those with haematological malignancies. However, patients with chronic myeloid leukaemia (CML) had better prognoses, suggesting that tyrosine kinase inhibitors (TKIs) may have a therapeutic effect against SARS-CoV-2. This study describes the clinical and epidemiological characteristics of patients with CML receiving the TKIs tested for SARS-CoV-2 in Tegucigalpa, Honduras. Methodology: An Analytical cross-sectional study was conducted. The sample included patients with Philadelphia chromosome-positive (Ph+) CML, who had been tested at least once for COVID-19 at the Emma Romero de Callejas Cancer Centre (CCERC). Sociodemographic and clinical variables were both analysed. Epi Info 7.2.4.0 and Stata/MP 16.0 were used to collect and analyse data. The COVID-19 positivity percentage and the association between severity and the TKI used were determined using Fisher's exact test and odds ratio (OR). Data were gathered from clinical records with approval of CCERC institutional management. Results: One hundred and forty-nine patients with Ph+ CML were included; 20.1% were COVID-19-positive; 56% were male; mean age was 46 years; 81% were receiving imatinib, with a mean treatment duration of 6 years; 55% achieved a BCR -ABL molecular response ≤ 0.1% (IS). Twenty-one percent had comorbidities. COVID-19 was asymptomatic in 38.7% of patients, mild in 35.5% and severe in 9.7%. One patient died, a fatality rate of 3.2%. No statistical association was found between disease severity and treatment with imatinib versus second-line TKI (OR: 0.833, p: 0.8493, 95% CI: 0.098-10.998). Conclusion: Despite high COVID-19 positivity in CML when compared with the literature, this study found a lower fatality rate. The type of TKI used or molecular response at the time of infection was not associated with case severity. Determining the effectiveness of imatinib or other TKIs as a COVID-19 treatment requires randomised clinical trials.

A Rapid Method for Detection of Antigen-Specific B Cells.

The global SARS-CoV-2 pandemic has united the efforts of many scientists all over the world to develop wet-lab techniques and computational approaches aimed at the identification of antigen-specific T and B cells. The latter provide specific humoral immunity that is essential for the survival of COVID-19 patients, and vaccine development has essentially been based on these cells. Here, we implemented an approach that integrates the sorting of antigen-specific B cells and B-cell receptor mRNA sequencing (BCR-seq), followed by computational analysis. This rapid and cost-efficient method allowed us to identify antigen-specific B cells in the peripheral blood of patients with severe COVID-19 disease. Subsequently, specific BCRs were extracted, cloned, and produced as full antibodies. We confirmed their reactivity toward the spike RBD domain. Such an approach can be effective for the monitoring and identification of B cells participating in an individual immune response.

Characterizing the cellular and molecular variabilities of peripheral immune cells in healthy recipients of BBIBP-CorV inactivated SARS-CoV-2 vaccine by single-cell RNA sequencing.

Over 3 billion doses of inactivated vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been administered globally. However, our understanding of the immune cell functional transcription and T cell receptor (TCR)/B cell receptor (BCR) repertoire dynamics following inactivated SARS-CoV-2 vaccination remains poorly understood. Here, we performed single-cell RNA and TCR/BCR sequencing on peripheral blood mononuclear cells at four time points after immunization with the inactivated SARS-CoV-2 vaccine BBIBP-CorV. Our analysis revealed an enrichment of monocytes, central memory CD4+ T cells, type 2 helper T cells and memory B cells following vaccination. Single-cell TCR-seq and RNA-seq comminating analysis identified a clonal expansion of CD4+ T cells (but not CD8+ T cells) following a booster vaccination that corresponded to a decrease in the TCR diversity of central memory CD4+ T cells and type 2 helper T cells. Importantly, these TCR repertoire changes and CD4+ T cell differentiation were correlated with the biased VJ gene usage of BCR and the antibody-producing function of B cells post-vaccination. Finally, we compared the functional transcription and repertoire dynamics in immune cells elicited by vaccination and SARS-CoV-2 infection to explore the immune responses under different stimuli. Our data provide novel molecular and cellular evidence for the CD4+ T cell-dependent antibody response induced by inactivated vaccine BBIBP-CorV. This information is urgently needed to develop new prevention and control strategies for SARS-CoV-2 infection. (ClinicalTrials.gov Identifier: NCT04871932).

Proof-of-Concept Analysis of B Cell Receptor Repertoire in COVID-19 Patients Undergoing ECMO by Single-Cell V(D)J and Gene Expression Sequencing.

SARS-CoV-2, which causes COVID-19, has altered human activities all over the world and has become a global hazard to public health. Despite considerable advancements in pandemic containment techniques, in which vaccination played a key role, COVID-19 remains a global threat, particularly for frail patients and unvaccinated individuals, who may be more susceptible to developing ARDS. Several studies reported that patients with COVID-19-related ARDS who were treated with ECMO had a similar survival rate to those with COVID-19-unrelated ARDS. In order to shed light on the potential mechanisms underlying the COVID-19 infection, we conducted this proof-of-concept study using single-cell V(D)J and gene expression sequencing of B cells to examine the dynamic changes in the transcriptomic BCR repertoire present in patients with COVID-19 at various stages. We compared a recovered and a deceased COVID-19 patient supported by ECMO with one COVID-19-recovered patient who did not receive ECMO treatment and one healthy subject who had never been infected previously. Our analysis revealed a downregulation of FXYD, HLA-DRB1, and RPS20 in memory B cells; MTATP8 and HLA-DQA1 in naïve cells; RPS4Y1 in activated B cells; and IGHV3-73 in plasma cells in COVID-19 patients. We further described an increased ratio of IgA + IgG to IgD + IgM, suggestive of an intensive memory antibody response, in the COVID ECMO D patient. Finally, we assessed a V(D)J rearrangement of heavy chain IgHV3, IGHJ4, and IGHD3/IGHD2 families in COVID-19 patients regardless of the severity of the disease.

Unexpected CD5+ B Cell Lymphocytosis during SARS-CoV-2 Infection: Relevance for the Pathophysiology of Chronic Lymphocytic Leukemia.

Recently, cases of fortuitous discovery of Chronic Lymphocytic Leukemia (CLL) during hospitalization for Coronavirus disease (COVID-19) have been reported. These patients did not show a monoclonal B cell expansion before COVID-19 but were diagnosed with CLL upon a sudden lymphocytosis that occurred during hospitalization. The (hyper)lymphocytosis during COVID-19 was also described in patients with overt CLL disease. Contextually, lymphocytosis is an unexpected phenomenon since it is an uncommon feature in the COVID-19 patient population, who rather tend to experience lymphopenia. Thus, lymphocytosis that arises during COVID-19 infection is a thought-provoking behavior, strikingly in contrast with that observed in non-CLL individuals. Herein, we speculate about the possible mechanisms involved with the observed phenomenon. Many of the plausible explanations might have an adverse impact on these CLL patients and further clinical and laboratory investigations might be desirable.

RAMIHM generates fully human monoclonal antibodies by rapid mRNA immunization of humanized mice and BCR-seq.

As a clinical vaccine, lipid nanoparticle (LNP) mRNA has demonstrated potent and broad antibody responses, leading to speculation about its potential for antibody discovery. Here, we developed RAMIHM, a highly efficient strategy for developing fully human monoclonal antibodies that employs rapid mRNA immunization of humanized mice followed by single B cell sequencing (scBCR-seq). We immunized humanized transgenic mice with RAMIHM and generated 15 top-ranked clones from peripheral blood, plasma B, and memory B cell populations, demonstrating a high rate of antigen-specificity (93.3%). Two Omicron-specific neutralizing antibodies with high potency and one broad-spectrum neutralizing antibody were discovered. Furthermore, we extended the application of RAMIHM to cancer immunotherapy targets, including a single transmembrane protein CD22 and a multi-transmembrane G protein-coupled receptor target, GPRC5D, which is difficult for traditional protein immunization methods. RAMIHM-scBCR-seq is a broadly applicable platform for the rapid and efficient development of fully human monoclonal antibodies against an assortment of targets.

Estudo De Descontinuacao De Fase I/Ii De Imatinibe Apos Pioglitazona (Edi-Pio) Em Pacientes Com Leucemia Mieloide Cronica Em Resposta Molecular Profunda Sustentada - Analise De 5 Anos De Seguimento

Objetivos: avaliar a eficacia e seguranca da pioglitazona utilizada concomitante ao imatinibe antes da descontinuacao do tratamento. Metodos: EDI-PIO (do Estudo de Descontinuacao de Imatinibe em Portugues apos Pioglitazona) e um estudo prospectivo, aberto, de braco unico, fase I/II de descontinuacao. Criterios de inclusao: LMC em fase cronica, tratada com imatinibe por pelo menos 3 anos, com MR 4.5 (Escala Internacional) por 2 anos. Os pacientes receberam pioglitazona 30 mg/dia por via oral por tres meses antes da descontinuacao do imatinibe. Apos a descontinuacao, os niveis de BCR-ABL foram medidos por PCR quantitativo em tempo real mensalmente por 12 meses, a cada dois meses no segundo ano e depois a cada tres meses. O tratamento com imatinibe foi reiniciado na recidiva molecular (amostra unica com valor de PCR >0,1% ou duas amostras consecutivas >0,01%). A sobrevida livre de terapia (SLT) foi calculada a partir da descontinuacao do imatinibe ate a recaida molecular, progressao ou morte por causas relacionadas a LMC. A sobrevida global (SG) foi calculada a partir da descontinuacao do imatinibe ate a ultima consulta ou data de obito por qualquer causa. Registro: Clinicaltrials.gov, NCT02852486. Resultados: Entre junho/2016 e janeiro/2019, foram incluidos 32 pacientes com LMC, com idade mediana de 54 anos (29-77), tratados com imatinibe por um tempo mediano de 9,5 anos (3-16). A duracao mediana de MR4 e MR4.5 foi de 106 e 93 meses, respectivamente. A data de corte desta analise foi 1de julho de 2022. Um paciente deixou o estudo antes da descontinuacao do imatinibe e nao foi analisado para SLT e SG. Nao houve eventos adversos de grau 3 ou 4 relacionados a pioglitazona. A mediana de seguimento dos 31 pacientes que descontinuaram a terapia foi de 61 meses (37-69). 15/31(48%) pacientes apresentaram sintomas relacionados a sindrome de retirada da medicacao. Doze pacientes apresentaram recidiva molecular apos uma mediana de 5 meses (2-30). Nove recaidas ocorreram nos primeiros seis meses e tres em 7, 13 e 30 meses apos a interrupcao do imatinibe. Todos os pacientes com recaida alcancaram resposta molecular maior numa mediana de 3 meses (1,8-4,1). Um paciente desenvolveu um adenocarcinoma do canal anal no terceiro ano apos a descontinuacao e foi tratado com cirurgia e quimioterapia. A SLT foi 71%, 67%, 61% e 61% aos 6,12, 30 e 60 meses, respectivamente. A SG aos 60 meses foi de 95% (IC 95%: 85-100%). Houve 5 casos de COVID-19 entre os 19 pacientes em descontinuacao (26%) e dois suspeitos. Quatro casos foram leves e um paciente em MR4.5 morreu devido a COVID-19 grave. O escore de Sokal baixo risco e a duracao do MR4.5 foram fatores significativos para SLT prolongada (P = 0,032 e 0,012, respectivamente). Discussao: A descontinuacao do tratamento com ITQ na LMC e bem-sucedida em aproximadamente 40-60% dos pacientes que atingem uma resposta molecular profunda e sustentada. Recaidas podem ocorrer devido a persistencia de celulas-tronco leucemicas quiescentes (CTL). A pioglitazona, um medicamento usado no tratamento do diabetes, e um agonista de PPAR gama e reduz a atividade de STAT5, e seus alvos a jusante, HIF2alpha e CITED2, principais guardioes das CTL quiescente. As CLT residuais podem ser gradualmente purgadas dos nichos da medula ossea pela pioglitazona, sendo o racional para a associacao da pioglitazona nesse estudo. Conclusoes: a combinacao de pioglitazona e imatinibe foi segura, sem eventos adversos graves. O seguimento a longo prazo de 5 anos demonstrou respostas moleculares duradouras e estaveis. Financiamento: CAPES (bolsa de mestrado ABPL). Copyright © 2022

Ponatinibe Para Tratamento De Lla Ph+ Recaida: Relato De Caso E Revisao Da Literatura

Objetivos: Descrever caso de paciente com leucemia linfoblastica aguda Ph+, nao elegivel a transplante alogenico de medula ossea, em uso de ponatinibe como terapia de resgate. Material e metodos: Avaliacao de prontuario e pesquisa em base de dados. Resultados: Paciente do sexo feminino, 65 anos de idade, com antecedente de obesidade, asma e nefrolitiase, em investigacao de plaquetopenia, tendo feito uso de prednisona sem incremento da contagem plaquetaria. Avaliacao medular evidenciou leucemia linfoblastica aguda B (LLA-B) com cariotipo normal. Iniciada quimioterapia de inducao com protocolo GRAAL e realizada coleta de BCR-ABL de sangue periferico, que resultou positivo (p190). Diante desse dado, e devido a pandemia de covid-19, com o objetivo de minimizar toxicidades e evitar internacao hospitalar, o tratamento foi modificado para o protocolo GIMEMA LAL, baseado em dasatinibe 140 mg/dia e prednisona. A paciente evoluiu com resposta citomorfologica completa, doenca residual mensuravel negativa e BCR-ABL indetectavel apos 3 meses de tratamento. Nesse periodo, apresentou multiplas intercorrencias - diverticulite aguda, colite pseudomembranosa e artrite (posteriormente diagnosticada com artrite reumatoide). Enviada para avaliacao com equipe de transplante de medula ossea, sendo considerada inelegivel naquele momento devido as comorbidades e decidido por manter inibidor de tirosino quinase. Evoluiu com recaida apos sete meses de uso de dasatinibe, sendo submetida a reinducao com inotuzumabe-mini-Hyper-CVAD. Apos o primeiro ciclo, alcancou resposta citomorfologica completa, porem evoluiu com cefaleia persistente e perda visual a direita. Evidenciada hemorragia intracraniana, sem indicacao de abordagem neurocirurgica. Devido a essa intercorrencia, foi considerada definitivamente inelegivel ao transplante alogenico. Nesse interim, foi pesquisada e detectada a mutacao t315i. Diante da nova informacao e do evento adverso grave, iniciou uso de ponatinibe 30 mg/dia em monoterapia. A paciente segue em uso do medicamento, mantendo resposta citomorfologica completa, com doenca residual mensuravel positiva (0,36% de blastos residuais) e BCR-ABL detectavel apos dois meses de tratamento. Apresenta boa tolerancia ao tratamento, sem toxicidade hematologica ou cardiovascular. Apresentou episodio de diverticulite aguda, sem necessidade de internacao hospitalar. Discussao: Historicamente, LLA-B Ph+ e considerada uma doenca de mau prognostico, com taxas de sobrevida em longo prazo inferiores a 20% na era anterior aos inibidores de tirosino quinase (ITK). Entretanto, com o advento dessa terapia-alvo, os desfechos de pacientes com LLA-B Ph+ tem se equiparado ou mesmo ultrapassado os daqueles sem essa alteracao molecular. No presente caso, a paciente atingiu resposta molecular completa com uso de terapia a base de corticoide e ITK de segunda geracao, tendo evoluido com recaida associada a mutacao T315i. Segue em resposta citomorfologica apos dois meses de ponatinibe, sendo necessario maior periodo de acompanhamento para avaliar a profundidade da melhor resposta, bem como sua duracao. Conclusao: Ponatinibe e opcao terapeutica eficaz e com boa tolerancia para paciente com LLA-B Ph+ (t315i) inelegivel a quimioterapia intensiva. Copyright © 2022

Leucemia Aguda De Fenotipo Misto - Apresentacao Inicial Atipica - Um Relato De Caso

Introducao: Com desafiador tratamento e de diagnostico criterioso, a leucemia aguda de fenotipo misto (LAFM) e uma entidade rara dentro do espectro das leucemias agudas. Requer a presenca imunofenotipica de marcadores de linhagem B (CD19, CD22, CD79a), T (CD3) em conjunto com a linhagem mieloide (mieloperoxidase e diferenciacao monocitica - CD11c, CD14, CD64 ou lisozima). Relato de caso: Paciente masculino, 30 anos, obeso e diabetico tipo 2, hipertrigliceridemia, inicia com febre (38C), dor abdominal em hipocondrio direito e fadiga. Com dois dias de sintomas procura atendimento sendo liberado com sintomaticos. No quarto dia de sintomas houve piora da febre (39degreeC) e da dor, surgindo maculas hiperemiadas pruriginosas pelo corpo, ictericia e coluria. Retornou ao hospital de sua cidade sendo prescrito azitromicina e liberado com suspeita de influenza. No sexto dia de sintomas notou piora da ictericia, procurando, novamente, atendimento. Encaminhado, entao ao servico de referencia da regiao. Interna inicialmente na equipe da gastroenterologia como suspeita de hepatite viral. Na chegada: Hb 14,9, leucocitos 6140 com 2793 neutrofilos, 442 monocitos e 2812 linfocitos, 53 mil plaquetas;AST 57, ALT 733, hiperbilirrubinemia as custas de bilirrubina direta. Com todos os marcadores virais negativos, prosseguiu a investigacao de hepatite. No dia em que realiza ressonancia magnetica, que indicava processo infiltrativo/inflamatorio em figado e rim esquerdo, alem de testar positivo para COVID-19, ha evolucao no hemograma: Hb 10,7, leucocitos 7450 com 1192 blastos, 60 neutrofilos, 2012 monocitos e 4187 linfocitos, 23 mil plaquetas. Com o aparecimento de blastos, piora dos niveis de bilirrubinas e das lesoes de pele, foi realizado imunofenotipagem de sangue periferico que indicava leucemia monocitica aguda. Transferido a equipe da hematologia, sendo realizada biopsia de medula e iniciado protocolo 7 + 3 com substituicao das antraciclinas em falta no mercado por doxorrubicina 45 mg/m2. No terceiro dia da inducao, foi liberado o resultado da imunofenotipagem que confirmava o diagnostico de leucemia aguda de fenotipo misto B/mieloide, marcando CD19, CD22 e CD79a, com diferenciacao monocitica (CD14 e CD64). Cariotipo nao houve crescimento e PCR BCR/ABL negativo. Optado por seguir tratamento com 7 + 3, apresentando medula no D14 aplasiada e medula no D28 com doenca residual minima (DRM) negativa. Realiza tres consolidacoes com altas doses de citarabina (3g/m2). Paciente sustenta DRM negativa, estando em remissao completa. Iniciado manutencao com vincristina, mercaptopurina, metotrexato e prednisona. Aguarda transplante de celulas tronco hematopoieticas (TCTH). Discussao: Com o diagnostico de LAFM, o tratamento requer o maior numero de quimioterapicos, sendo sugerido o uso de protocolos para leucemia linfoblastica aguda. Como ja havia sido instituido o tratamento com doxorrubicina e citarabina, foi optado por seguir protocolo e, na manutencao da remissao completa, terminar as consolidacoes e iniciar a manutencao prevista pelo protocolo HyperCVAD. Devido a ser uma leucemia de alto risco, a realizacao do TCTH e necessaria e, neste caso relatado, a manutencao sera mantida ate a realizacao do transplante. Conclusao: Contudo, por se tratar de doenca rara e com poucos estudos publicados, requer compartilhamento de conhecimentos e condutas para melhora da abordagem. Copyright © 2022

COVID-19 en pacientes con Leucemia Mieloide Cronica en tratamiento con inhibidores de la tirosina quinasa, un estudio observacional hondureno

Background In the first COVID-19 series, a larger percentage of severe and lethal cases were seen among oncologic patients, including those with malignant hematologic diseases. Nonetheless, patients with CML showed more favorable prognosis, posing that tyrosine-kinase inhibitors might have a therapeutic effect against SARS CoV2. In this study a clinical and epidemiological description was performed in patients with LMC on TKIs that were tested for COVD-19 in CCERC in Tegucigalpa. Methods Cross sectional analytic study. Sample were patients with CML with ph+ with at least one diagnostic test performed for COVID-19 in CCERC. Sociodemographic and clinical variables were analyzed. Data collection and statistical analysis was done in epiinfo 7.2.4.0 and STATA/MP 16.0. COVID-19 percent positivity was determined along with association between severity and TKI used and molecular response through Fisher exact test and OR. Data was extracted solely from clinical records. Approval was given by board of directors from the CCERC. Results 149 patients with ph+ CML were included;20.1% were positive for COVID-19, 56% were male, mean age was 46 years, 81% receiving imatinib, mean time of treatment of 6 years;55% in molecular response of BCR-ABL & LE;0.1% (IS). 21% had comorbidities. 38.7% had an asymptomatic COVID-19 disease, 35.5% had mild disease and 9.7% were severe, one patient died;lethality was 3.2%. No statistical significance was found between COVID-19 severity and treatment with imatinib vs other TKI. Conclusions Despite a high positivity rate of COVID-19 in CML patients compared to current evidence, a low lethality rate was found. The TKI used or the molecular response of CML were not associated with disease severity. The efficacy of TKIs for treatment of COVID-19 must be stablished by randomized-controlled trials.

Profiling the B cell immune response elicited by vaccination against the respiratory virus SARS-CoV-2.

B cells play a fundamental role in host defenses against viral infections. Profiling the B cell response elicited by SARS-CoV-2 vaccination, including the generation and persistence of antigen-specific memory B cells, is essential for improving the knowledge of vaccine immune responsiveness, beyond the antibody response. mRNA-based vaccines have shown to induce a robust class-switched memory B cell response that persists overtime and is boosted by further vaccine administration, suggesting that memory B cells are critical in driving a recall response upon re-exposure to SARS-CoV-2 antigens. Here, we focus on the role of the B cell response in the context of SARS-CoV-2 vaccination, offering an overview of the different technologies that can be used to identify spike-specific B cells, characterize their phenotype using machine learning approaches, measure their capacity to reactivate following antigen encounter, and tracking the maturation of the B cell receptor antigenic affinity.

IgTreeZ, A Toolkit for Immunoglobulin Gene Lineage Tree-Based Analysis, Reveals CDR3s Are Crucial for Selection Analysis.

Somatic hypermutation (SHM) is an important diversification mechanism that plays a part in the creation of immune memory. Immunoglobulin (Ig) variable region gene lineage trees were used over the last four decades to model SHM and the selection mechanisms operating on B cell clones. We hereby present IgTreeZ (Immunoglobulin Tree analyZer), a python-based tool that analyses many aspects of Ig gene lineage trees and their repertoires. Using simulations, we show that IgTreeZ can be reliably used for mutation and selection analyses. We used IgTreeZ on empirical data, found evidence for different mutation patterns in different B cell subpopulations, and gained insights into antigen-driven selection in corona virus disease 19 (COVID-19) patients. Most importantly, we show that including the CDR3 regions in selection analyses - which is only possible if these analyses are lineage tree-based - is crucial for obtaining correct results. Overall, we present a comprehensive lineage tree analysis tool that can reveal new biological insights into B cell repertoire dynamics.

CML-466 Asciminib Provides Durable Molecular Responses in Patients With Chronic Myeloid Leukemia in Chronic Phase (CML-CP) With the T315I Mutation: Updated Efficacy and Safety Data From a Phase I Trial

CONTEXT: In CML-CP, the BCR::ABL1 T315I mutation confers resistance to previously approved ATP-competitive tyrosine kinase inhibitors (TKIs), except ponatinib and olverembatinib. In a previous analysis of the phase I, dose-escalation trial X2101, asciminib-a BCR::ABL1 inhibitor that binds to the ABL myristoyl pocket-demonstrated efficacy and a favorable safety profile in heavily pretreated patients with T315I-mutated CML. We report updated efficacy and safety data in patients with CML-CP with the T315I mutation (data cutoff: January 6, 2021). OBJECTIVE: Provide updated safety and efficacy data for patients with T315I-mutated CML-CP after added exposure. DESIGN: Patients with T315I-mutated CML-CP and treated with >=1 prior TKI were enrolled and received asciminib 200mg twice daily (BID). RESULTS: 48 patients were included;25 patients (52.1%) received >=3 prior TKIs. At data cutoff, treatment was ongoing in 27 patients (56.3%). 45 of 48 patients were evaluable (BCR::ABL1IS >0.1% at baseline) for major molecular response (MMR);3 were excluded for BCR::ABL1 atypical transcripts. Among evaluable patients, 19 (42.2%) achieved MMR by week 24 and 22 (48.9%) by week 96. Evaluable patients included 26 ponatinib-pretreated and 19 ponatinib-naive patients;34.6% and 68.4%, respectively, achieved MMR by week 96. The probability of maintaining MMR for >=96 weeks was 84% (95% CI, 68.1%-100.0%). 23 of 37 patients (62.2%) with BCR::ABL1IS >1% at baseline achieved BCR::ABL1IS <=1% by week 96. The safety/tolerability profile of asciminib remained favorable after =9 months of added exposure (median duration of exposure, 2.08 years;range, 0.04-4.13 years). The most common (>=10%) grade >=3 adverse events (AEs) were lipase increase (18.8%, all asymptomatic elevations) and thrombocytopenia (14.6%). Arterial occlusive events occurred in 4 patients (8.3%);none led to dose adjustment/interruption/discontinuation. AEs leading to discontinuation occurred in 5 patients (10.4%). Only 2 study deaths, both due to COVID-19, occurred in this patient population. CONCLUSIONS: After a median exposure of >2 years, asciminib monotherapy 200mg BID exhibited a sustained, favorable safety profile and clinical efficacy in patients with T315I-mutated CML-CP-a population with high unmet medical need. This updated analysis confirms asciminib as a treatment option for patients with T315I-mutated CML-CP, including those previously treated with ponatinib.

The major role of junctional diversity in the horse antibody repertoire.

The antibody repertoire (Rep-seq) sequencing revolutionized the diversity of antigen B cell receptor studies, allowing deep and quantitative analysis to decipher the role of adaptive immunity in health and disease. Particularly, horse (Equus caballus) polyclonal antibodies have been produced and used since the century XIX to treat and prophylaxis diphtheria, tuberculosis, tetanus, pneumonia, and, more recently, COVID-19. However, our knowledge about the horse B cell receptors repertories is minimal. We present a deep horse antibody heavy chain repertoire (IGH) characterization of non-infected horses using NGS (Next generation sequencing). This study obtained a mean of 248,169 unique IgM clones and 66,141 unique IgG clones from four domestic adult horses. Rarefaction analysis showed sequence coverage was between 52 % and 82 % in IgM and IgG isotypes. We observed that besides horses antibody can use all functional IGHV genes, around 80 % of their antibodies use only three IGHV gene segments, and around 55 % use only one IGHJ gene segment. This limited VJ diversity seems to be compensated by the junctional diversity of these antibodies. We observed that the junctional diversity in horse antibodies is widespread, present in more than 90 % of horse antibodies. Besides this, the length of this region seems to be higher in horse antibodies than in other species. N1 and N2 nucleotides addition range from 0 to 111 nucleotides. In addition, around 45 % of the antibody clones have more than ten nucleotides in both the N1 and N2 junction regions. This diversity mechanism may be one of the most important in providing variability to the equine antibody repertoire. This study provides new insights regarding horse antibody composition, diversity generation, and particularities compared to other species, such as the frequency and length of N nucleotide addition. This study also points out the urgent need to better characterize TdT in horses and other species to better understand antibody repertoire characteristics.

COVID-19 IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA IN TREATMENT-FREE REMISSION: DISEASE SEVERITY AND IMPACT ON TFR STATUS

Background: Severe SARS-CoV-2 infections associated with high mortality rates are reported in a higher percentage of patients (pts) with hematologic malignancies compared to general population. In chronic myeloid leukemia (CML), pts with uncontrolled disease have a higher mortality risk. The impact of SARS-CoV-2 infection on CML pts in treatment-free remission (TFR) has not been studied so far. In particular, as immune control of residual disease may be important for TFR, the concern is that the infection could induce loss of TFR. Aims: To evaluate the outcome of SARS-CoV-2 infection in CML pts in TFR and assess any impact on maintenance of TFR. Methods: From March 2020 to December 2021, the CANDID study organized by the international CML Foundation has collected data on COVID-19 positive CML pts worldwide. Details on the registry were presented recently (Pagano ASH 2021). For this sub-analysis on pts in TFR additional information were collected including;molecular remission status (BCR::ABL1 ratios) before, during and after SARS-CoV-2 infection covering at least 6 months. For molecular analyses, BCR::ABL1 ratios were classified according to Cross et al (Leukemia 2015). In addition, ratios of 0% without indication of sensitivity were allocated as MR4 i.e. 0.01%IS. PCR outlier results were identified using the ROUT method by nonlinear regression with a maximum false discovery rate (FDR) of 1% (Motulsky et al 2006). Time to molecular relapse (MR) was measured from the date of COVID-19 diagnosis to the date of MR defined as loss of major molecular remission (MMR, BCR::ABL1 >0.1%IS) or the date of last molecular test. Molecular relapse-free survival (MRFS) and overall survival (OS) were estimated with the Kaplan-Meier method. The statistical difference between groups was performed using log-rank test. Results: By December 2021, 1050 COVID-19 positive CML pts were registered. 95 pts were in TFR at the time point of SARS-CoV-2 infection of which 89 (93.68%) recovered and 6 deceased (6.32%). Median age of TFR pts was 57 years, male were 51 (53.68%). Median time from CML diagnosis to reporting date was 13 years (range 3.7-27.0 years). TFR duration was 2.83 years in median (range 0.5 months - 10.1 years) including 19 pts with a duration < 1 year. From the 89 recovered TFR pts, 74 pts completed the 6-month follow up (83%), a further 6 pts with molecular follow-up of 3-5 months after COVID-19 diagnosis were still in TFR, 9 pts were lost to follow-up. Of 74 pts with complete reports, 69 pts remained in TFR (93%) and 5 pts lost TFR. For 71 pts, PCR results were obtained before, during and after infection. With the ROUT method 10 pts demonstrated outlier PCR tests, 61 pts demonstrated stable PCR results. There was no statistically significant difference in PCR results before and during/after infection (p>0.2). MRFS for these 71 pts 15 months after COVID-19 diagnosis was 86%. Probability of TFR loss was higher in pts with a TFR duration < 6 months compared to pts with TFR duration >6 months (27% vs 10%, Fig 1A). Additionally, there were no statistically differences in hospitalization rate (16% vs 23%, p=0.12) and severity of COVID-19 symptoms (12.6% vs 12%, p=0.87) comparing TFR and TKI treated pts. OS of COVID-19 positive TFR pts did not differ from COVID-19 positive pts on TKI therapy (HR 1.1, CI 0.47-2.54) (Fig 1B). Summary/Conclusion: In this sub-analysis of the CANDID study, CML pts in TFR had similar severity and survival to CML pts who were on TKI therapy and there was no evidence of an increased risk of TFR loss after SARS-CoV-2 infection.