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

Todos los Autores y su Filiación: Mariana Seija 1,2, Florencia Rammauro3,4, José Santiago1, Natalia Orihuela5, Catherine Zulberti5, Danilo Machado6, Cecilia Recalde6, Javier Noboa1,4, Victoria Frantchez7, Rossana Astesiano1, Federico Yandián1, Ana Guerisoli1, Álvaro Morra5, Daniela Cassinelli8, Cecilia Coelho8, Belén de Aramburu8, Paulina González-Severgnini8, Romina Moreno8, Aldana Pippolo8, Gabriela López9, Mónica Lemos9, Lorena Somariva9, Eliana López9, Soledad Fumero9, Carla Orihuela9, Rosalía Rodríguez6, Gonzalo Acuña6, Victoria Rabaza6, Nancy Perg6, Rossana Cordero6, Cristina Reisfeld6, Paula Olivera6, Paola Montero6, Cecilia Nogueira6, Catheryn Nalerio5, Sergio Orihuela5, Lilián Curi5, Ema Burgstaller6, Oscar Noboa1, Otto Pritsch3,4, Marcelo Nin1,5 and Sergio Bianchi 2,10 1Centro de Nefrología, Hospital de Clínicas, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay, 2Departamento de Fisiopatología, Hospital de Clínicas, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay, 3Laboratorio de Inmunovirología, Institut Pasteur de Montevideo, Montevideo, Uruguay, 4Departamento de Inmunobiología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay, 5Centro de Trasplante INU, Hospital Italiano, Montevideo, Uruguay, 6Centro de Trasplante, Hospital Evangélico, Montevideo, Uruguay, 7Cátedra de Enfermedades Infecciosas, Hospital de Clínicas, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay, 8Students of Scientific Methods 2, Medical Doctor Degree, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay, 9Departamento de Enfermería, Hospital de Clínicas, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay and 10Laboratorio de Genómica Funcional, Institut Pasteur de Montevideo, Montevideo, Uruguay ∗These authors contributed equally to this work. Correspondence to: Sergio Bianchi; E-mail: [email protected]. 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.Fondo para la Convergencia Estructural del Mercosur (FOCEM, COF 03/11); Agencia Nacional de Investigación e Innovación (ANII), Uruguay; y Fondo de Investigación en Nefrología (FOINE), Hospital de Clínicas, Uruguay

Humoral Response to Heterologous SARS-CoV-2 Vaccination in Kidney Transplant Patients Is Heterogeneous and Dose Dependent

Todos los Autores y su Filiación: Mariana Seija1,2,11, Florencia Rammauro3,4,11, Javier Noboa1,4 , José Santiago1 , Natalia Orihuela5 , Catherine Zulberti5 , Danilo Machado6 , Cecilia Recalde6 , Rossana Astesiano1 , Federico Yandián1 , Victoria Frantchez7 , Ana Guerisoli1 , Álvaro Morra5 , Daniela Cassinelli8 , Cecilia Coelho8 , Belén de Aramburu8 , Paulina González-Severgnini8 , Romina Moreno8 , Aldana Pippolo8 , Gabriela López9 , Mónica Lemos9 , Lorena Somariva9 , Eliana López9 , Soledad Fumero9 , Carla Orihuela9 , Ana Laura Suárez2 , Rosalía Rodríguez6 , Gonzalo Acuña6 , Victoria Rabaza6 , Nancy Perg6 , Rossana Cordero6 , Cristina Reisfeld6 , Paula Olivera6 , Paola Montero6 , Cecilia Nogueira6 , Catheryn Nalerio5 , Sergio Orihuela5 , Lilián Curi5 , Ema Bugstaller6 , Otto Pritsch3,4 , Marcelo Nin1,5 , Oscar Noboa1 and Sergio Bianchi2,10 1 Centro de Nefrología, Hospital de Clínicas, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; 2 Departamento de Fisiopatología, Hospital de Clínicas, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; 3 Laboratorio de Inmunovirología, Institut Pasteur de Montevideo, Montevideo, Uruguay; 4 Departamento de Inmubionobiología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; 5 Centro de Trasplante INU, Hospital Italiano, Montevideo, Uruguay; 6 Centro de Trasplante, Hospital Evangélico, Montevideo, Uruguay; 7 Cátedra de Enfermedades Infecciosas, Hospital de Clínicas, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; 8 Students of Scientific Methodology, Medical Doctor Degree, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; 9 Departamento de Enfermería, Hospital de Clínicas, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; and 10Laboratorio de Genómica Funcional, Institut Pasteur de Montevideo, Montevideo, Uruguay. Correspondence: Sergio Bianchi, Departamento de Fisiopatología, Hospital de Clínicas, Facultad de Medicina, Universidad de la República, Av. Italia s/n, Montevideo 11600, Uruguay. E-mail: [email protected] para la Convergencia Estructural del Mercosur (FOCEM, COF 03/11); Agencia Nacional de Investigación e Innovación (ANII), Uruguay; y Fondo de Investigación en Nefrología (FOINE), Hospital de Clínicas, Uruguay

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 Fo¨ rster 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.CSIC: I+D 201

Expression, purification, and characterization of bovine leukemia virus-like particles produced in Drosophila S2 cells

Material complementario: https://www.frontiersin.org/articles/10.3389/fviro.2021.756559/full#supplementary-materialBovine leukemia virus (BLV) is an oncogenic deltaretrovirus that infects cattle worldwide. In Uruguay, it is estimated that more than 70% of dairy cattle are infected, causing serious economic losses due to decreased milk production, increased calving interval, and livestock losses due to lymphosarcoma. Several attempts to develop vaccine candidates that activate protective immune responses against BLV were performed, but up to date, there is no vaccine that ensures efficient protection and/or decreased viral transmission. The development and application of new vaccines that effectively control BLV infection represent amajor challenge for countries with a high prevalence of infection. In this study, we generated two Drosophila melanogaster S2 stable cell lines capable of producing BLV virus-like particles (BLV-VLPs). One of them, BLV-VLP1, expressed both Gag and Env wild-type (Envwt) full-length proteins, whereas BLV-VLP2 contain Gag together with a mutant form of Env non-susceptible to proteolytic maturation by cellular furin type enzymes (EnvFm).We showed that Envwt is properly cleaved by cellular furin, whereas EnvFm is produced as a full-length gp72 precursor, which undergoes some partial cleavage. We observed that said mutation does not drastically affect its expression or its entry into the secretory pathway of S2 insect cells. In addition, it is expressed on the membrane and retains significant structural motifs when expressed in S2 insect cells. Morphology and size of purified BLV-VLPs were analyzed by transmission electron microscopy and dynamic light scattering, showing numerous non-aggregated and approximately spherical particles of variable diameter (70–200 nm) as previously reported for retroviral VLPs produced using different expression systems. Furthermore, we identified two N-glycosylation patterns rich in mannose in EnvFm protein displayed on VLP2. Our results suggest that the VLPs produced in Drosophila S2 cells could be a potential immunogen to be used in the development of BLV vaccines that might contribute, in conjunction with other control strategies, to reduce the transmission of the virus.CSIC I+D 2014ANII: ALI_1_2016_2_129851; POS_NAC_2015_1_109471PEDECIBA-FOCEM: COF 03/11CAP: BFPD_2020_1#2814383

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

Abordaje sistémico de la respuesta inmune mediada por anticuerpos contra el SARS-COV-2

El SARS-CoV-2 es un betacoranavirus causante de la enfermedad llamada de COVID- 19, la cual, desde la aparición de los primeros casos en diciembre de 2019 en China, ha causado numerosos impactos sanitarios, económicos y sociales. En este contexto, la aplicación de ensayos serológicos capaces de evidenciar la presencia de anticuerpos específicos ha sido muy útil. En nuestro país, se desarrolló una prueba de tipo ELISA con el objetivo de determinar la presencia de anticuerpos específicos dirigidos al dominio de unión al receptor (RBD) de la proteína S del SARS-CoV-2. En este trabajo describimos el desarrollo, validación y la aplicación de este ensayo en estudios de seroprevalencia, así como para la evaluación de plasmas hiperinmunes COVID-19. La vacunación contra el SARS-CoV-2 ha mostrado se una estrategia exitosa para prevenir las formas severas de enfermedad y muertes asociadas a COVID-19. CoronaVac (SinoVac) y BNT162b2 (Pfizer-BionTech) han sido las vacunas más empleadas a nivel global, y también en nuestro país. Como parte de este trabajo, evaluamos la respuesta inmune humoral, en particular los niveles de anticuerpos de tipo IgG dirigidos al RBD de la proteína S de SARS-CoV-2, inducida por la vacunación con CoronaVac y BNT162b2, y la aplicación de dosis de refuerzo en esquemas de vacunación homóloga y heteróloga, en una cohorte de población sana, trasplantados renales, trasplantados hepáticos y pacientes en diálisis crónica. En la cohorte de población sana evaluamos además la dinámica de decaimiento de los anticuerpos post-vacunación en el tiempo, y la actividad neutralizante, cinética de unión y capacidad de mediar funciones efectoras dependientes del Fc de estos anticuerpos. En la cohorte de individuos sanos todos mostraron niveles de anticuerpos detectables luego de la vacunación con dos dosis, independientemente de la vacuna empleada, aunque los niveles de anticuerpos alcanzados con BNT162b2 fueron significativamente mayores. La aplicación de una dosis de refuerzo con BNT162b2 en aquellos inmunizados con CoronaVac, aumento cerca de 20 veces los niveles de anticuerpos anti-RBD comparado con lo encontrado luego de la aplicación de dos dosis. La funcionalidad de las inmunoglobulinas mostró un comportamiento similar. En los grupos de trasplantados, se pudieron detectar anticuerpos en un porcentaje menor de individuos luego de la vacunación y los niveles de anticuerpos también fueron significativamente menores, independiente de la vacuna empleada, comparado con el grupo control. En los pacientes en diálisis si bien no se encontraron diferencias tan notables con respecto al porcentaje de individuos con presencia de anticuerpos específicos, los niveles alcanzados fueron si significativamente menores. La aplicación de dosis de refuerzo aumento de manera significativa tanto el número de individuos en los que se pudo detectar la presencia de anticuerpos como los niveles de estos, para todos los grupos estudiados. A pesar del impacto de la aplicación de dosis de refuerzo en estas poblaciones, algunos individuos continúan sin evidenciar respuesta a la vacunación, lo cual parece estar relacionado al estado de inmunosupresión

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

International audienceSevere 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 β-coronavirus. Tmem176b −/− mice infected with murine β-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 β-coronavirus disease

PD-1/PD-L1 blockade abrogates a dysfunctional innate-adaptive immune axis in critical β-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 β-coronavirus. Tmem176b-/- mice infected with murine β-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 β-coronavirus disease.Fil: Duhalde Vega, Maite. Institut Pasteur de Montevideo; Uruguay. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Olivera, Daniela. Institut Pasteur de Montevideo; Uruguay. Universidad de la República; UruguayFil: Davanzo, Gustavo Gastão. Universidade Estadual de Campinas; BrasilFil: Bertullo, Mauricio. Immunoregulation And Inflammation Lab; UruguayFil: Noya, Verónica. Sanatorio Americano; UruguayFil: de Souza, Gabriela Fabiano. Universidade Estadual de Campinas; BrasilFil: Muraro, Stéfanie Primon. Universidade Estadual de Campinas; BrasilFil: Castro, Icaro. Hospital Israelita Albert Einstein; BrasilFil: Arévalo, Ana Paula. Institut Pasteur de Montevideo; UruguayFil: Crispo, Martina. Institut Pasteur de Montevideo; UruguayFil: Galliussi, Germán. Institut Pasteur de Montevideo; UruguayFil: Russo, Sofía. Institut Pasteur de Montevideo; Uruguay. Universidad de la República; UruguayFil: Charbonnier, David. Institut Pasteur de Montevideo; UruguayFil: Rammauro, Florencia. Institut Pasteur de Montevideo; Uruguay. Universidad de la República; UruguayFil: Jeldres, Mathías. Institut Pasteur de Montevideo; Uruguay. Universidad de la República; UruguayFil: Alamón, Catalina. Institut Pasteur de Montevideo; UruguayFil: Varela, Valentina. Institut Pasteur de Montevideo; UruguayFil: Batthyany, Carlos. Institut Pasteur de Montevideo; UruguayFil: Bollati Fogolín, Mariela. Institut Pasteur de Montevideo; UruguayFil: Oppezzo, Pablo. Institut Pasteur de Montevideo; UruguayFil: Pritsch, Otto. Institut Pasteur de Montevideo; Uruguay. Universidad de la República; UruguayFil: Proença Módena, José Luiz. Universidade Estadual de Campinas; BrasilFil: Nakaya, Helder I.. Hospital Israelita Albert Einstein; BrasilFil: Trias, Emiliano. Institut Pasteur de Montevideo; UruguayFil: Barbeito, Luis. Institut Pasteur de Montevideo; UruguayFil: Anegon, Ignacio. Center For Research In Transplantation And Immunology; FranciaFil: Cuturi, María Cristina. Center For Research In Transplantation And Immunology; FranciaFil: Moraes Vieira, Pedro. Universidade Estadual de Campinas; BrasilFil: Segovia, Mercedes. Institut Pasteur de Montevideo; Uruguay. Universidad de la República; UruguayFil: Hill, Marcelo. Universidad de la República; Uruguay. Institut Pasteur de Montevideo; Urugua