RESUMO
Background: Nanocovax is a recombinant severe acute respiratory syndrome coronavirus 2 subunit vaccine composed of full-length prefusion stabilized recombinant SARS-CoV-2 spike glycoproteins (S-2P) and aluminium hydroxide adjuvant. Methods: We conducted a dose-escalation, open label trial (phase 1) and a randomized, double-blind, placebo-controlled trial (phase 2) to evaluate the safety and immunogenicity of the Nanocovax vaccine (in 25 mcg, 50 mcg, and 75 mcg doses, aluminium hydroxide adjuvanted (0·5 mg/dose) in 2-dose regime, 28 days apart (ClinicalTrials.gov number, NCT04683484). In phase 1, 60 participants received two intramuscular injection of the vaccine following dose-escalation procedure. The primary outcomes were reactogenicity and laboratory tests to evaluate the vaccine safety. In phase 2, 560 healthy adults received either vaccine doses similar in phase 1 (25 or 50 or 75 mcg S antigen in 0·5 mg aluminium per dose) or adjuvant (0·5 mg aluminium) in a ratio of 2:2:2:1. One primary outcome was the vaccine safety, including solicited adverse events for 7 day and unsolicited adverse events for 28 days after each injection as well as serious adverse event or adverse events of special interest throughout the study period. Another primary outcome was anti-S IgG antibody response (Index unit/ml). Secondary outcomes were surrogate virus neutralisation (inhibition percentage), wild-type SARS-CoV-2 neutralisation (dilution fold), and T-cell responses by intracellular staining for interferon gamma (IFNg). Anti-S IgG and neutralising antibody levels were compared with convalescent serum samples from symptomatic Covid-19 patients. Findings: For phase 1 study, no serious adverse events were observed for all 60 participants. Most adverse events were grade 1 and disappeared shortly after injection. For phase 2 study, after randomisation, 480 participants were assigned to receive the vaccine with adjuvant, and 80 participants were assigned to receive the placebo (adjuvant only). Reactogenicity was absent or mild in the majority of participants and of short duration (mean ≤3 days). Unsolicited adverse events were mild in most participants. There were no serious adverse events related to Nanocovax. Regarding the immunogenicity, Nanocovax induced robust anti-S antibody responses. In general, there humoral responses were similar among vaccine groups which reached their peaks at day 42 and declined afterward. At day 42, IgG levels of vaccine groups were 60·48 [CI95%: 51·12-71·55], 49·11 [41·26-58·46], 57·18 [48·4-67·5] compared to 7·10 [6·32-13·92] of convalescent samples. IgG levels reported here can be converted to WHO international standard binding antibody unit (BAU/ml) by multiplying them to a conversion factor of 21·8. Neutralising antibody titre of vaccine groups at day 42 were 89·2 [52·2-152·3], 80·0 [50·8-125.9] and 95·1 [63·1-143·6], compared to 55·1 [33·4-91·0] of the convalescent group. Interpretation: Up to day 90, Nanocovax was found to be safe, well tolerated, and induced robust immune responses. Funding: This work was funded by the Coalition for Epidemic Preparedness Innovations (CEPI), the Ministry of Science and Technology of Vietnam, and Nanogen Pharmaceutical Biotechnology JSC.
RESUMO
BACKGROUND: Polyclonal antithymocyte globulin (ATG) is widely used as an anti-T-cell agent for induction and treatment of acute cellular rejection in solid organ transplantation. The authors recently demonstrated that rabbit (r) ATG can be used in combination with plasmapheresis to effectively treat antibody-mediated renal allograft rejection. This observation suggested that rATG may have anti-B cell activity. METHODS: The authors tested the complement-independent, apoptosis-inducing properties of rATG on CD27- naive B cells, CD40 ligand-activated B cells, and plasma cells in vitro by annexin V staining, subdiploid DNA content, caspase activation, and loss of mitochondrial membrane polarity. Potential surface targets for rATG were assayed by competitive inhibition of monoclonal antibody binding. RESULTS: Rabbit ATG strongly induced apoptosis in vitro against naive, activated B cells and bone marrow resident plasma cells at clinically relevant concentrations (1-100 ng/mL). The authors found rATG activity against numerous B-cell surface proteins and observed that crosslinking of CD30, CD38, CD95, CD80, and HLA-DR likely accounts for this activity. F(ab)2 fragments of rATG showed 90% of the activity of the intact molecule, suggesting participation of the Fc fragment. Inhibition of caspase- and cathepsin-dependent apoptotic pathways partially inhibits rATG-induced B-cell apoptosis. Immunohistochemical staining of pediatric thymi demonstrated the presence of CD20+ B cells and CD138+ plasma cells within the thymic parenchyma, which accounts for the anti-B-cell activity in rATG. CONCLUSIONS: Polyclonal rATG induces complement-independent apoptosis of naive, activated, and plasma B cells. This effect appears to involve the caspase- and cathepsin-mediated apoptosis pathways.
Assuntos
Soro Antilinfocitário/farmacologia , Apoptose/efeitos dos fármacos , Linfócitos B/imunologia , Plasmócitos/imunologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/fisiologia , Humanos , Fragmentos Fab das Imunoglobulinas/sangue , Ativação Linfocitária , Plasmócitos/efeitos dos fármacos , Plasmócitos/fisiologia , Coelhos , Valores de ReferênciaRESUMO
Current monoclonal antibody therapies for multiple myeloma have had limited success, perhaps due to narrow target specificity. We have previously described the ability of polyclonal rabbit antithymocyte globulin (rATG) to induce caspase- and cathepsin-mediated apoptosis in human B and plasma cells. We now extend this observation to myeloma cells. Complement independent cell death was measured after addition of rATG (1-1000 microg/mL) to cultures of myeloma cell lines or primary CD138+ isolates from patient bone marrow aspirates. rATG induced significant levels of apoptosis in myeloma cells as assayed by caspase induction, annexin V binding, subdiploid DNA fragmentation, plasma-membrane permeability, and loss of mitochondrial-membrane potential. Addition of complement greatly augmented myeloma-cell death. Binding of rATG to individual myeloma cell-surface proteins, primarily CD38, CD52, CD126, and CD138, was demonstrated by competitive inhibition experiments with targeted monoclonal antibodies. Three pathways of cell death were identified involving caspase activation, cathepsin D, and the genistein sensitive tyrosine kinase pathway. Fab'2 fragments of rATG had reduced proapoptotic activity, which was restored by coincubation with Fc fragments, and anti-CD32 or anti-CD64 antibodies. We conclude that rATG is an effective agent for in vitro induction of apoptosis in multiple myeloma, and that exploratory clinical trials may be warranted.
Assuntos
Soro Antilinfocitário/farmacologia , Apoptose/efeitos dos fármacos , Mieloma Múltiplo/patologia , Animais , Anticorpos Antineoplásicos/imunologia , Soro Antilinfocitário/imunologia , Células da Medula Óssea/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , CoelhosRESUMO
A major impediment to repetitive monitoring of alloreactivity or tolerance is the limited supply of donor cells available for assays of host-versus-graft T- and B-cell reactivity. In this paper, we describe the use of CD40L stimulated CD19(+) B cells as targets or stimulators in flow cytometric crossmatching (FXM), mixed lymphocyte reactivity and IFN-gamma ELISPOT assays. Stimulated B cells (sBc) express high levels of MHC class I and II, as well as the costimulatory molecules CD80 and CD86. They can be polyclonally expanded and frozen for later use. We describe the use of sBc in ELISPOT, mixed lymphocyte cultures and FXM. CD4(+) T cells exposed to sBc express a similar cytokine profile as those stimulated with unfractionated PBMC. We further analyzed T- and B-cell responses in 14 patients on the renal transplant waiting list, finding that those with an elevated panel reactive antibody (PRA) (>60%) had higher alloreactive T-cell precursor frequencies as measured by CDFSE MLR and IFN-gamma ELISPOT. We conclude that sBc are a renewable source of donor-specific target/stimulator cells for use in repetitive and coordinate assays of B- and T-cell alloreactivity.
Assuntos
Linfócitos B/imunologia , Separação Celular/métodos , Transplante de Células/métodos , Anticorpos/química , Antígenos CD/biossíntese , Antígenos CD19/biossíntese , Linfócitos B/metabolismo , Antígeno B7-1/biossíntese , Complexo CD3/biossíntese , Linfócitos T CD4-Positivos/imunologia , Ligante de CD40/metabolismo , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo/métodos , Genótipo , Humanos , Tolerância Imunológica , Imunoglobulina G/química , Interferon gama/metabolismo , Transplante de Rim , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Linfonodos/patologia , Ativação Linfocitária , Linfócitos/imunologia , Linfócitos/metabolismo , Fenótipo , Receptores Fc/química , Receptores Imunológicos/biossíntese , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo , Doadores de TecidosAssuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Neuralgia/tratamento farmacológico , Neuralgia/fisiopatologia , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/fisiopatologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Doença Crônica , Ensaios Clínicos como Assunto/estatística & dados numéricos , Cultura , Inibidores de Ciclo-Oxigenase/farmacologia , Inibidores de Ciclo-Oxigenase/uso terapêutico , Modelos Animais de Doenças , Humanos , Inflamação/tratamento farmacológico , Inflamação/etiologia , Inflamação/fisiopatologia , Efeito Placebo , Prescrições/estatística & dados numéricos , Resultado do TratamentoRESUMO
Studies of memory T cell differentiation are hampered by a lack of quantitative models to test hypotheses in silico before in vivo experimentation. We created a stochastic computer model of CD4+ memory T cell generation that can simulate and track 10(1)-10(8) individual lymphocytes over time. Parameters for the model were derived from experimental data using naive human CD4+ T cells stimulated in vitro. Using discrete event computer simulation, we identified two key variables that heavily influence effector burst size and the persistent memory pool size: the cell cycle dependent probability of apoptosis, and the postactivation mitosis at which memory T cells emerge. Multiple simulations were performed and varying critical parameters permitted estimates of how sensitive the model was to changes in all of the model parameters. We then compared two hypotheses of CD4+ memory T cell generation: maturation from activated naive to effector to memory cells (model I) vs direct progression from activated naive to memory cells (model II). We find that direct progression of naive to memory T cells does not explain published measurements of the memory cell mass unless postactivation expansion of the memory cell cohort occurs. We conclude that current models suggesting direct progression of activated naive cells to the persistent memory phenotype (model II) do not account for the experimentally measured size of the postactivation CD4+, Ag-specific, memory T cell cohort.