Additional heterologous versus homologous booster vaccination in immunosuppressed patients without SARS-CoV-2 antibody seroconversion after primary mRNA vaccination: a randomised controlled trial

ObjectivesSARS‐CoV‐2-induced COVID-19 has led to exponentially rising mortality, particularly in immunosuppressed patients, who inadequately respond to conventional COVID-19 vaccination.MethodsIn this blinded randomised clinical trial, we compare the efficacy and safety of an additional booster vaccination with a vector versus mRNA vaccine in non-seroconverted patients. We assigned 60 patients under rituximab treatment, who did not seroconvert after their primary mRNA vaccination with either BNT162b2 (Pfizer–BioNTech) or mRNA-1273 (Moderna), to receive a third dose, either using the same mRNA or the vector vaccine ChAdOx1 nCoV-19 (Oxford–AstraZeneca). Patients were stratified according to the presence of peripheral B cells. The primary efficacy endpoint was the difference in the SARS-CoV-2 antibody seroconversion rate between vector (heterologous) and mRNA (homologous) vaccinated patients by week 4. Key secondary endpoints included the overall seroconversion and cellular immune response; safety was assessed at week 1 and week 4.ResultsSeroconversion rates at week 4 were comparable between vector (6/27 patients, 22%) and mRNA (9/28, 32%) vaccines (p=0.6). Overall, 27% of patients seroconverted; specific T cell responses were observed in 20/20 (100%) vector versus 13/16 (81%) mRNA vaccinated patients. Newly induced humoral and/or cellular responses occurred in 9/11 (82%) patients. 3/37 (8%) of patients without and 12/18 (67%) of the patients with detectable peripheral B cells seroconverted. No serious adverse events, related to immunisation, were observed.ConclusionsThis enhanced humoral and/or cellular immune response supports an additional booster vaccination in non-seroconverted patients irrespective of a heterologous or homologous vaccination regimen.

Additional heterologous versus homologous booster vaccination in immunosuppressed patients without SARS-CoV-2 antibody seroconversion after primary mRNA vaccination: a randomized controlled trial

ABSTRACTSevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-induced coronavirus disease 2019 (COVID-19) has led to exponentially rising mortality, particularly in immunosuppressed patients, who inadequately respond to conventional COVID-19 vaccination. In this blinded randomized clinical trial (EudraCT 2021-002348-57) we compare the efficacy and safety of an additional booster vaccination with a vector versus mRNA vaccine in non-seroconverted patients. We assigned 60 patients under rituximab treatment, who did not seroconvert after their primary mRNA vaccination with either BNT162b2 (Pfizer–BioNTech) or mRNA-1273 (Moderna), to receive a third dose, either using the same mRNA or the vector vaccine ChAdOx1 nCoV-19 (Oxford-AstraZeneca). Patients were stratified according to the presence of peripheral B-cells. The primary efficacy endpoint was the difference in the SARS-CoV-2 antibody seroconversion rate between vector (heterologous) and mRNA (homologous) vaccinated patients by week four. Key secondary endpoints included the overall seroconversion and cellular immune response; safety was assessed at weeks one and four.Seroconversion rates at week four were comparable between vector (6/27 patients, 22%) and mRNA (9/28, 32%) vaccine (p=0.6). Overall, 27% of patients seroconverted; specific T-cell responses were observed in 20/20 (100%) vector versus 13/16 (81%) mRNA vaccinated patients. Newly induced humoral and/or cellular responses occurred in 9/11 (82%) patients. No serious adverse events, related to immunization, were observed. This enhanced humoral and/or cellular immune response supports an additional booster vaccination in non-seroconverted patients irrespective of a heterologous or homologous vaccination regimen.

Requirement of Caspase-8 Versus Caspase-9 during Apoptosis in Multiple Myeloma Cells Induced by Bortezomib- or a Novel Proteasome Inhibitor NPI-0052.

Proteasome inhibition is an effective therapy for the treatment of relapsed/refractory multiple myeloma (MM); however, the sequence of events leading to apoptosis following proteasome inhibition is unclear. Here we defined the requirement for caspase-8 versus caspase-9 during Bortezomib (VelcadeTM)- or novel proteasome inhibitor NPI-0052-induced apoptosis in MM cells. Incubation of MM.1S cells with pan-caspase inhibitor (Z-VAD-FMK) markedly abrogates both NPI-0052- and Bortezomib-induced apoptosis. Inhibition of caspase-8 (IETD-FMK) led to a significant decrease in NPI-0052-triggered cell death, whereas inhibition of caspase-9 (LEHD-FMK) only moderately blocked NPI-0052-triggered decreased viability in MM.1S cells (P < 0.005). In contrast, Bortezomib-induced decrease in viability is equally blocked by either caspase-8 or caspase-9 inhibitors (P < 0.005). These biochemical data were further examined by genetic studies using dominant-negative (DN) strategies. Treatment of DN-caspase-8-transfected MM cells with NPI-0052 (IC50: 7 nM) markedly increases survival compared to cells transfected with DN-caspase-9. In contrast, treatment of either DN-caspase-8 or DN caspase-9-tranfected MM.1S cells with Bortezomib (IC50: 5 nM) increases survival to a similar extent. The proteolytic processing of pro-caspase-8 is mediated by Fas Associated Death-Domain (FADD) protein, and we next examined blockade of FADD with DN-FADD to further confirm the role of caspase-8. DN-FADD significantly attenuated NPI-0052-induced cytotoxicity compared to empty vector-transfected MM.1S cells (42 ± 2.0% viable cells in vector- versus 76 ± 5.1% viable cells in DN-FADD-transfected cells; P < 0.05). Importantly, treatment of DN-FADD-transfected MM.1S cells with Bortezomib results in only 16% increase in survival compared to vector-transfected cells (39 ± 2.4% viable cells in vector- versus 55 ± 4.1% viable cells in DN-FADD-transfected cells; P < 0.05). These data, coupled with caspase-8 or caspase-9 inhibition studies, suggest that NPI-0052 relies more on FADD-caspase-8 signaling than does Bortezomib, confirming a differential mechanism of action of NPI-0052 versus Bortezomib in MM cells. To further address this issue, we examined alterations in Bax, a proapoptotic protein which translocates from cytosol to mitochondria during apoptosis, inhibits Bcl-2, and facilitates release of cyto-c and activation of caspase-9. NPI-0052 induces little, if any, increase in Bax levels in mitochondria, whereas Bortezomib triggers a significant accumulation of Bax in mitochondria. Experiments using Bax wild type (WT) or knockout mouse embryonic fibroblast (MEFs) show that NPI-0052 decreases viability in both Bax (WT) and Bax (knock-out) MEFs, whereas deletion of Bax confers significant resistance to Bortezomib. These findings suggest a differential requirement for Bax during NPI-0052- versus Bortezomib-induced apoptosis. Collectively, our data suggest that 1) NPI-0052-induced MM cell apoptosis is predominantly mediated by caspase-8; and 2) Bortezomib-induced apoptosis requires both caspase-8 and caspase-9 activation. These data provide rationale for combining agents based on differential signaling cascades to amplify apoptosis and enhance anti-tumor activity.