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We investigated mRNA vaccines encoding a membrane-anchored receptor-binding domain (RBD), each a fusion of a variant RBD, the transmembrane (TM) and cytoplasmic tail fragments of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. In naive mice, RBD-TM mRNA vaccines against SARS-CoV-2 variants induced strong humoral responses against the target RBD. Multiplex surrogate viral neutralization (sVNT) assays revealed broad neutralizing activity against a range of variant RBDs. In the setting of a heterologous boost, against the background of exposure to ancestral whole-spike vaccines, sVNT studies suggested that BA.1 and BA.5 RBD-TM vaccines had the potential to overcome the detrimental effects of immune imprinting. A subsequent heterologous boost study using XBB.1.5 booster vaccines was evaluated using both sVNT and authentic virus neutralization. Geometric mean XBB.1.5 neutralization values after third-dose RBD-TM or whole-spike XBB.1.5 booster vaccines were compared with those after a third dose of ancestral spike booster vaccine. Fold-improvement over ancestral vaccine was just 1.3 for the whole-spike XBB.1.5 vaccine, similar to data published using human serum samples. In contrast, the fold-improvement achieved by the RBD-TM XBB.1.5 vaccine was 16.3, indicating that the RBD-TM vaccine induced the production of antibodies that neutralize the XBB.1.5 variant despite previous exposure to ancestral spike protein., Competing Interests: Two provisional patents (PCT/AU2022/050912 and PCT/AU2022/050913) covering the RBD-TM mRNA vaccine design and the LNP formulation used in this study, and underlying technology, have been submitted through Monash University, with C.W.P., H.A.W., and S.A.F. as co-inventors of 050912 and C.W.P., H.A.W., and J.K.H. as co-inventors of 050913. C.W.T. and L.-F.W. are co-inventors of a patent on the surrogate VNT test (sVNT) platform. T.N. receives research contracts to conduct clinical trials, with funding to institutions from Moderna, SanofiPasteur, GSK, Iliad Biotechnologies, Dynavax, Seqirus, Janssen, and MSD. T.N. receives consulting fees from GSK, Seqirus, MSD, SanofiPasteur, AstraZeneca, Moderna, BioNet, and Pfizer. T.N. serves on DSMBs for Seqirus, Clover, Moderna, Emergent, Serum Institute of India, SK Bioscience Korea, Emergent Biosolutions, and Novavax. S.R. is an employee of CSL Seqirus that is a maker of influenza vaccines. C.Y.W. is a shareholder of Ena Respiratory. D.I.G. has received research funding from CSL for an unrelated project., (Crown Copyright © 2024 Published by Elsevier Inc. on behalf of The American Society of Gene and Cell Therapy.)

HIV-1 persists indefinitely in people living with HIV (PLWH) on antiretroviral therapy (ART). If ART is stopped, the virus rapidly rebounds from long-lived latently infected cells. Using a humanized mouse model of HIV-1 infection and CD4 + T cells from PLWH on ART, we investigate whether antagonizing host pro-survival proteins can prime latent cells to die and facilitate HIV-1 clearance. Venetoclax, a pro-apoptotic inhibitor of Bcl-2, depletes total and intact HIV-1 DNA in CD4 + T cells from PLWH ex vivo. This venetoclax-sensitive population is enriched for cells with transcriptionally higher levels of pro-apoptotic BH3-only proteins. Furthermore, venetoclax delays viral rebound in a mouse model of persistent HIV-1 infection, and the combination of venetoclax with the Mcl-1 inhibitor S63845 achieves a longer delay in rebound compared with either intervention alone. Thus, selective inhibition of pro-survival proteins can induce death of HIV-1-infected cells that persist on ART, extending time to viral rebound., Competing Interests: Declaration of interests The Walter and Eliza Hall Institute receives milestone and royalty payments related to venetoclax and has a commercial collaboration with Servier with respect to Mcl-1 inhibitors under which it may receive future payments. M.P. is eligible for financial benefits related to these payments. S.R.L. has received investigator-initiated, industry-funded research support from Merck Sciences, Gilead Sciences, and ViiV and provision of reagents from Infinity Pharmaceuticals, Merck Sciences, and BMS for investigator-initiated research. S.R.L. and J.L.A. have research collaborations with Merck Sciences unrelated to this work., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The development of therapeutics to prevent or treat COVID-19 remains an area of intense focus. Protein biologics, including monoclonal antibodies and nanobodies that neutralize virus, have potential for the treatment of active disease. Here, we have used yeast display of a synthetic nanobody library to isolate nanobodies that bind the receptor-binding domain (RBD) of SARS-CoV-2 and neutralize the virus. We show that combining two clones with distinct binding epitopes within the RBD into a single protein construct to generate biparatopic reagents dramatically enhances their neutralizing capacity. Furthermore, the biparatopic nanobodies exhibit enhanced control over clinically relevant RBD variants that escaped recognition by the individual nanobodies. Structural analysis of biparatopic binding to spike (S) protein revealed a unique binding mode whereby the two nanobody paratopes bridge RBDs encoded by distinct S trimers. Accordingly, biparatopic nanobodies offer a way to rapidly generate powerful viral neutralizers with enhanced ability to control viral escape mutants., Competing Interests: Lin-Fa Wang and Chee Wah Tan are co-inventors of the surrogate virus neutralization test commercialized under the tradename cPass by GenScript Biotech., (© 2022 The Author(s).)

Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions., Competing Interests: Declaration of interests The authors declare that D.S.S., J.P., A.W., I.Y.K., M.R., J.P.C., K.C.D., S.H., H.A., M.D., Y.D., L.M., M.D., A.S.H., S.E.N., J.R.G., M.J.H., E.D.H., A.S., J.S., M.P., R.F., and J.E.V. are employees or former employees of the Walter and Eliza Hall Medical Institute, which receives milestone payments from Genentech and AbbVie for the development of ABT-199 for cancer therapy. J.E.V. sits on the advisory board of Avammune Therapeutics., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Potent neutralizing monoclonal antibodies are one of the few agents currently available to treat COVID-19. SARS-CoV-2 variants of concern (VOCs) that carry multiple mutations in the viral spike protein can exhibit neutralization resistance, potentially affecting the effectiveness of some antibody-based therapeutics. Here, the generation of a diverse panel of 91 human, neutralizing monoclonal antibodies provides an in-depth structural and phenotypic definition of receptor binding domain (RBD) antigenic sites on the viral spike. These RBD antibodies ameliorate SARS-CoV-2 infection in mice and hamster models in a dose-dependent manner and in proportion to in vitro, neutralizing potency. Assessing the effect of mutations in the spike protein on antibody recognition and neutralization highlights both potent single antibodies and stereotypic classes of antibodies that are unaffected by currently circulating VOCs, such as B.1.351 and P.1. These neutralizing monoclonal antibodies and others that bind analogous epitopes represent potentially useful future anti-SARS-CoV-2 therapeutics., Competing Interests: Declaration of interests The authors declare no competing interests. A provisional patent covering human mAbs isolated from convalescent donors has been submitted through the University of Melbourne. A provisional patent covering human mAbs isolated from phage display has been submitted through the Walter and Eliza Hall Institute., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)