Biology subjects

Gardner, M. R.

Publications and source records attributed to Gardner, M. R..

7 recordsLinked to original sources

bioRxiv2026

Co-delivered PD-L1 rescues the protective efficacy mediated by an AAV-expressed HIV-1 bNAb

Adeno-associated virus (AAV)-delivered anti-HIV-1 broadly neutralizing antibodies (bNAbs) could prevent and treat HIV-1 infection but are limited by host immune responses, specifically anti-drug antibodies (ADA). We tested whether PD-L1-mediated immune shielding could improve the consistency of AAV-delivered bNAb expression from muscle tissue in rhesus macaques. AAV9.PD-L1 co-delivery with AAV9.3BNC117 reduced the occurrence of ADA and T cell responses and improved the durability of 3BNC117 expression for one year post administration. Importantly, 5 of 6 macaques that received co-delivered AAV9.PD-L1 vectors were protected against ten repeated SHIVAD8-EO challenges. Histopathological and spatial transcriptomic profiling showed that AAV9.PD-L1 co-delivery prevented severe local inflammation, muscle injury, and tertiary lymphoid structure formation at the administration site. Thus, immune shielding could serve as a strategy to prolong transgene expression from muscle-directed AAV-delivered biologics.

immunology↗

bioRxiv2026

Overcoming host immune responses to an AAV-delivered HIV-1 bNAb in rhesus macaques mediated by co-delivery of PD-L1

Adeno-associated virus (AAV)-delivered anti-HIV-1 broadly neutralizing antibodies (bNAbs) have demonstrated promise for preventing and treating HIV-1 infection in preclinical models. However, host immune responses, specifically anti-drug antibodies (ADA), limit sustained bNAb expression. We have previously shown that PD-L1-mediated immune shielding improves the consistency of AAV-delivered bNAb 3BNC117 expression from muscle tissue in rhesus macaques. Here, we test the breadth of this approach with another bNAb, 10-1074. We show that AAV9.PD-L1 co-delivery with AAV9.10-1074 reduced the occurrence of ADA responses and improved the durability of bNAb expression for one year post administration. Notably 12 of 12 macaques that received AAV9.10-1074 vectors were protected against ten repeated SHIVAD8-EO challenges. Histopathological profiling showed that AAV9.PD-L1 co-delivery prevented severe local inflammation and tertiary lymphoid structure formation at the administration site. Thus, immune shielding could serve as a broad strategy to prolong transgene expression from muscle-directed AAV-delivered biologics.

immunology↗

bioRxiv2026

Characterization of expression elements for an AAV delivered antibody in nonhuman primates when co-delivered with PD-L1

Successful AAV-expressed antibody therapy for HIV-1 requires broadly neutralizing antibody (bNAbs) concentrations and reduced immune responses to sustain viral suppression without ART. We have previously demonstrated that co-delivery of AAV-expressed PD-L1 reduces immune responses against HIV-1 bNAbs in rhesus macaques. Here we systematically evaluated six AAV9 transgene cassettes encoding 10-1074 with different promoter/intron combinations (CMV, CMV/R, CBA, CASI, CB7, EF1) across in vitro systems, immune-deficient mice, and in rhesus macaques. We show that both promoter and species selection, leads to differences in 10-1074 concentrations with the CB7 promoter leading to greatest expression in mice and CMV/R promoter in macaques. In addition to differences observed, loss of 10-1074 serum concentrations in macaques resulted in higher anti-drug antibody responses and antigen specific IFN-y T cell responses were focused on the 10-1074 heavy-chain variable region. Furthermore, inclusion of the WPRE greatly impacted 10-1074 expression leading to higher concentrations in both mice and nonhuman primates. Lastly, circulating 10-1074 in macaques retained neutralizing activity against diverse HIV-1 pseudovirus isolates. Together these results demonstrate how expression elements influence AAV-expressed antibodies in the context of co-delivery and highlight the need for further improvements to AAV transgene cassettes when co-delivered with AAV expressed PD-L1.

microbiology↗

bioRxiv2026

Durable protection against SIV challenge by adeno-associated virus delivery of Env-specific antibodies

Conventional vaccines have so far failed to elicit the types of antibodies needed for protection against HIV. As an alternative, we evaluated adeno-associated virus (AAV) delivery of rhesus macaque antibodies to the SIV envelope glycoprotein for protection against SIV challenge. AAV vectors encoding a broadly neutralizing antibody (bnAb) and an antibody that only mediates antibody-dependent cellular cytotoxicity (ADCC) were administered individually or together to separate groups of rhesus macaques. Antibody expression was sustained for more than a year with minimal anti-drug antibody responses. All animals that received a control antibody or the ADCC-only antibody became infected after five low-dose, intrarectal challenges with SIVmac239. In contrast, 14 of 16 animals that received the bnAb resisted two rounds of twelve SIVmac239 challenges more than a year apart. Thus, AAV delivery of a single bnAb can afford durable protection against a pathogenic SIV strain that is notoriously difficult to protect against by vaccination.

immunology↗

bioRxiv2024

Adeno-associated viral delivery of Env-specific antibodies prevents SIV rebound after discontinuing antiretroviral therapy

An alternative to lifelong antiretroviral therapy (ART) is needed to achieve durable control of HIV-1. Here we show that adeno-associated virus (AAV)-delivery of two rhesus macaque antibodies to the SIV envelope glycoprotein (Env) with potent neutralization and antibody-dependent cellular cytotoxicity can prevent viral rebound in macaques infected with barcoded SIVmac239M after discontinuing suppressive ART. Following AAV administration, sustained antibody expression with minimal anti-drug antibody responses was achieved in all but one animal. After ART withdrawal, SIV replication rebounded within two weeks in all of the control animals but remained below the threshold of detection in plasma (<15 copies/mL) for more than a year in four of the eight animals that received AAV vectors encoding Env-specific antibodies. Viral sequences from animals with delayed rebound exhibited restricted barcode diversity and antibody escape. Thus, sustained expression of antibodies with potent antiviral activity can afford durable, ART-free containment of pathogenic SIV infection.

immunology↗

bioRxiv2020

A pentameric protein ring with novel architecture is required for herpesviral packaging

Genome packaging in large double-stranded DNA viruses requires a powerful molecular motor to force the viral genome into nascent capsids. This process appears mechanistically similar in two evolutionarily distant viruses, the herpesviruses and the tailed bacteriophages, which infect different kingdoms of life. While the motor and mechanism as a whole are thought to be conserved, accessory factors that influence packaging are divergent and poorly understood, despite their essential roles. An accessory factor required for herpesviral packaging is encoded by ORF68 in the oncogenic virus Kaposis sarcoma-associated herpesvirus (KSHV), whose homolog in Epstein Barr Virus (EBV) is BFLF1. Here, we present structures of both KSHV ORF68 and EBV BFLF1, revealing that these proteins form a highly similar homopentameric ring. The central channel of this ring is positively charged, and we demonstrate that this region of KSHV ORF68 binds double-stranded DNA. Mutation of individual positively charged residues within but not outside the channel ablates DNA binding, and in the context of KSHV infection these mutants fail to package the viral genome or produce progeny virions. Thus, we propose a model in which ORF68 facilitates the transfer of newly replicated viral genomes to the packaging motor.

microbiology↗

bioRxiv2018

Kaposi’s sarcoma-associated herpesvirus ORF68 is a DNA binding protein required for viral genome cleavage and packaging

Herpesviral DNA packaging into nascent capsids requires multiple conserved viral proteins that coordinate genome encapsidation. Here, we investigated the role of the ORF68 protein of Kaposis sarcoma-associated herpesvirus (KSHV), a protein required for viral DNA encapsidation whose function remains largely unresolved across the herpesviridae. We found that KSHV ORF68 is expressed with early kinetics and localizes predominantly to viral replication compartments, although it is dispensable for viral DNA replication and gene expression. However, in agreement with its proposed role in viral DNA packaging, KSHV-infected cells lacking ORF68 failed to cleave viral DNA concatemers, accumulated exclusively immature B-capsids, and released no infectious progeny virions. ORF68 has no predicted domains aside from a series of putative zinc finger motifs. However, in vitro biochemical analyses of purified ORF68 protein revealed that it robustly binds DNA and is associated with nuclease activity. These activities provide new insights into the role of KSHV ORF68 in viral genome encapsidation.\n\nImportanceKaposis sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposis sarcoma and several B-cell cancers, causing significant morbidity and mortality in immunocompromised individuals. A critical step in the production of infectious viral progeny is the packaging of the newly replicated viral DNA genome into the capsid, which involves coordination between at least seven herpesviral proteins. While the majority of these packaging factors have been well studied in related herpesviruses, the role of the KSHV ORF68 protein and its homologs remains unresolved. Here, using a KSHV mutant lacking ORF68, we confirm its requirement for viral DNA processing and packaging in infected cells. Furthermore, we show that the purified ORF68 protein directly binds DNA and is associated with a metal-dependent cleavage activity on double stranded DNA in vitro. These activities suggest a novel role for ORF68 in herpesviral genome processing and encapsidation.

molecular biology↗