Altered Response Pattern following AZD5582 Treatment of SIV-Infected, ART-Suppressed Rhesus Macaque Infants.

The "shock and kill" strategy for HIV-1 cure incorporates latency-reversing agents (LRA) in combination with interventions that aid the host immune system in clearing virally reactivated cells. LRAs have not yet been investigated in pediatric clinical or preclinical studies. Here, we evaluated an inhibitor of apoptosis protein (IAP) inhibitor (IAPi), AZD5582, that activates the noncanonical NF-κB (ncNF-κB) signaling pathway to reverse latency. Ten weekly doses of AZD5582 were intravenously administered at 0.1 mg/kg to rhesus macaque (RM) infants orally infected with SIVmac251 at 4 weeks of age and treated with a triple ART regimen for over 1 year. During AZD5582 treatment, on-ART viremia above the limit of detection (LOD, 60 copies/mL) was observed in 5/8 infant RMs starting at 3 days post-dose 4 and peaking at 771 copies/mL. Of the 135 measurements during AZD5582 treatment in these 5 RM infants, only 8 were above the LOD (6%), lower than the 46% we have previously reported in adult RMs. Pharmacokinetic analysis of plasma AZD5582 levels revealed a lower Cmax in treated infants compared to adults (294 ng/mL versus 802 ng/mL). RNA-Sequencing of CD4+ T cells comparing pre- and post-AZD5582 dosing showed many genes that were similarly upregulated in infants and adults, but the expression of key ncNF-κB genes, including NFKB2 and RELB, was significantly higher in adult RMs. Our results suggest that dosing modifications for this latency reversal approach may be necessary to maximize virus reactivation in the pediatric setting for successful "shock and kill" strategies. IMPORTANCE While antiretroviral therapy (ART) has improved HIV-1 disease outcome and reduced transmission, interruption of ART results in rapid viral rebound due to the persistent latent reservoir. Interventions to reduce the viral reservoir are of critical importance, especially for children who must adhere to lifelong ART to prevent disease progression. Here, we used our previously established pediatric nonhuman primate model of oral SIV infection to evaluate AZD5582, identified as a potent latency-reversing agent in adult macaques, in the controlled setting of daily ART. We demonstrated the safety of the IAPi AZD5582 and evaluate the pharmacokinetics and pharmacodynamics of repeated dosing. The response to AZD5582 in macaque infants differed from what we previously showed in adult macaques with weaker latency reversal in infants, likely due to altered pharmacokinetics and less inducibility of infant CD4+ T cells. These data supported the contention that HIV-1 cure strategies for children are best evaluated using pediatric model systems.

A structural homology approach to identify potential cross-reactive antibody responses following SARS-CoV-2 infection.

The emergence of the novel SARS-CoV-2 virus is the most important public-health issue of our time. Understanding the diverse clinical presentations of the ensuing disease, COVID-19, remains a critical unmet need. Here we present a comprehensive listing of the diverse clinical indications associated with COVID-19. We explore the theory that anti-SARS-CoV-2 antibodies could cross-react with endogenous human proteins driving some of the pathologies associated with COVID-19. We describe a novel computational approach to estimate structural homology between SARS-CoV-2 proteins and human proteins. Antibodies are more likely to interrogate 3D-structural epitopes than continuous linear epitopes. This computational workflow identified 346 human proteins containing a domain with high structural homology to a SARS-CoV-2 Wuhan strain protein. Of these, 102 proteins exhibit functions that could contribute to COVID-19 clinical pathologies. We present a testable hypothesis to delineate unexplained clinical observations vis-à-vis COVID-19 and a tool to evaluate the safety-risk profile of potential COVID-19 therapies.

Factor VIII-Fc Activates Natural Killer Cells via Fc-Mediated Interactions With CD16.

The most challenging complication associated with Factor VIII (FVIII) replacement therapy is the development of neutralizing anti-drug antibodies, or inhibitors, which occur in 23-35% of severe (FVIII level <1%) hemophilia A (HA) patients and are a serious hindrance to effective management of HA. Consequently, strategies that can either prevent anti-FVIII inhibitors from developing or "tolerize" individuals who develop such antibodies represent a clinically important unmet need. One intervention for patients with high-titer inhibitors is immune tolerance induction (ITI) therapy. Although ITI therapy is the only clinically proven strategy to eradicate anti-FVIII inhibitors, mechanisms of inhibitor reduction remain unknown. Factor VIII Fc-fusion (rFVIIIFc) is an enhanced half-life antihemophilic factor used in replacement therapy for HA. Fc-fusion is a successful protein bio-engineering platform technology. In addition to enhancement of plasma half-life via neonatal Fc receptor (FcRn) binding, other Fc-mediated interactions, including engagement with Fc gamma receptors (FcγR), may have immunological consequences. Several case reports and retrospective analyses suggest that rFVIIIFc offers superior outcomes with respect to ITI compared to other FVIII products. Previously we and others demonstrated rFVIIIFc interactions with activating FcγRIIIA/CD16. Here, we investigated if rFVIIIFc activates natural killer (NK) cells via CD16. We demonstrated rFVIIIFc signaling via CD16 independent of Von Willebrand Factor (VWF):FVIII complex formation. We established that rFVIIIFc potently activated NK cells in a CD16-dependent fashion resulting in IFNγ secretion and cytolytic perforin and granzyme B release. We also demonstrated an association between rFVIIIFc-mediated NK cell IFNγ secretion levels and the high-affinity (158V) CD16 genotype. Furthermore, we show that rFVIIIFc-activated CD16+ NK cells were able to lyse a B-cell clone (BO2C11) bearing an anti-FVIII B-cell receptor in an antibody-dependent cellular cytotoxicity (ADCC) assay. These in vitro findings provide an underlying molecular mechanism that may help explain clinical case reports and retrospective studies suggesting rFVIIIFc may be more effective in tolerizing HA patients with anti-FVIII inhibitors compared to FVIII not linked to Fc. Our in vitro findings suggest a potential use of Fc-fusion proteins acting via NK cells to target antigen-specific B-cells, in the management of unwanted immune responses directed against immunogenic self-antigens or therapeutic protein products.

Cas9-derived peptides presented by MHC Class II that elicit proliferation of CD4+ T-cells.

CRISPR-Cas9 mediated genome editing offers unprecedented opportunities for treating human diseases. There are several reports that demonstrate pre-existing immune responses to Cas9 which may have implications for clinical development of CRISPR-Cas9 mediated gene therapy. Here we use 209 overlapping peptides that span the entire sequence of Staphylococcus aureus Cas9 (SaCas9) and human peripheral blood mononuclear cells (PBMCs) from a cohort of donors with a distribution of Major Histocompatibility Complex (MHC) alleles comparable to that in the North American (NA) population to identify the immunodominant regions of the SaCas9 protein. We also use an MHC Associated Peptide Proteomics (MAPPs) assay to identify SaCas9 peptides presented by MHC Class II (MHC-II) proteins on dendritic cells. Using these two data sets we identify 22 SaCas9 peptides that are both presented by MHC-II proteins and stimulate CD4+ T-cells.

Performance of laser sintered Ti-6Al-4V implants with bone-inspired porosity and micro/nanoscale surface roughness in the rabbit femur.

Long term success of bone-interfacing implants remains a challenge in compromised patients and in areas of low bone quality. While surface roughness at the micro/nanoscale can promote osteogenesis, macro-scale porosity is important for promoting mechanical stability of the implant over time. Currently, machining techniques permit pores to be placed throughout the implant, but the pores are generally uniform in dimension. The advent of laser sintering provides a way to design and manufacture implants with specific porosity and variable dimensions at high resolution. This approach enables production of metal implants that mimic complex geometries found in biology. In this study, we used a rabbit femur model to compare osseointegration of laser sintered solid and porous implants. Ti-6Al-4V implants were laser sintered in a clinically relevant size and shape. One set of implants had a novel porosity based on human trabecular bone; both sets had grit-blasted/acid-etched surfaces. After characterization, implants were inserted transaxially into rabbit femora; mechanical testing, micro-computed tomography (microCT) and histomorphometry were conducted 10 weeks post-operatively. There were no differences in pull-out strength or bone-to-implant contact. However, both microCT and histomorphometry showed significantly higher new bone volume for porous compared to solid implants. Bone growth was observed into porous implant pores, especially near apical portions of the implant interfacing with cortical bone. These results show that laser sintered Ti-6Al-4V implants with micro/nanoscale surface roughness and trabecular bone-inspired porosity promote bone growth and may be used as a superior alternative to solid implants for bone-interfacing implants.