John W. Mellors

Background: COVID‐19 and other respiratory viral infections can cause cardiovascular complications. SARS‐CoV‐2 virions are found in the blood, and circulating viral RNA levels are associated with death. We hypothesized that viremia can induce thrombotic endotheliopathy that contributes to death and studied this relationship in patients hospitalized for COVID‐19 and enrolled in the ACTIV‐4a (Accelerating COVID‐19 Therapeutic Interventions and Vaccines) randomized trial of antithrombotic therapy. Methods: We quantified SARS‐CoV‐2 nucleocapsid RNA and protein in plasma and measured their associations with clinical outcomes and biomarkers of thromboinflammation and endotheliopathy. We used Cox regression and Fine–Gray competing risk models to analyze survival and thrombosis. We conducted causal mediation analysis to explore whether thrombotic endotheliopathy mediates the relationship between viral RNA and death. Results: In 93 patients, SARS‐CoV‐2 RNA and N‐antigen were higher in nonsurvivors. Baseline viral RNA levels were associated with increased 90‐day death (hazard ratio [HR], 1.31 [95% CI, 1.17–1.46]) and thrombosis (HR, 1.35 [95% CI, 1.24–1.47]). Viral RNA more effectively predicted survivorship than N‐antigen levels. Soluble thrombomodulin, a biomarker of thrombotic endotheliopathy, positively correlated with viral RNA levels. Mediation analysis revealed that soluble thrombomodulin accounts for 12.2% (95% CI, 0.1%–32.3%; P =0.048) after adjustment for age and sex of the relationship between viral RNA and the 90‐day mortality rate. Conclusions: Elevated plasma SARS‐CoV‐2 RNA levels are associated with death in ACTIV‐4a, which is causally mediated in part by soluble thrombomodulin. We propose that lung–blood viral dissemination is a potential mechanism for cardiovascular complications of respiratory viruses. Registration: URL: https://www.clinicaltrials.gov ; Unique Identifier: NCT04505774.

BACKGROUND: Approaches to achieving antiretroviral therapy–free (ART-free) remission from HIV-1 must consider that people over 50 years now comprise the majority of people with HIV (PWH) on ART in various regions, including the United States. METHODS: We report a double-blind, randomized trial in which PWH on ART, aged 21–60 years, received modified vaccinia Ankara–vectored (MVA-vectored) vaccines, MVA.tHIVconsv3 (M3) and MVA.tHIVconsv4 (M4), either alone or in combination ( n = 7/group), or saline placebo ( n = 3). M3 and M4 contain complementary HIVconsvX immunogens that each span the same regions in HIV-1 Gag and Pol but differ by approximately 8% at the amino acid level. RESULTS: M3, M4, and M3M4 regimens were well tolerated and all significantly increased both the frequency (peak median increase ~3-fold) and breadth of the HIVconsvX-specific T cell response while redirecting T cells to target conserved regions in HIV-1 for up to 10 weeks after vaccination. We also demonstrated that vaccination increased frequencies of T cells targeting participant autologous HIV-1 sequences. Vaccination mostly expanded preexisting HIV-1–specific T cells and did not impact CD4 ⁺ T cell activation, low-level viremia, or integrated HIV-1 provirus. Linear regression indicated that age was independently and negatively associated with the change in T cell frequency at 1, 2, and 10 weeks after vaccination (~1.41-fold decrease per 10 years older). After adjusting for age, years on ART was positively associated with HIVconsvX-specific T cell frequencies at 1 and 2 weeks following vaccination. CONCLUSION: In PWH receiving ART, MVA.HIVconsvX vaccines significantly increased T cells targeting conserved regions of HIV-1. Novel strategies may be required to enhance anti–HIV-1 immunity in older adults. TRIAL REGISTRATION: ClinicalTrials.gov NCT03844386 FUNDING: NIH National Institute of Allergy and Infectious Diseases (NIAID) grants U01AI131310, HHSN272201100021I/HHSN27200037 (subcontract OX-14007.004.0037-212), UM1TR004406, P30AI050410, and P30CA016086; International AIDS Vaccine Initiative; European and Developing Countries Clinical Trials Partnership SRIA2015-1066; European Commission’s Horizon 2020 Research and Innovation Programme 681137.

Treatment with latency-reversing agents (LRAs) alone has been ineffective in reducing HIV-1 reservoirs in people living with HIV-1 (PLWH) who are on antiretroviral therapy (ART), due to inefficiencies in reservoir reactivation and adaptive immune responses. However, NK cells activated with cytokines may be able to target HIV-1 reservoirs more effectively. To explore the therapeutic potential of NK cells, we expanded blood NK cells from multiple donors ex vivo into CD56 ^bright CD16 ⁺ “eNK” cells using artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL21. eNK cells express multiple activating receptors and are highly cytotoxic against specific target cells. They can also kill HIV-infected CD4+ T cells via antibody-dependent cell-mediated cytotoxicity (ADCC) using broadly neutralizing antibodies (bNAbs) against HIV-1 Env gp120/gp41. Notably, eNK cells from PLWH on ART efficiently killed autologous HIV-1+ T cells reactivated by a combination of vorinostat (SAHA) and IL-15 or an IL-15 superagonist (N-803), as evidenced by declines in proviral load, inducible HIV-1 mRNA, and virus release. Adoptive immunotherapy with eNK cells combined with LRA treatment thus presents a promising strategy to reduce the latent HIV-1 reservoir in PLWH. IMPORTANCE: Antiretroviral therapy (ART) lowers HIV levels in the blood to nearly undetectable amounts, but stopping therapy almost always leads to HIV rebounding in the bloodstream. DNA and RNA tests show that most people living with HIV (PLWH) on ART retain long-lasting HIV reservoirs that remain hidden from the immune system when no HIV is being produced. Eradicating HIV might look like “drug-free remission,” where HIV reservoirs are kept under control by the immune system even if ART is stopped indefinitely. Current strategies for this potential eradication include using HIV latency-reversing agents (LRAs), ex vivo expansion of natural killer (NK) cells, and improving the ability to kill infected cells with broadly neutralizing antibodies against HIV. Here, we demonstrate that NK cells from PLWH can be expanded outside the body into “eNK” cells that specifically attack HIV-infected cells without harming uninfected ones, significantly reducing HIV reservoirs in vitro after LRA treatment.

NK and T cells are key effectors that eliminate cancer cells, but upregulation of the inhibitory receptor NKG2A on these cells attenuates antitumor immune responses. To counteract NKG2A inhibitory signaling, we identified two specific fully human monoclonal anti-NKG2A antibodies that block HLA-E ligand binding. These antibodies activated NK cells and enhanced antibody-dependent cellular cytotoxicity of tumor-targeting IgG1s both in vitro and in vivo. Bispecific engagers (BiNKs), generated by fusing NKG2A antibodies with tumor targeting binders, promoted immune synapse formation and directed cytotoxicity of NK and CD8 ⁺ T cells toward cancer cells. In a human PBMC-engrafted NSG mouse xenograft lung cancer model, an anti-HER2 × anti-NKG2A BiNK markedly inhibited tumor growth as a monotherapy or in combination with pertuzumab. Cell depletion studies revealed that the BiNK enhanced antitumor activity of both NK and T cells. NKG2A blockade with potent and specific, fully human antibodies and BiNKs show promise for further development as cancer immunotherapeutics.

Several nucleic acid and antigen assays have been developed to detect HIV in plasma at levels below the detection limit of standard clinical assays; however, comparative assessment of performance of these ultrasensitive assays on identical panels has been limited. Here, we report the relative performance characteristics of three manual (ultracentrifugation concentration and laboratory-developed PCR) and two automated commercial assays for single-copy detection of HIV RNA, as well as two ultrasensitive HIV p24 assays, using blinded panels of plasma samples containing HIV at low concentrations. Independent sample sets were studied in two phases: in the first phase, qualification panels consisted of analytic standards in serial dilution and clinical plasma samples from virologically suppressed people with HIV (PWH). HIV detection in clinical samples was infrequent using the ultrasensitive p24 assays (mean 11%). In contrast, a higher proportion of the same samples were detected using single-copy RNA assays (mean 61%). In the second phase, evaluation panels of clinical plasma samples ( n = 80) from virally suppressed PWH and low-copy analytic standards assessed performance of one automated and two manual single-copy RNA assays with the highest qualification phase detection frequencies. The automated assay performed comparably in three separate laboratories and consistently detected HIV RNA in over half (mean 57%) of samples from virally suppressed PWH, whereas the manual assays detected HIV in ≤40% of the same samples. An automated single-copy assay provides a scalable method for measuring residual HIV viremia that may be broadly useful in pathogenesis and cure-directed studies. IMPORTANCE: Most people with HIV (PWH) on antiretroviral therapy have viral loads below the detection limit of clinical assays, yet virus is often present and detectable at very low levels using ultrasensitive research assays. Clinical trials evaluating curative interventions and interpreting outcomes of analytical treatment interruptions depend on reliable assays to assess and quantify changes in HIV persistence often at very low levels. We conducted a two-stage head-to-head, blinded comparison of multiple ultrasensitive HIV RNA and p24 assays, first using 50 low viral load plasma samples, then further evaluating the top-performing assays on a 144-member blinded panel composed of duplicate contrived and clinical specimens from well-suppressed PWH. Single-copy RNA methods performed better than p24 assays, and a fully automated, 9-replicate commercial RNA assay demonstrated high sensitivity, reproducibility across laboratories, and practical scalability that can be applied to measure the impact of interventions in HIV cure trials.

HIV-1 breakthrough on long-acting cabotegravir (CAB-LA) for HIV prevention is rare but could impact viral suppression on integrase strand transfer inhibitor (INSTI)–based antiretroviral therapy (ART). We report the first study of ART outcomes following CAB-LA breakthrough during routine clinical care. Three individuals acquired HIV-1 on CAB-LA despite on-time injections; 2 had low-frequency major INSTI resistance mutations. All started darunavir-based ART; 1 subsequently switched to bictegravir-based ART. All maintained plasma HIV-1 RNA <50 copies/mL through ≥5 months post–ART initiation, providing early evidence of virologic success with standard ART regimens and paving the way for longer-term studies on optimal ART after CAB-LA breakthrough.

Background: Macrophages play an important role in eliminating diseased and damaged cells through programmed cell death. Signal regulatory protein alpha (SIRPα) is a crucial immune checkpoint primarily expressed on myeloid cells and macrophages. It initiates a ‘do not eat me’ signal when engaged with CD47, which is typically expressed at elevated levels on multiple solid tumors. The phospholipase A2 Group 7 (PLA2G7), which is mainly secreted by macrophages, interacts with oxidized low-density lipoprotein (oxLDL) and associates with several vascular diseases and cancers. Methods: To identify potent fully human monoclonal antibodies (mAbs) against human SIRPα and PLA2G7, we conducted bio-panning of phage antibody libraries. Results: We isolated one human Fab (1B3) and VH (1A3) for SIRPα, as well as one human Fab (1H8) and one VH (1A9) for PLA2G7; the 1B3 Fab and 1A3 VH are competitively bound to SIRPα, interfering with CD47 binding. The 1B3 IgG and 1A3 VH-Fc augmented macrophage-mediated phagocytic activity when combined with the anti-EGFR antibody, cetuximab. The anti-PLA2G7 antibodies exhibited high specificity for the PLA2G7 antigen and effectively blocked the PLA2G7 enzymatic activity with half-maximal inhibitory concentrations (IC₅₀) in the single-digit nanomolar range. Additionally, 1H8 IgG and its derivative bispecific antibody exhibited the ability to block PLA2G7-mediated tumor cell migration. Conclusions: Our anti-SIRPα mAbs are expected to serve as potent and fully human immune checkpoint inhibitors of SIRPα, enhancing the antitumor responses of SIRPα-positive immune cells. Moreover, our anti-PLA2G7 mAbs represent promising fully human PLA2G7 enzymatic blockade antibodies with the potential to enhance both anti-tumor and anti-aging responses. Anti-SIRPα and PLA2G7 mAbs can modulate macrophage phagocytic activity and inflammatory responses against tumors.

Therapeutic antibody design has garnered widespread attention, highlighting its interdisciplinary importance. Advancements in technology emphasize the critical role of designing nanobodies and humanized antibodies in antibody engineering. However, current experimental methods are costly and time-consuming. Computational approaches, while progressing, faced limitations due to insufficient structural data and the absence of a standardized protocol. To tackle these challenges, our lab previously developed IsAb1.0, an in silico antibody design protocol. Yet, IsAb1.0 lacked accuracy, had a complex procedure, and required extensive antibody bioinformation. Moreover, it overlooked nanobody and humanized antibody design, hindering therapeutic antibody development. Building upon IsAb1.0, we enhanced our design protocol with artificial intelligence methods to create IsAb2.0. IsAb2.0 utilized AlphaFold-Multimer (2.3/3.0) for accurate modeling and complex construction without templates and employed the precise FlexddG method for in silico antibody optimization. Validated through optimization of a humanized nanobody J3 (HuJ3) targeting HIV-1 gp120, IsAb2.0 predicted five mutations that can improve HuJ3-gp120 binding affinity. These predictions were confirmed by commercial software and validated through binding and neutralization assays. IsAb2.0 streamlined antibody design, offering insights into future techniques to accelerate immunotherapy development.

The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length antibody including deeper tumor penetration, cost-effective production and fast washout from normal tissues. In this study, we identified a human immunoglobulin heavy chain variable domain (VH domain) (VH20) from an in-house phage library. VH20 exhibits good developability and high specificity with no off-target binding to ~6000 human membrane proteins. VH20 efficiently bound to the glycine-rich region of ALK with an EC₅₀ of 0.4 nM and a KD of 6.54 nM. Both VH20-based bispecific T cell engager (TCE) and chimeric antigen receptor T cells (CAR Ts) exhibited potent cytolytic activity to ALK-expressing tumor cells in an ALK-dependent manner. VH20 CAR Ts specifically secreted proinflammatory cytokines including IL-2, TNFα and IFNγ after incubation with ALK-positive cells. To our knowledge, this is the first reported human single-domain antibody against ALK. Our in vitro characterization data indicate that VH20 could be a promising ALK-targeting sdAb with potential applications in ALK-expressing tumors, including neuroblastoma (NBL) and non-small cell lung cancer.