Lisa H. Tostanoski

Allogeneic cell therapies can enable off-the-shelf products that address limitations of autologous therapies. Mesenchymal stem cells are a robust allogeneic source, but no bioengineered mesenchymal stem cell-based therapies exist. Here we use mRNA engineering to create an off-the-shelf immunotherapy that we term DC-25. DC-25 consists of a mesenchymal stem cell armed with three designed mRNA constructs encoding CXCR4 to direct migration, a T cell engager specific for B cell maturation antigen to target B cell maturation antigen-expressing plasma cells involved in cancer and autoimmunity, and interleukin-12 to potentiate pro-immune responses. DC-25 allows tunable expression of each gene, supporting a predictable pharmacokinetic profile. In vitro, DC-25 exhibits synergistic killing of target cells, and in a preclinical in vivo myeloma model, this therapy exhibits potent efficacy that surpasses T cell engager protein infusion. In a phase 1 safety study in patients with myeloma, DC-25 appears safe and generates interleukin-12 production after each infusion. This study motivates human cell therapies that exploit mRNA to achieve efficacy through induction of secreted or surface-bound therapeutic elements.

Medical interventions often require timed series of doses, thus necessitating accurate medical record-keeping. In many global settings, these records are unreliable or unavailable at the point of care, leading to less effective treatments or disease prevention. Here we present an invisible-to-the-naked-eye on-patient medical record-keeping technology that accurately stores medical information in the patient skin as part of microneedles that are used for intradermal therapeutics. We optimize the microneedle design for both a reliable delivery of messenger RNA (mRNA) therapeutics and the near-infrared fluorescent microparticles that encode the on-patient medical record-keeping. Deep learning-based image processing enables encoding and decoding of the information with excellent temporal and spatial robustness. Long-term studies in a swine model demonstrate the safety, efficacy and reliability of this approach for the co-delivery of on-patient medical record-keeping and the mRNA vaccine encoding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This technology could help healthcare workers make informed decisions in circumstances where reliable record-keeping is unavailable, thus contributing to global healthcare equity.

Nanomaterials offer unique opportunities to engineer immunomodulatory activity. In this work, we report the Toll-like receptor agonist activity of a nanoscale adjuvant zeolitic imidazolate framework–8 (ZIF-8). The accumulation of ZIF-8 in endosomes and the pH-responsive release of its subunits enable selective engagement with endosomal Toll-like receptors, minimizing the risk of off-target activation. The intrinsic adjuvant properties of ZIF-8, along with the efficient delivery and biomimetic presentation of a severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain trimer, primed rapid humoral and cell-mediated immunity in a dose-sparing manner. Our study offers insights for next-generation adjuvants that can potentially impact future vaccine development.

We report the direct comparison of monomeric, dimeric and trimeric RBD protein subunit vaccines to a virus-like particle (VLP) displaying RBD. After two and three doses, a RBD dimer and trimer elicited antibody levels in mice comparable to an RBD-VLP. Furthermore, an Omicron (BA.1) RBD hetero-dimer induced neutralizing activity similar to the RBD-VLP. A RBD hetero-dimer and RBD-VLP also shows comparable breadth to other SARS-CoV-2 variants-of-concern (VOCs).

Decentralized manufacture of thermostable mRNA vaccines in a microneedle patch (MNP) format could enhance vaccine access in low-resource communities by eliminating the need for a cold chain and trained healthcare personnel. Here we describe an automated process for printing MNP Coronavirus Disease 2019 (COVID-19) mRNA vaccines in a standalone device. The vaccine ink is composed of lipid nanoparticles loaded with mRNA and a dissolvable polymer blend that was optimized for high bioactivity by screening formulations in vitro. We demonstrate that the resulting MNPs are shelf stable for at least 6 months at room temperature when assessed using a model mRNA construct. Vaccine loading efficiency and microneedle dissolution suggest that efficacious, microgram-scale doses of mRNA encapsulated in lipid nanoparticles could be delivered with a single patch. Immunizations in mice using manually produced MNPs with mRNA encoding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain stimulate long-term immune responses similar to those of intramuscular administration.

Antigen-specific tolerance is a key goal of experimental immunotherapies for autoimmune disease and allograft rejection. This outcome could selectively inhibit detrimental inflammatory immune responses without compromising functional protective immunity. A major challenge facing antigen-specific immunotherapies is ineffective control over immune signal targeting and integration, limiting efficacy and causing systemic non-specific suppression. Here we use intra-lymph node injection of diffusion-limited degradable microparticles that encapsulate self-antigens with the immunomodulatory small molecule, rapamycin. We show this strategy potently inhibits disease during pre-clinical type 1 diabetes and allogenic islet transplantation. Antigen and rapamycin are required for maximal efficacy, and tolerance is accompanied by expansion of antigen-specific regulatory T cells in treated and untreated lymph nodes. The antigen-specific tolerance in type 1 diabetes is systemic but avoids non-specific immune suppression. Further, microparticle treatment results in the development of tolerogenic structural microdomains in lymph nodes. Finally, these local structural and functional changes in lymph nodes promote memory markers among antigen-specific regulatory T cells, and tolerance that is durable. This work supports intra-lymph node injection of tolerogenic microparticles as a powerful platform to promote antigen-dependent efficacy in type 1 diabetes and allogenic islet transplantation.

The COVID-19 pandemic marks the third coronavirus pandemic this century (SARS-CoV-1, MERS, SARS-CoV-2), emphasizing the need to identify and evaluate conserved immunogens for a pan-sarbecovirus vaccine. Here we investigate the potential utility of a T-cell vaccine strategy targeting conserved regions of the sarbecovirus proteome. We identified the most conserved regions of the sarbecovirus proteome as portions of the RNA-dependent RNA polymerase (RdRp) and Helicase proteins, both of which are part of the coronavirus replication transcription complex (RTC). Fitness constraints suggest that as SARS-CoV-2 continues to evolve these regions may better preserve cross-reactive potential of T-cell responses than Spike, Nucleocapsid, or Membrane proteins. We sought to determine if vaccine-elicited T-cell responses to the highly conserved regions of the RTC would reduce viral loads following challenge with SARS-CoV-2 in mice using a rhesus adenovirus serotype 52 (RhAd52) vector. The RhAd52.CoV.Consv vaccine generated robust cellular immunity in mice and led to significant reductions in viral loads in the nasal turbinates following challenge with a mouse-adapted SARS-CoV-2. These data suggest the potential utility of T-cell targeting of conserved regions for a pan-sarbecovirus vaccine.

There is a need to standardize pathologic endpoints in animal models of SARS-CoV-2 infection to help benchmark study quality, improve cross-institutional comparison of data, and assess therapeutic efficacy so that potential drugs and vaccines for SARS-CoV-2 can rapidly advance. The Syrian hamster model is a tractable small animal model for COVID-19 that models clinical disease in humans. Using the hamster model, the authors used traditional pathologic assessment with quantitative image analysis to assess disease outcomes in hamsters administered polyclonal immune sera from previously challenged rhesus macaques. The authors then used quantitative image analysis to assess pathologic endpoints across studies performed at different institutions using different tissue processing protocols. The authors detail pathological features of SARS-CoV-2 infection longitudinally and use immunohistochemistry to quantify myeloid cells and T lymphocyte infiltrates during SARS-CoV-2 infection. High-dose immune sera protected hamsters from weight loss and diminished viral replication in tissues and reduced lung lesions. Cumulative pathology scoring correlated with weight loss and was robust in distinguishing IgG efficacy. In formalin-infused lungs, quantitative measurement of percent area affected also correlated with weight loss but was less robust in non-formalin-infused lungs. Longitudinal immunohistochemical assessment of interstitial macrophage infiltrates showed that peak infiltration corresponded to weight loss, yet quantitative assessment of macrophage, neutrophil, and CD3+ T lymphocyte numbers did not distinguish IgG treatment effects. Here, the authors show that quantitative image analysis was a useful adjunct tool for assessing SARS-CoV-2 treatment outcomes in the hamster model.

Despite the rapid creation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines, the precise correlates of immunity against severe Coronavirus Disease 2019 (COVID-19) are still unknown. Neutralizing antibodies represent a robust surrogate of protection in early Phase III studies, but vaccines provide protection prior to the evolution of neutralization, vaccines provide protection against variants that evade neutralization, and vaccines continue to provide protection against disease severity in the setting of waning neutralizing titers. Thus, in this study, using an Ad26.CoV2.S dose-down approach in nonhuman primates (NHPs), the role of neutralization, Fc effector function, and T-cell immunity were collectively probed against infection as well as against viral control. While dosing-down minimally impacted neutralizing and binding antibody titers, Fc receptor binding and functional antibody levels were induced in a highly dose-dependent manner. Neutralizing antibody and Fc receptor binding titers, but minimally T cells, were linked to the prevention of transmission. Conversely, Fc receptor binding/function and T cells were linked to antiviral control, with a minimal role for neutralization. These data point to dichotomous roles of neutralization and T-cell function in protection against transmission and disease severity and a continuous role for Fc effector function as a correlate of immunity key to halting and controlling SARS-CoV-2 and emerging variants.

Authorized vaccines against SARS-CoV-2 remain less available in low- and middle-income countries due to insufficient supply, high costs, and storage requirements. Global immunity could still benefit from new vaccines using widely available, safe adjuvants, such as alum and protein subunits, suited to low-cost production in existing manufacturing facilities. Here, a clinical-stage vaccine candidate comprising a SARS-CoV-2 receptor binding domain–hepatitis B surface antigen virus–like particle elicited protective immunity in cynomolgus macaques. Titers of neutralizing antibodies (>10 ⁴ ) induced by this candidate were above the range of protection for other licensed vaccines in nonhuman primates. Including CpG 1018 did not significantly improve the immunological responses. Vaccinated animals challenged with SARS-CoV-2 showed reduced median viral loads in bronchoalveolar lavage (~3.4 log ₁₀ ) and nasal mucosa (~2.9 log ₁₀ ) versus sham controls. These data support the potential benefit of this design for a low-cost modular vaccine platform for SARS-CoV-2 and other variants of concern or betacoronaviruses.