Tuning the immune response: sulfated archaeal glycolipid archaeosomes as an effective vaccine adjuvant for induction of humoral and cell-mediated immunity towards the SARS-CoV-2 Omicron variant of concern.

Liposomes composed of sulfated lactosyl archaeol (SLA) have been shown to be a safe and effective vaccine adjuvant with a multitude of antigens in preclinical studies. In particular, SLA-adjuvanted SARS-CoV-2 subunit vaccines based on trimeric spike protein antigens were shown to be immunogenic and efficacious in mice and hamsters. With the continued emergence of SARS-CoV-2 variants, we sought to evaluate next-generation vaccine formulations with an updated antigenic identity. This was of particular interest for the widespread Omicron variant, given the abundance of mutations and structural changes observed within its spike protein compared to other variants. An updated version of our resistin-trimerized SmT1 corresponding to the B.1.1.529 variant was successfully generated in our Chinese Hamster Ovary (CHO) cell-based antigen production platform and characterized, revealing some differences in protein profile and ACE2 binding affinity as compared to reference strain-based SmT1. We next evaluated this Omicron-based spike antigen for its immunogenicity and ability to generate robust antigen-specific immune responses when paired with SLA liposomes or AddaS03 (a mimetic of the AS03 oil-in-water emulsion adjuvant system found in commercialized SARS-CoV-2 protein vaccines). Immunization of mice with vaccine formulations containing this updated antigen with either adjuvant stimulated neutralizing antibody responses favouring Omicron over the reference strain. Cell-mediated responses, which play an important role in the neutralization of intracellular infections, were induced to a much higher degree with the SLA adjuvant relative to the AddaS03-adjuvanted formulations. As such, updated vaccines that are better capable of targeting towards SARS-CoV-2 variants can be generated through an optimized combination of antigen and adjuvant components.

Immunogenicity of SARS-CoV-2 spike antigens derived from Beta & Delta variants of concern.

Using our strongly immunogenic SmT1 SARS-CoV-2 spike antigen platform, we developed antigens based on the Beta & Delta variants of concern (VOC). These antigens elicited higher neutralizing antibody activity to the corresponding variant than comparable vaccine formulations based on the original reference strain, while a multivalent vaccine generated cross-neutralizing activity in all three variants. This suggests that while current vaccines may be effective at reducing severe disease to existing VOC, variant-specific antigens, whether in a mono- or multivalent vaccine, may be required to induce optimal immune responses and reduce infection against arising variants.

Structure-based dual affinity optimization of a SARS-CoV-1/2 cross-reactive single-domain antibody.

The SARS coronavirus 2 (SARS-CoV-2) spike (S) protein binding to the human ACE2 receptor is the molecular event that initiates viral entry into host cells and leads to infection and virus replication. There is a need for agents blocking viral entry into host cells that are cross-reactive with emerging virus variants. VHH-72 is an anti-SARS-CoV-1 single-domain antibody that also exhibits cross-specificity with SARS-CoV-2 but with decreased binding affinity. Here we applied a structure-based approach to affinity-mature VHH-72 for the SARS-CoV-2 spike protein while retaining the original affinity for SARS-CoV-1. This was achieved by employing the computational platform ADAPT in a constrained dual-affinity optimization mode as a means of broadening specificity. Select mutants designed by ADAPT were formatted as fusions with a human IgG1-Fc fragment. These mutants demonstrated improved binding to the SARS-CoV-2 spike protein due to decreased dissociation rates. Functional testing for virus neutralization revealed improvements relative to the parental VHH72-Fc up to 10-fold using a SARS-CoV-2 pseudotyped lentivirus and 20-fold against the SARS-CoV-2 authentic live virus (Wuhan variant). Binding and neutralization improvements were maintained for some other SARS-CoV-2 variants currently in circulation. These improved VHH-72 mutants are predicted to establish novel interactions with the S antigen. They will be useful, alone or as fusions with other functional modules, in the global quest for treatments of COVID-19 infections.

Microenvironment-triggered multimodal precision diagnostics.

Therapeutic outcomes in oncology may be aided by precision diagnostics that offer early detection, localization and the opportunity to monitor response to therapy. Here, we report a multimodal nanosensor engineered to target tumours through acidosis, respond to proteases in the microenvironment to release urinary reporters and (optionally) carry positron emission tomography probes to enable localization of primary and metastatic cancers in mouse models of colorectal cancer. We present a paradigm wherein this multimodal sensor can be employed longitudinally to assess burden of disease non-invasively, including tumour progression and response to chemotherapy. Specifically, we showed that acidosis-mediated tumour insertion enhanced on-target release of matrix metalloproteinase-responsive reporters in urine. Subsequent on-demand loading of the radiotracer 64Cu allowed pH-dependent tumour visualization, enabling enriched microenvironmental characterization when compared with the conventional metabolic tracer 18F-fluorodeoxyglucose. Through tailored target specificities, this modular platform has the capacity to be engineered as a pan-cancer test that may guide treatment decisions for numerous tumour types.

Structure-based engineering of pH-dependent antibody binding for selective targeting of solid-tumor microenvironment.

Recent development of monoclonal antibodies as mainstream anticancer agents demands further optimization of their safety for use in humans. Potent targeting and/or effector activities on normal tissues is an obvious toxicity concern. Optimization of specific tumor targeting could be achieved by taking advantage of the extracellular acidity of solid tumors relative to normal tissues. Here, we applied a structure-based computational approach to engineer anti-human epidermal growth factor receptor 2 (Her2) antibodies with selective binding in the acidic tumor microenvironment. We used an affinity maturation platform in which dual-pH histidine-scanning mutagenesis was implemented for pH selectivity optimization. Testing of a small set of designs for binding to the recombinant Her2 ectodomain led to the identification of antigen-binding fragment (Fab) variants with the desired pH-dependent binding behavior. Binding selectivity toward acidic pH was improved by as much as 25-fold relative to the parental bH1-Fab. In vitro experiments on cells expressing intact Her2 confirmed that designed variants formatted as IgG1/k full-size antibodies have high affinity and inhibit the growth of tumor spheroids at a level comparable to that of the benchmark anti-Her2 antibody trastuzumab (Herceptin®) at acidic pH, whereas these effects were significantly reduced at physiological pH. In contrast, both Herceptin and the parental bH1 antibody exhibited strong cell binding and growth inhibition irrespective of pH. This work demonstrates the feasibility of computational optimization of antibodies for selective targeting of the acidic environment such as that found in many solid tumors.

Acidification of Tumor at Stromal Boundaries Drives Transcriptome Alterations Associated with Aggressive Phenotypes.

Acidosis is a fundamental feature of the tumor microenvironment, which directly regulates tumor cell invasion by affecting immune cell function, clonal cell evolution, and drug resistance. Despite the important association of tumor microenvironment acidosis with tumor cell invasion, relatively little is known regarding which areas within a tumor are acidic and how acidosis influences gene expression to promote invasion. Here, we injected a labeled pH-responsive peptide to mark acidic regions within tumors. Surprisingly, acidic regions were not restricted to hypoxic areas and overlapped with highly proliferative, invasive regions at the tumor-stroma interface, which were marked by increased expression of matrix metalloproteinases and degradation of the basement membrane. RNA-seq analysis of cells exposed to low pH conditions revealed a general rewiring of the transcriptome that involved RNA splicing and enriched for targets of RNA binding proteins with specificity for AU-rich motifs. Alternative splicing of Mena and CD44, which play important isoform-specific roles in metastasis and drug resistance, respectively, was sensitive to histone acetylation status. Strikingly, this program of alternative splicing was reversed in vitro and in vivo through neutralization experiments that mitigated acidic conditions. These findings highlight a previously underappreciated role for localized acidification of tumor microenvironment in the expression of an alternative splicing-dependent tumor invasion program. SIGNIFICANCE: This study expands our understanding of acidosis within the tumor microenvironment and indicates that acidosis induces potentially therapeutically actionable changes to alternative splicing.

Characterization of the expression of the pro-metastatic Mena(INV) isoform during breast tumor progression.

Several functionally distinct isoforms of the actin regulatory Mena are produced by alternative splicing during tumor progression. Forced expression of the Mena(INV) isoform drives invasion, intravasation and metastasis. However, the abundance and distribution of endogenously expressed Mena(INV) within primary tumors during progression remain unknown, as most studies to date have only assessed relative mRNA levels from dissociated tumor samples. We have developed a Mena(INV) isoform-specific monoclonal antibody and used it to examine Mena(INV) expression patterns in mouse mammary and human breast tumors. Mena(INV) expression increases during tumor progression and to examine the relationship between Mena(INV) expression and markers for epithelial or mesenchymal status, stemness, stromal cell types and hypoxic regions. Further, while Mena(INV) robustly expressed in vascularized areas of the tumor, it is not confined to cells adjacent to blood vessels. Altogether, these data demonstrate the specificity and utility of the anti-Mena(INV)-isoform specific antibody, and provide the first description of endogenous Mena(INV) protein expression in mouse and human tumors.

Ephrin-Eph signaling in embryonic tissue separation.

The physical separation of the embryonic regions that give rise to the tissues and organs of multicellular organisms is a fundamental aspect of morphogenesis. Pioneer experiments by Holtfreter had shown that embryonic cells can sort based on "tissue affinities," which have long been considered to rely on differences in cell-cell adhesion. However, vertebrate embryonic tissues also express a variety of cell surface cues, in particular ephrins and Eph receptors, and there is now firm evidence that these molecules are systematically used to induce local repulsion at contacts between different cell types, efficiently preventing mixing of adjacent cell populations.

Variable combinations of specific ephrin ligand/Eph receptor pairs control embryonic tissue separation.

Ephrins and Eph receptors are involved in the establishment of vertebrate tissue boundaries. The complexity of the system is puzzling, however in many instances, tissues express multiple ephrins and Ephs on both sides of the boundary, a situation that should in principle cause repulsion between cells within each tissue. Although co-expression of ephrins and Eph receptors is widespread in embryonic tissues, neurons, and cancer cells, it is still unresolved how the respective signals are integrated into a coherent output. We present a simple explanation for the confinement of repulsion to the tissue interface: Using the dorsal ectoderm-mesoderm boundary of the Xenopus embryo as a model, we identify selective functional interactions between ephrin-Eph pairs that are expressed in partial complementary patterns. The combined repulsive signals add up to be strongest across the boundary, where they reach sufficient intensity to trigger cell detachments. The process can be largely explained using a simple model based exclusively on relative ephrin and Eph concentrations and binding affinities. We generalize these findings for the ventral ectoderm-mesoderm boundary and the notochord boundary, both of which appear to function on the same principles. These results provide a paradigm for how developmental systems may integrate multiple cues to generate discrete local outcomes.

EpCAM controls actomyosin contractility and cell adhesion by direct inhibition of PKC.

Epithelial cell adhesion molecule (EpCAM) is a cell-surface protein highly expressed in embryonic tissues and in malignant carcinomas. We report that EpCAM acts as a potent inhibitor of novel protein kinase C (nPKC) in both embryos and cancer cells. We observed dramatic effects of loss of EpCAM on amphibian embryonic tissues, which include sequentially strong overstimulation of PKC activity and of the Erk pathway, leading to exacerbated myosin contractility, loss of cadherin-mediated adhesion, tissue dissociation, and, ultimately, cell death. We show that PKC inhibition is caused by a short segment of the EpCAM cytoplasmic tail. This motif resembles the pseudosubstrate inhibitory domains of PKCs and binds nPKCs with high affinity. A bioinformatics search reveals the existence of similar motifs in other plasma membrane proteins, most of which are cell-cell adhesion molecules. Thus, direct inhibition of PKC by EpCAM represents a general mode of regulation of signal transduction by cell-surface proteins.

A molecular base for cell sorting at embryonic boundaries: contact inhibition of cadherin adhesion by ephrin/ Eph-dependent contractility.

The mechanism responsible for subdividing the embryo into individual tissues is a fundamental, yet still poorly understood, question in developmental biology. Various general hypotheses have been proposed, involving differences in cell adhesion, contractility, or contact-mediated repulsion. However, the key parameter in tissue separation, i.e., the regulation of cadherin-based adhesion at the boundary, has not yet been investigated. We show that cadherin clustering is specifically inhibited at the vertebrate notochord-presomitic mesoderm boundary, preventing formation of adhesive bonds between cells of the two different types. This local regulation depends on differentially expressed ephrins and Eph receptors, which increase cell contractility and generate a membrane blebbing-like behavior along the boundary. Inhibiting myosin activity is sufficient to induce cadherin clustering and formation of stable contacts across the boundary, causing notochord and presomitic tissues to fuse. Local inhibition of cadherin adhesion explains how sharp separation can be achieved in response to cell-cell contact signals.

EphrinB/EphB signaling controls embryonic germ layer separation by contact-induced cell detachment.

BACKGROUND: The primordial organization of the metazoan body is achieved during gastrulation by the establishment of the germ layers. Adhesion differences between ectoderm, mesoderm, and endoderm cells could in principle be sufficient to maintain germ layer integrity and prevent intermixing. However, in organisms as diverse as fly, fish, or amphibian, the ectoderm-mesoderm boundary not only keeps these germ layers separated, but the ectoderm also serves as substratum for mesoderm migration, and the boundary must be compatible with repeated cell attachment and detachment. PRINCIPAL FINDINGS: We show that localized detachment resulting from contact-induced signals at the boundary is at the core of ectoderm-mesoderm segregation. Cells alternate between adhesion and detachment, and detachment requires ephrinB/EphB signaling. Multiple ephrinB ligands and EphB receptors are expressed on each side of the boundary, and tissue separation depends on forward signaling across the boundary in both directions, involving partially redundant ligands and receptors and activation of Rac and RhoA. CONCLUSION: This mechanism differs from a simple differential adhesion process of germ layer formation. Instead, it involves localized responses to signals exchanged at the tissue boundary and an attachment/detachment cycle which allows for cell migration across a cellular substratum.

Measuring learning gain during a one-day introductory bronchoscopy course.

BACKGROUND: Rigorous assessment of medical knowledge and technical skill inspires learning, reinforces confidence, and reassures the public. Identifying curricular effectiveness using objective measures of learning is therefore crucial for competency-oriented program development in a learner-centric educational environment. The aim of this study was to determine whether various measures of learning, including class-average normalized gain, can be used to assess the effectiveness of a one-day introductory bronchoscopy course curriculum. METHODS: We conducted a quasi-experimental one-group pre-test/post-test study at the University of California, Irvine. The group comprised 24 first-year pulmonary and critical care trainees from eight training institutions in southern California. Class-average normalized gain, single-student normalized gain, absolute gain, and relative gain were used as objective measures of cognitive knowledge and bronchoscopy technical skill learning. A class-average normalized gain of 30% was used to determine curricular effectiveness. Perceived educational value using Likert-scale surveys and post-course questionnaires was determined during and 3 months after course participation. RESULTS: Mean test scores of cognitive knowledge improved significantly from 48 to 66% (p = 0.043). Absolute gain for the class was 18%, relative gain was 37%, class average normalized gain was 34%, and the average of the single-student normalized gains g(ave) was 29%. Mean test scores of technical skill improved significantly from 43 to 77% (p = 0.017). Absolute gain was 34%, relative gain was 78%, class average normalized gain was 60%, and the average of the single-student normalized gains g(ave) was 59%. Statistically significant improvements in absolute gain were noted in all five elements of technical skill (p < 0.05). Likert-scale surveys, questionnaires, and surveys demonstrated strong perceived educational value. CONCLUSION: The effectiveness of a one-day introductory bronchoscopy curriculum was demonstrated using a pre-test/post-test model with calculation of normalized gain and related metrics.

Use of competency-based metrics to determine effectiveness of a postgraduate thoracoscopy course.

BACKGROUND: Despite the paradigm shift from process to competency-based education, no study has explored how competency-based metrics might be used to assess short-term effectiveness of thoracoscopy-related postgraduate medical education. OBJECTIVES: To assess the use of a single-group, pre-/post-test model comprised of multiple-choice questions (MCQ) and psychomotor skill measures to ascertain the effectiveness of a postgraduate thoracoscopy program. METHODS: A 37-item MCQ test of cognitive knowledge was administered to 17 chest physicians before and after a 2-day continued medical education-approved program. Pre- and post-course technical skills were assessed using rigid videothoracoscopy simulation stations. Competency-based metrics (mean relative gain, mean absolute gain, and class-average normalized gain ) were calculated. A >30% was used to determine curricular effectiveness. RESULTS: Mean cognitive knowledge score improved significantly from 20.9 to 28.7 (7.8 ± 1.3 points, p < 0.001), representing a relative gain of 37% and an absolute gain of 21%. Mean technical skill score improved significantly from 5.20 to 7.82 (2.62 ± 0.33 points, p < 0.001), representing a relative gain of 50% and an absolute gain of 33%. Non-parametric testing confirmed t test results (p < 0.001). Class-average normalized gains were 48 and 92%, respectively. CONCLUSION: Competency-based metrics, including class-average normalized gain, can be used to assess course effectiveness and to determine if a program meets predesignated objectives of knowledge acquisition and psychomotor technical skill.

Comparative effectiveness of low- and high-fidelity bronchoscopy simulation for training in conventional transbronchial needle aspiration and user preferences.

BACKGROUND: Conventional transbronchial needle aspiration (TBNA) can be learned using high-fidelity virtual-reality platforms and low-fidelity models comprised of molded silicone or excised animal airways. OBJECTIVES: The purpose of this study was to determine perceptions and preferences of learners and instructors regarding the comparative effectiveness of low-fidelity and high-fidelity bronchoscopy simulation for training in TBNA. METHODS: During the 2008 annual CHEST conference, a prospective randomized crossover design was used to train study participants in three methods of conventional TBNA using low- and high-fidelity models. Likert style questions were administered to learners and instructors in order to elicit preferences and opinions regarding educational effectiveness of the models. Results were tabulated and depicted in graphic format, with medians calculated. RESULTS: Learners felt that the models were equally enjoyable (13-13) and enthusiasm generating (low 17-high 15). There was preference for low-fidelity in terms of realism (23-17), ease of learning (20-6), and learning all three TBNA methods (31-7 for hub-against-wall, 31-6 for jabbing, 29-6 for piggyback). Low-fidelity was preferred as an ideal model overall (19-11). Instructors thought that low-fidelity was more useful in teaching TBNA (9-0 for all three methods). Instructors perceived the low-fidelity model overall as an ideal tool for learning TBNA (8-0) and a more effective teaching instrument (8-0). CONCLUSION: Based on learner and instructor perceptions, a low-fidelity model is superior to a high-fidelity platform for training in three methods of conventional TBNA.