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Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Patients with TNBC have poor overall survival because of limited molecular therapeutic targets. Recently, exosomes have been recognized as key mediators in cancer progression, but the molecular components and function of TNBC-derived exosomes remain unknown. The main goal of this study was to reveal the proteomic landscape of serum exosomes derived from ten patients with TNBC and 17 healthy donors to identify potential therapeutic targets. Using a tandem mass tag-based quantitative proteomics approach, we characterized the proteomes of individual patient-derived serum exosomes, identified exosomal protein signatures specific to patients with TNBC, and filtered out differentially expressed proteins. Most importantly, we found that the tetraspanin CD151 expression levels in TNBC-derived serum exosomes were significantly higher than those exosomes from healthy subjects, and we validated our findings with samples from 16 additional donors. Furthermore, utilizing quantitative proteomics approach to reveal the proteomes of CD151-deleted exosomes and cells, we found that exosomal CD151 facilitated secretion of ribosomal proteins via exosomes while inhibiting exosome secretion of complement proteins. Moreover, we proved that CD151-deleted exosomes significantly decreased the migration and invasion of TNBC cells. This is the first comparative study of the proteomes of TNBC patient-derived and CD151-deleted exosomes. Our findings indicate that profiling of TNBC-derived exosomal proteins is a useful tool to extend our understanding of TNBC, and exosomal CD151 may be a potential therapeutic target for TNBC.
Subject(s)
Exosomes/metabolism , Proteome/metabolism , Tetraspanin 24/metabolism , Triple Negative Breast Neoplasms/metabolism , Adult , Aged , Aged, 80 and over , Cell Line, Tumor , Female , Humans , Middle Aged , Protein Interaction Maps , Tetraspanin 24/genetics , Triple Negative Breast Neoplasms/bloodABSTRACT
In this work, a microwave resonator sensor with a unique configuration consisting of three resonators and two feedlines is proposed. This novel design aims to improve the performance and functionality of microwave resonator sensors for various applications. The frequency response of the sensor to materials with different dielectric constants is simulated. The results show that the most sensitive region of the sensor is located on the first interdigital structure, and placing the materials in other regions would enhance the linear correlation of its frequency response. The sensor also exhibits the ability to distinguish whether the same material has defects and the ability to qualitatively detect subtle changes in dielectric constant. Finally, the proposed sensor is fabricated and measured under the condition consistent with the simulation environment. The measured results are basically consistent with the simulation results, which confirms the potential of this sensor in detecting dielectric constants and resolving materials with defects, and the response of the sensor to the materials under test demonstrates its potential in measuring different thicknesses and loss tangents.
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In this study, a multifunctional high-vacuum system was established to measure the electro-optical conversion efficiency of metamaterial-based thermal emitters with built-in heaters. The system is composed of an environmental control module, an electro-optical conversion measurement module, and a system control module. The system can provide air, argon, high vacuum, and other conventional testing environments, combined with humidity control. The test chamber and sample holder are carefully designed to minimize heat transfer through thermal conduction and convection. The optical power measurements are realized using the combination of a water-cooled KBr flange, an integrating sphere, and thermopile detectors. This structure is very stable and can detect light emission at the µW level. The system can synchronously detect the heating voltage, heating current, optical power, sample temperatures (both top and bottom), ambient pressure, humidity, and other environmental parameters. The comprehensive parameter detection capability enables the system to monitor subtle sample changes and perform failure mechanism analysis with the aid of offline material analysis using scanning electron microscopy, energy dispersive X-ray spectroscopy, and X-ray diffraction. Furthermore, the system can be used for fatigue and high-low temperature impact tests.
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Gas sensing performance characterization systems are essential for the research and development of gas sensing materials and devices. Although existing systems are almost completely automatically operated, the accuracies of gas concentration control and of pressure control and the ability to simultaneously detect different sensor signals still require improvement. In this study, a high-precision gas sensing material characterization system is developed based on vacuum technology, with the objective of enabling the precise and simultaneous measurement of electrical responses. Because of the implementation of vacuum technology, the gas concentration control accuracy is improved more than 1600 times, whereas the pressure of the test ambient condition can be precisely adjusted between vacuum and 1.2 bar. The vacuum-assisted gas-exchanging mechanism also enables the sensor response time to be determined more accurately. The system is capable of performing sensitivity, selectivity, and stability tests and can control the ambient relative humidity in a precise manner. More importantly, the levels of performance of three different optical signal measurement set-ups were investigated and compared in terms of detection range, linearity, noise, and response time, based on which of their scopes of application were proposed. Finally, single-period and cyclical tests were performed to examine the ability of the system to detect optical and electrical responses simultaneously, both at a single wavelength and in a spectral region.
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OBJECTIVES: To investigate the promoting effects of biomimetic intrafibrillarly mineralized collagen (IMC) bone scaffold material on the osseointegration of a titanium implant simultaneously grafted into a critical-sized bone defect as well as the underlying mechanisms involved. MATERIALS AND METHODS: A critical-sized bone defect was created in the rat femur, and a titanium (Ti) implant surrounded by IMC or extrafibrillarly mineralized collagen (EMC) bone scaffold material was placed in the defect. A blank group and a natural bone group were included as controls. Osseointegration was assessed by micro-computed tomographic, histological, and biochemical evaluations at 12 weeks postoperatively. Microarray technology was applied for transcriptional profile analysis at days 7 and 14 postoperatively. RESULTS: Significant bone regeneration and osseointegration were observed in the IMC and EMC groups according to µ-CT and histological analyses. The bone volume (BV)/total volume (TV) fraction, bone-to-implant contact percentage, and bone area percentage as well as ultimate shear strength and maximal pull-out force were all significantly higher in the IMC group than in the EMC group (all p < 0.05). Transcriptional analysis revealed overexpression of genes mainly associated with cell proliferation, immuno-inflammatory response, skeletogenesis, angiogenesis, neurogenesis, and skeletogenesis-related pathways during the early process of osseointegration in the IMC group. CONCLUSION: Our data suggest that IMC placed simultaneously with a Ti implant may be a promising strategy in jawbone defect reconstruction. Several candidate genes that were found to be differentially expressed in the IMC group may be responsible for the superior osseointegration effects in this model.
Subject(s)
Osseointegration , Titanium , Animals , Biomimetics , Bone Regeneration , Collagen , RatsABSTRACT
Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation. More than 65% of class I phosphosites were novel when compared with PhosphoSitePlus database. Quantification results showed that the early and late stage of NSC differentiation differ greatly. We mapped 69 changed phosphosites on 20 proteins involved in Wnt signaling pathway, including S552 on catenin beta-1 (Ctnnb1) and S9 on glycogen synthase kinase 3ß (Gsk3ß). Western blotting and real-time PCR results proved that Wnt signaling pathway plays critical roles in NSC fate determination. Furthermore, inhibition and activation of PKA dramatically affected the phosphorylation state of Ctnnb1 and Gsk3ß, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self-renewal and differentiation of NSCs. Stem Cells 2016;34:2090-2101.
Subject(s)
Cell Differentiation , Cyclic AMP-Dependent Protein Kinases/metabolism , Glycogen Synthase Kinase 3 beta/metabolism , Neural Stem Cells/cytology , Phosphoproteins/metabolism , Proteomics/methods , beta Catenin/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Cell Differentiation/drug effects , Fetus/cytology , Gene Ontology , Mice , Molecular Sequence Annotation , Neural Stem Cells/drug effects , Neural Stem Cells/metabolism , Phosphoproteins/chemistry , Phosphorylation/drug effects , Rats , Tretinoin/pharmacology , Wnt Signaling Pathway/drug effectsABSTRACT
Neuroblastoma is the most common pediatric extracranial solid tumor with a broad spectrum of clinical behavior and poor prognosis. Despite intensive multimodal therapy, ongoing clinical trials, and basic science investigations, neuroblastoma remains a complex medical challenge with a long-term survival rate less than 40%. In our study, we found that resveratrol (3, 5, 4'-trihydroxystilbene, RSV), a naturally occurring phytoalexin, possesses an anticancer activity through blocking cell growth and inducing apoptosis in neuroblastoma cell line Neuro-2a (N-2a) cells. Using stable isotope labeling with amino acids in cell culture (SILAC) and quantitative proteomic analysis, we found that 395 proteins were up-regulated and 302 proteins were down-regulated in the nucleus of N-2a cells treated with RSV. Among these, the polycomb protein histone methyltransferase EZH2 was reduced significantly, which is aberrantly overexpressed in neuroblastoma and crucial to maintain the malignant phenotype of neuroblastoma by epigenetic repression of multiple tumor suppressor genes. EZH2 reduction further led to decreased H3K27me3 level and reactivation of neuroblastoma tumor suppressor genes CLU and NGFR. Disruption EZH2 expression by RNA interference-mediated knockdown or pharmacologic inhibition with DZNep triggered cellular apoptosis in N-2a cells. We found that the up-regulation of miR-137 level was responsible for reduced EZH2 level in tumor suppression induced by RSV. Inhibition of miR-137 expression rescued the cellular apoptosis phenotypes, EZH2 reduction, and CLU and NGFR reactivation, associated with RSV treatment. Taken together, our findings present for the first time, an epigenetic mechanism involving miR-137-mediated EZH2 repression in RSV-induced apoptosis and tumor suppression of neuroblastoma, which would provide a key potential therapeutic target in neuroblastoma treatment.
Subject(s)
Apoptosis/drug effects , MicroRNAs/metabolism , Neuroblastoma/metabolism , Nuclear Proteins/metabolism , Polycomb Repressive Complex 2/metabolism , Proteomics , Stilbenes/pharmacology , Animals , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Computational Biology , Enhancer of Zeste Homolog 2 Protein , Gene Knockdown Techniques , HEK293 Cells , Humans , Isotope Labeling , Mice , Neuroblastoma/pathology , Protein Biosynthesis/drug effects , Proteome/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of Results , Resveratrol , Time FactorsABSTRACT
In this work, we report a simple yet robust strategy for eliminating surface defects in red-emitting fluorides through solid-liquid interface friction under elevated temperature and high pressure. This method is minimally dependent on the solvent type and especially excels at stripping away the abundant surface defects caused by mechanical crushing.
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Micro/nanomotors (MNMs) emerge as a vital candidate for biosensing due to its nano-size structure, high surface-to-area ratio, directional mobility, biocompatibility, and ease of functionalization, therefore being able to detect objects with high efficiency, precision, and selectivity. The driving mode, nanostructure, materials property, preparation technique, and biosensing applications have been thoroughly discussed in publications. To promote the MNMs-based biosensors from in vitro to in vivo, it is necessary to give a comprehensive discussion from the perspective of sensing performances enhancement. However, until now, there is few reviews dedicated to the systematic discussion on the multiple performance enhancement schemes and the current challenges of MNMs-based biosensors. Bearing it in mind and based on our research experience in this field, we summarized the enhancement methods for biosensing properties such as sensitivity, selectivity, detection time, biocompatibility, simplify system operation, and environmental availability. We hope that this review provides the readers with fundamental understanding on performance enhancement schemes for MNMs-based biosensors.
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BACKGROUND: We previously demonstrated the safety and immunogenicity of an MF59-adjuvanted COVID-19 vaccine based on the SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a molecular clamp using HIV-1 glycoprotein 41 sequences. Here, we describe 12-month results in adults aged 18-55 years and ≥56 years. METHODS: Phase 1, double-blind, placebo-controlled trial conducted in Australia (July 2020-December 2021; ClinicalTrials.govNCT04495933; active, not recruiting). Healthy adults (Part 1: 18-55 years; Part 2: ≥56 years) received two doses of placebo, 5 µg, 15 µg, or 45 µg vaccine, or one 45 µg dose of vaccine followed by placebo (Part 1 only), 28 days apart (n = 216; 24 per group). Safety, humoral immunogenicity (including against virus variants), and cellular immunogenicity were assessed to day 394 (12 months after second dose). Effects of subsequent COVID-19 vaccination on humoral responses were examined. FINDINGS: All two-dose vaccine regimens were well tolerated and elicited strong antigen-specific and neutralising humoral responses, and CD4+ T-cell responses, by day 43 in younger and older adults, although cellular responses were lower in older adults. Humoral responses waned by day 209 but were boosted in those receiving authorised vaccines. Neutralising activity against Delta and Omicron variants was present but lower than against the Wuhan strain. Cross-reactivity in HIV diagnostic tests declined over time but remained detectable in most participants. INTERPRETATION: The SARS-CoV-2 molecular clamp vaccine is well tolerated and evokes robust immune responses in adults of all ages. Although the HIV glycoprotein 41-based molecular clamp is not being progressed, the clamp concept represents a viable platform for vaccine development. FUNDING: This study was funded by the Coalition for Epidemic Preparedness Innovations, the National Health and Medical Research Council of Australia, and the Queensland Government.
Subject(s)
COVID-19 , HIV Infections , Vaccines , Humans , Aged , SARS-CoV-2 , COVID-19 Vaccines/adverse effects , COVID-19/prevention & control , Spike Glycoprotein, Coronavirus , Adjuvants, Immunologic , HIV Infections/prevention & control , Glycoproteins , Double-Blind Method , Antibodies, Viral , Antibodies, NeutralizingABSTRACT
Recently, we have shown that fate of a vaccine is determined by the cytokine milieu in the innate immune compartment at the early stage of vaccination. Specifically, 24 h post-delivery, level of innate lymphoid cell type 2 (ILC2)-derived IL-13/IL-13Rα2 are the master regulators of DC and also different ILC subsets responsible for modulating the downstream immune outcomes. Here, we provide step-by-step details how to assess different ILC and DC subsets in lung and muscle following intranasal and intramuscular viral vector vaccination, respectively, using multi-color flow cytometry and confocal microscopy.
Subject(s)
Immunity, Innate , Viral Vaccines , Dendritic Cells , Lymphocytes , VaccinationABSTRACT
Janus nanoparticles refer to a kind of asymmetric-structured nanoparticles composed of two or more distinct sides with differences in chemical nature and/or polarity on each side and thus can integrate two or more properties in one single particle. Due to their unique structure and surface properties, Janus nanoparticles have shown broad application potentials in optics, nuclear magnetic resonance, multi-mode imaging, and other fields. Unlike traditional contrast agents used in biological imaging, Janus nanoparticles are asymmetrically and directionally oriented to ensure stable partitioning of individual nanoparticles while integrating more functions. Much advancement have been carried out in the past few years, with some studies partially covering bioimaging applications. However, to our best knowledge, there are still no review papers specifically dedicated to the bioimaging applications with Janus nanoparticles. Bearing this in mind and taking the current challenges in this field into consideration, herein, we discuss representative approaches orchestrated for bioimaging applications, with the focus on the improvement of imaging quality brought by Janus nanoparticles and the development of multifunctional nanoplatforms in biological imaging fields, such as theranostics and therapies. Finally, based on the research experience of our group in this field, prospects for future research trends are put forward to provide new ideas for designing new Janus nanoparticles for clinical bioimaging.
Subject(s)
Multifunctional Nanoparticles , Nanoparticles , Contrast Media/chemistry , Contrast Media/pharmacology , Diagnostic Imaging/methods , Nanoparticles/chemistry , Surface PropertiesABSTRACT
We have shown that manipulation of IL-13 and STAT6 signaling at the vaccination site can lead to different innate lymphoid cell (ILC)/dendritic cell (DC) recruitment, resulting in high avidity/poly-functional T cells and effective antibody differentiation. Here we show that permanent versus transient blockage of IL-13 and STAT6 at the vaccination site can lead to unique ILC-derived IL-13 and IFN-γ profiles, and differential IL-13Rα2, type I and II IL-4 receptor regulation on ILC. Specifically, STAT6-/- BALB/c mice given fowl pox virus (FPV) expressing HIV antigens induced elevated ST2/IL-33R+ ILC2-derived IL-13 and reduced NKp46+/- ILC1/ILC3-derived IFN-γ expression, whilst the opposite (reduced IL-13 and elevated IFN-γ expression) was observed during transient inhibition of STAT6 signaling in wild type BALB/c mice given FPV-HIV-IL-4R antagonist vaccination. Interestingly, disruption/inhibition of STAT6 signaling considerably impacted IL-13Rα2 expression by ST2/IL-33R+ ILC2 and NKp46- ILC1/ILC3, unlike direct IL-13 inhibition. Consistently with our previous findings, this further indicated that inhibition of STAT6 most likely promoted IL-13 regulation via IL-13Rα2. Moreover, the elevated ST2/IL-33R+ IL-13Rα2+ lung ILC2, 24 h post FPV-HIV-IL-4R antagonist vaccination was also suggestive of an autocrine regulation of ILC2-derived IL-13 and IL-13Rα2, under certain conditions. Knowing that IL-13 can modulate IFN-γ expression, the elevated expression of IFN-γR on lung ST2/IL-33R+ ILC2 provoked the notion that there could also be inter-regulation of lung ILC2-derived IL-13 and NKp46- ILC1/ILC3-derived IFN-γ via their respective receptors (IFN-γR and IL-13Rα2) at the lung mucosae early stages of vaccination. Intriguingly, under different IL-13 conditions differential regulation of IL-13/IL-13Rα2 on lung DC was also observed. Collectively these findings further substantiated that IL-13 is the master regulator of, not only DC, but also different ILC subsets at early stages of viral vector vaccination, and responsible for shaping the downstream adaptive immune outcomes. Thus, thoughtful selection of vaccine strategies/adjuvants that can manipulate IL-13Rα2, and STAT6 signaling at the ILC/DC level may prove useful in designing more efficacious vaccines against different/chronic pathogens.
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The ongoing coronavirus disease (COVID-19) pandemic urgently requires the availability of interventions that improve outcomes for those with severe disease. Since severe acute respiratory syndrome coronavirus 2 infection is characterized by dysregulated lung mucosae, and that mucosal homeostasis is heavily influenced by interleukin (IL)-13 activity, we explore recent findings indicating that IL-13 production is proportional to disease severity. We propose that excessive IL-13 contributes to the progression of severe/fatal COVID-19 by (1) promoting the recruitment of immune cells that express inflammatory cytokines, causing a cytokine storm that results in widespread destruction of lung tissue, (2) directly facilitating tissue-remodeling that causes airway hyperinflammation and obstruction, and (3) diverting the immune system away from developing high-quality cytotoxic T cells that confer effective anti-viral immunity. These factors may cumulatively result in significant lung distress, multi-organ failure, and death. Here, we suggest repurposing existing IL-13-inhibiting interventions, including antibody therapies routinely used for allergic lung hyperinflammation, as well as viral vector-based approaches, to alleviate disease. Since many of these strategies have previously been shown to be both safe and effective, this could prove to be a highly cost-effective solution. Relevance for Patients: There remains a desperate need to establish medical interventions that reliably improves outcomes for patients suffering from COVID-19. We explore the role of IL-13 in maintaining homeostasis at the lung mucosae and propose that its dysregulation during viral infection may propagate the hallmarks of severe disease - further exploration may provide a platform for invaluable therapeutics.
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IL-4 production is associated with low-avidity, poorly cytotoxic T cell induction that contributes to viral immune evasion and the failure of T cell-based vaccines. Yet, the precise mechanisms that regulate IL-4 signalling in T cells remain elusive. Mounting evidence indicates that cells can dynamically alter their IL-4/IL-13 receptor signature to modulate downstream immune outcomes upon pathogen encounter. Here, we describe how naïve (CD62L+CD44lo-mid) CD4 and CD8 T cells distinctly engage both STAT6 and STAT3 in response to IL-4. We further show that IL-4R⺠expression is both time- and IL-4 concentration-dependent. Remarkably, our findings reveal that STAT3 inhibition can ablate IL-4R⺠and affect transcriptional expression of other Stat and Jak family members. By extension, the loss of STAT3 lead to aberrant STAT6 phosphorylation, revealing an inter-regulatory relationship between the two transcription factors. Moreover, IL-4 stimulation down-regulated TGF-ß1 and IFN-γR1 expression on naïve T cells, possibly signifying the broad regulatory implications of IL-4 in conditioning lineage commitment decisions during early infection. Surprisingly, naïve T cells were unresponsive to IL-13 stimulation, unlike dendritic cells. Collectively, these findings could be exploited to inform more efficacious vaccines, as well as design treatments against IL-4/IL-13-associated disease conditions.
Subject(s)
Interleukin-4/metabolism , STAT3 Transcription Factor/physiology , Signal Transduction , T-Lymphocytes/metabolism , Animals , Biomarkers/metabolism , Female , Mice , Mice, Inbred BALB C , Phosphorylation , Receptors, Interleukin-4/metabolism , STAT6 Transcription Factor/metabolismABSTRACT
Subunit vaccines exhibit favorable safety and immunogenicity profiles and can be designed to mimic native antigen structures. However, pairing with an appropriate adjuvant is imperative in order to elicit effective humoral and cellular immune responses. In this study, we aimed to determine an optimal adjuvant pairing with the prefusion form of influenza haemagglutinin (HA) or respiratory syncytial virus (RSV) fusion (F) subunit vaccines in BALB/c mice in order to inform future subunit vaccine adjuvant selection. We tested a panel of adjuvants, including aluminum hydroxide (alhydrogel), QS21, Addavax, Addavax with QS21 (AdQS21), and Army Liposome Formulation 55 with monophosphoryl lipid A and QS21 (ALF55). We found that all adjuvants elicited robust humoral responses in comparison to placebo, with the induction of potent neutralizing antibodies observed in all adjuvanted groups against influenza and in AdQS21, alhydrogel, and ALF55 against RSV. Upon HA vaccination, we observed that none of the adjuvants were able to significantly increase the frequency of CD4+ and CD8+ IFN-γ+ cells when compared to unadjuvanted antigen. The varying responses to antigens with each adjuvant highlights that those adjuvants most suited for pairing purposes can vary depending on the antigen used and/or the desired immune response. We therefore suggest that an adjuvant trial for different subunit vaccines in development would likely be necessary in preclinical studies.
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BACKGROUND: Given the scale of the ongoing COVID-19 pandemic, the development of vaccines based on different platforms is essential, particularly in light of emerging viral variants, the absence of information on vaccine-induced immune durability, and potential paediatric use. We aimed to assess the safety and immunogenicity of an MF59-adjuvanted subunit vaccine for COVID-19 based on recombinant SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a novel molecular clamp (spike glycoprotein-clamp [sclamp]). METHODS: We did a phase 1, double-blind, placebo-controlled, block-randomised trial of the sclamp subunit vaccine in a single clinical trial site in Brisbane, QLD, Australia. Healthy adults (aged ≥18 to ≤55 years) who had tested negative for SARS-CoV-2, reported no close contact with anyone with active or previous SARS-CoV-2 infection, and tested negative for pre-existing SARS-CoV-2 immunity were included. Participants were randomly assigned to one of five treatment groups and received two doses via intramuscular injection 28 days apart of either placebo, sclamp vaccine at 5 µg, 15 µg, or 45 µg, or one dose of sclamp vaccine at 45 µg followed by placebo. Participants and study personnel, except the dose administration personnel, were masked to treatment. The primary safety endpoints included solicited local and systemic adverse events in the 7 days after each dose and unsolicited adverse events up to 12 months after dosing. Here, data are reported up until day 57. Primary immunogenicity endpoints were antigen-specific IgG ELISA and SARS-CoV-2 microneutralisation assays assessed at 28 days after each dose. The study is ongoing and registered with ClinicalTrials.gov, NCT04495933. FINDINGS: Between June 23, 2020, and Aug 17, 2020, of 314 healthy volunteers screened, 120 were randomly assigned (n=24 per group), and 114 (95%) completed the study up to day 57 (mean age 32·5 years [SD 10·4], 65 [54%] male, 55 [46%] female). Severe solicited reactions were infrequent and occurred at similar rates in participants receiving placebo (two [8%] of 24) and the SARS-CoV-2 sclamp vaccine at any dose (three [3%] of 96). Both solicited reactions and unsolicited adverse events occurred at a similar frequency in participants receiving placebo and the SARS-CoV-2 sclamp vaccine. Solicited reactions occurred in 19 (79%) of 24 participants receiving placebo and 86 (90%) of 96 receiving the SARS-CoV-2 sclamp vaccine at any dose. Unsolicited adverse events occurred in seven (29%) of 24 participants receiving placebo and 35 (36%) of 96 participants receiving the SARS-CoV-2 sclamp vaccine at any dose. Vaccination with SARS-CoV-2 sclamp elicited a similar antigen-specific response irrespective of dose: 4 weeks after the initial dose (day 29) with 5 µg dose (geometric mean titre [GMT] 6400, 95% CI 3683-11â122), with 15 µg dose (7492, 4959-11â319), and the two 45 µg dose cohorts (8770, 5526-13â920 in the two-dose 45 µg cohort; 8793, 5570-13â881 in the single-dose 45 µg cohort); 4 weeks after the second dose (day 57) with two 5 µg doses (102â400, 64â857-161â676), with two 15 µg doses (74â725, 51â300-108â847), with two 45 µg doses (79â586, 55â430-114â268), only a single 45 µg dose (4795, 2858-8043). At day 57, 67 (99%) of 68 participants who received two doses of sclamp vaccine at any concentration produced a neutralising immune response, compared with six (25%) of 24 who received a single 45 µg dose and none of 22 who received placebo. Participants receiving two doses of sclamp vaccine elicited similar neutralisation titres, irrespective of dose: two 5 µg doses (GMT 228, 95% CI 146-356), two 15 µg doses (230, 170-312), and two 45 µg doses (239, 187-307). INTERPRETATION: This first-in-human trial shows that a subunit vaccine comprising mammalian cell culture-derived, MF59-adjuvanted, molecular clamp-stabilised recombinant spike protein elicits strong immune responses with a promising safety profile. However, the glycoprotein 41 peptide present in the clamp created HIV diagnostic assay interference, a possible barrier to widespread use highlighting the criticality of potential non-spike directed immunogenicity during vaccine development. Studies are ongoing with alternative molecular clamp trimerisation domains to ameliorate this response. FUNDING: Coalition for Epidemic Preparedness Innovations, National Health and Medical Research Council, Queensland Government, and further philanthropic sources listed in the acknowledgments.
Subject(s)
Adjuvants, Immunologic/pharmacology , COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Squalene/immunology , Adult , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Australia , Female , Healthy Volunteers , Humans , Male , Pandemics/prevention & control , Polysorbates , Vaccination/adverse effects , Young AdultABSTRACT
OBJECTIVES: Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion-stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 'MF59C.1' (Seqirus, Parkville, Australia). METHODS: A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo-electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. RESULTS: In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells in vivo. In the Syrian hamster challenge model (n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. CONCLUSION: The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2-8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models.
ABSTRACT
OBJECTIVE: The aim of this study was to investigate the efficiency of a novel biomedical system that repairs facial nerve gaps in a miniature swine model. STUDY DESIGN: A collagen (COL)/nano-sized ß-tricalcium phosphate (nß-TCP) conduit combined with COL filaments and nerve growth factor (NGF) was prepared and used to bridge a 35-mm-long facial nerve gap in miniature swine. The functional recovery and axonal regeneration were evaluated by electrophysiologic and histologic assessments in the different groups at 6 months postoperatively. RESULTS: Morphologic analysis revealed that the COL+NGF, COL/nß-TCP+NGF, and autograft groups exhibited a superior recovery compared with the COL and COL/nß-TCP groups. The compound muscle action potential ratios were significantly greater in the COL/nß-TCP+NGF group compared to the COL+NGF and COL/nß-TCP groups (P < .01). Moreover, transmission electron microscopy demonstrated significantly larger axon diameters and myelin sheath thicknesses in the COL/nß-TCP+NGF group compared with the COL, COL+NGF, and COL/nß-TCP groups (P < .05). The expression of S-100 was significantly greater in the COL/nß-TCP+NGF group than in the COL+NGF and COL/nß-TCP groups (P < .05). CONCLUSIONS: The functional nerve biomedical system containing the COL/nß-TCP conduit combined with COL filaments and NGF could promote facial nerve regeneration, thus offering promising potential for clinical applications.
Subject(s)
Calcium Phosphates , Dental Implants , Facial Nerve , Nerve Growth Factor , Animals , Collagen , Facial Nerve/growth & development , Nerve Regeneration , Swine , Swine, MiniatureABSTRACT
A HIV vaccine that provides mucosal immunity is urgently needed. We evaluated an intranasal recombinant Fowlpox virus (rFPV) priming vaccine followed by intramuscular Modified Vaccinia Ankara (rMVA) booster vaccine, both expressing SIV antigens. The vaccination generated mucosal and systemic SIV-specific CD4+ T cell mediated immunity and was associated with partial protection against high-dose intrarectal SIVmac251 challenge in outbred pigtail macaques. Three of 12 vaccinees were completely protected and these animals elicited sustained Gag-specific poly-functional, cytotoxic mucosal CD4+ T cells, complemented by systemic poly-functional CD4+ and CD8+ T cell immunity. Humoral immune responses, albeit absent in completely protected macaques, were associated with partial control of viremia in animals with relatively weaker mucosal/systemic T cell responses. Co-expression of an IL-4R antagonist by the rFPV vaccine further enhanced the breadth and cytotoxicity/poly-functionality of mucosal vaccine-specific CD4+ T cells. Moreover, a single FPV-gag/pol/env prime was able to induce rapid anamnestic gp140 antibody response upon SIV encounter. Collectively, our data indicated that nasal vaccination was effective at inducing robust cervico-vaginal and rectal immunity, although cytotoxic CD4+ T cell mediated mucosal and systemic immunity correlated strongly with 'complete protection', the different degrees of protection observed was multi-factorial.