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In this study we utilised an in vitro model system to gain insight into the potential cellular interactions that quantum dot (QD) nanoparticles may experience while transiting the viable skin epidermis, and we consider the effects of UVB exposure. UVB skin exposure is known to induce a skin barrier defect that facilitates QD stratum corneum penetration. Primary human keratinocytes were cultured in low and high calcium to induce basal and differentiated phenotypes, respectively. Results suggest that differentiation state plays a role in keratinocyte response to UVB exposure and exposure to negatively charged CdSe/ZnS core/shell QD. QD cell uptake increased with QD dose but association with differentiated cells was significantly lower than the basal keratinocyte phenotype. Differentiated keratinocytes were also less sensitive to the cytotoxic effects of UVB exposure. We did not observe an effect of UVB preexposure on QD cytotoxicity level despite the fact that fluorescent microscopy and flow cytometry data suggest that UVB may slightly increase QD uptake in the basal cell phenotype. The implications of these findings for assessing potential risk of human skin exposure are discussed.

Ebola virus (EBOV) vaccines containing glycoprotein (GP) provide protection against severe Ebola virus disease (EVD). EBO vaccinations elicit antibodies that are detectable in Ebola serodiagnostic tests, as EBOV GP is a major target antigen. This vaccine-induced seropositivity presents issues with early detection of natural EBOV infections, following vaccination and during surveillance, leading to 'uninfected' vaccine trial participants being falsely diagnosed as 'EBOV infected' potentially resulting in long-term social and economic distress. Since mass vaccinations are being employed to curtail the recurrent EBOV epidemics in multiple African countries, it is, therefore, essential to differentiate vaccine-induced from natural infection-induced antibodies by a differential serodiagnosis assay for accurate detection of Ebola virus infections.To develop a serodiagnostic test that can differentiate between individuals with EBOV infection-induced antibodies and individuals with EBOV vaccine-induced antibodies, we analysed peptides of EBOV viral protein 40 (VP40), viral protein 35 (VP35) and nucleocapsid protein (NP) using an ELISA with a panel of 181 human sera collected from healthy controls, EBO vaccinees, and EBOV-infected survivors. Receiver Operating Characteristic (ROC) curve analysis was used to calculate sensitivity and specificity of the assay. A simple peptide-based serodiagnostic assay was used to evaluate detection of breakthrough EBOV infections in vaccinated non-human primates (NHP) in EBOV challenge studies.We identified conserved peptide sequences in EBOV VP40, VP35 and NP, produced soon after EBOV infection that are not part of the current EBO vaccine target antigens. The new ELISA-based differential serodetection assay termed 'EBOV-Detect' demonstrated94% specificity and 96% sensitivity for diagnosis of EBOV infection. Importantly, the uninfected vaccine-trial participants scored negative in 'EBOV-Detect' assay. The results from the NHPs EBOV challenge study established that post-EBO vaccination serum scored negative in 'EBOV-Detect' and all NHPs with Ebola breakthrough infections, following EBOV challenge, were serodiagnosed positively with EBOV-Detect.The new 'EBOV-Detect' is a simple and sensitive serodiagnostic assay that can specifically differentiate between natural Ebola virus infected and those with vaccine-induced immunity. This could potentially be implemented as a robust diagnostic tool for epidemiology and surveillance of EBOV infections during and after outbreaks, especially in countries with mass Ebola vaccinations.The antibody characterization work described in this manuscript was supported by FDA Office of Counterterrorism and Emerging Threats (OCET) - Medical Countermeasures initiative (MCMi) grant- OCET 2019-1018 and Defense Threat Reduction Agency (HDTRA1930447) funds to S.K.

Hospitalized COVID-19 patients often present with a large spectrum of clinical symptoms. There is a critical need to better understand the immune responses to SARS-CoV-2 that lead to either resolution or exacerbation of the clinical disease. Here, we examine longitudinal plasma samples from hospitalized COVID-19 patients with differential clinical outcome. We perform immune-repertoire analysis including cytokine, hACE2-receptor inhibition, neutralization titers, antibody epitope repertoire, antibody kinetics, antibody isotype and antibody affinity maturation against the SARS-CoV-2 prefusion spike protein. Fatal cases demonstrate high plasma levels of IL-6, IL-8, TNFα, and MCP-1, and sustained high percentage of IgA-binding antibodies to prefusion spike compared with non-ICU survivors. Disease resolution in non-ICU and ICU patients associates with antibody binding to the receptor binding motif and fusion peptide, and antibody affinity maturation to SARS-CoV-2 prefusion spike protein. Here, we provide insight into the immune parameters associated with clinical disease severity and disease-resolution outcome in hospitalized patients that could inform development of vaccine/therapeutics against COVID-19., SARS-CoV2 infection has been linked to a wide range of clinical severities and the immunopathology is still under intense scrutiny. Here, the authors uncover an association of antibody affinity maturation and plasma IgA levels with clinical outcome in patients with COVID-19 disease.

SARS-CoV-2–induced human antibody repertoire in patients with COVID-19 reveals immune markers of disease severity and recovery., Limited knowledge exists on immune markers associated with disease severity or recovery in patients with coronavirus disease 2019 (COVID-19). Here, we elucidated longitudinal evolution of SARS-CoV-2 antibody repertoire in patients with acute COVID-19. Differential kinetics was observed for immunoglobulin M (IgM)/IgG/IgA epitope diversity, antibody binding, and affinity maturation in “severe” versus “mild” COVID-19 patients. IgG profile demonstrated immunodominant antigenic sequences encompassing fusion peptide and receptor binding domain (RBD) in patients with mild COVID-19 who recovered early compared with “fatal” COVID-19 patients. In patients with severe COVID-19, high-titer IgA were observed, primarily against RBD, especially in patients who succumbed to SARS-CoV-2 infection. The patients with mild COVID-19 showed marked increase in antibody affinity maturation to prefusion SARS-CoV-2 spike that associated with faster recovery from COVID-19. This study revealed antibody markers associated with disease severity and resolution of clinical disease that could inform development and evaluation of effective immune-based countermeasures against COVID-19.

Limited knowledge exists in post-partum women regarding durability of SARS-CoV-2 vaccine-induced antibody responses and their neutralising ability against SARS-CoV-2 variants of concern (VOC).We elucidated longitudinal mRNA vaccination-induced antibody profiles of 13 post-partum and 13 non-post-partum women (control).The antibody neutralisation titres against SARS-CoV-2 WA-1 strain were comparable between post-partum and non-post-partum women and these levels were sustained up to four months post-second vaccination in both groups. However, neutralisation titers declined against several VOCs, including Beta and Delta. Higher antibody binding was observed against SARS-CoV-2 receptor-binding domain (RBD) mutants with key VOC amino acids when tested with post-second vaccination plasma from post-partum women compared with controls. Importantly, post-vaccination plasma antibody affinity against VOCs RBDs was significantly higher in post-partum women compared with controls.This study demonstrates that there is a differential vaccination-induced immune responses in post-partum women compared with non-post-partum women, which could help inform future vaccination strategies for these groups.The antibody characterisation work described in this manuscript was supported by FDA's Medical Countermeasures Initiative (MCMi) grant #OCET 2021-1565 to S.K and intramural FDA-CBER COVID-19 supplemental funds.

Background: Limited knowledge exists regarding antibody affinity maturation following mRNA vaccination in naïve vs. COVID-19 recovered individuals and potential sex differences. Methods: We elucidated post-vaccination antibody profiles of 69 naïve and 17 COVID-19 convalescent adults using pseudovirus neutralization assay (PsVNA) covering SARS-CoV-2 WA-1, variants of concern (VOCs) and variants of interest (VOIs). Surface Plasmon Resonance (SPR) was used to measure antibody affinity against prefusion spike and receptor binding domain (RBD) and RBD mutants. Findings: Higher neutralizing antibodies were observed in convalescent vs. naïve adults against, WA-1, VOCs, and VOIs. Antibody binding to RBD and RBD mutants showed lower binding of post-vaccination sera from naïve compared with convalescent individuals. Moreover, we observed early antibody affinity maturation in convalescent individuals after one vaccine dose and higher antibody affinity after two doses compared with the naïve group. Among the naïve participants, antibody affinity against the SARS-CoV-2 prefusion spike was significantly higher for males than females even though there were no difference in neutralization titers between sexes. Interpretation: This study demonstrates the impact of prior infection on vaccine-induced antibody affinity maturation and difference in antibody affinity between males and females. Further studies are needed to determine whether antibody affinity may contribute to correlates of protection against SARS-CoV-2 and its variants. Funding: The antibody characterization work described in this manuscript was supported by FDA's Medical Countermeasures Initiative (MCMi) grant #OCET 2021-1565 to S.K and intramural FDA-CBER COVID-19 supplemental funds. The SPARTA program was supported by the National Institute of Allergy and Infectious Diseases (NIAID), U.S. National Institutes of Health (NIH), Department of Health and Human Services contract 75N93019C00052, and the University of Georgia (US) grant UGA-001. T.M.R is also supported by the Georgia Research Alliance (US) grant GRA-001. The CTRU was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR002378.

Summary Evolution of antibody repertoire against the Ebola virus (EBOV) proteome was characterized in an acutely infected patient receiving supportive care alone to elucidate virus-host interactions over time. Differential kinetics are observed for IgM-IgG-IgA epitope diversity, antibody binding, and affinity maturation to EBOV proteins. During acute illness, antibodies predominate to VP40 and glycoprotein (GP). At day 13 of clinical illness, a marked increase in antibody titers to most EBOV proteins and affinity maturation to GP is associated with rapid decline in viral replication and illness severity. At one year, despite undetectable virus, a diverse IgM repertoire against VP40 and GP epitopes is observed suggesting occult viral persistence. Rabbit immunization experiments identify key immunodominant sites of GP, while challenge studies in mice found these epitopes induce EBOV-neutralizing antibodies and protect against lethal EBOV challenge. This study reveals markers of viral persistence and provides promising approaches for development and evaluation of vaccines and therapeutics.

Several Ebola vaccines and therapeutics are under clinical development. However, limited knowledge exists on the quality of antibody response generated by different Ebola vaccines. In this study, antibody repertoire induced by vaccination of transchromosomal bovine (TcB) with Ebola virus (EBOV) glycoprotein ([GP]; recombinant GP [rGP]) encoded by either deoxyribonucleic acid (DNA) or nanoparticle-based vaccine platform was analyzed using EBOV genome fragment phage display library and surface plasmon resonance (SPR)-based real-time kinetics assay to measure antibody affinity maturation to both native and partially denatured Ebola GP as well as GP containing the receptor binding domain but lacking the mucin-like domain. Immunoglobulin (IgG) obtained from rGP nanoparticle-vaccinated TcB demonstrated ~4-fold higher binding affinity compared with DNA-vaccinated TcB-induced IgG against the native rGP’s. The rGP nanoparticle vaccine generated a more robust and diverse antibody immune response to the native EBOV-GP compared with the DNA vaccine, which may explain the protective efficacy observed for these antibody preparations.

We report on the effect of surface charge and the ligand coating composition of CdSe/ZnS core/shell quantum dot (QD) nanoparticles on human keratinocyte toxicity using fluorescent microscopy, flow cytometry, transmission electron microscopy. Two commonly reported positive charged (cysteamine, polyethylenimine) and two negative charged (glutathione, dihydrolipoic acid) ligands were studied. The QDs were fully characterized by UV-vis absorption spectroscopy, fluorescence emission spectroscopy, dynamic light scattering and zeta potential. Differences in surface coatings and charges were evaluated against cellular uptake, ROS generation, cytotoxicity, and mitochondrial targeting. Results show that the negative charged QDs coated with GSH exhibit excellent water solubility, high quantum yield and low cytotoxicity. Ligand composition is more important in ROS generation than surface charge whereas surface charge is an important driver of cytotoxicity. Most importantly we observe the selective accumulation of glutathione coated QDs in vesicles in the mitochondria matrix. This observation suggests a new strategy for developing mitochondria-targeted nanomaterials for drug/gene delivery.

The increasing use of nanoparticles (NPs) in technological applications and in commercial products has escalated environmental health and safety concerns. The detection of NPs in the environment and in biological systems is challenged by limitations associated with commonly used analytical techniques. In this paper we report on the development and characterization of NP binding antibodies, termed NProbes. Phage display methodology was used to discover antibodies that bind NPs dispersed in solution. We present a proof-of-concept for the generation of NProbes and their use for detecting quantum dots and titanium dioxide NPs in vitro and in an ex vivo human skin model. Continued development and refinement of NProbes to detect NPs that vary in composition, shape, size, and surface coating will comprise a powerful tool kit that can be used to advance nanotechnology research particularly in the nanotoxicology and nanotherapeutics fields.

Development of an effective vaccine against Ebola virus is of high priority. However, knowledge about potential correlates of protection and the durability of immune response after vaccination is limited. Here, we elucidate the human antibody repertoire after administration of vesicular stomatitis virus (VSV)-Ebola vaccine at 3 million, 20 million and 100 million plaque-forming units (PFU) and homologous VSV-Ebola vaccine boost in healthy adult volunteers. Whole genome-fragment phage display libraries, expressing linear and conformational epitopes of Ebola glycoprotein (GP), showed higher diversity of antibody epitopes in individuals vaccinated with 20 million PFU than in those vaccinated with 3 million or 100 million PFU. Surface plasmon resonance kinetics showed higher levels of GP-binding antibodies after a single vaccination with 20 million or 100 million PFU than with 3 million PFU, and these correlated strongly with neutralization titers. A second vaccination did not boost antibody or virus neutralization titers, which declined rapidly, and induced only minimal antibody affinity maturation. Isotype analysis revealed a predominant IgM response even after the second vaccination, which contributed substantially to virus neutralization in vitro. These findings may help identify new vaccine targets and aid development and evaluation of effective countermeasures against Ebola.

Increases in the research and consumer applications of nanoparticles means an increased opportunity for human skin contact. As researchers work to evaluate the most important factors in assessing nanoparticle skin penetration risk, there are common pitfalls or noise factors that can easily complicate interpretation of results and induce ambiguity. We first look at the impact of time course on skin barrier impairment, as different treatments have immediate or delayed responses. The next important factor is the skin model, as the ability to relate animal model or ex vivo tissue data to in vivo human skin susceptibility to nanoparticle skin penetration varies depending on the treatment used to induce a barrier defect. Upon consideration of skin barrier status, the characterization and stability of applied nanoparticles- in our case quantum dots- is the next most important factor. Once accounting for the experimental inputs, evaluation techniques for assessing skin penetration have an additional set of specific challenges that must be met or accounted for. With proper consideration, the challenge of determining nanoparticle and skin barrier effect on human skin penetration can be met.

Studies have shown that UVB can slightly increase the penetration of nanoparticles through skin and significantly alter skin cell biology, thus it is important to understand if and how UVB may impact subsequent nanoparticle skin cell interactions. The research presented herein evaluates the effect of UVB on quantum dot (QD) uptake and reactive oxygen species (ROS) generation in primary keratinocytes, primary melanocytes, and related cell lines. QD exposure induced cell type dependent ROS responses increased by pre-exposing cells to UVB and correlated with the level of QD uptake. Our results suggest that keratinocytes may be at greater risk for QD induced ROS generation than melanocytes, and raise awareness about the differential cellular effects that topically applied nanomaterials may have on UVB exposed skin.

In this study, we utilize in vivo human skin and a viable ex-vivo human skin model to investigate the effect of a commercial depilatory agent on barrier function. Tape stripping was used as a positive control. The magnitude of skin barrier was quantified by measuring transepidermal water loss values on in vivo human skin and transepithelial electrical resistance measurements and tissue histology on ex vivo skin. The susceptibility to carboxylated quantum dot penetration through ex vivo skin was investigated using fluorescent microcopy analysis of microtomed skin sections and flow cytometry to quantify quantum dot association with live epidermal cells. Results show that depilatory treatment modifies the outside-in barrier sufficiently to allow quantum dots to penetrate the stratum corneum but to a lesser extent than tape stripping. The implications of these finding are discussed.