Need for strategic communications and stakeholder engagement to advance acceptability of an overdose preventing vaccine targeting fentanyl.

BACKGROUND: Fentanyl is a synthetic opioid, exposure to which has led to hundreds of thousands of overdose deaths. Novel vaccines are being developed that might protect against fentanyl overdose. Proactive attention to strategic communications and stakeholder engagement may smooth uptake of a novel vaccine given known challenges around vaccine hesitancy and concern for stigma related to substance use. METHODS: Qualitative interviews (N = 74) with a purposive sample of adolescents/young adults with opioid use disorder (OUD), family members of persons with OUD, experts in substance use treatment and harm reduction, and community members were conducted and thematically analyzed to discern attitudes toward a fentanyl vaccine, and directions for communications and engagement. RESULTS: Major themes reflected personal concerns for biomedical risk and system-level concerns for alignment and integration of an overdose preventing vaccine with prevailing beliefs about addiction and associated frameworks and philosophies for treatment and response. CONCLUSION: Acceptability and implementation of a novel fentanyl vaccine targeting overdose will need precision communications that address biomedical, moral/spiritual, and structural perspectives about the nature of addiction. Education about the purpose and limits of a fentanyl vaccine, partnerships with diverse stakeholders from throughout the opioid response ecosystem and interweaving of a vaccine strategy into comprehensive prevention and treatment are recommended.

BECC-engineered live-attenuated Shigella vaccine candidates display reduced endotoxicity with robust immunogenicity in mice.

Shigella spp. infection contributes significantly to the global disease burden, primarily affecting young children in developing countries. Currently, there are no FDA-approved vaccines against Shigella, and the prevalence of antibiotic resistance is increasing, making therapeutic options limited. Live-attenuated vaccine strains WRSs2 (S. sonnei) and WRSf2G12 (S. flexneri 2a) are highly immunogenic, making them promising vaccine candidates, but possess an inflammatory lipid A structure on their lipopolysaccharide (LPS; also known as endotoxin). Here, we utilized bacterial enzymatic combinatorial chemistry (BECC) to ectopically express lipid A modifying enzymes in WRSs2 and WRSf2G12, as well as their respective wild-type strains, generating targeted lipid A modifications across the Shigella backgrounds. Dephosphorylation of lipid A, rather than deacylation, reduced LPS-induced TLR4 signaling in vitro and dampened endotoxic effects in vivo. These BECC-modified vaccine strains retained the phenotypic traits of their parental strains, such as invasion of epithelial cells and immunogenicity in mice without adverse endotoxicity. Overall, our observations suggest that BECC-engineered live attenuated vaccines are a promising approach to safe and effective Shigella vaccines.

From hit to vial: Precision discovery and development of an imidazopyrimidine TLR7/8 agonist adjuvant formulation.

Vaccination can help prevent infection and can also be used to treat cancer, allergy, and potentially even drug overdose. Adjuvants enhance vaccine responses, but currently, the path to their advancement and development is incremental. We used a phenotypic small-molecule screen using THP-1 cells to identify nuclear factor-κB (NF-κB)-activating molecules followed by counterscreening lead target libraries with a quantitative tumor necrosis factor immunoassay using primary human peripheral blood mononuclear cells. Screening on primary cells identified an imidazopyrimidine, dubbed PVP-037. Moreover, while PVP-037 did not overtly activate THP-1 cells, it demonstrated broad innate immune activation, including NF-κB and cytokine induction from primary human leukocytes in vitro as well as enhancement of influenza and SARS-CoV-2 antigen-specific humoral responses in mice. Several de novo synthesis structural enhancements iteratively improved PVP-037's in vitro efficacy, potency, species-specific activity, and in vivo adjuvanticity. Overall, we identified imidazopyrimidine Toll-like receptor-7/8 adjuvants that act in synergy with oil-in-water emulsion to enhance immune responses.

Coherence of the frequency-difference autoproduct deduced from high-frequency acoustic fields scattered from a rough sea surfacea).

The prevalence of random scattering from a rough ocean surface increases with increasing χ=kh cos θ, where k is the acoustic wavenumber, h is the root-mean-square surface height, and θ is the incidence angle. Generally, when χ≫1, coherence between incident and surface-scattered fields is lost. However, such coherence may be recovered when χ≫1 by considering the frequency-difference autoproduct of the surface-scattered field, a quadratic product of complex fields at nearby frequencies. Herein, the autoproduct's coherent reflection coefficient for χ> 20 is determined from surface-scattered sound fields obtained from 50 independent realizations of the rough ocean surface measured in pelagic waters off the coast of California in January 1992. The recordings were made with a source at a depth of 147 m that broadcasted 30 and 40 kHz signals to a single receiver 576 m away at depth of 66 m. An analytic formula for the coherent reflection coefficient of the frequency-difference autoproduct, based on the Kirchhoff approximation and a Gaussian surface autocorrelation function, compares favorably with measurements. Improved agreement with the single-receiver measurements is possible via a minor adjustment to the surface autocorrelation length. The adjustment identified here matches that determined previously from horizontal spatial coherence estimates utilizing the experiment's eight-element receiving array.

Immune profiling of age and adjuvant-specific activation of human blood mononuclear cells in vitro.

Vaccination reduces morbidity and mortality due to infections, but efficacy may be limited due to distinct immunogenicity at the extremes of age. This raises the possibility of employing adjuvants to enhance immunogenicity and protection. Early IFNγ production is a hallmark of effective vaccine immunogenicity in adults serving as a biomarker that may predict effective adjuvanticity. We utilized mass cytometry (CyTOF) to dissect the source of adjuvant-induced cytokine production in human blood mononuclear cells (BMCs) from newborns (~39-week-gestation), adults (~18-63 years old) and elders (>65 years of age) after stimulation with pattern recognition receptors agonist (PRRa) adjuvants. Dimensionality reduction analysis of CyTOF data mapped the BMC compartment, elucidated age-specific immune responses and profiled PRR-mediated activation of monocytes and DCs upon adjuvant stimulation. Furthermore, we demonstrated PRRa adjuvants mediated innate IFNγ induction and mapped NK cells as the key source of TLR7/8 agonist (TLR7/8a) specific innate IFNγ responses. Hierarchical clustering analysis revealed age and TLR7/8a-specific accumulation of innate IFNγ producing γδ T cells. Our study demonstrates the application of mass cytometry and cutting-edge computational approaches to characterize immune responses across immunologically distinct age groups and may inform identification of the bespoke adjuvantation systems tailored to enhance immunity in distinct vulnerable populations.

Coherent reflection recovery in scattering from the ocean surface using the frequency-difference autoproducta).

The coherence of rough sea-surface-scattered acoustic fields decreases with increasing frequency. The frequency-difference autoproduct, a quadratic product of acoustic fields at nearby frequencies, mimics a genuine field at the difference frequency. In rough-surface scattering, the autoproduct's lower effective frequency decreases the apparent surface roughness, restoring coherent reflection. Herein, the recovery of coherent reflection in sea surface scattering via the frequency-difference autoproduct is examined for data collected off the coast of New Jersey during the Shallow Water '06 (SW06) experiment. An acoustic source at depth 40 m and receiver at depth 24.3 m and range 200 m interrogated 160 independent realizations of the ocean surface. The root mean square surface height h was 0.167 m, and broadcast frequencies were 14-20 kHz, so that 2.5 ≤kh cos θ≤ 3.7 for acoustic wavenumber k and incidence angle θ. Measured autoproducts, constructed from scattered constituent fields, show significant coherent reflection at sufficiently low difference frequencies. Theoretical results, using the Kirchhoff approximation and a non-analytic surface autocorrelation function, agree with experimental findings. The match is improved using a numerical strategy, exploiting the relationship between autoproduct-based coherence recovery, the ocean-surface autocorrelation function, and the ocean-surface height spectrum. Error bars computed from Monte Carlo scattering simulations support the validity of the measured coherence recovery.

Human in vitro modeling of adjuvant formulations demonstrates enhancement of immune responses to SARS-CoV-2 antigen.

Adjuvants can enhance vaccine immunogenicity, but their mechanism of action is often incompletely understood, hampering rapid applicability for pandemic vaccines. Herein, we characterized the cellular and molecular activity of adjuvant formulations available for pre-clinical evaluation, including several developed for global open access. We applied four complementary human in vitro platforms to assess individual and combined adjuvants in unformulated, oil-in-water, and liposomal delivery platforms. Liposomal co-formulation of MPLA and QS-21 was most potent in promoting dendritic cell maturation, selective production of Th1-polarizing cytokines, and activation of SARS-CoV-2 Spike-specific CD4+ and CD8+ T cells in a co-culture assay. Select formulations also significantly enhanced Spike antigen-specific humoral immunity in vivo. This study confirms the utility of the cumulative use of human in vitro tools to predict adjuvanticity potential. Thus, human in vitro modeling may advance public health by accelerating the development of affordable and scalable adjuvants for vaccines tailored to vulnerable populations.

Investigating the Impact of Tillage and Crop Rotation on the Prevalence of phlD-Carrying Pseudomonas Potentially Involved in Disease Suppression.

Winter oilseed rape (OSR) is becoming an increasingly popular crop in rotations as it provides a cash crop and reduces the incidence of take-all fungal disease (caused by Gaeumannomyces graminis) in subsequent wheat production. The exact mechanism of this inhibition of fungal pathogens is not fully understood; however, the selective recruitment of bacterial groups with the ability to suppress pathogen growth and reproduction is thought to play a role. Here we examine the effect of tillage practice on the proliferation of microbes that possess the phlD gene involved in the production of the antifungal compound 2,4-diacetylphloroglucinol (2,4-DAPG), in the rhizospheres of both winter oilseed rape and winter wheat grown in rotation over a two-year period. The results showed that conservation strip tillage led to a significantly greater phlD gene copy number, both in the soil and in the roots, of oilseed rape and wheat crops, whereas crop rotation of oilseed rape and wheat did not increase the phlD gene copy number in winter wheat.

Reduced SARS-CoV-2 mRNA vaccine immunogenicity and protection in mice with diet-induced obesity and insulin resistance.

BACKGROUND: Obesity and type 2 diabetes mellitus (T2DM) are associated with an increased risk of severe outcomes from infectious diseases, including coronavirus disease 2019. These conditions are also associated with distinct responses to immunization, including an impaired response to widely used severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines. OBJECTIVE: We sought to establish a connection between reduced immunization efficacy via modeling the effects of metabolic diseases on vaccine immunogenicity that is essential for the development of more effective vaccines for this distinct vulnerable population. METHODS: A murine model of diet-induced obesity and insulin resistance was used to model the effects of comorbid T2DM and obesity on vaccine immunogenicity and protection. RESULTS: Mice fed a high-fat diet (HFD) developed obesity, hyperinsulinemia, and glucose intolerance. Relative to mice fed a normal diet, HFD mice vaccinated with a SARS-CoV-2 mRNA vaccine exhibited significantly lower anti-spike IgG titers, predominantly in the IgG2c subclass, associated with a lower type 1 response, along with a 3.83-fold decrease in neutralizing titers. Furthermore, enhanced vaccine-induced spike-specific CD8+ T-cell activation and protection from lung infection against SARS-CoV-2 challenge were seen only in mice fed a normal diet but not in HFD mice. CONCLUSIONS: The study demonstrated impaired immunity following SARS-CoV-2 mRNA immunization in a murine model of comorbid T2DM and obesity, supporting the need for further research into the basis for impaired anti-SARS-CoV-2 immunity in T2DM and investigation of novel approaches to enhance vaccine immunogenicity among those with metabolic diseases.

A protocol for high-throughput screening for immunomodulatory compounds using human primary cells.

High-throughput screening is a powerful platform that can rapidly provide valuable cytotoxic, immunological, and phenotypical information for thousands of compounds. Human peripheral blood mononuclear cells (PBMCs) cultured in autologous plasma can model the human immune response. Here, we describe a protocol to stimulate PBMCs for 72 h and measure cytokine secretion via AlphaLISA assays and cell surface activation marker expression via flow cytometry. Cryopreserved PBMCs are incubated for 72 h with various small molecule libraries and the supernatants are harvested to rapidly measure secretion levels of key cytokines (tumor necrosis factor alpha, interferon gamma, interleukin 10) via the AlphaLISA assay. Almost simultaneously, the cells can be fixated and stained using antibodies against innate immune activation markers (CD80, CD86, HLA-DR, OX40) for analysis via flow cytometry. This multiplexed readout workflow can directly aid in the phenotypic identification and discovery of novel immunomodulators and potential vaccine adjuvant candidates. For complete details on the use and execution of this protocol, please refer to Chew et al.1.

Precision Vaccinology Approaches for the Development of Adjuvanted Vaccines Targeted to Distinct Vulnerable Populations.

Infection persists as one of the leading global causes of morbidity and mortality, with particular burden at the extremes of age and in populations who are immunocompromised or suffer chronic co-morbid diseases. By focusing discovery and innovation efforts to better understand the phenotypic and mechanistic differences in the immune systems of diverse vulnerable populations, emerging research in precision vaccine discovery and development has explored how to optimize immunizations across the lifespan. Here, we focus on two key elements of precision vaccinology, as applied to epidemic/pandemic response and preparedness, including (a) selecting robust combinations of adjuvants and antigens, and (b) coupling these platforms with appropriate formulation systems. In this context, several considerations exist, including the intended goals of immunization (e.g., achieving immunogenicity versus lessening transmission), reducing the likelihood of adverse reactogenicity, and optimizing the route of administration. Each of these considerations is accompanied by several key challenges. On-going innovation in precision vaccinology will expand and target the arsenal of vaccine components for protection of vulnerable populations.

Field scale biodegradation of total petroleum hydrocarbons and soil restoration by Ecopiles: microbiological analysis of the process.

Ecopiling is a method for biodegradation of hydrocarbons in soils. It derives from Biopiles, but phytoremediation is added to biostimulation with nitrogen fertilization and bioaugmentation with local bacteria. We have constructed seven Ecopiles with soil heavily polluted with hydrocarbons in Carlow (Ireland). The aim of the study was to analyze changes in the microbial community during ecopiling. In the course of 18 months of remediation, total petroleum hydrocarbons values decreased in 99 and 88% on average for aliphatics and aromatics, respectively, indicating a successful biodegradation. Community analysis showed that bacterial alfa diversity (Shannon Index), increased with the degradation of hydrocarbons, starting at an average value of 7.59 and ending at an average value of 9.38. Beta-diversity analysis, was performed using Bray-Curtis distances and PCoA ordination, where the two first principal components (PCs) explain the 17 and 14% of the observed variance, respectively. The results show that samples tend to cluster by sampling time instead of by Ecopile. This pattern is supported by the hierarchical clustering analysis, where most samples from the same timepoint clustered together. We used DSeq2 to determine the differential abundance of bacterial populations in Ecopiles at the beginning and the end of the treatment. While TPHs degraders are more abundant at the start of the experiment, these populations are substituted by bacterial populations typical of clean soils by the end of the biodegradation process. Similar results are found for the fungal community, indicating that the microbial community follows a succession along the process. This succession starts with a TPH degraders or tolerant enriched community, and finish with a microbial community typical of clean soils.

Localization of a remote source in a noisy deep ocean sound channel using phase-only matched autoproduct processing.

Long-range passive source localization is possible in the deep ocean using phase-only matched autoproduct processing (POMAP) [Geroski and Dowling (2021). J. Acoust. Soc. Am. 150, 171-182], an algorithm based on matched field processing that is more robust to environmental mismatch. This paper extends these prior POMAP results by analyzing the localization performance of this algorithm in the presence of environmental noise. The noise rejection performance of POMAP is assessed using both simulated and measured signal data, with noise data based on environmental noise measurements. Herein, signal and noise measurements are from the nominally one-year-long PhilSea10 ocean acoustic propagation experiment. All signals were recorded from a single moored source, placed near the ocean sound channel 129.4 km away from a nearly water-column-spanning distributed vertical line array. The source transmitted linear frequency modulated chirps with nominal bandwidth from 200 to 300 Hz. The noise measurements used in this study were collected in the months after this source stopped transmitting, and synthetic samples of noise are calculated based on the characteristics of this measured noise. The effect that noise rejection algorithms have on the source localization performance of POMAP is also evaluated, but only 1 dB of performance improvement is achieved using these.

At the nexus of ludology and narratology: Advances in reality-based story-driven games.

Story-driven games are growing in popularity across a wide range of genres. However, the narrative potential of video games is still being debated, particularly in light of the so-called tension between gameplay and storytelling. This study proposes that rules and game mechanics perform narrative semiotic functions, offering a ludic grammar of interactive storytelling. Case studies of four representative games show through exploratory player action shaped by rules, the medium of video games can generate meanings that traditional media cannot, thereby better achieving their narrative goals.

Carbohydrate fatty acid monosulphate: oil-in-water adjuvant enhances SARS-CoV-2 RBD nanoparticle-induced immunogenicity and protection in mice.

Development of SARS-CoV-2 vaccines that protect vulnerable populations is a public health priority. Here, we took a systematic and iterative approach by testing several adjuvants and SARS-CoV-2 antigens to identify a combination that elicits antibodies and protection in young and aged mice. While demonstrating superior immunogenicity to soluble receptor-binding domain (RBD), RBD displayed as a protein nanoparticle (RBD-NP) generated limited antibody responses. Comparison of multiple adjuvants including AddaVax, AddaS03, and AS01B in young and aged mice demonstrated that an oil-in-water emulsion containing carbohydrate fatty acid monosulphate derivative (CMS:O/W) most effectively enhanced RBD-NP-induced cross-neutralizing antibodies and protection across age groups. CMS:O/W enhanced antigen retention in the draining lymph node, induced injection site, and lymph node cytokines, with CMS inducing MyD88-dependent Th1 cytokine polarization. Furthermore, CMS and O/W synergistically induced chemokine production from human PBMCs. Overall, CMS:O/W adjuvant may enhance immunogenicity and protection of vulnerable populations against SARS-CoV-2 and other infectious pathogens.

Reduced SARS-CoV-2 mRNA vaccine immunogenicity and protection in mice with diet-induced obesity and insulin resistance.

Background: Obesity and Type 2 Diabetes Mellitus (T2DM) are associated with an increased risk of severe outcomes from infectious diseases, including COVID-19. These conditions are also associated with distinct responses to immunization, including an impaired response to widely used SARS-CoV-2 mRNA vaccines. Objective: To establish a connection between reduced immunization efficacy via modeling the effects of metabolic diseases on vaccine immunogenicity that is essential for the development of more effective vaccines for this distinct vulnerable population. Methods: We utilized a murine model of diet-induced obesity and insulin resistance to model the effects of comorbid T2DM and obesity on vaccine immunogenicity and protection. Results: Mice fed a high-fat diet (HFD) developed obesity, hyperinsulinemia, and glucose intolerance. Relative to mice fed a normal diet (ND), HFD mice vaccinated with a SARS-CoV-2 mRNA vaccine exhibited significantly lower anti-spike IgG titers, predominantly in the IgG2c subclass, associated with a lower type 1 response, along with a 3.83-fold decrease in neutralizing titers. Furthermore, enhanced vaccine-induced spike-specific CD8 + T cell activation and protection from lung infection against SARS-CoV-2 challenge were seen only in ND mice but not in HFD mice. Conclusion: We demonstrate impaired immunity following SARS-CoV-2 mRNA immunization in a murine model of comorbid T2DM and obesity, supporting the need for further research into the basis for impaired anti-SARS-CoV-2 immunity in T2DM and investigation of novel approaches to enhance vaccine immunogenicity among those with metabolic diseases. Capsule summary: Obesity and type 2 diabetes impair SARS-CoV-2 mRNA vaccine efficacy in a murine model.

Development of a TLR7/8 agonist adjuvant formulation to overcome early life hyporesponsiveness to DTaP vaccination.

Infection is the most common cause of mortality early in life, yet the broad potential of immunization is not fully realized in this vulnerable population. Most vaccines are administered during infancy and childhood, but in some cases the full benefit of vaccination is not realized in-part. New adjuvants are cardinal to further optimize current immunization approaches for early life. However, only a few classes of adjuvants are presently incorporated in vaccines approved for human use. Recent advances in the discovery and delivery of Toll-like receptor (TLR) agonist adjuvants have provided a new toolbox for vaccinologists. Prominent among these candidate adjuvants are synthetic small molecule TLR7/8 agonists. The development of an effective infant Bordetella pertussis vaccine is urgently required because of the resurgence of pertussis in many countries, contemporaneous to the switch from whole cell to acellular vaccines. In this context, TLR7/8 adjuvant based vaccine formulation strategies may be a promising tool to enhance and accelerate early life immunity by acellular B. pertussis vaccines. In the present study, we optimized (a) the formulation delivery system, (b) structure, and (c) immunologic activity of novel small molecule imidazoquinoline TLR7/8 adjuvants towards human infant leukocytes, including dendritic cells. Upon immunization of neonatal mice, this TLR7/8 adjuvant overcame neonatal hyporesponsiveness to acellular pertussis vaccination by driving a T helper (Th)1/Th17 biased T cell- and IgG2c-skewed humoral response to a licensed acellular vaccine (DTaP). This potent immunization strategy may represent a new paradigm for effective immunization against pertussis and other pathogens in early life.

mRNA booster vaccination protects aged mice against the SARS-CoV-2 Omicron variant.

The SARS-CoV-2 Omicron variant evades vaccine-induced immunity. While a booster dose of ancestral mRNA vaccines effectively elicits neutralizing antibodies against variants, its efficacy against Omicron in older adults, who are at the greatest risk of severe disease, is not fully elucidated. Here, we evaluate multiple longitudinal immunization regimens of mRNA BNT162b2 to assess the effects of a booster dose provided >8 months after the primary immunization series across the murine lifespan, including in aged 21-month-old mice. Boosting dramatically enhances humoral and cell-mediated responses with evidence of Omicron cross-recognition. Furthermore, while younger mice are protected without a booster dose, boosting provides sterilizing immunity against Omicron-induced lung infection in aged 21-month-old mice. Correlational analyses reveal that neutralizing activity against Omicron is strongly associated with protection. Overall, our findings indicate age-dependent vaccine efficacy and demonstrate the potential benefit of mRNA booster immunization to protect vulnerable older populations against SARS-CoV-2 variants.

Shaping Neonatal Immunization by Tuning the Delivery of Synergistic Adjuvants via Nanocarriers.

Adjuvanted nanocarrier-based vaccines hold substantial potential for applications in novel early-life immunization strategies. Here, via mouse and human age-specific in vitro modeling, we identified the combination of a small-molecule STING agonist (2'3'-cyclic GMP-AMP, cGAMP) and a TLR7/8 agonist (CL075) to drive the synergistic activation of neonatal dendritic cells and precision CD4 T-helper (Th) cell expansion via the IL-12/IFNγ axis. We further demonstrate that the vaccination of neonatal mice with quadrivalent influenza recombinant hemagglutinin (rHA) and an admixture of two polymersome (PS) nanocarriers separately encapsulating cGAMP (cGAMP-PS) and CL075 (CL075-PS) drove robust Th1 bias, high frequency of T follicular helper (TFH) cells, and germinal center (GC) B cells along with the IgG2c-skewed humoral response in vivo. Dual-loaded cGAMP/CL075-PSs did not outperform admixed cGAMP-PS and CL075-PS in vivo. These data validate an optimally designed adjuvantation system via age-selected small-molecule synergy and a multicomponent nanocarrier formulation as an effective approach to induce type 1 immune responses in early life.