The COVID-19 pandemic triggered the resurgence of synthetic RNA vaccine platforms allowing rapid, scalable, low-cost manufacturing, and safe administration of therapeutic vaccines. Self-amplifying mRNA (SAM), which self-replicates upon delivery into the cellular cytoplasm, leads to a strong and sustained immune response. Such mRNAs are encapsulated within lipid nanoparticles (LNPs) that act as a vehicle for delivery to the cell cytoplasm. A better understanding of LNP-mediated SAM uptake and release mechanisms in different types of cells is critical for designing effective vaccines. Here, we investigated the cellular uptake of a SAM-LNP formulation and subsequent intracellular expression of SAM in baby hamster kidney (BHK-21) cells using hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) microscopy and multiphoton-excited fluorescence lifetime imaging microscopy (FLIM). Cell classification pipelines based on HS-CARS and FLIM features were developed to obtain insights on spectral and metabolic changes associated with SAM-LNPs uptake. We observed elevated lipid intensities with the HS-CARS modality in cells treated with LNPs versus PBS-treated cells, and simultaneous fluorescence images revealed SAM expression inside BHK-21 cell nuclei and cytoplasm within 5 h of treatment. In a separate experiment, we observed a strong correlation between the SAM expression and mean fluorescence lifetime of the bound NAD(P)H population. This work demonstrates the ability and significance of multimodal optical imaging techniques to assess the cellular uptake of SAM-LNPs and the subsequent changes occurring in the cellular microenvironment following the vaccine expression.

Delivery of self-amplifying mRNA (SAM) has high potential for infectious disease vaccination due to its self-adjuvanting and dose-sparing properties. Yet a challenge is the susceptibility of SAM to degradation and the need for SAM to reach the cytosol fully intact to enable self-amplification. Lipid nanoparticles are successfully deployed at incredible speed for mRNA vaccination, but aspects such as cold storage, manufacturing, efficiency of delivery, and the therapeutic window can benefit from further improvement. To investigate alternatives to lipid nanoparticles, a class of >200 biodegradable end-capped lipophilic poly(beta-amino ester)s (PBAEs) that enable efficient delivery of SAM in vitro and in vivo as assessed by measuring expression of SAM encoding reporter proteins is developed. The ability of these polymers to deliver SAM intramuscularly in mice is evaluated, and a polymer-based formulation that yields up to 37-fold higher intramuscular (IM) expression of SAM compared to injected naked SAM is identified. Using the same nanoparticle formulation to deliver a SAM encoding rabies virus glycoprotein, the vaccine elicits superior immunogenicity compared to naked SAM delivery, leading to seroconversion in mice at low RNA injection doses. These biodegradable nanomaterials may be useful in the development of next-generation RNA vaccines for infectious diseases.

The goal of this study was to utilize a multimodal magnetic resonance imaging (MRI) and positron emission tomography (PET) imaging approach to assess the local innate immune response in skeletal muscle and draining lymph node following vaccination in rats using two different vaccine platforms (AS01 adjuvanted protein and lipid nanoparticle (LNP) encapsulated Self-Amplifying mRNA (SAM)). MRI and 18FDG PET imaging were performed temporally at baseline, 4, 24, 48, and 72 hr post Prime and Prime-Boost vaccination in hindlimb with Cytomegalovirus (CMV) gB and pentamer proteins formulated with AS01, LNP encapsulated CMV gB protein-encoding SAM (CMV SAM), AS01 or with LNP carrier controls. Both CMV AS01 and CMV SAM resulted in a rapid MRI and PET signal enhancement in hindlimb muscles and draining popliteal lymph node reflecting innate and possibly adaptive immune response. MRI signal enhancement and total 18FDG uptake observed in the hindlimb was greater in the CMV SAM vs CMV AS01 group (↑2.3 – 4.3-fold in AUC) and the MRI signal enhancement peak and duration were temporally shifted right in the CMV SAM group following both Prime and Prime-Boost administration. While cytokine profiles were similar among groups, there was good temporal correlation only between IL-6, IL-13, and MRI/PET endpoints. Imaging mass cytometry was performed on lymph node sections at 72 hr post Prime and Prime-Boost vaccination to characterize the innate and adaptive immune cell signatures. Cell proximity analysis indicated that each follicular dendritic cell interacted with more follicular B cells in the CMV AS01 than in the CMV SAM group, supporting the stronger humoral immune response observed in the CMV AS01 group. A strong correlation between lymph node MRI T2 value and nearest-neighbor analysis of follicular dendritic cell and follicular B cells was observed (r=0.808, P<0.01). These data suggest that spatiotemporal imaging data together with AI/ML approaches may help establish whether in vivo imaging biomarkers can predict local and systemic immune responses following vaccination.

RNA vaccines have demonstrated efficacy against SARS-CoV-2 in humans, and the technology is being leveraged for rapid emergency response. In this report, we assessed immunogenicity and, for the first time, toxicity, biodistribution, and protective efficacy in preclinical models of a two-dose self amplifying messenger RNA (SAM) vaccine, encoding a prefusion-stabilized spike antigen of SARS-CoV-2 Wuhan-Hu-1 strain and delivered by lipid nanoparticles (LNPs). In mice, one immunization with the SAM vaccine elicited a robust spike-specific antibody response, which was further boosted by a second immunization, and effectively neutralized the matched SARS-CoV-2 Wuhan strain as well as B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) variants. High frequencies of spike-specific germinal center B, Th0/Th1 CD4, and CD8 T cell responses were observed in mice. Local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. In hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following SARS-CoV-2 challenge. Therefore, the SARSCoV-2 SAM (LNP) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple SARS-CoV-2 variants, and has been advanced to phase 1 clinical evaluation (NCT04758962).

The efficacy of RNA-based vaccines has been recently demonstrated, leading to the use of mRNA-based COVID19 vaccines. The application of self-amplifying mRNA within these formulations may offer further enhancement to these vaccines, as self-amplifying mRNA replicons enable longer expression kinetics and more potent immune responses compared to non-amplifying mRNAs. To investigate the impact of administration route on RNAvaccine potency, we investigated the immunogenicity of a self-amplifying mRNA encoding the rabies virus glycoprotein encapsulated in different nanoparticle platforms (solid lipid nanoparticles (SLNs), polymeric nanoparticles (PNPs) and lipid nanoparticles (LNPs)). These were administered via three different routes: intramuscular, intradermal and intranasal. Our studies in a mouse model show that the immunogenicity of our 4 different saRNA vaccine formulations after intramuscular or intradermal administration was initially comparable; however, ionizable LNPs gave higher long-term IgG responses. The clearance of all 4 of the nanoparticle formulations from the intramuscular or intradermal administration site was similar. In contrast, immune responses generated after intranasal was low and coupled with rapid clearance for the administration site, irrespective of the formulation. These results demonstrate that both the administration route and delivery system format dictate self-amplifying RNA vaccine efficacy.

A Zika self-amplifying mRNA vaccine delivered by a flexible bedside mixing formulation induces protective immunity in NHPs. Zika virus (ZIKV) is the cause of a pandemic associated with microcephaly in newborns and Guillain-Barre syndrome in adults. Currently, there are no available treatments or vaccines for ZIKV, and the development of a safe and effective vaccine is a high priority for many global health organizations. We describe the development of ZIKV vaccine candidates using the self-amplifying messenger RNA (SAM) platform technology delivered by cationic nanoemulsion (CNE) that allows bedside mixing and is particularly useful for rapid responses to pandemic outbreaks. Two immunizations of either of the two lead SAM (CNE) vaccine candidates elicited potent neutralizing antibody responses to ZIKV in mice and nonhuman primates. Both SAM (CNE) vaccines protected these animals from ZIKV challenge, with one candidate providing complete protection against ZIKV infection in nonhuman primates. The data provide a preclinical proof of concept that a SAM (CNE) vaccine candidate can rapidly elicit protective immunity against ZIKV.

messenger RNA (mRNA)-based vaccines combine the positive attributes of both live-attenuated and subunit vaccines. In order for these to be applied for clinical use, they require to be formulated with delivery systems. However, there are limited in vivo studies which compare different delivery platforms. Therefore, we have compared four different cationic platforms: (1) liposomes, (2) solid lipid nanoparticles (SLNs), (3) polymeric nanoparticles (NPs) and (4) emulsions, to deliver a self-amplifying mRNA (SAM) vaccine. All formulations contained either the non-ionizable cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or dimethyldioctadecylammonium bromide (DDA) and they were characterized in terms of physico-chemical attributes, in vitro transfection efficiency and in vivo vaccine potency. Our results showed that SAM encapsulating DOTAP polymeric nanoparticles, DOTAP liposomes and DDA liposomes induced the highest antigen expression in vitro and, from these, DOTAP polymeric nanoparticles were the most potent in triggering humoral and cellular immunity among candidates in vivo.

Interest in value frameworks that account for values other than narrowly defined health-state utilities offers opportunities to apply established stated-preference methods to quantify community benefits of state-sponsored mental-health programs, including reduced homelessness, unemployment, and criminality. The aim of this study was to quantify the public’s perceived value for publicly funded programs to treat severe mental illness. A national sample of 10,000 US adults completed an online discrete-choice experiment survey. Respondents answered five 3-alternative trade-off questions consisting of status quo (no increased spending) and 2 budget-increase alternatives. Each budget profile included expanding a mental-health program plus 2 respondent-specific expansions randomly selected from 4 non-health programs. Statistical modeling accounted for latent-class preference heterogeneity, state size, region, and individual-specific covariates. We omitted from the analysis 23% of the sample who rejected any increase in taxes for any purpose. Depending on state size and region, latent classes for the remainder of the sample included groups of participants who had well-defined priorities for mental-health programs and were relatively sensitive (32%-42%) or insensitive (21% to 35%) to increased taxes. Other groups ignored tax increases (21%-23%) or provided incoherent preference data (12%-18%). We imputed zero willingness to pay additional taxes for the latter two classes and for tax objectors. For a mental-health program expansion that would treat an additional 1500 patients in medium-sized states in the Northeast, Midwest, South, and West regions of the US, the median acceptable increase in annual taxes was $218 (209, 227), $429 (415, 442), $252 (242, 263), and $216 (140, 293), respectively. Focusing narrowly on direct health outcomes understates the value of benefits to patients, their families, and communities of successfully treating severe mental illness. Even after accounting for a significant proportion of general-population respondents who indicated zero perceived benefits for improved mental-health treatment, we obtained significant values for total community benefits.

PURPOSE This in vitro study evaluated the fracture resistance and clinical prognosis of anterior lithium disilicate crowns (e.max Press and e.max CAD), following endodontic access and repair. The research design simulates intraoral loading conditions to produce clinically applicable results. MATERIALS AND METHODS Monolithic anterior crowns, based on #8 anatomy, were fabricated from e.max Press ingots and e.max CAD blocks and adhesively bonded on identical dies milled out of a dentin analog material (NEMA G10). Specimens were divided into 4 groups: intact pressed, repaired pressed, intact milled, and repaired milled (n = 15/group). Repaired pressed and repaired milled were prepared with a standardized endodontic access and repaired using a porcelain repair system and composite resin. All crowns were cyclically loaded under simulated oral conditions and then loaded to failure in water, using a universal testing machine. Data were interpreted using ANOVA/Tukey post-hoc test (α = 0.05). RESULTS Mean loads to failure ranged from 758.9 to 931.4 N for the 4 groups, indicating that both fabrication techniques, pressed and milled, yielded restorations that could reasonably withstand maximum masticatory forces. The pressed groups (923.7 N) exhibited significantly higher fracture resistance than the milled groups (797.5 N), p = 0.0002. When milled and pressed groups were categorized into intact and repaired subgroups, no difference was found in fracture resistance between the subgroups. Differences were noted in the modes of fracture, where the milled groups (intact and repaired) exhibited higher frequency of catastrophic fractures than the pressed groups. CONCLUSIONS Endodontic access preparation does not appear to affect fracture resistance of an anterior lithium disilicate restoration, suggesting that replacement may not be necessary. Fabrication technique had a significant effect on fracture resistance and fracture mode of lithium disilicate restorations. The pressed fabrication technique resulted in significantly greater crown strength and fracture resistance than the milled technique.

PURPOSE:This in vitro study evaluated and compared the vertical marginal gap of cast and milled full coverage gold copings using two margin designs (chamfer and chamfer bevel) before and after fitting adjustments.MATERIALS AND METHODS:Ten impressions were made of two metal master dies (one chamfer margin, one chamfer-bevel margin) and poured twice in Type IV stone. The 20 subsequent casts with 40 dies were split into four groups (n = 10); cast gold bevel, cast gold chamfer, milled gold bevel, and milled gold chamfer groups. The cast specimens received approximately 40 μm die relief no closer than 1 mm from the finish line. Cast copings were hand waxed, cast in a high noble gold alloy, chemically divested, and the sprues were removed. For milled gold copings, casts were scanned and copings designed using 3shape D900 scanner and software. Parameters were set to approximate analog fabrication (cement gap = 0.01 mm; extra cement gap = 0.04 mm, drill radius = 0.65 mm). Copings were milled from the same high noble alloy. All copings were seated on their respective master die in a custom scanning jig and measured using a measuring microscope at 90× (60 measurements per specimen, 15 per surface). Following initial measurements, all copings were adjusted on stone dies. The number of adjustment cycles was recorded and post-adjustment measurements were made using the same method. Data were analyzed using independent and paired t-tests.RESULTS:Milled gold copings with a beveled margin (11.7 ± 20.4 μm) had a significantly (p < 0.05) smaller marginal gap than cast gold copings with a beveled margin (43.6 ± 46.8 μm) after adjustment. Cast gold copings with a chamfer margin (22.7 ± 24.7 μm) had a significantly (p < 0.05) smaller marginal gap than milled gold copings with a chamfer margin (27.9 ± 31.6 μm) following adjustments. Adjustments significantly decreased marginal gap for both cast groups (p < 0.05) and the milled chamfer bevel group (p < 0.05) but had no significant effect on the milled chamfer group.CONCLUSIONS:Within the limitations of this study, results indicate that gold restorations milled with the tested parameters provide a vertical marginal gap that is an acceptable alternative to traditional gold crown casting techniques.

Statement of problem. Studies evaluating the marginal adaptation of available computer-aided design and computer-aided manufacturing (CAD-CAM) noble alloys for metal-ceramic prostheses are lacking. Purpose. The purpose of this in vitro study was to evaluate the vertical marginal adaptation of cast, milled, and direct metal laser sintered (DMLS) noble metal-ceramic 3-unit fixed partial denture (FDP) frameworks before and after fit adjustments. Material and method. Two typodont teeth were prepared for metal-ceramic FDP abutments. An acrylic resin pattern of the prepared teeth was fabricated and cast in nickel-chromium (Ni-Cr) alloy. Each specimen group (cast, milled, DMLS) was composed of 12 casts made from 12 impressions (n=12). A single design for the FDP substructure was created on a laboratory scanner and used for designing the specimens in the 3 groups. Each specimen was fitted to its corresponding cast by using up to 5 adjustment cycles, and marginal discrepancies were measured on the master Ni-Cr model before and after laboratory fit adjustments. Results. The milled and DMLS groups had smaller marginal discrepancy measurements than those of the cast group (P<.001). Significant differences were found in the number of adjustments among the groups, with the milled group requiring the minimum number of adjustments, followed by the DMLS and cast groups (F=30.643, P<.001). Conclusions. Metal-ceramic noble alloy frameworks fabricated by using a CAD-CAM workflow had significantly smaller marginal discrepancies compared with those with a traditional cast workflow, with the milled group demonstrating the best marginal fit among the 3 test groups. Manual refining significantly enhanced the marginal fit of all groups. All 3 groups demonstrated marginal discrepancies within the range of clinical acceptability.

SUMMARY Peptide-based polymers are promising vehicles for gene delivery due to the ability to incorporate peptides to specifically address various intracellular barriers, such as cellular uptake and endosomal escape. Recently, our group optimized a panel of statistical HPMA-oligolysine copolymers to investigate variations in oligolysine length and polymer molecular weight. We showed that comblike polymers synthesized using K10 (10-mers of lysines) were efficient at gene delivery whereas comparable polymers synthesized using K15 performed poorly with delivery efficiencies similar to poly(L)lysine. The goal of this study was to elucidate differences in transfection mechanisms between these two polymers in order to better identify key parameters that affect gene delivery. We demonstrate that polyplex morphology, unpackaging ability, and cellular uptake are important properties are to consider when designing polymers for gene delivery. Specifically, polyplexes with smaller aspect ratios, the ability to unpackage more easily, and high uptake efficiency correlated with higher in vitro transfection efficiencies in our HPMA-oligolysine system. INTRODUCTION Successful gene delivery requires vectors capable of overcoming several extracellular and intracellular barriers, such as cellular uptake, endosomal escape, effective unpackaging of therapeutic cargo, and nuclear import. Peptidebased polymers have been used as nucleic acid carriers to tackle these barriers by the incorporation of specific peptides for cell targeting, endosomal-lytic activity, and nuclear localization sequences, to name a few. These peptides can also be incorporated into well-defined narrowly-disperse statistical polymers, synthesized using living radical polymerization techniques, such as reversible-addition fragmentation polymerization (RAFT). Previously, our group reported the synthesis and characterization of a series of HPMA-oligolysine statistical copolymers for the delivery of plasmid DNA, optimizing oligolysine peptide length and polymer molecular weight. We found that a peptide length of ten lysines (K10) and a degree of polymerization of 150 (DP 150) performed almost as well as 25 kDa branched polyethylenimine (PEI), despite a lack of incorporated endosomal escape capabilities. Interestingly, a polymer with a 50% longer oligolysine length (K15) was inefficient at gene transfer. The goal of this work is to identify key differences between these two similar polymers that might affect transfection efficiency. These studies demonstrate that differences in polyplex morphology, unpackaging, and cellular uptake may lead to the increased transfection efficiency of pHK10. In addition, pHK10 and pHK15 polyplexes were taken up more efficiently in GAG-deficient cells, indicating possible extracellular inhibition of oligolysine-containing polyplexes. EXPERIMENTAL METHODS Synthesis of peptide monomers, methacrylated AhxK10 (MaAhxK10) and methacrylated AhxK15 (MaAhxK15) were synthesized on a solid support of Rink amide following standard Fmoc/tBu chemistry on an automated PS3 peptide synthesizer and cleaved off the solid support with a standard acidic cleavage cocktail (TFA/TIPS/DMB) for 2 h while gently shaking. Peptides were then precipitated in cold ether and lyophilized. The monomers were analyzed using RP-HPLC and MALDI-TOF mass spectrometry, and were shown to have greater than 95% purity after cleavage. Copolymers of HPMA and either MaAhxK10 or MaAhxK15 were synthesized via RAFT polymerization under aqueous conditions, using ECT as the chain transfer agent. The resulting polymers were dialyzed, lyophilized, and characterized by size exclusion chromatography and amino acid analysis. For uptake studies, luciferase-expressing plasmid DNA was radiolabeled using nick translation and 2’-deoxycytidine 5’-triphosphate, [5-H], according to manufacturer’s instructions. A mixture of unlabeled pDNA and [H]DNA was used to formulate polyplexes (N/P 5). HeLa cells were treated with radiolabeled polyplexes for up to 4 h in OptiMEM, and then chased for up to 20 h in complete media. At various time points, cells were washed with PBS, and incubated with CellScrub to remove membrane-bound polyplexes. Cells were then trypsinized and collected. All washes and cell samples were analyzed for radioactivity by scintillation counting (Beckman LS-6500). For microscopy and unpackaging studies, polyplexes were formulated at a charge (N/P) ratio of 5. Polyplex morphology was determined by electron microscopy on a hydrophilic surface, rendered by glow discharge for 45 s. Polyplexes were applied to 400-mesh copper/formvar grids for 30 min. The grids were then rinsed in dH2O, and negatively-stained with 4% (w/v) uranyl acetate. Images were taken with a JOEL 1010 transmission electron microscope (Fred Hutch CRC). Polyplex unpackaging was determined by treating polyplexes with various amounts of heparan sulfate for 5 min at RT. The treated sample was then mixed with loading buffer and applied to a 0.8% agarose gel. DNA was visualized with ethidium bromide using a UV transilluminator. RESULTS AND DISCUSSION Differences in polyplex uptake may account for differences in transfection efficiency. To determine cellular uptake of polyplexes, HeLa cells were treated with radiolabeled polyplexes for up to 4 h, and then chased for up to 20 h. Three lysine-based polymers were tested: poly(L)lysine (PLL), pHK10 and pHK15. pHK10 polyplexes showed the most efficient uptake PLL, pHK15, and DNA (Figure 1). These results indicate that despite polyplex association with the cell, lack of efficient cellular uptake may lead to reduced transfection efficiencies. Figure 1. Uptake of radiolabeled polyplexes over