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IntroductionDozens of vaccines have been approved or authorized internationally in response to the ongoing SARS-CoV-2 pandemic, covering a range of modalities and routes of delivery. For example, mucosal delivery of vaccines via the intranasal (i.n.) route has been shown to improve protective mucosal responses in comparison to intramuscular (i.m.) delivery. As we gain knowledge of the limitations of existing vaccines, it is of interest to understand if changes in product presentation or combinations of multiple vaccine modalities can further improve immunological outcomes.MethodsWe investigated a commercial-stage SARS-CoV-2 receptor binding domain (RBD) antigen adjuvanted with a clinical-stage TLR-7/8 agonist (3M-052) formulated on aluminum oxyhydroxide (Alum). In a murine immunogenicity model, we compared i.n. and i.m. dosing of the RBD-3M-052-Alum vaccine. We measured the magnitude of antibody responses in serum and lungs, the antibody-secreting cell populations in bone marrow, and antigen-specific cytokine-secreting splenocyte populations. Similarly, we compared different heterologous and homologous prime-boost regimens using the RBD-3M-052-Alum vaccine and a clinical-stage self-amplifying RNA (saRNA) vaccine formulated on a nanostructured lipid carrier (NLC) using the i.m. route alone. Finally, we developed a lyophilized presentation of the RBD-3M-052-Alum vaccine and compared it to the liquid presentation and a heterologous regimen including a previously characterized lyophilized form of the saRNA-NLC vaccine.Results and discussionWe demonstrate that i.n. dosing of the RBD-3M-052-Alum vaccine increased IgA titers in the lung by more than 1.5 logs, but induced serum IgG titers 0.8 logs lower, in comparison to i.m. dosing of the same vaccine. We also show that the homologous prime-boost RBD-3M-052-Alum regimen led to the highest serum IgG and bronchial IgA titers, whereas the homologous saRNA-NLC regimen led to the highest splenocyte interferon-γ response. We found that priming with the saRNA-NLC vaccine and boosting with the RBD-3M-052-Alum vaccine led to the most desirable immune outcome of all regimens tested. Finally, we show that the lyophilized RBD-3M-052-Alum vaccine retained its immunological characteristics. Our results demonstrate that the route of delivery and the use of heterologous regimens each separately impacts the resulting immune profile, and confirm that multi-product vaccine regimens can be developed with stabilized presentations in mind.

Abstract The development of broad-spectrum coronavirus vaccines is essential to prepare for future respiratory virus pandemics. We demonstrated broad neutralization by a trivalent subunit vaccine, formulating the receptor-binding domains of SARS-CoV, MERS-CoV, and SARS-CoV-2 XBB.1.5 with Alum and CpG55.2. Vaccinated mice produced cross-neutralizing antibodies against all three human Betacoronaviruses and others currently exclusive to bats, indicating the epitope preservation of the individual antigens during co-formulation and the potential for epitope broadening.

Introduction The development of a yeast-expressed recombinant protein-based vaccine technology co-developed with LMIC vaccine producers and suitable as a COVID-19 vaccine for global access is described. The proof-of-concept for developing a SARS-CoV-2 spike protein receptor-binding domain (RBD) antigen as a yeast-derived recombinant protein vaccine technology is described. Areas Covered Genetic Engineering: The strategy is presented for the design and genetic modification used during cloning and expression in the yeast system. Process and Assay Development: A summary is presented of how a scalable, reproducible, and robust production process for the recombinant protein COVID-19 vaccine antigen was developed. Formulation and Pre-clinical Strategy: We report on the pre-clinical and formulation strategy used for the proof-of-concept evaluation of the SARS-CoV-2 RBD vaccine antigen. Technology Transfer and Partnerships: The process used for the technology transfer and co-development with LMIC vaccine producers is described. Clinical Development and Delivery: The approach used by LMIC developers to establish the industrial process, clinical development, and deployment is described. Expert Opinion Highlighted is an alternative model for developing new vaccines for emerging infectious diseases of pandemic importance starting with an academic institution directly transferring their technology to LMIC vaccine producers without the involvement of multinational pharma companies.

Introduction: Since their introduction, adjuvanted recombinant subunit vaccines against COVID-19 have played a pivotal role in protecting global populations. Optimizing the immune response’s quality, amplitude, and durability to these vaccines depends on the appropriate adjuvant choice and dose in combination with the selected antigen.Methods: Here, we employed a preclinical mouse model to study the adaptive humoral and cellular immune responses to a SARS-CoV-2 receptor binding domain (RBD) antigen formulated with one of four different immune agonists [GLA, 3M-052, CpG-1826 (CpG), and dmLT], in combination with one of two different immune-stimulating formulations, a stabilized squalene emulsion (SE) or aluminum hydroxide (Alum). Using a weighted desirability index, we established an immunogenicity ranking for each adjuvant in combination with the RBD antigen.Results: We found that formulations of the RBD with Alum in combination with either 3M-052 or CpG led to at least a 2-log increase in serum IgG production and a 1.3- to 2.2-log increase in the number of bone marrow-derived antibody-secreting cells compared to the RBD formulated with Alum without an additional agonist. In contrast, the RBD formulated with SE in combination with 3M-052 or CpG did not elicit an IgG response greater than the unadjuvanted control. Additionally, RBD formulated with 3M-052 or CpG on Alum generated a 0.8- or 1.6-log lower splenocyte IL-5 response (a pro-Th2 marker), respectively, than Alum without an additional agonist. When formulated with 3M-052-Alum, a bivalent vaccine containing the original lineage (Wuhan-Hu-1) and the Delta variant (B.1.617.2) RBD antigens led to a more than 2-log increase in neutralizing antibodies against an Omicron variant (B.1.1.529) pseudovirus in vaccinated animals compared to animals that received the monovalent RBD antigen.Discussion: Our results suggest that optimal immune responses to subunit antigens may be achieved through an orthogonal approach that applies adjuvant formulation, antigen combination, and advances in rational vaccine development techniques.

(1) Background: We previously reported the development of a recombinant protein SARS-CoV-2 vaccine, consisting of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, adjuvanted with aluminum hydroxide (alum) and CpG oligonucleotides. In mice and non-human primates, our wild-type (WT) RBD vaccine induced high neutralizing antibody titers against the WT isolate of the virus, and, with partners in India and Indonesia, it was later developed into two closely resembling human vaccines, Corbevax and Indovac. Here, we describe the development and characterization of a next-generation vaccine adapted to the recently emerging XBB variants of SARS-CoV-2. (2) Methods: We conducted preclinical studies in mice using a novel yeast-produced SARS-CoV-2 XBB.1.5 RBD subunit vaccine candidate formulated with alum and CpG. We examined the neutralization profile of sera obtained from mice vaccinated twice intramuscularly at a 21-day interval with the XBB.1.5-based RBD vaccine, against WT, Beta, Delta, BA.4, BQ.1.1, BA.2.75.2, XBB.1.16, XBB.1.5, and EG.5.1 SARS-CoV-2 pseudoviruses. (3) Results: The XBB.1.5 RBD/CpG/alum vaccine elicited a robust antibody response in mice. Furthermore, the serum from vaccinated mice demonstrated potent neutralization against the XBB.1.5 pseudovirus as well as several other Omicron pseudoviruses. However, regardless of the high antibody cross-reactivity with ELISA, the anti-XBB.1.5 RBD antigen serum showed low neutralizing titers against the WT and Delta virus variants. (4) Conclusions: Whereas we observed modest cross-neutralization against Omicron subvariants with the sera from mice vaccinated with the WT RBD/CpG/Alum vaccine or with the BA.4/5-based vaccine, the sera raised against the XBB.1.5 RBD showed robust cross-neutralization. These findings underscore the imminent opportunity for an updated vaccine formulation utilizing the XBB.1.5 RBD antigen.

There is an urgent need for an accessible and low-cost COVID-19 vaccine suitable for low- and middle-income countries. Here, we report on the development of a SARS-CoV-2 receptor-binding domain (RBD) protein, expressed at high levels in yeast (Pichia pastoris), as a suitable vaccine candidate against COVID-19. After introducing two modifications into the wild-type RBD gene to reduce yeast-derived hyperglycosylation and improve stability during protein expression, we show that the recombinant protein, RBD219-N1C1, is equivalent to the wild-type RBD recombinant protein (RBD219-WT) in an in vitro ACE-2 binding assay. Immunogenicity studies of RBD219-N1C1 and RBD219-WT proteins formulated with Alhydrogel® were conducted in mice, and, after two doses, both the RBD219-WT and RBD219-N1C1 vaccines induced high levels of binding IgG antibodies. Using a SARS-CoV-2 pseudovirus, we further showed that sera obtained after a two-dose immunization schedule of the vaccines were sufficient to elicit strong neutralizing antibody titers in the 1:1,000 to 1:10,000 range, for both antigens tested. The vaccines induced IFN-γ IL-6, and IL-10 secretion, among other cytokines. Overall, these data suggest that the RBD219-N1C1 recombinant protein, produced in yeast, is suitable for further evaluation as a human COVID-19 vaccine, in particular, in an Alhydrogel® containing formulation and possibly in combination with other immunostimulants.

A SARS-CoV receptor-binding domain (RBD) recombinant protein was developed and manufactured under current good manufacturing practices in 2016. The protein, known as RBD219-N1 when formulated on Alhydrogel®, induced high-level neutralizing antibodies and protective immunity with minimal immunopathology in mice after a homologous virus challenge with SARS-CoV (MA15 strain). We examined published evidence in support of whether the SARS-CoV RBD219-N1 could be repurposed as a heterologous vaccine against Coronavirus Infectious Disease (COVID)-19. Our findings include evidence that convalescent serum from SARS-CoV patients can neutralize SARS-CoV-2. Additionally, a review of published studies using monoclonal antibodies (mAbs) raised against SARS-CoV RBD and that neutralizes the SARS-CoV virus in vitro finds that some of these mAbs bind to the receptor-binding motif (RBM) within the RBD, while others bind to domains outside this region within RBD. This information is relevant and supports the possibility of developing a heterologous SARS-CoV RBD vaccine against COVID-19, especially due to the finding that the overall high amino acid similarity (82%) between SARS-CoV and SARS-CoV-2 spike and RBD domains is not reflected in RBM amino acid similarity (59%). However, the high sequence similarity (94%) in the region outside of RBM offers the potential of conserved neutralizing epitopes between both viruses.

Background: To describe a novel operative technique resulting in extended durability and improved ease of use of current flexible ureteroscopes (FURS). A surgical method employing a modified technique of using an Olympus digital flexible URF-V ureteroscope was developed. Methods: We retrospectively studied 546 patients who underwent retrograde intrarenal surgery (RIRS) using this modified approach performed by a single surgeon at our hospital, and investigated the outcome and durability of the ureteroscope. Results: Through the study period, the URF-V ureteroscope required repair five times in total. During factory maintenance, distal working channel damage was noted twice, and outer bending rubber damage was noted once. The most recent two repairs were required due to laser penetration. Despite the damage and repairs, the deflection system was almost entirely intact after high-frequency use. The durability of FURS determines the efficacy of RIRS for renal stones. Conclusion: In this report, we described our modified upside-down technique for manipulation of FURS under unequal dual deflection in order to preserve the deflection apparatus, which yielded a greatly prolonged durability. Additionally, the use of mimic drive turning decreased the time needed to train urologists. Keywords: Flexible ureteroscopes, Renal stone, Retrograde intrarenal surgery

Objective: Management of patients with upper ureteral and renal urolithiasis is challenging for endourologists. Since the advancements in endourological equipment/devices, retrograde intrarenal surgery (RIRS) has become an attractive, widespread technique for upper urinary tract stone manipulation, owing to its advantage of being noninvasive with higher stone clearance rate and less pain compared with traditional shock wave lithotripsy (SWL) and percutaneous nephrolithotripsy (PNL). However, the outcome and complication of staged RIRS for large stone burden (> 2.0 cm) management for renal stones still need to be reported. A total of 172 cases of renal calculi were treated by RIRS in a single center; among these, 22 had renal stone size larger than 2.0 cm. In this work, we present our experience and treatment outcomes of these patients. Materials and methods: Between August 2015 and January 2016, we enrolled 172 patients with upper urinary tract calculi who underwent RIRS with holmium laser lithotripsy performed by one surgeon in a singer center. Based on a medical record review, we collected data pertaining to the outcomes and complications of RIRS. Results: A total of 172 patients with upper urinary tract stones were included in this study (113 male and 59 female patients). All patients underwent RIRS with holmium laser lithotripsy procedure. The overall stone-free rate (SFR) was 90.6%. The average stone size was 0.8 cm with average operation time of 89.7 minutes. Average hospital stay was 2.7 days. The average pain score was 2.38/10 by the Numerical Rating Scale. In the large stones size subgroups, the SFR of stone burden between 2.0 cm and 3.0 cm was 80.2%, and the SFR of stone burden over 3.0 cm was 45.0% by single RIRS. Ten of 22 patients underwent two-staged RIRS and their SFR improved from 45.0% to 76.5%. No major complications (Clavien III–V) were noted in the study groups. Conclusion: To the best of our knowledge, RIRS for large renal stone manipulation is an effective and safe treatment modality currently. In our study, the single RIRS SFR was superior to PNL or SWL even when the stone burden was between 2.0 cm and 3.0 cm (80.2%). For those patients whose stone burden was over 3.0 cm or for those with comorbidities, staged RIRS resulted in a lower complication rate, reduced hospital stay, and better SFR (76.5%).

To assess the effect of alfuzosin (XATRAL) 10 mg once daily on sexual function in men with moderate to severe lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH), patients with suggestive symptomatic BPH, an International Prostate Symptom Score (IPSS) >8 (range of scores, 0–35), and sexual attempts at least once per month were enrolled. All patients received alfuzosin 10 mg once daily for 24 weeks and were asked to complete the IPSS test and Male Sexual Health Questionnaire at weeks 0 (baseline), 1, 4, 12, and 24. Other assessments included the International Index of Erectile Function—five-item version (range of scores: 5–25), as well as onset of action and peak urinary flow rate (Qmax). From September 2006 to May 2008, 279 patients were enrolled from nine centers in Taiwan. At 24 weeks, alfuzosin effectively improved LUTS and quality of life, as demonstrated by a reduction in the IPSS total score (17.3 vs. 9.9, p

Objective: The goal of this paper is to evaluate the safety and efficacy of perioperative parecoxib, a cyclooxygenase-2 inhibitor in reducing transient postoperative pyelovenous backflow pain in patients undergoing retrograde intrarenal surgery (RIRS). Materials and Methods: We instituted a change in our institutional clinical practice starting in January 2016. Since January 2016, all patients undergoing RIRS were administered perioperative intravenous parecoxib. Patients with a history of chronic renal failure (Cr over 2.5) or coronary heart disease did not receive parecoxib. The numerical scale NRS (Numerical rating scale) was recorded after postoperative conscious recovery within 24 h of the postoperative hospitalization. The NRS values were compared with a matched cohort of patients treated with RIRS at our center before January 2016 initiation of parecoxib administration to RIRS patients. Results: A total of 116 patients received Parecoxib during RIRS between January 2016 and August 2016. We found that parecoxib infusion was associated with significantly decreased of NRS. Out of 243 patients, 160 patients treated with parecoxib received an NRS of 1.57 points versus 127 of 243 matched controls who had an NRS of 3.32 points (P < 0.0001). The proportion of NRS >4 score in the patients with parecoxib infusion is much lower than those patients who did not receive Parecoxib (9% vs. 30%). Conclusion: Use of perioperative parecoxib during RIRS is associated with a significant reduction in transient postoperative pyelovenous backflow pain and increased operation quality. [ABSTRACT FROM AUTHOR]

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by asmuch as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally. [ABSTRACT FROM AUTHOR]

The article presents information on a study which examines the removal conditions of iron and manganese ions from groundwater by using three methods, aeration, chlorine oxidation and microfiltration (MF). It analyzed the removal effects of iron and manganese under the conditions of oxidant doses, pH and reaction times in the Jar-test.

ABSTRACT The aim of this research was to investigate the removal of iron and manganese from groundwater by aeration, chlorine oxidation and microfiltration (MF). Pilot-scale experiments were performed at the Chang-Hua Water Treatment Plant in Taiwan. The raw waters contained soluble iron (0.4 mg/L) and manganese (0.5 mg/L) which were oxidized by chlorine and then filtered by hollow-fiber PTFE-MF. The oxidized particles on membranes under appropriate backwash were also investigated. The particle size and quality of treated water were examined. The experimental results indicated that the concentration of the residual manganese remained greater than 0.1 mg/L in treated water during the initial operation period. However, it decreased to below 0.01 mg/L after 2 weeks processing time. The scanning electron microscopy (SEM) energy dispersive spectroscopy analysis of oxidized metal particles under each process illustrated that the particles accumulated on membrane enhanced removal of iron and manganese, through autocatalytic activity in the accumulated layer on membrane. Moreover, the membrane operation pressure over 120 kPa was suggested for the active backwash process, where no structural damage on membrane was confirmed by SEM analysis. [ABSTRACT FROM AUTHOR]