CspZ FH-Binding Sites as Epitopes Promote Antibody-Mediated Lyme Borreliae Clearance.

Transmitted by ticks, the bacterium Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD), the most common vector-borne disease in the Northern hemisphere. No effective vaccines are currently available. B. burgdorferi sensu lato produces the CspZ protein that binds to the complement inhibitor, factor H (FH), promoting evasion of the host complement system. We previously showed that while vaccination with CspZ did not protect mice from B. burgdorferi infection, mice can be protected after immunization with CspZ-Y207A/Y211A (CspZ-YA), a CspZ mutant protein without FH-binding activity. To further study the mechanism of this protection, herein we evaluated both poly- and monoclonal antibodies recognizing CspZ FH-binding or non-FH-binding sites. We found that the anti-CspZ antibodies that recognize the FH-binding sites (i.e., block FH-binding activity) eliminate B. burgdorferi sensu lato in vitro more efficiently than those that bind to the non-FH-binding sites, and passive inoculation with anti-FH-binding site antibodies eradicated B. burgdorferi sensu lato in vivo. Antibodies against non-FH-binding sites did not have the same effect. These results emphasize the importance of CspZ FH-binding sites in triggering a protective antibody response against B. burgdorferi sensu lato in future LD vaccines.

Yeast-expressed recombinant SARS-CoV-2 receptor binding domain RBD203-N1 as a COVID-19 protein vaccine candidate.

SARS-CoV-2 protein subunit vaccines are currently being evaluated by multiple manufacturers to address the global vaccine equity gap, and need for low-cost, easy to scale, safe, and effective COVID-19 vaccines. In this paper, we report on the generation of the receptor-binding domain RBD203-N1 yeast expression construct, which produces a recombinant protein capable of eliciting a robust immune response and protection in mice against SARS-CoV-2 challenge infections. The RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity was evaluated in mice. Sera were evaluated for their efficacy using a SARS-CoV-2 pseudovirus assay. The RBD203-N1 protein was expressed with a yield of 492.9 ± 3.0 mg/L of fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 ± 3% (total yield of purified protein: 270.5 ± 13.2 mg/L fermentation supernatant). The protein was characterized to be a homogeneous monomer that showed a well-defined secondary structure, was thermally stable, antigenic, and when adjuvanted on Alhydrogel in the presence of CpG it was immunogenic and induced high levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. The characteristics of the RBD203-N1 protein-based vaccine show that this candidate is another well suited RBD-based construct for technology transfer to manufacturing entities and feasibility of transition into the clinic to evaluate its immunogenicity and safety in humans.

Reducing the incidence of stroke-associated pneumonia: an evidence-based practice.

BACKGROUND: Pulmonary infection is a frequent complication among stroke patients and adversely affects clinical outcomes, increases the length of hospitalization stay and costs, and aggravates the financial burden of the national medical health system. Early identification and management of high-risk patients are necessary and imperative to reduce the incidence of stroke-associated pneumonia (SAP). AIM: The evidence-based practice project evaluated the effectiveness of a standard care bundle intervention in preventing the occurrence of SAP. METHODS: The project was conducted in a neurology department of a teaching hospital. Given the variation in assessment and management standards, evidence-based practice (EBP) methodology was used to establish a process for quality improvement. A thorough literature search was conducted to identify evidence-based interventions to manage and prevent SAP. Thorough critiques of the literature and synthesis of the evidence were completed. A systematic management flow and care bundle interventions were established. The care bundle included interventions, such as the utilization of tools for SAP risk screening; dysphagia screening and rehabilitation; feeding modification, oral care, airway management, position management, and the nursing techniques of traditional Chinese medicine. RESULTS: A significant improvement was observed in preventing SAP in patients in the postimplementation group compared with those in the preimplementation group (14.0% vs. 37.2%, p = 0.025). In addition, significantly lower duration of hospitalization, lower rate of aspiration, and improvements in albumin and oral hygiene were found after the implementation of the care bundle. CONCLUSIONS: Evidence-based care bundles successfully empower nurses to reduce the incidence of SAP. The management flow of SAP prevention could be promoted to other units of the neurology department in the future. The results of the project reflect positively on the capacity to implement EBP in an acute care setting for stroke. The EBP methodology can be utilized to solve other clinical problems.

A scalable and reproducible manufacturing process for Phlebotomus papatasi salivary protein PpSP15, a vaccine candidate for leishmaniasis.

Cutaneous leishmaniasis is a parasitic and neglected tropical disease transmitted by the bites of sandflies. The emergence of cutaneous leishmaniasis in areas of war, conflict, political instability, and climate change has prompted efforts to develop a preventive vaccine. One vaccine candidate antigen is PpSP15, a 15 kDa salivary antigen from the sandfly Phlebotomus papatasi that facilitates the infection of the Leishmania parasite and has been shown to induce parasite-specific cell-mediated immunity. Previously, we developed a fermentation process for producing recombinant PpSP15 in Pichia pastoris and a two-chromatographic-step purification process at 100 mL scale. Here we expand the process design to the 10 L scale and examine its reproducibility by performing three identical process runs, an essential transition step towards technology transfer for pilot manufacture. The process was able to reproducibly recover 81% of PpSP15 recombinant protein with a yield of 0.75 g/L of fermentation supernatant, a purity level of 97% and with low variance among runs. Additionally, a freeze-thaw stability study indicated that the PpSP15 recombinant protein remains stable after undergoing three freeze-thaw cycles, and an accelerated stability study confirmed its stability at 37 °C for at least one month. A research cell bank for the expression of PpSP15 was generated and fully characterized. Collectively, the cell bank and the production process are ready for technology transfer for future cGMP pilot manufacturing.

Process development and scale-up optimization of the SARS-CoV-2 receptor binding domain-based vaccine candidate, RBD219-N1C1.

A SARS-CoV-2 RBD219-N1C1 (RBD219-N1C1) recombinant protein antigen formulated on Alhydrogel® has recently been shown to elicit a robust neutralizing antibody response against SARS-CoV-2 pseudovirus in mice. The antigen has been produced under current good manufacturing practices (cGMPs) and is now in clinical testing. Here, we report on process development and scale-up optimization for upstream fermentation and downstream purification of the antigen. This includes production at the 1-L and 5-L scales in the yeast, Pichia pastoris, and the comparison of three different chromatographic purification methods. This culminated in the selection of a process to produce RBD219-N1C1 with a yield of >400 mg per liter of fermentation with >92% purity and >39% target product recovery after purification. In addition, we show the results from analytical studies, including SEC-HPLC, DLS, and an ACE2 receptor binding assay that were performed to characterize the purified proteins to select the best purification process. Finally, we propose an optimized upstream fermentation and downstream purification process that generates quality RBD219-N1C1 protein antigen and is fully scalable at a low cost. KEY POINTS: • Yeast fermentation conditions for a recombinant COVID-19 vaccine were determined. • Three purification protocols for a COVID-19 vaccine antigen were compared. • Reproducibility of a scalable, low-cost process for a COVID-19 vaccine was shown. Graphical abstract.

Impact of the systematic use of the volume-viscosity swallow test in patients with acute ischaemic stroke: a retrospective study.

BACKGROUND: Dysphagia is common after stroke. Patients with dysphagia have a higher risk of stroke-associated pneumonia (SAP) and poor outcomes. Early detection of dysphagia is necessary to identify and manage patients at high risk of aspiration. The aim of the study was to assess the impact of the systematic administration of the volume-viscosity swallow test (V-VST) in patients with acute ischaemic stroke. METHODS: This was a retrospective observational study that enrolled patients with acute ischaemic stroke in two consecutive time periods: pre-V-VST, when the 30-mL water-swallowing test (WST) was systematically administered, and V-VST, when all patients underwent the WST and the V-VST test was systematically administered if the patient failed the WST. RESULTS: Two hundred and 42 patients were enrolled. The mean age of the participants was 68.8 ± 10.88 years, 61.2% were male, and the median National Institutes of Health Stroke Scale score was 3 (IQR, 1-6). A total of 147 patients were enrolled during the pre-V-VST period and 95 were enrolled during the V-VST period. There was a significant difference in the occurrence of SAP (21.8% vs. 10.5%, p = 0.024) and the rate of nasogastric tube feeding (25.9% vs. 14.7%, p = 0.040) between the two groups, and no differences were found in the length of hospital stay (p = 0.277) or the total cost of hospitalization (p = 0.846). CONCLUSIONS: The V-VST was a better clinical screening tool, and it can also provide detailed suggestions regarding dietary modifications to prevent aspiration and SAP.

Factors influencing health behaviour, blood pressure control, and disability in hypertensive ischaemic stroke patients after a comprehensive reminder intervention.

AIMS: To test prospective pathways of a Comprehensive Reminder System based on the Health Belief Model (CRS-HBM), stroke knowledge, health belief in health behaviour, blood pressure (BP) control, and disability in hypertensive ischaemic stroke patients at 6-month postdischarge. DESIGN: A nested cohort study design. METHODS: Data were derived from a randomized controlled trial evaluating the effects of the intervention (N = 174, performed during February 2015 - March 2016). Data were collected by questionnaires and analysed in structural equation modelling in Mplus software. RESULTS: The proposed model provided a good fit to the data. This model accounted for 51.5% of the variance in health behaviour, 34.1% in BP control, and 5.7% in modified Rankin Scale score at 6-month postdischarge. The CRS-HBM had: (a) direct positive effect (ß = .391, p < .001) and indirect positive effects (ß = .186, p = .002) on health behaviour; (b) direct positive effect (ß = .356, p < .001) and indirect positive effects (ß = .183, p = .009) on BP control; and (c) indirect negative effect (ß = -.146, p = .008) on disability. Being female was linked to better health behaviour. Higher education predicted higher level of stroke knowledge and health belief. CONCLUSIONS: The CRS-HBM can not only directly but also indirectly improve patients' health behaviours by improving their health knowledge or health belief. Better health behaviour can improve patients' BP control and reduce disability. Therefore, nurses need to pay more attention to not only patients' health knowledge but also their health belief when providing education. IMPACT: The CRS-HBM intervention accounted for 51.5% of variance in health behaviour, 34.1% in BP control, and 5.7% in modified Rankin Scale score at 6-month postdischarge. This research can help nurses improve health education strategies in postdischarge and community contexts to achieve better health results.

Glucose screening within six months postpartum among Chinese mothers with a history of gestational diabetes mellitus: a prospective cohort study.

BACKGROUND: Gestational diabetes mellitus (GDM) is a risk factor for diabetes mellitus. The 75-g, 2-h oral glucose tolerance test is recommended for mothers with a history of GDM to screen for diabetes in the postnatal period. The aim of this study was to investigate the rate of glucose screening within 6 months postpartum among Chinese mothers with a history of GDM, and to identify its predictors. METHODS: A prospective cohort study was conducted in a regional teaching hospital in Guangzhou, China, between July 2016 and June 2017. The participants were Chinese mothers (n = 237) who were diagnosed with GDM, were aged 18 years or older with no serious physical or mental disease and had not been diagnosed with type 1 or type 2 diabetes prior to their pregnancy. The revised Chinese version of the Champion's Health Belief Model Scale and social-demographic and perinatal characteristics factors were collected and used to predict postpartum glucose screening (yes or no). Adjust odds ratio (AOR) and 95% confidence interval (95% CI) were calculated. RESULTS: The mean age of the 237 mothers was 32.70 years (range from 22 to 44). Almost half of the mothers (45.6%) were college graduates or higher. Chinese mothers reported a high level of perceived benefits, self-efficacy, and health motivation towards postpartum glucose screening, with a mean score above 3.5. Chinese mothers were more likely to undertake postpartum glucose screening if they were a first-time mother [AOR 2.618 (95% CI: 1.398-4.901)], had a high perceived susceptibility score [AOR 2.173 (95% CI: 1.076-4.389)], a high perceived seriousness score [AOR 1.988 (95%CI: 1.020-3.875)] and high perceived benefits score [AOR 2.978 (95%CI: 1.540-5.759)]. CONCLUSION: The results of this study will lead to better identification of mothers with a history of GDM who may not screen for postpartum glucose abnormality. Health care professionals should be cognizant of issues that may affect postpartum glucose screening among mothers with a history of GDM, including parity, perceived susceptibility, perceived seriousness and perceived benefits.

Expression, purification, immunogenicity and protective efficacy of a recombinant nucleoside hydrolase from Leishmania donovani, a vaccine candidate for preventing cutaneous leishmaniasis.

The nucleoside hydrolase gene from Leishmania donovani was cloned and expressed in Escherichia coli as a full length 36-kDa protein (LdNH36). Following lysis and extraction, the protein was purified by anion exchange and gel filtration chromatography. The purified protein had a molecular mass of approximately 36-kDa and was confirmed to be >99% pure. Using a nucleoside hydrolase assay, the protein was found to exhibit a Km of 741 ± 246 µM. Protein integrity was confirmed by lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE), mass spectrometry (MS), and enzymatic assay. Analysis of antibody levels from immunized mice indicated that LdNH36 alone or in a stable emulsion with the Toll-like receptor-4 ligand glucopyranosyl lipid adjuvant (GLA-SE) as immunostimulant induced high levels of antigen-specific IgG antibodies. The cellular immune response indicated a Th1 response in mice immunized with LdNH36, but only when formulated with GLA-SE. Mice immunized with the LdNH36 antigen in combination with the GLA-SE adjuvant and challenged with Leishmania mexicana showed significant reductions (>20 fold) in parasite burden, confirming the protective efficacy of this vaccine candidate.

[Effect of Jian'erle Granule on Thl7/Treg Imbalance of Asthma Mice].

Objective To observe the levels of Th17 associated inflammatory factor IL-17 and Treg associated inflammatory factor IL-10 in serum and bronchoalveolar lavage fluid (BALF) of bronchial asthma mice, and to explore the mechanism of Jian'erle Granule (JG) for preventing and treating asth- ma. Methods Totally 40 Balb/c mice were divided into 4 groups according to random digit table, i.e., the normal control group, the asthma group, the post-excitation asthma group, the JG +asthma group, 10 in each group. Asthma model was established by 1% ovalbumin (OVA) (grade V) solution sensitization and excitation after intraperitoneal injection of antigen suspension in all groups except the normal control group. JG (0. 36 g/mL) was administrated to mice in the JG +asthma group after modeling. Equal volume of normal saline was administrated to mice in the rest 3 groups. All medication lasted for 14 successive days. After medication mice in the JG +asthma group and the asthma group were excited with 1% OVA (grade I) again for 40 min by atomization inhalation. Lung inflammation was examined by HE staining. Levels of IL-10 and IL-17 in BALF and serum were measured by ELISA. Results Bronchial structure in lung tissue was normal in mice of the normal control group, with no inflammatory infiltration seen. Mucosal epithelial cells of bronchial wall were injured in the asthma group and the post-excitation asthma group, with inflammatory infiltration seen. Inflammation around bronchus and blood vessels was obviously attenuated in the JG+asthma group. Compared with the normal control group,the IL-17 level in serum and BALF significantly increased, IL-10 level significantly decreased in the asthma group (P <0.01). Compared with the asthma group, IL-17 level in serum and BALF increased and IL-10 level in serum and BALF decreased in the post-excitation asthma group (P <0.01). Compared with the asthma group, the IL-17 level in serum and BALF significantly decreased, IL-10 level significantly increased in the JG +asth- ma group (P <0. 01). Compared with the post-excitation.asthma group,the IL-17 level in serun and BALF significantly decreased and IL-10 level in serum and BALF significantly increased in the JG + asthma group (P <0. 01). Conclusion The mechanism for JG preventing and treating asthma might be correla- ted with regulating Th17/Treg cytokine balance in serum and BALF.

Identification of immunodominant antigens for the laboratory diagnosis of toxocariasis.

OBJECTIVES: To identify immunodominant antigens of Toxocara canis recognised by Toxocara-infected sera as recombinant reagents for immunodiagnosis of toxocariasis. METHODS: Pooled sera from human cases of toxocariasis were used to identify immunodominant antigens by immunoscreening a T. canis larval expression cDNA library. The positive clones were sequenced to reveal the identity of the antigens. The recombinant proteins were expressed in E. coli and then used to confirm their immunoreaction with sera of humans with toxocariasis. Two chosen antigens were also used to differentiate Toxocara infection from other helminth infections in mice. RESULTS: Eleven antigens with immunodiagnostic potential were identified, including two C-type lectins (CTLs) that reacted strongly with the Toxocara-positive serum pool. The first CTL (Tc-CTL-1) is the same as TES-32, previously identified as a major immunodominant component of TES; the second CTL (Tc-CTL-2) is a novel C-type lectin sharing 83% amino acid sequence identity within the functional domain of Tc-CTL-1. The E. coli-expressed recombinant Tc-CTL-1 was strongly recognised by the Toxocara-positive serum pool or sera from animals experimentally infected with T. canis. Reactivity with recombinant Tc-CTL-1 was higher when the unreduced protein was used in an enzyme-linked immunosorbent assay (ELISA), dot-blot assay or Western blot test compared to the protein under reduced condition. Both recombinant Tc-CTL-1- and Tc-CTL-2-based ELISAs were able to differentiate T. canis infection from other helminth infections in experimentally infected mice. CONCLUSIONS: Both Tc-CTL-1 and Tc-CTL-2 were able to differentiate Toxocara infection from other helminth infections and could potentially be used as sensitive and specific immunodiagnostic antigens.

Natural disruption of two regulatory networks in serotype M3 group A Streptococcus isolates contributes to the virulence factor profile of this hypervirulent serotype.

Despite the public health challenges associated with the emergence of new pathogenic bacterial strains and/or serotypes, there is a dearth of information regarding the molecular mechanisms that drive this variation. Here, we began to address the mechanisms behind serotype-specific variation between serotype M1 and M3 strains of the human pathogen Streptococcus pyogenes (the group A Streptococcus [GAS]). Spatially diverse contemporary clinical serotype M3 isolates were discovered to contain identical inactivating mutations within genes encoding two regulatory systems that control the expression of important virulence factors, including the thrombolytic agent streptokinase, the protease inhibitor-binding protein-G-related α2-macroglobulin-binding (GRAB) protein, and the antiphagocytic hyaluronic acid capsule. Subsequent analysis of a larger collection of isolates determined that M3 GAS, since at least the 1920s, has harbored a 4-bp deletion in the fasC gene of the fasBCAX regulatory system and an inactivating polymorphism in the rivR regulator-encoding gene. The fasC and rivR mutations in M3 isolates directly affect the virulence factor profile of M3 GAS, as evident by a reduction in streptokinase expression and an enhancement of GRAB expression. Complementation of the fasC mutation in M3 GAS significantly enhanced levels of the small regulatory RNA FasX, which in turn enhanced streptokinase expression. Complementation of the rivR mutation in M3 GAS restored the regulation of grab mRNA abundance but did not alter capsule mRNA levels. While important, the fasC and rivR mutations do not provide a full explanation for why serotype M3 strains are associated with unusually severe invasive infections; thus, further investigation is warranted.

Incidence and risk factors for hypotension after carotid artery stenting: Systematic review and meta-analysis.

OBJECTIVE: Hypotension is recognized as a common complication after carotid artery stenting, but its incidence and the risk factors associated with it are uncertain. Therefore, we performed a systematic review and meta-analysis to investigate and identify risk factors for hypotension after surgery. METHODS: We retrieved risk factors from eight databases for case-control and cross-sectional studies of hypotension after carotid artery stenting according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines on 28 November 2022. Data were analyzed by using R4.2.1 and Review Manager 5.3. RESULTS: A total of 2843 samples were searched, and 17 publications were included in the analysis. The meta-analysis results showed that the incidence of hypotension after surgery was 28.6% (95% confidence interval [CI] (0.225, 0.347)). Age ⩾ 65 years (odds ratio [OR] = 4.55, 95% CI (2.50, 8.29), P < 0.00001), stenosis site (bulb) (OR = 4.41, 95% CI (2.50, 7.79), P < 0.00001), severe stenosis (OR = 3.56, 95% CI (1.62, 7.85), P = 0.002), stenosis proximity (⩽ 10 mm) to bifurcation (OR = 2.69, 95% CI (1.74, 4.15), P < 0.00001), calcified plaques (OR = 4.64, 95% CI (1.93, 11.14), P = 0.0006), post-balloon dilation (OR = 5.95, 95% CI (2.31, 15.31), P = 0.0002), bilateral carotid stenting (OR = 30.51, 95% CI (2.33, 399.89), P = 0.009), and intravenous fluid intake/mL on the first postoperative day (mean difference = 444.99, 95% CI (141.40, 748.59), P = 0.004) were risk factors for hypotension after surgery. CONCLUSIONS: A high incidence of hypotension was observed after carotid artery stenting. Age, stenosis site, severe stenosis, stenosis proximity to bifurcation, calcified plaques, post-balloon dilation, type of surgery, and intravenous fluid intake were identified as risk factors.

Anti-metastatic effects of AGS-30 on breast cancer through the inhibition of M2-like macrophage polarization.

AGS-30, a new andrographolide derivative, showed significant anticancer and anti-angiogenic characteristics. However, its role in controlling macrophage polarization and tumor immune response is unknown. Thus, the main goals of this study are to investigate how AGS-30 regulates macrophage polarization and how it suppresses breast cancer metastasis. AGS-30 inhibited IL-4 and IL-13-induced RAW 264.7 and THP-1 macrophages into M2-like phenotype. However, AGS-30 did not affect the LPS and IFN-γ-induced polarization of M1-like macrophages. AGS-30 reduced the mRNA expressions of CD206, Arg-1, Fizz-1, Ym-1, VEGF, IL-10, MMP2, and MMP9 in M2-like macrophages in a concentration-dependent manner. In contrast, andrographolide treatment at 5 µM did not affect M1-like and M2-like macrophage polarization. The conditioned medium from M2-like macrophages increased 4T1 breast cancer cell migration and invasion, whereas AGS-30 inhibited these effects. In the 4T1 breast tumor xenograft mice, the tumor volume and weight were reduced without affecting body weight after receiving AGS-30. AGS-30 treatment also reduced lung and liver metastasis, with reduced STAT6, CD31, VEGF, and Ki67 protein expressions. Moreover, the tumors had considerably fewer M2-like macrophages and Arg-1 expression, but the proportion of M1-like macrophages and iNOS expression increased after AGS-30 treatment. Same results were found in the tail vein metastasis model. In conclusion, this study shows that AGS-30 inhibits breast cancer growth and metastasis, probably through inhibiting M2-like macrophage polarization. Our findings suggest that AGS-30 may be a potential immunotherapeutic alternative for metastatic breast cancer.

Biophysical and biochemical characterization of a recombinant Lyme disease vaccine antigen, CspZ-YA.

Lyme disease, caused by Lyme Borrelia spirochetes, is the most common vector-borne illness in the United States. Despite its global significance, with an estimated 14.5 % seroprevalence, there is currently no licensed vaccine. Previously, we demonstrated that CspZ-YA protein conferred protection against Lyme Borrelia infection, making it a promising vaccine candidate. However, such a protein was tagged with hexahistidine, and thus not preferred for vaccine development; furthermore, the formulation to stabilize the protein was understudied. In this work, we developed a two-step purification process for tag-free E. coli-expressed recombinant CspZ-YA. We further utilized various bioassays to analyze the protein and determine the suitable buffer system for long-term storage and formulation as a vaccine immunogen. The results indicated that a buffer with a pH between 6.5 and 8.5 stabilized CspZ-YA by reducing its surface hydrophobicity and colloidal interactions. Additionally, low pH values induced a change in local spatial conformation and resulted in a decrease in α-helix content. Lastly, an optimal salinity of 22-400 mM at pH 7.5 was found to be important for its stability. Collectively, this study provides a fundamental biochemical and biophysical understanding and insights into the ideal stabilizing conditions to produce CspZ-YA recombinant protein for use in vaccine formulation and development.

RivR is a negative regulator of virulence factor expression in group A Streptococcus.

The bacterial pathogen group A Streptococcus (GAS) causes human diseases ranging from self-limiting pharyngitis (also known as strep throat) to severely invasive necrotizing fasciitis (also known as the flesh-eating syndrome). To control virulence factor expression, GAS utilizes both protein- and RNA-based mechanisms of regulation. Here we report that the transcription factor RivR (RofA-like protein IV) negatively regulates the abundance of mRNAs encoding the hyaluronic acid capsule biosynthesis proteins (hasABC; ∼7-fold) and the protein G-related α(2)-macroglobulin-binding protein (grab; ∼29-fold). Our data differ significantly from those of a previous study of the RivR regulon. Given that grab and hasABC are also negatively regulated by the two-component system CovR/S (control of virulence), we tested whether RivR functions through CovR/S. A comparison of riv and cov single and double mutant strains showed that RivR requires CovR activity for grab and hasABC regulation. Analysis of the upstream region of rivR identified a novel promoter the deletion of which reduced rivR mRNA abundance by 70%. A rivR mutant strain had a reduced ability to adhere to human keratinocytes relative to that of the parental and complemented strains, a phenotype that was abolished upon GAS pretreatment with hyaluronidase, highlighting the importance of capsule regulation by RivR during colonization. The rivR mutant strain was also attenuated for virulence in a murine model of bacteremia infection. Thus, we identify RivR as an important regulator of GAS virulence and provide new insight into the regulatory networks controlling virulence factor production in this pathogen.

The small regulatory RNA FasX controls pilus expression and adherence in the human bacterial pathogen group A Streptococcus.

Bacterial pathogens use cell surface-associated adhesion molecules to promote host attachment and colonization, and the ability to modulate adhesion expression is critical to pathogen success. Here, we show that the human-specific pathogen the group A Streptococcus (GAS) uses a small regulatory RNA (sRNA) to regulate the expression of adhesive pili. The fibronectin/fibrinogen-binding/haemolytic-activity/streptokinase-regulator-X (FasX) sRNA, previously shown to positively regulate expression of the secreted virulence factor streptokinase (SKA), negatively regulates the production of pili on the GAS cell surface. FasX base pairs to the extreme 5' end of mRNA from the pilus biosynthesis operon, and this RNA:RNA interaction reduces the stability of the mRNA, while also inhibiting translation of at least the first gene in the pilus biosynthesis operon (cpa, which encodes a minor pilin protein). The negative regulation of pilus expression by FasX reduces the ability of GAS to adhere to human keratinocytes. Our findings cement FasX sRNA as an important regulator of virulence factor production in GAS and identify that FasX uses at least three distinct mechanisms, positive (ska mRNA) and negative (pilus operon mRNA) regulation of mRNA stability, and negative regulation of mRNA translation (cpa mRNA), to post-transcriptionally regulate target mRNAs during infection.

Exploration of novel hexahydropyrrolo[1,2-e]imidazol-1-one derivatives as antiviral agents against ZIKV and USUV.

Zika virus (ZIKV) and Usutu virus (USUV) are two emerging flaviviruses mostly transmitted by mosquitos. ZIKV is associated with microcephaly in newborns and the less-known USUV, with its reported neurotropism and its extensive spread in Europe, represents a growing concern for human health. There is still no approved vaccine or specific antiviral against ZIKV and USUV infections. The main goal of this study is to investigate the anti-ZIKV and anti-USUV activity of a new library of compounds and to preliminarily investigate the mechanism of action of the selected hit compounds in vitro. Two potent anti-ZIKV and anti-USUV agents, namely ZDL-115 and ZDL-116, were discovered, both presenting low cytotoxicity, cell-line independent antiviral activity in the low micromolar range and ability of reducing viral progeny production. The analysis of the structure-activity relationship (SAR) revealed that introduction of 2-deoxyribose to 3-arene was fundamental to enhance the solubility and improve the antiviral action. Additionally, we demonstrated that ZDL-115 and ZDL-116 are significantly active against both viruses when added on cells for at least 24 h prior to viral inoculation or immediately post-infection. The docking analysis showed that ZDL-116 could target the host vitamin D receptor (VDR) and viral proteins. Future experiments will be focused on compound modification to discover analogues that are more potent and on the clarification of the mechanism of action and the specific drug target. The discovery and the development of a novel anti-flavivirus drug will have a significant impact in a context where there are no fully effective antiviral drugs or vaccines for most flaviviruses.

From concept to delivery: a yeast-expressed recombinant protein-based COVID-19 vaccine technology suitable for global access.

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.

A Recombinant Protein XBB.1.5 RBD/Alum/CpG Vaccine Elicits High Neutralizing Antibody Titers against Omicron Subvariants of SARS-CoV-2.

(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.