Repurposing of SARS-CoV nucleocapsid protein specific nuclease resistant RNA aptamer for therapeutics against SARS-CoV-2

COVID-19 pandemic is rapidly advancing among human population. Development of new interventions including therapeutics and vaccines against SARS-CoV-2 will require time and validation before it could be made available for public use. Keeping in view of the emergent and evolving situation the motive is to repurpose and test the immediate efficacy of available drugs and therapeutics against COVID-19. Through this article we propose and discuss the possibility of repurposing the available nuclease resistant RNA aptamer against the nucleocapsid protein of SARS-CoV as a potential therapeutic agent for COVID-19.     

Comparative analysis of the immunogenicity of SARS-CoV nucleocapsid DNA vaccine administrated with different routes in mouse model

The development of strategies to augment the immunogenicity of DNA vaccines is critical for improving their clinical utility. One such strategy involves using the different immune routes with DNA vaccines. In the present study, the immunogenicity of SARS-CoV nucleocapsid DNA vaccine, induced by using the current routine vaccination routes (intramuscularly, by electroporation, or orally using live-attenuated Salmonella typhimurium), was compared in mouse model. The comparison between the three vaccination routes indicated that immunization intramuscularly induced a moderate T cell response and antibody response. Mice administrated by electroporation induced the highest antibody response among the three immunization groups and a mid-level of cellular response. In contrast, the orally DNA vaccine evoked vigorous T cell response and a weak antibody production. These results indicated that the distinct types of immune responses were generated by the different routes of DNA immunization. In addition, our results also show that the delivery of DNA vaccines by electroporation and orally using live-attenuated Salmonella in vivo is an effective method to increase the immune responses. Further studies could be carried out using a combination strategy of both oral and electroporation immunizations to stimulate higher cellular and humoral immune responses.     

Induction of anti-viral immune responses by immunization with recombinant-DNA encoded avian coronavirus nucleocapsid protein.

Immune responses to the infectious bronchitis virus (IBV) nucleocapsid protein were studied using a recombinant-DNA expression product. In mice, a lymphocyte proliferative response and a delayed-type hypersensitivity reaction to IBV were induced upon immunization with this nucleocapsid protein. Next, we studied the role of the expressed nucleocapsid protein in induction of a protective immune response to IBV in chickens. Chickens were primed with nucleocapsid protein and subsequently boosted with inactivated IBV, strain M41. Proliferative responses of blood mononuclear cells corresponded with increased mean haemagglutination inhibition and virus neutralization titres. Finally, an increased tracheal protection against challenge with live IBV was observed. These results indicate that infectious bronchitis virus nucleocapsid protein is a relevant target for immune recognition in both the mouse and the chicken.     

Yeast-expressed Puumala hantavirus nucleocapsid protein induces protection in a bank vole model

Hantaviruses are rodent-borne agents that cause severe human diseases. The coding sequences for the authentic and a His-tagged Puumala hantavirus (PUUV) nucleocapsid (N) protein were expressed in yeast (Saccharomyces cerevisiae). N-specific monoclonal antibodies demonstrated native antigenicity of the two proteins. All bank voles vaccinated with the His-tagged N protein in Freund's adjuvant (n=12) were defined as completely protected against subsequent virus challenge, based on the absence of viral N protein, RNA and G2-specific antibodies. In the group vaccinated with the yeast-expressed authentic N protein in Freund's adjuvant, 2/6 animals were defined as completely protected and 4/6 as partially protected. Moreover, when animals were vaccinated with the His-tagged N protein in an adjuvant certified for human use (alum), all (n=8) were at least partially protected (six completely, two partially). The general advantages of the yeast expression system make the described recombinant proteins promising candidate vaccines against hantavirus infection.     

Variability of hemagglutinin-neuraminidase and nucleocapsid protein of vaccine and wild-type mumps virus strains.

The mumps virus (MuV) molecular evolution is characterized by the co-circulation of numerous distinct strains. Standardized phylogenetic analyses based on the nucleotide sequences of the SH gene are important for mumps surveillance, but lack the information regarding antigenic properties. So far, the location of antigenic epitopes has been determined for two MuV proteins, the hemagglutinin-neuraminidase (HN) and the nucleocapsid (N) protein. We performed multiple sequence comparisons of putative HN and N protein sequences in order to describe their diversity and plasticity, and to determine the level of similarity between vaccine and wild-type strains. The results of full-length HN or N protein phylogeny showed that MuV strains form a number of differing clades which are in concordance with grouping obtained by standard MuV genotyping. When vaccine strains are compared to all wild-type strains, the highest mean percentage of amino acid differences in both HN and N protein analysis was found for Jeryl Lynn 5 and Jeryl Lynn 2 strains while the lowest value was obtained for Leningrad-3 and L-Zagreb strains. When only 3 antigenic regions of the HN protein, comprising 45 amino acids in total, were investigated, the diversity is considerably diminished: 51.5% of all putative HN proteins show identical sequences (including those of vaccine strains L-Zagreb, Leningrad-3, Hoshino and Urabe). Another 26.5% proteins (including Miyahara vaccine strain) differ in only one amino acid, while the others differ in two to five amino acids from the most common sequence. Jeryl Lynn 2 and Jeryl Lynn 5 strains differ in four amino acids each. N protein antigenic sites have been mapped within its hypervariable C-terminus. Our results indicate that there might be genotype-specific amino acids residing in this antigenic region. The results of our study present the background information for investigations of MuV heterogeneity and antigenic diversity.     

Priming with rAAV encoding RBD of SARS-CoV S protein and boosting with RBD-specific peptides for T cell epitopes elevated humoral and cellular immune responses against SARS-CoV infection

Development of vaccines against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is crucial in the prevention of SARS reemergence. The receptor-binding domain (RBD) of SARS-CoV spike (S) protein is an important target in developing safe and effective SARS vaccines. Our previous study has demonstrated that vaccination with adeno-associated virus encoding RBD (RBD-rAAV) induces high titer of neutralizing antibodies. In this study, we further assessed the immune responses and protective effect of the immunization with RBD-rAAV prime/RBD-specific T cell peptide boost. Compared with the RBD-rAAV prime/boost vaccination, RBD-rAAV prime/RBD-peptide (RBD-Pep) boost induced similar levels of Th1 and neutralizing antibody responses that protected the vaccinated mice from subsequent SARS-CoV challenge, but stronger Th2 and CTL responses. No significant immune responses and protective effects were detected in mice vaccinated with RBD-Pep or blank AAV alone. Since T cell epitopes are highly conserved and boosting with peptides may induce the production of effector memory T cells, which may be effective against viruses with mutations in the neutralizing epitopes, our results suggest that the vaccination protocol used may be ideal for providing effective, broad and long-term protection against SARS-CoV infection.     

Identification and characterization of novel neutralizing epitopes in the receptor-binding domain of SARS-CoV spike protein: revealing the critical antigenic determinants in inactivated SARS-CoV vaccine

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is considered as a major antigen for vaccine design. We previously demonstrated that the receptor-binding domain (RBD: residues 318-510) of S protein contains multiple conformation-dependent neutralizing epitopes (Conf I to VI) and serves as a major target of SARS-CoV neutralization. Here, we further characterized the antigenic structure in the RBD by a panel of novel mAbs isolated from the mice immunized with an inactivated SARS-CoV vaccine. Ten of the RBD-specific mAbs were mapped to four distinct groups of conformational epitopes (designated Group A to D), and all of which had potent neutralizing activity against S protein-pseudotyped SARS viruses. Group A, B, C mAbs target the epitopes that may overlap with the previously characterized Conf I, III, and VI respectively, but they display different capacity to block the receptor binding. Group D mAb (S25) was directed against a unique epitope by its competitive binding. Two anti-RBD mAbs recognizing the linear epitopes (Group E) were mapped to the RBD residues 335-352 and 442-458, respectively, and none of them inhibited the receptor binding and virus entry. Surprisingly, most neutralizing epitopes (Groups A to C) could be completely disrupted by single amino acid substitutions (e.g., D429A, R441A or D454A) or by deletions of several amino acids at the N-terminal or C-terminal region of the RBD; however, the Group D epitope was not sensitive to the mutations, highlighting its importance for vaccine development. These data provide important information for understanding the antigenicity and immunogenicity of SARS-CoV, and this panel of novel mAbs can be used as tools for studying the structure of S protein and for guiding SARS vaccine design.     

Modifying the cellular transport of DNA-based vaccines alters the immune response to hantavirus nucleocapsid protein.

Puumala virus is a member of the hantavirus genus (family Bunyaviridae) and is one of the causative agents of hemorrhagic fever with renal syndrome (HFRS) in Europe. A genetic vaccination approach was conducted to investigate if the immune response could be modulated using different cellular secretion and/or localisation signals, and the immune responses were analysed in BALB/c mice and in a bank vole infectious model. Rodents vaccinated with DNA constructs encoding the antigen fused to an amino-terminal secretion signal raised significantly higher antibody levels when compared to using constructs lacking secretion signals. Furthermore, the ratios of the IgG subclasses (IgG2a/IgG1) were raised by the use of cellular localisation signals, indicating a more pronounced Th1-type of immune response. The majority of the mice, or bank voles, immunised with DNA encoding a secreted form of the antigen showed a positive lymphoproliferative response and were protected against challenge with Puumala virus (strain Kazan-wt).     

发热伴血小板减少综合征患者核衣壳蛋白特异性IgM水平与病情严重程度的相关性

目的探讨核衣壳蛋白(N蛋白)特异性Ig M抗体水平与发热伴血小板减少综合征(SFTS)疾病病情严重程度的相关性。方法回顾性分析某三甲医院30例SFTS患者的临床特点及实验室检查结果,比较N蛋白特异性Ig M(+)组与N蛋白特异性Ig M(-)组患者的临床特点及预后转归;根据患者疾病严重程度分为轻症组和重症组,比较轻症组患者与重症组患者Ig M抗体滴度与发热伴血小板减少综合征病毒(SFTSV) RNA载量的相关性;从而观察分析N蛋白特异性Ig M抗体滴度、SFTSV-RNA载量与患者病情严重程度的相关性。结果 N蛋白特异性Ig M(-)组中有神经症状、死亡及病重例数均明显较Ig M(+)组多(均P 0. 05); N蛋白特异性Ig M抗体水平与SFTSV-RNA载量、凝血酶原时间(PT)、乳酸脱氢酶(LDH)、天门冬氨酸氨基转移酶(AST)均呈负相关(r值分别为-0. 495、-0. 440、-0. 367、-0. 280,均P 0. 05);而与血小板(PLT)呈正相关(r=0. 335,P=0. 002)。SFTSVRNA载量与PT、LDH、AST均呈正相关(r值分别为0. 606、0. 604、0. 587,均P 0. 001);而与PLT呈负相关(r=-0. 384,P 0. 001)。发病第10～13天时轻症组患者N蛋白特异性Ig M抗体滴度高于重症组患者(P 0. 05)。结论N蛋白特异性Ig M抗体的出现及滴度的升高有助于SFTSV的清除,且对患者凝血功能及肝损伤、心肌损伤有修复作用; N蛋白特异性Ig M抗体可能是预测患者预后的重要因素。     

Serological response after SARS-CoV2 vaccination in healthcare workers: a multicenter study

Introduction:Characterizing immunological response following COVID-19 vaccination is an important public health issue. The objectives of the present analysis were to investigate the proportion, level and the determinants of humoral response from 21 days to three months after the first dose in vaccinated healthcare workers (HCWs).Methods:We abstracted data on level of anti-SARS-CoV-2 Spike antibodies (IgG) and sociodemographic characteristics of 17,257 HCWs from public hospitals and public health authorities from three centers in Northern Italy who underwent COVID-19 vaccination (average 70.6 days after first dose). We fitted center-specific multivariate regression models and combined them using random-effects meta-analyses.Results:A humoral response was elicited in 99.3% of vaccinated HCW. Female sex, young age, and previous COVID-19 infection were predictors of post-vaccination antibody level, and a positive association was also detected with pre-vaccination serology level and with time between pre- and post-vaccination testing, while a decline of antibody level was suggested with time since vaccination.Conclusions:These results stress the importance of analyzing retrospective data collected via occupational health surveillance of HCWs during the COVID-19 epidemic and following vaccination. They need to be confirmed in larger series based on prospectively collected data.     

Evidence from the evolutionary analysis of nucleotide sequences for a recombinant history of SARS-CoV.

The origins and evolutionary history of the Severe Acute Respiratory Syndrome (SARS) coronavirus (SARS-CoV) remain an issue of uncertainty and debate. Based on evolutionary analyses of coronavirus DNA sequences, encompassing an approximately 13kb stretch of the SARS-TOR2 genome, we provide evidence that SARS-CoV has a recombinant history with lineages of types I and III coronavirus. We identified a minimum of five recombinant regions ranging from 83 to 863bp in length and including the polymerase, nsp9, nsp10, and nsp14. Our results are consistent with a hypothesis of viral host jumping events, concomitant with the reassortment of bird and mammalian coronaviruses, a scenario analogous to earlier outbreaks of influenzae.     

不同物理学性状金属铝尘的溶血活性

用溶血试验测定不同物理学性状铝尘的生物学作用。实验用铝尘有5μm片状铝,1、5、7.5、10、15μm粒状铝共6种。结果表明,不同粒径的粒状铝溶血活性有显著差异,铝尘粒径越小其溶血活性越强;在相同粒径情况下,片状铝溶血活性强于粒状铝。相同表面积、不同重量铝尘的溶血试验结果显示粒径、剂量对铝尘溶血活性的影响比表面积、粒子数更为重要。铝尘溶血指数高低与其体内致肺脏纤维化能力基本平行。     

Predictors of Sarcopenia in Outpatients with Post-Critical SARS-CoV2 Disease. Nutritional Ultrasound of Rectus Femoris Muscle,a Potential Tool

Background and Objectives: The loss of muscle mass in post-critical COVID-19 outpatients is difficult to assess due to the limitations of techniques and the high prevalence of obesity. Ultrasound is an emerging technique for evaluating body composition. The aim is to evaluate sarcopenia and its risk factors, determining ultrasound usefulness as a potential tool for this purpose according to established techniques, such as the bioimpedance vector analysis (BIVA), handgrip strength, and timed up-and-go test. Methods: This is a transversal study of 30 post-critical COVID-19 outpatients. We evaluated nutritional status by ultrasound (Rectus Femoris-cross-sectional-area (RF-CSA), thickness, and subcutaneous-adipose-tissue), BIVA, handgrip strength, timed up-and-go test, and clinical variables during admission. Results: According to The European Society for Clinical Nutrition and Metabolism and the European Association for the Study of Obesity (ESPEN&EASO) Consensus for Sarcopenic and Obesity, in terms of excess fat mass and decreased lean mass, the prevalence of class-1 sarcopenic obesity was 23.4% (n = 7), and class-2 sarcopenic obesity was 33.3% (n = 10) in our study. A total of 46.7% (n = 14) of patients had a handgrip strength below the 10th percentile, and 30% (n = 9) achieved a time greater than 10s in the timed up-and-go test. There were strong correlations between the different techniques that evaluated the morphological (BIVA, Ultrasound) and functional measurements of muscle. Intensive care unit stay, mechanical ventilation, and age all conditioned the presence of sarcopenia in COVID-19 outpatients (R² = 0.488, p = 0.002). Predictive models for sarcopenic diagnosis based on a skeletal muscle index estimation were established by RF-CSA (R² 0.792, standard error of estimate (SEE) 1.10, p < 0.001), muscle-thickness (R² 0.774, SEE 1.14, p < 0.001), and handgrip strength (R² 0.856, SEE 0.92, p < 0.001). RF-CSA/weight of 5.3 cm²/kg × 100 was the cut-off value for predicting sarcopenia in post-critical COVID-19 outpatients, with 88.2 sensitivity and 69.2% specificity. Conclusion: More than half of the post-critical COVID-19 survivors had sarcopenic obesity and functional impairment of handgrip strength. Intensive care unit stay, age, and mechanical ventilation all predict sarcopenia. An ultrasound, when applied to the assessment of body composition in post-critical COVID-19 patients, provided the possibility of assessing sarcopenia in this population.     

Undernutrition in sheep. The effect of supplementation with protein on protein accretion

In a comparative-slaughter experiment, individually rationed wether lambs initially of 42 kg were given 235, 362 or 456 kJ metabolizable energy (ME)/kg live weight (LW)0.75 per d as sodium hydroxide-treated barley straw with urea (six lambs per treatment), or NaOH-treated barley straw with urea plus 125 g/d white-fish meal to give 307 or 488 kJ ME/kg LW0.75 per d (seven lambs per treatment) for 92 d. All unsupplemented lambs lost both fat and body protein. The changes in fat were -3.53, -2.75 and -1.40 (SE 0.59) kg (initial value 8.6 kg), and the changes in body protein were -0.47, -0.09 and -0.14 (SE 0.13) kg (initial value 4.9 kg) for the three unsupplemented groups respectively. When supplemented with fish meal, fat was again lost as -1.53 and -0.93 (SE 0.55) kg, but wool-free body protein was increased, and gains were 0.48 and 0.89 (SE 0.12) kg for the two supplemented groups respectively. All animals lost wool-free body energy, total changes being -150, -111, -59 and -49 and -16 MJ respectively. When corrected to an equal ME intake the supplemented lambs, when compared with the unsupplemented lambs, gained (instead of losing) body protein (P less than 0.001) and lost less fat (P less than 0.05). Wool growth did not respond to supplemental protein, but was related to ME intake with an increase of 0.78 g wool fibre for each additional MJ ME. The maintenance requirements of the unsupplemented and supplemented groups respectively were estimated by regression analysis to be 554 and 496 kJ ME/kg LW0.75 per d. The apparent utilization of ME below energy equilibrium (km) was 0.31 (SE 0.08) for the unsupplemented animals, and 0.12 (SE 0.10) for the supplemented animals, well below a km of 0.70 which current UK standards (Agricultural Research Council, 1980) would predict. Most of these differences could be reconciled if basal metabolism was assumed not to be constant. It is concluded that lambs in negative energy balance can continue lean body growth at the expense of body fat, provided sufficient dietary protein is available. It is also concluded that since the animals at the lowest ME intakes required less ME than predicted by current feeding standards, the effect was that it would have been difficult to distinguish between the apparent utilization of ME for maintenance (km) and for fattening (kf).     

Intrauterine contraception with the copper-T device. 4. Influence on protein and copper concentrations and enzyme activities in uterine washings

The concentrations of protein and copper and the activities of acid and alkaline phosphatase and beta-glucuronidase were measured in the uterine fluid of 8 25-38 year old women using the copper-T (Cu-T) 200 device for intrauterine contraception. Specimens were obtained in the proliferative phase on Cycle Days 10-12 of 1 menstrual cycle, and in the secretory phase on Days 20-23 during the next cycle prior to the insertion of the Cu-T, and during the same cycle days in Cycles 2 or 3 or 6 or 7 following insertion. Uterine fluid was obtained by irrigating the uterine cavity with physiological saline, while endometrial biopsies were taken for histological dating of the endometrium. The protein concentration of the uterine washings did not change significantly as a result of the Cu-T insertion. There was a significant difference (p.001) in Cycles 2 or 3 and 6 or 7 following insertion. Acid phosphatase activity was not influenced by the presence of the device. The betaglucuronidase in the fluid obtained during the proliferative phase showed a significant increase (p .001) DURING THE TIME WHEN the device was situ. The device caused a significant increase in the copper concentration in both phases, while the copper level in the blood serum remained unchanged. There was as increased number of white blood cells in the washings obtained during the secretory phase. The increase in the copper concentration of the uterine fluid might be the cause of the Cu-T antifertility effect due to a spermatotoxic and/or blastotoxic effect, as may the enzymic changes and increase of white blood cells.     

The environment as a health surveillance issue: analysis of nursing activities

The fact that the need for environmental conservation has been on the national and international agenda determined the development of this study, which aims to examine how the environment is an issue for nurses working in health surveillance services, according to their testimonies. From July to August 1999, eight female nurses described their routine. Their reports were studied by means of the discourse analysis technique. It was concluded that, in general, their actions were not aimed at environmental conservation and were limited to factual and emergency issues.