A Vaccine Based on the Receptor-Binding Domain of the Spike Protein Expressed in Glycoengineered Pichia pastoris Targeting SARS-CoV-2 Stimulates Neutralizing and Protective Antibody Responses

In 2020 and 2021,severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),a novel coronavirus,caused a global pandemic.Vaccines are expected to reduce the pressure of prevention and control,and have become the most effective strategy to solve the pandemic crisis.SARS-CoV-2 infects the host by binding to the cellular receptor angiotensin converting enzyme 2(ACE2)via the receptor-binding domain(RBD)of the surface spike(S)glycoprotein.In this study,a candidate vaccine based on a RBD recombinant subunit was prepared by means of a novel glycoengineered yeast Pichia pastoris expression system with characteristics of glycosylation modification similar to those of mammalian cells.The candidate vaccine effectively stimulated mice to produce high-titer anti-RBD specific antibody.Furthermore,the specific antibody titer and virus-neutralizing antibody(NAb)titer induced by the vaccine were increased significantly by the combination of the double adjuvants Al(OH)_(3) and CpG.Our results showed that the virus-NAb lasted for more than six months in mice.To summarize,we have obtained a SARS-CoV-2 vaccine based on the RBD of the S glycoprotein expressed in glycoengineered Pichia pastoris,which stimulates neutralizing and protective antibody responses.A technical route for fucose-free complex-type N-glycosylation modified recombinant subunit vaccine preparation has been established.

Identification of Dual Receptor-binding Specific Strains of Human H5N1 Viruses in China

Objective Both the 2, 6 linkage and its topology on target cells are critical for the recognition by human influenza virus. The binding preference of avian flu virus H5N1 HA to the 2, 3-1inked sialylated glycans is considered the major factor limiting its efficient infection and transmission in humans. To monitor potential adaptation of H5N1 virus in human population, the surveillance of receptor-binding specificity was undertaken in China. Methods The binding specificity of 32 human H5N1 virus strains isolated from 2003 to 2009 was tested by 2, 3-specific sialidase-treated chicken red blood cell （CRBC） agglutination assay and a solid-phase direct binding assay with synthetic sialylglycopolymers. Results Dual binding preference to 2, 3 and 2, 6-glycans were found in two strains： A/Guangdong/1/06 （A/GD/1/06） and A/Guangxi/1/08 （A/GX/1/08）. Though minor effect of short-2, 6-binding was detected in A/GX/1/08 at a low virus titer, both showed high affinity to the oligosaccharide at a high load. Notably both are of the long-2, 6-recognition, with the same topology as that of human H1N1 and H3N2 viruses. Conclusion The findings suggest that human H5N1 virus in China likely acquired the potential human-adaptation ability. Further research and surveillance on receptor-binding specificity of H5N1 viruses are required.

Real role of growth factor receptor-binding protein 10:Linking lipid metabolism to diabetes cardiovascular complications

Cardiovascular complications of patients with type 2 diabetes mellitus(T2DM)threaten the health and life of numerous individuals.Recently,growth factor receptor-binding protein 10(GRB10)was found to play a pivotal role in vascular complications of T2DM,which participates in the regulation of lipid metabolism of T2DM patients.The genetic variation of GRB10 rs1800504 is closely related to the risk of coronary heart disease in patients with T2DM.The development of GRB10 as a key mediator in the association of lipid metabolism with cardiovascular complications in T2DM is detailed in and may provide new potential concerns for the study of cardiovascular complications in T2DM patients.

Characterization of the receptor-binding domain(RBD)of 2019 novel coronavirus:implication for development of RBD protein as a viral attachment inhibitor and vaccine

The outbreak of Coronavirus Disease 2019(COVID-19)has posed a serious threat to global public health,calling for the development of safe and effective prophylactics and therapeutics against infection of its causative agent,severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),also known as 2019 novel coronavirus(2019-nCoV).The CoV spike(S)protein plays the most important roles in viral attachment,fusion and entry,and serves as a target for development of antibodies,entry inhibitors and vaccines.Here,we identified the receptor-binding domain(RBD)in SARS-CoV-2 S protein and found that the RBD protein bound strongly to human and bat angiotensin-converting enzyme 2(ACE2)receptors.SARS-CoV-2 RBD exhibited significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD and could block the binding and,hence,attachment of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells,thus inhibiting their infection to host cells.SARS-CoV RBD-specific antibodies could crossreact with SARS-CoV-2 RBD protein,and SARS-CoV RBD-induced antisera could cross-neutralize SARS-CoV-2,suggesting the potential to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV-2 and SARS-CoV infection.

Identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against Middle East respiratory syndrome coronavirus

Middle East respiratory syndrome （MERS）, an emerging infectious disease caused by MERS coronavirus （MERS-CoV）, has garnered worldwide attention as a consequence of its continuous spread and pandemic potential, making the development of effective vaccines a high priority. We previously demonstrated that residues 377-588 of MERS-CoV spike （S） protein receptor-binding domain （RBD） is a very promising MERS subunit vaccine candidate, capable of inducing potent neutralization antibody responses. In this study, we sought to identify an adjuvant that optimally enhanced the immunogenicity of S377-588 protein fused with Fc of human IgG （S377-588-Fc）. Specifically, we compared several commercially available adjuvants, including Freund＇s adjuvant, aluminum, Monophosphoryl lipid A, Montanide ISA51 and MF59 with regard to their capacity to enhance the immunogenicity of this subunit vaccine. In the absence of adjuvant, S377-588-Fc alone induced readily detectable neutralizing antibody and T-cell responses in immunized mice. However, incorporating an adjuvant improved its immunogenicity. Particularly, among the aforementioned adjuvants evaluated, MF59 is the most potent as judged by its superior ability to induce the highest titers of IgG, IgG1 and IgG2a subtypes, and neutralizing antibodies. The addition of MF59 significantly augmented the immunogenicity of S377-588-Fcto induce strong IgG and neutralizing antibody responses as well as protection against MERS-CoV infection in mice, suggesting that MF59 is an optimal adjuvant for MERS-CoV RBD-based subunit vaccines.

An ultrapotent pan-β-coronavirus lineage B(β-CoV-B)neutralizing antibody locks the receptor-binding domain in closed conformation by targeting its conserved epitope

New threats posed by the emerging circulating variants of SARS-CoV-2 highlight the need to find conserved neutralizing epitopes for therapeutic antibodies and efficient vaccine design.Here,we identified a receptorbinding domain(RBD)-binding antibody,XG014,which potently neutralizesβ-coronavirus lineage B(β-CoV-B),including SARS-CoV-2,its circulating variants,SARSCoV and bat SARSr-CoV WIV1.Interestingly,antibody family members competing with XG014 binding show reduced levels of cross-reactivity and induce antibodydependent SARS-CoV-2 spike(S)protein-mediated cellcell fusion,suggesting a unique mode of recognition by XG014.Structural analyses reveal that XG014 recognizes a conserved epitope outside the ACE2 binding site and completely locks RBD in the non-functional“down”conformation,while its family member XG005 directly competes with ACE2 binding and position the RBD“up”.Single administration of XG014 is effective in protection against and therapy of SARS-CoV-2 infection in vivo.Our findings suggest the potential to develop XG014 as pan-β-CoV-B therapeutics and the importance of the XG014 conserved antigenic epitope for designing broadly protective vaccines againstβ-CoV-B and newly emerging SARS-CoV-2 variants of concern.

Licorice-saponin A3 is a broad-spectrum inhibitor for COVID-19 by targeting viral spike and anti-inflammation

Currently,human health due to corona virus disease 2019(COVID-19)pandemic has been seriously threatened.The coronavirus severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike(S)protein plays a crucial role in virus transmission and several S-based therapeutic approaches have been approved for the treatment of COVID-19.However,the efficacy is compromised by the SARS-CoV-2 evolvement and mutation.Here we report the SARS-CoV-2 S protein receptor-binding domain(RBD)inhibitor licorice-saponin A3(A3)could widely inhibit RBD of SARS-CoV-2 variants,including Beta,Delta,and Omicron BA.1,XBB and BQ1.1.Furthermore,A3 could potently inhibit SARS-CoV-2 Omicron virus in Vero E6 cells,with EC50 of 1.016μM.The mechanism was related to binding with Y453 of RBD determined by hydrogen-deuterium exchange mass spectrometry(HDX-MS)analysis combined with quantum mechanics/molecular mechanics(QM/MM)simulations.Interestingly,phosphoproteomics analysis and multi fluorescent immunohistochemistry(mIHC)respectively indicated that A3 also inhibits host inflammation by directly modulating the JNK and p38 mitogen-activated protein kinase(MAPK)pathways and rebalancing the corresponding immune dysregulation.This work supports A3 as a promising broad-spectrum small molecule drug candidate for COVID-19.

Therapeutic Implications of Monoclonal Antibody

Background: The coronavirus disease 2019 (COVID-19) pandemic is a distinct public health issue that calls for the quick development of novel treatments and viral detection. Due to their high specificity and reliability, monoclonal antibodies (mAbs) have emerged as useful diagnostic and therapeutic tools for a variety of diseases. As a result, several scientists have jumped right into developing Ab-based assays for the identification of SARS-CoV-2 and Ab drugs for use as COVID-19 therapy agents. Since the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is essential for viral infection and has a known precise structure, it has become a key target for the creation of therapeutic antibodies. The use of Ab cocktails is anticipated to be a key component of an efficient COVID-19 treatment plan since SARS-CoV-2 is an RNA virus with a high mutation rate, particularly when subjected to the selection pressure of aggressively applied preventive vaccinations and neutralizing Abs. Furthermore, SARS-CoV-2 infection could provoke an overzealous immune response, leading to a cytokine storm that accelerates the onset of a severe disease. Abs to counteract cytokine storms are also actively being researched as COVID-19 therapies. Abs are now used in SARS-CoV-2 detection assays, including immunoglobulin and antigen tests, in addition to their use as medicines. In order to stop the spread of COVID-19, such Ab-based detection tests are essential surveillance tools. In this article, we’ll go over several important ideas related to mAb-based COVID-19 pandemic detection tests and treatments. Objective: To understand the role of hybridoma technology in therapeutic implications. 1) To study the basic concepts and options in hybridoma technology;2) To study the applications of hybridoma technology;3) To explore how hybridoma technology is applied in diagnostic histopathology. Method: For this method generally there is use of mouse or mammals are transfect with the Ags to find out the formation of antibody afterwards isolate the antibody which has been formed after injecting the antigens for a number of weeks. Following are the steps for mAbs: Step 1: In this step immunization of mouse is done;Step 2: Spleen is used for the isolation of B cells;Step 3: Cultivation of cancerous cells;Step 4: Merging of B cells with Myeloma cells;Step 5: This step cell lines are separated;Step 6: in the next step screening the suitable cell lines;Step 7: observation of multiplication in vitro as well as in vivo;Step 8: Harvesting. Discussion: Now a day there are many diseases which has been cured easily at the mean time it’s very difficult to diagnose and get the treatment. Due to advancement of monoclonal antibodies are used in the diagnosis and treatments such as COVID-19, SARS and SARS COV-2. Therefore important part of the monoclonal antibodies are its used in the diagnosis as well as in the treatment tools.

Understanding neutralising antibodies against SARS-CoV-2 and theirimplications in clinical practice

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is a newly identified member of the coronavirus family that has caused the coronavirus disease 2019 (COVID-19) pandemic. This rapidly evolving and unrelenting SARS-CoV-2has disrupted the lives and livelihoods of millions worldwide. As of 23 August 2021, a total of 211,373,303 COVID-19cases have been confirmed globally with a death toll of 4,424,341. A strong understanding of the infection pathway of SARS-CoV-2, and how our immune system responds to the virus is highly pertinent for guiding the development and improvement of effective treatments. In this review, we discuss the current understanding of neutralising antibodies(NAbs) and their implications in clinical practice. The aspects include the pathophysiology of the immune response,particularly humoral adaptive immunity and the roles of NAbs from B cells in infection clearance. We summarise the onset and persistence of IgA, IgM and IgG antibodies, and we explore their roles in neutralising SARS-CoV-2, their persistence in convalescent individuals, and in reinfection. Furthermore, we also review the applications of neutralising antibodies in the clinical setting—from predictors of disease severity to serological testing to vaccinations, and finally in therapeutics such as convalescent plasma infusion.

Pharmacokinetic-Pharmacodynamic modeling of the analgesic effect of bupredermTM, in mice

Purpose: BupredermTM-Buprenorphine transdermal delivery system (BTDS) was developed for the treatment of post-operative and chronic pains. This study examined the relationship between the plasma concentration of buprenorphine and its analgesic effect (tail flick test) in order to assess the usefulness of pharmacokinetic-pharmacodynamic (PK-PD) modeling in describing this relationship. Methods: After patch application, plasma concentrations of bu- prenorphine in mice were measured for 72 hours with a validated LC/MS/MS system, and the analgesic effects were assessed by tail flick test for the period of 24 hours. A modified two- compartment open model was used to explain the PK properties of BTDS, and the PD model was characterized by slow receptor binding. Results: The peak buprenorphine level in plasma was achieved at 1-24 h and the effective therapeutic drug concentration was maintained for 72 hours. BupredermTM induced prolongation of tail-flick latency in a dose and time dependent manner. Maximum analgesic effect was attained at 3-6 h and was maintained for 24 h after patch application. Counter-clockwise hysteresis between the plasma concentration and the analgesic efficacy of BTDS was observed after BupredermTM application, indicating there was a delay between plasma concentrations and the effect observed. From the developed PK-PD model, Kd values (0.69-0.82 nM) that were derived from the pharmacodynamic parameters (Kon and Koff) are similar to the reported values (Kd = 0.76 ± 0.14 nM). Good agreement between the predicted and observed values was noted for the rate of change in analgesic effect data (R2 = 0.822, 0.852 and 0.774 for 0.24, 0.8 and 2.4 mg/patch, respectively). Conclusions: The established PK- PD model successfully described the relationship between plasma concentration of buprenorphine and its analgesic efficacy measured by the tail flick test. Our model might be useful in estimation and prediction of onset, magnitude and time course of concentration and pharmacological effects of BTDS and will be useful to simulate PK-PD profiles with clinical regimens.

基于RBD抗原性的五种SARS-CoV-2血清型分类

新冠病毒(SARS-CoV-2)的不断进化带来了大量的变异株,特别是Omicron变异株及其众多的亚型.这些变异株表现出越来越强的免疫逃逸能力,使现有疫苗和治疗抗体的效力不断下降.目前,众多变异株已表现出血清交叉中和作用减弱的现象,表明新冠病毒可能已进化出多种血清型.因此,我们选取新冠病毒的主要抗原,即刺突(S)蛋白的受体结合域(RBD)对其进行血清分型.我们首先选择了23个具有代表性的新冠病毒毒株,涵盖了前Omicron变异株和Omicron变异株的多种亚型.通过对RBD抗原性的系统评估,我们将23种变异株分为5种血清型,每种血清型都包含了数种基因型不同的变异株.具体而言,Ⅰ型涵盖了所有前Omicron变异株(含两种亚型),而其余四种血清型均包含处于不同进化阶段的Omicron亚型.本文中的血清分型可以为新型变异株的快速评估奠定基础,并指导未来针对新冠病毒的广谱疫苗和中和抗体的开发.

Evidence for a mouse origin of the SARS-CoV-2 Omicron variant

The rapid accumulation of mutations in the SARS-CoV-2 Omicron variant that enabled its outbreak raises questions as to whether its proximal origin occurred in humans or another mammalian host. Here, we identified 45 point mutations that Omicron acquired since divergence from the B.1.1 lineage. We found that the Omicron spike protein sequence was subjected to stronger positive selection than that of any reported SARS-CoV-2 variants known to evolve persistently in human hosts, suggesting a possibility of hostjumping. The molecular spectrum of mutations(i.e., the relative frequency of the 12 types of base substitutions) acquired by the progenitor of Omicron was significantly different from the spectrum for viruses that evolved in human patients but resembled the spectra associated with virus evolution in a mouse cellular environment. Furthermore, mutations in the Omicron spike protein significantly overlapped with SARS-CoV-2 mutations known to promote adaptation to mouse hosts, particularly through enhanced spike protein binding affinity for the mouse cell entry receptor. Collectively, our results suggest that the progenitor of Omicron jumped from humans to mice, rapidly accumulated mutations conducive to infecting that host,then jumped back into humans, indicating an inter-species evolutionary trajectory for the Omicron outbreak.

Crystal structural studies of destripeptide (B28-B30) insulin

Single crystals of destripeptide (B28-B30) insulin (DTRI) in three forms were obtained by hanging-drop vapor diffusion method. Form 1 belongs to P21 space group with cell parameters a-4.77 nm, b=6.19 nm, c=6.12 nm, β=110.3°. Form 2 belongs to P4122 or P4322 space group with cell parameters a= 6.45 nm, c=12.07 nm. Form 3 belongs to P212121 space group with cell parameters a=4.98 nm, b=5.16 nm, c=10.06 nm. The structure of form 1 crystal was determined by molecular replacement method and refined at 0.23 nm resolution. The R-factor of the final model is 18.8% with r.m.s. deviations of 0.001 5 nm and 3.3?for the bond lengths and the bond angles, respectively. Studies on the crystal structure show that the removal of B28 Pro has brought DTRI structural changes which made it dissociate more easily than native insulin although DTRI can still form a hexamer.

Generation and Characterization of a Nanobody Against SARS-CoV

The sudden emergence of severe acute respiratory syndrome coronavirus(SARS-CoV) has caused global panic in 2003,and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available;thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain(RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensinconverting enzyme 2(ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.