Construction of SARS-CoV-2 receptor binding domain molecular probe and its application in the isolation of neutralizing antibodies / 中国热带医学

@#Abstract: Objective　To construct SARS-CoV-2 receptor binding domain molecular probe for monoclonal memory B cell sorting and obtain RBD specific neutralizing antibodies from peripheral blood mononuclear cells (PBMCs) of COVID-19 convalescents by single-cell sorting. Methods　The SARS-CoV-2 RBD sequence was downloaded from GenBank, and the Avi tag and 6-histidine tags were added at the C-terminal. After codon optimization, it was chemically synthesized, cloned into the pDRVI1.0 vector, expressed after transfection of 293F cells, and biotinylated consequently. RBD-specific B cells were sorted out with this probe1 from the PBMCs of convalescents recovered from COVID-19. After B cells were lysed, the variable regions of heavy chain and light chain were amplified, cloned into the antibody expression vector, and transfected into 293F cells to express the antibody. Then the antibody was purified from the supernatant using protein A column and SARS-CoV-2 pseudovirus was used to test their neutralizing activity. Results　RBD-Avi probe was produced and successfully biotinylated sequentially with an efficiency of 30%-50%. Western blot analysis revealed that the biotinylated probe was recognized by the antibodies purified from COVID-19 convalescent plasma. Using this probe, 7 and 16 RBD-specific memory B cells were successfully isolated from the PBMCs of two convalescent individuals, accounting for 0.24% and 0.17% of the total cell population, respectively. After amplifying the variable regions of antibody heavy and light chains from the lysed B cells, 7 and 12 pairs of antibody heavy-light chains were obtained. A total of 16 antibodies were expressed in the convalescent individuals, and most of the purified antibodies showed neutralizing activity against the pseudovirus, with IC50 values of 6 antibodies below 1 μg/mL. The IC50 values of XJ-A9 and SCF-F1 against the wild-type pseudovirus were 0.07 μg/mL and 0.35 μg/mL, respectively. Conclusion　The SARS-CoV-2 RBD molecular probe constructed in this study has good antigenicity, and the isolated antibodies present neutralizing activity against wild-type SARS-CoV-2 pseudovirus.

Viral resistance to VRC01-like antibodies with mutations in loop D and V5 from an HIV-1 B' subtype infected individual with broadly neutralization activity.

Recently we identified the VRC01-like antibody DRVIA7(A7) from an HIV-1 B' subtype-infected individual (DRVI01) with broad neutralization activity, and almost all viruses from the individual were resistant to both VRC01 and A7 lineage antibodies. Here, we identified and characterized a panel of HIV-1 variants with resistance to VRC01 and A7 using site-directed mutagenesis and swapping amino acid fragments of gp120. Site-directed mutagenesis revealed that E279D/R282K/N460A/T464N of gp120 from DRVI01 produced VRC01-susceptible variants. Multiple mutations significantly increased the neutralization sensitivity to VRC01. Residues N464 located at the tip of the V5 loop were considered irrelevant to the neutralization of VRC01. For DRVI01-derived viruses, the single N464T change fully produced VRC01-resistant variants; conversely, a single T464N mutation generated VRC01-susceptible variants. Alanine scanning revealed that the N464 residue plays a vital role in binding with VRC01. Neutralizing assays against A7 lineage antibodies showed that DRVI01-derived viruses with multiple mutations could be neutralized by A7 lineage antibodies with different neutralizing breadths. Combining the changes in loops D and V5 produced variants that were totally sensitive variants to A7 lineage antibodies.

Comparison of Antibody Immune responses to different HIV-1 Envelope Glycoprotein Mutants.

The identification of immunogens is crucial for human immunodeficiency virus type 1 (HIV-1) vaccine development. In our previous study, we demonstrated that HIV-1 envelope glycoprotein mutants based on the equine infectious anemia virus (EIAV)attenuated vaccine enhance immunogenicity, both for DNA immunization alone and as a combined DNA prime-vaccinia boost immunization. An RV144 clinical trial has demonstrated that an envelope protein boost may provide some degree of protection against HIV-1 infection. In order to explore the antibody immune responses to two HIV-1 envelope glycoprotein mutants based on the EIAV vaccine and wild-type envelope glycoprotein, mice and guinea pigs were immunized using a DNA prime-protein boost immunization strategy. The result showed, compared with wild-type gp140, gp140 2M (which contained 2 sites amino acid mutations) and gp140 5M (which contained 5 sites amino acid mutations) increased env-specific IgG and IgG3 binding antibody titers.Gp140 2M resulted in a slight improvement in the neutralizing antibody response against sensitive HIV-1 isolates compared with gp140. These findings have implications for HIV-1 vaccine development based on the HIV-1 CN54 envelope glycoprotein.

[A novel immunization strategy to induce strong humoral responses against HIV-1 using combined DNA, recombinant vaccinia virus and protein vaccines].

To optimize the immunization strategy against HIV-1, a DNA vaccine was combined with a recombinant vaccinia virus (rTV) vaccine and a protein vaccine. Immune responses against HIV-1 were detected in 30 female guinea pigs divided into six groups. Three groups of guinea pigs were primed with HIV-1 DNA vaccine three times, boosted with rTV at week 14, and then boosted with gp140 protein at intervals of 4, 8 or 12 weeks. Simultaneously, the other three groups of animals were primed with rTV vaccine once, and then boosted with gp140 after 4, 8 or 12 weeks. The HIV-1 specific binding antibody and neutralizing antibody, in addition to the relative affinity of these antibodies, were detected at different time points after the final administration of vaccine in each group. The DNA-rTV-gp140 immune regimen induced higher titers and affinity levels of HIV-1 gp120/gp140 antibodies and stronger V1V2-gp70 antibodies than the rTV-gp140 regimen. In the guinea pigs that underwent the DNA-rTV-gp140 regimen, the highest V1V2-gp70 antibody was induced in the 12-week-interval group. However, the avidity of antibodies was improved in the 4-week-interval group. Using the rTV-gp140 immunization strategy, guinea pigs boosted at 8 or 12 weeks after rTV priming elicited stronger humoral responses than those boosted at 4 weeks after priming. In conclusion, this study shows that the immunization strategy of HIV-1 DNA vaccine priming, followed by rTV and protein vaccine boosting, could strengthen the humoral response against HIV-1. Longer intervals were better to induce V1V2-gp70-specific antibodies, while shorter intervals were more beneficial to enhance the avidity of antibodies.

[Enteromorpha prolifera underwater spectral research based on simulation of radiation transmission].

The accumulation of Enteromorpha prolifera in huge amount in the Yellow sea in June, 2008 draws the attention from all over the world. It is an urgent requirement to monitor the wide range of Enteromorpha prolifera distribution by remote sensing. As to the Enteromorpha prolifera floating on the sea surface, effective monitoring by optical remote sensing has been basically achieved. As far as the underwater suspended Enteromorpha prolifera is concerned, the present paper carried out the radiative transfer simulation research on the above water spectral response, its variation with the suspending depth, the water turbidity and environmental conditions. It was found that with the increase in Enteromorpha prolifera suspending depth and water turbidity as well as the decrease in the thickness of Enteromorpha prolifera, the Enteromorpha prolifera information contained in the surface spectra would decrease. The influence of environmental factors such as water-gas interface roughness, cloud cover extent and sun zenith angle on the underwater suspended Enteromorpha prolifera spectra can be ignored. The maximum Enteromorpha prolifera depth that can cause surface spectrum changes is about 30 m in clean water and about 1 m in turbidity water.

[The cardioprotection of estrogen on solated global myocardial ischemia/reperfusion injury in ovariectomized rats].

AIM: To investigated the effect of estrogen on global myocardial ischemia/reperfusion (I/R) injury in ovariectomized (Ovx) rats. METHODS: Sprague-Dawley rats were randomly repartitioned into three groups including sham-operated(Sham), ovariectomized (Ovx), or ovariectomized and then given 17beta-estradiol (Ovx + E2). Hearts were excised, mounted on the Langendorff. After the initial stabilization period, all of the three group hearts were randomly divided into normal perfusion subgroup (Control) and I/R perfusion subgroups. Control, perfused for 60 min after stabilization. I/R perfusion subgroups divided into 10 min I + 30 min R, 20 min I + 30 min R, 30 min I + 0 min R, 30 min I + 5 min R, 30 min I + 15 min R and 30 min I + 30 min R. And then, every group hearts were isolated into the single cardiomyocyte. The cardiomyocytes basal contraction and isoproterenol(ISO) stimulation contraction were measured. The viability and yield of cardiomyocytes were counted. LDH and CK concentrations in coronary effluent were assayed with assay kit. RESULTS: The viability and yield of cardiomyocytes were significantly decreased in the conditions of 30 min ischemia followed by different times of reperfusion. The releases of LDH and CK in coronary effluent were significantly increased in the conditions of 30 min ischemia followed by different times of reperfusion. Except the 10 min and 20 min ischemia, the releases of LDH and CK were significantly increased in Ovx during I/R. Ovx + E2 could abate the heart injury through decreasing the releases of LDH and CK. Besides the control and the 10 min I + 30 min R groups, the myocardial basal and ISO stimulation contraction were higher from Ovx than Sham, and the effect was reversed by Ovx + Ez. CONCLUSION: The results indicate estrogen plays a cardioprotective role in global myocardial ischemia/reperfusion injury in ovariectomized (Ovx) rats.

Oestrogen changed cardiomyocyte contraction and beta-adrenoceptor expression in rat hearts subjected to ischaemia-reperfusion.

Women with functional ovaries have a lower cardiovascular risk than men and postmenopausal women. However, oestrogen replacement therapy remains controversial. This study examined the effect of ovarian hormone deficiency and oestrogen replacement on ventricular myocyte contractile function and expression of beta-adrenoceptors (beta-ARs). Female Sprague-Dawley rats were subjected to bilateral ovariectomy (OVX) or sham operation (Sham). A subgroup of OVX rats received oestrogen (E2) replacement (40 microg kg(-1) day(-1)) for 4 weeks. Cardiomyocyte shortening was evaluated in basal conditions and in the presence of isoprenaline (ISO). The expression of beta-ARs was assessed by Western blotting. The presence of lactate dehydrogenase (LDH) activity in the coronary effluent was determined. Ovariectomy promoted body weight gain associated with reduced serum E2 and uterine weight, all of which were abolished by treatment with E2. Ovariectomy increased the amplitude of both basal and ISO-stimulated contractions, increased LDH release, upregulated beta1-AR expression and downregulated beta2-AR expression, all of which were restored by treatment with E2. A beta1-AR antagonist, CGP20712A, but not a beta2-AR antagonist, ICI118,551, significantly decreased the amplitude of ventricular myocyte shortening. Oestrogen decreased cardiomyocyte contraction and the expression of beta1-AR, and increased expression of beta2-AR, and all these effects were abolished by the E2 receptor antagonist, ICI182,780. These data suggest that oestrogen plays a cardioprotective role in female rat hearts subjected to ischaemia-reperfusion injury, and the effects of oestrogen are associated with decreased cardiomyocyte contraction and expression of beta1-AR, and increased expression of beta2-AR.

[Application of ICP-MS/ICP-AES to detection of 22 trace elements in fruits of elm].

Fruit of elm has been a popular food in Chinese county for many years. With the rapid development of food nutrition and food safety, more and more people begin to pay attention to its content of trace elements and heavy metals. The wild fruit of elm was studied by ICP-MS/ICP-AES to detect the 22 trace elements. The results showed that the fruit of elm contained many trace elements which are necessary to human health: The concentrations of Mg, Ca, Mn, Fe, Cu, Zn, Sr and Rb were 224.57, 269.73, 9.23, 64.93, 1.68, 4.79, 7.68 and 2.21 microg x g(-1) (FW) respectively; while those of Li, B, V, Co, Ni, Se, Br, Mo and Sn were 318.43, 518.83, 265.52, 108.50, 411.21, 34.51, 51.72, 109.90 and 31.51 ng x g(-1) (FW), respectively. Except the wholesome trace elements, contents of heavy metals (As, Cr, Pb and Cd) are also the important standard to identify the quality of food, and the results showed that the concentrations of heavy metals, Pb, Cd, As, Hg and Cr were respectively 557.87, 8.81, 345.55, 0.78 and 347.97 ng x g(-1) (FW) in fruit of elm, which meet the national hygiene standards.

[Expression in Pichia pastoris, fermentation and purification of HIV-1 CN54 Gag antigen].

OBJECTIVE: To express the Gag protein of HIV-1 strain CN54 in Pichia pastoris (P.pastoris), optimize fermentation parameters and purify Gag antigen. METHODS: The Gag gene was subcloned into downstream of aox1 promoter of Pichia expression vector pPS1.0, an integrative vector which possesses an identical 5' untranslated region as the natural aox1 gene and employs both in vitro construction and in vivo selection for multi-copy integrants. The recombinant vector was introduced into P.pastoris strain GS115 by electroporation and selected with G418 for Gag gene integration. Super G418 resistant clones were selected and screened for Gag expression. The engineered P.pastoris was cultured to high cell density (>300 A600 Units/ml) in a 5L fermentor. Through methanol induction, the expression level of Gag reached 120 mg/L. Intracellularly expressed Gag was released by high-pressure homogenization and purified through Sepharose FF and DEAE Sepharose FF column chromatography, the purity of Gag reached up to 90%. RESULTS: Western-blotting suggested that purified Gag expressed in P.pastoris could react specifically with serum from HIV infected individual. CONCLUSION: Gag antigen expressed in P.pastoris has provided a good basis for the development of a new generation of HIV vaccine candidates against some Chinese prevalent strains.

Cloning of delta6-desaturase from Mucor circinelloides and its high expression in Saccharomyces cerevisiae.

The aim of this study is to obtain Saccharomyces cerevisiae engineering strain with high gamma-linolenic acid (GLA, gamma-C18:3), which is a nutritionally important fatty acid that plays a vital role in biological structure and cell functions. As the first step,we cloned gamma6-desaturase gene from fungus mucor circinelloides by RT-PCR; delta6-desaturase is responsible for the transformation of linoleic acid into GLA. The PCR product was subcloned into yeast expression vector pYES2 to generate a recombinant plasmid pYES412. Transformation of S. cerevisiae strain INVSc1 was done by the lithium acetate method and the recombinant yeast cells were selected on a uracil-deficient medium. On appropriate medium and temperature,linoleic acid was provided as a substrate to yeast cultures,and the level of gamma-linolenic acid reached 50.07%. So far,the result we obtained is the best in terms of the level of expression of delta6-desaturase gene in Saccharomyces cerevisiae.