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@#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.
ABSTRACT
<p><b>OBJECTIVE</b>To evaluate the influence of long-term highly active antiretroviral therapy (HAART) on bone metabolism in patients with acquired immune deficiency syndrome (AIDS).</p><p><b>METHODS</b>A total of 82 AIDS patients who received HAART in the First Affiliated Hospital of Zhejiang University Medical School during January 2012 and May 2016 were enrolled in the study. Patients were divided into TDF group (n=41) and AZT group (n=41), and glomerular filtration rate (GFR), serum calcium, serum phosphate and serum ALP levels in 6, 12, 18, 24 and 30-month during the treatment were analyzed in both groups.</p><p><b>RESULTS</b>Both TDF-based and AZT-based therapies significantly improved CD4(+) T-cell levels of the patients, and no significant difference was observed between the groups. Compared with the baseline, GFR [(120.71±62.85) vs(110.08±39.18) mL/min] and serum phosphate [(1.25±0.19) vs(1.22±0.21) mmol/L] levels rose after 6-month treatment in TDF group, but the difference was not statistically significant (P>0.05); Serum ALP [99.0(79.5-124.0) U/L vs 80.0(60.5-96.0) U/L] and serum calcium [(2.32±0.12) vs(2.25±0.17) mmol/L] levels rose significantly (P<0.05). As treatment continued, the levels of GFR, serum calcium and serum phosphate declined, while the serum ALP number was still increasing. After 30 months of HAART, the level of serum calcium [(2.16±0.15) vs(2.25±0.17) mmol/L], serum phosphate [(1.06±0.17) vs(1.22±0.21) mmol/L], GFR [(98.13±30.43) vs (110.08±39.18) mL/min] declined significantly (P<0.05) in TDF group, while serum ALP [110.0(98.5-120.5) U/L vs 80.0(60.5-96.0) U/L, P<0.05] increased. In AZT group, serum calcium and serum ALP levels rose and GFR level was declined (P<0.05), while serum phosphate level was not significantly changed during the treatment (P>0.05). Compared with AZT group, there were greater changes on the levels of GFR, serum calcium, serum phosphate and serum ALP in TDF group.</p><p><b>CONCLUSION</b>HAART is effective for patients with AIDS, and TDF-based therapy may have significant impact on bone metabolism of the patients, which needs close monitoring and timely intervention or adjustment if necessary.</p>
ABSTRACT
Latent reservoir (LR) of HIV is the cells (such as CD4(+)T cell) where HIV is able to hide. These cellular reservoirs are located throughout the body, including the spleen, lymph nodes, gastrointestinal lymphoid tissues, and become the major obstacle to cure HIV infection. To truly cure patients, a new strategy "shock and kill" was put forward by scientists, which is to shock HIV-infected cells out of hidden reservoirs in the body and then kill them. Quantitatively evaluating the size of long-lived LR is essential to this strategy. This paper reviews assays that measure the magnitude of the latent HIV reservoir, including Alu-gag PCR, quantitative viral outgrowth assay (Q-VOA) and tat/rev induced limiting dilution assay(TILDA). Alu-gag PCR can differentiate the integrated and un-integrated HIV DNA, however, it cannot distinguish defective virus from competent virus, leading to overestimate the real size of LR. Q-VOA is based on cell culture, and is the golden standard for measuring the LR since it provides a definitive minimal estimate of reservoir size. Its disadvantages are being more costly, large amount of blood sample required, and underestimating the true size, which was resulted from particles being not released after one round of stimulation. TILDA measures cells with inducible msRNA as these transcripts are absent in latently infected cells but induced upon viral reactivation. It requires small blood sample size, does not need extraction of viral nucleic acids, can be completed in 2 d and covers a wide dynamic range of reservoir sizes, but has the disadvantage of overestimating the true size of LR.