HIV-1 env gene mutations outside the targeting probe affects IPDA efficiency.

The intact proviral DNA assay (IPDA) based on droplet digital PCR was developed to identify intact proviral DNA and quantify HIV-1 latency reservoirs in patients infected with HIV-1. However, the genetic characteristics of different HIV-1 subtypes are non-consistent due to their high mutation and recombination rates. Here, we identified that the IPDA based on the sequences features of an HIV-1 subtype could not effectively detect different HIV-1 subtypes due to the high diversity of HIV-1. Furthermore, we demonstrated that mutations in env gene outside the probe binding site affect the detection efficiency of IPDA. Since mutations in env gene outside the probe binding site may also lead to the formation of stop codons, thereby preventing the formation of viruses and ultimately overestimating the number of HIV-1 latency reservoirs, it is important to address the effect of mutations on the IPDA.

Liver macrophages and sinusoidal endothelial cells execute vaccine-elicited capture of invasive bacteria.

Vaccination has substantially reduced the morbidity and mortality of bacterial diseases, but mechanisms of vaccine-elicited pathogen clearance remain largely undefined. We report that vaccine-elicited immunity against invasive bacteria mainly operates in the liver. In contrast to the current paradigm that migrating phagocytes execute vaccine-elicited immunity against blood-borne pathogens, we found that invasive bacteria are captured and killed in the liver of vaccinated host via various immune mechanisms that depend on the protective potency of the vaccine. Vaccines with relatively lower degrees of protection only activated liver-resident macrophage Kupffer cells (KCs) by inducing pathogen-binding immunoglobulin M (IgM) or low amounts of IgG. IgG-coated pathogens were directly captured by KCs via multiple IgG receptors FcγRs, whereas IgM-opsonized bacteria were indirectly bound to KCs via complement receptors of immunoglobulin superfamily (CRIg) and complement receptor 3 (CR3) after complement C3 activation at the bacterial surface. Conversely, the more potent vaccines engaged both KCs and liver sinusoidal endothelial cells by inducing higher titers of functional IgG antibodies. Endothelial cells (ECs) captured densely IgG-opsonized pathogens by the low-affinity IgG receptor FcγRIIB in a "zipper-like" manner and achieved bacterial killing predominantly in the extracellular milieu via an undefined mechanism. KC- and endothelial cell-based capture of antibody-opsonized bacteria also occurred in FcγR-humanized mice. These vaccine protection mechanisms in the liver not only provide a comprehensive explanation for vaccine-/antibody-boosted immunity against invasive bacteria but also may serve as in vivo functional readouts of vaccine efficacy.

Unmethylated CpG motif-containing genomic DNA fragments of bacillus calmette-guerin improves immune response towards a DNA vaccine for COVID-19.

The development of an effective vaccine to control the global coronavirus disease-2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus- 2 (SARS-CoV-2) is of utmost importance. In this study, a synthetic DNA-based vaccine candidate, known as pSV10-SARS-CoV-2, expressing the SARS-CoV-2 spike protein was designed and tested in 39 BALB/c mice with BC01, an adjuvant derived from unmethylated CpG motif-containing DNA fragments from the Bacillus Calmette-Guerin genome. Mice vaccinated with pSV10-SARS-CoV-2 with BC01 produced early neutralizing antibodies and developed stronger humoral and cellular immune responses compared to mice that received the DNA vaccine only. Moreover, sera from mice vaccinated with pSV10-SARS-CoV-2 with BC01 can neutralize certain variants, including 614G, 614G + 472 V, 452R, 483A, 501Y.V2, and B.1.1.7. The results of this study demonstrate that the addition of BC01 to a DNA-vaccine for COVID-19 could elicit more effective neutralizing antibody titers for disease prevention.

Effect of freezing on recombinant hepatitis E vaccine.

Studies have revealed that vaccines are more often exposed to sub-zero temperatures during cold chain transportation than what was previously known. Such exposure might be detrimental to the potency of temperature-sensitive vaccines. The aim of this study was to evaluate the impact of exposure to freezing on the physicochemical properties and biological activities of recombinant hepatitis E (rHE) vaccine. Changes in rHE vaccine due to freezing temperatures were analyzed with regard to sedimentation rate, antigenicity, and antibody affinity and potency. The freezing temperature of rHE was measured, then rHE vaccine was exposed to freezing temperatures below -10°C.Significant increase of sedimentation rate was noted, according to shake test and massed precipitates. In addition, the binding affinity of rHE vaccine to six specific monoclonal antibodies was significantly reduced and the in vivo potency for eliciting a protective IgG response was also partially lost, especially for anti-HEV neutralizing antibodies. Altogether, our work indicates that exposure of rHE vaccine to a temperature below -10°C results in the loss of structural integrity and biological potency of rHE vaccine.

Development of a pseudovirus-based assay for measuring neutralizing antibodies against Coxsackievirus A10.

Coxsackievirus A10 (CV-A10) has recently emerged as a major pathogen of hand, foot, and mouth disease in children worldwide. Currently no effective treatments are available; development of anti-CV-A10 vaccine is a most cost-effective way for CV-A10 prevention. Robust assay to measure neutralizing antibody (NtAb) titres elicited by vaccination would greatly prompt anti-CV-A10 vaccine development. Compare to the traditional neutralization assay based on inhibition of cytopathic effects (herein after referred to as cNT) which is time-consuming and labor-intensive, in this study we developed an efficient high-throughput neutralization antibody assay based on CV-A10 pseudoviruses (herein after referred to as pNT). In the pNT, anti-CV-A10 NtAb titre was negatively corresponded with the relative luminescent unit (RLU) produced by luciferase reporter gene incorporated in pseudovirus genome. As described in this study, the NtAb against CV-A10 could be detected within 10-16 h, anti- CV-A10 NtAb in 67 human serum samples were measured in parallel with pNT and cNT assays, a good correlation (r = 0.83,p < .0001) and good agreement(97%) were shown between cNT and pNT, indicating that the pNT provides a rapid and convenient procedure for measuring NtAb production against anti-CV-A10 NtAb measurement.

Construction and Identification of Recombinant HEK293T Cell Lines Expressing Non-structural Protein 1 of Zika Virus.

Background: Zika virus (ZIKV) infection has become a major public health problem all around the world. Early diagnosis of Zika infection is important for better management of the disease. Non-structural protein 1 (NS1) is a potential biomarker for ZIKV infections. The purpose of this study was to produce the ZIKV NS1 protein for establishing serological diagnostic methods for ZIKV. Methods: The cDNA fragment encoding a chimeric protein composed of murine Igκ signal peptide, NS1 and histidine tag was synthesized and cloned into the lentiviral expression vector pLV-eGFP. The resulting expression vector pLV-eGFP-ZIKV-NS1 was packaged and transduced into human embryonic kidney (HEK) 293T cells and clonal cell lines with NS1 gene were generated from the tranduced cells by limiting dilution. Over expressed recombination NS1 (rNS1) fusion protein was purified by nickel affinity chromatography. Mice immunization and enzyme-linked immunosorbent assay (ELISA) were carried out to evaluate the immunogenicity of rNS1. Results: Western blot analysis revealed that the reconstituted cells stably expressed and secreted high levels of approximately 45-kDa NS1, and no significant changes were observed in green fluorescent protein (GFP) fluorescence ratio and fluorescence intensity. The scanned gels showed that the purity of the purified rNS1 was 99.42%. BALB/c mice were then immunized with purified rNS1 and a high level of antibodies against NS1 was elicited in the mice. Conclusion: Overall, recombinant NS1 proteins were successfully purified and their antigenicity was assessed. Immunization of mice with recombinant proteins demonstrated the immunogenicity of the NS1 protein. Thus, the generated recombinant NS1 can be potentially used in the development of serological diagnostic methods for ZIKV.

Angiotensin-(1-7) Decreases Cell Growth and Angiogenesis of Human Nasopharyngeal Carcinoma Xenografts.

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous, heptapeptide hormone acting through the Mas receptor (MasR), with antiproliferative and antiangiogenic properties. Recent studies have shown that Ang-(1-7) has an antiproliferative action on lung adenocarcinoma cells and prostate cancer cells. In this study, we report that MasR levels were significantly upregulated in nasopharyngeal carcinoma (NPC) specimens and NPC cell lines. Viral vector-mediated expression of Ang-(1-7) dramatically suppressed NPC cell proliferation and migration in vitro. These effects were completely blocked by the specific Ang-(1-7) receptor antagonist A-779, suggesting that they are mediated by the Ang-(1-7) receptor Mas. In this study, Ang-(1-7) not only caused a significant reduction in the growth of human nasopharyngeal xenografts, but also markedly decreased vessel density, suggesting that the heptapeptide inhibits angiogenesis to reduce tumor size. Mechanistic investigations revealed that Ang-(1-7) inhibited the expression of the proangiogenic factors VEGF and PlGF. Taken together, the data suggest that upregulation of MasR could be used as a diagnostic marker of NPC and Ang-(1-7) may be a novel therapeutic agent for nasopharyngeal cancer therapy because it exerts significant antiangiogenic activity.

Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells.

OBJECTIVES: Lentiviral vectors have been used successfully to rapidly produce decigram quantities of active recombinant proteins in mammalian cell lines. To optimize the protein production platform, the roles of Ubiquitous Chromatin Opening Element (UCOE), an insulator, and selected promoters were evaluated based on efficiency and stability of foreign gene expression mediated by lentiviral vectors. METHODS: Five lentiviral vectors, pFIN-EF1α-GFP-2A-mCherH-WPRE containing EF1α promoter and HS4 insulator, p'HR.cppt.3'1.2kb-UCOE-SFFV-eGFP containing SFFV promoter and UCOE, pTYF-CMV(ß-globin intron)-eGFP containing CMV promoter and ß-globin intron, pTYF-CMV-eGFP containing CMV promoter, and pTYF-EF1α-eGFP with EF1α promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell). The transduced cells were passaged once every three days at a ratio of 1:10. Expression level and stability of the foreign gene, green fluorescence protein (GFP), was evaluated using fluorescent microscopy and flow cytometry. Furthermore, we constructed a hepatitis C virus (HCV) E1 recombinant lentiviral vector, pLV-CMV-E1, driven by the CMV promoter. This vector was packaged and transduced into 293T cells, and the recombinant cell lines with stable expression of E1 protein were established by limiting dilution. RESULTS: GFP expression in 293T cells transduced with the five lentiviral vectors peaked between passages 3 and 5 and persisted for more than 5 weeks. The expression was prolonged in the cells transduced with TYF-CMV (ß-globin intron)-eGFP or TYF-CMV-eGFP, demonstrating less than a 50% decrease even at 9 weeks post transduction (p>0.05). The TYF-CMV-eGFP-transduced cells began with a higher level of GFP expression than other vectors did. The percentage of GFP positive cells for any of the five lentiviral vectors sustained over time. Moreover, the survival rates of all transfected cells exceeded 80% at both 5 and 9 weeks post transduction. Surprisingly, neither the HS4 insulator nor the UCOE sequence improved the GFP expression level or stability. Clonal cell lines with HCV E1 gene were generated from LV-CMV-E1 vector-infected 293T cells. A representative recombinant cell line maintained stable E1expression for at least 9 weeks without significant difference in morphology compared with untreated 293T cells. CONCLUSION: The results suggest that all five vectors can stably transduce 293T cells, producing long term transgene expression with different efficiencies. However, neither the insulator nor the UCOE improved the GFP expression. The vectors containing the promoter CMV or CMV (ß-globin intron) generated the highest gene expressions, manifesting as more favorable candidates for recombinant protein production in HEK293T cells.

Safety and immunogenicity of a diphtheria, tetanus, acellular pertussis and Haemophilus influenzae Type b combination vaccine compared with separate administration of licensed equivalent vaccines in Chinese infants and toddlers for primary and booster immunization.

To evaluate the safety and immunogenicity of a diphtheria, tetanus, acellular pertussis and Haemophilus infuenzae Type b (DTaP/Hib) combination vaccine first developed by a Chinese manufacturer, a randomized, two-stage, parallel controlled, single center clinical trial was conducted in Dafeng, Jiangsu Province of China. A total of 720 infants were enrolled and randomly assigned to two groups with a 2:1 allocation. In Stage I, 480 subjects in Group T were administered with 3 doses of the DTaP/Hib vaccine at 3, 4 and 5 months of age, respectively, while 240 subjects in Group C received separate licensed DTaP vaccine and Hib conjugate vaccine on the same schedule. In Stage II, 633 primed toddlers (431 of Group T and 202 of Group C) were given a booster dose at 18 months of age. Sera samples were collected at pre-dose 1, 4 weeks post-dose 3, pre-dose 4 and 4 weeks post-dose 4, respectively. Levels of protective antibodies were measured by Enzyme Linked Immunoadsorbent Assay (ELISA). Immunogenicity was evaluated with regard to geometric mean concentrations (GMCs), seroconversion rates and seroprotection rates of the antibodies. Solicited adverse reactions were recorded for 3 days after each dose; unsolicited adverse events and serious adverse events were monitored for 28 days after vaccination. Results showed that seroconversion rates of anti-pertussis toxoid (PT), anti-filamentous hemagglutinin (FHA), anti-diphtheria toxoid (DT), anti-tetanus toxid (TT) and anti-polyribosyl-ribitol-phosphate (PRP) in Group T (Stage I: 98.06%, 97.33%, 100%, 100%, 98.79%; Stage II: 99.18%, 83.42%, 99.18%, 63.32%, 85.05%) were comparable to that of Group C (Stage I: 95.26%, 93.16%, 100%, 100%, 98.42%; Stage II: 98.89%, 83.89%, 98.33%, 53.89%, 76.67%). Nearly 100% of the subjects in both groups achieved seroprotective levels of anti-DT (> or = 0.1IU/ml), anti-TT (> or = 0.1IU/ml) and anti-PRP (> or = 0.15 microg/ml) after primary and booster vaccination. The frequencies of local induration, swelling and redness as well as general reactions such as fever, diarrhea and anaphylaxis were low and acceptable in both groups. In conclusion, the DTaP/Hib vaccine was demonstrated to be non-inferior to the control vaccines on safety and immunogenicity. There could be a bright future for the DTaP/Hib vaccine to be widely used in the universal vaccination of Chinese children.