Anti-OX40 Antibody Combined with HBc VLPs Delays Tumor Growth in a Mouse Colon Cancer Model.

Objective: Combination immunotherapy strategies targeting OX40, a co-stimulatory molecule that can enhance antitumor immunity by modulating the proliferation, differentiation, and effector function of tumor-infiltrating T cells, have attracted much attention for their excellent therapeutic effects. In this study, we aimed to evaluate the antitumor efficacy of combined anti-OX40 and hepatitis B core virus-like particles (HBc VLPs) therapy using a mouse colon cancer model. Methods: Humanized B-hOX40 mice were injected subcutaneously with MC38 colon tumor cells and treated with HBc VLPs+anti-hOX40 antibody. Tumor growth was monitored. Flow cytometric analysis was performed to evaluate the populations of T cell subsets in the tumors. Results: The combination of anti-OX40 with HBc VLPs resulted in a significant delay in tumor growth, suggesting that a potent antitumor immunity was induced by the combination therapy. Further studies revealed that HBc VLPs+anti-OX40 treatment induced a significant increase in effector T cells (Teffs) and a significant decrease in regulatory T cells (Tregs) in the tumor microenvironment (TME), which accounted for the synergistic antitumor effect of anti-OX40 in combination with HBc VLPs. Conclusion: Combination therapy of anti-hOX40 and HBc VLPs provides synergistic antitumor activity in colon cancer-bearing mice, which may represent a potential design strategy for cancer immunotherapy.

Recombinant SARS-CoV-2 RBD with a built in T helper epitope induces strong neutralization antibody response.

Without approved vaccines and specific treatments, COVID-19 is spreading around the world with above 26 million cases and approximately 864 thousand deaths until now. An efficacious and affordable vaccine is urgently needed. The Val308 - Gly548 of spike protein of SARS-CoV-2 linked with Gln830 - Glu843 of Tetanus toxoid (TT peptide) (designated as S1-4) and without TT peptide (designated as S1-5) were expressed and renatured. The antigenicity and immunogenicity of S1-4 were evaluated by Western Blotting (WB) in vitro and immune responses in mice, respectively. The protective efficiency was measured preliminarily by microneutralization assay (MN50). The soluble S1-4 and S1-5 protein was prepared to high homogeneity and purity. Adjuvanted with Alum, S1-4 protein stimulated a strong antibody response in immunized mice and caused a major Th2-type cellular immunity supplemented with Th1-type immunity. Furthermore, the immunized sera could protect the Vero E6 cells from SARS-CoV-2 infection with neutralizing antibody titer 256. Recombinant SARS-CoV-2 RBD with a built in T helper epitope could stimulate both strong humoral immunity supplemented with cellular immunity in mice, demonstrating that it could be a promising subunit vaccine candidate.

Epidemiological distribution of hepatitis B virus genotypes in 1-29-year-olds in the mainland of China.

BACKGROUND: To analyze the epidemiological distribution of Hepatitis B virus (HBV) genotype in the mainland of China following the implementation of effective preventive measures. METHODS: Five hundred and seventeen HBsAg-positive subjects aged 1-29 years surveyed in the 2014 national HBV sero-survey in the mainland of China were enrolled in the study. The full-length HBV genome was obtained by PCR amplification and sequencing. The HBV genotype was determined by phylogenetic analysis. Combined with questionnaire information, HBV genotype distribution was analyzed. RESULTS: Of the 517 HBsAg-positive subjects, 369 (71.4%) were included in the analysis. HBV genotypes found were B (45.0%), C (36.6%), D (6.0%), C/D (9.8%), B/C (2.2%), and I (0.5%). Geographic differences in HBV genotype were significant for seven regions. Three serotypes were found: adw (47.2%), adr (35.5%), and ayw (17.3%). B2 (43.9%) and C2 (25.2%) were the two major subgenotypes. The predominant genotypes differed between the Han group and the other ethnic groups. No statistical differences in genotype distribution were found by gender, age group, or hepatitis B (HepB) vaccination history. CONCLUSION: The prevalence of HBV genotype B was higher than that of genotype C with subgenotypes B2 and C2 endemic in 1-29-year-olds in the mainland of China, after HBV prevalence has reduced significantly due to the implementation of preventive measures. HepB vaccination or other factors did not interfere with HBV genotype distribution. The surveillance of HBV genotype was essential for responding to the potential changes and impact on the preventive policies in the future.

The biological characteristics of SARS-CoV-2 spike protein Pro330-Leu650.

SARS-CoV-2 is the cause of the worldwide outbreak of COVID-19 that has been characterized as a pandemic by the WHO. Since the first report of COVID-19 on December 31, 2019, 179,111 cases were confirmed in 160 countries/regions with 7426 deaths as of March 17, 2020. However, there have been no vaccines approved in the world to date. In this study, we analyzed the biological characteristics of the SARS-CoV-2 Spike protein, Pro330-Leu650 (SARS-CoV-2-SPL), using biostatistical methods. SARS-CoV-2-SPL possesses a receptor-binding region (RBD) and important B (Ser438-Gln506, Thr553-Glu583, Gly404-Aps427, Thr345-Ala352, and Lys529-Lys535) and T (9 CD4 and 11 CD8 T cell antigenic determinants) cell epitopes. High homology in this region between SARS-CoV-2 and SARS-CoV amounted to 87.7%, after taking the biological similarity of the amino acids into account and eliminating the receptor-binding motif (RBM). The overall topology indicated that the complete structure of SARS-CoV-2-SPL was with RBM as the head, and RBD as the trunk and the tail region. SARS-CoV-2-SPL was found to have the potential to elicit effective B and T cell responses. Our findings may provide meaningful guidance for SARS-CoV-2 vaccine design.

Intranasal Immunization Using CTA1-DD as a Mucosal Adjuvant for an Inactivated Influenza Vaccine.

OBJECTIVE: To evaluate the effect of intranasal immunization with CTA1-DD as mucosal adjuvant combined with H3N2 split vaccine. METHODS: Mice were immunized intranasally with PBS (negative control), or H3N2 split vaccine (3 µg/mouse) alone, or CTA1-DD (5 µg/mouse) alone, or H3N2 split vaccine (3 µg/mouse) plus CTA1-DD (5 µg/mouse). Positive control mice were immunized intramuscularly with H3N2 split vaccine (3 µg/mouse) and alum adjuvant. All the mice were immunized twice, two weeks apart. Then sera and mucosal lavages were collected. The specific HI titers, IgM, IgG, IgA, and IgG subtypes were examined by ELISA. IFN-γ and IL-4 were test by ELISpot. In addition, two weeks after the last immunization, surivival after H3N2 virus lethal challenge was measured. RESULTS: H3N2 split vaccine formulated with CTA1-DD could elicit higher IgM, IgG and hemagglutination inhibition titers in sera. Furthermore, using CTA1-DD as adjuvant significantly improved mucosal secretory IgA titers in bronchoalveolar lavages and vaginal lavages. Meanwhile this mucosal adjuvant could enhance Th-1-type responses and induce protective hemagglutination inhibition titers. Notably, the addition of CTA1-DD to split vaccine provided 100% protection against lethal infection by the H3N2 virus. CONCLUSION: CTA1-DD could promote mucosal, humoral and cell-mediated immune responses, which supports the further development of CTA1-DD as a mucosal adjuvant for mucosal vaccines.

HIV prevalence in suspected Ebola cases during the 2014-2016 Ebola epidemic in Sierra Leone.

BACKGROUND: The 2014-2016 Ebola virus epidemic in West Africa was the largest outbreak of Ebola virus disease (EVD) in history. Clarifying the influence of other prevalent diseases such as human immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS) will help improve treatment and supportive care of patients with EVD. CASE PRESENTATION: We examined HIV and hepatitis C virus (HCV) antibody prevalence among suspected EVD cases from the Sierra Leone-China Friendship Biological Safety Laboratory during the epidemic in Sierra Leone. HIV and HCV antibodies were tested in 678 EVD-negative samples by enzyme-linked immunosorbent assay. A high HIV prevalence (17.6%) and low HCV prevalence (0.22%) were observed among the suspected cases. Notably, we found decreased HIV positive rates among the suspected cases over the course of the epidemic. This suggests a potentially beneficial effect of an improved public health system after assistance from the World Health Organization and other international aid organizations. CONCLUSIONS: This EVD epidemic had a considerable impact on the public health system and influenced the prevalence of HIV found among suspected cases in Sierra Leone, but also provided an opportunity to establish a better surveillance network for infectious diseases.

Intranasal vaccination with ebola virus GP amino acids 258-601 protects mice against lethal challenge.

Ebola virus (EBOV) disease (EVD) leads to lethal hemorrhagic fever with a case fatality rate as high as 90%, thus posing a serious global public health concern. However, while several vaccines based on the EBOV glycoprotein have been confirmed to be effective in animal experiments, no licensed vaccines or effective treatments have been approved since the first outbreak was reported in 1976. In this study, we prepared the extracellular domain of the EBOV GP protein (designated as N20) by prokaryotic expression and purification via chromatography. Using CTA1-DD (designated as H45) as a mucosal adjuvant, we evaluated the immunogenicity of N20 by intranasal administration and the associated protective efficacy against mouse-adapted EBOV challenge in mice. We found that intranasal vaccination with H45-adjuvanted N20 could stimulate humoral immunity, as supported by GP-specific IgG titers; Th1 cellular immunity, based on IgG subclasses and IFN-γ/IL-4 secreting cells; and mucosal immunity, based on the presence of anti-EBOV IgA in vaginal lavages. We also confirmed that the vaccine could completely protect mice against a lethal mouse-adapted EBOV (MA-EBOV) challenge with few side effects (based on weight loss). In comparison, mice that received N20 or H45 alone succumbed to lethal MA-EBOV challenge. Therefore, mucosal vaccination with H45-adjuvanted N20 represents a potential vaccine candidate for the prevention of EBOV in an effective, safe, and convenient manner.

Efficient Humoral and Cellular Immune Responses Induced by a Chimeric Virus-like Particle Displaying the Epitope of EV71 without Adjuvant.

OBJECTIVE: To eliminate the side effects of aluminum adjuvant and His-tag, we constructed chimeric VLPs displaying the epitope of EV71 (SP70) without His-tagged. Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant. METHODS: The fusion protein was constructed by inserting SP70 into the MIR of truncated HBcAg sequence, expressed in E. Coli, and purified through ion exchange chromatography and density gradient centrifugation. Mice were immunized with the VLPs and sera were collected afterwards. The specific antibody titers, IgG subtypes and neutralizing efficacy were detected by ELISA, neutralization assay, and EV71 lethal challenge. IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay. RESULTS: HBc-SP70 proteins can self-assemble into empty VLPs. After immunization with HBc-SP70 VLPs, the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge. There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not. The specific IgG subtypes were mainly IgG1 and IgG2b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4. CONCLUSION: The fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation. In the absence of adjuvant, they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant. Furthermore, the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.

Expression and Purification of Recombinant Hepatitis Delta Virus (HDV) Antigen for Use in a Diagnostic ELISA for HDV Infection Using the High-Density Fermentation Strategy in Escherichia coli.

OBJECTIVE: Hepatitis Delt a Virus (HDV) antigen is widely used as a capture antigen in ELISAs for the identification of HDV infection; large amounts of recombinant HDV antigen with active antigenicity are required for this purpose. METHODS: Reconstruct the gene of HDV antigen based on the bias code of Escherichia coli, the recombinant protein expresses by high-density fermentation with fed-batch feeding strategy, and purify by immobilized metal chromatography. The sensitivity and specificity of this antigen detect by ELISA method. RESULTS: The expression of HDV antigen can reach 20% of the total cell mass in the soluble form. The recombinant HDV antigen can be conveniently purified (98%) by immobilized metal ion affinity chromatography (IMAC) using the interaction between a His-tag and nickel ions. Production of recombinant HDV antigen can reach 0.5 g/L under conditions of high-density cell fermentation. Applied to the diagnostic ELISA method, the recombinant HDV antigen shows excellent sensitivity (97% for IgM and 100% for IgG) and specificity (100% for IgG and IgM) for the detection of anti-HDV antibodies. CONCLUSION: Expression and purification the recombinant HDV antigen as a candidate protein for application in a diagnostic ELISA for HDV infection. Large-scale production of the protein can be achieved using the high-density fermentation strategy.

[Prokaryotic soluble expression of protein D of Haemophilus influenzae type b].

OBJECTIVE: To express the recombinant D protein in prokaryotic expression system solubly and make preparation for producing D-carrier conjugate vaccine next step. METHODS: The hpd gene fragment removed of signal peptide from genomic DNA of Hib CMCC was inserted into pET43. 1a. The recombinant plasmid was transformed to competent E. coli BL21 (DE3) for expression under induction of IPTG. The expressed recombination protein was precipitated with ammonium sulfate, purified by DEAE anion exchange column chromatography and identified for reactogenicity by Western Blot. RESULTS: The expressed recombination protein, in a soluble form, constained about 50% of total somatic protein and showed specific reaction with the HIB antisera after preliminary purification. CONCLUSION: The D protein recombined expression plasmid was constructed successfully and expressed D protein in prokaryotic cells in a solube form.

[Construction of recombinant CHO cell strain for high expression of HBsAg].

OBJECTIVE: To overexpress hepatitis B virus S gene in CHO cells cultured in serum-free media. METHOD: Plasmid was constructed by cloning of HBV S gene and then it was transfected into CHO cells. After cell screen, the positive clones were identified and isolated into a serum-free media followed by the serological and morphological characterization of the expression product. RESULT: CHO cell strains which can express HBsAg efficiently and stably were obtained. Spherical and filamentous HBsAg could be detected under electronic microscope. The titer of the expression product was up to 1:5000. CONCLUSION: Serum-free media cultured CHO cell strain for overexpression of HBsAg was successfully constructed and the expression product was high antigenic.

[Gene optimization, protein expression, purification and characterization of the HDV antigen for diagnosis].

OBJECTIVE: To prepare HDAg with biological activities as a candidate of diagnostic reagent. METHODS: To synthesize HDV gene fragment after codon optimization. To construct a thio-fused recombinant plasmid based on M48 expression vector. To express in E. coli induced by IPTG. To purify the protein by affinity chromatography followed by characterization in ELISA: RESULTS: Plasmid construction was verified by enzyme digestion. SDS-PAGE indicated the molecular weight of the protein was the same as we expectation. ELISA proved its affinity with HDV antibodies. CONCLUSION: HDAg was obtained successfully and it will pave the road to the research of HDV diagnostic reagent.

[Detection of hepatitis A virus RNA with an improved loop-mediated isothermal amplification assay].

OBJECTIVE: To establish a novel improved loop-mediated isothermal amplification (LAMP) technique to detect hepatitis A virus (HAV). METHODS: A novel improved LAMP assay based on the addition of an acceleration primer was developed for hepatitis A virus nucleotide acid detection. RESULTS: Precision and reproducibility analysis proved its high stability and reliability. Comparison between the improved and conventional LAMP assays revealed that the former was more sensitive with a detection limit of 5 TCID50/ml. The novel detection method displayed 100% consistency with the TaqMan real-time PCR assay when applied to clinical specimens collected from patients with confirmed acute HAV infection. CONCLUSION: This novel technique is widely applicable as a simple diagnostic tool in the clinical field as well as for the surveillance and investigation of the infectious disease in developing countries where HAV is endemic.

[Effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering].

OBJECTIVE: To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering. METHODS: Based on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized. Both the original gene and the optimized gene expressed in prokaryotic cells, SDS-PAGE was made to analyze the protein expression yield and to decide which protein expression style was more proportion than the other. Furthermore, two antigens were purified by chromatography in order to compare the purity by SDS-PAGE and Image Lab software. RESULTS: SDS-PAGE indicated that the molecular weight of target proteins from two groups were the same as we expected. Gene optimization resulted in the higher yield and it could make the product more soluble. After chromatography, the purity of target protein from optimized gene was up to 96.3%, obviously purer than that from original gene. CONCLUSION: Gene optimization could increase the protein expression yield and solubility of genetic engineering HDV small antigen. In addition, the product from the optimized gene group was easier to be purified for diagnosis usage.

[The investigation of hepatitis D virus infection situation in the human with HBsAg in Foshan of Guangdong province].

OBJECTIVE: To investigate the seroprevalence of hepatitis D virus in Foshan of Guangdong province, to provide the data for the study about it in China. METHODS: ELISA kits from two different companies were used for detecting anti-HDV IgG of all the serum samples, and then RT-PCR was carried out about the selected serum to ensure the results. All the serum samples were collected in 2011 in The First People's Hospital of Foshan. RESULTS: The results from two ELISA kits and RT-PCR were identical. Eight samples were positive. CONCLUSIONS: The seroprevalence rate of HDV in Foshan is higher than that in China. It has no statistically significant difference between female and male. Morever, the older with HBsAg are susceptible to HDV.