Impaired long-term anti-HBs responses in choronic hepatitis C patients: Results from a five-year follow-up study with healthy control.

Although hepatitis B virus (HBV) vaccination is recommended for hepatitis C virus (HCV)-infected individuals to avoid HBV superinfection, the persistence of their humoral and cell-mediated immunity responses to HBV vaccination is still under investigation. Patients with chronic hepatitis C (CHC) and matched healthy controls, who completed three doses of hepatitis B vaccine (HepB) in 2014, were followed up five years later. One booster dose of HepB was given to those with antibody against hepatitis B surface antigen (anti-HBs) lower than 10mIU/mL. Anti-HBs was tested at follow-up and on the 14th day after the booster dose, as well as HBsAg specific spot-forming cells of interferon γ and interleukin (IL) 2, 4, 5, and 6. At five years, only 56.58% of the CHC patients had sero-protective titers (≥10mIU/mL) of anti-HBs, compared to 70.83% in the controls (P < .05). Similarly, the geometric mean concentration (GMC) of anti-HBs in CHC patients was significantly lower than that in controls (16.95 vs 37.34 mIU/mL, P < .05). After the booster, both GMC and the rate of anamnestic response increased to a very high level in the two groups and the difference between them disappeared (P > .05). Multivariable analysis showed that HCV infection was an independent predictor factor to anti-HBs level at follow-up. HBsAg specific IL-6 was stronger in the CHC patients compared to the controls (P < .05). The data indicate that the durability of protective anti-HBs is poorer in CHC patients compared to healthy individuals, and impaired long-term anti-HBs responses might be associated with the increased HBsAg specific IL-6 responses.

B21 DNA vaccine expressing ag85b, rv2029c, and rv1738 confers a robust therapeutic effect against latent Mycobacterium tuberculosis infection.

Latent tuberculosis infection (LTBI) treatment is known to accelerate the decline in TB incidence, especially in high-risk populations. Mycobacterium tuberculosis (M. tb) expression profiles differ at different growth periods, and vaccines protective and therapeutic effects may increase when they include antigenic compositions from different periods. To develop a post-exposure vaccine that targets LTBI, we constructed four therapeutic DNA vaccines (A39, B37, B31, and B21) using different combinations of antigens from the proliferation phase (Ag85A, Ag85B), PE/PPE family (Rv3425), and latent phase (Rv2029c, Rv1813c, Rv1738). We compared the immunogenicity of the four DNA vaccines in C57BL/6j mice. The B21 vaccine stimulated the strongest cellular immune responses, namely Th1/Th17 and CD8+ cytotoxic T lymphocyte responses. It also induced the generation of strengthened effector memory and central memory T cells. In latently infected mice, the B21 vaccine significantly reduced bacterial loads in the spleens and lungs and decreased lung pathology. In conclusion, the B21 DNA vaccine can enhance T cell responses and control the reactivation of LTBI.

Preexisting antibodies targeting SARS-CoV-2 S2 cross-react with commensal gut bacteria and impact COVID-19 vaccine induced immunity.

The origins of preexisting SARS-CoV-2 cross-reactive antibodies and their potential impacts on vaccine efficacy have not been fully clarified. In this study, we demonstrated that S2 was the prevailing target of the preexisting S protein cross-reactive antibodies in both healthy human and SPF mice. A dominant antibody epitope was identified on the connector domain of S2 (1147-SFKEELDKYFKNHT-1160, P144), which could be recognized by preexisting antibodies in both human and mouse. Through metagenomic sequencing and fecal bacteria transplant, we demonstrated that the generation of S2 cross-reactive antibodies was associated with commensal gut bacteria. Furthermore, six P144 reactive monoclonal antibodies were isolated from naïve SPF mice and were proven to cross-react with commensal gut bacteria collected from both human and mouse. A variety of cross-reactive microbial proteins were identified using LC-MS, of which E. coli derived HSP60 and HSP70 proteins were confirmed to be able to bind to one of the isolated monoclonal antibodies. Mice with high levels of preexisting S2 cross-reactive antibodies mounted higher S protein specific binding antibodies, especially against S2, after being immunized with a SARS-CoV-2 S DNA vaccine. Similarly, we found that levels of preexisting S2 and P144-specific antibodies correlated positively with RBD binding antibody titers after two doses of inactivated SARS-CoV-2 vaccination in human. Collectively, our study revealed an alternative origin of preexisting S2-targeted antibodies and disclosed a previously neglected aspect of the impact of gut microbiota on host anti-SARS-CoV-2 immunity.

Immune Correlates of Disseminated BCG Infection in IL12RB1-Deficient Mice.

Interleukin-12 receptor ß1 (IL12RB1)-deficient individuals show increased susceptibilities to local or disseminated BCG infection and environmental mycobacteria infection. However, the low clinical penetrance of IL12RB1 deficiency and low recurrence rate of mycobacteria infection suggest that protective immunity still exists in this population. In this study, we investigated the mechanism of tuberculosis suppression using the IL12RB1-deficient mouse model. Our results manifested that Il12rb1-/- mice had significantly increased CFU counts in spleens and lungs, especially when BCG (Danish strain) was inoculated subcutaneously. The innate TNF-a and IFN-γ responses decreased, while the IL-17 responses increased significantly in the lungs of Il12rb1-/- mice. We also found that PPD-specific IFN-γ release was impaired in Il12rb1-/- mice, but the specific TNF-a release was not compromised, and the antibody responses were significantly enhanced. Moreover, correlation analyses revealed that both the innate and PPD-specific IFN-γ responses positively correlated with CFU counts, whereas the innate IL-12a levels negatively correlated with CFU counts in Il12rb1-/- mice lungs. Collectively, these findings proved that the adaptive immunities against mycobacteria are not completely nullified in Il12rb1-/- mice. Additionally, our results imply that IFN-γ responses alone might not be able to contain BCGitis in the setting of IL12RB1 deficiency.

Omicron Booster in Ancestral Strain Vaccinated Mice Augments Protective Immunities Against Both Delta and Omicron Variants.

A booster vaccination is called for constraining the evolving epidemic of SARS-CoV-2. However, the necessity of a new COVID-19 vaccine is currently unclear. To compare the effect of an Omicron-matched S DNA vaccine and an ancestral S DNA vaccine in boosting cross-reactive immunities, we firstly immunized mice with two-dose of a DNA vaccine encoding the spike protein of the ancestral Wuhan strain. Then the mice were boosted with DNA vaccines encoding spike proteins of either the Wuhan strain or the Omicron variant. Specific antibody and T cell responses were measured at 4 weeks post boost. Our data showed that the Omicron-matched vaccine efficiently boosted RBD binding antibody and neutralizing antibody responses against both the Delta and the Omicron variants. Of note, antibody responses against the Omicron variant elicited by the Omicron-matched vaccine were much stronger than those induced by the ancestral S DNA vaccine. Meanwhile, CD8+ T cell responses against both the ancestral Wuhan strain and the Omicron strain also tended to be higher in mice boosted by the Omicron-matched vaccine than those in mice boosted with the ancestral S DNA vaccine, albeit no significant difference was observed. Our findings suggest that an Omicron-matched vaccine is preferred for boosting cross-protective immunities.

Functional analysis and enzyme characterization of mannose-1-phosphate guanylyl transferase (ManB) from Mycobacterium tuberculosis.

Mycobacterium tuberculosis cell wall consist variety of mannose containing glycoconjugates including lipomannan (LM) and lipoarabinomannan (LAM). These lipoglycans are involved in cell wall integrity and play role in virulence of M. tuberculosis by modulating host immune response. GDP-mannose, required for the synthesis of lipoglycans, is catalyzed by enzyme Mannose-1-phosphate guanylyl transferase (ManB). The enzyme with similar function has been studied in variety of species of prokaryotes and eukaryotes. However, biological role of ManB and its enzymatic activity remains uncharacterized in M. tuberculosis. In present study, we elucidated the role of enzyme by constructing manB knockdown strain of M. tuberculosis H37Ra. The manB knockdown decreased the cell growth and also effected the morphology of M. tuberculosis by altering the permeability of cell membrane. These findings provide the understanding on ManB function and suggesting that ManB could be the potential target for novel anti-tuberculosis drug. Furthermore, we also characterized ManB enzyme by establishing 96 well plate colorimetric assay and determined the kinetic properties including initial velocity, optimum temperature, optimum pH and other kinetic parameters. Our established assay will be helpful for further high throughput screening of potential inhibitors against ManB.

Immunogenicity of an inactivated SARS-CoV-2 vaccine in people living with HIV-1: a non-randomized cohort study.

BACKGROUND: Inactivated COVID-19 vaccines are safe and effective in the general population with intact immunity. However, their safety and immunogenicity have not been demonstrated in people living with HIV (PLWH). METHODS: 42 HIV-1 infected individuals who were stable on combination antiretroviral therapy (cART) and 28 healthy individuals were enrolled in this open-label two-arm non-randomized study at Hubei Provincial Center for Disease Control and Prevention, China. Two doses of an inactivated COVID-19 vaccine (BBIBP-CorV) were given on April 22, 2021 and May 25, 2021, respectively. The reactogenicity of the vaccine were evaluated by observing clinical adverse events and solicited local and systemic reactions. Humoral responses were measured by anti-spike IgG ELISA and surrogate neutralization assays. Cell-mediated immune responses and vaccine induced T cell activation were measured by flow cytometry. FINDINGS: All the HIV-1 infected participants had a CD4+ T cell count >200 cells/µL both at baseline (659·0 ± 221·9 cells/µL) and 4 weeks after vaccination (476·9 ± 150·8 cells/µL). No solicited adverse reaction was observed among all participants. Similar binding antibody, neutralizing antibody and S protein specific T cell responses were elicited in PLWH and healthy individuals. PLWH with low baseline CD4+/CD8+ T cell ratios (<0·6) generated lower antibody responses after vaccination than PLWH with medium (0·6∼1·0) or high (≥1·0) baseline CD4+/CD8+ T cell ratios (P<0·01). The CD3+, CD4+ and CD8+ T cell counts of PLWH decreased significantly after vaccination (P<0·0001), but it did not lead to any adverse clinical manifestation. Moreover, we found that the general HIV-1 viral load among the PLWH cohort decreased significantly after vaccination (P=0·0192). The alteration of HIV-1 viral load was not significantly associated with the vaccine induced CD4+ T cell activation (P>0·2). INTERPRETATION: Our data demonstrated that the inactivated SARS-CoV-2 vaccine was safe, immunogenic in PLWH who are stable on cART with suppressed viral load and CD4+ T cell count > 200 cells/µL. However, the persistence of the vaccine-induced immunities in PLWH need to be further investigated.

Effect of Protein O-Mannosyltransferase (MSMEG_5447) on M. smegmatis and Its Survival in Macrophages.

Protein O-mannosyltransferase (PMT) catalyzes an initial step of protein O-mannosylation of Mycobacterium tuberculosis (Mtb) and plays a crucial role for Mtb survival in the host. To better understand the role of PMT in the host innate immune response during mycobacterial infection, in this study, we utilized Mycobacterium smegmatis pmt (MSMEG_5447) gene knockout strain, ΔM5447, to infect THP-1 cells. Our results revealed that the lack of MSMEG_5447 not only impaired the growth of M. smegmatis in 7H9 medium but also reduced the resistance of M. smegmatis against lysozyme and acidic stress in vitro. Macrophage infection assay showed that ΔM5447 displayed attenuated growth in macrophages at 24 h post-infection. The production of TNF-α and IL-6 and the activation of transcription factor NF-κB were decreased in ΔM5447-infected macrophages, which were further confirmed by transcriptomic analysis. Moreover, ΔM5447 failed to inhibit phagosome-lysosome fusion in macrophages. These findings revealed that PMT played a role in modulating the innate immune responses of the host, which broaden our understanding for functions of protein O-mannosylation in mycobacterium-host interaction.

Mycobacterium tuberculosis Rv1987 protein induces M2 polarization of macrophages through activating the PI3K/Akt1/mTOR signaling pathway.

Mycobacterium tuberculosis (Mtb) can subvert host immune responses and survive in macrophages. Specific Mtb antigens play a critical role in this process. Rv1987, a secretory protein encoded by the gene rv1987 in the region of difference-2 (RD2) of the Mtb genome, is specifically expressed in pathogenic mycobacteria. Our previous work proved that Rv1987 induced a Th2 response in mice and enhanced mycobacterial survival in mouse lungs, but its effect on macrophages, the most important effector immune cell involved in killing Mtb, remains unclear. In this study, we used an M. smegmatis strain overexpressing Rv1987 protein to infect alveolar macrophages and the macrophage cell line RAW264.7 and analyzed the effect of Rv1987 protein on macrophage polarization. Rv1987 induced M2 polarization in macrophages both in vivo and in vitro. The bactericidal ability of these M2 polarized macrophages decreased remarkably, which resulted in the increased survival of bacteria in macrophages. Proteomics, RT-qPCR and western blotting results revealed that the PI3K/Akt1/mTOR signaling pathway was activated in Rv1987-induced M2 macrophages. Meanwhile, the SHIP molecule, a negative regulator of the PI3K/Akt1/mTOR signaling pathway, was significantly downregulated. These results suggest that Rv1987 plays an important role in modulating the host immune response and could be established as a potential drug target.

Action of Dicumarol on Glucosamine-1-Phosphate Acetyltransferase of GlmU and Mycobacterium tuberculosis.

Mycobacterium tuberculosis is one of most pathogenic microorganisms in the world. Previously, the bifunctional enzyme GlmU with glucosamine-1-phosphate acetyltransferase activity and N-acetylglucosamine-1-phosphate uridyltransferase activity has been suggested as a potential drug target; therefore, discovering compounds targeting GlmU acetyltransferase is necessary. The natural products were tested for inhibition of GlmU acetyltransferase activity. We found that dicumarol exhibited inhibitory effects on GlmU acetyltransferase, with a concentration achieving a 50% inhibition (IC50) value of 4.608 µg/ml (13.7 µM). The inhibition kinetics indicated that dicumarol uncompetitively inhibited acetyl CoA and showed mixed-type inhibition for glucosamine-1-phosphate (GlcN-1-P). The activity of dicumarol against M. tuberculosis H37Ra was evaluated with a minimum inhibitory concentration (MIC) value of 6.25 µg/ml (18.55 µM) in the Alamar blue assay. Dicumarol also exhibited inhibitory effects on several clinically sensitive M. tuberculosis strains and drug-resistant strains, with a range of MIC value of 6.25 to >100 µg/ml. Dicumarol increased the sensitivity of anti-tuberculosis drugs (isoniazid and rifampicin) when dicumarol was present at a low concentration. The transcriptome and proteome data of M. tuberculosis H37Ra treated by dicumarol showed that the affected genes were associated with cell wall synthesis, DNA damage and repair, metabolic processes, and signal transduction. These results provided the mechanism of dicumarol inhibition against GlmU acetyltransferase and M. tuberculosis and also suggested that dicumarol is a potential candidate for TB treatment.

The Inhibitory Effect of GlmU Acetyltransferase Inhibitor TPSA on Mycobacterium tuberculosis May Be Affected Due to Its Methylation by Methyltransferase Rv0560c.

Mycobacterium tuberculosis bifunctional enzyme GlmU is a novel target for anti-TB drugs and is involved in glycosyl donor UDP-N-acetylglucosamine biosynthesis. Here, we found that TPSA (2-[5-(2-{[4-(2-thienyl)-2-pyrimidinyl]sulfanyl}acetyl)-2-thienyl]acetic acid) was a novel inhibitor for GlmU acetyltransferase activity (IC50: 5.3 µM). The interaction sites of GlmU and TPSA by molecular docking were confirmed by site-directed mutagenesis. TPSA showed an inhibitory effect on Mtb H37Ra growth and intracellular H37Ra in macrophage cells (MIC: 66.5 µM). To investigate why TPSA at a higher concentration (66.5 µM) was able to inhibit H37Ra growth, proteome and transcriptome of H37Ra treated with TPSA were analyzed. The expression of two methyltransferases MRA_0565 (Rv0558) and MRA_0567 (Rv0560c) were markedly increased. TPSA was pre-incubated with purified Rv0558 and Rv0560c in the presence of S-adenosylmethionine (methyl donor) respectively, resulting in its decreased inhibitory effect of GlmU on acetyltransferase activity. The inhibition of TPSA on growth of H37Ra with overexpressed Rv0558 and Rv0560c was reduced. These implied that methyltransferases could modify TPSA. The methylation of TPSA catalyzed by Rv0560c was subsequently confirmed by LC-MS. Therefore, TPSA as a GlmU acetyltransferase activity inhibitor may offer a structural basis for new anti-tuberculosis drugs. TPSA needs to be modified further by some groups to prevent its methylation by methyltransferases.

Characterization of serine acetyltransferase (CysE) from methicillin-resistant Staphylococcus aureus and inhibitory effect of two natural products on CysE.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major hospital-acquired infective pathogen that has developed resistance to many antibiotics. It is imperious to develop novel anti-MRSA drugs to control the emergence of drug resistance. The biosynthesis of cysteine in bacteria is catalyzed by CysE and CysK. CysE was predicted to be important for bacterial viability, it could be a potential drug target. The serine acetyltransferase activity of CysE was detected and its catalytic properties were also determined. CysE homology model was built to investigate interaction sites between CysE and substrate L-Ser or inhibitors by molecular docking. Docking data showed that residues Asp94 and His95 were essential for serine acetyltransferase activity of CysE, which were confirmed by site-directed mutagenesis. Colorimetric assay was used to screen natural products and six compounds which inhibited CysE activity (IC50 ranging from 29.83 µM to 203.13 µM) were found. Inhibition types of two compounds 4 (11-oxo-ebracteolatanolide B) and 30 ((4R,4aR)-dihydroxy-3-hydroxymethyl-7,7,10a-trimethyl-2,4,4a,5,6,6a,7,8,9,10,10a,l0b-dodecahydrophenanthro[3,2-b]furan-2-one) on CysE were determined. Compounds 4 and 30 showed inhibitory effect on MRSA growth (MIC at 12.5 µg/ml and 25 µg/ml) and mature biofilm. The established colorimetric assay will facilitate further high-throughput screening of CysE inhibitors from different compound libraries. The compounds 4 and 30 may offer structural basis for developing new anti-MRSA drugs.

An integrated course based on two dimensional-electrophoresis and mass spectrometry analysis to teach proteomic techniques for undergraduate students.

Here, we developed an integrated course based on two dimensional-electrophoresis and spectrometry mass (2DE-MS) technique for undergraduate students to help them learn proteomic techniques. The soluble proteins in wild type and gene knockout bacteria were separated by 2DE and the differently expressed proteins were identified by MS analysis. The proteomic data was finally confirmed by RT-PCR detection. The separated experiments of 2DE, MS, RNA isolation, RT-PCR, as well as essential bioinformatic analysis, were integrated into a one-week course, which provided students an opportunity to systematically understand the proteomic techniques and their applications in current scientific research. © 2018 by The International Union of Biochemistry and Molecular Biology, 46:354-360, 2018.

Alterations of the Gut Microbiome in Hypertension.

Introduction: Human gut microbiota is believed to be directly or indirectly involved in cardiovascular diseases and hypertension. However, the identification and functional status of the hypertension-related gut microbe(s) have not yet been surveyed in a comprehensive manner. Methods: Here we characterized the gut microbiome in hypertension status by comparing fecal samples of 60 patients with primary hypertension and 60 gender-, age-, and body weight-matched healthy controls based on whole-metagenome shotgun sequencing. Results: Hypertension implicated a remarkable gut dysbiosis with significant reduction in within-sample diversity and shift in microbial composition. Metagenome-wide association study (MGWAS) revealed 53,953 microbial genes that differ in distribution between the patients and healthy controls (false discovery rate, 0.05) and can be grouped into 68 clusters representing bacterial species. Opportunistic pathogenic taxa, such as, Klebsiella spp., Streptococcus spp., and Parabacteroides merdae were frequently distributed in hypertensive gut microbiome, whereas the short-chain fatty acid producer, such as, Roseburia spp. and Faecalibacterium prausnitzii, were higher in controls. The number of hypertension-associated species also showed stronger correlation to the severity of disease. Functionally, the hypertensive gut microbiome exhibited higher membrane transport, lipopolysaccharide biosynthesis and steroid degradation, while in controls the metabolism of amino acid, cofactors and vitamins was found to be higher. We further provided the microbial markers for disease discrimination and achieved an area under the receiver operator characteristic curve (AUC) of 0.78, demonstrating the potential of gut microbiota in prediction of hypertension. Conclusion: These findings represent specific alterations in microbial diversity, genes, species and functions of the hypertensive gut microbiome. Further studies on the causality relationship between hypertension and gut microbiota will offer new prospects for treating and preventing the hypertension and its associated diseases.