Medical Microbiology and Immunology最新文献

nKlebsiella pneumoniae is a common pathogen of healthcare-associated infections expressing a plethora of antimicrobial resistance loci, including ADP-ribosyltransferase coding genes (arr), able to mediate rifampicin resistance. The latter has activity against a broad range of microorganisms by inhibiting DNA-dependent RNA polymerases. This study aims to characterise the arr distribution and genetic context in 138 clinical isolates of K. pneumoniae and correlate these with rifampicin resistance. All isolates were subjected to whole-genome sequencing for species identification, typing and AMR genes identification, along with the determination of the minimum inhibitory concentration (MIC) of rifampicin. Molecular detection of arr genes and class 1 integrons was performed for rifampicin-resistant isolates. Efflux activity was investigated as a possible determinant of rifampicin resistance in isolates devoid of known genetic determinants. Twelve isolates exhibited high rifampicin MICs (≥ 64 mg/L), 124 showed intermediate MICs (16-32 mg/L) and two displayed low (8 mg/L) MICs. Two arr allelic variants, arr-2 and arr-3, were found across one and nine K. pneumoniae isolates, respectively, all within class 1 integrons, including a newly described integron, and all associated with high rifampicin MICs  (≥ 64 mg/L). Elevated resistance levels were additionally linked to increased arr-2/3 expression and closer proximity to the promoter. No arr gene or rpoB mutations were found across the remaining two isolates and no correlation between efflux activity and high-level rifampicin resistance was found for both isolates. In conclusion, this study demonstrates that arr genes confer high levels of rifampicin resistance in K. pneumoniae highlighting its widespread dissemination within class 1 integrons.

Toxoplasma gondii infects approximately 30% of the population, and there is currently no approved vaccine. Identifying immunogenic peptides with high affinity to different HLA molecules is a promising vaccine strategy. This study used an in silico approach using artificial neural networks to identify T. gondii peptides restricted to HLA-A*02, HLA-A*24, and HLA-B*35 alleles. Proteomes from seven T. gondii strains and transcriptomic data of overexpressed genes from T. gondii-RH in human PBMC were also used. Parasite protein sequences were analyzed with R 'Epitope Prediction' library. Peptide candidates were evaluated in the artificial neural networks based on the probabilities of output neurons (p > 0.5). The IFN-γ responses in PBMC from T. gondii seronegative and seropositive individuals were evaluated by ELISpot. Peptides with higher IFN-γ induction were evaluated to identify cytotoxic response in CD8⁺ T cells (CD107a). In silico analysis identified 36 peptides from T. gondii proteins with predicted affinity to HLA-A*02, A*24, and B*35 alleles. Experiments with PBMCs revealed that a peptide restricted to HLA-A02 (P1: FLFAWITYV) induced a significant increase in IFN-γ-producing cells (p = 0.004). For HLA-A24, a peptide (P8: VFAFAFAFFLI) also induced a significant IFN-γ response (p = 0.004), while for the HLA-B*35 allele, the P6 peptide (YPIAPSFAM) induced a response that differed significantly from the control (p = 0.05). These peptides induced also a significant percentage of central memory CD8 + T cells expressing the degranulation marker CD107a (p < 0.05). Finally, we identified three T. gondii peptides that induced IFN-γ response, and a cytotoxic response measured by CD107a expression on CD45RAneg-CD8 cells. These peptides could be considered part of a multi-epitope vaccine against toxoplasmosis in humans.

Introduction: While the general immune response to Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) is well-understood, the long-term effects of Human Immunodeficiency Virus-1/Severe Acute Respiratory Syndrome-Coronavirus-2 (HIV-1/SARS-CoV-2) co-infection on the immune system remain unclear. This study investigates the immune response in people with HIV-1 (PWH) co-infected with SARS-CoV-2 to understand its long-term health consequences.

Methods: A retrospective longitudinal study of PWH with suppressed viral load and SARS-CoV-2 infection was conducted. Cryopreserved peripheral blood mononuclear cells and plasma samples were collected at three time-points: HIV-1/pre-SARS-CoV-2 (n = 18), HIV-1/SARS-CoV-2 (n = 46), and HIV-1/post-SARS-CoV-2 (n = 36). Plasma levels of 25 soluble cytokines and chemokines, and anti-S/anti-N-IgG-SARS-CoV-2 antibodies were measured. Immunophenotyping of innate and adaptive immune components and HIV-1 and SARS-CoV-2-specific T/B-cell responses were assessed by flow cytometry.

Results: HIV-1/SARS-CoV-2 co-infection was associated with long-lasting immune dysfunction, characterized by elevated levels of pro-inflammatory cytokines and a decrease in the MIG-IP10-ITAC chemokine axis at the HIV/SARS-CoV-2 time-point, which persisted one year later. Additionally, alterations in the distribution of subsets and increased activation (NKG2D/NKG2C) and maturation (TIM3) markers of NK and dendritic cells were observed at the HIV-1/SARS-CoV-2 time-point, persisting throughout the study. Effector memory CD4 T-cell subsets were decreased, while exhaustion/senescence (PD1/TIM3/CD57) markers were elevated at all three time-points. SARS-CoV-2-specific T/B-cell responses remained stable throughout the study, while HIV-1-specific T-cell responses decreased at the HIV-1/SARS-CoV-2 time-point and remained so.

Conclusions: Persistent immune dysfunction in HIV-1/SARS-CoV-2 co-infection increases the risk of future complications, even in PWH with mild symptoms. Exacerbated inflammation and alterations in immune cells may contribute to reduce vaccine efficacy and potential reinfections.

Dengue virus (DENV) mediated disease severity leads to fatality among infected patients. Immune sentinels recognize DENV thereby secreting inflammatory mediators, endothelial biomarkers and anticoagulation factors. Absence of any diagnostic biomarkers for early identification of severe dengue (SD) patients has hindered disease management. Present study is aimed to evaluate diagnostic potential of these biomarkers along with their therapeutic targets for disease severity. Dengue infection was screened among 214 symptomatic patients and 25 healthy individuals by qRT-PCR, NS1-antigen, anti-dengue-IgM, anti-dengue-IgG ELISA and categorized them according to WHO-classification, 2009. Dengue viral-load and serotypes were determined by qRT-PCR. Serum-protein concentrations of inflammatory mediators (MIF, PAF, MMP2, MMP9, MCP1, RANTES, STNFRI, ST2, EOTAXIN), endothelial biomarkers (SDC1, VEGF, ANGPT2), anticoagulation factors (sTM, vWF, TF, PAI) were determined by sandwich ELISA. Statistical, PPI-network, hub-proteins, drug prediction analysis were performed by GraphPad-Prism⁹, STRING, Cytoscape-cytoHubba, DrugBank online, TTD, respectively. Among 81 dengue infected patients, significantly higher levels of MIF, PAF, sTNFRI, MMP9, VEGF, ANGPT2, MMP2, RANTES, SDC1 were detected among SD patients compared to non-severe ones, with excellent and good diagnostic potential of first (> 77.11, > 57.57 ng/ml, > 3226 pg/ml) and next three (> 105.3 ng/ml, > 12,380, > 8284 pg/ml) biomarkers, respectively. Serum MIF, PAF, MMP9, sTNFRI levels were significantly higher among hospitalized (p-value: 0.0081-0.0499), high-viral-load (p-value: 0.0266-0.0466) and DENV-2, 4 (p-value: < 0.0001-0.0298) infected patients. PPI-network analysed MMP9, PAI, vWF, ANGPT2, sTM, sTNFRI, MIF as hub-proteins targeted by FDA-approved/experimental drugs. This study recognized serum-biomarkers: MIF, PAF, sTNFRI, MMP9, VEGF, ANGPT2 to have significant diagnostic potential for identification of SD cases.

Mucosal infections normally cause an immune response including activation of antigen-specific B cells in regional mucosa-associated lymphoid tissue. After recirculation of plasmablasts, and maturation at mucosal surfaces or bone marrow, plasma cells produce secretory or systemic IgA. It remains uncertain to what extent secretory and systemic IgA share the same target specificities. For vaccine candidate optimization, it is important to know whether IgA targeting of glycosylated epitopes of a protein antigen vary between mucosal and systemic sites. We evaluated glycosylated epitope specificity of systemic and mucosally secreted IgA against YghJ, a potential vaccine candidate antigen secreted by most pathogenic Escherichia coli. IgA from intestinal lavage, saliva, serum, and blood-derived antibody in lymphocyte supernatants (ALS) were collected from 21 volunteers following experimental infection with enterotoxigenic E. coli. Methods for preparing IgA from saliva and ALS were developed, and multiplex bead flow cytometric immunoassays were used to determine levels of IgA targeting natively glycosylated YghJ and estimating what proportion of these antibodies specifically targeted glycosylated epitopes. Following infection, anti-YghJ IgA levels increased substantially for most volunteers across all four specimen types. Target specificity of ALS IgA correlated well with serum IgA, but not with mucosally secreted IgA. Furthermore, glycosylation-specific proportion of salivary IgA was higher than, and did not correlate with, intestinally secreted IgA. These results indicate a new degree of complexity to our understanding of epitope-targeting and tissue specificity of mucosal antibody responses. Our findings also suggest that all features of an intestinal IgA response may not be well reflected in serum, saliva, or ALS, which are commonly used proxy specimens for evaluating intestinal immune responses.

Marburg virus (MARV) is a zoonotic virus that can infect humans and non-human primates (NHPs) and lead to a fatal Marburg hemorrhagic fever (MHF), while there is no approved vaccine or antiviral treatment for MHF. The nucleic acid vaccine has unique advantages, including fast and simple preparation, easy to follow the virus mutation situation, and less adverse reactions. Therefore, we constructed the DNA and mRNA candidate vaccines based on codon-optimized MARV glycoprotein sequence, and evaluated the immune effect in mice through ELISA, ELISpot, and Flow cytometry. After the second booster immunization, both of the candidate vaccines induced strong humoral immune response, enhanced T cell response, and elicited neutralizing antibodies. Notably, DNA candidate vaccine induced stronger humoral immune response, while mRNA candidate vaccine elicited higher levels of IFN-γ and IL-4. In addition, transcriptome analysis revealed that the candidate vaccines activated immune response related pathways. Our study shed new light on the nucleic acid vaccines for MARV and further confirmed the potential of nucleic acid vaccine for future MHF prevention and control.

Hypervirulent Klebsiella pneumoniae (hvKp), an emerging Kp subtype, has become a serious global pathogen. However, the information regarding host interactions and innate immune responses during hvKp infection is limited. Here, we found that hvKp clinical strains increased triacylglycerol synthesis, resulting in lipid droplets (LDs) formation via the mammalian target of rapamycin signaling pathway in RAW264.7 cells. Treatment with rapamycin, an inhibitor of this pathway, affected LDs formation and antimicrobial responses against clinical hvKp infections. In accordance with the role of LDs in modulating inflammation, the pharmacological inhibition of lipogenesis reduced proinflammatory cytokine expression during hvKp infections. In addition, inhibition of LDs formation using pharmacological inhibitors and knockdown of lipogenesis regulators decreased the intracellular survival of hvKp in macrophages. Moreover, inhibiting LDs biogenesis reduced mortality, weight loss, and bacterial loads in hvKp-infected mice. Collectively, these data suggest that LDs biogenesis is crucial in linking host immune responses to clinical hvKp infections.

The recent outbreak of monkeypox virus (MPXV) has caused global concern. How the virus evades the interferon (IFN) response is still poorly understood. We analyzed type I/II IFN (IFN-I/II) expression in clinical samples from MPXV-infected patients and measured IFN-I kinetics in MPXV-infected cells. We also evaluated the anti-MPXV activity of IFN-I/II in A549, HeLa and Vero-E6 cell lines. IFN-I/II mRNA expression was detected in skin lesions, anal swabs, nasopharyngeal samples and peripheral blood mononuclear cells (PBMC), with the highest levels in skin lesions (p < 0.05). High MPXV DNA levels in clinical samples were associated with increased IFN-I levels. In vitro, MPXV infection induced a peak of IFN-I between 48 and 72 h post-infection (p < 0.01). Pre-treatment of the A549, HeLa and Vero-E6 cells with high concentrations (≥ 100,000 International Unit, IU/ml) of IFN-α and IFN-ω did not inhibit or had little effect on MPXV replication, while IFN-β moderately reduced MPXV replication by 2.7-1.5 log₁₀ at 100,000 IU/ml. In clinical samples there was a trend for elevated levels of IFN-γ in association with lower MPXV load and in vitro IFN-γ (3,600 IU/ml) strongly reduced viral titers by 3.4-1.6 log₁₀. There were no significant differences in expression of select IFN-stimulated genes (ISGs) in MPXV infection in vitro. This study shows that MPXV delays IFN-I induction and inhibits expression of selected ISGs in vitro and is associated with an IFN-I resistance phenotype in vivo. However, MPXV is less resistant to IFN-γ in vivo and is sensitive to IFN-γ treatment in vitro, suggesting a potential therapeutic role for IFN-γ.

The concern of COVID-19 persists due to the continuous emergence of variants and the potential spillover of animal coronaviruses. The broad-spectrum neutralizing antibodies play a pivotal role in the prevention and treatment of coronavirus (CoV) infections. Here, we constructed 18 bi-specific antibodies (bsAbs) using 9 antibodies isolated from COVID-19 convalescents and vaccinated individuals, designed as dual variable domain immunoglobulin (DVD-Ig). A bsAb 5-HI showed a high binding capability to the S1 subunit of spike and exhibited breadth and potency against pseudotyped SARS-CoV-2 variants of concerns (VOCs) and SARS-related-CoVs (SARSr-CoVs), with half maximal effective concentration (EC₅₀) of 0.028-3.444 nM and 50% inhibitory concentration (IC₅₀) of 0.008-0.800 nM. In addition, it retained neutralization potency against the peudotyped virus of recently prevalent JN.1 strain (IC₅₀, 12.74 nM). We found that the parental antibodies showed weak or no binding to the receptor binding domain (RBD) of the SARS-CoV, EG.5.1, and JN.1. However, the 5-HI maintained the binding with RBD and prevented the binding between hACE2 and RBD (IC₅₀ for the RBD of SARS-CoV, 1.067 nM; EG.5.1, 0.423 nM; JN.1, 0.223 nM). In neutralization assays with the authentic virus, we found that the 5-HI effectively neutralized Omicron variants XBB.1.5 (IC₅₀, 0.308 nM), EG.5.1 (IC₅₀, 0.129 nM), and JN.1 (IC₅₀, 13.692 nM), while its parental antibodies showed weakened or no neutralization. Therefore, the 5-HI represents a promising candidate for further development in the treatment and prevention of ongoing evolved SARS-CoV-2 VOCs and other SARSr-CoVs that potentially emerge in the future.

Giardia duodenalis, an important zoonotic protozoan parasite, adheres to host intestinal epithelial cells (IECs) via the ventral disc and causes giardiasis characterized mainly by diarrhea. To date, it remains elusive how excretory-secretory products (ESPs) of Giardia enter IECs and how the cells respond to the entry. Herein, we initially demonstrated that ESPs evoked IEC endocytosis in vitro. We indicated that ESPs contributed vitally in triggering intrinsic apoptosis, pro-inflammatory responses, tight junction (TJ) protein expressional changes, and autophagy in IECs. Endocytosis was further proven to be implicated in those ESPs-triggered IEC responses. Ten predicted virulent excretory-secretory proteins of G. duodenalis were investigated for their capability to activate clathrin/caveolin-mediated endocytosis (CME/CavME) in IECs. Pyridoxamine 5'-phosphate oxidase (PNPO) was confirmed to be an important contributor. PNPO was subsequently verified as a vital promoter in the induction of giardiasis-related IEC apoptosis, inflammation, and TJ protein downregulation. Most importantly, this process seemed to be involved majorly in PNPO-evoked CME pathway, rather than CavME. Collectively, this study identified Giardia ESPs, notably PNPO, as potentially important pathogenic factors during noninvasive infection. It was also noteworthy that ESPs-evoked endocytosis might play a role in triggering giardiasis-inducing cellular regulation. These findings would deepen our understanding about the role of ESPs, notably PNPO, in the pathogenesis of giardiasis and the potential attributed endocytosis mechanism.