Autodisplay of an avidin with biotin-binding activity on the surface of Escherichia coli.

OBJECTIVES: To display a recombinant avidin fused to the autotransporter ShdA to bind biotinylated molecules on the surface of Escherichia coli. RESULTS: Two chimeric protein constructs containing avidin fused to the autotransporter ShdA were expressed on the surface of Escherichia coli DH5α. One fusion protein contained 476 amino acids of the ShdA α and ß domains, whereas the second consisted of a 314 amino acid from α and truncated ß domains. Protein production was verified by SDS-PAGE using an antibody to the molecular FLAG-tag. The surface display of the avidin-shdA fusion protein was confirmed by confocal microscopy and flow cytometry analysis, and the biotin-binding activity was evaluated by fluorescence microscopy and flow cytometry using biotin-4-fluorescein and biotinylated-ovalbumin (OVA). CONCLUSIONS: Expression of a recombinant avidin with biotin-binding activity on the surface of E. coli was achieved using the autotransporter ShdA. This system is an alternative to bind biotinylated molecules to E. coli.

The percentage of CD39+ monocytes is higher in pregnant COVID-19+ patients than in nonpregnant COVID-19+ patients.

Current medical guidelines consider pregnant women with COVID-19 to be a high-risk group. Since physiological gestation downregulates the immunological response to maintain "maternal-fetal tolerance", SARS-CoV-2 infection may constitute a potentially threatening condition to both the mother and the fetus. To establish the immune profile in pregnant COVID-19+ patients, a cross-sectional study was conducted. Pregnant women with COVID-19 (P-COVID-19+; n = 15) were analyzed and compared with nonpregnant women with COVID-19 (NP-COVID-19+; n = 15) or those with physiological pregnancy (P-COVID-19-; n = 13). Serological cytokine and chemokine concentrations, leucocyte immunophenotypes, and mononuclear leucocyte responses to polyclonal stimuli were analyzed in all groups. Higher concentrations of serological TNF-α, IL-6, MIP1b and IL-4 were observed within the P-COVID-19+ group, while cytokines and chemokines secreted by peripheral leucocytes in response to LPS, IL-6 or PMA-ionomicin were similar among the groups. Immunophenotype analysis showed a lower percentage of HLA-DR+ monocytes in P-COVID-19+ than in P-COVID-19- and a higher percentage of CD39+ monocytes in P-COVID-19+ than in NP-COVID-19+. After whole blood polyclonal stimulation, similar percentages of T cells and TNF+ monocytes between groups were observed. Our results suggest that P-COVID-19+ elicits a strong inflammatory response similar to NP-COVID19+ but also displays an anti-inflammatory response that controls the ATP/adenosine balance and prevents hyperinflammatory damage in COVID-19.

Secondary rearrangements and hypermutation generate sufficient B cell diversity to mount protective antiviral immunoglobulin responses.

Variable (V) region gene replacement was recently implicated in B cell repertoire diversification, but the contribution of this mechanism to antibody responses is still unknown. To investigate the role of V gene replacements in the generation of antigen-specific antibodies, we analyzed antiviral immunoglobulin responses of "quasimonoclonal" (QM) mice. The B cells of QM mice are genetically committed to exclusively express the anti-(4-hydroxy-3-nitrophenyl) acetyl specificity. However, approximately 20% of the peripheral B cells of QM mice undergo secondary rearrangements and thereby potentially acquire new specificities. QM mice infected with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus, or poliovirus mounted virus-specific neutralizing antibody responses. In general, kinetics of the antiviral immunoglobulin responses were delayed in QM mice; however, titers similar to control animals were eventually produced that were sufficient to protect against VSV-induced lethal disease. VSV neutralizing single-chain Fv fragments isolated from phage display libraries constructed from QM mice showed VH gene replacements and extensive hypermutation. Thus, our data demonstrate that secondary rearrangements and hypermutation can generate sufficient B cell diversity in QM mice to mount protective antiviral antibody responses, suggesting that these mechanisms might also contribute to the diversification of the B cell repertoire of normal mice.

Lipopopeptidephosphoglycan from Entamoeba histolytica activates human macrophages and dendritic cells and reaches their late endosomes.

Lipopopeptidephosphoglycan (LPPG) is a complex macromolecule from the surface of Entamoeba histolytica trophozoites. We analysed the interaction between LPPG and human macrophages and dendritic cells (DCs) and found that LPPG is internalized by these cells and activates them. The internalization process involves intracellular traffic from the cell membrane to late endosomes, as shown by co-localization of LPPG with late endosomes marked with FITC-dextran and LAMP-1. LPPG-activated DCs have increased expression of co-stimulatory molecules CD80, CD86 and CD40 and produce pro-inflammatory cytokines TNF-alpha, IL-8 and IL-12. Taken together, these results show that LPPG activates antigen-presenting cells and reaches intracellular compartments that are involved in antigen presentation.

Triggering receptor expressed on myeloid cells (TREM-1) is regulated post-transcriptionally and its ligand is present in the sera of some septic patients.

Inflammation is necessary for survival, but it is also an important cause of human morbidity and mortality, as exemplified by sepsis. During inflammation, cells of the innate immune system are recruited and activated in response to infection, trauma or injury. These cells are activated through receptors, such as Toll-like receptors (TLRs), which recognize microbial ligands such as lipopolysaccharide (LPS). Triggering receptor expressed on myeloid cells (TREM)-1 amplifies the inflammatory response initiated by TLRs, and its expression on the surface of monocytes increases in the presence of TLR ligands. Here we have shown that in monocytes TREM-1 mRNA levels, measured by reverse transcription-polymerase chain reaction (RT-PCR), remained unchanged and TREM-1 protein levels, measured by flow cytometry, increased, indicating that LPS increases TREM-1 expression by a post-transcriptional mechanism. We also showed that TREM-1/Fc fusion protein decreased the ability of the sera of some patients with sepsis to activate monocytes, indicating that the TREM-1 ligand, whose identity is unknown, may be present in the sera of some of these patients. We describe a mechanism for the regulation of TREM-1 expression on monocytes and the possible presence of its ligand in serum; these findings help to explain the contribution of TREM-1 during systemic inflammation.

The innate immune response to Entamoeba histolytica lipopeptidophosphoglycan is mediated by toll-like receptors 2 and 4.

Entamoeba histolytica is a human pathogen that may invade the intestinal mucosa, causing amoebic colitis or hepatic abscesses when the trophozoites travel through the portal circulation to the liver. Lipopeptidophosphoglycan (LPPG) is a molecular pattern of E. histolytica recognized by the human immune system. Here we report that LPPG is exposed on the cell surface of E. histolytica trophozoites, and is recognized by the host through toll-like receptor (TLR) 2 and TLR4. Correspondingly, human embryonic kidney (HEK)-293 cells were rendered LPPG responsive through overexpression of TLR2 or TLR4/MD2. Moreover, co-expression of CD14 enhanced LPPG signal transmission through TLR2 and TLR4. The interaction of LPPG with TLR2 and TLR4 resulted in activation of NF-kappaB and release of interleukin (IL)-10, IL-12p40, tumour necrosis factor (TNF)-alpha, and IL-8 from human monocytes. Consistent with these findings, responsiveness of mouse macrophages lacking TLR2 expression (TLR2-/-) or functional TLR4 (TLR4d/d) to E. histolytica LPPG challenge was impaired while double deficient macrophages were unresponsive. In contrast to wild-type control and TLR2-/- animals succumbing to lethal shock syndrome, TLR4d/d mice were resistant to systemic LPPG challenge-induced pathology.

Antiviral protection after DNA vaccination is short lived and not enhanced by CpG DNA.

In this study, we investigated the potential of a DNA vaccine expressing the minimal cytotoxic T lymphocyte (CTL) epitope gp33 of the lymphocytic choriomeningitis virus glycoprotein to protect against infection of a non-lymphoid organ and compared this to protection against a systemic infection. Furthermore, since immune stimulatory sequences have been shown to augment CTL responses, we examined the capacity of CpG DNA to enhance CTL memory. The data show that DNA vaccination with a gp33-based gene construct induced short-lived gp33-specific CTL which protected against a systemic infection but not against a peripheral infection. Immune stimulatory sequences were incapable of either prolonging CTL memory or promoting protection against infection of a peripheral organ.

Virus neutralization by germ-line vs. hypermutated antibodies.

Mice infected with vesicular stomatitis virus (VSV), a cytopathic virus closely related to rabies virus, mount a virus-neutralizing antibody response protecting against lethal disease. VSVneutralizing monoclonal IgGs isolated from primary immune responses were devoid of somatic mutations, whereas most secondary and all hyperimmune response IgGs tested were hypermutated. A comparative analysis of recombinant single-chain antibody fragments (scFv-Ckappa) revealed that even the germ-line precursor of one hypermutated antibody bound and neutralized VSV. Four somatic amino acid substitutions in V(H) increased by 300-fold the binding strength of monovalent scFv-Ckappa. The multivalent binding avidity of germ-line scFv-Ckappa was increased by more than 10-fold compared with the monovalent binding strength. In contrast, hypermutated scFv-Ckappa did not show such avidity effects. Thus the overall binding difference between the germ-line and the hypermutated VSV-neutralizing antibody was only 10- to 15-fold. This may explain why primary germ-line antibodies and secondary hypermutated antibodies directed against pathogens such as viruses and bacteria expressing repetitive antibody determinants show rather similar binding qualities, whereas monovalently binding hapten-specific antibodies can show "affinity maturation" effects of up to 1000-fold.

Induction of optimal anti-viral neutralizing B cell responses by dendritic cells requires transport and release of virus particles in secondary lymphoid organs.

Dendritic cells (DC) are sentinels of the immune system, transporting antigens from the periphery to secondary lymphoid organs. This study investigates the interactions of DC with B cells for the induction of anti-viral neutralizing antibody responses. Using the vesicular stomatitis virus glycoprotein (VSV-G) as a model antigen, we show that DC contain infection with cytopathic VSV in the presence of a functional IFN system, facilitating transport and release of low levels of live virus in secondary lymphoid organs. DC exposed to live virus induced efficient neutralizing anti-viral B cell responses. In contrast, DC transporting UV-inactivated viral antigens were poor activators of anti-viral B cells, although they were capable of very efficiently inducing virus-specific Th cells. Transgenic DC expressing a membrane-bound form of VSV-G induced neutralizing B cell responses; however, this DC-induced, Th-dependent B cell response was significantly slower than the anti-viral B cell response induced by DC infected with live VSV, and was strongly dependent on concomitant priming of T help. These results suggest that DC may play a double role during infection with cytopathic virus: they transport and release live virus in secondary lymphoid tissues for optimal direct B cell induction and offer MHC class II-associated determinants for induction of T help.

Antiviral B cell memory in the absence of mature follicular dendritic cell networks and classical germinal centers in TNFR1-/- mice.

TNFR1-/- mice have been shown to lack networks of mature follicular dendritic cells (FDCs) and they do not form germinal centers. With nonreplicating Ags, IgG titers were inefficiently induced and not maintained. In this study, the neutralizing Ab response and the establishment of B cell memory in TNFR1-/- mice after infection with vesicular stomatitis virus (VSV) were analyzed histologically and functionally. Immunization with VSV-derived protein Ags without adjuvant induced only IgM but no IgG Abs in TNFR1-/- mice, whereas VSV glycoprotein emulsified in CFA or IFA induced IgM and IgG responses that were short-lived and of moderate titer. However, infection with live VSV induced excellent neutralizing IgM and IgG responses in TNFR1-/- mice, and adoptively transferable B cell memory was generated and persisted for more than 300 days. In contrast, IgG levels and Ab-forming cells in the bone marrow declined within 300 days by 90-95% compared with controls. These findings suggest that 1) increased Ag dose and time of Ag availability can substitute for FDC-stored Ab-complexed Ag in the induction of efficient IgG responses in TNFR1-/- mice devoid of classical germinal centers; 2) the induction and maintenance of adoptively transferable B cell memory can occur in the absence of Ag bound to mature FDCs; and 3) the long-term maintenance of elevated IgG titers is largely dependent on FDC-associated persisting Ag. However, about 5-10% of the Ab production remained in the absence of detectable persisting Ag in TNFR1-/- mice, probably either due to immature FDCs being partially functional and/or due to long-lived plasma cells.

Correlation of anti-viral B cell responses and splenic morphology with expression of B cell-specific molecules.

This study attempted to evaluate and compare the role of various B cell-specific markers for anti-viral immune responses in mouse strains lacking molecules belonging to the B cell receptor (BCR) complex (IgM, Ig alpha and C(kappa)), the co-stimulatory molecules (CD19 and CD22), the protein kinases [Bruton's tyrosine kinase (Btk)] or the transcription factors (OBF-1). These mice were tested in two model infections [vesicular stomatitis virus (VSV) and lymphocytic choriomeningitis virus (LCMV)] using T cell-independent (TI) or T cell-dependent (TD) antigens. All mice controlled an LCMV infection indicating that cytotoxic T cell functions were within normal ranges. In contrast, OBF-1(-/-) mice were partially protected and mb-1(delta c/delta c) mice not at all protected against VSV infection, a virus that is controlled virtually exclusively by neutralizing antibodies. Susceptibility to VSV infection was correlated with structural defects in the spleen: absence of mature B cells and follicles with marginal zone macrophages and absence of germinal centers with follicular dendritic cells correlated with lack or substantial reduction of protective IgM and IgG responses respectively. The lack of kappa light chain did not affect the neutralizing response, indicating that it could easily be replaced by the lambda chain. Absence of the co-stimulatory molecules CD19 and CD22 or of the signaling molecule Btk had modulating effects, but did not increase susceptibility to VSV or LCMV. Our findings suggest that there are crucial molecules for B cell activation at the beginning (BCR complex) and the end (transcription) of the signaling cascade, whereas fine-tuning factors modulating the response in between exhibit considerable functional overlap.