Recombinant outer membrane vesicles carrying Chlamydia muridarum HtrA induce antibodies that neutralize chlamydial infection in vitro

Background: Outer membrane vesicles (OMVs) are spheroid particles released by all Gram-negative bacteria as a result of the budding out of the outer membrane. Since they carry many of the bacterial surface-associated proteins and feature a potent built-in adjuvanticity, OMVs are being utilized as vaccines, some of which commercially available. Recently, methods for manipulating the protein content of OMVs have been proposed, thus making OMVs a promising platform for recombinant, multivalent vaccines development. Methods: Chlamydia muridarum DO serine protease HtrA, an antigen which stimulates strong humoral and cellular responses in mice and humans, was expressed in Escherichia coli fused to the OmpA leader sequence to deliver it to the OMV compartment. Purified OMVs carrying HtrA (CM rHtrA-OMV) were analyzed for their capacity to induce antibodies capable of neutralizing Chlamydia infection of LLC-MK2 cells in vitro. Results: CM rHtrA-OMV immunization in mice induced antibodies that neutralize Chlamydial invasion as judged by an in vitro infectivity assay. This was remarkably different from what observed with an enzymatically functional recombinant HtrA expressed in, and purified from the E. coli cytoplasm (CM rHtrA). The difference in functionality between anti-CM rHtrA and anti-CM rHtrA-OMV antibodies was associated to a different pattern of protein epitopes recognition. The epitope recognition profile of anti-CM HtrA-OMV antibodies was similar to that induced in mice during Chlamydial infection. Conclusions: When expressed in OMVs HtrA appears to assume a conformation similar to the native one and this results in the elicitation of functional immune responses. These data further support the potentiality of OMVs as vaccine platform

Human immunoglobulins are transported to HCMV viral envelope by viral Fc gamma receptors-dependent and independent mechanisms

Human cytomegaloviruses (HCMVs) employ many different mechanisms to escape and subvert the host immune system, including expression of the viral IgG Fcγ receptors (vFcγRs) RL11 (gp34), RL12 (gp95), RL13 (gpRL13), and UL119 (gp68) gene products. The role of vFcγRs in HCMV pathogenesis has been reported to operate in infected cells by interfering with IgG-mediated effector functions. We found that gp34 and gp68 are envelope proteins that bind and internalize human IgGs on the surface of infected cells. Internalized IgGs are then transported on the envelope of viral particles in a vFcR-dependent mechanism. This mechanism is also responsible for the incorporation on the virions of the anti-gH neutralizing antibody MSL-109. Intriguingly, we show that gp68 is responsible for MSL-109 incorporation, but it is dispensable for other anti-HCMV antibodies that do not need this function to be transported on mature virions. HCMV-infected cells grown in presence of anti-HCMV monoclonal antibodies generate a viral progeny still infective and possible to be neutralized. This is the first example of a virus carrying neutralizing IgGs on its surface and their possible role is discussed

A deoxyribonucleotidase in mitochondria: Involvement in regulation of dNTP pools and possible link to genetic disease

Three cytosolic and one plasma membrane-bound 5′-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5′-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5′- and 2′(3′)-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith–Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease

Human immunoglobulins are transported to HCMV viral envelope by viral Fc gamma receptors-dependent and independent mechanisms

: Human cytomegaloviruses (HCMVs) employ many different mechanisms to escape and subvert the host immune system, including expression of the viral IgG Fcγ receptors (vFcγRs) RL11 (gp34), RL12 (gp95), RL13 (gpRL13), and UL119 (gp68) gene products. The role of vFcγRs in HCMV pathogenesis has been reported to operate in infected cells by interfering with IgG-mediated effector functions. We found that gp34 and gp68 are envelope proteins that bind and internalize human IgGs on the surface of infected cells. Internalized IgGs are then transported on the envelope of viral particles in a vFcR-dependent mechanism. This mechanism is also responsible for the incorporation on the virions of the anti-gH neutralizing antibody MSL-109. Intriguingly, we show that gp68 is responsible for MSL-109 incorporation, but it is dispensable for other anti-HCMV antibodies that do not need this function to be transported on mature virions. HCMV-infected cells grown in presence of anti-HCMV monoclonal antibodies generate a viral progeny still infective and possible to be neutralized. This is the first example of a virus carrying neutralizing IgGs on its surface and their possible role is discussed

Shigella impairs T lymphocyte dynamics in vivo

International audienceThe Gram-negative enteroinvasive bacterium Shigella flexneri is responsible for the endemic form of bacillary dysentery, an acute rectocolitis in humans. S. flexneri uses a type III secretion system to inject effector proteins into host cells, thus diverting cellular functions to its own benefit. Protective immunity to reinfection requires several rounds of infection to be elicited and is short-lasting, suggesting that S. flexneri interferes with the priming of specific immunity. Considering the key role played by T-lymphocyte trafficking in priming of adaptive immunity, we investigated the impact of S. flexneri on T-cell dynamics in vivo. By using two-photon microscopy to visualize bacterium-T-cell cross-talks in the lymph nodes, where the adaptive immunity is initiated, we provide evidence that S. flexneri, via its type III secretion system, impairs the migration pattern of CD4(+) T cells independently of cognate recognition of bacterial antigens. We show that bacterial invasion of CD4(+) T lymphocytes occurs in vivo, and results in cell migration arrest. In the absence of invasion, CD4(+) T-cell migration parameters are also dramatically altered. Signals resulting from S. flexneri interactions with subcapsular sinus macrophages and dendritic cells, and recruitment of polymorphonuclear cells are likely to contribute to this phenomenon. These findings indicate that S. flexneri targets T lymphocytes in vivo and highlight the role of type III effector secretion in modulating host adaptive immune responses

The Shigella flexneri Type Three Secretion System Effector IpgD Inhibits T Cell Migration by Manipulating Host Phosphoinositide Metabolism.

International audienceShigella, the Gram-negative enteroinvasive bacterium that causes shigellosis, relies on its type III secretion system (TTSS) and injected effectors to modulate host cell functions. However, consequences of the interaction between Shigella and lymphocytes have not been investigated. We show that Shigella invades activated human CD4(+) T lymphocytes. Invasion requires a functional TTSS and results in inhibition of chemokine-induced T cell migration, an effect mediated by the TTSS effector IpgD, a phosphoinositide 4-phosphatase. Remarkably, IpgD injection into bystander T cells can occur in the absence of cell invasion. Upon IpgD-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)), the pool of PIP(2) at the plasma membrane is reduced, leading to dephosphorylation of the ERM proteins and their inability to relocalize at one T cell pole upon chemokine stimulus, likely affecting the formation of the polarized edge required for cell migration. These results reveal a bacterial TTSS effector-mediated strategy to impair T cell function

Moraxella catarrhalis evades neutrophil oxidative stress responses providing a safer niche for nontypeable Haemophilus influenzae

Moraxella catarrhalis and nontypeable Haemophilus influenzae (NTHi) are pathogenic bacteria frequently associated with exacerbation of chronic obstructive pulmonary disease (COPD), whose hallmark is inflammatory oxidative stress. Neutrophils produce reactive oxygen species (ROS) which can boost antimicrobial response by promoting neutrophil extracellular traps (NET) and autophagy. Here, we showed that M. catarrhalis induces less ROS and NET production in differentiated HL-60 cells compared to NTHi. It is also able to actively interfere with these responses in chemically activated cells in a phagocytosis and opsonin-independent and contact-dependent manner, possibly by engaging host immunosuppressive receptors. M. catarrhalis subverts the autophagic pathway of the phagocytic cells and survives intracellularly. It also promotes the survival of NTHi which is otherwise susceptible to the host antimicrobial arsenal. In-depth understanding of the immune evasion strategies exploited by these two human pathogens could suggest medical interventions to tackle COPD and potentially other diseases in which they co-exist

The respiratory syncytial virus (RSV) prefusion F‐protein functional antibody repertoire in adult healthy donors

Abstract Respiratory syncytial virus (RSV) is the leading cause of death from lower respiratory tract infection in infants and children, and is responsible for considerable morbidity and mortality in older adults. Vaccines for pregnant women and elderly which are in phase III clinical studies target people with pre‐existing natural immunity against RSV. To investigate the background immunity which will be impacted by vaccination, we single cell‐sorted human memory B cells and dissected functional and genetic features of neutralizing antibodies (nAbs) induced by natural infection. Most nAbs recognized both the prefusion and postfusion conformations of the RSV F‐protein (cross‐binders) while a smaller fraction bound exclusively to the prefusion conformation. Cross‐binder nAbs used a wide array of gene rearrangements, while preF‐binder nAbs derived mostly from the expansion of B‐cell clonotypes from the IGHV1 germline. This latter class of nAbs recognizes an epitope located between Site Ø, Site II, and Site V on the F‐protein, identifying an important site of pathogen vulnerability