Combination immunotherapy with two attenuated Listeria strains carrying shuffled HPV-16 E6E7 protein causes tumor regression in a mouse tumor model.

Cervical cancer continues to impose a heavy burden worldwide, and human papilloma virus (HPV) infection, especially persistent infection with type 16 (HPV-16), is known to be the primary etiological factor. Therapeutic vaccines are urgently needed because prophylactic vaccines are ineffective at clearing pre-existing HPV infection. Here, two recombinant Listeria strains (LMΔ-E6E7 & LIΔ-E6E7) with deletions of the actA and plcB genes, expressing the shuffled HPV-16 E6E7 protein were constructed. The strains were delivered into the spleen and liver by intravenous inoculation, induced antigen-specific cellular immunity and were eliminated completely from the internal organs several days later. Intravenously treating with single strain for three times, or with both strains alternately for three times significantly reduced the tumor size and prolonged the survival time of model mice. Combination immunotherapy with two strains seemed more effective than immunotherapy with single strain in that it enhanced the survival of the mice, and the LMΔ-E6E7-prime-LIΔ-E6E7-boost strategy showed significant stronger efficacy than single treatment with the LIΔ-E6E7 strain. The antitumor effect of this treatment might due to its ability to increase the proportion of CD8+ T cells and reduce the proportion of T regulatory cells (Tregs) in the intratumoral milieu. This is the first report regarding Listeria ivanovii-based therapeutic vaccine candidate against cervical cancer. Most importantly we are the first to confirm that combination therapy with two different recombinant Listeria strains has a more satisfactory antitumor effect than administration of a single strain. Thus, we propose a novel prime-boost treatment strategy.

PRAK Promotes the Pathogen Clearance by Macrophage Through Regulating Autophagy and Inflammasome Activation.

The p38 regulated/activated protein kinase (PRAK) is a protein kinase downstream of p38MAPK. The present study investigated its function in the macrophage. Myeloid-specific deletion of Prak resulted in a significant reduction in F4/80+CD11b+ peritoneal macrophages with decreased expression of MHC-II and CD80. Upon infection with Listeria monocytogenes, Prak-deficient mice demonstrated an increased mortality, which was accompanied by a higher bacterial load in multiple tissues and elevated levels of proinflammatory cytokines in the serum. While the Prak-deficient macrophage showed similar potency in phagocytosis assays, its bactericidal activity was severely impaired. Moreover, Prak deficiency was associated with defects in ROS production, inflammasome activation as well as autophagy induction. Therefore, PRAK critically contributes to the clearance of intracellular pathogens by affecting multiple aspects of the macrophage function.

A Comparison Between Recombinant Listeria GAPDH Proteins and GAPDH Encoding mRNA Conjugated to Lipids as Cross-Reactive Vaccines for Listeria, Mycobacterium, and Streptococcus.

Cross-reactive vaccines recognize common molecular patterns in pathogens and are able to confer broad spectrum protection against different infections. Antigens common to pathogenic bacteria that induce broad immune responses, such as the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of the genera Listeria, Mycobacterium, or Streptococcus, whose sequences present more than 95% homology at the N-terminal GAPDH1-22 peptide, are putative candidates for universal vaccines. Here, we explore vaccine formulations based on dendritic cells (DC) loaded with two molecular forms of Listeria monocytogenes GAPDH (LM-GAPDH), such as mRNA carriers or recombinant proteins, and compare them with the same molecular forms of three other antigens used in experimental vaccines, listeriolysin O of Listeria monocytogeness, Ag85A of Mycobacterium marinum, and pneumolysin of Streptococcus pneumoniae. DC loaded with LM-GAPDH recombinant proteins proved to be the safest and most immunogenic vaccine vectors, followed by mRNA encoding LM-GAPDH conjugated to lipid carriers. In addition, macrophages lacked sufficient safety as vaccines for all LM-GAPDH molecular forms. The ability of DC loaded with LM-GAPDH recombinant proteins to induce non-specific DC activation explains their adjuvant potency and their capacity to trigger strong CD4+ and CD8+ T cell responses explains their high immunogenicity. Moreover, their capacity to confer protection in vaccinated mice against challenges with L. monocytogenes, M. marinum, or S. pneumoniae validated their efficiency as cross-reactive vaccines. Cross-protection appears to involve the induction of high percentages of GAPDH1-22 specific CD4+ and CD8+ T cells stained for intracellular IFN-γ, and significant levels of peptide-specific antibodies in vaccinated mice. We concluded that DC vaccines loaded with L. monocytogenes GAPDH recombinant proteins are cross-reactive vaccines that seem to be valuable tools in adult vaccination against Listeria, Mycobacterium, and Streptococcus taxonomic groups.

Combination Adjuvants Affect the Magnitude of Effector-Like Memory CD8 T Cells and Protection against Listeriosis.

The development of T cell-based subunit protein vaccines against diseases such as tuberculosis and malaria remains a challenge for immunologists. Here, we have identified a nanoemulsion adjuvant, Adjuplex (ADJ), which enhanced dendritic cell (DC) cross-presentation and elicited effective memory T cell-based immunity to Listeria monocytogenes. We further evaluated whether cross-presentation induced by ADJ can be combined with the immunomodulatory effects of Toll-like receptor (TLR) agonists (CpG or glucopyranosyl lipid adjuvant [GLA]) to evoke systemic CD8 T cell-based immunity to L. monocytogenes. Mechanistically, vaccination with ADJ, alone or in combination with CpG or GLA, augmented activation and antigen uptake by CD103+ migratory and CD8α+ resident DCs and upregulated CD69 expression on B and T lymphocytes in vaccine-draining lymph nodes. By engaging basic leucine zipper ATF-like transcription factor 3-dependent cross-presenting DCs, ADJ potently elicited effector CD8 T cells that differentiated into granzyme B-expressing CD27LO effector-like memory CD8 T cells, which provided effective immunity to L. monocytogenes in the spleen and liver. CpG or GLA alone did not elicit effector-like memory CD8 T cells and induced moderate protection in the spleen but not in the liver. Surprisingly, combining CpG or GLA with ADJ reduced the number of ADJ-induced memory CD8 T cells and compromised protective immunity to L. monocytogenes, especially in the liver. Taken together, the data presented in this study provide a glimpse of protective CD8 T cell memory differentiation induced by a nanoemulsion adjuvant and demonstrate the unexpected negative effects of TLR signaling on the magnitude of CD8 T cell memory and protective immunity to L. monocytogenes, a model intracellular pathogen.

Heterologous Boosting With Listeria-Based Recombinant Strains in BCG-Primed Mice Improved Protection Against Pulmonary Mycobacterial Infection.

While Baccillus Calmette-Guerin (BCG) is used worldwide, tuberculosis (TB) is still a global concern due to the poor efficacy of BCG. Novel vaccine candidates are therefore urgently required. In this study, two attenuated recombinant Listeria strains, LMΔ-msv and LIΔ-msv were constructed by deletion of actA and plcB and expression of a fusion protein consisting of T cell epitopes from four Mycobacterium tuberculosis (Mtb) antigens (Rv2460c, Rv2660c, Rv3875, and Rv3804c). The safety and immunogenicity of the two recombinant strains were evaluated in C57BL/6J mice. After intravenous immunization individually, both recombinant strains entered liver and spleen but eventually were eliminated from these organs after several days. Simultaneously, they induced antigen-specific cell-mediated immunity, indicating that the recombinant Listeria strains were immunogenic and safe in vivo. LMΔ-msv immunization induced stronger cellular immune responses than LIΔ-msv immunization, and when boosted with LIΔ-msv, antigen-specific IFN-γ CD8+ T cell responses were notably magnified. Furthermore, we evaluated the protection conferred by the vaccine candidates against mycobacterial infection via challenging the mice with 1 × 107 CFU of BCG. Especially, we tested the feasibility of application of them as heterologous BCG supplement vaccine by immunization of mice with BCG firstly, and boosted with LMΔ-msv and LIΔ-msv sequentially before challenging. Combination immune strategy (LMΔ-msv prime-LIΔ-msv boost) conferred comparable protection efficacy as BCG alone. More importantly, BCG-vaccinated mice acquired stronger resistance to Mycobacterial challenge when boosted with LMΔ-msv and LIΔ-msv sequentially. Our results inferred that heterologous immunization with Listeria-based recombinant strains boosted BCG-primed protection against pulmonary mycobacterial infection.

Pyruvate dehydrogenase complex-enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies.

The genus Listeria comprises ubiquitous bacteria, commonly present in foods and food production facilities. In this study, three different phage display technologies were employed to discover targets, and to generate and characterize novel antibodies against Listeria: antibody display for biomarker discovery and antibody generation; ORFeome display for target identification; and single-gene display for epitope characterization. With this approach, pyruvate dehydrogenase complex-enzyme 2 (PDC-E2) was defined as a new detection target for Listeria, as confirmed by immunomagnetic separation-mass spectrometry (IMS-MS). Immunoblot and fluorescence microscopy showed that this protein is accessible on the bacterial cell surface of living cells. Recombinant PDC-E2 was produced in E. coli and used to generate 16 additional antibodies. The resulting set of 20 monoclonal scFv-Fc was tested in indirect ELISA against 17 Listeria and 16 non-Listeria species. Two of them provided 100% sensitivity (CI 82.35-100.0%) and specificity (CI 78.20-100.0%), confirming PDC-E2 as a suitable target for the detection of Listeria. The binding region of 18 of these antibodies was analyzed, revealing that ≈ 90% (16/18) bind to the lipoyl domains (LD) of the target. The novel target PDC-E2 and highly specific antibodies against it offer new opportunities to improve the detection of Listeria.

Type 3 Innate Lymphoid Cells Direct Goblet Cell Differentiation via the LT-LTßR Pathway during Listeria Infection.

As a specialized subset of intestinal epithelial cells (IECs), goblet cells (GCs) play an important role during the antibacterial response via mucin production. However, the regulatory mechanisms involved in GC differentiation and function during infection, particularly the role of immune cell-IEC cross-talk, remain largely unknown. In this study, using Villin∆Ltbr conditional knockout mice, we demonstrate that LTßR, expressed on IECs, is required for GC hyperplasia and mucin 2 (MUC2) expression during Listeria infection for host defense but not homeostatic maintenance in the naive state. Analysis of single gene-deficient mice revealed that the ligand lymphotoxin (LT), but not LIGHT, and type 3 innate lymphoid cells (ILC3s), but not conventional T cells, are required for MUC2-dependent Listeria control. Conditional deficiency of LT in ILC3s further confirmed the importance of LT signals derived from ILC3s. Lack of ILC3-derived LT or IEC-derived LTßR resulted in the defective expression of genes related to GC differentiation but was not correlated with IEC proliferation and cell death, which were found to be normal by Ki-67 and Annexin V staining. In addition, the alternative NF-κB signaling pathway (involving RelB) in IECs was found to be required for the expression of GC differentiation-related genes and Muc2 and required for the anti-Listeria response. Therefore, our data together suggest a previously unrecognized ILC3-IEC interaction and LT-LTßR-RelB signaling axis governing GC differentiation and function during Listeria infection for host defense.

Oxysterols provide innate immunity to bacterial infection by mobilizing cell surface accessible cholesterol.

Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene that converts cholesterol to the oxysterol 25-hydroxycholesterol (25HC). Circulating 25HC modulates essential immunological processes including antiviral immunity, inflammasome activation and antibody class switching; and dysregulation of CH25H may contribute to chronic inflammatory disease and cancer. Although 25HC is a potent regulator of cholesterol storage, uptake, efflux and biosynthesis, how these metabolic activities reprogram the immunological state of target cells remains poorly understood. Here, we used recently designed toxin-based biosensors that discriminate between distinct pools of plasma membrane cholesterol to elucidate how 25HC prevents Listeria monocytogenes from traversing the plasma membrane of infected host cells. The 25HC-mediated activation of acyl-CoA:cholesterol acyltransferase (ACAT) triggered rapid internalization of a biochemically defined fraction of cholesterol, termed 'accessible' cholesterol, from the plasma membrane while having little effect on cholesterol in complexes with sphingomyelin. We show that evolutionarily distinct bacterial species, L. monocytogenes and Shigella flexneri, exploit the accessible pool of cholesterol for infection and that acute mobilization of this pool by oxysterols confers immunity to these pathogens. The significance of this signal-mediated membrane remodelling pathway probably extends beyond host defence systems, as several other biologically active oxysterols also mobilize accessible cholesterol through an ACAT-dependent mechanism.

Role of a noncanonical disulfide bond in the stability, affinity, and flexibility of a VHH specific for the Listeria virulence factor InlB.

A distinguishing feature of camel (Camelus dromedarius) VHH domains are noncanonical disulfide bonds between CDR1 and CDR3. The disulfide bond may provide an evolutionary advantage, as one of the cysteines in the bond is germline encoded. It has been hypothesized that this additional disulfide bond may play a role in binding affinity by reducing the entropic penalty associated with immobilization of a long CDR3 loop upon antigen binding. To examine the role of a noncanonical disulfide bond on antigen binding and the biophysical properties of a VHH domain, we have used the VHH R303, which binds the Listeria virulence factor InlB as a model. Using site directed mutagenesis, we produced a double mutant of R303 (C33A/C102A) to remove the extra disulfide bond of the VHH R303. Antigen binding was not affected by loss of the disulfide bond, however the mutant VHH displayed reduced thermal stability (Tm = 12°C lower than wild-type), and a loss of the ability to fold reversibly due to heat induced aggregation. X-ray structures of the mutant alone and in complex with InlB showed no major changes in the structure. B-factor analysis of the structures suggested that the loss of the disulfide bond elicited no major change on the flexibility of the CDR loops, and revealed no evidence of loop immobilization upon antigen binding. These results suggest that the noncanonical disulfide bond found in camel VHH may have evolved to stabilize the biophysical properties of the domain, rather than playing a significant role in antigen binding.

Immuno-detection and differentiation of Listeria monocytogenes and Listeria ivanovii in stone fruits.

AIMS: The aim of this study was to develop a rapid detection and differentiation method for pathogenic Listeria species in stone fruits. METHODS AND RESULTS: We utilized activated charcoal enrichment media (ACM) to induce overexpression and hypersecretion of pathogenic Listeria virulence proteins which can subsequently be detected via immunoblot analysis. Plum and nectarine slices spiked with either L. monocytogenes or L. ivanovii were incubated in pre-enrichment broth followed by enrichment in ACM. Secreted proteins were precipitated and subjected to SDS-PAGE and immunoblot analysis using a combination of L. monocytogenes-specific antibody (α-listeriolysin O) and antibody specific for both L. monocytogenes and L. ivanovii (α-Internalin C). As low as 1 CFU per gram of L. monocytogenes in plum and nectarine was detected, whereas a detection limit of 10 CFU per gram was achieved for L. ivanovii in each food tested following a 20-h enrichment period. Nonpathogenic Listeria species and non-Listeria bacterial pathogens tested were negative. CONCLUSIONS: These results demonstrate the highly sensitive and specific nature of the detection method for pathogenic Listeria in stone fruits using activated charcoal enrichment as well as the capability to discriminate between L. monocytogenes and L. ivanovii. SIGNIFICANCE AND IMPACT OF THE STUDY: This method is the first to identify and differentiate L. monocytogenes and L. ivanovii in select stone fruit enrichments within 24 h using immunological techniques. The rapidity and sensitivity of the method could aid in the reduction of exposure to the public in the event of an outbreak and expedite the administration of appropriate antibiotics to infected individuals.

Recognition of Listeria Infection by Germline Elements of the Vγ1.1 Vδ6.3 TCR.

γδNKT cells are an abundant γδT cell population with restricted Vγ1.1 Vδ6.3 gene usage and phenotypic and functional similarity to conventional αß-invariant NKT cells. The γδNKT population responds to Listeria infections, but specific ligands are not known. In this work, we studied the CDR3 requirements of the γδNKT TCR, Vγ1.1Vδ6.3 for recognizing naive macrophages, and macrophages infected with Listeria We expressed four different variants of the Vγ1.1Vδ6.3 TCR in TCR-deficient hybridomas, one with germline-encoded sequences and three with nongermline-encoded sequences. All of the hybridomas were activated when cultured in the presence of macrophages, and the activation was increased when the macrophages were infected with Listeria This indicates that these TCRs can recognize a self-ligand present in macrophages and suggests that the ligand is modified or upregulated when the cells are infected with Listeria One of the three nongermline-encoded Vγ1.1 variants induced a lower activation level compared with the other variants tested in this study, suggesting that recognition of the Listeria-induced ligand involves the CDR3γ region of the TCR.

The heme-regulated inhibitor is a cytosolic sensor of protein misfolding that controls innate immune signaling.

Multiple cytosolic innate sensors form large signalosomes after activation, but this assembly needs to be tightly regulated to avoid accumulation of misfolded aggregates. We found that the eIF2α kinase heme-regulated inhibitor (HRI) controls NOD1 signalosome folding and activation through a process requiring eukaryotic initiation factor 2α (eIF2α), the transcription factor ATF4, and the heat shock protein HSPB8. The HRI/eIF2α signaling axis was also essential for signaling downstream of the innate immune mediators NOD2, MAVS, and TRIF but dispensable for pathways dependent on MyD88 or STING. Moreover, filament-forming α-synuclein activated HRI-dependent responses, which suggests that the HRI pathway may restrict toxic oligomer formation. We propose that HRI, eIF2α, and HSPB8 define a novel cytosolic unfolded protein response (cUPR) essential for optimal innate immune signaling by large molecular platforms, functionally homologous to the PERK/eIF2α/HSPA5 axis of the endoplasmic reticulum UPR.

Anti-pancreatic tumor efficacy of a Listeria-based, Annexin A2-targeting immunotherapy in combination with anti-PD-1 antibodies.

BACKGROUND: Immune checkpoint inhibitors are not effective for pancreatic ductal adenocarcinoma (PDAC) as single agents. Vaccine therapy may sensitize PDACs to checkpoint inhibitor treatments. Annexin A2 (ANXA2) is a pro-metastasis protein, previously identified as a relevant PDAC antigen that is expressed by a GM-CSF-secreting allogenic whole pancreatic tumor cell vaccine (GVAX) to induce an anti-ANXA2 antibody response in patients with PDAC. We hypothesized that an ANXA2-targeting vaccine approach not only provokes an immune response but also mounts anti-tumor effects. METHODS: We developed a Listeria-based, ANXA2-targeting cancer immunotherapy (Lm-ANXA2) and investigated its effectiveness within two murine models of PDAC. RESULTS: We show that Lm-ANXA2 prolonged the survival in a transplant model of mouse PDACs. More importantly, priming with the Lm-ANXA2 treatment prior to administration of anti-PD-1 antibodies increased cure rates in the implanted PDAC model and resulted in objective tumor responses and prolonged survival in the genetically engineered spontaneous PDAC model. In tumors treated with Lm-ANXA2 followed by anti-PD-1 antibody, the T cells specific to ANXA2 had significantly increased INFγ expression. CONCLUSIONS: For the first time, a listeria vaccine-based immunotherapy was shown to be able to induce a tumor antigen-specific T cell response within the tumor microenvironment of a "cold" tumor such as PDAC and sensitize the tumor to checkpoint inhibitor therapy. Moreover, this combination immunotherapy led to objective tumor responses and survival benefit in the mice with spontaneously developed PDAC tumors. Therefore, our study supports developing Lm-ANXA2 as a therapeutic agent in combination with anti-PD-1 antibody for PDAC treatment.

Cas13-induced cellular dormancy prevents the rise of CRISPR-resistant bacteriophage.

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci in prokaryotes are composed of 30-40-base-pair repeats separated by equally short sequences of plasmid and bacteriophage origin known as spacers1-3. These loci are transcribed and processed into short CRISPR RNAs (crRNAs) that are used as guides by CRISPR-associated (Cas) nucleases to recognize and destroy complementary sequences (known as protospacers) in foreign nucleic acids4,5. In contrast to most Cas nucleases, which destroy invader DNA4-7, the type VI effector nuclease Cas13 uses RNA guides to locate complementary transcripts and catalyse both sequence-specific cis- and non-specific trans-RNA cleavage8. Although it has been hypothesized that Cas13 naturally defends against RNA phages8, type VI spacer sequences have exclusively been found to match the genomes of double-stranded DNA phages9,10, suggesting that Cas13 can provide immunity against these invaders. However, whether and how Cas13 uses its cis- and/or trans-RNA cleavage activities to defend against double-stranded DNA phages is not understood. Here we show that trans-cleavage of transcripts halts the growth of the host cell and is sufficient to abort the infectious cycle. This depletes the phage population and provides herd immunity to uninfected bacteria. Phages that harbour target mutations, which easily evade DNA-targeting CRISPR systems11-13, are also neutralized when Cas13 is activated by wild-type phages. Thus, by acting on the host rather than directly targeting the virus, type VI CRISPR systems not only provide robust defence against DNA phages but also prevent outbreaks of CRISPR-resistant phage.

Necroptosis mediators RIPK3 and MLKL suppress intracellular Listeria replication independently of host cell killing.

RIPK3, a key mediator of necroptosis, has been implicated in the host defense against viral infection primary in immune cells. However, gene expression analysis revealed that RIPK3 is abundantly expressed not only in immune organs but also in the gastrointestinal tract, particularly in the small intestine. We found that orally inoculated Listeria monocytogenes, a bacterial foodborne pathogen, efficiently spread and caused systemic infection in Ripk3-deficient mice while almost no dissemination was observed in wild-type mice. Listeria infection activated the RIPK3-MLKL pathway in cultured cells, which resulted in suppression of intracellular replication of Listeria Surprisingly, Listeria infection-induced phosphorylation of MLKL did not result in host cell killing. We found that MLKL directly binds to Listeria and inhibits their replication in the cytosol. Our findings have revealed a novel functional role of the RIPK3-MLKL pathway in nonimmune cell-derived host defense against Listeria invasion, which is mediated through cell death-independent mechanisms.

Versatile nano-platform for tailored immuno-magnetic carriers.

Custom immuno-magnetic devices are desirable tools for biomedical and biotechnological applications. Herein, surface active maghemite nanoparticles (SAMNs) are proposed as a versatile platform for developing tailored immuno-magnetic nano-carriers by simple wet reactions. Two examples for conjugating native and biotinylated antibodies were presented along with their successful applications in the recognition of specific foodborne pathogens. Nanoparticles were functionalized with rhodamine B isothiocyanate (RITC), leading to a fluorescent nano-conjugate, and used for binding anti-Campylobacter fetus antibodies (SAMN@RITC@Anti-Cf). The microorganism was selectively captured in the presence of two other Campylobacter species (C. jejuni and C. coli), as verified by PCR. Alternatively, SAMNs were modified with avidin, forming a biotin-specific magnetic nano-carrier and used for the immobilization of biotinylated anti-Listeria monocytogenes antibodies (SAMN@avidin@Anti-Lm). This immuno-magnetic carrier was integrated in piezoelectric quartz crystal microbalance (QCM) sensor for the detection of L. monocytogenes in milk, showing a detection limit of 3 bacterial cells. The present work presents a new category of customized immuno-magnetic nano-carriers as a competitive option for suiting specific applications. Graphical abstract ᅟ.

STING: From Mammals to Insects.

STING is a key mediator of mammalian innate immunity in response to pathogens. In this issue of Cell Host & Microbe and the June 19th issue of Cell Reports, Liu et al. (2018) and Martin et al. (2018) reveal that Drosophila STING is required for antiviral and antibacterial defenses, respectively.

Conjugation of wildtype and hypoallergenic mugwort allergen Art v 1 to flagellin induces IL-10-DC and suppresses allergen-specific TH2-responses in vivo.

Allergies to weed pollen including members of the Compositae family, such as mugwort, ragweed, and feverfew are spreading worldwide. To efficiently treat these newly arising allergies, allergen specific immunotherapy needs to be improved. Therefore, we generated novel vaccine candidates consisting of the TLR5-ligand Flagellin A from Listeria and the major mugwort allergen Art v 1 including either the wild type Art v 1 sequence (rFlaA:Artv1) or a hypoallergenic variant (rFlaA:Artv1hyp) with reduced IgE-binding capacity. Immune modulating capacity of these constructs and respective controls was evaluated in vitro and in vivo. Incorporation of hypoallergenic Art v 1 derivative did not interfere with the resulting fusion proteins' immune stimulatory capacity. Both rFlaA:Artv1 and rFlaA:Artv1hyp induced a prominent, mTOR-dependent, IL-10 secretion from murine dendritic cells, and suppressed allergen-specific TH2-cytokine secretion in vitro and in vivo. Both conjugates retained the capacity to induce rFlaA-specific antibody responses while efficiently inducing production of Art v 1-specific IgG1 and IgG2a antibodies in mice. Interestingly, only the suppression of TH2-cytokine secretion by rFlaA:Artv1 (but not rFlaA:Artv1hyp) was paralleled by a strong secretion of IFN-γ. In summary, we provided evidence that incorporating hypoallergens into flagellin:allergen fusion proteins is a suitable strategy to further improve these promising vaccine candidates.

Prevalence of pregnancy-relevant infections in a rural setting of Ghana.

BACKGROUND: Although infectious diseases still account for a high burden of morbidity and mortality in sub-Saharan Africa, simultaneous investigations on multiple infections affecting maternal and child health are missing. METHODS: We conducted a cross-sectional, single-centre pilot study in a rural area of Ghana to assess the infectiological profile during pregnancy. Screening of 180 expectant mothers was done by vaginal swabs and serology to detect the most common pregnancy-relevant infections. They were also interviewed for potential risk factors, outcome of previous pregnancies, and socio-economic aspects. RESULTS: We found a high prevalence of infections caused by hepatitis B virus (16.7% HBs antigen positive). In contrast, infections caused by hepatitis C virus (1.1% anti-HCV) and HIV (0.6%) were rare. Maternal malaria was frequent (10.6%), despite increasing acceptance of intermittent preventive treatment during pregnancy (IPTp). Group B streptococci were present in 10.6% of all pregnant women. Absence of antibodies against varicella zoster virus in 43.2%, Toxoplasma gondii in 26.8%, parvovirus B19 in 20.0%, and rubella virus in 15.7% makes a significant proportion of pregnant women susceptible for acquiring primary infections. Whereas all study participants had specific IgG antibodies against human cytomegalovirus, infections with Listeria, Brucella, or Neisseria gonorrhoeae as well as active syphilis were absent. CONCLUSIONS: Our pilot study in a rural community in Ghana indicates an urgent need for action in dealing at least with high-prevalent pregnancy-relevant infections, such as hepatitis B, malaria and those caused by group B streptococci. In addition, the resulting prevalence rates of various other infections may offer guidance for health officials to prioritize possible future intervention schemes.

A20 Curtails Primary but Augments Secondary CD8+ T Cell Responses in Intracellular Bacterial Infection.

The ubiquitin-modifying enzyme A20, an important negative feedback regulator of NF-κB, impairs the expansion of tumor-specific CD8+ T cells but augments the proliferation of autoimmune CD4+ T cells. To study the T cell-specific function of A20 in bacterial infection, we infected T cell-specific A20 knockout (CD4-Cre A20fl/fl) and control mice with Listeria monocytogenes. A20-deficient pathogen-specific CD8+ T cells expanded stronger resulting in improved pathogen control at day 7 p.i. Imaging flow cytometry revealed that A20-deficient Listeria-specific CD8+ T cells underwent increased apoptosis and necroptosis resulting in reduced numbers of memory CD8+ T cells. In contrast, the primary CD4+ T cell response was A20-independent. Upon secondary infection, the increase and function of pathogen-specific CD8+ T cells, as well as pathogen control were significantly impaired in CD4-Cre A20fl/fl mice. In vitro, apoptosis and necroptosis of Listeria-specific A20-deficient CD8+ T cells were strongly induced as demonstrated by increased caspase-3/7 activity, RIPK1/RIPK3 complex formation and more morphologically apoptotic and necroptotic CD8+ T cells. In vitro, A20 limited CD95L and TNF-induced caspase3/7 activation. In conclusion, T cell-specific A20 limited the expansion but reduced apoptosis and necroptosis of Listeria-specific CD8+ T cells, resulting in an impaired pathogen control in primary but improved clearance in secondary infection.