Discovery of highly neutralizing human antibodies targeting Pseudomonas aeruginosa.

Drug-resistant Pseudomonas aeruginosa (PA) poses an emerging threat to human health with urgent need for alternative therapeutic approaches. Here, we deciphered the B cell and antibody response to the virulence-associated type III secretion system (T3SS) in a cohort of patients chronically infected with PA. Single-cell analytics revealed a diverse B cell receptor repertoire directed against the T3SS needle-tip protein PcrV, enabling the production of monoclonal antibodies (mAbs) abrogating T3SS-mediated cytotoxicity. Mechanistic studies involving cryoelectron microscopy identified a surface-exposed C-terminal PcrV epitope as the target of highly neutralizing mAbs with broad activity against drug-resistant PA isolates. These anti-PcrV mAbs were as effective as treatment with conventional antibiotics in vivo. Our study reveals that chronically infected patients represent a source of neutralizing antibodies, which can be exploited as therapeutics against PA.

Single priming and booster dose of ten-valent and 13-valent pneumococcal conjugate vaccines and Streptococcus pneumoniae colonisation in children in South Africa: a single-centre, open-label, randomised trial.

BACKGROUND: Pneumococcal conjugate vaccine (PCV) immunisation has reduced vaccine-serotype colonisation and invasive pneumococcal disease in South Africa, providing the opportunity to consider transitioning from a two-dose (2 + 1) to one-dose (1 + 1) primary series and a booster dose. METHODS: In this single-centre, open-label, randomised trial done in South Africa, infants aged 35-49 days without HIV infection, without childhood immunisations except for BCG and polio, and with gestation age at delivery of at least 37 weeks of age, a birthweight of at least 2500 g, and weight of at least 3500 g at the time of enrolment were randomly assigned (1:1:1:1:1:1), through block randomisation (block size of 30), to receive a single priming dose of ten-valent PCV (PCV10) or 13-valent PCV (PCV13) at either 6 weeks (6-week 1 + 1 group) or 14 weeks (14-week 1 + 1 group), compared with two priming doses at 6 weeks and 14 weeks (2 + 1 group), followed by a booster dose at 9 months of age in all groups. The primary objective of the trial has been published previously. We report the secondary objective of the effect of alternative doses of PCV10 and PCV13 on serotype-specific Streptococcus pneumoniae colonisation at 9 months, 15 months, and 18 months of age and a further exploratory analysis in which we assessed non-inferiority of serotype-specific serum IgG geometric mean concentrations 1 month after the booster (10 months of age) and the percentage of participants with serotype-specific IgG titre above the putative thresholds associated with a risk reduction of serotype-specific colonisation between the 1 + 1 and 2 + 1 groups for both vaccines. Non-inferiority was established if the lower limit of the 95% CI for the difference between the proportion of participants (1 + 1 group vs 2 + 1 group) above the putative thresholds was greater than or equal to -10%. All analyses were done in the modified intention-to-treat population, which included all participants who received PCV10 or PCV13 according to assigned randomisation group and for whom laboratory results were available. The trial is registered with ClinicalTrials.gov, NCT02943902. FINDINGS: 1564 nasopharyngeal swabs were available for molecular serotyping from 600 infants who were enrolled (100 were randomly assigned to each of the six study groups) between Jan 9 and Sept 20, 2017. There was no significant difference in the prevalence of overall or non-vaccine serotype colonisation between all PCV13 or PCV10 groups. PCV13 serotype colonisation was lower at 15 months of age in the 14-week 1 + 1 group than in the 2 + 1 group (seven [8%] of 85 vs 17 [20%] of 87; odds ratio 0·61 [95% CI 0·38-0·97], p=0·037), but no difference was seen at 9 months (nine [11%] of 86 vs ten [11%] of 89; 0·92 [0·60-1·55], p=0·87) or 18 months (nine [11%] of 85 vs 11 [14%] of 87; 0·78 [0·45-1·22], p=0·61). Compared with the PCV13 2 + 1 group, both PCV13 1 + 1 groups did not meet the non-inferiority criteria for serotype-specific anti-capsular antibody concentrations above the putative thresholds purportedly associated with risk reduction for colonisation; however, the PCV10 14-week 1 + 1 group was non-inferior to the PCV10 2 + 1 group. INTERPRETATION: The serotype-specific colonisation data reported in this study together with the primary immunogenicity endpoints of the control trial support transitioning to a reduced 1 + 1 schedule in South Africa. Ongoing monitoring of colonisation should, however, be undertaken immediately before and after transitioning to a PCV 1 + 1 schedule to serve as an early indicator of whether PCV 1 + 1 could lead to an increase in vaccine-serotype disease. FUNDING: The Bill & Melinda Gates Foundation.

Tauroursodeoxycholic Acid Inhibits Clostridioides difficile Toxin-Induced Apoptosis.

C. difficile infection (CDI) is a highly inflammatory disease mediated by the production of two large toxins that weaken the intestinal epithelium and cause extensive colonic tissue damage. Antibiotic alternative therapies for CDI are urgently needed as current antibiotic regimens prolong the perturbation of the microbiota and lead to high disease recurrence rates. Inflammation is more closely correlated with CDI severity than bacterial burden, thus therapies that target the host response represent a promising yet unexplored strategy for treating CDI. Intestinal bile acids are key regulators of gut physiology that exert cytoprotective roles in cellular stress, inflammation, and barrier integrity, yet the dynamics between bile acids and host cellular processes during CDI have not been investigated. Here we show that several bile acids are protective against apoptosis caused by C. difficile toxins in Caco-2 cells and that protection is dependent on conjugation of bile acids. Out of 20 tested bile acids, taurine conjugated ursodeoxycholic acid (TUDCA) was the most potent inhibitor, yet unconjugated UDCA did not alter toxin-induced apoptosis. TUDCA treatment decreased expression of genes in lysosome associated and cytokine signaling pathways. TUDCA did not affect C. difficile growth or toxin activity in vitro whereas UDCA significantly reduced toxin activity in a Vero cell cytotoxicity assay and decreased tcdA gene expression. These results demonstrate that bile acid conjugation can have profound effects on C. difficile as well as the host and that conjugated and unconjugated bile acids may exert different therapeutic mechanisms against CDI.

Blocking Borrelia burgdorferi transmission from infected ticks to nonhuman primates with a human monoclonal antibody.

Disrupting transmission of Borrelia burgdorferi sensu lato complex (B. burgdorferi) from infected ticks to humans is one strategy to prevent the significant morbidity from Lyme disease. We have previously shown that an anti-OspA human mAb, 2217, prevents transmission of B. burgdorferi from infected ticks in animal models. Maintenance of a protective plasma concentration of a human mAb for tick season presents a significant challenge for a preexposure prophylaxis strategy. Here, we describe the optimization of mAb 2217 by amino acid substitutions (2217LS: M428L and N434S) in the Fc domain. The LS mutation led to a 2-fold increase in half-life in cynomolgus monkeys. In a rhesus macaque model, 2217LS protected animals from tick transmission of spirochetes at a dose of 3 mg/kg. Crystallographic analysis of Fab in complex with OspA revealed that 2217 bound an epitope that was highly conserved among the B. burgdorferi, B. garinii, and B. afzelii species. Unlike most vaccines that may require boosters to achieve protection, our work supports the development of 2217LS as an effective preexposure prophylaxis in Lyme-endemic regions, with a single dose at the beginning of tick season offering immediate protection that remains for the duration of exposure risk.

Interactions of Antibodies to the Gram-Negative Gastric Bacterium Helicobacter pylori with the Synaptic Calcium Sensor Synaptotagmin 5, Correlate to Impaired Vesicle Recycling in SiMa Human Neuroblastoma Cells.

Due to molecular mimicry, maternal antibacterial antibodies are suspected to promote neurodevelopmental changes in the offspring that finally can cause disorders like autism and schizophrenia. Using a human first trimester prenatal brain multiprotein array (MPA), we demonstrate here that antibodies to the digestive tract bacteria Helicobacter pylori (α-HPy) and Campylobacter jejuni (α-CJe) interact with different synaptic proteins, including the calcium sensor synaptotagmin 5 (Syt5). Interactions of both antisera with Syt5 were confirmed by Western blot with a HEK293-cells overexpression lysate of this protein. Immunofluorescence and Western blotting revealed SiMa cells to express Syt5, which also co-migrated with a band/spot labeled by either α-HPy or α-CJe. Functionally, a 12-h pretreatment of SiMa cells with 10 µg/ml of either α-HPy or α-CJe resulted in a significant reduction of acetylcholine(ACh)-dependent calcium signals as compared to controls. Also ACh-dependent vesicle recycling was significantly reduced in cells pretreated with either α-HPy or α-CJe. Similar effects were observed upon pretreatment of SiMa cells with Syt5-specific antibodies. In conclusion, the present study supports the view that prenatal maternal antibacterial immune responses towards HPy and by this to Syt5 are able to cause functional changes, which in the end might contribute also to neurodevelopmental disorders.

Fighting Staphylococcus aureus infections with light and photoimmunoconjugates.

Infections caused by multidrug-resistant Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), are responsible for high mortality and morbidity worldwide. Resistant lineages were previously confined to hospitals but are now also causing infections among healthy individuals in the community. It is therefore imperative to explore therapeutic avenues that are less prone to raise drug resistance compared with today's antibiotics. An opportunity to achieve this ambitious goal could be provided by targeted antimicrobial photodynamic therapy (aPDT), which relies on the combination of a bacteria-specific targeting agent and light-induced generation of ROS by an appropriate photosensitizer. Here, we conjugated the near-infrared photosensitizer IRDye700DX to a fully human mAb, specific for the invariantly expressed staphylococcal antigen immunodominant staphylococcal antigen A (IsaA). The resulting immunoconjugate 1D9-700DX was characterized biochemically and in preclinical infection models. As demonstrated in vitro, in vivo, and in a human postmortem orthopedic implant infection model, targeted aPDT with 1D9-700DX is highly effective. Importantly, combined with the nontoxic aPDT-enhancing agent potassium iodide, 1D9-700DX overcomes the antioxidant properties of human plasma and fully eradicates high titers of MRSA. We show that the developed immunoconjugate 1D9-700DX targets MRSA and kills it upon illumination with red light, without causing collateral damage to human cells.

Characterization of Tissue Distribution, Catabolism, and Elimination of an Anti-Staphylococcus aureus THIOMAB Antibody-Antibiotic Conjugate in Rats.

Invasive Staphylococcus aureus infection is a leading cause of infectious disease-related deaths because S. aureus survives within host phagocytic cells, from which the bacteria are not adequately eliminated using current antibiotic treatments. Anti-S. aureus THIOMAB antibody-antibiotic conjugate (TAC), an anti-S. aureus antibody conjugated with antibiotic payload dmDNA31, was designed to deliver antibiotics into phagocytes, thereby killing intracellular S. aureus Herein, we present the distribution, metabolism/catabolism, and elimination properties for this modality. The tissue distribution of TAC and the release and elimination of its payload dmDNA31 were characterized in rats using multiple approaches. Intravenous injection of unconjugated [14C]dmDNA31 to rats resulted in a rapid clearance in both systemic circulation and tissues, with biliary secretion as the major route of elimination. Six major metabolites were identified. When [14C]dmDNA31 was conjugated to an antibody as TAC and administered to rat intravenously, a sustained exposure was observed in both systemic circulation and tissues. The dmDNA31 in blood and tissues mainly remained in conjugated form after administering TAC, although minimal deconjugation of dmDNA31 from TAC was also observed. Several TAC catabolites were identified, which were mainly eliminated through the biliary-fecal route, with dmDNA31 and deacetylated dmDNA31 as the most abundant catabolites. In summary, these studies provide a comprehensive characterization of the distribution, metabolism/catabolism, and elimination properties of TAC. These data fully support further clinical development of TAC for the invasive and difficult-to-treat S. aureus infection. SIGNIFICANCE STATEMENT: The present studies provide a comprehensive investigation of the absorption, distribution, metabolism/catabolism, and elimination of the first antibody-antibiotic conjugate developed for the treatment of an infectious disease. Although many antibody-drug conjugates are in development for various disease indications, only a limited amount of absorption, distribution, metabolism/catabolism, and elimination information is available in the literature. This study demonstrates the use of radiolabeling technology to delineate the absorption, distribution, metabolism/catabolism, and elimination properties of a complex modality and help address the key questions related to clinical pharmacological studies.

Characterization of Actinobacillus seminis biofilm formation.

Actinobacillus seminis is an autochthonous gram-negative bacterium that affects reproductive organs, causing epididymitis, low fertility, and occasional abortions in ovine and goats. The virulence factors and the pathogenicity mechanisms of A. seminis have not been clearly elucidated yet. In this work, biofilm production by A. seminis in in vitro assays is described and characterized. After 48-h incubation at 37 °C in trypticase soy broth, A. seminis formed biofilms containing an extracellular matrix comprised mainly of fibrillar material. Microaerophilia or the presence of calcium diminished biofilm formation in approximately 50% and 70%, respectively, but low iron concentrations increased it 40%. Through enzymatic digestion, it was found that proteins were the main component of these biofilms. Structural observations through scanning electron microscopy indicated the presence of a high amount of fibrillar material in which bacteria were immersed. Antibodies against different bacterial surface proteins, such as anti-biofilm matrix and anti-adhesin, diminished biofilm formation in 70% and 25%, respectively; whereas furanone C-30 and LED-209, compounds described as quorum-sensing inhibitors, completely inhibited biofilm formation. In conclusion, environmental conditions can influence strongly biofilm formation in A. seminis, and this could be an advantageous strategy that allows bacteria to persist inside a host.

Antibodies targeting Mycobacterium tuberculosis peptides inhibit mycobacterial entry to infection target cells.

The humoral immunity regarding tuberculosis can contribute towards controlling the mycobacteria and the disease. Antigens mediating such type of immunity should thus be evaluated for formulating anti-tuberculosis vaccines. The antigen recognition of seven peptides derived from proteins on Mtb H37Rv envelope and a further seven peptides modified from them was evaluated in sera taken from people suffering Mtb infection and others free from it. Peptide sequences' ability to inhibit Mtb entry to human macrophages was determined in vitro and, after isolating peptide-specific IgG antibodies, it was ascertained which ones were exercising such inhibitory function. Aotus were inoculated with the modified peptides for evaluating the activity of the antibodies so produced. Human QTF+ and QTF- sera recognised some of the peptides and inhibited Mtb entry. The same effect was seen with peptide-specific IgG regarding all the native sequences and modified ones. Sera taken from inoculated Aotus was also able to reduce the pathogen's entry. The data showed that some peptides evaluated in this study could induce antibodies able to inhibit the pathogen's entry to human macrophages, i.e. they could represent candidates for part of an anti-tuberculosis vaccine. The methodology used here complements the evaluation of promising antigens for designing effective vaccines.

Selective Killing of Shiga Toxin-Producing Escherichia coli with Antibody-Conjugated Chitosan Nanoparticles in the Gastrointestinal Tract.

Shiga toxin-producing Escherichia coli (STEC) are critical foodborne pathogens, which cause serious human health issues, including hemolytic uremic syndrome. Illnesses caused by STEC lack effective treatments that target the elimination of these bacteria from the gastrointestinal tract without causing an adverse effect. Reducing this pathogen from a reservoir of STEC is an effective strategy, but the challenges remain due to the lack of efficient, selective antimicrobial agents. We developed specific antibody-conjugated chitosan nanoparticles (CNs) to selectively target and treat STEC in the gastrointestinal tract. Given the great broad-spectrum antimicrobial activity of CN, we conjugated antibodies to CN. Antibodies were raised and purified from egg yolks after immunization of hens with seven different O-side-chain antigens isolated from STEC (O26, O45, O103, O111, O121, O145, and O157). We prepared CN-immunoglobulin Y (IgY) conjugates by forming amide bonds at different ratios of CN:IgY (10:1, 10:2, and 10:4). The CN-IgY conjugated at a 10:2 ratio demonstrated significantly enhanced antimicrobial activity against E. coli O157:H7. Conjugates of CN and anti-STEC IgY antibodies killed corresponding STEC serotypes specifically and selectively, while showing no significant impact on nontargeted bacteria, including Salmonella enterica and Lactobacillus plantarum. The enhanced antimicrobial activity of CN-IgY against STEC was also confirmed in synthetic intestinal fluid, as well as an in vivo animal model of Caenorhabditis elegans. These results suggest that the CN-IgY conjugates have strong and specific antimicrobial activity and that they are also great candidates to eliminate pathogens selectively in the gastrointestinal tract without inhibiting beneficial bacteria.

Monoclonal antibodies against LipL32 confer prophylactic protection against lethal leptospirosis challenge in animal model.

Leptospirosis is a widespread zoonotic disease caused by pathogenic spirochetes of the genus Leptospira. The commercially available vaccines are bacterins that offer limited protection, short-term effect, and serovar-specific immunity. The development of novel immunization strategies is crucial to control the infection and decrease the chances of new outbreaks. In this study, purified monoclonal antibodies (mAbs) anti-LipL32 (1D9 and mAb3) were evaluated by their capacity to bind and neutralize the pathogen improving host survival. For that, an in vitro growth inhibition assay, and in vivo passive immunization were performed in animal model. Syrian hamsters were passively immunized by three different strategies. Hamsters immunized with mAb3 6 h prior to the lethal challenge showed a significantly higher survival rate of 61.1%, and a significant reduction in tissue damage in the lungs. Cumulatively, our results showed that anti-LipL32 mAbs inhibited the growth of L. interrogans in vitro, and that passive immunization offered significant protection in animal model when administered prior to infection.

Functional characterization and evaluation of protective efficacy of EA752-862 monoclonal antibody against B. anthracis vegetative cell and spores.

The most promising means of controlling anthrax, a lethal zoonotic disease during the early infection stages, entail restricting the resilient infectious form, i.e., the spores from proliferating to replicating bacilli in the host. The extractible antigen (EA1), a major S-layer protein present on the vegetative cells and spores of Bacillus anthracis, is highly immunogenic and protects mice against lethal challenge upon immunization. In the present study, mice were immunized with r-EA1C, the C terminal crystallization domain of EA1, to generate a neutralizing monoclonal antibody EA752-862, that was evaluated for its anti-spore and anti-bacterial properties. The monoclonal antibody EA752-862 had a minimum inhibitory concentration of 0.08 mg/ml, was bactericidal at a concentration of 0.1 mg/ml and resulted in 100% survival of mice against challenge with B. anthracis vegetative cells. Bacterial cell lysis as observed by scanning electron microscopy and nucleic acid leakage assay could be attributed as a possible mechanism for the bactericidal property. The association of mAb EA752-862 with spores inhibits their subsequent germination to vegetative cells in vitro, enhances phagocytosis of the spores and killing of the vegetative cells within the macrophage, and subsequently resulted in 90% survival of mice upon B. anthracis Ames spore challenge. Therefore, owing to its anti-spore and bactericidal properties, the present study demonstrates mAb EA752-862 as an efficient neutralizing antibody that hinders the establishment of early infection before massive multiplication and toxin release takes place.

Immunogenicity and protective efficacy of Burkholderia pseudomallei BLF1-N and BLF1-C terminal domains against BLF1 toxin.

Burkholderia lethal factor 1 (BLF1), a glutamine deamidase, is a key virulence factor that plays significant role in B. pseudomallei pathogenesis. To elucidate the BLF1 immunological responses, two truncated BLF1 structural units, BLF1-C (90-211 amino acids) with structural similarity to T. maritima Chemoreceptor glutamine deamidase (CheD) protein, and BLF1-N (1-89 amino acids) disparate to CheD were identified from the 23 kDa BLF1 protein. Both the components were devoid of toxicity in mice and elicited an antibody titer of 1:16,000 that reacted with the respective truncated proteins and BLF1. A549 cell lines supplemented with anti BLF1-N and BLF1-C antibodies exhibited 73.47% and 83.24% survival when treated with BLF1 toxin. Passive i.p. transfer with antibodies elicited by BLF1-C that contained LSGC active site resulted in 80% protection while anti BLF1-N (devoid of LSGC) antibodies provided 51.4% protection, establishing the role of BLF1-N terminal also in deamidase action. The truncated proteins also elicited cell mediated immune responses through proliferation of CD4+ T cells, IFN-γ and IL-4 cytokines but with bias towards Th2 subsets. BLF1-C and BLF1-N immunization resulted in 80% and 60% active protection when challenged with BLF1 toxin while the sham immunized mice exhibited severe histopathological changes like necrosis in liver, lung, spleen and kidney similar to that observed in melioidosis and were killed within 7 days post challenge. The higher level of active and passive protection by BLF1-C protein could be attributed to the comparatively higher level of immune responses and inclusion of LSGC residues.

Monoclonal antibody anti-PBP2a protects mice against MRSA (methicillin-resistant Staphylococcus aureus) infections.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant bacterium responsible for serious nosocomial and community-acquired infections worldwide. Since few antibiotics are effective for treating MRSA infections, the development of new therapies is of great importance. Previous studies demonstrated that PBP2a is a target that generates protective antibodies against MRSA. A murine monoclonal antibody (MAb) that recognizes PBP2a from MRSA strains was previously isolated and characterized. In this report, we evaluated the biodistribution of this MAb in blood and tissues, as well as the extent of protection conferred using prophylactic and therapeutic assays compared to vancomycin treatment. Biodistribution was evaluated 12-96 h after MAb administration. It predominantly remained in the serum, but it was also detectable in the kidneys, lungs, and spleen at low concentrations (about 4.5% in the kidneys, 1.9% in the lungs, and 0.7% the spleen) at all observed timepoints. Prophylactic studies in a murine model demonstrated a significant bacterial load reduction in the kidneys of the groups treated with either with IgG (greater than 3 logs) or F(ab')2 (98%) when compared to that of the control groups (untreated). Mice were challenged with a lethal dose, and the survival rate was higher in the treated mice. Treatment with the MAb resulted in a bacterial load reduction in the kidneys similar to that of mice treated with vancomycin, and a MAb/vancomycin combination therapy was also effective. These results demonstrate that an anti-PBP2a MAb may be a promising therapeutic for treating MRSA infections.

Different mechanisms of two anti-anthrax protective antigen antibodies and function comparison between them.

BACKGROUND: Bacillus anthracis causes a highly lethal infectious disease primarily due to toxin-mediated injury. Antibiotics are no longer effective to treat the accumulation of anthrax toxin, thereby new strategies of antibody treatment are essential. Two anti- anthrax protective antigen (PA) antibodies, hmPA6 and PA21, have been reported by our lab previously. METHODS: The mechanisms of the two antibodies were elucidated by Electrophoresis, Competitive Enzyme-linked immune sorbent assay, Western blot analysis and immunoprecipitation test, and in vitro, in vivo (F344 rats) treatment test. The epitopes of the two antibodies were proved by Western blot and Enzyme-linked immune sorbent assay with different domains of PA. RESULTS: In this study, we compared affinity and neutralization of these two antibodies. PA21 was better in protecting cells and rats, whereas hmPA6 had higher affinity. Furthermore, the neutralization mechanisms of the two antibodies and their recognition domains of PA were studied. The results showed that hmPA6 recognized domain IV, thus PA could not bind to cell receptors. Conversely, PA21 recognized domain II, thereby limiting heptamer oligomerization of PA63 in cells. CONCLUSIONS: Our studies elucidated the mechanisms and epitopes of hmPA6 and PA21. The present investigation can advance future use of the two antibodies in anthrax treatment or prophylaxis, and potentially as a combination treatment as the antibodies target different epitopes.

Sustained activity of novel THIOMAB antibody-antibiotic conjugate against Staphylococcus aureus in a mouse model: Longitudinal pharmacodynamic assessment by bioluminescence imaging.

Staphylococcus aureus (S. aureus) infections are a leading cause of death by an infectious agent. Survival within host phagocytic cells is one mechanism by which S. aureus evades antibiotic treatment. A novel THIOMAB™ antibody-antibiotic conjugate (TAC) strategy was developed to kill S. aureus intracellularly and mitigate the spread of infection. In this report, we used a longitudinal whole-body bioluminescence imaging method to study the antibacterial dynamics of TAC alone or in combination with vancomycin in a mouse infection model. Injections of stably luminescent S. aureus bacteria into mice resulted in exponential increases in whole body bioluminescence with a reduction in body weight and survival rate. Vancomycin, a standard-of-care antibiotic, suppressed bacterial growth in mice. However, bacterial growth rebounded in these animals once treatment was discontinued. In contrast, single dose of TAC showed rapid reduction of bioluminescence intensity, which persisted for up to 19 days. The combination of TAC and vancomycin achieved a more sustained and significantly greater reduction of bioluminescence compared with vancomycin alone. In summary, the present study showed an imaging method to longitudinally assess antibacterial drug dynamics in mice and demonstrated that TAC monotherapy or in combination with vancomycin had superior and sustained activity compared to vancomycin alone.

Inhibition of Francisella tularensis phagocytosis using a novel anti-LPS scFv antibody fragment.

Francisella tularensis (Ft), the causative agent of lethal tularemia, is classified as a category A biological warfare threat agent. While Ft infection is treatable by antibiotics, many failed antibiotic treatments were reported, highlighting the need for effective new treatments. It has been demonstrated that binding of antibody-coated bacteria to the Fc receptor located on phagocytic cells is a key process needed for efficient protection against Ft. Yet, Ft utilizes the same receptor to enter the phagocytic cells in order to escape the immune system. To address the question whether an anti-Ft LPS antibody lacking the ability to bind the Fc receptor may inhibit the entry of Ft into host cells, a soluble scFv (TL1-scFv) was constructed from an anti Ft-LPS antibody (TL1) that was isolated from an immune single-chain (scFv) phage-display library. Bacterial uptake was assessed upon infection of macrophages with Ft live attenuated strain (LVS) in the presence of either TL1 or TL1-scFv. While incubation of LVS in the presence of TL1 greatly enhanced bacterial uptake, LVS uptake was significantly inhibited in the presence of TL1-scFv. These results prompt further experiments probing the therapeutic efficacy of TL1-scFv, alone or in combination with antibiotic treatment.

PerioVax3, a key antigenic determinant with immunoprotective potential against periodontal pathogen.

Treponema (T.) denticola is one of the key etiological agents in the development of periodontitis. The major outer sheath protein (Msp) of T. denticola has been shown to mediate pathogenesis and to facilitate adhesion of T. denticola to mucosal surfaces. This study aimed to find short polypeptides in the amino acid sequence of Msp which may be immunogenic and might elicit protective antisera against T. denticola. The complete msp sequence was divided into six fragments and the corresponding genes were cloned and expressed. Antisera against the polypeptides were raised in rabbits and fragment 3 (F3), hereinafter called PerioVax3 was the most potent fragment of the Msp in terms of yielding high titer antiserum. An adhesion assay was done to examine the inhibitory effects of antisera on the attachment of T. denticola to human gingival fibroblasts (HGFs) and human fibronectin. Antiserum against PerioVax3 significantly inhibited attachment of T. denticola to the substratum. Also, antiserum against PerioVax3 inhibited detachment of HGFs upon T. denticola exposure. To begin examining the clinical relevance of this work, blood samples from 12 sever periodontitis patients were collected and the sera were used in western blotting against the recombinant polypeptides. Periodontitis patient antisera exclusively detected PerioVax3 in western blotting. The data suggest that PerioVax3 carries epitopes that may trigger humoral immunity against T. denticola, which may protect against its adhesion functions. The complexity of periodontitis suggests that PerioVax3 may be considered for testing as a component of an experimental multivalent periodontal vaccine in further preclinical and clinical studies.

Evaluation of a Recombinant Flavobacterium columnare DnaK Protein Vaccine as a Means of Protection Against Columnaris Disease in Channel Catfish (Ictalurus punctatus).

Flavobacterium columnare causes substantial losses among cultured finfish species. The Gram-negative bacterium is an opportunistic pathogen that manifests as biofilms on the host's mucosal surfaces as the disease progresses. We previously demonstrated that the dominant mucosal IgM antibody response to F. columnare is to the chaperone protein DnaK that is found in the extracellular fraction. To establish the efficacy of using recombinant protein technology to develop a new vaccine against columnaris disease, we are reporting on two consecutive years of vaccine trials using a recombinant F. columnare DnaK protein (rDnaK). In year one, three groups of channel catfish (n = 300) were immunized by bath immersion with a live attenuated F. columnare isolate, rDnaK or sham immunized. After 6 weeks, an F. columnare laboratory challenge showed a significant increase in survival (>30%) in both the live attenuated and rDnaK vaccines when compared to the non-immunized control. A rDnaK-specific ELISA revealed significant levels of mucosal IgM antibodies in the skin of catfish immunized with rDnaK at 4- and 6-weeks post immunization. In the second year, three groups of channel catfish (n = 300) were bath immunized with rDnaK alone or with rDnaK after a brief osmotic shock or sham immunized. After 6 weeks a laboratory challenge with F. columnare was conducted and showed a significant increase in survival in the rDnaK (> 25%) and in rDnaK with osmotic shock (>35%) groups when compared to the non-immunized control. The rDnaK-specific ELISA demonstrated significant levels of mucosal IgM antibodies in the skin of catfish groups immunized with rDnaK at 4- and 6-weeks post immunization. To further understand the processes which have conferred immune protection in the rDnaK group, we conducted RNA sequencing of skin samples from the non-immunized (n = 6) and rDnaK treated channel catfish at 1-week (n = 6) and 6 weeks (n = 6) post immunization. Significantly altered gene expression was identified and results will be discussed. Work to further enhance the catfish immune response to F. columnare rDnaK is underway as this protein remains a promising candidate for additional optimization and experimental trials in a production setting.

Detection and characterization of bacterial polysaccharides in drug-resistant enterococci.

Enterococcus faecium (E. faecium) has emerged as one of today's leading causes of health care-associated infections that is difficult to treat with the available antibiotics. These pathogens produce capsular polysaccharides on the cell surface which play a significant role in adhesion, virulence and evasion. Therefore, we aimed at the identification and characterization of bacterial polysaccharide antigens which are central for the development of vaccine-based prophylactic approaches. The crude cell wall-associated polysaccharides from E. faecium, its mutant and complemented strains were purified and analyzed by a primary antibody raised against lipoteichoic acid (LTA) and diheteroglycan (DHG). The resistant E. faecium strains presumably possess novel capsular polysaccharides that allow them to avoid the evasion from opsonic killing. The E. faecium U0317 strain was very well opsonized by anti-U0317 (~95%), an antibody against the whole bacterial cell. The deletion mutant showed a significantly increased susceptibility to opsonophagocytic killing (90-95%) against the penicillin binding protein (anti-PBP-5). By comparison, in a mouse urinary tract and rat endocarditis infection model, respectively, there were no significant differences in virulence. In this study we explored the biological role of the capsule of E. faecium. Our findings showed that the U0317 strain is not only sensitive to anti-LTA but also to antibodies against other enterococcal surface proteins. Our findings demonstrate that polysaccharides capsule mediated-resistance to opsonophagocytosis. We also found that the capsular polysaccharides do not play an important role in bacterial virulence in urinary tract and infective endocarditis in vivo models.