The C-terminal fragment of the internal 110-kilodalton passenger domain of the Hap protein of nontypeable Haemophilus influenzae is a potential vaccine candidate

Nontypeable Haemophilus influenzae is a major causative agent of bacterial otitis media in children. H. influenzae Hap autotransporter protein is an adhesin composed of an outer membrane Hapβ region and a moiety of an extracellular internal 110-kDa passenger domain called Hap(S). The Hap(S) moiety promotes adherence to human epithelial cells and extracellular matrix proteins, and it also mediates bacterial aggregation and microcolony formation. A recent work (D. L. Fink, A. Z. Buscher, B. A. Green, P. Fernsten, and J. W. St. Geme, Cell. Microbiol. 5:175-186, 2003) demonstrated that Hap(S) adhesive activity resides within the C-terminal 311 amino acids (the cell binding domain) of the protein. In this study, we immunized mice subcutaneously with recombinant proteins corresponding to the C-terminal region of Hap(S) from H. influenzae strains N187, P860295, and TN106 and examined the resulting immune response. Antisera against the recombinant proteins from all three strains not only recognized native Hap(S) purified from strain P860295 but also inhibited H. influenzae Hap-mediated adherence to Chang epithelial cells. Furthermore, when mice immunized intranasally with recombinant protein plus mutant cholera toxin CT-E29H were challenged with strain TN106, they were protected against nasopharyngeal colonization. These observations demonstrate that the C-terminal region of Hap(S) is capable of eliciting cross-reacting antibodies that reduce nasopharyngeal colonization, suggesting utility as a vaccine antigen for the prevention of nontypeable H. influenzae diseases

Genomics of Loa loa, a Wolbachia-free filarial parasite of humans

Loa loa, the African eyeworm, is a major filarial pathogen of humans. Unlike most filariae, Loa loa does not contain the obligate intracellular Wolbachia endosymbiont. We describe the 91.4 Mb genome of Loa loa, and the genome of the related filarial parasite Wuchereria bancrofti, and predict 14,907 Loa loa genes based on microfilarial RNA sequencing. By comparing these genomes to that of another filarial parasite, Brugia malayi, and to several other nematode genomes, we demonstrate synteny among filariae but not with non-parasitic nematodes. The Loa loa genome encodes many immunologically relevant genes, as well as protein kinases targeted by drugs currently approved for humans. Despite lacking Wolbachia, Loa loa shows no new metabolic synthesis or transport capabilities compared to other filariae. These results suggest that the role played by Wolbachia in filarial biology is more subtle than previously thought and reveal marked differences between parasitic and non-parasitic nematodes

Rapid Molecular Assays for Specific Detection and Quantitation of Loa loa Microfilaremia

Loa loa is a filarial nematode that infects over 10 million people in Africa. Most infections cause no symptoms, but individuals with large numbers of blood-stage microfilariae are at risk for fatal reactions to ivermectin, an antiparasitic agent used to treat and prevent infections with Onchocerca volvulus, a related filarial parasite that may occur alongside L. loa. To address the urgent need for a point-of-care L. loa diagnostic assay, we screened a Loa microfilaria gene expression library and identified 18 Loa-specific DNA targets. From two targets, we developed a novel, rapid quantitative PCR assay for estimating L. loa microfilaria burden. The assay is highly sensitive (detects a single microfilaria in 20 µL of blood) and correlates well with microfilaria counts obtained with conventional microscopic techniques. The assay is species-specific for L. loa compared with related filarial parasites (including O. volvulus) and can be used in its current form in resource-rich areas as a diagnostic tool for L. loa infection. Although modifications will be required to make point-of-care use feasible, our assay provides a proof of concept for a potentially valuable tool to identify individuals at risk for adverse reactions to ivermectin and to facilitate the implementation of filarial control programs

Chromosomal Expression of the Haemophilus influenzae Hap Autotransporter Allows Fine-Tuned Regulation of Adhesive Potential via Inhibition of Intermolecular Autoproteolysis

The Haemophilus influenzae Hap autotransporter is a nonpilus adhesin that promotes adherence to respiratory epithelial cells and selected extracellular matrix proteins and facilitates bacterial aggregation and microcolony formation. Hap consists of a 45-kDa outer membrane translocator domain called Hapβ and a 110-kDa extracellular passenger domain called HapS. All adhesive activity resides within HapS, which also contains protease activity and directs its own secretion from the bacterial cell surface via intermolecular autoproteolysis. In the present study, we sought to determine the relationship between the magnitude of Hap expression, the efficiency of Hap autoproteolysis, and the level of Hap-mediated adherence and aggregation. We found that a minimum threshold of Hap precursor was required for autoproteolysis and that this threshold approximated expression of Hap from a chromosomal allele, as occurs in H. influenzae clinical isolates. Chromosomal expression of wild-type Hap was sufficient to promote significant adherence to epithelial cells and extracellular matrix proteins, and adherence was enhanced substantially by inhibition of autoproteolysis. In contrast, chromosomal expression of Hap was sufficient to promote bacterial aggregation only when autoproteolysis was inhibited, indicating that the threshold for Hap-mediated aggregation is above the threshold for autoproteolysis. These results highlight the critical role of autoproteolysis and an intermolecular mechanism of cleavage in controlling the diverse adhesive activities of Hap

The Haemophilus influenzae Hap Autotransporter Binds to Fibronectin, Laminin, and Collagen IV

Nontypeable Haemophilus influenzae (NTHI) initiates infection by colonizing the upper respiratory tract mucosa. NTHI disease frequently occurs in the context of respiratory tract inflammation, where organisms encounter damaged epithelium and exposed basement membrane. In this study, we examined interactions between the H. influenzae Hap adhesin and selected extracellular matrix proteins. Hap is an autotransporter protein that undergoes autoproteolytic cleavage, with release of the adhesive passenger domain, Hap(s), from the bacterial cell surface. We found that Hap promotes bacterial adherence to purified fibronectin, laminin, and collagen IV and that Hap-mediated adherence is enhanced by inhibition of autoproteolysis. Adherence is inhibited by pretreatment of bacteria with a polyclonal antiserum recognizing Hap(s). Purified Hap(s) binds with high affinity to fibronectin, laminin, and collagen IV but not to collagen II. Binding of Hap(s) to fibronectin involves interaction with the 45-kDa gelatin-binding domain but not the 30-kDa heparin-binding domain of fibronectin. Taken together, these observations suggest that interactions between Hap and extracellular matrix proteins may play an important role in NTHI colonization of the respiratory tract

Identification of Wb123 as an Early and Specific Marker of <em>Wuchereria bancrofti</em> Infection

<div><h3>Background</h3><p>The current antibody tests used for monitoring in lymphatic filariasis (LF) elimination programs suffer from poor specificity because of the considerable geographical overlap with other filarial infections such as <em>Loa loa (</em>Ll<em>)</em>, <em>Onchocerca volvulus (</em>Ov<em>)</em>, and <em>Mansonella</em> perstans (Mp).</p> <h3>Methods</h3><p>Using bioinformatics to assemble into contigs 2048 expressed sequence tags (ESTs) from the L3 infective larvae of <em>W. bancrofti</em> (Wb), these were next assessed for homology to known proteins and nucleotides and to similar assemblies of L3 larval ESTs of <em>B. malayi</em> (Bm – n = 5068), Ov (n<em> = </em>4166), and Ll (n<em> = </em>3315). Nineteen potential L3- and Wb- and/or Bm-specific antigens were identified. Sixteen of the 19 antigens could be expressed as fusion proteins with Renilla luciferase (Ruc); these were used in a rapid Luciferase Immunopreciptation System (LIPS) assay.</p> <h3>Results</h3><p>One of the 16 expressed antigens (Wb123) was both highly immunogenic and specific for Wb. Using Wb123-based IgG and IgG4 LIPS assays on well-defined sera from normal North Americans and those infected exclusively with intestinal helminths, we could detect all of the Wb-infected individuals (from diverse geographic regions) with 100% sensitivity and 100% specificity. Using sera from exclusively Ll-infected, Ov-infected Mp-infected or Bm-infected subjects as the negative comparator, the sensitivities were between 98–100% and the specificities ranged between 84–100% (for IgG anti-Wb123) and between 98–100% (for IgG4 anti-Wb123). Blinded assessments using panels of sera from various Wb-, Bm- or non-Wb helminth-infected subjects demonstrated equally high degrees of sensitivity and specificity.</p> <h3>Significance</h3><p>We have identified a Wb-encoded antigen that can be used both as a rapid, high throughput tool to diagnose individual <em>Wb</em> infections and as a sensitive method for early detection of recrudescent infections in areas of control and for mapping new areas of Wb transmission.</p> </div

Real-time PCR (qPCR) assays incorporating LLMF72 and LLMF269 targets.

<p>Shown are the results of Taqman qPCR assays using primer/probe sets for LLMF72 (circles) and LLMF269 (squares). Each point represents the mean and standard deviation of duplicate assays. Panel A: limiting dilutions of cDNA template created by reverse transcription of <i>L. loa</i> total RNA. Panel B: limiting dilutions of highly purified <i>L. loa</i> genomic DNA template. Panel C: total DNA extracted from whole blood spiked with limiting dilutions of intact <i>L. loa</i> microfilariae.</p

Antibodies to Wb123 fail to normalize following definitive anti-filarial treatment.

<p>IgG (Top panel) and IgG4 (Middle panel) anti-Wb123 antibodies in two individual patients followed longitudinally over an 8 (red line) or 17 (blue line) year period following definitive anti-filarial chemotherapy. Circulating antigen levels in the same two subjects is shown (Bottom panel). Gray shaded boxes show the normal range for each assay.</p

Wb123-specific IgG and IgG4 in <i>W. bancrofti</i> and related helminth infections.

<p>IgG and IgG4 antibodies to Wb123 distinguish <i>Wuchereria bancrofti</i>-infected subjects from those with related filarial and non-filarial helminth infection. IgG (Left panel) or IgG4 (Right panel) antibodies to Wb123 in individual serum samples from those with <i>Wuchereria bancrofti</i> (Wb), <i>Onchocerca volvulus</i> (Ov), <i>Loa loa</i> (Ll), <i>Mansonella perstans</i> (Mp), other helminth infections, or no infection (Normal). Each dot represents an individual sample and the horizontal line is the geometric mean (GM). The dashed line represents the cutoff between negative and positive based on ROC analysis.</p