Linear epitopes in Onchocerca volvulus vaccine candidate proteins and excretory-secretory proteins.

In our previous study, a proteome-wide screen was conducted to identify linear epitopes in this parasite's proteome, resulting in the discovery of three immunodominant motifs. Here, we investigated whether such antigenic peptides were found in proteins that were already known as vaccine candidates and excretome/secretome proteins for Onchocerca volvulus This approach led to the identification of 71 immunoreactive stretches in 46 proteins. A deep-dive into the immunoreactivity profiles of eight vaccine candidates that were chosen as most promising candidates for further development (Ov-CPI-2, Ov-ALT-1, Ov-RAL-2, Ov-ASP-1, Ov-103, Ov-RBP-1, Ov-CHI-1, and Ov-B20), resulted in the identification of a poly-glutamine stretch in Ov-RAL-2 that has properties for use as a serodiagnostic marker for O. volvulus infection. A peptide ELISA was developed, and the performance of this assay was evaluated. Based on this assessment, it was found that this assay has a sensitivity of 75.0% [95% CI: 64.9%-83.5%] and a specificity of 98.5% [95% CI: 94.6%-99.8%]. Furthermore, 8.7% reactivity in Asian parasite-infected individuals (8 out of 92) was observed. Besides this identification of a linear epitope marker, the information on the presence of linear epitopes in vaccine candidate proteins might be useful in the study of vaccines for river blindness.

Evaluation of the Diagnostic Performance of Onchocerca volvulus Linear Epitopes in a Peptide Enzyme-Linked Immunosorbent Assay.

Diagnostic tools for the detection of infection with Onchocerca volvulus are presently limited to microfilaria detection in skin biopsies and serological assessment using the Ov16 immunoglobulin G4 (IgG4) rapid test, both of which have limited sensitivity. We have investigated the diagnostic performance of a peptide enzyme-linked immunosorbent assay (ELISA) based on immunodominant linear epitopes previously discovered. Peptides that were used in these assays were designated O. volvulus motif peptides (OvMP): OvMP-1 (VSV-EPVTTQET-VSV), OvMP-2 (VSV-KDGEDK-VSV), OvMP-3 (VSV-QTSNLD-VSV), and the combination of the latter two, OvMP-23 (VSV-KDGEDK-VSV-QTSNLD-VSV). Sensitivity (O. volvulus infection), specificity (non-helminth infections), and cross-reactivity (helminth infections) were determined using several panels of clinical plasma isolates. OvMP-1 was found to be very sensitive (100%) and specific (98.7%), but showed substantial cross-reactivity with other helminths. Of the other peptides, OvMP-23 was the most promising peptide with a sensitivity of 92.7%, a specificity of 100%, and a cross-reactivity of 6%. It was also demonstrated that these peptides were immunoreactive to IgG but not IgG4, and there is no correlation with the Ov16 IgG4 status, making them promising candidates to complement this already available test. Combination of the Ov16 IgG4 rapid test and OvMP-23 peptide ELISA led to a sensitivity of 97.3% for the detection of O. volvulus infection, without compromising specificity and with minimal impact on cross-reactivity. The available results open the opportunity for a "clinical utility use case" discussion for improved O. volvulus epidemiological mapping.

Characterizing Reactivity to Onchocerca volvulus Antigens in Multiplex Bead Assays.

Multiplex bead assays (MBAs) may provide a powerful integrated tool for monitoring, evaluation, and post-elimination surveillance of onchocerciasis and co-endemic diseases; however, the specificity and sensitivity of Onchocerca volvulus antigens have not been characterized within this context. An MBA was developed to evaluate three antigens (OV-16, OV-17, and OV-33) for onchocerciasis. Receiver operating characteristics (ROC) analyses were used to characterize antigen performance using a panel of 610 specimens: 109 O. volvulus-positive specimens, 426 non-onchocerciasis controls with filarial and other confirmed parasitic infection, and 75 sera from patients with no other parasitic infection. The IgG and IgG4 assays for OV-16 demonstrated sensitivities of 95.4% and 96.3%, and specificities of 99.4% and 99.8%, respectively. The OV-17 IgG and IgG4 assays had sensitivities of 86.2% and 76.1% and specificities of 79.2% and 82.8%. For OV-33, the IgG and IgG4 assays had sensitivities of 90.8% and 96.3%, and specificities of 96.8% and 98.6%. The OV-16 IgG4-based MBA had the best assay characteristics, followed by OV-33 IgG4. The OV-16 IgG4 assay would be useful for monitoring and evaluation using the MBA platform. Further evaluations are needed to review the potential use of OV-33 as a confirmatory test in the context of program evaluations.

Stage-Specific Transcriptome and Proteome Analyses of the Filarial Parasite Onchocerca volvulus and Its Wolbachia Endosymbiont.

Onchocerciasis (river blindness) is a neglected tropical disease that has been successfully targeted by mass drug treatment programs in the Americas and small parts of Africa. Achieving the long-term goal of elimination of onchocerciasis, however, requires additional tools, including drugs, vaccines, and biomarkers of infection. Here, we describe the transcriptome and proteome profiles of the major vector and the human host stages (L1, L2, L3, molting L3, L4, adult male, and adult female) of Onchocerca volvulus along with the proteome of each parasitic stage and of its Wolbachia endosymbiont (wOv). In so doing, we have identified stage-specific pathways important to the parasite's adaptation to its human host during its early development. Further, we generated a protein array that, when screened with well-characterized human samples, identified novel diagnostic biomarkers of O. volvulus infection and new potential vaccine candidates. This immunomic approach not only demonstrates the power of this postgenomic discovery platform but also provides additional tools for onchocerciasis control programs. IMPORTANCE: The global onchocerciasis (river blindness) elimination program will have to rely on the development of new tools (drugs, vaccines, biomarkers) to achieve its goals by 2025. As an adjunct to the completed genomic sequencing of O. volvulus, we used a comprehensive proteomic and transcriptomic profiling strategy to gain a comprehensive understanding of both the vector-derived and human host-derived parasite stages. In so doing, we have identified proteins and pathways that enable novel drug targeting studies and the discovery of novel vaccine candidates, as well as useful biomarkers of active infection.

Criteria for the differentiation between young and old Onchocerca volvulus filariae.

Drugs exist that show long-lasting inhibition of embryogenesis and microfilaria production or macrofilaricidal activity against Onchocerca volvulus. Therefore, the patients have to be followed-up for several years. Clinical drug trials have to be performed in areas with ongoing transmission to assess the efficacy on younger worms. In addition, future vaccine trials may also require demonstrating efficacy against establishment of new worms. For the evaluation of the efficacy, it is necessary to differentiate between older worms, which were exposed to the drug, and younger worms newly acquired after drug treatment or vaccination. Here, we describe criteria for the differentiation between young and old filariae based on histological studies of worms with a known age from travellers, or from children, or patients living in areas with interrupted transmission in Burkina Faso, Ghana or Uganda. Older worms were larger and presented degenerated tissues. Gomori's iron stain showed that the worms accumulated more iron with increasing age, first in the gut and later in other organs. Using an antibody against O. volvulus lysosomal aspartic protease, the gut of young worms was stained only weakly; whereas, it was stronger labelled in older worms, accompanied by additional staining of hypodermis and epithelia. Using morphological and immunohistological criteria, it was possible to differentiate young (1-3 years old) from older females and to identify young males.

Immunohistological studies on Onchocerca volvulus paramyosin.

Paramyosin is a muscular protein exclusively found in invertebrate species, which has been proposed as a vaccine candidate against infections with Schistosoma mansoni and Brugia malayi. Here, we report the studies on the distribution of Onchocerca volvulus paramyosin, designated OvPmy, in different O. volvulus stages by immunohistochemistry using rabbit antibodies raised against the recombinant OvPmy protein as well as the induction of the human humoral immune response to OvPmy. To conduct the studies, OvPmy was expressed in Escherichia coli as a fusion protein to raise the rabbit antibodies. The recombinant OvPmy was tested in immunoblots using sera from individuals living in an area hyperendemic for onchocerciasis in Liberia, West Africa. The antibodies used here localised paramyosin exclusively in the muscle tissue of O. volvulus as well as Onchocerca ochengi. No extracellular compartments, such as the cuticle or the lumina of the pseudocoeloma cavity, were labelled; however, labelling was seen in microfilarial fragments taken up by host immune cells, such as giant cells. It was recognised by anti-paramyosin antibodies of a group of onchocerciasis patients.

The nematode parasite Onchocerca volvulus generates the transforming growth factor-beta (TGF-beta).

Transforming growth factor-beta (TGF-beta) is a highly conserved cytokine that has a well-known regulatory role in immunity, but also in organ development of most animal species including helminths. Homologous tgf-b genes and mRNA have been detected in the filaria Brugia malayi. The in situ protein expression is unknown for filariae. Therefore, we examined several filariae for the expression and localization of latent (stable) TGF-beta in adult and larval stages. A specific goat anti-human latency associated protein (LAP, TGF-beta 1) antibody, purified by affinity chromatography, was used for light and electron microscopic immunohistochemistry. Adult Onchocerca volvulus, Onchocerca gibsoni, Onchocerca ochengi, Onchocerca armillata, Onchocerca fasciata, Onchocerca flexuosa, Wuchereria bancrofti, Dirofilaria sp., B. malayi, and infective larvae of W. bancrofti reacted with the antibody. Labeling of worm tissues varied between negative and all degrees of positive reactions. Latent TGF-beta was strongly expressed adjacent to the cell membranes of the hypodermis, epithelia, and muscles and adjacent to many nuclei in all organs. TGF-beta was well expressed in worms without Wolbachia endobacteria eliminated by doxycycline treatment. Pleomorphic neoplasms in O. volvulus were also labeled. We conclude that latent TGF-beta protein is expressed by filariae independently of Wolbachia, possibly regulating worm tissue homeostasis.

Characterization of a novel filarial serine protease inhibitor, Ov-SPI-1, from Onchocerca volvulus, with potential multifunctional roles during development of the parasite.

A novel filarial serine protease inhibitor (SPI) from the human parasitic nematode Onchocerca volvulus, Ov-SPI-1, was identified through the analysis of a molting third-stage larvae expressed sequence tag dataset. Subsequent analysis of the expressed sequence tag datasets of O. volvulus and other filariae identified four other members of this family. These proteins are related to the low molecular weight SPIs originally isolated from Ascaris suum where they are believed to protect the parasite from host intestinal proteases. The two Ov-spi transcripts are up-regulated in the molting larvae and adult stages of the development of the parasite. Recombinant Ov-SPI-1 is an active inhibitor of serine proteases, specifically elastase, chymotrypsin, and cathepsin G. Immunolocalization of the Ov-SPI proteins demonstrates that the endogenous proteins are localized to the basal layer of the cuticle of third-stage, molting third-stage, and fourth-stage larvae, the body channels and multivesicular bodies of third-stage larvae and the processed material found between the two cuticles during molting. In O. volvulus adult worms the Ov-SPI proteins are localized to the sperm and to eggshells surrounding the developing embryos. RNA interference targeting the Ov-spi genes resulted in the specific knockdown of the transcript levels of both Ov-spi-1 and Ov-spi-2, a loss of native proteins, and a significant reduction in both molting and viability of third-stage larvae. We suggest the Ov-SPI proteins play a vital role in nematode molting by controlling the activity of an endogenous serine protease(s). The localization data in adults also indicate that these inhibitors may be involved in other processes such as embryogenesis and spermatogenesis.

Phosphocholine-containing, zwitterionic glycosphingolipids of adult Onchocerca volvulus as highly conserved antigenic structures of parasitic nematodes.

Human Onchocerca volvulus infection sera were found to recognize zwitterionic glycolipids of O. volvulus and to cross-react with those of other parasitic nematodes (Ascaris suum, Setaria digitata and Litomosoides sigmodontis). By the use of an epitope-specific monoclonal antibody, zwitterionic glycolipids of all these nematode species were observed to contain the antigenic determinant phosphocholine. A hyperimmune serum specific for arthro-series glycolipid structures reacted with the various neutral glycolipids of all these nematodes, which demonstrated that their oligosaccharide moieties belonged to the arthro-series of protostomial glycolipids. These results indicated that arthro-series glycosphingolipids carrying, in part, phosphocholine substituents, represent highly conserved, antigenic glycolipid markers of parasitic nematodes. Three glycolipid components of the O. volvulus zwitterionic fraction were structurally characterized by matrix-assisted laser-desorption/ionization time-of-flight MS, methylation analysis and exoglycosidase treatment. Their chemical structures were elucidated to be phosphocholine-6GlcNAc(beta1-3)Man(beta1-4)Glc(1-1)ceramide, GalNAc(beta1-4)[phosphocholine-6]GlcNAc(beta1-3)Man(beta1-4)Glc(1-1) ceramide and Gal(alpha1-3)GalNAc(beta1-4)[phosphocholine-6]GlcNAc(beta1-3)Man(beta 1-4)Glc(1-1)ceramide for the zwitterionic ceramide tri-, tetra- and penta-hexosides respectively. The ceramide composition was found to be dominated by 2-hydroxylated docosanoic (C(22h:0)), tricosanoic (C(23h:0)) and tetracosanoic (C(24h:0)) acids, and C(17) sphingosine (C(d17:1)) (where (h) is hydroxylated and (d) is dihydroxylated).

Characterization of the heat-shock protein 60 chaperonin from Onchocerca volvulus.

Chaperonin 60 (cpn60) belongs to the group of ubiquitous molecular chaperones that comprise the heat shock proteins, nucleoplasmins and chaperonins. Antibodies to recombinant CPN60 from humans was used to screen a cDNA library of Onchocerca volvulus and antigen-positive clones were selected. Sequencing of the DNA inserts confirmed their identity as cpn60 transcripts. These are distinct from a cpn60 sequence recorded previously from O. volvulus (GenBank accession number Y09416) that appears to be of endobacterial origin, rather than derived from the parasite itself. The full-length sequence of the cDNA (designated Ov-cpn60) codes for a protein of 64.3kDa (598 amino acid residues) and shares significant identity with homologous gene products from Caenorhabditis elegans (72%), humans (69%), yeast (53%) and Escherichia coli (50%). The endobacterial and parasite sequences are 41% conserved. Ov-CPN60 migrates with an apparent molecular mass of 65kDa on SDS-PAGE and is present in all life-cycle stages, as determined by immunoblotting with rabbit antibodies raised against the recombinant protein. Immunogold electron microscopy identified the protein within mitochondria, as expected, but also in extra-mitochondrial sites, including inclusion bodies of the glandular oesophagus (in infective larvae), the uterine wall, cytosol of developing spermatids, and the hypodermis and cuticle. Endobacteria were also labelled, indicating cross-reactivity between CPN60 from the parasite and its intracellular symbiont. In human infections, serum antibodies to Ov-CPN60 were present in only 11% of cases from Ecuador, but in 81-89% of subjects in three separate foci from West Africa. There was no relationship between antibody levels and age, sex, or infection intensity, and no consistent association between the serological response and immune status. An evaluation of antibody specificities in individual sera revealed a mixture of parasite-specific and host crossreactive anti-CPN60 antibodies, the ratio of which varied amongst geographic areas. It is concluded that antibody responses to Ov-CPN60 are unlikely to contribute either to host protection or pathology in onchocerciasis.

Reports from the cutting edge of parasitic genome analysis.

This new feature in Parasitology Today will host reports from the laboratories involved in genomics of parasites, be that sequencing, mapping or 'functional genomics' - the mining and analysis of the sequence datasets, and the development of postgenomics tools to examine gene expression, response to drugs and population variability. It will publicize new technology to wider audiences, let communities of researchers know about novel resources (particularly those available through the World Wide Web) and highlight significant advances in the understanding of parasitic genomes through functional genomics.

Structural studies of N-glycans of filarial parasites. Conservation of phosphorylcholine-substituted glycans among species and discovery of novel chito-oligomers.

N-Type glycans containing phosphorylcholine (PC-glycans), unusual structures found in the important human pathogens filarial nematodes, represent a novel target for chemotherapy. Previous work in our laboratories produced compositional information on the PC-glycan of ES-62, a secreted protein of the rodent parasite Acanthocheilonema viteae. In particular, we established using fast atom bombardment mass spectrometry (MS) analysis that PC was attached to a glycan with a trimannosyl core, with and without core fucosylation, carrying between one and four additional N-acetylglucosamine residues. In the present study, we demonstrate that this structure is conserved among filarial nematodes, including the parasite of humans, Onchocerca volvulus, for which new drugs are most urgently sought. Furthermore, by employing a variety of procedures, including collision-activated dissociation MS-MS analysis and matrix-assisted laser desorption MS analysis, we reveal that surprisingly, filarial nematodes also contain N-linked glycans, the antennae of which are composed of chito-oligomers. To our knowledge, this is the first report of such structures in a eukaryotic glycoprotein.

Onchocerca volvulus: immunolocalization of the extracellular CuZn superoxide dismutase using antibodies raised against a 15-mer epitope of this enzyme.

The study describes the immunohistological localization of the extracellular CuZn superoxide dismutase (SOD2) in the parasitic nematode Onchocerca volvulus. Using specific antiserum raised against a 15-amino-acid peptide from the N-terminal region of the mature protein, this enzyme is detected primarily in the intestinal epithelium of the adult worms and to a lesser extent in the muscle cells of the uterine wall. A blocking experiment with the SOD2 peptide reduced the staining significantly, confirming specificity. The localization profile of SOD2 correlates extremely well with the localization of iron deposits in the gut and uterine muscle cells of adult O. volvulus. The detection of SOD2 in the functional intestine of O. volvulus, together with the evidence that it is a secreted protein, indicates that this enzyme in parasitic nematodes is in a position to interact with host molecules. It also demonstrates the accessibility of the parasite enzyme to an inhibitor or blocking antibody.

The peroxidoxin 2 protein of the human parasite Onchocerca volvulus: recombinant expression, immunolocalization, and demonstration of homologous molecules in other species.

The peroxidoxin protein of the filarial parasite Onchocerca volvulus (OvPXN-2) belongs to a group of highly conserved antioxidant molecules. For a more detailed characterization of this protein and for determination of its expression pattern the OvPXN-2 protein was recombinantly expressed as a His-tagged protein. Under reducing conditions the recombinant protein had an apparent molecular mass of 28 kDa. Considering the size of the His-tag and the FLAG epitope introduced to the recombinant protein, this size is in agreement with that of the native protein identified in O. volvulus extract. Antiserum raised against the recombinant protein was used for immunolocalization. In O. volvulus the antigen is predominantly expressed in the hypodermis and particularly the lateral and median chords show high levels of expression. The protein is also expressed strongly in the hypodermis of infective larvae and more weakly in microfilariae. Related cross-reacting proteins were detected in several Onchocerca species and other filariae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with Western blotting revealed proteins with almost identical mobility in extracts prepared from O. ochengi, O. gibsoni, and Dirofilaria immitis.

Cloning and characterization of cDNAs encoding beta-tubulin from Dirofilaria immitis and Onchocerca volvulus.

Beta-Tubulin is the target for the benzimidazole anthelmintics. Unfortunately, none of these drugs is clinically useful against adult filariae. However, beta-tubulin has been shown to be a target for antibody-based toxicity to Brugia pahangi. We cloned and characterized cDNAs encoding beta-tubulin from 2 filariae, Dirofilaria immitis and Onchocerca volvulus, to explore possible explanations for benzimidazole insensitivity among adult filariae and the likelihood that epitopes of beta-tubulin could be used as antigens for a broad-spectrum filarial vaccine. The proteins predicted by these cDNAs were almost identical to the beta-tubulin previously reported from B. pahangi but were less similar to a beta-tubulin cDNA from Onchocerca gibsoni. We cloned the genomic locus for the O. volvulus beta-tubulin cDNA and compared its organization to the reported genomic loci for beta-tubulin in B. pahangi and O. gibsoni. The comparison reinforces the conclusion that the published O. gibsoni gene is in a different family, possibly the beta2 family previously described in B. pahangi. The substitution of tyr for phe at position 200 of beta-tubulin is associated with benzimidazole resistance. All 4 filarial beta-tubulins are predicted to encode phe at this position, suggesting that filarial beta-tubulin is not inherently insensitive to the benzimidazoles. A monoclonal antibody that recognizes the COOH terminus of B. pahangi beta-tubulin is lethal to this parasite in culture. The COOH terminal region is the most variable among the different isotypes of beta-tubulin and distinguishes mammalian from nematode tubulins. This region is highly conserved in 3 of the filarial beta-tubulins.

The Ov20 protein of the parasitic nematode Onchocerca volvulus. A structurally novel class of small helix-rich retinol-binding proteins.

Ov20 is a major antigen of the parasitic nematode Onchocerca volvulus, the causative agent of river blindness in humans, and the protein is secreted into the tissue occupied by the parasite. DNA encoding Ov20 was isolated, and the protein was expressed in Escherichia coli. Fluorescence-based ligand binding assays show that the protein contains a high affinity binding site for retinol, fluorescent fatty acids (11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid, dansyl-DL-alpha-aminocaprylic acid, and parinaric acid) and, by competition, oleic and arachidonic acids, but not cholesterol. The fluorescence emission of dansylated fatty acids is significantly blue-shifted upon binding in comparison to similarly sized beta-sheet-rich mammalian retinol- and fatty acid-binding proteins. Secondary structure prediction algorithms indicate that a alpha-helix predominates in Ov20, possibly in a coiled coil motif, with no evidence of beta structures, and this was confirmed by circular dichroism. The protein is highly stable in solution, requiring temperatures in excess of 90 degrees C or high denaturant concentrations for unfolding. Ov20 therefore represents a novel class of small retinol-binding protein, which appears to be confined to nematodes. The retinol binding activity of Ov20 could possibly contribute to the eye defects associated with onchocerciasis and, because there is no counterpart in mammals, represents a strategic target for chemotherapy.

Onchocera volvulus: characterization of a highly immunogenic Gln-rich protein.

A pool of sera from individuals classified as putatively immune (PI) to Onchocerca volvulus infection was employed in the screening of a fourth-stage larval cDNA expression library. A highly immunogenic clone, encoding the Ov 53/80 protein, was identified. The full length cDNA of clone 4.21 contained 2527 nucleotides encoding 769 amino acids of which 100 are glutamine residues (13%). Antibodies raised against recombinant protein encoded by a partial cDNA sequence (clone 73-k) recognized a 53 and 80 kDa protein in O. volvulus larval and adult parasite extracts, respectively. The antibodies localized the native protein in the cuticle, hypodermis, secretory vesicles and in granules of the glandular esophagus of larvae and in the hypodermis and the cuticle of adult worms. The recombinant 73-k polypeptide (r73) was recognized by 90-100% of sera from PI and infected individuals from Liberia, but only by 67% of similar groups from Ecuador. r73 specific IgG2 and IgG3 levels in the PI from Liberia and Ecuador, respectively, were significantly lower than in the infected, whereas the r73 specific IgG1/IgG3 or IgG1/IgG2 in the PI and the infected individuals from Liberia or Ecuador, respectively, were similar. The IgG4 specific antibody response in the PI from Liberia and Ecuador were lower than in the infected. The T-cell proliferative responses to r73 in infected individuals from Cameroon were found to be inversely correlated with their levels of microfilariae.

OvB20, an Onchocerca volvulus-cloned antigen selected by differential immunoscreening with vaccination serum in a cattle model of onchocerciasis.

A cDNA of Onchocerca volvulus has been isolated by differential immunoscreening of an adult worm expression library using sera raised in cattle against the related species, O. lienalis. It was selected because of its recognition by antibodies from cattle immunized with irradiated third-stage (L3) larvae and not by antibodies from animals infected with non-irradiated larvae. The original 311-bp clone was used to isolate a 1478-bp cDNA. Designated OvB20, this codes for 460 amino acid residues, hybridizes with a approximately 1.6 kBp transcript and appears to be transcribed from a filarial-specific, single copy gene. It is expressed in developing stages from embryo to L4 larva, but not in the adult. The product of OvB20 appears to undergo co- or post-translational processing: in vitro transcription and translation give rise to a polypeptide consistent with the deduced amino acid sequence (approximately 52 kDa), whilst products of 52 and 65 kDa are detected in larvae by immunoblotting and following in vitro translations to which exogenous microsomes have been added. A 42-kDa protein was also detected in all in vitro translations. No homologous genes were found in the computer databases, although there are regions of weak sequence similarity with C-reactive proteins. The functional role of OvB20 may warrant further attention, as it has recently been shown that the recombinant protein confers host protection against a related rodent filaria following active immunization (Taylor, M.J., Abdel-Wahab, N., Wu, Y., Jenkins, R.E. and Bianco, A.E. (1995) Onchocerca volvulus larval antigen, OvB20 induces partial protection in a rodent model of onchocerciasis. Infect. Immun. 63, 4417-4422).