Salivary Antigens Research Articles

CpG oligodeoxynucleotide vaccination suppresses IgE induction but may fail to down-regulate ongoing IgE responses in mice.

Antigen-specific IgE plays an important role in the pathogenesis of allergic disorders. Immunostimulatory CpG motifs (CpG) in bacterial DNA or synthesized oligodeoxynucleotides (ODN) are gaining recognition as potential immunomodulators for switching on protectiveT(h)1-mediated immunity and preventing or potentially inhibiting T(h)2-dependent allergic responses. To date, allergic models used in CpG ODN studies have been established by immunization of mice with allergen in the presence of adjuvant. This, in addition to failure to assess specific IgE production in most of the studies, has limited understanding of the role of CpG ODN vaccination in allergic responses. Here, we examine the effects of synthesized CpG ODN on both developing and ongoing IgE responses in mice sensitized using a recombinant mosquito salivary antigen (rAed a 2) without adjuvant. Pretreatment of mice with CpG ODN mixed with rAed a 2 successfully inhibited subsequent induction of serum rAed a 2-specific IgE (but not IgG1) and antigen-induced IL-4 and IL-5 production in spleen cells. This was associated with an increase of serum IgG2a and IL-12, and increased IFN-gamma and IL-12 production by spleen cells. In this model, however, co-administration of CpG ODN with rAed a 2 to presensitized mice failed to down-regulate ongoing IgE responses despite significant up-regulation of serum IL-12 and specific IgG2a. Strikingly, a transient skin delayed-type hypersensitivity reaction occurred in CpG ODN-treated mice. These observations provide a new insight into the potential therapeutic application of CpG ODN to allergic disorders.

Species-specific antigens in salivary glands of phlebotomine sandflies.

Saliva inoculated by sandfly females during feeding stimulated production of high levels of anti-saliva antibodies. To determine whether 3 species of the genus Phlebotomus have species-specific salivary antigens we performed dot-blots and immunoblots using sera from mice, hamsters and rabbits repeatedly bitten by sandflies. Important differences were found in the antigen components of the salivary gland lysates (SGL) of Phlebotomus papatasi, P. perniciosus and P. halepensis. In total 4-9 species-specific antigens were detected in each species by immunoblotting. Cross-reactivity was not detected between P. papatasi and the other species tested; in the SGL of P. papatasi sera from animals bitten by this species recognized 5-7 major antigens while sera from animals bitten by other species did not react. A weak cross-reaction was observed between P. perniciosus and P. halepensis; in SGL from P. perliciosus, the sera from rabbits and hamsters bitten by this species recognized about 8 intense bands while sera from animals bitten by P. halepensis reacted weakly with up to 4 saliva polypeptides.

Boophilus annulatus (Acari: Ixodidae): cattle-acquired humoral and cell-mediated immunity against tick gut and salivary gland antigens

Assays of antibodies and cell-mediated immunity (CMI) against specific antigens in Boophilus (B.) annulatus Say gut and salivary glands were performed using Bos indicus cattle infested for prolonged periods as a model for naturally acquired immunity to ticks. It was shown that cattle sera contained several antibodies that were reactive with gut proteins (45,66,116,150, and 274 KDa Mol. Wt.) and with salivary gland proteins (66,97,150 and 205 KDa Mol. Wt.) by an enzyme-linked immunosorbent assay (ELISA) and dot immunoblot assays. CMI positive responses to both gut proteins (2000 and 150 KDa Mol. Wt.) and salivary gland protein (150 KDa Mol. Wt) were observed by a macrophage migration inhibition (MMI) test. The potential usefulness of B. annulatus antigenic proteins for vaccination of local cattle breeds is the subject of an ongoing study.

Influence of repeated infestations with pathogen-free Ixodes scapularis (Acari: Ixodidae) on in vitro lymphocyte proliferation responses of C3H/HeN mice.

In the United States, Ixodes scapularis Say has been implicated as the vector of at least three human pathogens. Tick induced modulation of host immunity is increasingly recognized as an important factor in successful transmission or establishment of tick-borne pathogens. This study was conducted to determine the effects of repeated infestations with pathogen-free I. scapularis nymphs on in vitro proliferative responses of splenic lymphocytes from C3H/HeN mice. Lymphocytes from repeatedly infested and uninfested mice were exposed to concanavalin A (Con A), Escherichia coli Castellini & Chalmers lipopolysaccharide (LPS), or I. scapularis salivary gland soluble proteins (SGSP), to determine if lymphocyte responses differed between tick-exposed and nonexposed mice. Female C3H/HeN mice were infested one to four times with pathogen-free I. scapularis nymphs, with a 14-d tick-free period between each exposure. After each infestation, tick biology parameters were measured and lymphocyte proliferative responses assessed. Acquired resistance to I. scapularis was not evident in mice subjected to tick feeding. Significant differences in the responses of lymphocytes exposed to I. scapularis SGSP were observed between infested and noninfested mice. In contrast, few differences between infested and noninfested mice were evident for lymphocytes exposed to Con A or LPS. Our results suggest that repeated exposure to I. scapularis nymphs does not affect Con A or LPS-induced proliferation of splenic lymphocytes, but significantly effects lymphocyte responses to tick salivary gland antigens.

Diagnosis of invasive amebiasis by enzyme-linked immunosorbent assay of saliva to detect amebic lectin antigen and anti-lectin immunoglobulin G antibodies.

Saliva from subjects with amebic liver abscess (ALA), acute amebic colitis, asymptomatic infection with Entamoeba histolytica or Entamoeba dispar, and uninfected controls was tested by enzyme-linked immunosorbent assay (ELISA) for the presence of E. histolytica galactose-inhibitable lectin antigen and salivary immunoglobulin (IgG) antibodies to a recombinant cysteine-rich lectin-derived protein (LC3). Salivary lectin antigen was found in 65.8% of subjects with acute colitis, compared to 22.2% of those convalescent from ALA, 10.0% with asymptomatic E. histolytica infection, 9.8% with E. dispar infection, and 2.6% of controls (subjects from the United States and study patients with nonamebic diarrhea) (P < 0.001 for each compared to values for subjects with colitis). Salivary anti-LC3 IgG antibodies were found in 92% of ALA patients regardless of duration of illness and in 83.3% of colitis patients who were symptomatic for at least 7 days (P < 0.001 compared to other study groups). Serum anti-LC3 IgG antibodies were detected in 56.3% of subjects with acute colitis, 100% of subjects with ALA or prolonged colitis, 45% of subjects with asymptomatic E. histolytica infection, 32.3% of subjects with E. dispar infection, and 23.4% of diarrhea controls. In comparison to ELISA for serum anti-LC3 IgG antibodies, the salivary lectin antigen assay is a more sensitive and specific test for acute amebic colitis. Detection of salivary anti-LC3 IgG antibodies by ELISA is an effective means for the diagnosis of ALA and prolonged cases of amebic colitis.

Salivary Film Expresses a Complex, Macromolecular Binding Site for Streptococcus sanguis

Teeth in the oral cavity are coated with a salivary film or pellicle, which lacks apparent intermolecular organization. This heterogeneous film facilitates binding of early commensal colonizing bacteria, including Streptococcus sanguis. To test the hypothesis that sufficient intermolecular organization exists in salivary films to form binding sites for S. sanguis, an in vitro model of saliva-coated teeth was probed with murine anti-idiotypical monoclonal antibodies (mAb2, anti-ids). The anti-ids were harvested from hybridomas that were developed in response to first generation murine hybridomas that produced anti-S. sanguis adhesin monoclonal antibodies (mAb1). The anti-ids (i) reacted with experimental salivary films and inhibited S. sanguis adhesion in a dose-dependent fashion. In Western blots, the anti-ids (ii) recognized a high molecular weight salivary antigen and (iii) secretory IgA (sIgA) light chain and alpha-amylase. After isolation by gel filtration from whole saliva or mixed secretory IgA and alpha-amylase, the high molecular weight component, containing amylase activity and sIgA, bound to hydroxyapatite to promote adhesion of S. sanguis. Therefore, a complex enriched in secretory immunoglobulin A and alpha-amylase forms a S. sanguis-binding site.

SALP16, a gene induced in Ixodes scapularis salivary glands during tick feeding.

Guinea pigs infested with Ixodes scapularis acquire antibody-mediated resistance to tick bites, a phenomenon known as tick-immunity. An I. scapularis salivary gland cDNA expression library was therefore probed with sera from tick-immune guinea pigs to identify antigens that elicit humoral responses in the host. Sera from sensitized guinea pigs strongly recognized 3 of 4,500 library clones in an initial screening. The open reading frames of all 3 clones encoded a putative 16.4-kD acidic protein, designated Salp16, with an N-terminal signal sequence and signal peptidase cleavage sites specific for secretory proteins. The salp16 mRNA and Salp16 protein were detected in the salivary glands of engorged, but not unfed, nymphal and adult ticks, and Salp16 was also found in the saliva of engorged ticks. Immunization with recombinant Salp16 induced high antibody titers in guinea pigs, but did not elicit tick-immunity. Salp16 is the first feeding inducible gene that has been cloned from L. scapularis. Molecular characterization of I. scapularis salivary antigens that are induced upon tick feeding should help to facilitate our understanding of tick-host interactions.

吸血に伴うオオサシガメの唾液腺タンパク質の変化および宿主動物に誘導される特異抗体の変動

吸血に伴うオオサシガメの唾液腺タンパク質の変化および宿主動物に誘導される特異抗体の変動

Kinetics and cross-species comparisons of host antibody responses to lone star ticks and American dog ticks (Acari: Ixodidae).

Understanding of the animal antibody response to tick salivary gland proteins is necessary to identify candidate antibodies that may have use as species- and feeding-duration-specific biomarkers of tick exposure in humans. The kinetics of the humoral immune response of rabbits to challenge feeding by 2 tick species [Amblyomma americanum (L.) and Dermacentor variabilis (Say)] was characterized by both enzyme-linked immunosorbent assay and immunoblot. Western blot analysis revealed that rabbits produced antibodies against both A. americanum and D. variabilis tick salivary gland antigens, with molecular weights ranging from 12.2 to 125 kDa; the antibody response against the saliva of both tick species possessed both unique and common aspects. The presence of antibody against low-molecular-mass (< 20 kDa) salivary gland antigens of A. americanum may be specific for A. americanum exposure. Antibodies directed against D. variabilis salivary gland antigens of 111, 86.3, and 85 kDa may be specific for D. variabilis exposure. The data suggest that host antibodies directed against specific tick salivary gland proteins might have use as species-specific biologic markers of tick exposure.

Characterization of Amblyomma variegatum tick saliva and salivary gland antigens inducing anti-tick immunity in boran cattle

The sequence of proteins synthesized during the feeding of Amblyomma variegatum and humoral resistance acquired by Boran cattle when exposed to A. variegatum infestations were studied. Resistance was induced by repeatedly infested cattle with all stages of the tick. Salivary gland proteins possibly responsible for the acquired immunity were studied by analysing homogenates of salivary glands (SGA) on 5-20% gradient Sodium dodecylsulphate Polyacrylamide gel electrophoresis (SDS-PAGE) and comparing them to those in the tick saliva (SA) and nymph homogenates (NH). The proteins with Mr 18, 49.5, 55, 67.5 and 160 kDs were found unique for the tick saliva. On the other hand the proteins with molecular weights of about 17, 33, 27 and 66 kDs, which seemed to have been synthesized at different times during the feeding process, were only found in SGA. When SDS-PAGE was followed with immunobloting, IgG1 subclass isolated from infestation sera recognized prominently two SGA proteins with Mr of about 58 and 53 kD and a third high molecular weight protein, less prominently. Apart from the high molecular weight protein, the rest were not recognized by the IgG2 also from the same infestation sera.

Autoantibodies in salivary hypofunction in the NOD mouse.

The nonobese diabetic (NOD) mouse exhibits spontaneous salivary gland infiltration and loss of salivary function independent of its propensity to develop diabetes, and thus can serve as a model for salivary hypofunction in Sjögren's syndrome (SS). Studies by others have indicated that this pathology depends on lymphocytes and thus may be autoimmune mediated. We have found that NOD mice four months of age and older exhibit a 90-95% reduction in pilocarpine-stimulated salivary flow (5.8 +/- 6.6 mg/5 min) as compared to age- and sex-matched C57B1/10 controls (98.4 +/- 52.3 mg/5 min). These mice simultaneously possess only sparse mononuclear cell infiltrates (averaging approximately one small and one large focus per whole-gland section) in the submandibular glands, suggesting that loss of salivary function does not require massive infiltration of the salivary glands. NOD serum autoantibodies to salivary gland proteins are demonstrable by Western blotting, and, as in a subset of SS patients, some NOD serum autoantibodies recognize neural antigens. Splenic T-cell reactivity to salivary and neural proteins can also be observed. Transfer experiments using NOD mouse serum suggest that loss of salivary function, evaluated as a decrease in pilocarpine-stimulated flow rate, can be transferred by humoral factors, possibly autoantibodies. These results suggest that autoimmune mechanisms dependent on both autoantibody and autoreactive T cells can mediate loss of salivary function, and that salivary and/or neural antigens may serve as a target for these autoimmune reactions.

Identification of a salivary vasodilator in the primary North American vector of bluetongue viruses, Culicoides variipennis.

Several species of Culicoides biting midges are important pests and vectors of pathogens affecting humans and other animals. Bluetongue is the most economically important arthropod-borne animal disease in the United States. Culicoides variipennis is the primary North American vector of the bluetongue viruses. A reddish halo surrounding a petechial hemorrhage was noticed at the site of C. variipennis blood feeding in previously unexposed sheep and rabbits. Salivary gland extracts of nonblood-fed C. variipennis injected intradermally into sheep and rabbits induced cutaneous vasodilation in the form of erythema. A local, dose-dependent erythema, without edema or pruritus, was noted 30 min after injection. Erythema was inapparent with salivary gland extracts obtained after blood feeding. This observation suggested that the vasodilatory activity was inoculated into the host skin at the feeding site. The vasodilatory activity was insoluble in ethanol and destroyed by trypsin or chymotrypsin, which indicated that vasodilation was due to a protein. The association of cutaneous vasodilation with a salivary protein was corroborated by reversed-phase, high-performance liquid chromatography (HPLC). Fractionation of salivary gland extracts by molecular sieving HPLC resulted in maximal vasodilatory activity that coeluted with a protein having a relative molecular weight (MWr) of 22.45 kD. The C. variipennis vasodilator appears to be biologically active at the nanogram level. This vasodilator likely assists C. variipennis during feeding by increasing blood flow from host superficial blood vessels surrounding the bite site. The identification of a salivary vasodilator in C. variipennis may have implications for the transmission of Culicoides-borne pathogens and in the development of dermatitis resulting from the sensitization of humans and animals to Culicoides salivary antigens.

Identification of an Ixodes ricinus salivary gland fraction through its ability to stimulate CD4 T cells present in BALB/c mice lymph nodes draining the tick fixation site.

BALB/c mice infested with larvae or nymphs of Ixodes ricinus develop in their lymph nodes a T cell-specific immune response triggered by salivary gland soluble antigens (SGA). SGA are apparently conserved in the 3 biological stages of I. ricinus ticks and are species specific. SGA derived from partially fed females I. ricinus stimulate lymph node T cells from mice infested with I. ricinus larvae or nymphs. In contrast, lymph node cells from mice infested with Amblyomma hebraeum nymphs do not respond. A chromatographic fraction enriched with a 65 kDa protein (IrSG65) isolated from salivary glands of I. ricinus partially fed females induces in vitro a specific T cell proliferation of lymph node cells from mice infested with I. ricinus nymphs. The depletion of CD4+ T cells drastically reduces the ability of lymphocytes from infested mice to proliferate after IrSG65 stimulation.

Comparative immunizing power of infections, salivary extracts, and intestinal extracts of Hyalomma marginatum marginatum in cattle

Tick concealed antigens have been successful in producing immunity that inhibits tick fertility, but require periodic revaccination and are little effective in preventing tick feeding, which is critical to stop pathogen transmission. Tick natural salivary antigens also induce important immunity, but revaccination may be unnecessary in enzootic areas. In addition these antigens may inhibit tick feeding. We immunized groups of three tick-naive calves with four prior infestations with Hyalomma marginatum marginatum, a salivary extract (SE), or an intestinal extract (IE) of the ticks. The calves were challenged with 100 pairs of homologous ticks and characteristics representing tick feeding or fertility were recorded and compared between groups. The percentage of attachment was inhibited by 46% by the infestation-generated immunity, 47% by the SE-generated immunity, and 0% by the IE-generated immunity. The percentage of engorgement was reduced 40% by the infestations, 57% by the SE, and 29% by the IE. The length of feeding was prolonged 92% by the infestations, shortened 44% by the SE, and not affected by the IE. The weight of the engorged females was decreased 67% by the infestations, 64% by the SE, and 31% by the IE. The percentage of engorged ticks that oviposited was inhibited 52% by the infestations, 27% by the SE, and 63% by the IE. The preoviposition period was prolonged 160% by the infestations, 80% by the SE, and 140% by the IE. The egg weight was reduced 60% by the infestations, 60% by the SE, and 66% by the IE. Taking into account mortality before oviposition, fertility was inhibited 88.2% by the infestations, 87.5% by SE, and 91.4% by the IE. The effect of IE immunization on tick feeding was not significant statistically.

Accelerated onset of age-related autoimmune lesions in MRL/+ mice by ovariectomy

MRL/Mp +/+ (MRL/+) mice, not bearing the lpr gene, are known to have age-related autoimmune lesions in several organs such as pancreas, salivary and lacrimal glands at 30-weeks-old or more. In this study, MRL/+ mice were ovariectomized at 4-weeks-old, and their natural histories were analysed. Ovariectomy (Ovx) of MRL/+ mice led to marked acceleration of organ-specific autoimmune lesions exclusively in the salivary and lacrimal glands at 8-weeks-old or more, whereas no significant inflammatory change was observed in the pancreas. In the vast majority of inflammatory infiltrates, CD3 +CD4 + T cells were predominant in both the salivary and lacrimal glands of Ovx-MRL/+ mice. Up-regulated expression of cytokine genes including IL-1 β, TNF- α, IL-2, interferon (IFN)- γ, and IL-6 was detected in the salivary gland of Ovx-MRL/+ mice by reverse transcriptase (RT)-PCR analysis. FACS analysis of spleen cells of Ovx-mice revealed increase in I-A k expression on B220 + cells, and autoantibody production against the salivary gland-specific antigen in sera from Ovx-MRL/+ mice, but not in control mice. These results suggest that age-related autoimmunity in the salivary and lacrimal glands were accelerated in Ovx-MRL/+ mice, and that autoreactive Th1 cells were activated associated with organ-specific autoantibody production.

Salivary antigens of the cat flea, Ctenocephalides felis felis.

The cat flea, Ctenocephalides felis felis, is the major cause of flea bite hypersensitivity (FBH) in dogs and cats, yet little progress has been reported on identifying the antigens responsible. We obtained flea salivary antigens by washing secretions from containers probed by the mouthparts of fleas, and by extracting whole flea salivary glands. Mice were exposed to feeding fleas to generate antibodies to salivary antigens injected in vivo. The sera were tested for antibodies against the salivary antigens described and against a whole flea extract; in indirect ELISA, antibodies to salivary secretions were detected in 60% of the sera from mice exposed to feeding fleas. These sera identified four protein bands at apparent MW 56, 54, 42 and 40 K which corresponded to prominent protein bands in whole salivary gland extracts identified by protein staining after SDS-PAGE. Fixed sections of whole fleas exposed to the antisera showed that only structures within the salivary glands were identified. The salivary secretions and gland extracts are now being used to study immune responses of dogs suffering from FBH.

Immunity Induced by Vaccination with Rhipicephalus appendiculatus Salivary Gland Antigens Does Not Augment Protective Immunity Acquired Naturally by Exposing Rabbits to Adult Ticks

A study was conducted using rabbits to ascertain the effects of immunity induced with salivary gland antigens (SGA) on naturally acquired host resistance, which was confirmed by exposing groups of rabbits to adult Rhipicephalus appendiculatus (Neumann) ticks. A reciprocal experiment was conducted to establish the effect of naturally acquired resistance on vaccination with SGA. After the acquisition of resistance by either method of vaccination, rabbits were then challenged with the 3 life stages of the tick. Results of the experiment demonstrated 3 phenomena: infestation of rabbits with 60 adult ticks leads to high protection in terms of reduction in the engorged weight against adult ticks, larvae, and nymphs (88.6, 31.5, and 55.9%, respectively); vaccination alone provides 53.9, 29.7, and 35.7% reduction in adult, larval, and nymphal ticks, respectively; and vaccination of rabbits already exposed to adult tick infestation appeared to have no additive immunological benefit above that already provided by adult ticks. Sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analyses revealed that antibodies with high avidity to SGA were directed to a 39-kDa polypeptide. This polypeptide was not revealed by sera from rabbits that were first infested with adult ticks. Sera from rabbits that were first vaccinated with SGA consistently reacted with the 39-kDa polypeptide. Sera from rabbits that were infested recognized strongly a 42-kDa polypeptide among 5 polypeptides in the SGA. Results of the experiment show that resistance resulting from adult tick infestation is not augmented by immunity caused by vaccination with SGA.

Acquired resistance and antibody response of raccoons ( Procyon lotor) to sequential feedings of Ixodes scapularis (Acari: Ixodidae)

Captive-bred raccoons ( Procyon lotor) developed immune resistance to infestation by the larval stage of the ixodid tick, Ixodes scapularis, the vector of Borrelia burgdorferi, following repeated applications of both nymphs and larvae. Resistance was expressed as a significant decrease in the proportion of engorged larvae recovered from each cohort. Resistance to nymphs was not noted, but, only two such cohorts were applied. Utilizing an enzyme-linked immunosorbent assay (ELISA) developed to detect raccoon serum antibodies to tick salivary gland antigens, raccoons evidenced a two to ten-fold increase in anti-tick salivary extract antibody titer following the application of two cohorts of nymphs and eights cohorts of larvae. The tick saliva antigens recognized by both pre- and post-exposure raccoon sera were evaluated by Western blotting. The production of antibodies correlated with the development of resistance to infestation, suggesting that the resistance was immune-mediated and could be measured by anti-tick salivary extract antibody titers. Resistance in exposed raccoons prevents nearly 90% of larvae from prolonged feeding. Prolonged feeding is required for engorgement and the transmission of various infectious agents, such as B. burgdorferi.

Immunological cross-reactivity between salivary gland proteins of Hyalomma anatolicum anatolicum and Boophilus microplus ticks

Immunological cross-reactivity was established between salivary gland extract antigens of Hyalomma anatolicum anatolicum (H.a.a) and Boophilus microplus (B.m.), using native-polyacrylamide gel electrophoresis (native-PAGE), immunoblotting, DOT-enzyme immunoassay (DOT-EIA) and in vivo intra-dermal skin test revealing immediate type hypersensitivity swelling reaction. Native-PAGE revealed six proteins in common of molecular weights of 60, 66, 148, 264, 300 and > 300 kDa in the salivary gland extract (SGE) of both the ticks. Four additional bands of molecular weights of 56, 64, 120 and 220 kDa were present in H.a.a. but absent in B.m. Immunoblot assay of native-PAGE transblotted proteins of H.a.a., using mice anti- H.a.a. SGE and anti- B.m. SGE immune serum revealed a common protein of 66 kDa. In DOT-EIA, anti- H.a.a. or anti- B.m. SGE immune serum gave similar antibody titres against the SGE antigens of either tick species. Intradermal inoculation of the SGE antigens of either species on cross-bred calves ( Bos indicus × Bos taurus) immunized to H.a.a. or B.m. ticks produced a strong, immediate hypersensitivity skin swelling reaction.

Isolation and Characterization of Mouse Monoclonal Antibodies Reactive with Soft Tick (Acari: Argasidae) Salivary Antigens of High Molecular Weight

The purpose of this study was to immunologically characterize soft tick salivary antigens. BALB/c mice hyperimmunized with salivary gland extract prepared from Ornithodoros talaje (Guérin-Méneville) were observed to develop high titers of antitick salivary antigen antibodies. Subsequent fusion of splenic lymphocytes from the hyperimmunized mice with SP-2/0-AG14 myeloma cells resulted in the production of 10 antitick IgM-producing hybridoma clones. Partial characterization of the respective tick antigens by gel filtration and SDS-PAGE demonstrated all 10 monoclonal antitick antibodies to be reactive with a salivary gland extract fraction containing proteins 50-110 kDa in molecular weight. Cross-reactivity assays and electrophoretic comparison of salivary gland extract specimens demonstrated similar proteins in several ixodid tick genera and species.