3D structure prediction, dynamic investigation and rational construction of an epitope-masked thermostable bovine hyaluronidase.

Hyaluronidase (HAase) from bovine testes (BTH) has long been used in broad pharmaceutical areas, while it is associated with drawbacks in aspects of solubility, immunogenicity and pharmacokinetics. These issues can be addressed by gaining structural insights and designing rational modifications to the enzyme structure, as proposed in this study. A 3D structural model was built for HAase and underwent 40 ns of molecular dynamic simulation to examine its thermostability under normal, melting, and extreme conditions. The enzyme activity of BTH was measured against temperature and pH by kinetic assays. The interaction of bovine HAase with HA and chondroitin was defined by molecular docking. Furthermore, immunogenic properties of the enzyme were explored by immunoinformatics. Thermal effects on bovine HAase structural model and the HAase interactions with its substrates were described. We identified some B- and T-cell epitopes and showed that the protein could be recognized by human immune receptor molecules. Epitope masking by adding polyethylene glycol (PEG) to amine groups of residues presenting on the surface of the protein structure was adopted as a surface modification to enhance pharmacological properties of BTH. Assays showed that PEGylated BTH had higher thermostability and similar activity compared to the native enzyme.

Evaluation of multi-epitope recombinant protein as a candidate for a contraceptive vaccine.

Contraceptive vaccine (CV) is a valuable, non-invasive, and alternative method for purposeful contraception. Sperm antigens are useful targets for producing CVs due to their specialized expression in sperm. In this study, a recombinant protein containing three main sperm epitopes (IZUMO1, SACA3, and PH-20) was designed and evaluated as CV to control fertility in male mice. The chimeric recombinant protein was expressed and purified in E. coli. Male mice were immunized by 100 µg purified protein and sera were collected to assess IgG antibodies. Evaluating the reproductive performance, immunized male mice mated with normal-fertile female mice and mating rate and the number of newborns was studied. Immunized mice were sacrificed and necropsy and histopathology studies were conducted. The results revealed that the designed chimeric protein stimulated the immune system of the mice effectively. The level of IgG antibody was significantly higher in vaccinated mouse rather than control mouse. Eighty percent of the vaccinated mice became infertile and in the remaining ones, the number of children decreased to 4-6 offspring instead of 10-12 in normal mice. Histopathological studies showed that no organs including heart, brain, lung, liver, kidney and intestine were damaged. However, Normal spermatogenesis has been disrupted and necrotic spermatogonia cells were reported in Seminiferous tubules. We concluded that the designed chimeric protein containing IZUMO1, SACA3, and PH-20 epitopes can stimulate the immune system and cause male contraception without any side effects.

Neuropatía óptica compresiva secundaria a reacción alérgica a hialuronidasa / Compressive optic neuropathy secondary to an allergic reaction to hyaluronidase

Una mujer de 58 años presentó quemosis intensa y oftalmoparesia en el ojo izquierdo 8 h después de cirugía de catarata no complicada bajo anestesia subtenoniana. Tras tratamiento corticoideo y antihistamínico, se observó recuperación de la motilidad extrínseca pero se apreciaron un edema de papila no hemorrágico y un defecto concéntrico de campo visual. El caso evolucionó a atrofia papilar con agudeza visual central preservada pero con una contracción significativa del campo visual. El estudio etiológico reveló una alergia a la hialuronidasa, usada como adyuvante a la anestesia. Esta complicación debe ser diagnosticada y tratada precozmente, puesto que el edema de los tejidos orbitarios puede dañar el nervio óptico

A 58 year-old woman presented with severe chemosis and ophthalmoparesis on her left eye 8 hours after uncomplicated cataract surgery under sub-tenon anaesthesia. Recovery of extrinsic motility was observed after corticosteroid and antihistamine treatment, but a non-haemorrhagic papillary oedema and a concentric defect of visual field were found. It progressed to papillary atrophy with preserved central vision, but with a significant visual field constriction. The aetiological study revealed an allergy to hyaluronidase that was used as adjuvant to the anaesthesia. This complication needs to be promptly diagnosed and treated, as the swelling of the orbital tissues can cause damage to the optic nerve

Exopolysaccharide Produced by Lactobacillus paracasei IJH-SONE68 Prevents and Improves the Picryl Chloride-Induced Contact Dermatitis.

Allergic disease is one of the most important and common health problems worldwide. We have previously demonstrated that a fig leaf-derived lactic acid bacterium Lactobacillus (Lb.) paracasei IJH-SONE68 produces a novel exopolysaccharide (EPS). Furthermore, we have shown that the EPS inhibits the catalytic activity of hyaluronidase (EC 3.2.1.36) promoting inflammatory reactions. To evaluate the anti-allergy and anti-inflammatory effects of the EPS, in the present study, we employed the picryl-chloride-induced delayed-type (type IV) allergy model mice, which is used to evaluate the contact dermatitis. Oral administration of the EPS was observed to reduce the ear swelling in the model mice. We also observed that the overexpression of ear interleukin-4 (T helper (Th) 2 cytokine) mRNA and the increase in serum immunoglobulin E (IgE) are repressed. However, the expression of interferon-γ (Th1 cytokine) was not accelerated in all of the allergen-challenged model mice. The improvement may be responsible for the Th2 downregulation rather than the Th1 upregulation. In addition, the symptom of immediate-type (type I) allergy model mice was improved by oral administration of the IJH-SONE68 cell (data not shown). We can conclude that the IJH-SONE68-derived EPS is useful to improve the type I and IV allergies including atopic dermatitis.

LL-37 alone and in combination with IL17A enhances proinflammatory cytokine expression in parallel with hyaluronan metabolism in human synovial sarcoma cell line SW982-A step toward understanding the development of inflammatory arthritis.

LL-37 is the only human cathelicidin-family host defense peptide and has been reported to interact with invading pathogens causing inflammation at various body sites. Recent studies showed high levels of LL-37 in the synovial-lining membrane of patients with rheumatoid arthritis, a common type of inflammatory arthritis. The present study aims to investigate the role of LL-37 on mechanisms associated with pathogenesis of inflammatory arthritis. The effects of LL-37 on the expression of proinflammatory cytokines, hyaluronan (HA) metabolism-related genes, cell death-related pathways, and cell invasion were investigated in SW982, a human synovial sarcoma cell line. Time-course measurements of proinflammatory cytokines and mediators showed that LL-37 significantly induced IL6 and IL17A mRNA levels at early time points (3-6 hr). HA-metabolism-related genes (i.e., HA synthase 2 (HAS2), HAS3, hyaluronidase 1 (HYAL1), HYAL2, and CD44) were co-expressed in parallel. In combination, LL-37 and IL17A significantly enhanced PTGS2, TNF, and HAS3 gene expression concomitantly with the elevation of their respective products, PGE2, TNF, and HA. Cell invasion rates and FN1 gene expression were also significantly enhanced. However, LL-37 alone or combined with IL17A did not affect cell mortality or cell cycle. Treatment of SW982 cells with both LL-37 and IL17A significantly enhanced IKK and p65 phosphorylation. These findings suggest that the chronic production of a high level of LL-37 may synchronize with its downstream proinflammatory cytokines, especially IL17A, contributing to the co-operative enhancement of pathogenesis mechanisms of inflammatory arthritis, such as high production of proinflammatory cytokines and mediators together with the activation of HA-metabolism-associated genes and cell invasion.

Platelet hyaluronidase-2 regulates the early stages of inflammatory disease in colitis.

Platelets are specialized cells essential for hemostasis that also function as crucial effectors capable of mediating inflammatory and immune responses. These sentinels continually survey their environment and discriminate between homeostatic and danger signals such as modified components of the extracellular matrix. The glycosaminoglycan hyaluronan (HA) is a major extracellular matrix component that coats the vascular lumen and, under normal conditions, restricts access of inflammatory cells. In response to tissue damage, the endothelial HA matrix enhances leukocyte recruitment and regulates the early stages of the inflammatory response. We have shown that platelets can degrade HA from the surface of activated endothelial cells via the enzyme hyaluronidase-2 (HYAL2) and that HYAL2 is deficient in platelets isolated from patients with inflammatory bowel disease (IBD). Platelets are known to be involved in the pathogenesis of several chronic disease states, including IBD, but they have been largely overlooked in the context of intestinal inflammation. We therefore wanted to define the mechanism by which platelet HYAL2 regulates the inflammatory response during colitis. In this study, we provide evidence that HA catabolism is disrupted in human intestinal microvascular endothelial cells isolated from patients with IBD. Furthermore, mice deficient in HYAL2 are more susceptible to an acute model of colitis, and this increased susceptibility is abrogated by transfusion of HYAL2-competent platelets. Finally, we show that platelets, via HYAL2-dependent degradation of endothelial HA, regulate the early stages of inflammation in colitis by limiting leukocyte extravasation.

Compressive optic neuropathy secondary to an allergic reaction to hyaluronidase. / Neuropatía óptica compresiva secundaria a reacción alérgica a hialuronidasa.

A 58 year-old woman presented with severe chemosis and ophthalmoparesis on her left eye 8hours after uncomplicated cataract surgery under sub-tenon anaesthesia. Recovery of extrinsic motility was observed after corticosteroid and antihistamine treatment, but a non-haemorrhagic papillary oedema and a concentric defect of visual field were found. It progressed to papillary atrophy with preserved central vision, but with a significant visual field constriction. The aetiological study revealed an allergy to hyaluronidase that was used as adjuvant to the anaesthesia. This complication needs to be promptly diagnosed and treated, as the swelling of the orbital tissues can cause damage to the optic nerve.

Immunity to LuloHya and Lundep, the salivary spreading factors from Lutzomyia longipalpis, protects against Leishmania major infection.

Salivary components from disease vectors help arthropods to acquire blood and have been shown to enhance pathogen transmission in different model systems. Here we show that two salivary enzymes from Lutzomyia longipalpis have a synergist effect that facilitates a more efficient blood meal intake and diffusion of other sialome components. We have previously shown that Lundep, a highly active endonuclease, enhances parasite infection and prevent blood clotting by inhibiting the intrinsic pathway of coagulation. To investigate the physiological role of a salivary hyaluronidase in blood feeding we cloned and expressed a recombinant hyaluronidase from Lu. longipalpis. Recombinant hyaluronidase (LuloHya) was expressed in mammalian cells and biochemically characterized in vitro. Our study showed that expression of neutrophil CXC chemokines and colony stimulating factors were upregulated in HMVEC cells after incubation with LuloHya and Lundep. These results were confirmed by the acute hemorrhage, edema and inflammation in a dermal necrosis (dermonecrotic) assay involving a massive infiltration of leukocytes, especially neutrophils, in mice co-injected with hemorrhagic factor and these two salivary proteins. Moreover, flow cytometry results showed that LuloHya and Lundep promote neutrophil recruitment to the bite site that may serve as a vehicle for establishment of Leishmania infection. A vaccination experiment demonstrated that LuloHya and Lundep confer protective immunity against cutaneous leishmaniasis using the Lu. longipalpis-Leishmania major combination as a model. Animals (C57BL/6) immunized with LuloHya or Lundep showed minimal skin damage while lesions in control animals remained ulcerated. This protective immunity was abrogated when B-cell-deficient mice were used indicating that antibodies against both proteins play a significant role for disease protection. Rabbit-raised anti-LuloHya antibodies completely abrogated hyaluronidase activity in vitro. Moreover, in vivo experiments demonstrated that blocking LuloHya with specific antibodies interferes with sand fly blood feeding. This work highlights the relevance of vector salivary components in blood feeding and parasite transmission and further suggests the inclusion of these salivary proteins as components for an anti-Leishmania vaccine.

Recombinant Human PH20: Baseline Analysis of the Reactive Antibody Prevalence in the General Population Using Healthy Subjects.

BACKGROUND: Recombinant human PH20 (rHuPH20) is used to depolymerize hyaluronan in the subcutaneous space, increasing the dispersion and absorption of co-administered drugs. While ~ 5 to 10% of rHuPH20 treatment-naïve healthy volunteers have demonstrated rHuPH20-reactive antibodies, associations with age, sex, fertility, and immune disorders remain unknown. OBJECTIVES: Using demographically diverse healthy volunteers, we assessed the prevalence of rHuPH20-reactive antibodies in the general population and potential associations with fertility and autoimmunity diseases. METHODS: In total, 896 subjects aged ≥ 12 years (767 adults; 129 children) without prior exposure to rHuPH20 were enrolled. A demographic and limited medical history review was performed, and K3-EDTA-anticoagulated plasma was analyzed for rHuPH20-reactive antibodies using a bridging immunoassay. RESULTS: Adult and pediatric positivity rates for rHuPH20-reactive antibodies were 5.2% (40/767) and 1.6% (2/129), respectively. Titers ranged from 5 to 2560 (median 30). In five antibody-positive subjects from whom repeated samples were available, antibody titers remained unchanged or decreased fourfold over periods up to 590 days. The prevalence of rHuPH20-reactive antibodies significantly increased with age (p = 0.0006) and was significantly higher in males than in females (p = 0.0010). Men who had fathered children had a significantly increased prevalence of rHuPH20-reactive antibodies than men who had not (p = 0.0036), whereas the rate of childbearing was not significantly different between rHuPH20 antibody-positive and -negative women. The prevalence between racial/ethnic groups was not significantly different, nor was the presence/absence of an autoimmune disorder. CONCLUSIONS: Approximately 1/20 of the adult population had rHuPH20-reactive antibodies. The reason remains unknown; however, no evidence for a negative effect on fertility or association with autoimmune disease was demonstrated.

IgE cross-reactivity of phospholipase A2 and hyaluronidase of Apis dorsata (Giant Asian Honeybee) and Apis mellifera (Western Honeybee) venom: Possible use of A. mellifera venom for diagnosis of patients allergic to A. dorsata venom.

Diagnostic and therapeutic reagents are unavailable for anaphylaxis arising from stings by Apis dorsata. Venom profiles and cross-reactivity of A. dorsata and Apis mellifera were compared, to ascertain whether venom of A. mellifera can be used for diagnosis in A. dorsata allergy. Both venom profiles were similar by High Performance Liquid Chromatography and SDS-PAGE. Sera of 29 of 30 (96.7%) patients with anaphylaxis to A. dorsata stings had IgE to the phospholipase-2 (PLA2) doublet (15 and 16 kDa) of A. dorsata venom by immunoblot, compared to 26 of 30 (86.7%) with the PLA2 of A. mellifera and a purified preparation of PLA2. Twelve patients (40%) with severe anaphylaxis had IgE reactivity to a 39 kDa protein band of venom of both species, a third band, identified in immunoblot as hyaluronidase. The cross-reactivity of PLA2 and hyaluronidase of A. dorsata and A. mellifera were further confirmed by immunoblot inhibition results. Twenty five of 30 (83.3%) of our patients had positive venom specific IgE (>0.35 KUA/L) reactivity to Phadia ImmunoCAPs of A. mellifera venom. The observed IgE cross reactivity suggests the possibility of using A. mellifera venom as a diagnostic test for A. dorsata venom allergy.

Crosstalk Between T Lymphocytes and Lung Fibroblasts: Generation of a Hyaluronan-Enriched Extracellular Matrix Adhesive for Monocytes.

In immunity and inflammation, T cells are often associated with stromal mesenchymal cells such as fibroblasts. Hyaluronan and proteins that associate with hyaluronan such as versican and tumor necrosis factor-inducible gene-6 (TSG-6) are extracellular matrix (ECM) components that promote leukocyte adhesion, accumulation, and activation. However, the factors responsible for producing this specialized ECM and its impact on inflammatory events are not well understood. In this study, we explored the role of T cells in stimulating lung fibroblasts to produce an ECM that impacts monocyte adhesion. We found that CD3/CD28-activated human CD4+ T cells when co-cultured with human lung fibroblasts stimulated the expression of mRNA for hyaluronan synthase 2 (HAS2) and decreased the expression of hyaluronidase 2 (HYAL2). This led to an increase in the deposition of hyaluronan that formed cable-like structures within the ECM. Co-culturing activated T cells with fibroblasts also led to increased expression and accumulation of TSG-6. Surprisingly, addition of activated CD4+ T cells to the fibroblasts reduced the expression of mRNA for versican, and increased the expression of enzymes that degrade versican, such as ADAMTS4 and ADAMTS9 (a disintegrin and metalloproteinase with a thrombospondin type-1 motif) leading to a decrease in versican in the ECM of the co-cultures. Furthermore, addition of human monocytes to these co-cultures resulted in elevated monocyte adhesion to the cable-like structures in the ECM when compared to controls. These results illustrate the importance of crosstalk between T cells and fibroblasts in promoting the generation of a matrix that is adhesive for monocytes. J. Cell. Biochem. 118: 2118-2130, 2017. © 2016 Wiley Periodicals, Inc.

The Nuts and Bolts of Immunoglobulin Treatment for Antibody Deficiency.

Immunoglobulin therapy is a key element in the management of most patients with primary immunodeficiency disease. Allergist/immunologists should be familiar with the appropriate evaluation of candidates for immunoglobulin, the characteristics of immunoglobulin products, and how to use them to provide the best care to their patients. Available immunoglobulin products appear to be equally efficacious, but they are not interchangeable. Minimizing the risk of serious adverse events and controlling minor side effects is important to ideal patient care. Immunoglobulin may be administered intravenously or subcutaneously. Individualizing the choice of immunoglobulin product, mode of administration, and site of care can optimize the clinical outcome and minimize the burden of care.

Hyaluronan: More than just a wrinkle filler.

Dermatology is a field that strives not only to alleviate skin disease (therapeutics) but also to improve the perception of wellness (cosmetics). Thus, in this special issue of Glycobiology, it seems appropriate to discuss the biology of a glycosaminoglycan, called hyaluronic acid (hyaluronan, or HA), that has become the most popular agent today for intradermal injections to improve wrinkles and other cosmetic defects. HA is a simple linear polymer in which a simple disaccharide is repeated thousands of time, thereby creating a huge hydrophilic molecule that confers a large volume of hydration and contributes to the turgor and flexibility of healthy skin. Beyond cosmetic considerations, however, HA also has important biological and physiological functions that were largely under-appreciated until recently. New research has confirmed that HA is dynamically produced by most skin cells, not only fibroblasts (the cells that make most of the skin's extracellular matrix) but also by keratinocytes in the outer protective layer (epidermis). For both fibroblasts and keratinocytes, HA plays a regulatory role in controlling cell physiology through interaction of extracellular HA with a major cell-surface receptor, CD44. This interaction mediates intracellular signaling both directly and indirectly, through CD44 interactions with the cytoskeleton and with EGF and TGFß receptors. Furthermore, degradation of HA by specific hyaluronidase enzymes produces HA fragments that can help to regulate inflammatory processes. In this review, current knowledge about the role of HA in skin inflammation and wound healing are reviewed and possible future applications of such knowledge discussed.

Vaginal Inflammation: Association between Leukocyte Concentration and Levels of Immune Mediators.

PROBLEM: A wide variety of mediators are involved in inflammatory processes. However, the identity of those participating in vaginal immune responses has not been established. We correlated extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinase-8 (MMP-8), hyaluronan (HA), hyaluronidase-1 (Hyal-1), human ß-defensin-2 (hBD2), and neutrophil gelatinase-associated lipocalin (NGAL) concentrations with the extent of leukocyte infiltration into the vagina and suggest their participation in vaginal inflammation. METHODS OF STUDY: Vaginal fluid was obtained from 233 women seen at the outpatient clinic in the Department of Obstetrics and Gynecology at Campinas University, Brazil. The magnitude of vaginal inflammation was determined by the leukocyte count on vaginal smears and categorized as no inflammation (0 leukocytes/field), moderate inflammation (1-4 leukocytes/field), and intense inflammation (>4 leukocytes/field). Concentrations of EMMPRIN, MMP-8, HA, Hyal-1, hBD2, and NGAL were determined in vaginal fluid by ELISA. RESULTS: EMMPRIN, MMP-8, HA, hBD2, and NGAL concentration increased with elevated leukocyte numbers (P < 0.05), while Hyal-1 did not. EMMPRIN concentrations were correlated with HA and MMP-8 levels. CONCLUSION: EMMPRIN, MMP-8, HA, ß-defensin, and NGAL are elevated in women with vaginal inflammation.

Discovery of human posterior head 20 (hPH20) and homo sapiens sperm acrosome associated 1 (hSPACA1) immunocontraceptive epitopes and their effects on fertility in male and female mice.

The key goals of immunocontraception research are to obtain full contraceptive effects using vaccines administered to both males and females. Current research concerning human anti-sperm contraceptive vaccines is focused on delineating infertility-related epitopes to avoid autoimmune disease. We constructed phage-display peptide libraries to select epitope peptides derived from human posterior head 20 (hPH20) and homo sapiens sperm acrosome associated 1 (hSPACA1) using sera collected from infertile women harbouring anti-sperm antibodies. Following five rounds of selection, positive colonies were reconfirmed for reactivity with the immunoinfertile sera. We biopanned and analysed the chemical properties of four epitope peptides, named P82, Sa6, Sa37 and Sa76. Synthetic peptides were made and coupled to either bovine serum albumin (BSA) or ovalbumin. We used the BSA-conjugated peptides to immunise BALB/c mice and examined the effects on fertility in female and male mice. The synthetic peptides generated a sperm-specific antibody response in female and male mice that caused a contraceptive state. The immunocontraceptive effect was reversible and, with the disappearance of peptide-specific antibodies, there was complete restoration of fertility. Vaccinations using P82, Sa6 and Sa76 peptides resulted in no apparent side effects. Thus, it is efficient and practical to identify epitope peptide candidates by phage display. These peptides may find clinical application in the specific diagnosis and treatment of male and female infertility and contraceptive vaccine development.

Male fertility and apoptosis in normal spermatogenesis are regulated by vacuolar-ATPase isoform a2.

The a2 isoform of vacuolar-ATPase (ATP6V0A2, referred to as a2V) is required for normal spermatogenesis and maturation of sperm. Treatment of male mice with anti-a2V disturbs the testicular cytokine/chemokine balance and leads to severe deficiencies of spermatogenesis. The aim of the present study was to investigate the role of a2V in male fertility and in the regulation of apoptotic pathways required for normal spermatogenesis in mice. To study the role of a2V single dose of anti-a2V monoclonal antibody or mouse IgG isotype (3µg/animal) was injected i.p. into males on alternate days for 10 days. The expression of sperm maturation-related molecules and pro-apoptotic molecules was measured by real-time PCR or immunohistochemistry in control and anti-a2V-treated testes. The caspase levels and their activity were measured by western blot and fluorometry. We found that the expression of the sperm maturation-related molecules SPAM1, ADAM1, and ADAM2 was significantly decreased in testes from anti-a2V-treated males. The expression of pro-apoptotic molecules (Bax, p53, and p21) and molecules involved in the intrinsic pathway of apoptosis (caspase-9, caspase-3, and PARP), which are crucial for normal spermatogenesis was significantly reduced in testes from anti-a2V-treated males compared with the control. The total ATP level was significantly lower in anti-a2V-treated testes. The data provide novel evidence showing that a2V can regulate the apoptotic pathways, an essential testicular feature, and is necessary for efficient spermatogenesis.

Clinical Immunogenicity of rHuPH20, a Hyaluronidase Enabling Subcutaneous Drug Administration.

Recombinant human PH20 hyaluronidase (rHuPH20) is used to facilitate dispersion of subcutaneously delivered fluids and drugs. This report summarizes rHuPH20 immunogenicity findings from clinical trials where rHuPH20 was co-administered with SC human immunoglobulin, trastuzumab, rituximab, or insulin. Plasma samples were obtained from evaluable subjects participating in ten different clinical trials as well as from healthy plasma donors. A bridging immunoassay and a modified hyaluronidase activity assay were used to determine rHuPH20-reactive antibody titers and neutralizing antibodies, respectively. rHuPH20-binding antibody populations from selected subjects with positive titers were affinity-purified and subjected to further characterization such as cross-reactivity with endogenous PH20. Among individual trials, the prevalence of pre-existing rHuPH20-reactive antibodies varied between 3 and 12%, excepting the primary immunodeficiency (PID) studies. Incidence of treatment-induced rHuPH20 antibodies was 2 to 18%, with the highest titers (81,920) observed in PID. No neutralizing antibodies were observed. Within most trials, the kinetics of antibody responses were comparable between pre-existing and treatment-induced antibody responses, although responses classified as persistent were more common in subjects with pre-existing titers. There was no association between antibody positivity and either local or systemic adverse events. Pre-existing and treatment-induced antibody populations were of similar immunoglobulin isotypes and cross-reacted to endogenous PH20 to similar extents. No cross-reactivity to PH20 paralogs was detected. rHuPH20 induces only modest immunogenicity which has no association with adverse events. In addition, antibodies purified from baseline-positive individuals are qualitatively similar to those purified from individuals developing rHuPH20-reactive antibodies following exposure to the enzyme.