Generalized urticaria induced by the Na-ASP-2 hookworm vaccine: implications for the development of vaccines against helminths.

BACKGROUND: Necator americanus Ancylostoma-secreted protein 2 (Na-ASP-2) is secreted by infective hookworm larvae on entry into human hosts. Vaccination of laboratory animals with recombinant Na-ASP-2 provides significant protection against challenge infections. In endemic areas antibodies to Na-ASP-2 are associated with reduced risk of heavy N americanus infections. OBJECTIVE: To assess the safety and immunogenicity of recombinant Na-ASP-2 adjuvanted with Alhydrogel in healthy Brazilian adults previously infected with N americanus. METHODS: Participants were randomized to receive Na-ASP-2 or hepatitis B vaccine. Major IgG and IgE epitopes of the Na-ASP-2 molecule were mapped by using sera from these same subjects. Seroepidemiologic studies in adults and children residing in hookworm-endemic areas were conducted to assess the prevalence of IgE responses to Na-ASP-2. RESULTS: Vaccination with a single dose of Na-ASP-2 resulted in generalized urticarial reactions in several volunteers. These reactions were associated with pre-existing Na-ASP-2-specific IgE likely induced by previous hookworm infection. Surveys revealed that a significant proportion of the population in hookworm-endemic areas had increased levels of IgE to Na-ASP-2. Epitope mapping demonstrated sites on the Na-ASP-2 molecule that are uniquely or jointly recognized by IgG and IgE antibodies. CONCLUSION: Infection with N americanus induces increased levels of total and specific IgE to Na-ASP-2 that result in generalized urticaria on vaccination with recombinant Na-ASP-2. These data advance knowledge of vaccine development for helminths given their propensity to induce strong T(H)2 responses. Study data highlight the important differences between the immune responses to natural helminth infection and to vaccination with a recombinant helminth antigen.

Mycobacterium-Induced Th1, Helminths-Induced Th2 Cells and the Potential Vaccine Candidates for Allergic Asthma: Imitation of Natural Infection.

Bronchial asthma is one of the most chronic pulmonary diseases and major public health problems. In general, asthma prevails in developed countries than developing countries, and its prevalence is increasing in the latter. For instance, the hygiene hypothesis demonstrated that this phenomenon resulted from higher household hygienic standards that decreased the chances of infections, which would subsequently increase the occurrence of allergy. In this review, we attempted to integrate our knowledge with the hygiene hypothesis into beneficial preventive approaches for allergic asthma. Therefore, we highlighted the studies that investigated the correlation between allergic asthma and the two different types of infections that induce the two major antagonizing arms of T cells. This elucidation reflects the association between various types of natural infections and the immune system, which is predicted to support the main objective of the current research on investigating of the benefits of natural infections, regardless their immune pathways for the prevention of allergic asthma. We demonstrated that natural infection with Mycobacterium tuberculosis (Mtb) prevents the development of allergic asthma, thus Bacille Calmette-Guérin (BCG) vaccine is suggested at early age to mediate the same prevention particularly with increasing its efficiency through genetic engineering-based modifications. Likewise, natural helminth infections might inhabit the allergic asthma development. Therefore, helminth-derived proteins at early age are good candidates for designing vaccines for allergic asthma and it requires further investigation. Finally, we recommend imitation of natural infections as a general strategy for preventing allergic asthma that increased dramatically over the past decades.

Efficacy of the EG95 hydatid vaccine in a macropodid host, the tammar wallaby.

In Australia, macropodids are common intermediate hosts for the cestode Echinococcus granulosus, and sylvatic transmission is maintained via wild dogs. The parasite causes mortality in a number of macropodid species and the sylvatic cycle provides a source of infection to domestic livestock and humans. We determined the efficacy of the hydatid vaccine, EG95 in the tammar wallaby, Macropus eugenii, challenging either 1 or 9 months post-vaccination. EG95 provides similar protection to that seen in sheep (96-100%). Control tammars were significantly more likely to become infected (odds ratio 29.44; CI 4.13, 209.97; P=0.001) and to develop more cysts (count ratio 26.69; CI 5.83, 122.19; P<0.001). The vaccination may be beneficial if administered pre-release in captive breeding programmes for endangered macropodids. Further work to develop oral delivery methods may enable vaccine administration of wild animals and thereby a reduction in sylvatic transmission.

Dose-response study of recombinant factor VIIa/tissue factor inhibitor recombinant nematode anticoagulant protein c2 in prevention of postoperative venous thromboembolism in patients undergoing total knee replacement.

BACKGROUND: With the best prophylactics now available, venous thromboembolism after total knee replacement remains substantial (25% to 27%). Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent inhibitor of factor VIIa/tissue factor complex that has the potential to reduce this risk. The present study was performed to determine an efficacious and safe dose of rNAPc2 for prevention of venous thromboembolism after elective, unilateral total knee replacement. METHODS AND RESULTS: This open-label, sequential dose-ranging study was conducted in 11 centers in Canada, Europe, and the United States. Five regimens were tested. Injections were administered subcutaneously on the day of surgery (day 1) and days 3, 5, and optionally, day 7. Primary efficacy outcome was a composite of overall deep vein thrombosis based on mandatory unilateral venography (day 7+/-2) and confirmed symptomatic venous thromboembolism recorded

Recombinant nematode anticoagulant protein c2, an inhibitor of the tissue factor/factor VIIa complex, in patients undergoing elective coronary angioplasty.

OBJECTIVES: We investigated the safety and pharmacodynamics of escalating doses of recombinant nematode anticoagulant protein c2 (rNAPc2) in patients undergoing elective coronary angioplasty. BACKGROUND: Recombinant NAPc2 is a potent inhibitor of the tissue factor/factor VIIa complex, which has the potential to reduce the risk of thrombotic complications in coronary artery disease. METHODS: In a randomized, double-blinded, dose-escalation, multicenter trial, 154 patients received placebo or rNAPc2 at doses of 3.5, 5.0, 7.5, and 10.0 microg/kg body weight as a single subcutaneous administration 2 to 6 h before angioplasty. All patients received aspirin, unfractionated heparin during angioplasty, and clopidogrel in case of stent implantation. RESULTS: Minor bleeding rates for the doses 3.5 to 7.5 microg/kg were comparable to that with placebo (6.7%), whereas an incidence of 26.9% was observed at the 10.0 microg/kg dose level (p < 0.01). Major bleedings occurred in the 5.0 microg/kg (n = 3) and 7.5 microg/kg (n = 1) dose groups. The three patients in the 5.0 microg/kg dose group also received a glycoprotein IIb/IIIa receptor inhibitor at the moment of major bleeding. Systemic thrombin generation, as measured by prothrombin fragment 1+2 (F(1+2)), was suppressed in all rNAPc2 dose groups to levels below pretreatment values for at least 36 h. In the placebo group, a distinct increase of F(1+2) levels was observed following cessation of heparin. CONCLUSIONS: Inhibition of the tissue factor/factor VIIa complex with rNAPc2, at doses up to 7.5 microg/kg, in combination with aspirin, clopidogrel, and unfractionated heparin appears to be a safe and effective strategy to prevent thrombin generation during coronary angioplasty.

UK-279,276, a neutrophil inhibitory glycoprotein, in acute stroke: tolerability and pharmacokinetics.

BACKGROUND AND PURPOSE: UK-279,276, a recombinant glycoprotein, binds selectively to the CD11b/CD18 integrin on neutrophils and has the potential to modulate the neuroinflammation associated with acute stroke. After preclinical evidence of neuroprotection, UK-279,276 has entered clinical development. The purposes of this study were to evaluate the safety and tolerability of UK-279,276 and to examine its pharmacokinetics and pharmacodynamics (binding to neutrophil CD11b) in patients with acute stroke. METHODS: This was a multicenter, double-blind, dose-escalation study in 176 patients randomized to a single intravenous dose of UK-279,276 (6 cohorts: 0.06, 0.1, 0.2, 0.5, 1.0, 1.5 mg/kg) or placebo (3:1 randomization within each cohort) within 12 hours of stroke onset. RESULTS: Age and stroke severity were well balanced across groups, with a mean age of 70 years (range, 39 to 92 years) and moderate baseline stroke severity (mean Scandinavian Stroke Scale score, 36.5 to 43.2; mean National Institutes of Health Stroke Scale score, 6.3 to 8.5). UK-279,276 was well tolerated at doses up to 1.5 mg/kg. There was no evidence of a relationship between dose of UK-279,276 and adverse events or clinical chemistry or hematology laboratory tests, or of an increased incidence of infection-related adverse events with the study drug. A dose-dependent UK-279,276-specific IgG antibody response was observed in patients treated with the 1.0- and 1.5-mg/kg doses. UK-279,276 displayed nonlinear pharmacokinetics across the dose range investigated. The duration of CD11b saturation was dose dependent, with >80% saturation achieved for at least 7 days after treatment with UK-279,276 1.0 and 1.5 mg/kg. CONCLUSIONS: UK-279,276 was well tolerated in acute stroke patients at single doses up to 1.5 mg/kg. Further clinical investigation of UK-279,276 is ongoing.

Recombinant factor VIIa as an antidote for anticoagulant treatment.

Increasing knowledge on the function of the hemostatic system in vivo and limitations of currently available anticoagulant agents have led to the development of a new generation of anticoagulants. These new agents have a greater specificity towards activated coagulation pathways and factors and are presently being evaluated in clinical studies. The new generation anticoagulants include specific inhibitors of factor IIa (melagatran), factor Xa (pentasaccharides), and agents that interfere with tissue factor (TF) activity. A limitation of this new class of anticoagulants may be the lack of an appropriate strategy to reverse the effect if a bleeding event occurs. Recombinant factor VIIa (rFVIIa) is a potent prohemostatic agent and may represent an interesting option for consideration when an antidote is required. Indeed, rFVIIa has proven to be efficacious in the reversal of anticoagulant treatment with vitamin K antagonists. Studies in healthy subjects have also revealed that rFVIIa administration corrects coagulation time and can induce thrombin generation during anticoagulation with pentasaccharides or TF-inhibiting therapy. These results indicate that rFVIIa infusion results in a prohemostatic response in vivo in patients receiving treatment with factor Xa- or TF-specific anticoagulants. This suggests that rFVIIa may be a good candidate as an antidote for new anticoagulants in cases of (severe) bleeding or in patients scheduled for emergency surgery.

Recombinant nematode anticoagulant protein c2, an inhibitor of tissue factor/factor VIIa, attenuates coagulation and the interleukin-10 response in human endotoxemia.

The tissue factor-factor (F)VIIa complex (TF/FVIIa) is responsible for the initiation of blood coagulation under both physiological and pathological conditions. Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent inhibitor of TF/FVIIa, mechanistically distinct from tissue factor pathway inhibitor. The first aim of this study was to elucidate the pharmacokinetics and pharmacodynamics of a single intravenous (i.v.) dose of rNAPc2. The second aim was to study its effect on endotoxin-induced coagulation and inflammation. Initially, rNAPc2 was administered to healthy volunteers in three different doses. There were no safety concerns and the pharmacokinetics were consistent with previous studies, in which rNAPc2 was administered subcutaneously. rNAPc2 elicited a dose-dependent reduction of the endogenous thrombin potential and a selective prolongation of prothrombin time. Subsequently, the effect on endotoxin-induced coagulation and inflammation was studied. The administration of rNAPc2 completely blocked the endotoxin-induced thrombin generation, as measured by plasma prothrombin fragment F1+2. The endotoxin-induced effect on fibrinolytic parameters such as plasmin-antiplasmin complexes and plasminogen activator inhibitor type 1 was not affected by rNAPc2. The administration of rNAPc2 attenuated the endotoxin-induced rise in interleukin (IL)-10, without affecting the rise in other cytokines. In conclusion, rNAPc2 is a potent inhibitor of TF/FVIIa, which was well tolerated and could safely be used intravenously in this Phase I study in healthy male volunteers. A single i.v. dose rNAPc2 completely blocked endotoxin-induced thrombin generation without affecting the fibrinolytic response. In addition, rNAPc2 attenuated the endotoxin-induced rise in IL-10, without affecting the rises in other cytokines.

Maternal antibody parameters of cattle and calves receiving EG95 vaccine to protect against Echinococcus granulosus.

Cattle may act as hosts for the transmission of the cestode parasite Echinococcus granulosus and play a role in transmission of the parasite leading to human cystic echinococcosis (CE). The recombinant EG95 vaccine has been shown to be able to protect cattle and other intermediate host species against CE. Ideally the immunisation of bovines against E. granulosus, using EG95 vaccine, should occur early in life so as to provide maximum protection against the establishment of hydatid cysts. Maternally derived antibody from vaccinated cows may provide some protection for the neonate, but may also interfere with the active response to vaccination. Experiments were undertaken to determine the optimal regime for protection of young cattle against CE. One group of pregnant cattle received 2 vaccinations of EG95 antigen+Quil A adjuvant two months and one month prior to calving. The control group of pregnant cattle were not vaccinated. Calves were either challenged with E. granulosus eggs at 4, 9, 13 or 17 weeks post-birth or were given their first vaccination at 8, 12 or 16 weeks post-birth. Sera obtained at regular intervals were tested by ELISA to assess the immunological response. All calves were experimentally challenged with E. granulosus eggs and subsequent necropsy confirmed the levels of protection. Maternal antibody was shown to protect calves to some extent for at least 17 weeks. Calves from vaccinated cows responded well serologically if the first vaccination was given at 8 or 12 weeks, but full protection against a challenge infection was achieved only if the first vaccination was delayed until 16 weeks after birth. Calves from non-vaccinated cattle also were not fully protected if the first vaccination was at 8 or 12 weeks, but were fully protected if the first vaccination was given when they were 16 weeks old. This suggests that immunological maturity is not acquired in calves until 4 or 5 months of age. No safety problems were observed following two vaccinations of 40 pregnant cows or 30 suckling calves.

Can helminths or helminth-derived products be used in humans to prevent or treat allergic diseases?

Recent epidemiological and experimental data indicate that infection with helminths can protect humans from the development of allergic disorders by immunosuppressive mechanisms that involve the induction of IL-10 and/or regulatory T cells. Furthermore, helminth-derived immune modulators suppress allergic responses in mice. Trichuris suis therapy has been shown to be safe and efficacious in treating inflammatory bowel disease in humans. Has the time come to treat patients who have allergic diseases or healthy humans who are at risk of developing these diseases with helminths or helminth-derived products? Here, I discuss the pros and cons of such an approach.

Randomized, placebo-controlled, double-blind trial of the Na-ASP-2 hookworm vaccine in unexposed adults.

Necator americanus Ancylostoma Secreted Protein-2 (Na-ASP-2) is a leading larval-stage hookworm vaccine candidate. Recombinant Na-ASP-2 was expressed in Pichia pastoris and formulated with Alhydrogel. In a phase 1 trial, 36 healthy adults without history of hookworm infection were enrolled into 1 of 3 dose cohorts (n=12 per cohort) and randomized to receive intramuscular injections of either Na-ASP-2 or saline placebo. Nine participants in the first, second and third cohorts were assigned to receive 10, 50 and 100 microg of Na-ASP-2, respectively, on study days 0, 56 and 112, while 3 participants in each cohort received placebo. The most frequent adverse events were mild-to-moderate injection site reactions; in 8 participants these were delayed and occurred up to 10 days after immunization. No serious adverse events occurred. Anti-Na-ASP-2 IgG endpoint titers as determined by ELISA increased from baseline in all vaccine groups and peaked 14 days after the third injection, with geometric mean titers of 1:7066, 1:7611 and 1:11,593 for the 10, 50 and 100 microg doses, respectively, compared to <1:100 for saline controls (p<0.001). Antibody titers remained significantly elevated in all vaccine groups until the end of the study, approximately 8 months after the third vaccination. In vitro stimulation of PBMCs collected from participants with Na-ASP-2 resulted in robust proliferative responses in those who received vaccine, which increased with successive immunizations and remained high in the 50 and 100 microg dose groups through the end of the study. This first trial of a human hookworm vaccine demonstrates that the Na-ASP-2 vaccine is well-tolerated and induces a prolonged immune response in adults not exposed to hookworm, justifying further testing of this vaccine in an endemic area.

Vaccination against Schistosoma mansoni infection using 74 kDa Schistosoma protein antigen.

An IgG2a anti-Schistosoma mansoni mouse monoclonal antibody was shown to passively protect Swiss mice. The 74 kDa target antigen was isolated from antigenic extracts of S. mansoni adult worms. Swiss and C57 BL/6J mice were immunized with 30, 50, 100 and 200 microg antigen/mouse doses with and without Freund's adjuvant. Sera of immunized mice showed high reactivity against 74 kDa antigen. The highest protection level (76.6% in Swiss mice and 50.1% in C57 BL/6J mice) was obtained using the 50 microg antigen dose with and without Freund's adjuvant. A marked reduction in granuloma number and intensity of collagen and reticular granuloma fibers was observed. The 74 kDa antigen has the ability to protect mice of different strains and to modulate the host immune system.

Interaction of filarial proteins on growth regulation of normal lung epithelial cells in vitro.

An in vitro model to examine the effects of filarial proteins on lung epithelial cells has been developed. Several of these proteins appear in circulation of infected individuals. A close association between tropical pulmonary eosinophilia (TPE) and filariasis has been reported by several workers. [3H]-thymidine studies do indicate that when optimum concentration of these filarial proteins were added to lung cultures in proliferating and basal/maintenance media a further increase in growth stimulation was observed early in culture. However, on longer exposures and at higher concentrations an inhibitory effect with distinct morphological changes were noted. The dual role of these proteins on lung epithelial cells in vitro may highlight the possibility of a direct interaction of these proteins with lung cells during disease also contributing to tissue damage.