Safety and immunogenicity of the Na-GST-1 hookworm vaccine in Brazilian and American adults.

Necator americanus Glutathione-S-Transferase-1 (Na-GST-1) plays a role in the digestion of host hemoglobin by adult N. americanus hookworms. Vaccination of laboratory animals with recombinant Na-GST-1 is associated with significant protection from challenge infection. Recombinant Na-GST-1 was expressed in Pichia pastoris and adsorbed to aluminum hydroxide adjuvant (Alhydrogel) according to current Good Manufacturing Practice. Two Phase 1 trials were conducted in 142 healthy adult volunteers in the United States and Brazil, first in hookworm-naïve individuals and then in residents of a N. americanus endemic area in Brazil. Volunteers received one of three doses of recombinant Na-GST-1 (10, 30, or 100 µg) adjuvanted with Alhydrogel, adjuvanted with Alhydrogel and co-administered with an aqueous formulation of Glucopyranosyl Lipid A (GLA-AF), or the hepatitis B vaccine. Vaccinations were administered via intramuscular injection on days 0, 56, and 112. Na-GST-1/Alhydrogel was well tolerated in both hookworm-naïve and hookworm-exposed adults, with the most common adverse events being mild to moderate injection site pain and tenderness, and mild headache and nausea; no vaccine-related severe or serious adverse events were observed. Antigen-specific IgG antibodies were induced in a dose-dependent fashion, with increasing levels observed after each vaccination in both trials. The addition of GLA-AF to Na-GST-1/Alhydrogel did not result in significant increases in specific IgG responses. In both the US and Brazil studies, the predominant IgG subclass induced against Na-GST-1 was IgG1, with lesser amounts of IgG3. Vaccination of both hookworm-naïve and hookworm-exposed adults with recombinant Na-GST-1 was safe, well tolerated, and resulted in significant antigen-specific IgG responses. Based on these results, this vaccine will be advanced into clinical trials in children and eventual efficacy studies. TRIAL REGISTRATION: ClinicalTrials.gov (NCT01261130 for the Brazil trial and NCT01385189 for the US trial).

A Comparison of the Quality of Informed Consent for Clinical Trials of an Experimental Hookworm Vaccine Conducted in Developed and Developing Countries.

Informed consent is one of the principal ethical requirements of conducting clinical research, regardless of the study setting. Breaches in the quality of the informed consent process are frequently described in reference to clinical trials conducted in developing countries, due to low levels of formal education, a lack of familiarity with biomedical research, and limited access to health services in these countries. However, few studies have directly compared the quality of the informed consent process in developed and developing countries using the same tool and in similar clinical trials. This study was conducted to compare the quality of the informed consent process of a series of clinical trials of an investigational hookworm vaccine that were performed in Brazil and the United States. A standardized questionnaire was used to assess the ethical quality of the informed consent process in a series of Phase 1 clinical trials of the Na-GST-1/Alhydrogel hookworm vaccine that were conducted in healthy adults in Brazil and the United States. In Brazil, the trial was conducted at two sites, one in the hookworm non-endemic urban area of Belo Horizonte, Minas, and one in the rural, resource-limited town of Americaninhas, both in the state of Minas Gerais; the American trial was conducted in Washington, DC. A 32-question survey was administered after the informed consent document was signed at each of the three trial sites; it assessed participants' understanding of information about the study presented in the document as well as the voluntariness of their decision to participate. 105 participants completed the questionnaire: 63 in Americaninhas, 18 in Belo Horizonte, and 24 in Washington, DC. Overall knowledge about the trial was suboptimal: the mean number of correct answers to questions about study objectives, methods, duration, rights, and potential risks and benefits, was 45.6% in Americaninhas, 65.2% in Belo Horizonte, and 59.1% in Washington, DC. Although there was no difference in the rate of correct answers between participants in Belo Horizonte and Washington, DC, there was a significant gap between participants at these two locations compared to Americaninhas (p = 0.0002 and p = 0.0001, respectively), which had a lower percentage of correct answers. Attitudes towards participating in the clinical trial also differed by site: while approximately 40% had doubts about participating in Washington, DC and Belo Horizonte, only 1.5% had concerns in Americaninhas. Finally, in Belo Horizonte and Washington, high percentages cited a desire to help others as motivation for participating, whereas in Americaninhas, the most common reason for participating was personal interest (p = 0.001). Understanding of information about a Phase 1 clinical trial of an experimental hookworm vaccine following informed consent was suboptimal, regardless of study site. Although overall there were no differences in knowledge between Brazil and the US, a lower level of understanding about the trial was seen in participants at the rural, resource-limited Brazilian site. These findings demonstrate the need for educational interventions directed at potential clinical trial participants, both in developing and developed countries, in order to improve understanding of the informed consent document.

The potential economic value of a hookworm vaccine.

Hookworm infection is a significant problem worldwide. As development of hookworm vaccine proceeds, it is essential for vaccine developers and manufacturers, policy makers, and other public health officials to understand the potential costs and benefits of such a vaccine. We developed a decision analytic model to evaluate the cost-effectiveness of introducing a hookworm vaccine into two populations in Brazil: school-age children and non-pregnant women of reproductive age. Results suggest that a vaccine would provide not only cost savings, but potential health benefits to both populations. In fact, the most cost-effective intervention strategy may be to combine vaccine with current drug treatment strategies.

Multivalent anthelminthic vaccine to prevent hookworm and schistosomiasis.

Hookworm infection and schistosomiasis are two of the world's most important human parasitic infections, affecting hundreds of millions of people in developing countries. Measured together in disability-adjusted life years, hookworm infection and schistosomiasis rank closely behind malaria as the most prevalent human parasitic diseases. A major approach for the control of these two helminth infections relies on periodic, mass chemotherapy with anthelminthics. However, high rates of post-treatment reinfection, the declining efficacy with repeated treatment, rebound morbidity (in the case of schistosomiasis) and the potential for the emergence of anthelminthic drug resistance threaten the sustainability of mass drug administration as the only form of control. Hence, there is a strong rationale for developing a vaccine that simultaneously targets both hookworms and schistosomes because of similarities in the pathobiology of both parasites, the ability of both helminths to cause anemia and their coendemicity in sub-Saharan Africa, Brazil and East Asia. A multivalent anthelminthic vaccine for hookworm infection and schistosomiasis would represent an important new tool for combating disease and poverty.

Vaccines against blood-feeding nematodes of humans and livestock.

This paper summarises the progress towards vaccine development against the major blood-feeding nematodes of man and livestock, the hookworms and Haemonchus contortus, respectively. The impact of the diseases and the drivers for vaccine development are summarized as well as the anticipated impact of the host immune response on vaccine design. The performance requirements are discussed and progress towards these objectives using defined larval and adult antigens, many of these being shared between species. Specific examples include the Ancylostoma secreted proteins and homologues in Haemonchus as well as proteases used for digestion of the blood meal. This discussion shows that many of the major vaccine candidates are shared between these blood-feeding species, not only those from the blood-feeding stages but also those expressed by infective L3s in the early stages of infection. Challenges for the future include: exploiting the expanding genome information for antigen discovery, use of different recombinant protein expression systems, formulation with new adjuvants, and novel methods of field testing vaccine efficacy.

Immunologic responses to common antigens in helminthic infections and allergic disease.

PURPOSE OF REVIEW: It is estimated that over 1 billion individuals are infected with helminth parasites worldwide. Epidemiologic studies have pointed to a protective role of helminthic infections in the development of allergy and asthma; however, evidence for this inverse association has not been consistently established. The focus of this review is to discuss the potential role of shared antigens between parasites and environmental allergens in modulating allergic immune responses, specifically tropomyosin. RECENT FINDINGS: Tropomyosin has been identified as a highly conserved molecule in invertebrates. In populations exposed concomitantly to mites, cockroach, Ascaris, and shrimp and other crustaceans and mollusks, IgE antibody responses to tropomyosin are found in over 50% of individuals. Evidence suggests that IgE cross-reactivity to tropomyosin has clinical relevance. SUMMARY: Mechanisms underlying the immunomodulatory effects of parasites in allergy and asthma remain poorly understood. Identification of molecules in intestinal parasites, particularly Schistosoma mansoni and Ascaris lumbricoides, associated with protection from or promotion of allergy and asthma, could provide the basis for novel forms of treatment or prevention of these diseases. Prospective studies will be necessary to clarify the role of tropomyosin and other parasite antigens shared with inhalant or food allergens in the development of allergic diseases.

Factors affecting high and low human IgE responses to schistosome worm antigens in an area of Brazil endemic for Schistosoma mansoni and hookworm.

We have previously examined the antibody isotype responses to schistosome worm and egg antigens in human populations living in areas of Kenya and the Philippines endemic for Schistosoma mansoni and S. japonicum, respectively. Here, we have analyzed antibody isotype responses to S. mansoni adult worm (AW) antigen and soluble egg antigen (SEA) in more than 500 Brazilian individuals, and found similar relationships with age and sex as in the Kenyan and Filipino populations. Isotype responses to AW antigen broadly increased with age whereas isotype responses to SEA decreased, and a higher proportion of males than females had detectable IgE against AW antigen. Most isotype responses to AW antigen and SEA correlated positively with intensity of infection with S. mansoni except AW antigen-specific IgG2, which correlated negatively. The overall prevalence of infection with S. mansoni in this area was relatively low at only 39.5%; hookworm prevalence was higher at 57.4%. The majority of those infected with S. mansoni were also infected with hookworm (76%). Those individuals with high IgE responses to AW antigen were matched for sex, age, and total IgG to AW antigen as closely as possible with individuals with low levels of AW antigen-specific IgE. The two groups were compared for factors potentially influential in IgE production. No difference was found between the high and low IgE responders for 1) intensity or prevalence of infection with S. mansoni, 2) relative exposure to S. mansoni, 3) number of previous treatments for schistosomiasis, or 4) prevalence of infection with hookworm, but differences were found in other isotype responses to S. mansoni. The high IgE responders had higher IgA and IgG4 against both AW antigen and SEA but lower IgG3 responses to AW antigen than the low IgE responders. The IgE responses to three S. mansoni antigens (paramyosin, Sm22.6, and a 12-kD AW antigen band) were detected in individuals with IgE against AW antigen only.

Ultrastructural observations on the in vitro interaction between rat eosinophils and some parasitic helminths (Schistosoma mansoni, Trichinella spiralis and Nippostrongylus brasiliensis).

Rat eosinophils form an intimate association with the surfaces of parasitic helminths, in vitro, in the presence of immune serum. The parasite presents a non-phagocytosable surface to the cell. The initial response of the eosinophil is degranulation which leads to the formation of large cytoplasmic vacuoles. Peroxidase, an enzyme localized in the matrix of the crystalloid secretion granules, is discharged into these vacuoles as a consequence of degranulation. The vacuoles eventually become connected to the adherent basal plasma membrane of the eosinophil, and peroxidase is secreted directly onto the surface of the parasite. There is no morphological evidence to suggest that this particular secretion affects the integrity of the parasite surface.

Immune mechanism of rats on Nippostrongylus brasiliensis in vitro. II. The influence of lymphocytes and peritoneal cells.

Nippostrongylus were collected from the intestines of rats 6 days p.i. and kept under sterile conditions in cultures. Serum, lymphocytes and peritoneal cells of immune or non-infected animals were added in various combinations to the culture media. The culture media were changed 2-3 times in an experimental period of 10 days, resp. serum and cells were added. The lymphocytes were isolated from the peripheral blood or from the mesenterial lymph nodes whereas the mononuclear cells were obtained from the peritoneal cavity. Serum and lymphocytes from the peripheral blood both from immune and non-infected rats, had no increased lethal effect on Nippostrongylus. The highest lethality rate of adults (65-68%) was achieved in cultures with peritoneal cells and lymphocytes from the lymph nodes of sensitized rats. Serum of infected or non-infected animals had no influence on adult Nippostrongylus in cultures with these cell combinations. In the controls without any cell-supplements the survival rate of the parasites was up to 88%.

[Immune mechanism of rats on Nippostrongylus brasiliensis in vitro (author's transl)]. / Immunmechanismus der Ratte gegen Nippostrongylus brasiliensis in vitro. I. Einfluss von humoralen und sekretorischen Antikörpern sowie Mastzellen

The influence of humoral and secretory antibodies as well as cell supplements on Nippostrongylus brasiliensis was tested in vitro. Adult Sprague-Dawley-rats approximately 12 weeks of age were used in these experiments. For the in vitro tests the following culture media were used: 25% chicken-embryo-extract, sodium casein, pig liver extract and rat serum for the larval stages. The medium for the cultures of adult N. brasiliensis consisted of 5% yeast extract, 15% casein, 30% Krebs-Ringer-solution and 50% rat serum. Secretory antibodies were isolated from the rinsing fluid of the rat intestines by high pressure filtration (10 to 15 micron), then cleaning of the fluid through a Sephadex G 15 column and finally narrowing down through collodene capsules. Mast cells were isolated from the peritoneal cavity by Ficoll-gradient-centrifugation. Various test series were conducted with the addition of serum or secretory antibodies of repeatedly infected and immune rats to the medium. In these tests there was never a difference in the influence on growth nor a higher mortality rate of larval or adult N. brasiliensis in contrast to cultures where serum and secretory antibodies of non-infected animals were used. A 100%, degranulation of mast cells from infected rats occurred already within 14 to 22 hours in the cultures of adult N. brasiliensis. Variations were not noticed in the influence on the viability of N. brasiliensis kept in media for 10 days without or with cell supplements as well as sera of infected or not infected rats. The results from our experiments demonstrated that there was no variation in the influence on the development and a higher mortality rate of the larval stages and adult Nippostrongylus in media containing either sera and secretory antibodies of infected or not infected rats.

Protection of rats against nippostrongylus brasiliensis with worm antigens by oral administration.

Different types of antigens collected from adult Nippostrongylus brasiliensis were used to vaccinate rats against this parasite: somatic worm homogenate, secretory antigens and whole fixed worms. After challenge infection there was a significant reduction of worm number in immunized rats. However, vaccination with secretory antigens only induced a high level of immunity, indicating that protective antigens were probably worm metabolites.

Nippostrongylus brasiliensis in the rat: immune response in serum and intestinal secretions.

The kinetics of local and serum hemagglutinins production was observed following infections of rats with N. brasiliensis. Antibodies which appeared in the gut immediately after parasite localization had occured there 6 days following infection with 3,000 larvae. Serum antibodies were detected on day 18. Parasites were still being expelled at this time from the intestine. When rats were infected with 83 larvae, which is the smallest immunizing dose, local antibodies appeared in the same manner as in the first experiment, serum antibodies appearing only after challenge with 3,000 larvae. Local antibody activity is mainly due to IgA molecules. The presence of parasites and, at the same time, of local antibodies suggests an hypothesis for the mechanism of worm expulsion.

Carrier effect by Nippostrongylus brasiliensis infection on rat antihapten IgE antibody response.

Nippostrongylus brasiliensis infection was found to be highly effective in inducing a carrier-specific enhancing effect on primary and secondary antihapten IgE antibody response in the rat, when animals were immunized i.p. with 10 mug of dinitrophenylated N. brasiliensis protein (DNP-Nb) plus 10 mg Al(OH)3 2 weeks after the infection. The carrier effect by the infection was much greater than that obtained by any other supplementary immunization with the carrier (Nb) plus adjuvant, so far examined, in terms of the IgE antibody response. The results, together with our previous observation in the mouse, provide an explanation for the reason why antiworm IgE antibodies are so easily detectable in helminth infections.

Studies on immune responses to parasite antigens in mice. III. Nippostrongylus brasiliensis infections in hypothymic nu/nu mice.

Normal mice of several strains reject the nematode worm Nippostronglyus brasiliensis from the intestine within 14 days (and most often within 10 days) of injection of high numbers of infective third stage larvae (L3). Previously-infected mice are markedly resistant to a second infection. Hypothymic, nu/nu ('nude') mice continue to harbor worms long after intact mice have rejected the worm burden and an inoculum of T cells at the time of, or several days after L3 injection, leads to worm elimination within 14 days in nu/nu mice. As yet no evidence is available to indicate whether or not T cells with specificity for parasite antigens are required in a reconstitutive inoculum in nu/nu mice. Pretreatment of normal mice with cyclophosphamide, at doses reported to lead to temporary B cell hypofunction, does not result in delayed rejection of worms but the participation of B cell products (antibodies) cannot be discounted on the basis of this result. The nu/nu mouse - N. brasiliensis system will be useful in the search for, and characterization of, parasite antigens which gain access to the lymphoid organs and circulation of parasitized mice and in the analysis of T cell subtypes (both functional and surface Thy and Ly alloantigenic subtypes) involved in worm elimination.

Levels of prostaglandins in the small intestine of rats during primary and secondary infection with Nippostrongylus brasiliensis.

The level of PGE is increased 10-fold in intestinal tissues during primary infection of the rat with Nippostrongylus brasiliensis. Peak levels (ca. 7,000 pg/cm) were assayed in the jejunal site of infection on day 7 of infection and similar levels were recorded in 'post-infection' (ileal) segments at this time. The level of PGE in 'post-infection' segments showed further increase to 12,000 pg/cm on day 10. The level of PGE also increased in 'pre-infection' (duodenal) but this was delayed by 4-5 days. The level of PGF also increased during primary infection (from about 100 to 950 pg/cm) but this occurred after expulsion. Increase in the level of PGE occurred earlier (at 3-4 days) during secondary challenge given 19 days after primary infection, but the PGE levels followed the primary response when challenge was given 10 weeks after primary infection. It is suggested that PGE plays a dual role in parasite immunity. (1) PGE may directly affect metabolism of the parasite. In this event it is also suggested that protective antibodies cause the release of PG. (2) Elevated levels of PGE act indirectly by affecting gastrointestinal function which alters the microenvironment at the site of infection. The duodenal migration of parasites may be due to this effect of PGE.

[Hemagglutinating antibodies in the serum and intestinal secretions of rats infested by Nippostrongylus brasiliensis]. / Mise en évidence d'anticorps hémagglutinants dans le sérum et les sécrétions intestinales de rats infestés par Nippostrongylus brasiliensis

Nippostrongylus brasiliensis infected rats responded with the formation of hemagglutinating antibodies in both serum and intestinal secretions. After the first infection seric antibody titers were quite weak but increased sharply after challenge. Intestinal secretion hemagglutinines remained at constant level after both infections. This result represents a new approach in our understanding of the immune mechanism towards this parasite in rats.