Ancylostoma ceylanicum: immunization with soluble worm extract and responses to challenge infection of dogs

When dogs were immunized with soluble extract of adult Ancylostoma ceylanicum antigen, they were partially resistant to challenge infection in this model of human hookworm infection. Two immunizing doses, each of 1 mg protein suspended in Freund's complete adjuvant, were administered to one group of animals 1 and 3 weeks prior to infection with 5000 larvae. When compared with control dogs given the same infective dose, fecal egg excretion and intestinal adult worm burden in the immunized animals were reduced by 59 and 74%, respectively. Infection had no significant effect on hemoglobin concentrations, mean red cell volumes, total white cell counts, platelet levels, or spontaneous and phytohemagglutinin-induced lymphocyte transformations in both control and immunized animals. Both groups developed an eosinophilia, and lymphocytes from the immunized dogs responded transiently to stimulation with both larval and adult worm antigens. Specific IgM antibodies were transitory in both groups of dogs following infection. IgG antibodies developed significantly 2 weeks after infection in the immunized group; however, they did not appear until 4 weeks after infection in the control group. Both groups developed IgA antibodies 1 week after infection. They were maintained in the control dogs, in contrast to the levels in immunized animals which subsided rapidly 4 weeks after infection. Therefore, when animals are injected with soluble adult worm antigen prior to infection, specific protective immunity is acquired.     

Schistosoma japonicum: Immunological response to circulating polysaccharide antigens in rabbits with a light infection

To study the detectability of circulating polysaccharide antigens and the immunological response to such antigens in rabbits with a light Schistosoma japonicum infection, sera of five rabbits infected with 50 cercariae were studied up to 29 weeks post infection (p.i.). While one rabbit developed no worm burden, the other rabbits developed low worm burdens (4 to 16 worms). In the sera of these rabbits, the only polysaccharide antigen demonstrable with immunoelectrophoresis (IEF), was the circulating anodic antigen (CAA). With the enzyme-linked immunosorbent assay (ELISA), CAA was detectable from 5 to 6 weeks p.i. in the sera of the two rabbits with the highest number of worm couples. The lowest CAA level which was detectable in unconcentrated sera from which serum proteins had been removed was 125 ng CAA/ml, corresponding with a worm burden of 4.5 worm/kg body wt. During the entire infection, CAA-specific immune complexes were only demonstrable in very low concentrations. Antibodies against polysaccharide antigens were assessed with immunofluorescent antibody (IFA) on Rossman's fixed sections of adult worms, with the ELISA, and with IEF. Specific IgA, IgG, and IgM antibodies were detectable from 2 to 3 weeks p.i. with IFA and ELISA. These early antibodies were shown to be directed against gut-associated antigens, while antibodies against parenchyma-associated antigens were found later in the infection. With IEF, antibodies against two trichloroacetic acid (TCA)-soluble antigens were detectable, including the major, S. japonicum-specific antigen 2.     

Bge snail cell-line antigens: ineffectiveness as antischistosomal vaccine in mice.

Mice and rabbits were immunized with antigens derived from Bge cells, Biomphalaria glabrata hemolymph, or Schistosoma mansoni. Antisera from mice given molluscan antigens did not form immunoprecipitates with soluble antigen from adult worms, but their binding to surfaces of sporocysts, cercariae, and schistosomules suggests the presence of cross-reacting determinants. In vitro, cell-mediated immune responses to Bge antigens were not demonstrable in infected nor in immunized mice. Mice immunized with Bge cell-line antigens and challenged with S. mansoni cercariae showed no reduction in worm burden when compared with control mice.     

Early stage-specific immune responses in primary experimental human hookworm infection

To determine the cellular immune response during different stages of hookworm infection, we infected two human volunteers with Necator americanus and followed their immune responses against stage-specific, crude antigen extracts through larval migration, pre-patency, and early patency. After chemotherapy, the volunteers were followed for an additional 6 months. Low-dose N. americanus infection resulted in mild clinical signs and peripheral blood eosinophilia occurred during the estimated time of arrival of juvenile worms in the intestine. After an initial rise in proliferative responses against larval and adult worm antigens, the cellular reactivity decreased until the end of pre-patency, rose again during patency, and dropped after chemotherapy. Before infection and during the course of infection, elevated concentrations of TNF-alpha were detected in supernatants of peripheral blood mononuclear cells (PBMC) stimulated in vitro with third-stage (L3) or adult worm excretory-secretory (ES) antigens, which dropped off after chemotherapy. After stimulation with L3 antigen, elevated concentrations of CCL17 were detected in supernatants during pre-patency and patency. Interestingly, a prominent rise in IL-10 secretion was detected in ES antigen-stimulated PBMC cultures during late pre-patency. During reinfection studies in endemic areas, such distinct cytokine and chemokine profiles might be additional markers to better classify egg-negative patients.     

Serological responses to Ascaris suum adult worm antigens in Iberian finisher pigs

Adult Ascaris suum were dissected to obtain different worm components (body wall, body fluid, ovaries, uterus and oesophagus) which were used as antigens when testing 95 sera of naturally A. suum-infected Iberian pigs by enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). Pigs with patent Ascaris infections had significantly lower ELISA optical density values than pigs without adult worms when using the body fluid and the body wall as antigens. A poor negative correlation was found between adult intestinal worm burden or eggs in faeces and specific antibody responses, measured by ELISA and WB using all antigens. By WB, the recognition of specific bands was variable, but three groups of bands with molecular weights of 97 kDa, 54-58 kDa and 42-44 kDa were generally recognized by sera from naturally infected pigs as well as from hyperimmunized pigs when using the five antigen extracts. The ELISA and WB techniques may be used for immunodiagnosis, using somatic adult worm antigens, to declare young pigs to be Ascaris-free but cannot be used for individual Ascaris-diagnosis in adult Iberian pigs.     

Induction of protective immunity against Schistosoma mansoni by a nonliving vaccine. III. Correlation of resistance with induction of activated larvacidal macrophages

Mice protected against Schistosoma mansoni infection by intradermal (i.d.) immunization with nonliving larval or adult worm antigens plus bacterial adjuvant developed 24-hr skin test responsiveness to schistosome antigens with the histologic features of delayed hypersensitivity. Intraperitoneal antigen injection elicited a mononuclear cell-enriched exudative population containing macrophages activated for direct cytotoxicity against schistosomula and tumor cell targets. This was likely to be due to in vivo exposure to macrophage-activating lymphokines, since these cells were unresponsive to further lymphokine stimulation in vitro and splenocytes from immunized mice reacted to specific in vitro antigen challenge by production of lymphokines capable of conferring larvacidal activity upon control macrophages. In contrast, mice treated with schistosome antigens by i.v. injection, which were not protected against challenge infection, failed to develop delayed hypersensitivity or activated macrophages in response to specific antigen challenge in vivo, and the titers of macrophage-activating lymphokine produced by in vitro antigen-stimulated splenocytes from these mice were threefold to fourfold lower than those produced by cells from animals immunized by the i.d. route. Thus, sensitization for cell-mediated immune responses including lymphokine production and macrophage activation correlated with induction of resistance to S. mansoni in this model of vaccination.     

Acquired immunity in rats against Angiostrongylus cantonensis infection

Acquired immunity against Angiostrongylus cantonensis was induced by immunizing rats with somatic antigens from fifth-stage larvae and adult worms and live third-stage larvae. Rats immunized twice had significantly fewer worms than rats immunized three times. Fewer worms were recovered from rats immunized with 200 live third-stage larvae than from any other groups. Rats immunized with somatic antigens had higher enzyme-linked immunosorbent assay (ELISA) antibody levels than rats immunized with live larvae. Rats immunized with live third-stage larvae of Angiostrongylus cantonensis were more strongly protected against challenge infections (62-92%) than rats immunized with antigens extracted from fifth-stage larvae (0-30%) and adult worms (11-24%).     

IL-6 is required for protective immune responses against early filarial infection

IL-6 has a wide range of biological activities that includes anti- and pro-inflammatory aspects. In this study, we investigated the role of IL-6 in immune responses to the rodent filarial nematode Litomosoides sigmodontis, a model for human filarial infections. IL-6^?/? mice had a significantly increased worm burden after natural infection compared with wild type controls at early time points p.i. Given that the worm burden in IL-6^?/? mice was already increased at the time point the infective larvae reached the pleural cavity, immune responses that may facilitate the migration from the site of infection (skin) via the lymphatics to the pleural cavity were analysed. Increased vascular permeability may facilitate larval migration, but blocking of histamine receptors had no effect on worm burden and vascular permeability was similar between IL-6^?/? mice and wild type controls. In contrast, blocking mast cell degranulation reduced the worm burden in IL-6^?/? mice partially, suggesting that release of mast cell-derived mediators improves larval migration to some degree. Protective immune responses within the skin were involved, as bypassing the skin barrier by inoculating infective L3s subcutaneously resulted in a comparable worm recovery in both mouse strains. Analysis of the cellular composition by flow cytometry and PCR array in the skin after exposure to filarial extract or L3s, respectively, indicate that the absence of IL-6 results in a delayed recruitment of neutrophils and macrophages to the site of initial infection. These results demonstrate that IL-6 is essentially involved in protective immune responses within the skin that impair migration of infective L3s.     

Cross-reactions with Ascaris suum antigens of sera from mice infected with A. suum, Toxocara canis, and Angiostrongylus cantonensis

Ascaris suum larval excretory-secretory (AsES) antigen and larval (AsLA) as well as adult somatic antigen (AsAA) which were thought to be possibly helpful in the diagnosis of visceral larva migrans (VLM) due to A. suum infection were investigated in the present study. Serum taken from mice orally inoculated with approximately 250 embryonated eggs of A. suum or Toxocara canis, or 40 third-stage larvae of Angiostrongylus cantonensis were assessed by enzyme-linked immunosorbent assay (ELISA) using the AsES antigen, AsLA or AsAA at 1, 2, 3, 4, 6 and 8 weeks post infection (WPI). The titer of serum IgG from mice infected with A. suum increased from 1 WPI and a peak at 4 WPI was observed when it reached approximately three times the level of uninfected control mice. Thereafter, it decreased gradually but remained high as found from 6 to 8 WPI. No cross-reactions of heterologous serum IgG against AsES antigen was observed, whereas heterologous serum IgM exhibited significant cross-reactions to AsES antigen. Cross-reactivities to AsLA and AsAA by heterologous serum IgG as well as IgM antibodies were also observed in the trial. Altogether, the AsES antigen apparently seemed to be superior to the other two somatic antigens when used in the diagnosis of A. suum-induced VLM with serum IgG as tested by ELISA. Moreover, it was the first report to test the possibly antigenic cross-reactivity between A. suum and A. cantonensis.     

The intensity and duration of primary Heligmosomoides polygyrusinfection in TO mice modify acquired immunity to secondary challenge

The effect of dose and duration of immunizing infections of Heligmosomoides polygyrus on protection against homologous challenge was studied in female TO mice. Primary infections were terminated at various levels with pyrantel embonate (adult infections) or ivermectin (larval infections) and mice were then challenged with 500 infective larvae (L3). The level of protection to secondary challenge positively correlated with the intensity of the primary immunizing infection but truncation of larval infection produced significantly better protection than termination of the adult nematode infection. The duration of the primary larval infection (1-6 days) positively correlated with the level of protection to secondary challenge, antibody responses and the proportion of circulating eosinophils. Histological changes in the gastrointestinal tract, peripheral leucocytic changes and antibody responses of the mice to H. polygyrus adult somatic antigens indicate both a cellular and humoral basis of host immunity to secondary challenge. Although the TO mice are slow responders in that they harbour chronic infections, immunization by intramucosal killing of the larval stage produced strong protection against secondary challenge infection. The presence of dead immunogenic larval stages within the intestinal wall may well be an important factor, since it exposes the host to stage specific antigens at an appropriate location. The implications of the findings for the control of gastrointestinal nematode infections are also discussed.     

Brugia malayi: antibody responses to larval antigens in infected and immunized jirds.

Vaccination with irradiated third stage Brugia malayi larvae (L3) has been reported to induce partial protective immunity to L3 challenge in jirds. The purpose of this study was to identify antigens that may be targets of protective immunity in this model. Jirds were immunized by s.c. injection of irradiated L3 and challenged either s.c. or i.p. Necropsy was performed 11 wk after challenge. Partial protection was achieved in s.c. challenged animals; worm recovery was only 41% of that observed in unvaccinated controls, and worms recovered from immunized animals were stunted. Worm recoveries in immunized animals that were challenged i.p. did not differ from those of unimmunized controls. Group differences in parasite antigen levels in sera collected 2-11 wk after larval challenge were consistent with parasitological findings obtained at necropsy. Antibody studies compared prechallenge sera from immunized animals to sera from infected (unimmunized) controls. Antibody responses to L3 surface antigens (assessed by IFA) were much stronger after immunization than after infection. Immunoblot studies showed preferential recognition of several L3 antigens (97, 54, 48, and 40 kDa) by antibodies in sera from immunized animals. Additional studies are needed to determine whether immunization with such preferentially recognized antigens can induce protection to larval challenge comparable to or better than that observed with live vaccines.     

Dirofilaria immitis: Parasite-specific humoral and cellular immune responses in experimentally infected dogs

Humoral and cellular immune responses to adult antigens of Dirofilaria immitis were evaluated in experimentally infected dogs during the chronic phase of infection. All infected dogs had significantly elevated IgG (enzyme-linked immunosorbent assay) and IgE (passive cutaneous anaphylaxis) titers against D. immitis adult antigens. However, there was little difference between infected dogs and uninfected controls in cellular-immune responses to D. immitis adult antigen or phytohemagglutinin as assessed by the lymphocyte transformation assay. Although neither cellular nor humoral responses correlated with worm burdens, cellular responses among infected dogs correlated inversely with IgG titers to D. immitis adult antigen. These results are consistent with observations in other nematode and trematode systems which suggest that in chronic tissue helminth infections there is suppression of cellular immune responses to parasite antigens while humoral responses to the same antigens remain relatively preserved.     

Induction of protective immunity against Schistosoma mansoni by a non living vaccine. I. Partial characterization of antigens recognized by antibodies from mice immunized with soluble schistosome extracts

A single intradermal injection of frozen and thawed schistosomula in conjunction with the bacterial adjuvant Mycobacterium bovis strain Bacille Calmette Guerin, Phipps substrain (BCG) induced significant levels of resistance to challenge Schistosoma mansoni infection in C57BL/6 mice. Immunization with the aqueous fraction remaining after 100,000 X G centrifugation of the larval lysate was also protective under these conditions, suggesting that some immunogenic determinants may not be membrane associated. Frozen-thawed cercariae and soluble components of adult worms also protected against challenge infection in these experiments. These observations indicate that soluble immunogens are present in both early and late developmental stages of the parasite, and therefore may be good candidate antigens for an immunochemically defined vaccine against schistosomiasis. Induction of humoral reactivity against soluble or membrane antigens was examined in mice protected against cercarial challenge by prior exposure to frozen-thawed larvae, soluble larval, or soluble adult antigens plus BCG. Animals that were immunized with frozen-thawed larvae produced low but significant levels of antibodies against larval surface antigens when examined by indirect immunofluorescence or by immunoprecipitation of surface-labeled schistosomula. Mice immunized with soluble antigens, however, showed negligible antibody reactivity against surface membrane antigens. Because mice immunized with soluble antigens were resistant to challenge infection, these results strongly suggest that anti-surface membrane reactivity is not required in the mechanism of protective immunity in this model. Sera from mice immunized with either total freeze-thaw larval lysate or soluble schistosome extracts all showed strong reactivity against soluble antigens, as detected by ELISA. Western blot analysis showed these antisera to react with a restricted number of high m.w. antigens that were present both in schistosomula and in adult worms. These antigens are therefore likely to play a major role in the development of resistance in this model as immunogens and/or as targets of protective immune response.     

Onchocerca volvulus: immunization of chimpanzees with X-irradiated third-stage (L3) larvae.

To provide a theoretical basis for the potential development of vaccines against Onchocerca volvulus (Ov) a trial has been conducted to assess the protective efficacy of immunization of chimpanzees with X-irradiated L3 larvae. Approximately 1000 larvae were injected at 0, 1, and 7 months. The immunized animals, and unimmunized controls, were then challenged with 250 live L3. In order to provide possibly protective exposure to the immunologically distinct L4 epicuticle, a radiation dose (45 krad) was chosen which preserved about 50% of the molting ability of unirradiated larvae. Despite the presence of a strong immune response to crude adult worm extracts, and to cloned Ov antigens, at the time of challenge little or no significant protection against patent infection was observed: three of four immunized animals developed patent infection as compared to four of four controls. One immunized animal failed to become patent or to manifest the late antibody response to adult worm antigens seen in both subpatent and patent infections in this model, and may have been protected from infection. The implications of these studies for future attempts to immunize against O. volvulus are discussed.     

Schistosoma japonicum: protective immunity induced by schistosomulum-derived cells in a mouse model

We previously reported that immunization with intact live cells from schistosomula of Schistosoma japonicum (S.j) partially protected the Kunming strain of mice from challenge infection. In the present work, 2 immune protective experiments were designed to further validate the protective effect induced by this type of vaccine and to optimize the immunization protocol, including the number of inoculations and parasite stages from which immunogenic cells were derived. Three antigens derived from 18-day-old postinfection live (LLC) and dead (DLC) larval worm cells and from dead 42-day-old postinfection adult worm cells (DAC) were used as immunogens. Our results demonstrate that live cells from 18-day-old worms are capable of inducing significant protection in mice using a murine-Sj challenge model as shown by reduction rates of worm recoveries and egg burdens. The development of adult worms was stunted. A Th1-biased immune response was reflected in the protected groups as evidenced by the ratio of IgG2a/IgG1. A 38-kDa polypeptide was recognized by sera from LLC immunized animals. We demonstrate that live parasite cells are a source of novel protective antigens that can be exploited for vaccine development.     

Studies on suspected clinical and experimental angiostrongyliasis: serological responses

Serum antibodies in suspected angiostrongyliasis patient were detected by ELISA. The antibody titre was 1:51,200 in the serum and 1:6,400 in CSF with preadult A. cantonensis antigen. Other tests like AGD and CIEP failed to show any positive reaction with both preadult and adult worm antigens. Experimental infection with 100 A. cantonensis larvae in albino rats indicated positive CIEP reaction in serum from the day 5 to 375 after infection. No precipitin line was seen on the other hand, in AGD during observation period. Different rat groups infected with larval doses of 100, 500, 2,000, and 5,000 showed positive CIEP reaction, on the 21st day of infection when preadult worms were seen in CNS. There was no CIEP reaction when a low dose of 15 larvae was used. Cerebral fluid of rats infected with heavy dose of 5,000 larvae showed positive CIEP reaction on the 21st day.     

Filarial-specific IgG4 response correlates with active Wuchereria bancrofti infection

The filarial-specific humoral immune response of adult residents of two areas of Papua New Guinea, differing in transmission of Wuchereria bancrofti infection was compared. The majority of residents of the village of Bonahoi, in an area where transmission of filariasis had been interrupted by a 20-year insecticide spray program to control malaria, showed no parasitologic signs of active W. bancrofti infection and were negative for both circulating phosphorylcholine Ag and peripheral blood microfilariae. In contrast, adult residents of the village of Nanaha were in an area exposed to infection, and were phosphorylcholine-Ag- and microfilariae-positive. The antibody response of these two groups to both adult worm excretory/secretory (ES) Ag and somatic antigen extract was examined to determine which components of the filarial-specific immune response were dependent on active infection. Identification of these immune responses may point to immunologic methods to evaluate control programs for lymphatic filariasis. Adults from Bonahoi were found to have significant immune responses to [35S] methionine-labeled ES Ag by immunoprecipitation and to adult somatic antigen extracts by ELISA and by immunoblotting. This result is consistent with the fact that these individuals were previously exposed to and/or infected with W. bancrofti. Similarly, residents of the endemic village had detectable immune responses to these Ag irrespective of if they were microfilaremic. The most striking immunologic difference observed between the two groups was that residents of Bonahoi had a dramatically reduced filarial-specific IgG4 antibody response to both adult somatic Ag and adult ES Ag. These data suggest that longitudinal measurement of filarial-specific IgG4 levels may be a useful seroepidemiologic indicator of changes in W. bancrofti infection status.     

Tyvelose and protective responses to the intestinal stages of Trichinella spiralis

The unusual sugar tyvelose is the immunodominant portion of the major larval glycoprotein antigens of Trichinella spiralis, which play an important role in generating immunity against the intestinal stages of infection. The possibility that the tyvelose component itself may have a host- or parasite-protective role in the intestine was tested by following the outcome of challenge infections in mice primed and boosted with tyvelose-BSA, or in mice primed with tyvelose-BSA before boosting with larval antigen. Although antibody responses were raised against tyvelose there was no evidence of protective immunity against the intestinal stages, as assessed by total adult worm recovery or by size and fecundity of female worms in immunized mice. Equally, priming with tyvelose-BSA before boosting with larval antigen had no effect on the expression of immunity against a challenge infection. The predominant antibody isotype recorded in all immunized mice was IgG1, suggesting the induction of type 2 T cell responses, and this was confirmed by cytokine analysis, mesenteric node lymphocytes of all mice showing production of IL-5 but not IFN-gamma. Clearly immunization with tyvelose had no significant effect on T cell polarization. The data show that, with the experimental design employed, there was no evidence for a functional role of tyvelose in either host- or parasite-protection during the intestinal phase of infection.     

Schistosoma mansoni-New Zealand rabbit model: resistance induced by infection followed by active immunization with protective antigens

In previous studies, rabbits immunized with adult worm antigens released from fresh adult schistosomes incubated in saline media showed a significant level of protection against challenge parasites. Focusing on the rabbit-Schistosoma mansoni model, concomitant immunity was investigated. A peculiar form of response to cercarial infection was observed: rabbits subjected to percutaneous infection and similar reinfections at different times after primary infection killed schistosomula from the challenge infection as well as established parasites from the primary infection. In this study the challenge infection stimulus was replaced by active immunization with an adult worm-derived protective antigenic mixture. The results show that immunization of New Zealand rabbits with an adult worm antigenic extract is capable of inducing a response that results in a significant reduction of the mean worm burden of the primary infection earlier than did homologous infection, as compared to worm reduction due to a second infection.     

The evaluation of recombinant hookworm antigens as vaccines in hamsters (Mesocricetus auratus) challenged with human hookworm, Necator americanus

We have previously reported the successful adaptation of human hookworm Necator americanus in the golden hamster, Mesocricetus auratus. This animal model was used to test a battery of hookworm (N. americanus and Ancylostoma caninum) recombinant antigens as potential vaccine antigens. Hamsters immunized a leading vaccine candidate N. americanus-Ancylostoma secreted protein 2 (Na-ASP-2) and challenged with N. americanus infective larvae (L3), resulted in 30-46.2% worm reduction over the course of three vaccine trials, relative to adjuvant controls. In addition, significant reduction of worm burdens was also observed in the hamsters immunized with adult hookworm antigens A. caninum aspartic protease 1 (Ac-APR-1); A. caninum-glutathione-S transferase 1 (Ac-GST-1) and Necator cysteine proteases 2 (Na-CP-2) (44.4%, 50.6%, and 29.3%, respectively). Our data on the worm burden reductions afforded by these hookworm antigens approximate the level of protection reported previously from dogs challenged with A. caninum L3, and provide additional evidence to support these hookworm antigens as vaccine candidates for human hookworm infection. The hamster model of N. americanus provides useful information for the selection of antigens to be tested in downstream vaccine development.