Basophils help establish protective immunity induced by irradiated larval vaccination for filariasis.

Basophils are increasingly recognized as playing important roles in the immune response toward helminths. In this study, we evaluated the role of basophils in vaccine-mediated protection against filariae, tissue-invasive parasitic nematodes responsible for diseases such as elephantiasis and river blindness. Protective immunity and immunological responses were assessed in BALB/c mice vaccinated with irradiated L3 stage larvae and depleted of basophils with weekly injections of anti-CD200R3 antibody. Depletion of basophils after administration of the vaccination regimen but before challenge infection did not alter protective immunity. In contrast, basophil depletion initiated prior to vaccination and continued after challenge infection significantly attenuated the protective effect conferred by vaccination. Vaccine-induced cellular immune responses to parasite antigen were substantially decreased in basophil-depleted mice, with significant decreases in CD4(+) T-cell production of IL-4, IL-5, IL-10, and IFN-γ. Interestingly, skin mast cell numbers, which increased significantly after vaccination with irradiated L3 larvae, were unchanged after vaccination in basophil-depleted mice. These findings demonstrate that basophils help establish the immune responses responsible for irradiated L3 vaccine protection.

Vaccination against filarial nematodes with irradiated larvae provides long-term protection against the third larval stage but not against subsequent life cycle stages.

Sustainable control of human filariasis would benefit enormously from the development of an effective vaccine. The ability to vaccinate experimental animals, with reductions in worm burden of over 70%, suggests this aim is possible. However, in experimental vaccinations the challenge is usually administered 2 weeks after the immunisation phase and thus the protection obtained is likely to be biased by persisting inflammation. Using the murine model Litomosoides sigmodontis, we increased the time between immunisation with irradiated larvae and challenge with fully infective L3 to 5 months. Significant protection was achieved (54-58%) and the reduced worm burden was observed by 10 days p.i. The developmental stage targeted was the L3, since no nematodes died once they reached the pleural cavity of vaccinated mice, as has been previously shown in short-term protocols. However, larval developmental rate was faster in vaccinated than in primary-infected mice. Immunological assessments were made prior to challenge and then from 6 h to 34 days post-challenge. Samples were taken from the subcutaneous tissue where the larvae were inoculated, the lymph nodes through which they migrate and the pleural cavity in which they establish. Eosinophils were still present although scarce in the subcutaneous tissue of vaccinated mice before challenge. Cytokine and specific antibody production of vaccinated and challenged mice were L3-specific and Th2-biased and greatly exceeded the response of primary-infected mice. The heightened Th2 response may explain the faster development of the filarial worms in vaccinated mice. Thus, long-term vaccination protocols generated a strong memory response that led to significant but incomplete protection that was limited to the infective larval stage suggesting alternative vaccination strategies are needed.

Parasitology and immunology of mice vaccinated with irradiated Litomosoides sigmodontis larvae.

This study was performed with Litomosoides sigmodontis, the only filarial species which can develop from the infective larvae to the patent phase in immunocompetent laboratory BALB/c mice. Parasitological features and immune responses were analysed up to 3 months before and after challenge inoculation, by comparing 4 groups of mice: vaccinated challenged, challenged only, vaccinated only, and naive mice. Male larvae were very susceptible to irradiation and only female irradiated larvae survived in vivo. Protection, assessed by a lower recovery rate, was confirmed and was established within the first 2 days of challenge. This early reduction of the recovery rate in vaccinated challenged mice was determined by their immune status prior to the challenge inoculation. This was characterized by high specific IgM and IgG subclass (IgG1, IgG2a and IgG3) levels, high specific IL-5 secretion from spleen cells in vitro and a high density of eosinophils in the subcutaneous connective tissue. Six h after the challenge inoculation, most tissue eosinophils were degranulated in vaccinated challenged mice. Thus, in the protocol of vaccination described, protection appeared mainly to result from the stimulation of a Th2 type response and eosinophils seemed to be the main effectors for the increased killing of infective larvae in vaccinated challenged mice. Two months after challenge inoculation, the percentage of microfilaraemic mice was lower in vaccinated challenged mice as a consequence of this overall reduction in the worm load. In both vaccinated challenged and challenged only groups, the in vitro splenocyte proliferative capacity was reduced in microfilaraemic mice.

Early reduction of the challenge recovery rate following immunization with irradiated infective larvae in a filaria mouse system.

The filaria Litomosoides sigmodontis, which develops a patent infection in BALB/c mice, was used to determine the fate of a challenge inoculum following immunization of mice with irradiation attenuated infective larvae (3 subcutaneous inoculations at weekly intervals with 25 L3 irradiated at 60 krad, and challenge with 25 L3 two weeks after the final immunization). The adult worm burden of vaccinated mice was reduced to 50% of that of controls although the pattern of larval migration and microfilaraemia were not affected. Necropsies showed that the increased killing of the filariae of the challenge inoculum occurred at the L3 stage within the first 2 days of challenge. This result draws attention on the protective mechanisms operating very early and probably in the subcutaneous region.

203Hg and other gamma-emitting radio-isotopes as labels for Dirofilaria immitis microfilariae.

The in vitro uptake of gamma-emitting radionuclides by microfilariae of Dirofilaria immitis was investigated. Radionuclides tested were 133Ba, 207Bi, 82Br, 109Cd, 51Cr, 60Co, 59Fe, 203Hg, 125I, 54Mn, 32P, 125Sb, 46Sc, 75Se and 65Zn. Only 207Bi, 59Fe, 203Hg, 54Mn and 46Sc showed more than 2% of the available radioactivity to bind to the microfilariae. When tested for retention in vitro only 203Hg showed retention levels approaching 90%. Moreover, when dimethyl-sulphoxide was incorporated into the medium at levels of 1% (v/v) the uptake of 203Hg could be increased by 3-5 times; no other radio-isotope tested responded in this manner. The uptake of 203Hg was directly related to temperature and time of incubation. Mercury, as mercuric chloride, was toxic to the microfilariae and represents an impediment to the incorporation of high levels of 203Hg in microfilariae.

The development of normal and gamma-irradiated Litomosoides carinii larvae in cotton rats.

The development of infective, stage 3 larvae of Litomosoides carinii irradiated at either 2.7, 5.4, 10.8, 16.2, 21.6 or 40 krads in a cobalt 60 source was compared with that of normal larvae following injection into cotton rats. Worm recovery decreased as the radiation dose increased, male worms being more affected than female worms. Worms developing from irradiated larvae were shorter than normal and at 16.2 krads and above they failed to develop into adults despite reaching the pleural cavity and moulting to stage 4 larvae. Development had ceased by 35 and 20 days post infection for larvae irradiated at 21.6 and 40 krads respectively. 10 krads and below allowed larvae to moult into adults but they were stunted and retarded developmentally. Microfilariae were absent from the pleural cavity and peripheral circulation of all cotton rats injected with irradiated larvae.

Studies with Brugia pahangi. 15. Cobalt 60 irradiation of the worm.

Infective larvae of Brugia pahangi were irradiated at 10, 25 or 45 krads by means of a Cobalt 60 source. In cats, 10 krads caused the worms to be stunted and sterile but allowed them to become 5th stage, migrate posteriorly into the afferent lymphatic, and produce pathology. 25 krads prevented the worms from developing beyond the early fourth stage and from migrating away from the popliteal lymph node. No gross pathological reacions were evident. 45 krads produced the same effects as 25 krads but the longevity of the worms was much reduced.