Adult 175 kDa collagenase antigen of Setaria cervi in immunoprophylaxis against Brugia malayi in jirds

A 175 kDa antigen fraction with collagenase activity was isolated and purified from somatic extracts of adult Setaria cervi females using column chromatography involving consecutive steps of DEAE-Sepharose CL6B and Sephadex G-100. The optimum pH for 175 kDa collagenase was found to be pH 7.0. Sensitivities to a variety of inhibitors and activators indicated that the 175 kDa coIlagenolytic enzyme was metalloserine in nature. The enzyme hydrolysed a variety of protein substrates such as haemoglobin, casein, azocasein (general substrates) and collagen, FALGPA (furanoyl-acryloyl-leu-gly-pro-ala), the specific substrate of collagenase. The enzyme showed 57% inhibition by jird anti-somatic collagenase antibodies and reacted insignificantly with normal jird sera. Further analysis was undertaken on the immunoprophylactic potential of 175 kDa collagenase in inducing immunity against Brugia malayi (a human filarial parasite) in jirds (Meriones unguiculatus) in vitro and in situ. Immune sera of jirds raised against this antigen promoted partial adherence of peritoneal exudate cells to B. malayi microfilariae (mf) and infective larvae (L3) in vitro and induced partial cytotoxicity to the parasites within 48 h. The anti-S. cervi 175 kDa antigen serum was more effective in inducing cytotoxicity to B. malayi L3, than mf. In the microchambers implanted inside immune jirds, host cells could migrate and adhere to the mf and infective larvae thereby killing them partially within 48 h.     

Induction of protection against Brugia malayi infection in jirds by microfilarial antigens

The development of immunologic methods to reduce transmission of human lymphatic filariasis depends on measures that will enhance the host's ability to eliminate infective larvae, adult worms, or blood-borne microfilariae (mf). The present study was designed to assess the capacity of a crude extract of Brugia malayi mf to decrease the level of microfilaremia and adult worm burden in jirds inoculated with infective larvae, and to identify the filarial antigens that elicit antibody responses in these animals. Thirty weeks after subcutaneous inoculation with 75 infective larvae, 100% of control jirds were patent (i.e., had microfilaremia) compared with 60% of the group immunized with 10 micrograms of crude microfilarial extract (p less than 0.05). In addition, microfilaremia was lower in patent immunized animals compared with controls (p less than 0.05). The mean total number of adult female B. malayi per jird recovered at necropsy in control animals was 16.0 vs 7.0 in immunized jirds (p less than 0.05). Serum of immunized jirds contained anti-mf antibodies with an end titer of 1:8000, a value similar to that of animals with chronic B. malayi infection. Microfilarial antigens of Mr approximately 150,000, 75,000, 42,000, and 25,000 were identified in immunoblotting studies by reactivity with antibodies in sera of immunized jirds. Antibodies induced by immunization with microfilarial extract were not specific for this stage of the parasite life cycle, as jird anti-mf antibodies reacted with a Mr approximately 150,000 and several Mr 50,000 to 110,000 antigens derived from immature and mature adult parasites of both sexes. These data indicate that immunization of jirds with a water soluble microfilarial extract enhances the host's ability to eliminate adult worms and blood-borne mf. The filarial antigens that induce antibodies in immunized jirds have been identified.     

Seroreactivity of purified Brugia malayi microfilarial soluble and excretory-secretory antigens in different clinical presentations of bancroftian filariasis

Brugia malayi microfilarial excretory-secretory (mf ES) and phosphate buffer saline soluble (mf S) antigens were fractionated by fast protein liquid chromatography (FPLC) on superdex 200 HR 10/30 gel filtration column. The active antigen fractions were identified and explored in comparison with whole mf ES and mf S antigens to detect filarial IgG antibodies in different groups viz microfilaraemics, acute, chronic and occult filarial cases of Wuchereria bancrofti infection and endemic and non-endemic normals. One of the fractions of mf ES antigen (ESF-6) and two fractions of mf S antigen (SF-2 & 3) were identified to be useful to detect filarial antibodies. A pooled preparation of these antigen fractions gave a sensitivity of 86.6% (for microfilaraemic cases) and a specificity of 95% to detect filarial IgG antibodies by indirect ELISA. The pooled FPLC purified mf antigens also showed 55-88% of cases of different grades of clinical filariasis and 65% of tropical pulmonary eosinophilia cases as positive for filarial antibodies. The pooled FPLC purified B. malayi mf antigens with higher specificity are preferable to whole mf ES and mf S antigens to detect active filarial infection in microfilaraemia and as well in different clinical entities of bancroftian filariasis.     

A combination of two Brugia malayi filarial vaccine candidate antigens (BmALT-2 and BmVAH) enhances immune responses and protection in jirds

In this study filarial recombinant protein or DNA vaccine constructs encoding BmALT-2 and BmVAH as single or as cocktail antigens were evaluated. Male jirds were immunized intramuscularly with DNA vaccine constructs or were immunized intraperitoneally with protein vaccine. The single and bicistronic DNA constructs induced substantial interferon-γ responses in spleen cells; antigen-specific responses were higher following immunization with the bicistronic cocktail construct and evoked a significant protective response of 57% in jirds challenged with Brugia malayi that was similar in the antibody-dependent cellular cytotoxicity (ADCC) assay and micropore chamber experiment. The cocktail protein vaccines induced a mixture of IgG2a (Th1) and IgG1 (Th2) responses with 80% protective response when challenged with B. malayi infective larvae. However, the single protein vaccine rALT-2 induced Th2 (IgG1/IgG3) with a 70% protective response and rVAH induced Th1 (IgG2a) with a lower proliferative response with 60% protection following challenge with B. malayi infective larvae. These results suggest that filarial cocktail protein vaccines are able to elicit substantial immune and protective responses when compared with single antigen vaccination in suitably vaccinated jirds.     

Paramyosin-enhanced clearance of Brugia malayi microfilaremia in mice

Progress in development of a vaccine against human filariasis has been hampered by lack of knowledge of the biochemical structure of specific Ag that induce protective immunity in experimental hosts. In the current study, antiserum to infective third-stage larvae of Brugia malayi was used to select potentially protective Ag shared by microfilariae (mf) and adult worms. A major Ag of 97 kDa (Bm 97) was identified by immunoblotting and isolated by electroelution. Immunization of mice with 2 micrograms electroeluted Bm 97 induced partial resistance to subsequent i.v. challenge with live B. malayi mf (40 to 60% reduction in parasitemia compared to controls, p less than 0.05). Immunoblot studies of B. malayi mf and adult worm lysates showed reactivity of a 97-kDa molecule with monospecific antiserum to Schistosoma mansoni paramyosin. In addition, mouse antibody to Bm 97 reacted with a 97-kDa molecule contained in wild-type Caenorhabditis elegans but not in two mutant strains deficient for paramyosin. Subcutaneous injection of mice with paramyosin (5 micrograms twice at a 2-wk interval) purified from C. elegans or B. malayi by salt precipitation induced resistance to microfilaremia (21 to 60% lower intensities than controls, p less than 0.01). These data indicate that the invertebrate muscle protein paramyosin enhances clearance of blood-borne stages of lymphatic filariae. Examination of the ability of paramyosin to induce resistance in third-stage larvae-challenged hosts is warranted.     

Human antibody responses to Brugia malayi antigens in brugian filariasis.

Human antibody responses to Brugia malayi antigens were studied with sera from a Brugia endemic area in South India. Patients with clinical filariasis had significantly higher IgE and lower IgG4 levels to adult worm antigens than people with asymptomatic microfilaraemia. Intermediate antibody levels were observed in endemic normals. A majority of sera from each clinical group contained IgG antibodies to surface antigens of infective larvae (L3) by IFAT. IgG immunoblot studies did not reveal group differences in L3 antigen recognition. IgE antibodies bound to a subset of antigens bound by IgG. IgE antibodies in sera from clinical filariasis patients preferentially bound to L3 antigens at 200, 97, 68 and 58 kDa compared with sera from microfilaria carriers. These results are consistent with prior studies of antibody responses in filariasis and add new information on the targets of IgG and IgE antibodies to L3 antigens in brugian filariasis.     

A 43-kDa circulating filarial antigen fraction of Wuchereria bancrofti in immunoprophylaxis against Brugia malayi in jirds

A 43 kiloDaltan (kDa) antigen fraction (CFA₂-6) isolated from microfilaraemic plasma of bancroftian filarial patients showed selective reactivity with sera samples collected from endemic normals. Antibodies raised against this antigen showed a strong reactivity with the surface of Brugia malayi infective larvae as well as microfilariae. Similar antigenic determinants were detected in the parasite extracts, but not in the excretory–secretory products. Further analysis was done on the immunoprophylactic potential of CFA₂-6 in inducing immunity against Brugia malayi in Meriones unguiculatus (jird) in vivo. A strong protective response of approximately 84% was observed against the development of the filarial parasite in the jirds immunized with CFA₂-6. The immunized jirds also showed a significant clearance (87%) of microfilariae inoculated intravenously. Approximately 65% of infective larvae failed to survive in jirds transferred with anti-CFA₂-6 serum compared to the jirds transferred with sera from the control jirds. Passive transfer of anti-CFA₂-6 antibody to the jirds followed by intravenous inoculation of microfilariae resulted in the reduction of 77% of circulating microfilariae. This study suggests that the 43-kDa CFA₂-6 could stimulate a strong protective immune response against infective larvae and microfilariae in experimental animals.     

Development of antigen detection ELISA for the diagnosis of brugian and bancroftian filariasis using antibodies to recombinant filarial antigens Bm-SXP-1 and Wb-SXP-1

Antibodies specific to recombinant filarial antigens Wb-SXP-1 and Bm-SXP-1 have been used to develop a sandwich ELISA for the detection of circulating filarial antigen (CFA) in sera from patients with lymphatic filariasis caused by Wuchereria bancrofti of Brugia malayi. In patients with W. bancrofti infections, a high proportion of microfilaria (mf) positive (MF) and low proportions of patients with chronic pathology (CP) and endemic normals (EN) showed the presence of CFA. Similarly in patients with brugian infections a high proportion of mf positive individuals contained CFA while none of the patients with chronic pathology or endemic normals showed the presence of CFA. Sera from patients with other parasitic infections (OPI) like O. volvulus, Loa loa, Ascaris lumbricoides and from individuals residing in areas non-endemic to filariasis did not exhibit any reactivity. This assay shows promise for the detection of microfilaremic infections in lymphatic filariasis and its usefulness as a diagnostic tool especially in B. malayi infections, needs to be further evaluated.     

Brugia pahangi: stage-specific antigens on microfilariae detected by serum from infected jirds or by monoclonal antibodies

Experiments were carried out to determine whether there are stage-specific antigens on microfilariae of Brugia pahangi, using sera from Mongolian jirds infected with B. pahangi and monoclonal antibodies against microfilariae of B. pahangi. These studies showed that microfilariae have both stage-specific and nonspecific antigens. The nonspecific antigens were also present on adult worms and on infective larvae. Among monoclonal antibodies, 6 out of 14 clones produced antibodies against the microfilarial stage-specific antigens, and 8 clones produced antibodies against nonspecific antigens. These monoclonal antibodies could not distinguish between adults, microfilariae, or infective larvae of B. malayi and B. pahangi.     

Setaria cervi: immunoprophylactic potential of glutathione-S-transferase against filarial parasite Brugia malayi

Glutathione-S-transferase (GST) has been detected in the adult female Setaria cervi, a bovine filarial parasite. The role of S. cervi GST antigen in inducing immunity in the host against Brugia malayi microfilariae and infective larvae was studied by in vitro antibody dependent cell mediated reaction as well as in situ inoculation of filarial parasites within a microchamber in Mastomys. The immune sera from glutathione-S-transferase immunized Mastomys promoted the adherence of peritoneal exudate cells to B. malayi microfilariae and infective larvae in vitro inducing 80.7 and 77.6% cytotoxicity, respectively in 72 h. In the microchambers implanted in the immunized Mastomys host cells could migrate and adhere to the microfilariae and infective larvae and induced 77.8 and 75% cytotoxicity to B. malayi microfilariae and infective larvae in 72 h, respectively. These results suggest that native GST from S. cervi is effective in inducing protection against heterologous B. malayi filarial parasite and thus has potential in immunoprophylaxis.     

Immunoprophylaxis against filarial parasite,Brugia malayi: potential of excretory-secretory antigens in inducing immunity

The role of excretory-secretory antigens in inducing immunity in the host againstBrugia malayi microfilariae and infective larvae was studied byin vitro antibody dependent cell-mediated reaction as well asin vivo inoculation of filarial parasites within a microchamber in the host. The immune sera of jirds raised againstBrugia malayi microfilarial and infective larval excretory-secretory antigens(Bm Mf ESA andBm L₃ ESA) promoted the adherence of peritoneal exudate cells toBrugia malayi microfilariae and infective larvaein vitro and induced cytotoxicity to the parasites within 48 h. The antiBm Mf ESA serum was more effective than antiBm L3 ESA serum in inducing cytotoxicity to microfilariae and both antisera had a similar cytotoxic effect on infective larvae. In the microchambers implanted in the immune jirds, host cells could migrate and adhere to the microfilariae and infective larvae and kill them within 48–72 h. Further,Mastomys natalensis immunized againstBm Mf ESA and L₃ ESA generated a high degree of protective response against circulating microfilariae. These results suggest that excretory-secretory antigens are effective in inducing resistance against filarial parasites and thus have potential in immunoprophylaxis.     

The endemic normal in lymphatic filariasis: A static concept

Residents of areas endemic for lymphatic filariasis are continually exposed to infection with mosquito-transmitted infective larvae (L3), some of which survive to become adult worms and subsequently produce micro filarial (mf) transmission stages. The question of whether naturally acquired resistance occurs in adult residents of endemic areas has recently become of interest as the development of molecular vaccines against filarial parasites is being considered(1,2). There have been two epidemiological approaches to demonstrate acquired resistance to Filariasis in human populations. In this review Karen Day examines both approaches in the context of an immunoepidemiological study of bancroftian filariasis in Papua New Guinea (PNG). The merits of each as a conceptual framework for studies of protective immunity in lymphatic filariasis will be discussed.     

Brugia malayi: detection of parasite antigen in sera from infected jirds

Sera from Brugia malayi-infected jirds were demonstrated to contain a heat-stable, 95- to 105-kDa parasite antigen by immunoblot with rabbit antibody to the parasite and with a monoclonal antibody that binds to phosphorylcholine. This antigen is a major component of B. malayi adult worm excretory/secretory antigen, and it is present in lavage fluid obtained from ip-infected animals. The antigen was detected by enzyme immunoassay in all sera collected from jirds 9-54 weeks after sc injection with 100 or 300 infective larvae (L3). Parasite antigen titers were higher in animals infected with the higher L3 dose. Antiphosphorylcholine antibodies were present in jird sera for the first 12 weeks after larval injection, but thereafter, antibody titers decreased to undetectable levels. Parasite antigen was not detected by immunoblot or enzyme immunoassay in sera from 21 human subjects with B. malayi microfilaremia. Antigen may be cleared from human sera by antiphosphorylcholine antibodies, which were present in all sera tested. The practical significance of B. malayi antigen detection in the jird is that it provides a sensitive means of noninvasively monitoring the status of infection in this important experimental filariasis model.     

Brugia malayi: rat cell interactions with infective larvae mediated by complement

Albino rat macrophages and neutrophils, in the presence of fresh normal rat serum as a source of complement, adhered to and promoted killing of Brugia malayi infective larvae in vitro. Eosinophils, by themselves, were marginally cytotoxic at a high cell-target ratio but promoted cytotoxicity when mixed with macrophages. Eosinophil culture supernatants enhanced the macrophage mediated killing of infective larvae. The complement of fresh normal rat serum was found to act by the alternate pathway. Fresh normal rat serum depleted of alternate pathway complement activity by treatment with zymosan A, or of Factor B by heating at 50 C for 20 min, or of Factor D by passing through Sephadex G75 column, failed to promote cell adherence to the parasite. C3 molecules were detected on the surface of infective larvae by immunofluorescence. There was a significant consumption of complement when Brugia malayi infective larvae were incubated in fresh normal rat serum. Albino rat cells were more potent in inducing cytotoxicity to infective larvae in vitro than those from jird or Mastomys natalensis, which may reflect the greater resistance offered by the rat to B. malayi infection. There was much less cellular infiltration on introduction of Brugia malayi infective larvae into the peritoneal cavity of jirds compared to rats and Mastomys natalensis indicating the greater susceptibility of jirds to intraperitoneally induced infections.     

Localization of Brugia malayi (sub-periodic) adults in different organs of Mastomys coucha and its influence on microfilaraemia and host antibody response

Lymphatic filariasis caused by nematode parasites Wuchereria bancrofti or Brugia malayi is a spectral disease and produces wide range of immune responses and varying levels of microfilaraemia in infected individuals. The relationship between the immune response of host and the developmental stage of the parasite as well as the microfilariae (mf) density and specific location of the adult worms is yet to be understood. As an experimental model, B. malayi adapted in the experimental animal Mastomys coucha has been used widely for various studies in filariasis. The present study was to assess microfilaraemia as well as the humoral immune response of M. coucha during various stages of B. malayi development and their localization in different organs. The result showed that the density of mf in the circulating blood of the experimental animal depended upon the number of female worms as well as the location and co-existence of male and female worms. The mf density in the blood increased with the increase in the number of females. The clearance of inoculated infective stage (L3) or single sex infection or segregation of male and female to different organs of infected host resulted in a microfilaraemic condition. With respect to antibody response, those animals cleared L3 after inoculation and those with adult worm as well as mf showed low antibody levels. But those with developmental fourth stage and/or adult worms without mf showed significantly higher antibody levels.     

Immunocytochemical localization of antigens recognized by human antisera in infective larvae of Wuchereria bancrofti

Ultrathin sections of L3 of Wuchereria bancrofti embedded in hydrophilic resin were incubated with antisera pools from individuals (1) asymptomatic microfilaremic with different microfilaria (mf) densities (1-100, 101-500, and >1,000 mf/ml); (2) chronic with hydrocele or lymphedema; and (3) with no evidence of microfilaremia or clinical filariasis but residing in an endemic area. The groups of microfilaremic subjects studied presented differences relative to the intensity of labeling, with the density of gold particles per square micrometer proportional to microfilaremia. Incubation of ultrathin sections of W. bancrofti L3 larvae in the presence of antisera from patients exhibiting chronic obstructive lymphatic pathology of hydrocele and from individuals with clear clinical evidence of lymphedema exhibited a strong reaction in the same tissues. Except for the endemic normal group, all groups studied showed reactivity against epitopes in all tissues of infective larvae of W. bancrofti. The cuticle presented an intense labeling, suggesting a possible target structure for immune response.     

Effect of CGP 20376 on Brugia malayi and parasite antigenemia in jirds

This study was designed to investigate the activity of CGP 20376, a benzothiazole derivative, against Brugia malayi in jirds and to illustrate the utility of parasite antigen detection as a means of monitoring drug efficacy in filariasis. Drug treatment was 100% effective in jirds treated 3 or 24 days after infection. Microfilaria and adult worm counts were reduced (relative to counts in sham-treated control animals) by 96% and 95%, respectively, in animals treated 153 days after infection. Four of 6 animals in this treatment group cleared their microfilaremias and were free of adult worms 5 mo after treatment. Thus, CGP 20376 was effective against all life cycle stages of B. malayi in jirds. Parasite antigen levels in jird sera were consistent with parasitological results in all treatment groups, but antigen clearance was incomplete in some cases after apparently successful treatment of mature and immature infections.     

Cryopreservation of infective larvae of Dipetalonema viteae.

infective larvae of Dipetalonema viteae produced infections in Mongolian jirds (Meriones unguiculatus) after storage of infected ticks (Ornithodoros tartakovskyi) in the presence of dimethyl sulfoxide (DMSO, 5%) for 7 or 595 days in liquid nitrogen (-196 C). Infectivity of these larvae was only partially impaired. Microfilaremias of test jirds were generally lower than those of control jirds given nonfrozen larvae; however, the majority of test jirds developed microfilarial counts suitable for use in infecting ticks. In contradistinction, larvae frozen free of the tick failed to retain infectivity. Apparently the tick, in conjunction with DMSO, protects the larvae during freezing and thawing.     

Brugia pahangi: Antibodies and microfilaremias in Lewis rats

Male and female Lewis rats were inoculated subcutaneously in the left groin with 75 infective larvae of Brugia pahangi and microfilaremias were followed for as long as 420 days postinoculation. Patent infections developed in 64% of the female rats and 95% of the male rats. Mean prepatent periods were similar (65.9 and 63.9 days, respectively), but mean microfilaremias in males rose much higher, to a mean of 218 mf/0.25 ml blood at 270 days postinoculation. IgG titers, as measured by enzyme-linked immunosorbent assay (ELISA), to adult worm somatic antigen were higher than those to microfilariae in almost all rats. For both sexes, the most consistently microfilaremic rats had highest titers to these antigens. Granulomas with degenerating microfilaria were present in the spleen of male rats with high microfilaremias (>100–300 mf/0.25 ml blood). Ouchterlony precipitin reactions suggested that most rats with spleen granulomas responded to microfilarial antigen components to which most rats without granulomas did not. Neither spleen granulomas nor antibody responses measured in this study appeared to have protective (microfilaremia-lowering) value. As measured by microfilaremias, the male Lewis rat is not as susceptible as some conventional hosts of B. pahangi, but it does consistently become infected and remains microfilaremic for more than a year. Preferential male susceptibility indicates that this model may be useful for studying this aspect of human lymphatic filariasis.     

The transmission of an avian filarioid Cardiofilaria nilesi to the jird, Meriones unguiculatus

This paper reports the experimental transmission of a bird parasite into jirds. Infective larvae of Cardiofilaria nilesi obtained from laboratory colonized Coquillettidia crassipes mosquitoes which had fed on an infected chicken were inoculated subcutaneously into jirds. The number of larvae per jird varied from 10 to 228. Microfilaraemia appeared 22 to 89 days after inoculation of the infective larvae. Experiments were carried out with 24 jirds through six generations extending over a period of 22 months and 17 produced patent infections. At present 8 infected jirds are being maintained in the laboratory; their patent periods ranging from 6 to 13 months. However, the longest patent period observed was about thirteen months. The percentage of adults recovered in autopsied jirds ranged from 0 to 40 with an average of 16. The chicken showed a microfilarial periodicity with the peak microfilarial density around 2200 hours. However, in jirds there was a change in sub-periodicity. This model in the jird may be very useful for the screening of filaricides and in immunological work.