Brugia Malayi Infective Larvae Research Articles

Brugia malayi infective larvae fail to activate Langerhans cells and dermal dendritic cells in human skin.

Filarial infection in humans is initiated when a mosquito deposits third-stage parasite larvae (L3) in the skin. Langerhans cells (LCs) and dermal dendritic cells (DDCs) are the first cells that the parasite encounters, and L3s must evade these highly effective antigen-presenting cells to establish infection. To assess LC and DDC responses to L3 in human skin, we employed three models of increasing physiologic relevance: in vitro-generated LCs, epidermal blister explants and full-thickness human skin sections. In vitro-generated LCs expressed TLR1-10 and robustly produced IL-6 and TNF-α in response to PolyI:C, but pre-exposure to L3s did not alter inflammatory cytokine production or TLR expression. L3s did not modulate expression of LC markers CDH1, CD207, or CD1a, or the regulatory products TSLP or IDO in epidermal explants or in vitro-generated LC. LC, CD14+ DDC, CD1c+ DC and CD141+ DC from human skin sections were analysed by flow cytometry. While PolyI:C potently induced CCL22 production in LC, CD1c+ DC, and CD141+ DC, and IL-10 production in LC, L3s did not modulate the numbers of or cytokine production by any skin DC subset. L3s broadly failed to activate or modulate LCs or DDCs, suggesting filarial larvae expertly evade APC detection in human skin.

Evaluation of a multivalent vaccine against lymphatic filariasis in rhesus macaque model.

Lymphatic filariasis affects 120 million people worldwide and another 1.2 billion people are at risk of acquiring the infection. Chemotherapy with mass drug administration is substantially reducing the incidence of the infection. Nevertheless, an effective vaccine is needed to prevent the infection and eradicate the disease. Previously we reported that a multivalent fusion protein vaccine (rBmHAT) composed of small heat shock proteins 12.6 (HSP12.6), abundant larval transcript-2 (ALT-2) and large extracellular domain of tetraspanin (TSP LEL) could confer >95% protection against the challenge infection with Brugia malayi infective larvae (L3) in mouse and gerbil models. In this study we evaluated the immunogenicity and efficacy of rBmHAT fusion protein vaccine in a rhesus macaque model. Our results show that rBmHAT is highly immunogenic in rhesus macaques. All the vaccinated monkeys developed significant titers of antigen-specific IgG antibodies against each of the component antigens (16,000 for rBmHSP12.6), (24,000 for rBmALT-2) and (16,000 for rBmTSP-LEL). An in vitro antibody dependent cellular cytotoxicity (ADCC) assay performed using the sera samples from vaccinated monkeys showed that the anti-rBmHAT antibodies are functional with 35% killing of B. malayi L3s. Vaccinated monkeys also had antigen responding cells in the peripheral blood. Vaccine-induced protection was determined after challenging the monkeys with 500 B. malayi L3. Following challenge infection, 3 out of 5 vaccinated macaques failed to develop the infection. These three protected macaques had high titers of IgG1 antibodies and their PBMC secreted significantly high levels of IFN-γ in response to the vaccine antigens. The two vaccinated macaques that picked the infection had slightly low titers of antibodies and their PBMC secreted high levels of IL-10. Based on these findings we conclude that the rBmHAT vaccine is highly immunogenic and safe and can confer significant protection against challenge infections in rhesus macaques.

In vivo transfection of developmentally competent Brugia malayi infective larvae

Transient transfection of isolated Brugia malayi embryos by biolistics has proven to be useful in defining promoter structure and function in this parasite. However, isolated transfected embryos are developmentally incompetent. A method of producing developmentally competent transfected parasites is therefore needed. We report that L3 parasites can be chemically transfected in situ in the peritoneal cavity of a gerbil with a construct consisting of a secreted luciferase reporter gene containing a promoter, the 3′ untranslated region and first intron derived from the B. malayi 70kDa heat shock protein gene. The in situ chemically transfected parasites are developmentally competent, producing adult parasites with an efficiency similar to that obtained from implanted untreated L3s. Cultured adult parasites and progeny microfilariae (mf) derived from L3s transfected with this construct secreted luciferase into the culture medium. When the transfected mf were fed to mosquitoes and the resulting L3s collected, the L3s also secreted luciferase into the culture medium. Progeny mf from transgenic adult parasites contained transgenic DNA, and the transgenic mRNA produced in these parasites was found to be correctly cis- and trans-spliced. In situ chemical transformation thus results in developmentally competent transfected B. malayi in which the transgenic sequences remain transcriptionally active in all life cycle stages and are present in the subsequent generation.

Effects of gamma radiation on Brugia malayi infective larvae and their intracellular Wolbachia bacteria

Prior studies have shown that irradiated filarial larvae are developmentally stunted but capable of inducing partial immunity to filariasis in animals. The mechanisms for these effects are poorly understood. Recent studies suggest that intracellular Wolbachia bacteria are necessary for the normal development, reproduction and survival of filarial nematodes. The purpose of this study was to examine the effects of irradiation on Wolbachia in Brugia malayi infective larvae (L3) and on L3 development. The L3 were exposed to 0, 25, 35, 45, 55, 65 or 75 krad of gamma irradiation from a (137) Cesium source and cultured in vitro at 37 degrees C in NCTC/IMDM medium with 10% FCS for 12 days. Irradiation prevented molting of L3 to the L4 stage in a dose-dependent manner. Electron microscopy studies showed that irradiation damaged Wolbachia (25 krad) or cleared them from worm tissues (45 krad). In addition, majority of the irradiated L3s failed to develop the L4 cuticle. Real-time PCR studies showed that irradiation reduced Wolbachia DNA in worm tissues. Parallel in vivo studies confirmed decreased development of irradiated L3 in jirds, with associated effects on Wolbachia. Jirds injected s.c with normal L3 developed antibodies to Wolbachia surface protein (wsp) shortly after the onset of microfilarial patency. In contrast, jirds injected with irradiated L3 did not develop microfilaremia or antibodies to wsp. Additional studies are needed to test the hypothesis that irradiation retards growth and development of filarial L3 by killing Wolbachia.

Experimental infection of the leaf-monkey,Presbytis cristata, with subperiodicBrugia malayi

Presbytis cristata monkeys infected through the inoculation of between 200 and 400 subperiodic Brugia malayi infective larvae (L3) in the right thigh, in both thighs or in the dorsum of the right foot were followed up for varying periods of up to about 8 months after infection. All 148 inoculated animals became patent, with mean prepatent periods being between 66 and 76 days. In animals injected in the thigh, the patterns of microfilaraemia were similar, there being a rapid rise in the geometric mean counts (GMCs) of microfilariae during the first 10-12 weeks of patency, which then plateaued at levels of greater than 1000/ml. Adult worm recovery, expressed as the percentage of the infective dose, was significantly higher in animals injected with 100 L3 in each thigh, being 9.4% as compared with 2.8%-4.8% in other groups. It is therefore recommended that animals should be injected with 100 L3 in each thigh and that the testing of potential filaricides in this model be carried out during the phase of rapid increase in microfilaraemia to ensure that any microfilaricidal effect can easily be detected.

Protective immunity against Brugia malayi infective larvae in mice. II. Induction by a T cell-dependent antigen isolated by monoclonal antibody affinity chromatography and SDS-PAGE.

A mAb directed against filarial worm secretory/excretory product and reactive with Brugia malayi larval worm surface was used in conjunction with preparative SDS-PAGE to isolate protective Ag from extracts of adult B. malayi. The IgM mAb OVH bound to a repeating carbohydrate epitope present in adult, infective, and fourth stage larvae and microfilariae of B. malayi, and on the surface of fourth stage larvae. Ag bearing this epitope were also present in the sera of hosts infected with a variety of helminths, including Brugia, Onchocerca, Dirofilaria, and Paragonimus. Affinity chromatography of SDS extract of adult Brugia, using mAb OVH immobilized on agarose beads, isolated several Ag that separated into multiple protein staining bands on SDS-PAGE. In comparing SDS-PAGE-fractionated Ag from the crude SDS extract with fractionated mAb OVH-isolated Ag for the ability to protect BALB/c mice from challenge with B. malayi-infective larvae, it was found that of the mAb OVH-isolated Ag only those at a molecular mass of 26 to 32 kDa were protective while the original SDS extract yielded protective Ag at the following molecular mass: greater than 200, 170 to 200, 40 to 44, 33 to 36, 23 to 28, 20 to 22, and 17 to 19 kDa. Although Ag isolated by mAb OVH were highly protective, they failed to induce high antibody levels against the immunogen or SDS extracts compared to crude SDS extract immunized mouse sera, as determined by immunoblot and ELISA. Transfer of nylon wool non-adherent T cells from BALB/c mice immunized with the 26- to 28-kDa fraction of mAb OVH-isolated Ag to naive mice just before challenge with infective larvae of B. malayi resulted in a 70% reduction in larvae recovered 14 days after challenge.

Protective immunity against Brugia malayi infective larvae in mice. I. Parameters of active and passive immunity.

Protective immunity against infective larvae of Brugia malayi was studied in different strains of mice using various sources of antigens. The following strains of mice were susceptible to infective larvae development for 2 weeks after primary ip challenge: BALB/c, C3H/HeJ, C3H/NeN, C3H/HeJms, C57BL/6Jms, and DDD. In comparison to gerbils, BALB/c mice developed stronger resistance to infective larvae after immunization with irradiation attenuated larvae or with killed microfilariae (mf). However, killed mf failed to enhance resistance in C3H/HeJ mice, although C3H/HeN mice were strongly protected and C3H/HeJms mice were protected to a lesser degree by this antigen. Extracts of mf with phosphate buffered saline and sodium dodecyl sulfate both induced high levels of resistance in BALB/c mice. Transfer of resistance from BALB/c mice immunized with attenuated infective larvae to naive mice was accomplished at a high level at protection with nylon wool nonadherent spleen cells (T cells) but not with adherent cells treated with anti-Thy 1.2 serum and complement. In contrast, sera from immunized mice were much less protective.

Radiolabeling of Brugia malayi infective larvae in mosquitoes with 75Se-methionine and detection of these larvae in tissues of the Mongolian jird by autoradiography.

Through preliminary experiments, an effective method for radiolabeling Brugia malayi-infected mosquitoes in order to produce labeled infective Brugia larvae was developed. Starting on the 6th day after the infective blood meal, mosquitoes were fed a 7% sucrose solution containing 100 microCi/ml 75Se-L-methionine for 5 days. Infective larvae, retrieved 2 days after this labeling period, averaged 381 +/- 136 counts/min. Jirds were infected with these infective, labeled larvae either by allowing infected mosquitoes to feed on uninfected jirds for 30 min or by inoculating jirds subcutaneously in the groin with washed larvae recovered from mosquitoes. Jirds were killed at various times after infection and were sliced into approximately 0.5 mm thick sagittal sections, which were dried and placed on X-ray film. Autoradiograms were developed after 30-60 days at 5 degrees C. In a sample of 26 inoculated jirds, approximately 30% of the infecting larvae could subsequently be accounted for as Ag degrees foci on autoradiograms. The Ag degrees foci representing larvae were apparent up to 2.5 weeks after infection. In jirds infected by mosquito feeding, the Ag degrees associated with the feeding site persisted for more than 6 weeks after infection.

Immune responses in human Brugia malayi infections: serum dependent cell-mediated destruction of infective larvae in vitro

Serum dependent cell-mediated cytotoxity of subperiodic Brugia malayi infective larvae in vitro was investigated. In vitro cellular adherence of normal human buffy coat cells to infective larvae of B. malayi was promoted by sera from patients with elephantiasis, tropical pulmonary eosinophilia (TPE) and amicrofilaraemic symptomatic filariasis, as well as by sera from normal subjects from filariasis endemic areas. However, strongest adherence was observed with TPE sera. Scanning electron microscopic (SEM) studies confirmed that the cellular adherence resulted in gross surface damage to the infective larvae. Studies with the indirect fluorescent antibody test (IFAT) suggested that IgG and/or IgM might be involved in the process of adherence. Complement did not appear to be important.

Distribution of Developing and Mature Brugia malayi in Rhesus Monkeys and in the Jird, Meriones unguiculatus, after a Single Infection

Ten rhesus monkeys (Macaca mulatta) and 20 jirds (Meriones unguiculatus) were each given a single inoculation of subperiodic Brugia malayi infective larvae in the rear foot by a puncture wound technique. The monkeys were killed after a period varying from 5 days to 25 weeks and the jirds from 2 to 32 weeks following inoculation. The individual recovery of worms from monkeys ranged from 0% to 40% with an average of 12%. Out of an overall total of 121 worms recovered from the 10 monkeys, 78 were found in the lymph nodes of the inguinal area, and 21 in the lymph vessels of the infected leg distal to the popliteal node. In jirds the individual recovery varied from 3% to 40% of the administered inoculum with an overall average of 207%; up to 6 weeks following infection, the majority were found in the lymph vessels distal to the popliteal node; from 7 to 32 weeks following infection, the majority were located in the heart and lungs with some worms recovered from the testes. This study confirms the production of patent infections of Brugia malayi in rhesus monkeys and in jirds. However, the levels of larval and adult recovery in both of these species is relatively low when compared with previously reported results using cats. This low rate of recovery, together with the more general distribution pattern in monkeys and the apparently pronounced migration in jirds, may be of importance in the selection of an experimental host for the study of Malayan filariasis. Of the several different vertebrates in which patent infection with Brugia malayi has been demonstrated, the domestic cat has most frequently been used for experimental studies. Recently we have described techniques for infection and recovery of this parasite that should facilitate future experimental studies using the cat as a model. However, at times it would be advantageous to have other models to use instead of, or in conjunction with, cats. It is commonly accepted that results from experiments using primates can more readily be extrapolated to man. Thus it was felt that if the techniques found to be useful in cats were found to be equally successful in a primate, certain studies such as preclinical drug studies or pathological studies could be carried out more effectively. Orihel and Pacheco (1966) demonstrated that at least the Philippine strain of B. malayi persisted in the rhesus monkey and therefore it might serve as a useful experimental host. On the other hand, a small animal Received for publication 20 May 1971. * This investigation was supported by the United States-Japan Cooperative Medical Science Program administered by the NIAID of the NIH, Department of Health, Education and Welfare. Grant No. AI 08260. t Permanent address: Box 32, Khartoum North, Sudan. model which would provide an inexpensive, easily handled host which could be used in drug screening programs would also be valuable. The jird, Meriones unguiculatus, has been shown to be a suitable host for Brugia malayi by Ash and Riley (1970). The present paper describes the results of distribution studies in rhesus monkeys and in jirds using methods previously found to facilitate observation and recovery of developing and mature Brugia malayi in cats. MATERIALS AND METHODS