|Year : 2017 | Volume
| Issue : 1 | Page : 29-36
Stage-specific antibody response against two larval stages of Brugia malayi in different clinical spectra of brugian filariasis
Praveen Kumar Tripathi1, Ramesh Chander Mahajan1, Nancy Malla1, Naveen Kumar1, Shailja Misra Bhattacharya2, Ranganatha Krishna Shinoy3, Abhishek Mewara1, Rakesh Sehgal1
1 Department of Medical Parasitology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Division of Parasitology, CDRI, Lucknow, Uttar Pradesh, India
3 Department of Medicine, Filariasis Chemotherapy Unit, TD Medical College Hospital, Alappuzha, Kerala, India
|Date of Acceptance||20-Oct-2014|
|Date of Web Publication||16-Mar-2017|
Department of Medical Parasitology, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012
| Abstract|| |
Context: T-cell hypo-responsiveness in microfilaria (Mf) carriers against the microfilarial stage antigen of Brugia malayi has been described, but no study has been carried out to assess antibody dynamics against stage-specific antigens.
Aim: The work was carried out with the aim to assess stage-specific antibody responses against L3 and microfilarial stage antigens in brugian filariasis in an endemic area.
Setting and Design: Patients with different clinical spectra of brugian filariasis were recruited to evaluate antibody responses to brugian antigens.
Subjects and Methods: Serum samples were collected from patients with different clinical spectra and antibody response was evaluated for total immunoglobulin G (IgG), IgG isotypes (IgG1, IgG2, IgG3, IgG4) and immunoglobulin E (IgE) response to L3 and microfilarial stage by enzyme-linked immunosorbent assay.
Statistical Analysis: Paired t-test and one-way analysis of variance were carried out to analyze the data.
Results: L3 and microfilarial stage antigens showed almost similar antibody responses in adenolymphangitis (ADL) and chronic pathology (CP) patients, however, diminished antibody response was observed with Mf stage antigen, especially with microfilaraemia. ADL patients had minimum antibody levels of all isotypes except IgG2 on day 0 which showed an increase subsequently, indicating suppression of antibody response during filarial fever. CP patients showed increase in IgE and decrease in IgG4 antibodies on day 365 indicating that these differences may be due to recent conversion into CP.
Conclusion: A prominent hyporesponsiveness in microfilaraemic individuals against microfilarial stage, but not against the L3 stage of the same parasite was observed, concluding stage-specificity in humoral immune response in brugian filariasis.
Keywords: Brugian filariasis, L3 and microfilarial antigens, stage-specific antibody responses
|How to cite this article:|
Tripathi PK, Mahajan RC, Malla N, Kumar N, Bhattacharya SM, Shinoy RK, Mewara A, Sehgal R. Stage-specific antibody response against two larval stages of Brugia malayi in different clinical spectra of brugian filariasis. Trop Parasitol 2017;7:29-36
|How to cite this URL:|
Tripathi PK, Mahajan RC, Malla N, Kumar N, Bhattacharya SM, Shinoy RK, Mewara A, Sehgal R. Stage-specific antibody response against two larval stages of Brugia malayi in different clinical spectra of brugian filariasis. Trop Parasitol [serial online] 2017 [cited 2017 Mar 29];7:29-36. Available from: http://www.tropicalparasitology.org/text.asp?2017/7/1/29/202298
| Introduction|| |
Human lymphatic filariasis (LF) affects over 120 million people world-wide. World Health Organization has targeted the year 2020 for global elimination of LF. The disease is marked by two phases – hypo-responsiveness during active infection and chronic lymphatic disease (though initiated by parasite and immune factors, but not with active infection). Among the three main life-cycle stages of Brugia malayi – L3 (infective stage), adult worm (AW) and the microfilaria (Mf), it is the L3 stage which initiates infection in humans and so is the likely primary target of protective immune response. Yet, relatively few studies have been carried out to examine antibody responses against this stage of the filarial parasite.
The immune responses in brugian filariasis using AW stage and L3 stage antigens have been shown to have no significant difference in total immunoglobulin G (IgG) amongst microfilaraemic individuals, patients with chronic pathology (CP) and endemic normal (EN) controls. In contrast, sera from people with microfilaraemia have been shown to have significantly higher levels of specific IgG4 antibodies as compared to sera from CP patients and EN controls. Furthermore, AW specific immunoglobulin E (IgE) antibodies have been reported to be significantly higher in CP patients compared with those with microfilaremia. Using Mf excretory-secretory antigen, the microfilremics have been shown to have significantly elevated levels of IgG4 and IgG3 antibodies compared to EN controls, while acute filarial cases have been shown to have pronounced IgG1 antibodies, Grade I CP cases to have high levels of IgG3 and IgG4 antibodies, and occult filarial cases shown to have higher levels of IgG4, IgG3 and IgG1 antibodies. IgE antibodies have been found to be elevated in microfilaremic as well as other clinical filarial groups., Thus, the knowledge about immune response dynamics in filariasis remains inconclusive because of varying results from different studies.
There have been very few reports on stage-specific humoral immune responses in brugian filariasis. Most of the studies have used AW antigens, few have used L3 antigen and rarely Mf antigens have been used. Moreover, most studies have not used uniform methodology in terms of parasite stages, types of antigens (crude AW antigen, L3 surface antigen, Mf excretory-secretary, or heterologous antigen, i.e., B. malayi antigen used for Wuchereria bancrofti patients samples), and methodology (one stage antigen used in enzyme-linked immunosorbent assay (ELISA) while another used in immuno-fluorescence, enzyme-linked immuno-electro transfer blotting or immuno-blots). The variability in methodology may lead to discrepancies in the findings in such studies. Keeping the above issues in context, the present study was carried out to determine antibody responses in serum samples from patients with different clinical spectra along with endemic and non-endemic controls, at different time points, using L3 and Mf stage antigens of B. malayi.
| Subjects and Methods|| |
Study population and samples
The study was carried out in 101 adult subjects (≥18 years) including 68 males and 33 females. Venous blood samples were collected from subjects with different clinical spectra (Groups I-III as below) EN controls (Group IV) from TD Medical College Hospital, Alleppey, Kerala (endemic area) and from non-endemic normal (NEN) controls (Group V) from Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, after obtaining written informed consent. Microfilarial count was done on night blood samples in all the subjects. The study was approved by Institute Ethics Committee (IEC) of TD Medical College Hospital, Alleppey, Kerala, and by IEC and Animal Ethics Committee of PGIMER, Chandigarh. The following groups were made:
- Group I (n = 21): Patients with microfilaraemia but without signs or symptoms of filariasis or any other chronic disease (i.e., microfilaraemic but asymptomatic)
- Group II (n = 20): Patients with acute lymphangitis (adenolymphangitis [ADL]) with or without underlying filarial edema
- Group III (n = 20): Patients with chronic filarial edema (CP) without any history of acute lymphangitis during the last 6 months. This group comprised of patients with lymphedema (Grade I, II, III or IV)
- Group IV (n = 20): EN controls comprised of apparently healthy persons from Alleppey without signs or symptoms of filarial infection or any other chronic infection and who had been found negative for Mf by peripheral blood smear examination on three consecutive nights
- Group V (n = 20): NEN controls comprised of apparently healthy subjects from Chandigarh, who were Mf negative and did not give any history of travel to any endemic area in the past 5 years.
After collecting blood samples on day 0, all individuals in Group I were given diethylcarbamazine (DEC) treatment (6 mg/kg body weight, single dose), while ADL and CP patients were not given any anti-filarial drug treatment. In Group I, blood samples were collected sequentially on days 1, 30, 60, 90, 180 and 365 following treatment, and pre- and post-treatment microfilarial counts were recorded. In ADL and CP patients, blood samples were collected on days 0, 90, 180 and 365. Serum samples collected at Alleppey were stored at −20°C and transported to PGIMER, Chandigarh, under refrigerated conditions. Sera were further stored at −80°C (Heto Ultra Freeze, Denmark) till used.
Maintenance of B. malayi strain
The B. malayi strain was maintained between the Black-eyed susceptible strain of Aedes aegypti mosquito (originally developed by Prof. WW Macdonald, Liverpool School of Tropical Medicine, UK), and the 'GRA' Giessen strain of Mastomys natalensis rats with bright yellow body color and red eyes (both strains procured from Central Drug Research Institute, Lucknow, India and propagated in the Department of Medical Parasitology, PGIMER, Chandigarh). The infective larvae (L3) were separated from infected mosquitoes while microfilarial stage of the parasite was obtained from rats.,
Extracts/soluble antigens from L3 and Mf stages of the parasite were prepared by disruption of parasites in phosphate-buffered saline (PBS) pH 7.2 containing following protease inhibitors: 1 mM ethylene diamine tetraacetic acid, 1 mM ethylene glycol bis-tetraacetic acid, 0.2 mM tosyl lysine chloromethyl ketone, 0.1 mM tosyl phenylalanine chloromethyl ketone and 1 mM phenyl methyl sulphonyl fluoride (Sigma-Aldrich, Bangalore, India).
L3 antigen preparation
Briefly, 103 L3 larvae in 1 ml of PBS were disrupted in a ground glass homogenizer. The suspension was sonicated on ice with an ultrasonic homogenizer (Soniprep 150, MSE, London, UK), six cycles of 30 s each at 20 kHz with 30 s intermittent cooling. The sonicate was centrifuged at 10,000g at 4°C for 30 min and the supernatant was passed through 0.22 µm filter. The filtrate was aliquoted and protein estimation done by Lowry's method. The antigen was stored at –80°C till use.,
Mf antigen preparation
Briefly, 106 microfilariae/ml of buffer pH 7.2, were directly sonicated (without disrupting in ground glass homogenizer). Rest of the procedure was similar as mentioned above for L3 antigen.
Indirect ELISA for antibody detection
Specific total IgG, IgG isotypes (IgG1, IgG2, IgG3 and IgG4) and IgE responses against L3 and Mf stages were detected in serum samples by ELISA as described previously,,, with minor modifications wherever required.
ELISA for total IgG
Briefly, the 96 well microtitre plates (Dynatech, NY, USA) were coated with 100 µl of optimal dilutions of L3 or Mf antigen diluted in coating buffer (pH 9.6) and incubated overnight at 4°C. Next morning the plate was washed thrice with washing buffer, phosphate buffered saline-Tween 20 (PBS-T20) and 100 µl of 1% bovine serum albumin (BSA; HiMedia, Mumbai, India) was added to each well to bind the remaining sites of the wells. After 1 h of incubation at 37°C, the plate was again washed thrice and the diluted test and control sera (diluted in PBS-T20) were added in duplicate wells (100 µl/well) followed by incubation at 37°C for 1 h. After similar washing step, 100 µl of the optimally diluted HRP conjugated anti-human IgG (Sigma-Aldrich, Bangalore, India) was added to each well. Again after similar incubation and washing steps, 100 µl of optimally diluted H2O2-OPD (substrate-chromogen) was added to each well. The plate was incubated at room temperature for 15-30 min for development of color. The reaction was stopped with 100 µl of 6% H2 SO4 and the absorbance values (optical densities [OD]) were read at 490 nm in an ELISA reader (Emax ™, California, USA).
ELISA for IgG subclasses (IgG1, IgG2, IgG3 and IgG4)
The coating, washing and sera charging methodology were essentially similar as above. Further, 100 µl of the optimally diluted Biotin-labeled anti-human IgG1 (Clone: 8c/6-39)/IgG2 (Clone: HP6014)/IgG3 (Clone: HP6050)/IgG4 (Clone: HP6025) conjugate (Sigma, USA) were added to each well. The plate was again incubated at 37°C for 1 h, washed thrice and 100 µl of optimally diluted Avidin-HRP conjugate (Sigma, USA) was added to each well, followed by incubation at 37°C for 30 min. After washing, 100 µl of optimally diluted H2O2-OPD was added and after development of color the reaction was stopped with 100 µl of 6% H2 SO4 and absorbance read as above.
ELISA for IgE
The coating, washing and sera charging methodology were essentially similar as above. Further, 100 µl of the optimally diluted alkaline phosphatase (AP) conjugated mouse monoclonal anti-human IgE (Clone: GE-1, Sigma, USA) was added to each well, followed by incubation at 37°C for 1 h. After washing, 100 µl of optimally diluted substrate, para-nitrophenyl phosphate was added to each well. After development of color the reaction was stopped with 100 µl of 2 M Na2 CO3 and the OD values read at 405 nm.
All samples were tested in duplicate and the mean of two optical densities (OD values) was taken as absorbance value for that sample. ELISA-ratio (or E-ratio) system was adopted to minimize batch to batch or plate to plate variations. The individual OD values were divided by the mean + 2 standard deviation (SD) of normal controls run in the same plate, the ratios thus obtained known as E-ratios. These E-ratios, unlike OD values, remain almost unaffected by the variations due to plates, temperature, incubation periods, reagents or other common factors. Before statistical analyses, all OD values were converted to E-ratios and using the above methodology the cut-off E-ratios were calculated. The statistical analyses were performed using GraphPad Prism software (GraphPad Software, CA, USA). Paired t-test and one way analysis of variance were carried out to find significance of difference within individual groups and among various groups of patients and controls, respectively. The statistical differences with P ≤ 0.05 were considered to be significant.
| Results|| |
Mf carrier group
The mean total IgG response on day 0, i.e., before giving the DEC treatment, against L3 antigen was 1.528 (SD, 0.161), which after treatment showed a significant gradual decrease upto day 365 (1.354, SD 0.186, P = 0.042). At the baseline (day 0), the filaria-specific total IgG response in EN controls was significantly lower than the microfilaraemic group (P < 0.03). On the other hand, a significantly diminished antibody response was observed against Mf antigen as compared to that against L3 antigen on day 0 (P = 0.0017), 365 (P = 0.009), as well as other time points. Although a post-treatment decrease was observed against Mf, it was not statistically significant between day 0 and day 365 (P = 0.45). All the filarial-specific antibody responses in the NEN control group were significantly less as compared to all the endemic categories including the EN control group (P < 0.001) [Figure 1].
|Figure 1: Antibody response against L3 and Mf antigens in microfilaremic individuals. (a) Total immunoglobulin G (IgG), (b) IgG1, (c) IgG2, (d) IgG3, (e) IgG4, (f) immunoglobulin E. EN: Endemic normal controls; Mf: Microfilaria; NEN: Non-endemic normal controls; SD: Standard deviation|
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A statistically significant decrease in antibody titer from day 0 to day 365 against L3 antigen was shown by IgG4 (P < 0.0001) and IgE (P = 0.05), but not for IgG1 (P = 0.085), IgG2 (P = 0.081), and IgG3 (P = 0.078). Moreover, a drastic fall was observed in case of IgG4, where the difference became significant as early as on day 30 (P = 0.0027) and was highly significant on day 365 (P < 0.0001). A different pattern was observed, however, against Mf antigen. Overall, the antibody responses were significantly lower than L3 antigen (P < 0.01), and no significant difference at day 365 was observed in total IgG, IgG1, IgG2, IgG3 and IgE responses (P > 0.05). Secondly, the post-treatment decreases were not as prominent as against L3 antigen, except for IgG4 antibodies, which showed significant difference between day 0 and day 365 (P = 0.0076), although the difference was not so drastic as was observed for the corresponding responses against L3 antigen (P < 0.0001).
In serum samples from ADL patients much suppressed antibody responses were observed on day 0, i.e., at the time of acute filarial episode. The mean total IgG, IgG1, IgG3, IgG4 and IgE response to L3 as well as Mf antigen showed significant increase between day 0 and day 90 (P value for all antibodies tested ≤0.05) [Figure 2], thereafter on day 90, 180 and 365, the responses were rather stable against both the antigens. IgG2 antibody response, however, demonstrated an opposite pattern. On day 90, against both L3 and Mf, it showed significant decrease in titer when compared to day 0 (P = 0.031 and 0.018, respectively). Thereafter, it also showed stable response. Another observation in the ADL group was that higher antibody responses against Mf antigen were observed on day 0 when compared to L3 responses. At all other time-points, the response against L3 remained higher for total IgG, IgG1, IgG2, IgG3 and IgE antibodies. Only IgG4 antibody showed higher responses against Mf antigen compared to L3 antigen at all follow-up time-points in ADL patients. All the antibody responses in ADL patients were higher than EN controls (P ≤ 0.045) except IgE which was significantly higher in EN controls against both the antigens (P ≤ 0.041). All the differences of ADL group patients remained higher than NEN controls (P < 0.0001).
|Figure 2: Antibody response against L3 and Mf antigens in adenolymphangitis patients. (a) Total immunoglobulin G (IgG), (b) IgG1, (c) IgG2, (d) IgG3, (e) IgG4, (f) immunoglobulin E. EN: Endemic normal controls; Mf: Microfilaria; NEN: Non-endemic normal controls; SD: Standard deviation|
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The CP patients can be divided into lymphoedema Grade I, II, III and IV. Due to less number of patients in Grade I (n = 3) and II (n = 4), these were combined with those of Grade III (n = 6) and IV (n = 7) for analysis. Serum samples from this group showed relatively stable antibody responses. The total IgG, IgG1 and IgG2 antibody responses remained stable from day 0 to day 365 against both antigens. IgG3 and IgE antibodies exhibited an increasing tendency but did not reach any significance upto day 365 (P = 0.054), however, IgG4 antibody response showed significant difference between day 0 and day 365 (P = 0.046) against L3 antigen [Figure 3]. All the antibody responses against L3 antigen were higher than the responses against Mf antigen. When this comparison was made for Grade (I + II) versus (III + IV), IgG3 and IgE antibodies showed significant increase (P = 0.041 and 0.038 respectively). Similarly the decreasing tendency of IgG4 against L3 and Mf antigens exhibited significant differences on day 365 when compared to day 0 (P = 0.039 and 0.049 respectively). Highly significant differences were observed amongst all the responses between CP patients and NEN controls (P < 0.0001).
|Figure 3: Antibody response against L3 and Mf antigens in chronic pathology patients. (a) Total immunoglobulin G (IgG), (b) IgG1, (c) IgG2, (d) IgG3, (e) IgG4, (f) immunoglobulin E. EN: Endemic normal controls; Mf: Microfilaria; NEN: Non-endemic normal controls; SD: Standard deviation|
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Pre- and post-DEC treatment for Mf carrier group
On day 0, i.e., before giving DEC, the total Mf count of all 21 patients in night blood sample was 7632. It was treated as 100% and the post-treatment Mf counts were calculated accordingly. A fall of count from 100% to 2.15% was observed on day 1. On day 30, the counts again increased to 12.68% and thereafter showed a gradual decrease till day 365, however, the Mf levels in blood were never completely eliminated [Table 1].
|Table 1: Microfilarial counts pre- and post-DEC treatment in microfilaria carriers (group I)|
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| Discussion|| |
In the present study, we assessed immune responses against different life-stage antigens of B. malayi amongst the major clinical filariasis groups. A significantly suppressed level of all the antibodies (total IgG, IgG1, IgG2, IgG3, IgG4 and IgE) was observed against Mf stage antigen in microfilaraemic individuals as compared to L3 stage. This phenomenon may be due to anergy or hypo-responsiveness characteristic of active infection. Anergy has largely been described in Th1-cell population, although the down-regulation of specific IgE response in microfilariae carriers appears to be determined by a more elusive modulation by parasite factors., A single dose of DEC (6 mg/kg body weight) was given to the microfilaraemic individuals after taking the first blood sample. All the antibody responses to L3 antigen showed a gradual decrease up to day 365 and these differences were significant for total IgG (P = 0.042), IgG4 (P < 0.0001) and IgE (P = 0.05) antibodies. This is in agreement with a previous study which reported significant decreases in IgG1, IgG4 and IgE antibodies after treatment with DEC. The corresponding response against Mf antigen was surprisingly low in our study, wherein only IgG4 showed significant decrease post-treatment. This is a clear evidence of antibody hypo-responsiveness in Mf carriers against Mf antigen. A drastic decrease in microfilaremia on day 1 post-treatment was observed and the levels remained decreased thereafter, indicating a positive correlation between Mf clearance and the levels of specific IgG4 antibody levels. However, the Mf counts were found to have increased after day 1, showing that one-dose DEC treatment may not kill all AWs. This may partly explain why some of the antibody sub-classes remained elevated even after treatment.
In LF, recurrent episodes of acute phases of ADL are characterized by pain and inflammation of the affected extremity usually accompanied by high grade fever or chills., In our study, the ADL patients showed several important features. Firstly, all the antibody responses except IgG2, against both antigens, showed significantly low responses on day 0, i.e., at the time of acute episode of ADL. This could be due to antigen-antibody immune complex formation at this stage of illness. From day 90 to day 365, all the immunoglobulin responses were found to be stable, when these patients did not have any ADL episode. Secondly, contrary to total IgG, IgG1, IgG3, IgG4 and IgE, the IgG2 responses were significantly raised against L3 as well as Mf antigen at the time of filarial fever when compared to day 90 responses. Thirdly, unlike responses in all other groups, in ADL group, all the antibody responses to Mf antigen were higher than those against the L3 antigen on day 0, indicating some microfilarial involvement in ADL patients, which are otherwise thought to be amicrofilaraemic. In concordance, another study has reported 11.11% Mf positivity in ADL patients. Furthermore, the prominent reactivity of IgG2 in ADL patients during filarial fever observed in our study is similar to a previous study which assessed IgG1, IgG2, IgG3 and IgG4 antibody responses against the pro-inflammatory molecular fraction F6 of B. malayi AWs and concluded that IgG2 antibody may be related to pathogenesis in filariasis.
Overall, unlike other clinical groups, the CP group exhibited almost steady levels of all the antibodies against both antigens. However, whereas the total IgG, IgG1 and IgG2 showed stable levels, IgG3 and IgE antibodies showed an increasing tendency up to day 365, which was not statistically significant. This higher level of non-IgG4 antibodies in CP group observed in our study has been noted by other studies also. This tendency of slow increase in IgG3 and IgE levels, and decrease in IgG4, may be displayed in early grade lymphedema patients who may have recently progressed to pathology group from microfilaraemic status. Higher IgE antibody levels observed in CP patients when compared to other groups makes the role of IgE debatable that whether these antibodies play role in protection or lead to pathogenesis or lymphedema.
| Conclusion|| |
In this study, two larval stages, L3 and microfilarial stage of B. malayi were used to assess the dynamics of immune response in LF. In microfilariae carriers a significantly high difference between antibody responses of L3 and Mf stages of the same parasite was surprising. A gradual decrease in the total IgG, IgG1, IgG2, IgG3, IgG4 and IgE was observed post-treatment. In ADL patients, a significant increase in antibody response on day 90 were observed, however, IgG2 showed a reverse pattern, i.e., highest responses on day 0. This differential behavior of IgG2 remains to be investigated. In CP group, overall, almost similar antibody responses were observed, with high levels of IgG2, IgG3 and IgE responses. In future studies, which are the common antigenic fractions between the two larvae and what makes them so different, being from the same lineage, are a few important questions which require further investigation.
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[Figure 1], [Figure 2], [Figure 3]