Arch Virol DOI 10.1007/s00705-015-2519-7
ORIGINAL ARTICLE
Higher levels of dengue-virus-specific IgG and IgA during predefervescence associated with primary dengue hemorrhagic fever Rupali Bachal1 • Kalichamy Alagarasu1 • Anand Singh1 • Asha Salunke1 Paresh Shah1 • Dayaraj Cecilia1
•
Received: 30 April 2015 / Accepted: 28 June 2015 Ó Springer-Verlag Wien 2015
Abstract Dengue hemorrhagic fever (DHF), although predominantly associated with secondary infections, has also been reported in primary infections. An enhanced immune response including antibodies and cytokines is implicated in the pathogenesis of secondary DHF. However, the factors operating in primary DHF are poorly understood. To understand the role of the antibody response, the relative levels of different antibody isotypes during the acute phase of infection in primary and secondary dengue infections were determined. Levels of DENV-specific IgM, IgG, IgA and IgE were measured in the serum samples of 200 dengue patients and 20 denguenaı¨ve individuals. Samples were collected within 15 days of onset of illness. The DENV-specific IgM levels were significantly higher in DF cases compared to DHF, which was more evident in secondary infections and in post-defervescence samples. The levels of IgG, IgA and IgE were higher in DHF cases, with greater significance in primary infections. A higher level of IgG in DHF cases was evident in pre-defervescence samples, whilst the IgE level was higher in pre- and post-defervescence samples. There was a significant correlation of IgG titres with platelet counts, with higher titres associated with lower platelet counts. It is speculated that IgG, IgA and IgE produced in response to primary infections may contribute to pathogenesis, whilst IgM produced in response to secondary infections may protect against progression to severe disease.
& Dayaraj Cecilia [email protected] 1
Dengue Group, National Institute of Virology, 20-A Dr. Ambedkar Road, Pune 411001, India
Introduction Dengue virus (DENV) causes 50 to 100 million infections each year in over 100 countries and has recently spread to Europe. Of these infections, 500,000 cases are severe, requiring hospitalization, and 2.5 % of them are fatal [1]. DENV causes a spectrum of disease including subclinical infections, dengue fever (DF), an acute febrile illness, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), which is severe and occasionally lifethreatening [2]. There are four serotypes of DENV, and people experiencing their first DENV infection develop a robust antibody response that is cross-reactive with other serotypes [3] but protective only against the homologous serotype [4]. Severe disease is associated more often with secondary infections but also occurs during primary infections [5]. Development of severe disease is due to multiple factors, including inflammatory cytokines [6], antibody-dependent enhancement (ADE) [7], antibodydependent cell-mediated cytotoxicity (ADCC) [8], and ADE due to antibodies produced against the prM protein [9]. An additional factor is auto-reactive antibodies produced against platelets, endothelial cells and coagulation molecules due to molecular mimicry between DENV proteins and host proteins [10]. Several studies have shown the involvement of complement factors in progression to DHF [11–13]. Considering the role of antibodies in disease pathogenesis and the different functions attributed to each isotype [14], the relative concentrations and kinetics of the antibody isotypes in dengue need to be defined in the context of different host and virus populations. There have been several studies on this aspect, but most of them have included children. Koraka et al. determined the isotype profile in dengue-virus-infected children in Indonesia [15, 16]. They reported increased
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levels of IgA, IgG1, IgG4 and IgE in DHF and DSS cases. Another study, carried out in children in Myanmar, found higher levels of IgG1 in DHF cases [17]. Vasquez et al. compared primary DF, secondary DF and secondary DHF in Cuba (age not mentioned) and in El Salvador (in children) and reported significantly higher levels of IgM in DF compared to DHF in secondary infections and higher levels of IgA and IgE in secondary infections compared to primary infections [18, 19]. Our earlier study revealed that the age profile and clinical presentation of dengue in India differ from that of South East Asian countries, where large numbers of children are affected and the clinical presentation is more severe [20]. Our recent studies have shown that the association of human leukocyte antigen alleles with clinical outcomes of DENV infection is different from that of studies from other regions [21, 22]. The virus populations circulating in India are also different from those in South East Asia [23]. The present study investigates the antibody isotype response in a predominantly adult population and compares DF and DHF patients in the context of their immune status for the first time. It is also the first time that the response in the pre- and post-defervescence phase of disease has been compared.
Materials and methods Clinical samples A total of 200 samples from confirmed dengue patients during annual seasonal outbreaks of dengue in Pune during 2008-10 were included in the study. The National Institute of Virology (NIV) is a WHO Collaborating Centre for Arbovirus and Haemorrhagic Fever Reference and Research and Rapid Diagnosis of Viral Diseases. The cases were confirmed to be dengue, either by the presence of DENV-specific IgM (using an NIV MAC-ELISA DEN Kit, which is used in the national surveillance programme) or multiplex reverse transcriptase PCR. Serum samples collected from each patient were stored in aliquots at –80 °C. For each patient, the day of onset of fever was designated as the first day of illness. Serum samples collected 2-5 days after onset of illness were designated as ‘pre-defervescence’, and those collected 6-15 days after onset of illness, as ‘post-defervescence’ samples. Twenty samples from healthy blood donors who were naı¨ve to dengue were included as negative controls. This study was approved by the Human Ethics Committee of NIV. Waiver of the informed consent requirement was granted by the committee on the basis of ‘‘Use of leftover specimens after clinical investigation’’ under the Indian Council of Medical Research Guidelines 2006.
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Cells and viruses DENV-2 (Indian strain 803347, obtained from the Virus Repository of NIV) was used in all ELISA experiments. Virus was propagated in porcine kidney stable (PS) cell line (procured from National Centre for Cell Sciences, India), which was maintained in Dulbecco’s modified Eagle’s medium (GIBCO BRL) with 10 % fetal bovine serum. Preparation of DENV-2 antigen for ELISA PS cell monolayers in 75-cm2 flasks were infected with DENV-2 at a multiplicity of infection (MOI) of 0.1. On the fourth day postinfection, when the cells showed a cytopathic effect (CPE), the infected cultures were freeze/thawed and the lysate was collected. The lysate was centrifuged at 2000 rpm for 15 minutes to remove cell debris. The clarified lysate, stored at -80 °C in convenient aliquots, was used as DENV antigen for the IgM/IgA/IgE /IgG ELISAs. Mock-infected cell culture clarified lysate was used as control antigen. DENV-specific IgM/IgA/IgE capture ELISA Immunosorp strips (NUNC) were coated with a 1:5000 dilution of anti-human IgM (Dako Cytomation)/anti-human IgA (Sigma)/anti-human IgE (Sigma) diluted in 0.05 M carbonate buffer (pH 9.6) and incubated overnight at 4 °C. The strips were blocked with 300 lL of 1 % milk powder solution in 0.01 M PBS (pH 7.2) for 2 hours at 37 °C. The test sample, 50 lL diluted 1:100 in PBS (pH 7.2) with 0.1 % Tween (PBST) and 1 % BSA, was added to each well and incubated for 1 hour at 37 °C. This was followed by the addition of 50 lL of DENV-2 antigen/control antigen and incubation for 1 hour at 37 °C. The bound antigen was detected using anti-DENV envelope-protein-specific mouse monoclonal antibody GE9 (a kind gift from Dr. G. Sapkal, NIV) conjugated to horseradish peroxidase (HRP) (1:1000, 50 lL/well), with incubation for 1 hour at 37 °C. After the addition of 100 lL of the substrate tetramethyl benzedine, the plate was incubated for 10 minutes at room temperature in the dark, and the reaction was stopped with 1 N H2SO4. The optical density (OD) was read on an ELISA reader at 450 nm. The mean of the readings obtained with DENVnegative samples tested on DENV antigen was taken as mean negative control. A P/N (sample OD/mean OD of the negative controls) ratio of C2.1 was considered positive. The ELISA plates were washed five times with PBST after each step. The common steps of ELISA, i.e., coating, blocking, incubation and washing, as well as the diluents used, were similar for all ELISAs. ELISA titres were determined for IgM and IgG by testing tenfold dilutions of the serum samples in the antibody capture ELISAs, The reciprocal of the sample dilution yielding a P/N ratio of 2.1
Antibody isotypes in dengue hemorrhagic fever
was considered the titre. For samples that were negative (P/ N \ 2.1), an arbitrary value of 1 was given for calculation purposes with regard to antibody titres. DENV-specific IgG capture ELISA ELISA plates were coated with purified GE9 IgG diluted 1:50 in carbonate buffer (pH 9.6). After blocking, 50 lL of neat DENV-2-infected tissue culture fluid was added per well and incubated for 1 hour. This was followed by addition of the test sample at 1:100 dilution. The IgG captured on the antigen was detected with goat anti-human IgG-HRP conjugate (Sigma) diluted 1:30,000. The plate was developed as described above. Primary/secondary detection by IgG capture ELISA To detect primary and secondary infections, IgG Capture ELISA E-DEN02G (Panbio Diagnostics) was used. The test was performed as per the manufacturer’s protocol. The results were interpreted based on the calculations provided. Statistical analysis Using the Statcalc program (Epi info version 6.0.4), the chi-square test with Yates correction or Fisher’s exact test (when any cell value was less than 5) was performed to examine differences in demographic and clinical characteristics of the dengue patients. Age and platelet counts were compared using Student’s t-test. Mean P/N ratios of antibodies of different patient categories were compared using Student’s t-test or one-way ANOVA. P-values less than 0.05 were considered significant. All statistical analyses were performed using Graphpad prism (version 4).
Results Demographic characteristics of dengue cases A total of 200 dengue cases were included in the study. Based on the criteria defined by WHO [24], 126 (63 %) Table 1 Influence of age on disease severity in dengueinfected patients
patients were classified as DF and 74 (37 %) as DHF. The average age of dengue patients was 27.5 ±14.92 (SD) yrs. When the cases were divided into five age groups: 1-10, 11-20, 21-30, 31-40 and [41 yrs, the largest number of dengue cases fell into the 21-30 yrs age group (34.3 %) as compared to the other age groups (10.0 % to 21 %) (Table 1). A larger number of DHF cases were seen in children B10 yrs (55 %) (p = 0.11, OR = 2.32) and adults above 40 yrs (51.4 %) (p = 0.069, OR = 2.12). The occurrence of DHF in other age groups was around 30 %. None of the differences were significant. Clinical characteristics of dengue cases Clinical symptoms common to DF and DHF were analyzed in the context of disease category (Table 2). Nausea/ vomiting (p = 0.03), and maculopapular rash/petechiae (p = 0.02) were found to be more prevalent in DHF cases, along with abdominal pain (p = 0.11) which was not statistically significant. Thrombocytopenia was also observed in a significantly larger number of DHF patients (85.1 %) compared to DF patients (38.1 %) (p \ 0.0001). Among the 74 DHF cases, gastrointestinal bleeding; melena (n = 14) or hematemesis (n = 13) was observed in 40.7 % of cases. Hematuria, gum bleeding and epistaxis were less common. Plasma leakage was observed in 13 (16 %) patients – either as ascites or as pleural effusion. Only two patients presented with shock. Correlation of isotype with disease severity The performance of all of the DENV-specific isotype detection ELISAs was first assessed with a panel of 10 DENV-positive and 10 DENV-negative sera tested with DENV-2 antigen and control antigen from mock-infected cells. The mean P/N ratios for the negative controls, i.e., DENV-negative sera with DENV-2 antigen and DENVpositive sera with control antigen, ranged from 0.8 to 1.5. The IgM, IgG, IgA and IgE levels were compared among DF and DHF cases as P/N ratios (Fig. 1) and for IgM and IgG, titres were also compared. The average P/N ratio for IgM in healthy controls was 1.01 (range, 0.57-1.8).
Age in years*
DHF cases
1-10
11
11-20
12
21-30
21
46
31-40 [40
DF cases
P-value
Odds ratio
CI 95 %
9
0.11
2.32
0.82-6.70
30
0.298
0.63
0.27-1.39
0.343
0.70
0.35-1.37
9
21
0.541
0.71
0.27-1.73
18
17
0.069
2.12
0.094-4.75
* For 6 patients, age details were not available. The chi square test was used to compare the number of DF and DHF cases in each age group. The odds ratio with 95 % confidence interval (CI) was calculated using the Statcalc program
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R. Bachal et al. Table 2 Clinical characteristics of dengue patients
Clinical characteristics
DF cases (n = 126)
DHF cases (n = 74)
P-value
Odds ratio
CI 95 %
Fever
126 (100 %)
74 (100 %)
1
-
-
Headache
80 (89.49 %)
42 (56.75 %)
0.4279
0.75
0.40-1.42
Body pain
67 (53.17 %)
37 (50 %)
0.7738
0.88
0.48-1.63
Arthralgia
35 (27.77 %)
18 (24.32 %)
0.7126
0.84
0.40-1.69
Nausea/vomiting
11 (8.73 %)
15 (20.27 %)
0.0335
2.66
1.06-6.80
Abdominal pain Retro-orbital pain
5 (3.96 %)
8 (10.81 %)
0.1100
2.93
0.80-11.81
12 (9.52 %)
7 (9.45 %)
0.8143
0.99
0.31-2.89
Rash
13 (10.31 %)
17 (22.97 %)
0.0267
2.59
1.09-6.22
Platelet count \1 lakh
48 (38.09 %)
63 (85.13 %)
0.0000
9.31
4.28-21.34
The chi square test was used to compare the clinical characteristics of DF and DHF cases. The odds ratio with 95 % confidence interval (CI) was calculated using the Statcalc program
Fig. 1 Influence of antibody isotypes on disease severity. P/N ratios of antibody isotypes are presented in the form of box whisker plots with the median in the centre of box, the 25th and 75th percentiles as borders of the box, and minimal and maximal values as whiskers for DF and DHF cases. Data for IgM, IgG, IgA and IgE were compared among DF and DHF cases using Student’s t-test
The DF cases had significantly higher P/N ratios (mean value, 9.364) for IgM than DHF (mean value, 7.653) (p = 0.0396) but the titre was similar in both groups of patients (*4.5 logs). The average P/N ratio for IgG in healthy controls was 1.32 (range, 0.83 to 1.60). The mean P/N ratio for IgG was significantly higher in DHF cases (mean value, 7.86) compared to DF cases (mean value, 6.04) (p = 0.0001), and the titre was also significantly higher in DHF group (5.09 logs) compared to the DF group (4.4 logs) (p = 0.0070). In healthy controls, the mean P/N for IgA was 1.02 (range, 0.90 to 0.126). DENV-specific IgA antibodies were
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not present in all dengue samples. The number of samples positive for IgA was higher in the DHF group (51.3 %) compared to the DF group (30.9 %) (p = 0.006, OR = 2.35, CI 95 % = 1.25 to 4.44). The P/N ratio of IgA was also higher in DHF (p = 0.0024). For IgE, the mean P/N ratio for healthy controls was 0.82 (range, 0.63 to 1.07). The number of DHF samples positive for IgE (54 %) was higher than the number of DF samples (35.7 %) (p = 0.017, OR = 2.12, CI 95 % = 1.10 to 3.96) accompanied by higher P/N ratios in DHF samples (p = 0.0001). Samples were not titrated for IgA and IgE, as the mean P/N ratio observed for the positive samples tested at 1/100 was \3.0.
Antibody isotypes in dengue hemorrhagic fever
Correlation of isotype profile with immune status of patients By testing 200 samples in the E-DEN02G ELISA, it was determined that 61 were primary infections and that 137 were secondary infections. Two samples had equivocal results and were excluded from the analysis. A significantly larger number of secondary cases was observed in the DHF group (78.4 %) than in the DF group (63.7 %) (p = 0.045, OR with 95 % CI 2.06 [1.02-4.30]). The antibody levels were then compared in 61 primary and 137 secondary infections classified into DF and DHF cases (Fig. 2, Table 3). In primary cases, antiDENV IgM antibody levels were not significantly different among DF and DHF. However, in secondary cases, the IgM levels were significantly higher in DF compared to DHF cases (p \ 0.0019). In contrast, anti DENV IgG antibody levels were higher in primary DHF cases than in primary DF cases (p = 0.0061), whereas in secondary cases, the difference was not statistically significant.
A larger number of DHF cases were positive for antiDENV IgA compared to DF; however, the difference (50 % versus 17.8 %) was significant in primary cases (p = 0.02, OR 4.63, CI 95 % = 1.11-19.03) and tending to significance in secondary cases (50 % versus 35.4 %). This was reflected in the IgA levels, which were higher in both primary and secondary DHF cases compared to DF with significance evident only in primary infections (p = 0.0004). More cases were positive for anti-DENV IgE in primary DHF (50.0 %) than in primary DF cases (20.0 %) (p = 0.047, OR 4.00, CI 95 % = 0.98-16.02). The anti-DENV IgE levels were also significantly higher in DHF cases than in DF, irrespective of whether they were primary (p \ 0.0001) or secondary (p = 0.034). Correlation of isotype profile with day of sample collection and disease severity The results were further analyzed in the context of the day of sampling and disease severity for 160 samples for which the requisite data were available (Fig. 3). The anti DENV
Fig. 2 Influence of antibody isotypes on disease severity in patients with different immune status. P/N ratios of isotypes are presented in the form of box whisker plots with the median in the centre of box, the 25th and 75th percentiles as borders of the box, and minimal and maximal values as whiskers. Data for IgM, IgG, IgA and IgE were compared among primary DF and DHF cases and secondary DF and DHF cases using Student’s t-test
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R. Bachal et al. Table 3 Comparison of isotype levels in DF versus DHF cases in the context of immune status Isotype
Primary DF (n = 45)
Primary DHF (n = 16)
P-value
Secondary DF (n = 79)
Secondary DHF (n = 58)
P-value
IgM
6.78 ± 0.62*
7.68 ± 0.96
0.46
10.81 ± 0.80
7.55 ± 0.49
0.0019
IgG
4.17 ± 0.40
7.20 ± 1.39
0.0061
7.18 ± 0.26
8.04 ± 0.40
0.62
IgA
1.42 ± 0.12
2.60 ± 0.41
0.0004
1.98 ± 0.13
2.24 ± 0.17
0.23
IgE
1.43 ± 0.09
2.35 ± 0.22
\0.0001
1.96 ± 0.12
2.42 ± 0.20
0.034
* Mean values of P/N ratios ± standard error. Student’s t-test was used to compare the mean values of P/N ratios for the different isotypes among primary DF and DHF cases and among secondary DF and DHF cases
Fig. 3 Influence of antibody isotypes on disease severity depending on the day of sample collection. P/N ratios of antibody isotypes are presented in the form of box whisker plots with the median in the centre of box, the 25th and 75th percentiles as borders of the box, and minimal and maximal values as whiskers. Data for IgM, IgG, IgA and IgE were compared among DF and DHF cases for samples collected within 5 days of onset of illness (pre-defervescence) and for samples collected after 5 days of onset of illness (postdefervescence) using Student’s t-test
IgM levels were higher in DF cases in the post-defervescence samples (p = 0.0363), whilst anti-DENV IgG levels were higher in DHF cases in the pre-defervescence samples (p = 0.0004). In pre-defervescence samples, titres of antiDENV IgA were significantly higher in DHF cases (p = 0.0486) whilst those of IgE were higher in DHF samples from both the pre (p = 0.057) and post-defervescence period (p = 0.0036).
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Correlation of antibody titres and platelet counts The levels of IgM and IgG were also correlated to thrombocytopenia. There were 20 samples that were negative for IgG, which were arbitrarily assigned a value of 1.0. All of those samples were from cases with primary infection. There was significant correlation in the levels of IgG with platelet count. The lower the platelet count, the
Antibody isotypes in dengue hemorrhagic fever
higher the levels of IgG (r2 = 0.148, p \ 0.0001). There was no correlation with IgM (Fig. 4).
Discussion In the present study, the levels of DENV-specific IgM, IgG, IgA and IgE antibodies were compared among DF and DHF cases in the context of immune status and time of sample collection. The patients were classified using the older WHO definition [24] because the study was retrospective and initiated in 2005. The present study was different from earlier studies, as it included mostly adults (174/194 individuals) and cases of DF and DHF with no representation of DSS. The IgM level in DF cases was higher than in DHF cases, and this was significant in secondary cases and in post-defervescence samples rather than pre-defervescence. A similar observation of higher IgM levels in secondary DF cases was reported by Vasquez et al. [18, 19]. IgM has
Fig. 4 Correlation between IgM and IgG titres and platelet counts. Platelet counts were plotted against log10 titres of antibody isotypes IgM (n = 151) and IgG (n = 147). Samples with log10 titres \2.0 were given a value of 1.0. The samples (n = 20) with no detectable IgG were from primary cases
been shown to participate in the neutralization response against dengue viruses [25]. The lower levels in DHF cases may result in higher viral load and slow clearance of virus, which has been reported in DHF cases [26, 27]. In contrast to IgM, significantly higher levels of IgG were observed in DHF compared to DF cases. It was noteworthy that the difference was more evident in primary infections. The boosted IgG response in secondary infections has been considered responsible for severe disease through the mechanism of ADE [7]. The evidence of higher levels of IgG in primary DHF cases suggests additional mechanisms of immune pathogenesis. There have been reports of complement-mediated cell lysis and ADCC being important factors in dengue pathogenesis [8, 28, 29]. The higher level of IgG in DHF in the pre-defervescence phase suggests that it may be the early antibody response that contributes to ADE and results in the increased viremia reported in DHF [30]. The presence of IgA in a significantly larger number of DHF cases at significantly higher levels that was evident during pre-defervescence suggests IgA involvement in the pathogenesis of DHF. The difference was more evident in primary DHF. Higher levels of DENV-specific IgA in patients who developed shock has been reported earlier [15, 31]. Deposition of IgA-containing immune complexes on endothelial cells is reported to result in apoptosis and production of pro-inflammatory cytokines, which can enhance vascular permeability [32]. A case study of glomeruli deposits of IgA and subsequent mesangioproliferative glomerulonephritis in DENV infection has been reported [33]. The IgE response was higher in both primary and secondary DHF cases. The implication of IgE in increased vascular permeability has been reported in the past [34]. Proinflammatory cytokines, such as IL-1b and tumor necrosis factor-a, triggered by IgE-containing immune complexes may contribute to this phenomenon [35, 36]. A shift from Th1 to Th2 responses during dengue virus infection might lead to elevated IgE antibodies in serum [37]. The main strength of our study was that primary DHF samples were included, and these were lacking in other studies [15–19]. To conclude, the present study suggests that higher titres of anti-DENV IgG, IgA and IgE during primary infection are associated with DHF. The presence of higher titres of anti-DENV IgM in DF cases during the post-defervescence phase in secondary infections is associated with reduced risk of DHF. To the best of our knowledge, this is the first study to correlate the levels of different isotypes of antibody with dengue disease severity in the context of immune status in a predominantly adult population from India. Conflict of interest
The authors declare no conflicts of interest.
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primary and secondary dengue 4 infected salvadorian children. J Med Virol. doi:10.1002/jmv.23833 Priyadarshini D, Gadia RR, Tripathy A, Gurukumar KR, Bhagat A, Patwardhan S et al (2010) Clinical findings and pro-inflammatory cytokines in dengue patients in western india: a facilitybased study. PLoS ONE 5:e8709 Alagarasu K, Mulay AP, Singh R, Gawade VB, Shah PS, Cecilia D (2013) Association of HLA-DRB1 and TNF genotypes with dengue hemorrhagic fever. Hum Immunol 74:610–661 Alagarasu K, Mulay AP, Mohsen S, Rashmika D, Shah PS, Cecilia D (2013) Profile of human leukocyte antigen class I alleles in patients with dengue infection from Western India. Hum Immunol 74:1624–1628 Cecilia D (2014) Current status of dengue and chikungunya. WHO South-East Asia J Public Health 3:22–27 World Health Organisation (1999): Prevention and control of Dengue and Dengue haemorrhagic fever: Comprehensive guidelines. WHO Regional Publication SEARO No. 29, pp 11–19 Puschnik A, Lau L, Cromwell EA, Balmaseda A, Zompi S, Harris E (2013) Correlation between dengue-specific neutralizing antibodies and serum avidity in primary and secondary dengue virus 3 natural infections in humans. PLoS Negl Trop Dis 7:e2274 Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S, Suntayakorn S et al (2000) Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis 181:2–9 Libraty DH, Endy TP, Houng HS, Green S, Kalayanarooj S, Suntayakorn S et al (2002) Differing influences of virus burden and immune activation on disease severity in secondary dengue-3 virus infections. J Infect Dis 185:1213–1221 Falgout B, Bray M, Schlesinger JJ, Lai CJ (1990) Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis. J Virol 64:4356–4363 Laoprasopwattana K, Libraty DH, Endy TP, Nisalak A, Chunsuttiwat S, Ennis FA et al (2007) Antibody-dependent cellular cytotoxicity mediated by plasma obtained before secondary dengue virus infections: potential involvement in early control of viral replication. J Infect Dis 195:1108–1116 Guzman MG, Alvarez M, Halstead SB (2013) Secondary infection as a risk factor for dengue hemorrhagic fever/dengue shock syndrome: an historical perspective and role of antibodydependent enhancement of infection. Arch Virol 158:1445– 1459 De Rivera IL, Parham L, Murillo W, Moncada W, Vazquez S (2008) Humoral immune response of dengue hemorrhagic fever cases in children from Tegucigalpa, Honduras. Am J Trop Med Hyg 79:262–266 Yang YH, Huang YH, Lin YL, Wang LC, Chuang YH, Yu HH et al (2006) Circulating IgA from acute stage of childhood Henoch-Scho¨nlein purpura can enhance endothelial interleukin (IL)-8 production through MEK/ERK signalling pathway. Clin Exp Immunol 144:247–253 Upadhaya BK, Sharma A, Khaira A, Dinda AK, Agarwal SK, Tiwari SC (2010) Transient IgA nephropathy with acute kidney injury in a patient with dengue fever. Saudi J Kidney Dis Transpl 21:521–525 Boesiger J, Tsai M, Maurer M, Yamaguchi M, Brown LF, Claffey KP et al (1998) Mast cells can secrete vascular permeability factor/vascular endothelial cell growth factor and exhibit enhanced release after immunoglobulin E-dependent upregulation of fc epsilon receptor I expression. J Exp Med 188:1135–1145
Antibody isotypes in dengue hemorrhagic fever 35. Borish L, Mascali JJ, Rosenwasser LJ (1991) IgE-dependent cytokine production by human peripheral blood mononuclear phagocytes. J Immunol 146:63–67 36. Chaturvedi UC, Agarwal R, Elbishbishi EA, Mustafa AS (2000) Cytokine cascade in dengue hemorrhagic fever: implications for pathogenesis. FEMS Immunol Med Microbiol 28:183–188
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ORIGINAL ARTICLE
Higher levels of dengue-virus-specific IgG and IgA during predefervescence associated with primary dengue hemorrhagic fever Rupali Bachal1 • Kalichamy Alagarasu1 • Anand Singh1 • Asha Salunke1 Paresh Shah1 • Dayaraj Cecilia1
•
Received: 30 April 2015 / Accepted: 28 June 2015 Ó Springer-Verlag Wien 2015
Abstract Dengue hemorrhagic fever (DHF), although predominantly associated with secondary infections, has also been reported in primary infections. An enhanced immune response including antibodies and cytokines is implicated in the pathogenesis of secondary DHF. However, the factors operating in primary DHF are poorly understood. To understand the role of the antibody response, the relative levels of different antibody isotypes during the acute phase of infection in primary and secondary dengue infections were determined. Levels of DENV-specific IgM, IgG, IgA and IgE were measured in the serum samples of 200 dengue patients and 20 denguenaı¨ve individuals. Samples were collected within 15 days of onset of illness. The DENV-specific IgM levels were significantly higher in DF cases compared to DHF, which was more evident in secondary infections and in post-defervescence samples. The levels of IgG, IgA and IgE were higher in DHF cases, with greater significance in primary infections. A higher level of IgG in DHF cases was evident in pre-defervescence samples, whilst the IgE level was higher in pre- and post-defervescence samples. There was a significant correlation of IgG titres with platelet counts, with higher titres associated with lower platelet counts. It is speculated that IgG, IgA and IgE produced in response to primary infections may contribute to pathogenesis, whilst IgM produced in response to secondary infections may protect against progression to severe disease.
& Dayaraj Cecilia [email protected] 1
Dengue Group, National Institute of Virology, 20-A Dr. Ambedkar Road, Pune 411001, India
Introduction Dengue virus (DENV) causes 50 to 100 million infections each year in over 100 countries and has recently spread to Europe. Of these infections, 500,000 cases are severe, requiring hospitalization, and 2.5 % of them are fatal [1]. DENV causes a spectrum of disease including subclinical infections, dengue fever (DF), an acute febrile illness, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), which is severe and occasionally lifethreatening [2]. There are four serotypes of DENV, and people experiencing their first DENV infection develop a robust antibody response that is cross-reactive with other serotypes [3] but protective only against the homologous serotype [4]. Severe disease is associated more often with secondary infections but also occurs during primary infections [5]. Development of severe disease is due to multiple factors, including inflammatory cytokines [6], antibody-dependent enhancement (ADE) [7], antibodydependent cell-mediated cytotoxicity (ADCC) [8], and ADE due to antibodies produced against the prM protein [9]. An additional factor is auto-reactive antibodies produced against platelets, endothelial cells and coagulation molecules due to molecular mimicry between DENV proteins and host proteins [10]. Several studies have shown the involvement of complement factors in progression to DHF [11–13]. Considering the role of antibodies in disease pathogenesis and the different functions attributed to each isotype [14], the relative concentrations and kinetics of the antibody isotypes in dengue need to be defined in the context of different host and virus populations. There have been several studies on this aspect, but most of them have included children. Koraka et al. determined the isotype profile in dengue-virus-infected children in Indonesia [15, 16]. They reported increased
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levels of IgA, IgG1, IgG4 and IgE in DHF and DSS cases. Another study, carried out in children in Myanmar, found higher levels of IgG1 in DHF cases [17]. Vasquez et al. compared primary DF, secondary DF and secondary DHF in Cuba (age not mentioned) and in El Salvador (in children) and reported significantly higher levels of IgM in DF compared to DHF in secondary infections and higher levels of IgA and IgE in secondary infections compared to primary infections [18, 19]. Our earlier study revealed that the age profile and clinical presentation of dengue in India differ from that of South East Asian countries, where large numbers of children are affected and the clinical presentation is more severe [20]. Our recent studies have shown that the association of human leukocyte antigen alleles with clinical outcomes of DENV infection is different from that of studies from other regions [21, 22]. The virus populations circulating in India are also different from those in South East Asia [23]. The present study investigates the antibody isotype response in a predominantly adult population and compares DF and DHF patients in the context of their immune status for the first time. It is also the first time that the response in the pre- and post-defervescence phase of disease has been compared.
Materials and methods Clinical samples A total of 200 samples from confirmed dengue patients during annual seasonal outbreaks of dengue in Pune during 2008-10 were included in the study. The National Institute of Virology (NIV) is a WHO Collaborating Centre for Arbovirus and Haemorrhagic Fever Reference and Research and Rapid Diagnosis of Viral Diseases. The cases were confirmed to be dengue, either by the presence of DENV-specific IgM (using an NIV MAC-ELISA DEN Kit, which is used in the national surveillance programme) or multiplex reverse transcriptase PCR. Serum samples collected from each patient were stored in aliquots at –80 °C. For each patient, the day of onset of fever was designated as the first day of illness. Serum samples collected 2-5 days after onset of illness were designated as ‘pre-defervescence’, and those collected 6-15 days after onset of illness, as ‘post-defervescence’ samples. Twenty samples from healthy blood donors who were naı¨ve to dengue were included as negative controls. This study was approved by the Human Ethics Committee of NIV. Waiver of the informed consent requirement was granted by the committee on the basis of ‘‘Use of leftover specimens after clinical investigation’’ under the Indian Council of Medical Research Guidelines 2006.
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Cells and viruses DENV-2 (Indian strain 803347, obtained from the Virus Repository of NIV) was used in all ELISA experiments. Virus was propagated in porcine kidney stable (PS) cell line (procured from National Centre for Cell Sciences, India), which was maintained in Dulbecco’s modified Eagle’s medium (GIBCO BRL) with 10 % fetal bovine serum. Preparation of DENV-2 antigen for ELISA PS cell monolayers in 75-cm2 flasks were infected with DENV-2 at a multiplicity of infection (MOI) of 0.1. On the fourth day postinfection, when the cells showed a cytopathic effect (CPE), the infected cultures were freeze/thawed and the lysate was collected. The lysate was centrifuged at 2000 rpm for 15 minutes to remove cell debris. The clarified lysate, stored at -80 °C in convenient aliquots, was used as DENV antigen for the IgM/IgA/IgE /IgG ELISAs. Mock-infected cell culture clarified lysate was used as control antigen. DENV-specific IgM/IgA/IgE capture ELISA Immunosorp strips (NUNC) were coated with a 1:5000 dilution of anti-human IgM (Dako Cytomation)/anti-human IgA (Sigma)/anti-human IgE (Sigma) diluted in 0.05 M carbonate buffer (pH 9.6) and incubated overnight at 4 °C. The strips were blocked with 300 lL of 1 % milk powder solution in 0.01 M PBS (pH 7.2) for 2 hours at 37 °C. The test sample, 50 lL diluted 1:100 in PBS (pH 7.2) with 0.1 % Tween (PBST) and 1 % BSA, was added to each well and incubated for 1 hour at 37 °C. This was followed by the addition of 50 lL of DENV-2 antigen/control antigen and incubation for 1 hour at 37 °C. The bound antigen was detected using anti-DENV envelope-protein-specific mouse monoclonal antibody GE9 (a kind gift from Dr. G. Sapkal, NIV) conjugated to horseradish peroxidase (HRP) (1:1000, 50 lL/well), with incubation for 1 hour at 37 °C. After the addition of 100 lL of the substrate tetramethyl benzedine, the plate was incubated for 10 minutes at room temperature in the dark, and the reaction was stopped with 1 N H2SO4. The optical density (OD) was read on an ELISA reader at 450 nm. The mean of the readings obtained with DENVnegative samples tested on DENV antigen was taken as mean negative control. A P/N (sample OD/mean OD of the negative controls) ratio of C2.1 was considered positive. The ELISA plates were washed five times with PBST after each step. The common steps of ELISA, i.e., coating, blocking, incubation and washing, as well as the diluents used, were similar for all ELISAs. ELISA titres were determined for IgM and IgG by testing tenfold dilutions of the serum samples in the antibody capture ELISAs, The reciprocal of the sample dilution yielding a P/N ratio of 2.1
Antibody isotypes in dengue hemorrhagic fever
was considered the titre. For samples that were negative (P/ N \ 2.1), an arbitrary value of 1 was given for calculation purposes with regard to antibody titres. DENV-specific IgG capture ELISA ELISA plates were coated with purified GE9 IgG diluted 1:50 in carbonate buffer (pH 9.6). After blocking, 50 lL of neat DENV-2-infected tissue culture fluid was added per well and incubated for 1 hour. This was followed by addition of the test sample at 1:100 dilution. The IgG captured on the antigen was detected with goat anti-human IgG-HRP conjugate (Sigma) diluted 1:30,000. The plate was developed as described above. Primary/secondary detection by IgG capture ELISA To detect primary and secondary infections, IgG Capture ELISA E-DEN02G (Panbio Diagnostics) was used. The test was performed as per the manufacturer’s protocol. The results were interpreted based on the calculations provided. Statistical analysis Using the Statcalc program (Epi info version 6.0.4), the chi-square test with Yates correction or Fisher’s exact test (when any cell value was less than 5) was performed to examine differences in demographic and clinical characteristics of the dengue patients. Age and platelet counts were compared using Student’s t-test. Mean P/N ratios of antibodies of different patient categories were compared using Student’s t-test or one-way ANOVA. P-values less than 0.05 were considered significant. All statistical analyses were performed using Graphpad prism (version 4).
Results Demographic characteristics of dengue cases A total of 200 dengue cases were included in the study. Based on the criteria defined by WHO [24], 126 (63 %) Table 1 Influence of age on disease severity in dengueinfected patients
patients were classified as DF and 74 (37 %) as DHF. The average age of dengue patients was 27.5 ±14.92 (SD) yrs. When the cases were divided into five age groups: 1-10, 11-20, 21-30, 31-40 and [41 yrs, the largest number of dengue cases fell into the 21-30 yrs age group (34.3 %) as compared to the other age groups (10.0 % to 21 %) (Table 1). A larger number of DHF cases were seen in children B10 yrs (55 %) (p = 0.11, OR = 2.32) and adults above 40 yrs (51.4 %) (p = 0.069, OR = 2.12). The occurrence of DHF in other age groups was around 30 %. None of the differences were significant. Clinical characteristics of dengue cases Clinical symptoms common to DF and DHF were analyzed in the context of disease category (Table 2). Nausea/ vomiting (p = 0.03), and maculopapular rash/petechiae (p = 0.02) were found to be more prevalent in DHF cases, along with abdominal pain (p = 0.11) which was not statistically significant. Thrombocytopenia was also observed in a significantly larger number of DHF patients (85.1 %) compared to DF patients (38.1 %) (p \ 0.0001). Among the 74 DHF cases, gastrointestinal bleeding; melena (n = 14) or hematemesis (n = 13) was observed in 40.7 % of cases. Hematuria, gum bleeding and epistaxis were less common. Plasma leakage was observed in 13 (16 %) patients – either as ascites or as pleural effusion. Only two patients presented with shock. Correlation of isotype with disease severity The performance of all of the DENV-specific isotype detection ELISAs was first assessed with a panel of 10 DENV-positive and 10 DENV-negative sera tested with DENV-2 antigen and control antigen from mock-infected cells. The mean P/N ratios for the negative controls, i.e., DENV-negative sera with DENV-2 antigen and DENVpositive sera with control antigen, ranged from 0.8 to 1.5. The IgM, IgG, IgA and IgE levels were compared among DF and DHF cases as P/N ratios (Fig. 1) and for IgM and IgG, titres were also compared. The average P/N ratio for IgM in healthy controls was 1.01 (range, 0.57-1.8).
Age in years*
DHF cases
1-10
11
11-20
12
21-30
21
46
31-40 [40
DF cases
P-value
Odds ratio
CI 95 %
9
0.11
2.32
0.82-6.70
30
0.298
0.63
0.27-1.39
0.343
0.70
0.35-1.37
9
21
0.541
0.71
0.27-1.73
18
17
0.069
2.12
0.094-4.75
* For 6 patients, age details were not available. The chi square test was used to compare the number of DF and DHF cases in each age group. The odds ratio with 95 % confidence interval (CI) was calculated using the Statcalc program
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R. Bachal et al. Table 2 Clinical characteristics of dengue patients
Clinical characteristics
DF cases (n = 126)
DHF cases (n = 74)
P-value
Odds ratio
CI 95 %
Fever
126 (100 %)
74 (100 %)
1
-
-
Headache
80 (89.49 %)
42 (56.75 %)
0.4279
0.75
0.40-1.42
Body pain
67 (53.17 %)
37 (50 %)
0.7738
0.88
0.48-1.63
Arthralgia
35 (27.77 %)
18 (24.32 %)
0.7126
0.84
0.40-1.69
Nausea/vomiting
11 (8.73 %)
15 (20.27 %)
0.0335
2.66
1.06-6.80
Abdominal pain Retro-orbital pain
5 (3.96 %)
8 (10.81 %)
0.1100
2.93
0.80-11.81
12 (9.52 %)
7 (9.45 %)
0.8143
0.99
0.31-2.89
Rash
13 (10.31 %)
17 (22.97 %)
0.0267
2.59
1.09-6.22
Platelet count \1 lakh
48 (38.09 %)
63 (85.13 %)
0.0000
9.31
4.28-21.34
The chi square test was used to compare the clinical characteristics of DF and DHF cases. The odds ratio with 95 % confidence interval (CI) was calculated using the Statcalc program
Fig. 1 Influence of antibody isotypes on disease severity. P/N ratios of antibody isotypes are presented in the form of box whisker plots with the median in the centre of box, the 25th and 75th percentiles as borders of the box, and minimal and maximal values as whiskers for DF and DHF cases. Data for IgM, IgG, IgA and IgE were compared among DF and DHF cases using Student’s t-test
The DF cases had significantly higher P/N ratios (mean value, 9.364) for IgM than DHF (mean value, 7.653) (p = 0.0396) but the titre was similar in both groups of patients (*4.5 logs). The average P/N ratio for IgG in healthy controls was 1.32 (range, 0.83 to 1.60). The mean P/N ratio for IgG was significantly higher in DHF cases (mean value, 7.86) compared to DF cases (mean value, 6.04) (p = 0.0001), and the titre was also significantly higher in DHF group (5.09 logs) compared to the DF group (4.4 logs) (p = 0.0070). In healthy controls, the mean P/N for IgA was 1.02 (range, 0.90 to 0.126). DENV-specific IgA antibodies were
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not present in all dengue samples. The number of samples positive for IgA was higher in the DHF group (51.3 %) compared to the DF group (30.9 %) (p = 0.006, OR = 2.35, CI 95 % = 1.25 to 4.44). The P/N ratio of IgA was also higher in DHF (p = 0.0024). For IgE, the mean P/N ratio for healthy controls was 0.82 (range, 0.63 to 1.07). The number of DHF samples positive for IgE (54 %) was higher than the number of DF samples (35.7 %) (p = 0.017, OR = 2.12, CI 95 % = 1.10 to 3.96) accompanied by higher P/N ratios in DHF samples (p = 0.0001). Samples were not titrated for IgA and IgE, as the mean P/N ratio observed for the positive samples tested at 1/100 was \3.0.
Antibody isotypes in dengue hemorrhagic fever
Correlation of isotype profile with immune status of patients By testing 200 samples in the E-DEN02G ELISA, it was determined that 61 were primary infections and that 137 were secondary infections. Two samples had equivocal results and were excluded from the analysis. A significantly larger number of secondary cases was observed in the DHF group (78.4 %) than in the DF group (63.7 %) (p = 0.045, OR with 95 % CI 2.06 [1.02-4.30]). The antibody levels were then compared in 61 primary and 137 secondary infections classified into DF and DHF cases (Fig. 2, Table 3). In primary cases, antiDENV IgM antibody levels were not significantly different among DF and DHF. However, in secondary cases, the IgM levels were significantly higher in DF compared to DHF cases (p \ 0.0019). In contrast, anti DENV IgG antibody levels were higher in primary DHF cases than in primary DF cases (p = 0.0061), whereas in secondary cases, the difference was not statistically significant.
A larger number of DHF cases were positive for antiDENV IgA compared to DF; however, the difference (50 % versus 17.8 %) was significant in primary cases (p = 0.02, OR 4.63, CI 95 % = 1.11-19.03) and tending to significance in secondary cases (50 % versus 35.4 %). This was reflected in the IgA levels, which were higher in both primary and secondary DHF cases compared to DF with significance evident only in primary infections (p = 0.0004). More cases were positive for anti-DENV IgE in primary DHF (50.0 %) than in primary DF cases (20.0 %) (p = 0.047, OR 4.00, CI 95 % = 0.98-16.02). The anti-DENV IgE levels were also significantly higher in DHF cases than in DF, irrespective of whether they were primary (p \ 0.0001) or secondary (p = 0.034). Correlation of isotype profile with day of sample collection and disease severity The results were further analyzed in the context of the day of sampling and disease severity for 160 samples for which the requisite data were available (Fig. 3). The anti DENV
Fig. 2 Influence of antibody isotypes on disease severity in patients with different immune status. P/N ratios of isotypes are presented in the form of box whisker plots with the median in the centre of box, the 25th and 75th percentiles as borders of the box, and minimal and maximal values as whiskers. Data for IgM, IgG, IgA and IgE were compared among primary DF and DHF cases and secondary DF and DHF cases using Student’s t-test
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R. Bachal et al. Table 3 Comparison of isotype levels in DF versus DHF cases in the context of immune status Isotype
Primary DF (n = 45)
Primary DHF (n = 16)
P-value
Secondary DF (n = 79)
Secondary DHF (n = 58)
P-value
IgM
6.78 ± 0.62*
7.68 ± 0.96
0.46
10.81 ± 0.80
7.55 ± 0.49
0.0019
IgG
4.17 ± 0.40
7.20 ± 1.39
0.0061
7.18 ± 0.26
8.04 ± 0.40
0.62
IgA
1.42 ± 0.12
2.60 ± 0.41
0.0004
1.98 ± 0.13
2.24 ± 0.17
0.23
IgE
1.43 ± 0.09
2.35 ± 0.22
\0.0001
1.96 ± 0.12
2.42 ± 0.20
0.034
* Mean values of P/N ratios ± standard error. Student’s t-test was used to compare the mean values of P/N ratios for the different isotypes among primary DF and DHF cases and among secondary DF and DHF cases
Fig. 3 Influence of antibody isotypes on disease severity depending on the day of sample collection. P/N ratios of antibody isotypes are presented in the form of box whisker plots with the median in the centre of box, the 25th and 75th percentiles as borders of the box, and minimal and maximal values as whiskers. Data for IgM, IgG, IgA and IgE were compared among DF and DHF cases for samples collected within 5 days of onset of illness (pre-defervescence) and for samples collected after 5 days of onset of illness (postdefervescence) using Student’s t-test
IgM levels were higher in DF cases in the post-defervescence samples (p = 0.0363), whilst anti-DENV IgG levels were higher in DHF cases in the pre-defervescence samples (p = 0.0004). In pre-defervescence samples, titres of antiDENV IgA were significantly higher in DHF cases (p = 0.0486) whilst those of IgE were higher in DHF samples from both the pre (p = 0.057) and post-defervescence period (p = 0.0036).
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Correlation of antibody titres and platelet counts The levels of IgM and IgG were also correlated to thrombocytopenia. There were 20 samples that were negative for IgG, which were arbitrarily assigned a value of 1.0. All of those samples were from cases with primary infection. There was significant correlation in the levels of IgG with platelet count. The lower the platelet count, the
Antibody isotypes in dengue hemorrhagic fever
higher the levels of IgG (r2 = 0.148, p \ 0.0001). There was no correlation with IgM (Fig. 4).
Discussion In the present study, the levels of DENV-specific IgM, IgG, IgA and IgE antibodies were compared among DF and DHF cases in the context of immune status and time of sample collection. The patients were classified using the older WHO definition [24] because the study was retrospective and initiated in 2005. The present study was different from earlier studies, as it included mostly adults (174/194 individuals) and cases of DF and DHF with no representation of DSS. The IgM level in DF cases was higher than in DHF cases, and this was significant in secondary cases and in post-defervescence samples rather than pre-defervescence. A similar observation of higher IgM levels in secondary DF cases was reported by Vasquez et al. [18, 19]. IgM has
Fig. 4 Correlation between IgM and IgG titres and platelet counts. Platelet counts were plotted against log10 titres of antibody isotypes IgM (n = 151) and IgG (n = 147). Samples with log10 titres \2.0 were given a value of 1.0. The samples (n = 20) with no detectable IgG were from primary cases
been shown to participate in the neutralization response against dengue viruses [25]. The lower levels in DHF cases may result in higher viral load and slow clearance of virus, which has been reported in DHF cases [26, 27]. In contrast to IgM, significantly higher levels of IgG were observed in DHF compared to DF cases. It was noteworthy that the difference was more evident in primary infections. The boosted IgG response in secondary infections has been considered responsible for severe disease through the mechanism of ADE [7]. The evidence of higher levels of IgG in primary DHF cases suggests additional mechanisms of immune pathogenesis. There have been reports of complement-mediated cell lysis and ADCC being important factors in dengue pathogenesis [8, 28, 29]. The higher level of IgG in DHF in the pre-defervescence phase suggests that it may be the early antibody response that contributes to ADE and results in the increased viremia reported in DHF [30]. The presence of IgA in a significantly larger number of DHF cases at significantly higher levels that was evident during pre-defervescence suggests IgA involvement in the pathogenesis of DHF. The difference was more evident in primary DHF. Higher levels of DENV-specific IgA in patients who developed shock has been reported earlier [15, 31]. Deposition of IgA-containing immune complexes on endothelial cells is reported to result in apoptosis and production of pro-inflammatory cytokines, which can enhance vascular permeability [32]. A case study of glomeruli deposits of IgA and subsequent mesangioproliferative glomerulonephritis in DENV infection has been reported [33]. The IgE response was higher in both primary and secondary DHF cases. The implication of IgE in increased vascular permeability has been reported in the past [34]. Proinflammatory cytokines, such as IL-1b and tumor necrosis factor-a, triggered by IgE-containing immune complexes may contribute to this phenomenon [35, 36]. A shift from Th1 to Th2 responses during dengue virus infection might lead to elevated IgE antibodies in serum [37]. The main strength of our study was that primary DHF samples were included, and these were lacking in other studies [15–19]. To conclude, the present study suggests that higher titres of anti-DENV IgG, IgA and IgE during primary infection are associated with DHF. The presence of higher titres of anti-DENV IgM in DF cases during the post-defervescence phase in secondary infections is associated with reduced risk of DHF. To the best of our knowledge, this is the first study to correlate the levels of different isotypes of antibody with dengue disease severity in the context of immune status in a predominantly adult population from India. Conflict of interest
The authors declare no conflicts of interest.
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