Lambda-free light chain: A serum marker of dengue disease via NS3 protease-mediated antibody cleavage

Figures & data

Figure 1. Positive correlation of serum λ and κ FLCs in dengue patients.

Pearson correlation of serum λ and κ FLCs concentration in dengue patients (n = 88). Correlations are shown as linear regression, R² and p-value are indicated for the analysis. Significance is denoted as *p < 0.05.

Figure 2. Higher serum λ free light chain expression in dengue patients with varying severity.

The serum levels of free λ light chains (a) and free κ light chains (b) were detected in healthy donors (n = 21) and patients with dengue fever (n = 88). The expression level of free λ light chains (c) and free κ light chains (d) by varying clinical severity, Dengue w/o WS (n = 49), Dengue with WS (n = 27), and Severe dengue (n = 12). Dengue w/o WS: dengue patients without warning signs; Dengue with WS: dengue patients with warning signs. Mann–Whitney U test was performed, and data were calculated and shown with mean ± SEM. Significance is indicated as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3. Lambda (λ) free light chain as a potential serum marker of dengue disease and severe dengue classification.

ROC curve of serum FLCs. Univariate logistic regression analysis was conducted. Performance of ROC curves of serum λ and κ FLCs for differentiating dengue patients. (a) ROC curve compared between dengue patients and healthy subjects. (b) ROC curve compared between Dengue ± WS and SD groups. Dengue ± WS: dengue patients with and without warning signs; SD: severe dengue. Significance is indicated as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Table 1. Area under ROC curve of serum free light chains to discriminate dengue patients.

	Lambda light chain
	AUC	P-value	Criterion	Youden index	Sensitivity (%)	95% CI	Specificity (%)	95% CI
Healthy vs DENV	0.7825	<0.0001, ****	>12.09	0.51	80	70.86% − 86.81%	71.43	50.04% − 86.19%
DENV ± WS vs SD	0.8102	0.0005, ***	>29.55	0.54	75	46.77% − 91.11%	79.52	69.62% − 86.80%
	Kappa light chain
	AUC	P-value	Criterion	Youden index	Sensitivity (%)	95% CI	Specificity (%)	95% CI
Healthy vs DENV	0.5911	0.1918	>12.28	0.27	55.67	45.76% − 65.15%	71.43	50.04% − 86.19%
DENV ± WS vs SD	0.7216	0.0125,*	>14.31	0.42	91.67	64.61% − 99.57%	50.52	40.74% − 60.25%

Figure 4. Lambda (λ) free light chain appears upon co-incubation of IgG and DENV protease.

(a) Sera from 10 acute dengue patients and 2 healthy donors were used to determine serum DENV NS3 level. Cell extracts of DENV-infected and uninfected Vero cells (n = 3) served as positive and negative controls. (b) Recombinant DENV proteases NS2B-NS3pro and NS3pro were purified from Rosetta 2 competent cells. The desalting column profile of the peak fraction indicated the expression of DENV protease. (c) Purified human IgG (0.8 μg/μL) was reacted with NS1 (0.33 μg/μL), NS2B-NS3pro (0.33 μg/μL), and NS3pro (0.33 μg/μL), respectively, at 37°C for 60 minutes. SDS-PAGE was utilized for protein expression pattern analysis. The NS2B-NS3pro, light chain, and NS3pro are represented by the green, red, and purple arrows, respectively. Mann – Whitney U test was performed and data were calculated and shown with mean ± SEM. Significance is indicated as *p < 0.05; **p < 0.01; ***p < 0.001.

Table 2. Protein composition revealing light chain product after NS2B-NS3pro treatment.

Protein name	MW [kDa]	PSMs^#	Relative abundance (%)
Immunoglobulin lambda-like polypeptide 5 isoform 1	23.0	108	90.8
Mucosa-associated lymphoid tissue lymphoma translocation protein 1 isoform X1	48.3	5	4.2
Immunoglobulin lambda-like polypeptide 1 isoform a precursor	22.9	2	1.7
Albumin preproprotein	69.3	2	1.7
Immunoglobulin heavy variable 4-38-2-like	23.8	2	1.7

^#PSMs: Peptide-spectrum match.

Table 3. Protein composition revealing light chain product after NS3pro treatment.

Protein name	MW [kDa]	PSMs^#	Relative abundance (%)
Immunoglobulin lambda-like polypeptide 5 isoform 1	23.0	105	92.1
FAS-associated death domain protein	23.3	3	2.6
Immunoglobulin lambda-like polypeptide 1 isoform a precursor	22.9	2	1.8
Albumin preproprotein	69.3	2	1.8
Immunoglobulin heavy variable 4-38-2-like	23.8	1	0.9
Protein KIAA0100 isoform 7	178.4	1	0.9

^#PSMs: Peptide-spectrum match.

Figure 5. Lambda (λ) free light chain increases in a trend of dose-dependent manner after DENV protease cleaves human IgG.

Purified human IgG (8 μg/mL) was reacted with NS1 (control) and DENV proteases NS2B-NS3pro (a) and NS3pro (b) in different amounts (0.05, 0.1, and 0.2 μg/μL). Western blotting was performed to detect immunoglobulin λ light chain. His-tag protein was utilized as a marker for both the NS2B-NS3pro and NS3pro. Mann–Whitney U test was performed and data were calculated and shown with mean ± SEM (n = 3). Significance is indicated as *p < 0.05; **p < 0.01.

Figure 6. DENV protease cleaves DENV2-neutralizing antibodies resulting in λ free light chain increase and neutralizing efficacy reduction.

DENV2 neutralizing monoclonal antibody (3H5-1 clone, 16 μg/mL) was reacted with NS1 (control), and purified DENV proteases NS2B-NS3pro (a) and NS3pro (b) in different amounts (0.1, 0.2, and 0.4 μg/μL). Western blotting was performed to detect immunoglobulin λ light chain. His-tag protein is a marker for both the NS2B-NS3pro and NS3pro. (c) The neutralizing efficacy of the DENV2 neutralizing antibody (3H5-1 clone) against DENV infection after DENV protease treatment was tested by FRNT assay. The working concentration of the DENV2 neutralizing antibody (3H5-1 clone) was at 0.32 μg/μL, and DENV proteases were at 0.4 μg/μL. Mann–Whitney U test was performed, and data were calculated and shown with mean ± SEM (n = 3). Significance is indicated as *p < 0.05; **p < 0.01.

Data Availability statement

The supporting data for this study can be obtained from the corresponding author upon reasonable request.