Joint Research Projects

Study In Thailand

Investigation of the mechanisms by which DENV non-structure protein-1 induced reduction of antibody-secreting cell in hematological diseases





A protective dengue vaccine remains at large since the antibody response in subjects previously exposed to the dengue virus (DENV) is resumed to the profile of primary status upon re-infection. Abnormal bone marrow (BM) populations in dengue patients and that hematopoietic stem cells are permissive to DENV have been known. People recovered from dengue are at higher risk of developing leukemia. Thirty-two leukemia BM were collected; among them, 12 were found to be NS1+. NS1+ or NS1- specimens were subjected to FACS analysis for antibody-secreting B cells (ASCs) and T cells. FACS results showed that ASCs significantly decreased in NS1+ BM compared to NS1- BM, while no alter was found in T cells. BM cells from NS1+ or NS1- were stimulated by DENV or NS1 protein directly. Results demonstrated that the ASCs were decreased after the stimulation. Pilot data indicated that mature plasma cells were decreased in the NS1+ BM samples. However, mature plasma cells were come from the maturation of memory B cells (MBCs). We therefore hypothesize that MBCs may be affected during DENV infection and further influence the generation of plasma cell within BM. As a results, we proposed to dissect the mechanism by which how DENV infection result in the reduction of plasma cells.


The BM specimens were collected after signing the informed consent. All cells including the plasma cells were collected after brief centrifugation. The serological status of bone marrows was tested by using CTK dengue rapid diagnostic kit. DENV NS1 (non-structure protein 1) antigens were mainly detected during the viremia period. IgM antibodies appeared after NS1 antigens in acute infection representing the recent infection. IgG antibodies represent late infection which meant the subjects were infected by DENV previously. Cell pellets were used for immune cell analysis by multicolor staining and analyzed by flow cytometry. Cell surface marker fluorescence antibodies were added with the recommended volume and mixed gently with the cells. After 30 minutes of incubation, the antibodies-stained cells were washed with excessive staining buffer and centrifuged to remove the non-binding antibodies. The resuspended cells were analyzed using flow cytometry (LSR II, BD). The collected data was further analyzed by Kaluza software to identify different cell populations.



We collected 32 BM from leukemia patients, and 12 of these BM were NS1+(Table 1). ASCs were analyzed by FACS in NS1+ or NS1- BM. Results showed that ASCs were significantly decreased in NS1+ BM, while T cells were not altered when compared to that of NS1- BM (Figure 1).

In addition, NS1+ BMs were stimulated with DENV or NS1 protein directly. FACS analysis was performed at the indicated times after the stimulation. Results showed that the subset of ACS populations, plasma cells appeared to be decreased (Figure 2A), while plasmablasts were increased after the stimulation according to the days of stimulation (Figure 2B). The results demonstrated that the mainly decreased population of functional ASCs was plasma cells after DENV or NS1 protein stimulation.

Previous work from pull-down assay in our lab found that BET family transcriptional factor interacts with DENV NS1 protein. Moreover, literature suggests that BRD2, one of BET transcriptional factors, interacts with cyclin A which is important for the expansion and mitogenesis of B cell. Hence, this line of evidence demonstrates that transcriptional factors regulating the proliferation and differentiation of B cells may be interfered during DENV infection. Therefore, we suppose the interaction between DENV NS1 and BRD2 affect on the B cell differentiation and proliferation resulting in the alteration in plasma cells (Figure 3).

Table 1. Rapid test results for DENV uninfected/infected bone marrow specimen





Figure 1. Analysis of lymphocyte populations in DENV NS1- or NS1+ Bone marrows

Cells were collected from NS1- or NS1+ bone marrows and analyzed by multicolor flow cytometry with cell surface markers.

(A)The population of T cells including CD8+ effector T cells, Naïve T cells, effector memory T cells, central memory T cells as well as CD4+ effector T cells, Naïve T cells, effector memory T cells, central memory T cells were shown. (B) The population of B cell including Naïve B cells, Resting memory B cells, Tissue-like memory B cells, Active memory B cells, Antibody-secreting cells (ASCs). (C) Subpopulation of ASCs including plasma cells and plasmablast cells. The data was shown with mean ± SEM.



Figure 2. B cell populations after DENV2 or NS1 recombinant protein stimulation in DENV NS1 positive bone marrows

Cells were collected from DENV NS1+ bone marrows and stimulated with DENV2 (MOI=1) or NS1 recombinant protein (100 ng). Cells were collected at indicated times and subjected to FACS analysis for B cell populations. (A) Subset of ASCs, plasma cells, DENV2 or NS1 recombinant protein stimulation at indicated times. (B) Subset of ASCs, Plasmablast cells, after DENV2 or NS1 recombinant protein stimulation at indicated times. The data was shown with mean ± SEM

Figure 3. Proposed mechanism of impaired MBCs mitogenesis after DENV infection via DENV NS1 protein interact with Brd2 transcription factor



The results cumulated from the human BM studies suggest that the reduction of ACSs in BM was due to the effect of DENV infection and that the NS1 protein may be responsible for the hindrance of plasmablast to differentiate into mature plasma cells. In addition, the antibody response observed in subjects who are previously exposed to the DENV and resume to the primary profile upon re-infection may result from the reduction of plasma cells in BM. In future work, we will attempt to validate the mechanism of how DENV NS1 protein interact with BET family transcription factors and further alters the plasma cell generation from MBCs. Hope the research outcomes can provide new insight into DENV vaccine development.