Short communication: Feasibility of dengue vaccine to infect different human cell lines: An alternative potency test using HEK293T cells

Abstract

Dengue is caused by an arbovirus that belongs to the Flaviviridae family and there are four distinct, but close related, circulating serotypes. Dengue disease is of great importance for global public health, with vaccination being its main prophylactic measure. However, there is a paucity of biological models for evaluating tetravalent dengue vaccines. The aim of this study was to evaluate the susceptibility of human cell lines HEK293T and THP-1 to a commercial dengue vaccine and test the feasibility of this approach in the development of a potency assay with human cell lines, as a methodological alternative to the golden standard potency assay with VERO cells. In this context, we used a batch of the commercial vaccine Dengvaxia® (CYD-TDV) for the infection tests. We evaluated the presence of the vaccine virus in THP-1 cells, differentiated into macrophages (dTHP-1), and in HEK293T by confocal microscopy, using 4G2 pan-flavivirus antibody. Vaccine infectivity and potency were determined by immunocolorimetric assay using monoclonal antibodies specific for each serotype. The results indicated that the human strain HEK293T was responsive to the tetravalent vaccine, as shown by the presence of virus particles in the cell cytoplasm in a pattern similar to the one observed with VERO cells. Moreover, it was possible to determine the infectivity and potency values of each vaccine virus serotype in the HEK293T, with serotype 4 prevailing over the others. Thus, the human cell line HEK293T provides a potential candidate to be used in assays to determine potency and identity of tetravalent dengue vaccines.

Fig 1. Confocal immunofluorescence of VERO, HEK293T and PMA-treated THP-1 cells infected or not with dengue vaccine.

Cells were stained with anti-4G2 (pan-flavivirus antibody, pink fluorescence) and PAFI (DNA staining, blue fluorescence). Negative control (A), (D) and (G), infected cells with 10⁻¹ vaccine dilution for 7 days (B), (E) and (H) show positivity for 4G2 in VERO (B) and HEK293T (E) cells. Representative graph of fluorescence intensity (y-axis) against the distance in micrometer (x-axis) of infected cells (C), (F) and (I). Representative images obtained under confocal microscope Leica TCS-SP8 at 200X magnification. n = 2.

Fig 2. Dengue vaccine infectivity in VERO and HEK293T cells by immunocolorimetric assay.

VERO and HEK293T cells incubated with dengue vaccine, 10⁻¹ vaccine dilution, for 7 days. Control VERO cells (A) and infected VERO cells (B) stained with anti-E DENV-2 mAb. Control HEK293T cells (C) and infected HEK293T cells (D) stained with anti-E DENV-2 mAb. Black arrows indicate viral infection. Representative images obtained under Olympus microscope at 100X magnification. n = 4.

Fig 3. Dose-response curve showing VERO and HEK293T cells response after 7 days of infection with Dengvaxia.

VERO (black circles) and HEK293T (black squares) cells infected with dengue vaccine (4,5–6,0 log₁₀ CCID₅₀/dose) in dilutions of 10^−2.6–10^−6.8, 1:4 dilution factor, and 10^−1.3–10^−3.4, 1:2 dilution factor, respectively, and analyzed by immunocolorimetric assay after 7 days of incubation. Infection of serotype 1 (A), serotype 2 (B), serotype 3 (C) and serotype 4 (D) evaluated by the presence of staining after incubation with serotype-specific mAb—positive wells. Linear regression (straight lines) of infectious responses in VERO and HEK293T cells for serotypes 1 (R²_VERO = 0.887 and R²_HEK293T = 0.793), serotype 2 (R²_VERO = 0.883 and R²_HEK293T = 0.801), serotype 3 (R²_VERO = 0.903 and R²_HEK293T = 0.767) and serotype 4 (R²_VERO = 0.913 and R²_HEK293T = 0.917). Data presented as mean ± SEM. n = 4.

Fig 4. Comparison of the infectivity of the four distinct DENV serotypes in VERO and HEK293T cells.

CCID₅₀ (mean ± SD) of each serotype measured by immunocolorimetric assay after 7 days of infection. Two-Way ANOVA performed for group comparison and significant differences observed among cell types (p<0.0001) and serotypes 4 and 3 (p<0.01) and serotypes 4 and 1 (p<0.01) measured in HEK293T cells. No significance detected in VERO cell groups. n = 4.