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. 2022 May 6;17(5):e0267653. doi: 10.1371/journal.pone.0267653

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

Renata Faria de Carvalho 1,2, Lucas de Siqueira Penna Quintaes 1, Thaís de Cássia de Souza Su 1, Leticia Mitiko Kobayashi 1, Ana Cristina Martins de Almeida Nogueira 2,3,*
Editor: Henning Ulrich4
PMCID: PMC9075668  PMID: 35522661

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.

Introduction

Dengue, an arthropod-borne illness, occurring in tropical and subtropical areas, is an important public health burden, with approximately 40% of the world population at risk of infection [1]. It is estimated that 390 million cases of dengue virus (DENV) infection occur annually, while 96 million are clinically manifest [2]. DENV circulates as four distinct serotypes (DENV-1 to 4) and the illness can evolve from a mild dengue fever to a severe form. The severe manifestation of dengue disease is often hasty and it is known as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). This complication is manifested by thrombocytopenia, hemorrhagic symptoms and increased vascular permeability, leading frequently to death [3]. Due to the major epidemiological burden that dengue illness characterizes, the development of an effective vaccine against the etiological agent of this disease is considered a worldwide priority [4].

A dengue vaccine should protect against all four serotypes of DENV simultaneously, provide long-term protection, be safe and well tolerated. Additionally, antibody-dependent enhancement phenomenon (ADE) must be strictly avoided in any vaccine proposed for the prevention of DENV infection [5]. Up to the present day, Sanofi Pasteur´s Dengvaxia® tetravalent dengue vaccine (CYD-TDV) is the only one registered worldwide [6]. However, there are several other candidate tetravalent vaccines in preclinical and clinical development, such as the TV003/TV005 (US NIH/Brazil Butantan Institute) and DENVax (Japan Takeda laboratory) [7].

Robust and reproducible production processes guarantee the quality of vaccines. The recommended quality control tests that ensure the effectiveness of the dengue tetravalent vaccine are: identity, potency and thermostability [8]. Potency, as defined by the International Council for Harmonisation (ICH), is the ability of a product to generate a specific biological activity that can be quantified [9]. The determination of CYD-TDV potency is based on a 50% cell culture infective dose assay (CCID50) essentially performed in VERO cells (African green monkey kidney epithelial cells). However, as established by WHO, the potency of tetravalent vaccines against dengue can also be determined on other cell lineages, as soon as these cells are susceptible to the vaccine virus [10].

Assays performed in VERO cells demonstrated that the CYD-TDV resulted in a balanced viral and antibody titers for the four viral serotypes of DENV [11–15]. Immunogenicity studies, however, point to unbalanced viral and antibody titers, with a strong dominance of serotype 4 (DENV-4) over the other serotypes (DENV-1, DENV-2 and DENV-3) [16–19]. Despite the historical application of VERO cells to determine infectivity of virus, those findings underlined the necessity of considering other cell lineages that would still be passive of infection, but also able to point differences in response to each DENV serotype. Therefore, we aimed to investigate the infectivity, along with the determination of potency of CYD-TDV in two different human cell lines and then to compare the results obtained with the human lines with the VERO cell assay. For that purpose, we have chosen a monocyte/macrophage like human leukemic cell lineage, THP-1, due to the fact that those cells respond to wild DENV infection, as published elsewhere. Besides, as a macrophage like cell, it is closer to the physiological situation than VERO cells, since it may act as an antigen presenting cell [20]. The second cell line elected to this study was the human embryonic kidney 293T lineage, HEK293T. That cell is widely used in virus research, because of its high susceptibility to transfections, easy genetic manipulation and permissibility to virus infection [21]. Out of those facts, we stood by the motivating hypothesis that THP-1 (differentiated THP-1 or dTHP-1) and HEK293T cells lineages, other than VERO cells, can be used to determine differences in the infectivity of each serotype of CYD-TDV. The results showed that infection of HEK293T cells was possible with all four vaccine serotypes. Furthermore, after establishing a vaccine dose-response curve in these cells, we observed that the potency accessed in HEK293T cells varied for each serotype, while no significant variation was detected in VERO cells.

Materials and methods

Cell culture

VERO cells (ATCC Cat No. CCL-81) were cultured at 37° C ± 1° C and 5% CO2 in MEM (Gibco) supplemented with 5% FBS (Hyclone, Thermo Scientific), 2 mM L-glutamine (Gibco), 26 mM NaHCO3 (Eurobio) and 100 U/mL of penicillin and 100 μg/mL of dihydrostreptomycin (Eurobio). For the infectivity assays, VERO cells were kept in the same culture medium but supplied with 10% FBS. 2% HEPES were added to the cell medium once cells were exposed to the vaccine dilutions.

The HEK293T cells were kindly donated by Prof Dr Raquel Ciuvalschi Maia from the Laboratory of Cellular and Molecular Hemato-Oncology (INCA, Rio de Janeiro, Brazil). They were cultured in DMEM (Sigma-Aldrich) supplemented with 10% FBS (Hyclone, Thermo Scientific), 2 mM L-glutamine (Gibco), 26mM NaHCO3 (Eurobio), 25 mM HEPES (Gibco), 100 U/mL of penicillin and 100 μg/mL of dihydrostreptomycin (Eurobio), at 37° C ± 1° C and 5% CO2.

THP-1 cells, obtained at Rio de Janeiro Cell Bank—BCRJ—accession number 0234, were cultured in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% FBS (Hyclone, Thermo Scientific), 26 mM NaHCO3 (Eurobio), 100 U/mL of penicillin and 100 μg/mL of dihydrostreptomycin (Eurobio), 1 mM of sodium pyruvate (Gibco), at 37° C ± 1° C and 5% CO2. For the infectivity assays, THP-1 cells were previously treated with 20 ng/mL of PMA for 48 hours to obtain macrophage-like THP-1 cells (dTHP-1), as described elsewhere [22].

Vaccine and antibodies

Commercially available dengue vaccine (Dengvaxia® from Sanofi Pasteur, France) was used in all experiments described in this manuscript. The pan-flavivirus antibody (4G2), kindly provided by BioManguinhos (FIOCRUZ, Rio de Janeiro, Brazil), was used for determining the positivity of infection at the immunofluorescence assay. Determination of vaccine dose-response and potency were performed using individually murine anti-Envelope DENV protein monoclonal antibodies (mAb) from each serotype (anti-CYD1 D2-1F1-3, Batch No. VT150630-3176; anti-CYD2 3H5-1-12 Batch No. VT170329-4270; anti-CYD3 8A1-2F12, Batch No. VT150713-3176; anto-CYD4 1H10-6-7, Batch No. VT150701-3176), kindly donated by Sanofi Pasteur, and the polyclonal goat anti-mouse IgG secondary antibody conjugated to human alkaline-phosphatase (Lot. No. E2518-PK49F; Cat. No. 1030–04; SouthernBiotech).

Immunofluorescence

HEK293T, dTHP-1 and VERO cells were seeded to the Nunc® Lab-Tek® (Thermo Scientific) cell culture 8 chamber slides in concentrations of 3x105 cells/mL, 1x106 cells/mL and 3x105 cells/ml, respectively. Then, 100 μL of dengue vaccine in 10−1 dilution was added to the chambers and the slides were incubated for 7 days at 36° C ± 1° C, 5% ± 1% CO2.

After the incubation period, culture medium was discarded and the cells were gently washed with 200 μL of PBS (Eurobio). Then, the cells were fixed with 100 μL of acetone (85% v/v; Carlo-Erba Reagents) for 2 minutes at room temperature. After fixation, the cells were gently washed three times with 100 μL of PBS and then incubated for 20 minutes at 5° C ± 3° C in 100 μL of PBS / BSA 1% (Sigma-Aldrich) for blocking. After that, cells were labeled with 60 μL of 4G2 antibody conjugated to Alexa-Fluor 647, at a 1:200 dilution in 1% PBS / BSA + 0.1% Triton X-100 solution (Sigma-Aldrich), for 1 hour at 5° C ± 3° C, under light protection. After labeling, the cells were gently washed twice with 100 μL of PBS and then the slide culture chambers were removed. Then, 10 μL of Hoechst’s dye 33258 (Sigma-Aldrich), for nucleus staining, and 5 μL of oil were added to each condition.

The slides were analyzed using a confocal microscope (Leica TCS-SP8) in 200x optical magnification. The analyzes were performed using the LAS X software.

Immunocolorymetry

To assess the infectivity of dengue vaccine, 100 μL of HEK293T and VERO cells, at a concentration of 3x105 cells/mL, were seeded in a 96-well microplate (Falcon). Then, in 10 replicates, 100 μL of the vaccine dilutions, prepared with culture medium, was added at concentrations of 10−1, 10−2 and 10−2.6, and 100 μL of culture medium was added as negative control. The plates were incubated for 7 days at 36° C ± 1° C and 5% CO2.

To determine dengue vaccine titer, 100 μL of HEK293T and VERO cells at a concentration of 3x105 cells/mL were seeded in a 96-well microplate (Falcon). Then, in 10 replicates, 100 μL of the vaccine was added to the wells in dilutions of 10−1.3 to 10−3.4, 1:2 dilution factor, for HEK293T cells, and 10−2.6 to 10−6.8, 1:4 dilution factor, for VERO cells. 100 μL of culture medium was added to negative controls. The plates were incubated for 7 days at 36° C ± 1° C and 5% CO2.

After incubation, the cells were fixed with 250 μL of acetone (85% v/v) for 15 minutes at -20° C. After fixation, the plates were left to dry and, after dryness, 100 μL of saturation buffer (PBS with 2.5% skimmilk (Difco™ BD), 0.5% Triton X-100 (Sigma-Aldrich), 0.05% Tween-20 (Merck)) was distributed to the wells, followed by a 30 minutes incubation at 36° C ± 1° C. After that, saturation buffer was removed and 50 μL of primary anti-DENV-1, anti-DENV-2, anti-DENV-3, anti-DENV-4 diluted to a concentration of 1/5000 in saturation buffer, was added to the wells and plates were incubated for 45 minutes at 36° C ± 1° C. Then, the plates were washed twice with washing buffer (PBS with 0.05% Tween-20) and 50 μL of secondary antibody conjugated to alkaline-phosphatase, diluted to a concentration of 1/1000 in saturation buffer, was added to plates, followed by incubation for 45 minutes at 36° C ± 1° C. After incubation, the plates were washed twice and 50 μL of NBT/BCIP substrate (Sigma-Aldrich) was distributed to the wells, followed by incubation for 60–90 minutes at room temperature under light protection, while the pigmentation developed. After that, the substrate was removed, and the plates were washed twice with distilled water and left to dry.

The wells were evaluated for the presence of staining, using an inverted optical microscope (IMT, Olympus), at a magnification of 100X, indicating infection, for each dilution used. The CCID50/dose values in log10 were used to determine viral titers.

Statistical analysis

Data is expressed as mean ± SD or SEM and evaluated with GraphPad prism software v5.1 (La Jolla, USA). To determine the linearity of the results found in the dose-response curve, a linear regression was performed for both dilutions used in VERO and HEK293T cells.

Viral titers were calculated by CombiStats® v5.0 program (European Directorate for the Quality of Medicines and HealthCare/Council of Europe, Strasbourg, France) using the Spearman & Karber method. Titers are expressed in log10 CCID50 per dose. To verify statistical differences in the potency of the different experimental groups, we applied the two-way ANOVA test with a post Bonferroni test. Statistical significant differences were defined with probability (p) values inferior or equal to 0.05.

Results

Infectivity of different human cell lines with DENV vaccine

To investigate whether a human cell line would be susceptible to infection by DENV vaccine, we chose two human derived cell lines, dTHP-1 and HEK293T. After 7 days of incubation with 10−1 DENV vaccine dilution, we performed immunofluorescence assay by confocal microscopy. VERO cells, widely used in infectivity tests, functioned as a positive control.

As expected, we observed viral infectivity in the cytoplasm of VERO cells, as determined by the positive staining with pan-Flavivirus antibody 4G2 (Fig 1B). The fluorescence intensity increased considerably in infected VERO cells once compared with the controls (Figs 1C and S1). Regarding HEK293T cell line, a clear staining of 4G2 antibody detected in the cytoplasm of these cells, determined their susceptibility to infection with DENV vaccine, under our experimental conditions (Fig 1E). In contrast to the scattered fluorescence in the cytoplasm of VERO cells, the fluorescence signal detected in infected HEK293T cells was mostly evidenced in the perinuclear region (Figs 1F and S2). In the differentiated THP-1 cells, however, we did not detect any staining of 4G2 antibody as evidenced in both confocal image and graph analysis (Figs 1H, 1I and S3). As negative controls, cells were also stained with the 4G2 antibody in the absence of DENV vaccine and, as expected, showed no detectable signal (Fig 1A, 1D and 1G).

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

Fig 1

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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.

Quantification of infection of DENV vaccine in VERO cells and in HEK293T cells

After determining the susceptibility of HEK293T to DENV vaccine, we tested whether these cells would respond to the same infectivity assay used in VERO cells to quantify infection and determine potency of DENV vaccine. For this purpose, we performed the infection assay with a 10−1 vaccine dilution and determined infectivity using a monoclonal antibody specific for dengue virus serotype 2 (CYD-2). VERO cell, positive control, and HEK293T infection assay was run together and precisely under the same conditions.

Both cells showed positivity to CYD-2 antibody, indicating that infection of HEK293T cells with at least vaccine virus serotype 2 was measurable using immunocolorimetric assay. The distinctive dark staining of infected cells (Fig 2B and 2D), when compared to non-infected cell controls, evidenced this finding (Fig 2A and 2C).

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

Fig 2

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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.

The next step was to obtain an optimal vaccine dilution curve, to confirm the feasibility of HEK293T cells to respond to different DENV vaccine dilutions. HEK293T cells were exposed to different DENV vaccine dilutions from 10−1.3 to 10−3.4, following a dilution factor of 1:2, whereas VERO cells were subjected to dilutions from 10−2.6 to 10−6.8, with a dilution factor of 1:4, during 7 days of incubation. In this set of experiments, specific monoclonal antibodies for each serotype determined separately the degree of infectivity of the DENV vaccine both in VERO and HEK293T cells. We observed infection of HEK293T cells only at the most concentrated dilutions of the vaccine, as determined by the presence of dark color after incubation with monoclonal antibody specific to each dengue serotype (positive wells). Even though, the DENV vaccine dilutions used to infect VERO cells were less concentrated when compared to the ones used to infect HEK293T cells, VERO cells confirmed their higher susceptibility to the infection with this vaccine under our experimental conditions.

The dose-response curves and the linear coefficient of the regression equation of infectious responses in VERO and HEK293T cells for each serotype are shown in Fig 3 (serotype 1 R2VERO = 0.887 and R2HEK293T = 0.793, Fig 3A; serotype 2 R2VERO = 0.883 and R2HEK293T = 0.801, Fig 3B; serotype 3 R2VERO = 0.903 and R2HEK293T = 0.767, Fig 3C and serotype 4 R2VERO = 0.913 and R2HEK293T = 0.917, Fig 3D). All regression results showed over 75 percent of probability, thus supporting the idea that a dose curve response for the DENV vaccine in HEK293T cells is feasible.

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

Fig 3

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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 (R2VERO = 0.887 and R2HEK293T = 0.793), serotype 2 (R2VERO = 0.883 and R2HEK293T = 0.801), serotype 3 (R2VERO = 0.903 and R2HEK293T = 0.767) and serotype 4 (R2VERO = 0.913 and R2HEK293T = 0.917). Data presented as mean ± SEM. n = 4.

Potency of the 4 different DENV vaccine serotypes evaluated in HEK293T and VERO cells

One important feature regarding the assessment of the quality of a vaccine, whether in the developmental or in the control phase, is to verify the potency. Therefore, the final step of this experimental set up was to calculate the estimated potencies among the 4 different serotypes in the same cell line and compare the results obtained in VERO cells with the ones in HEK293T cells. The result of the calculated potency, illustrated in Fig 4 in log10 CCID50/dose, in HEK293T cells for each serotype was 2.49 ± 0.19 (CYD-1), 3.01 ± 0.26 (CYD-2), 2.6 ± 0.27 (CYD-3) and 3.39 ± 0.46 (CYD-4) and in VERO cells, the estimated potencies were 5.49 ± 0.33 (CYD-1), 5.38 ± 0.28 (CYD-2), 5.34 ± 0.47 (CYD-3) and 5.42 ± 0.37 (CYD-4). Interestingly, we observed a different behavior on the potency estimated for each serotype using VERO or HEK293T cells. Whereas in VERO cells the estimated potency was approximately the same for each serotype, in HEK293T cells the serotypes showed different results. Regarding serotype 4, the potency showed a significantly higher result when compared to the ones detected for serotypes 1 and 3 in HEK293T cells (p<0.01). Additionally, the serotype 2 showed a slightly higher potency result when compared to the one obtained with serotype 1 and 3, but this tendency wasn´t statistically significant (Fig 4).

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

Fig 4

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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.

Comparisons of the estimated potencies for each serotype in VERO and HEK293T cell lines demonstrated that DENV vaccine achieved a significant higher potency result in the usual VERO cell model once compared to the HEK293T cell model proposed here (p<0.0001; Fig 4).

Discussion

In this work, we investigated the feasibility of dengue vaccine to infect two human cell lines and subsequently the viability of these cells in estimating each serotype potency as an alternative or a complement to the VERO cells assay.

VERO cells are widely used in the development and control of vaccines due to their low cost and easy handling, but above all features one should stress that they are highly susceptible to infection by most viruses [23]. As matter of fact, once the addressed problem regards virus titers, to sought for a highly permissive cell is certainly a necessity. However, since this high permissibility may possibly be associated to the absence of an effective antiviral response, this ought to be considered before drawing conclusions from data obtained solely with those cells. In fact, VERO cells lack antiviral response mediated by type 1 interferons [23,24]. If this response is by some means important for the experimental approach, the use of VERO cells alone might lead to misinterpretation of the results.

In the context of dengue, VERO cells are used in the production of attenuated virus tetravalent vaccines due to the high susceptibility to DENV [25,26]. Despite its widespread use, the high susceptibility to a heterologous virus vaccine may lead to overestimated infectivity and therefore give as an outcome not necessarily tangible potency value of each serotype, which may turn out not to be consistent to observations in vivo [15–17]. To test our hypothesis, we evaluated the responsiveness of HEK293T and THP-1 cell lines to the attenuated viral vaccines by measuring their susceptibility to infection and analyzing virus vaccine serotypes individually.

HEK293T cells have been used in the research, production and control of viral vaccines, mainly viral vector and nucleic acid vaccines, for example, the COVID-19 and Influenza vaccines, respectively [27–29]. Regarding dengue, HEK293T cells were shown to be susceptible to infection by wild DENV and other members of the Flaviviridae family [30]. Both the report that HEK293T is used in control trials of virus vaccines and that it is susceptible to wild dengue virus, led us to consider these cells as a candidate for our proposal. In fact, in the present study, we showed that the vaccine viruses constituting the tetravalent vaccine against dengue (Dengvaxia®) yield infection in these cells, under our experimental conditions, as proven by the observation of viral particles in cell cytoplasm. To our knowledge, this is the first report showing the susceptibility of these cells to DENV vaccine. In many aspects, this is an interesting finding, since it also indicates potential novel paths for studying mechanisms of action of Dengvaxia®. But for our purpose in this study, we needed to comparatively evaluate the use of HEK293T and VERO cells to measure potency. The determination of vaccine potency consists of evaluating the response of the vaccine product to a given biological substrate in order to estimate the potential of a vaccine to generate the desired final effect [31]. For attenuated vaccines, potency is generally expressed in terms of infectious units contained in a dose, as established in clinical studies [32]. According to WHO, for tetravalent vaccines against dengue, potency must be evaluated in terms of individual titers of each of the four serotypes in viral titration assays by plaque, CCID50 or immunofocus in culture of VERO cells or other sensitive cells [32]. Since VERO cells are the golden standard in this field, the sought of another cell line might be performed in comparison with them.

Our results demonstrated that the HEK293T cell line is permissive to infection by the Dengvaxia® vaccine, even though, as expected, less susceptible than the VERO cell line. Hence, this study showed the viability of HEK293T cells as an alternative or complementary cell line for determining infection of DENV vaccine in different concentrations, using the immunoassay recommended by the vaccine producer. As a matter of fact, the dose-dependent curve obtained with these cells was found to be similar to the one observed with VERO cells and, therefore, this model could be recommended as an additional cell model.

Moreover, by using HEK293T cells it was possible to determine potency values for each of the serotypes present in the vaccine, in CCID50 values, as recommended by WHO. Interestingly enough, HEK293T cells responded differently for each virus vaccine serotype with respect to their potency levels, as higher values were obtained associated to serotype 4 when compared to the others. VERO cells, in contrast, showed no significant differences between the virus vaccine serotypes regarding potency levels. In both pre- and clinical trials concerning the immunogenicity to the vaccine, a dominance of antibodies against the serotype 4 in comparison to the others was observed in the serum samples of vaccinated non-human primates and volunteers [16,17], despite the tests of potency in VERO cells had shown no differences between the serotypes. Some studies, investigating the virus vaccine viremia of Dengvaxia® in individuals serum negative, showed a predominance for the CYD-4 and highest titer of DENV-4 neutralizing antibodies after vaccination. Regarding the vaccine TV003/TV005 from US NIH/Butantan Institute, consisted of a chimera of DENV-2 attenuated virus (backbone DENV-4) and attenuated DENV-1, DENV-3 and DENV-4, different formulations were tested in an attempt to balance the serotypes antibody titers. Thus, this study demonstrated that balanced infectivity correlated with the production of homotypic antibody [33]. Another vaccine approach developed by Takeda, used DENV-2 as the backbone virus to build a chimeric tetravalent vaccine. Again, differences in seroconversion were detected, but in this case, DENV-2 immunogenicity was higher once compared to the other serotypes. Alteration of concentrations of the vaccine virus like particles lead to a shift on the response. However, the increase of one specific serotype concentration–DENV-4 –with respect to the others resulted in a decrease on the seroconversion of DENV-1 serotype [34]. Yet, though the concentrations play an important role on seroconversion, other factors like cellular response and the virus strategy in the vaccine formulations have also to be explored [35].

Indeed, it might be helpful to use a cell that may better predict infectivity fluctuations and, consequently participate to the development of more uniform vaccines regarding their immunogenicity. Even though our work is a first effort on this token, it already shows that HEK293T might be interesting for testing heterologous DENV vaccines, for their ability to measure differences in serotypes infectivity. Whether this cell could also be a tool for studying cellular responses remains an open subject.

Our hypothesis was also explored by testing another cell line, the THP-1 cells. Since these cells can be differentiated to macrophages and macrophages are target cells on a natural DENV infection [36], we wanted to verify whether these cells would be permissive to vaccine virus as well. In fact, the THP-1 cell line has been widely used to study the mechanisms of DENV infection [37–39]. We observed no cellular susceptibility of differentiated THP-1 to infection by the CYD-TDV tetravalent vaccine at the time and vaccine concentrations studied. The absence of infection might be related to viral attenuation, which in turn leads to a loss in the virulence of vaccine viruses, added to the resistance of these cells to infections by DENV [34–41]. Herein, we concluded that THP-1 cells might be very useful for mechanism elucidation purposes rather than for potency assays.

The importance of developing vaccines against dengue capable of generating a balanced and effective immune response against the four viral serotypes, indicates the necessity of applying more sensitive in vitro and in vivo models during the establishment of those vaccines. In our work, we demonstrated the feasibility of CYD-TDV to infect the HEK293T cell line and that potency could be determined. Therefore, we considered the HEK293T cell line as a promising candidate in the development of potency assay and identity of tetravalent vaccines against dengue. As far as the simultaneous observation of different potencies for each serotype might be very useful in the development and production of heterologous dengue vaccine, our results reinforce the eligibility of HEK293T cell line as the basis for forthcoming assays.

Supporting information

S1 Fig. Confocal immunofluorescence graph of VERO cells infected with dengue vaccine.

Cells were stained with anti-4G2 (pan-flavivirus antibody, pink fluorescence) and PAFI (DNA staining, blue fluorescence). Representative graph of fluorescence intensity (y-axis) against the distance in micrometer (x-axis) of infected VERO cells, obtained from confocal representative image (confocal microscope Leica TCS-SP8 at 200X magnification). n = 2.

(TIF)

Click here for additional data file. (126.2KB, tif)
S2 Fig. Confocal immunofluorescence graph of HEK293T cells infected with dengue vaccine.

Cells were stained with anti-4G2 (pan-flavivirus antibody, pink fluorescence) and PAFI (DNA staining, blue fluorescence). Representative graph of fluorescence intensity (y-axis) against the distance in micrometer (x-axis) of infected HEK293T cells, obtained from confocal representative image (confocal microscope Leica TCS-SP8 at 200X magnification). n = 2.

(TIF)

Click here for additional data file. (141.1KB, tif)
S3 Fig. Confocal immunofluorescence graph of PMA-treated THP-1 cells infected with dengue vaccine.

Cells were stained with anti-4G2 (pan-flavivirus antibody, pink fluorescence) and PAFI (DNA staining, blue fluorescence). Representative graph of fluorescence intensity (y-axis) against the distance in micrometer (x-axis) of infected PMA-treated THP-1 cells, obtained from confocal representative image (confocal microscope Leica TCS-SP8 at 200X magnification). n = 2.

(TIF)

Click here for additional data file. (114.2KB, tif)
S1 Data

(XLSX)

Click here for additional data file. (64.2KB, xlsx)

Acknowledgments

We are grateful to Dr Arnon Jurberg, from Universidade Estácio de Sá (UNESA), for his assistance with the immunofluorescence analyses. We also thank Dr Raquel Ciuvalschi Maia, from Instituto Nacional de Câncer (INCA), and Diagnostic Technology Laboratory (Lated/Biomanguinhos, Oswaldo Cruz Foundation), for the donation of HEK293T cell line and antibodies, respectively. In frame of our strong collaboration, we thank Sanofi Pasteur for kindly providing specific monoclonal antibodies against dengue serotypes.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported by INCQS and IOC, both FIOCRUZ. ACMAN and RFC received institutional grant. This work was part of the doctoral thesis of RFC at the Post-Graduation Program in Sanitary Surveillance. The funders had no role in the study.

References

  • 1.WHO. Dengue. 2016. [cited 2021 April 09]. Available from: https://www.who.int/immunization/diseases/dengue/en/. [Google Scholar]
  • 2.WHO. Dengue and Severe dengue. 2021. [cited 2021 April 09]. Available from: https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue. [Google Scholar]
  • 3.Hadinegoro SRS. The revised WHO dengue case classification: does the system need to be modified? Paediatrics and International Child Health. 2012; 32:sup1: 33–38. doi: 10.1179/2046904712Z.00000000052 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Reiter P. Yellow fever and dengue: a threat to Europe? Euro Surveill. 2010; 15(10):pii:19509. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19509. [PubMed] [Google Scholar]
  • 5.Murphy BR, Whitehead SS. Immune response to Dengue virus and prospects for a vaccine. Annu Rev Immunol. 2011; 29:587–619. doi: 10.1146/annurev-immunol-031210-101315 [DOI] [PubMed] [Google Scholar]
  • 6.WHO. Vaccines and Diseases. Immunization, Vaccines and Biologicals. Revised SAGE recommendation on use of dengue vaccine. 2018. [cited 2019 July 30]. Available from: https://www.who.int/immunization/diseases/dengue/revised_SAGE_recommendations_dengue_vaccines_apr2018/en/. doi: 10.1016/j.vaccine.2018.09.063 [DOI] [PubMed] [Google Scholar]
  • 7.Swaminathan S, Khanna N. Dengue vaccine development: Global and Indian scenarios. Int J Infect Dis. 2019; 84: S80–S86. doi: 10.1016/j.ijid.2019.01.029 [DOI] [PubMed] [Google Scholar]
  • 8.WHO. Technical Report Series, No. 932. Guidelines on the quality, safety and efficacy of dengue tetravalent vaccines (live, attenuated). Proposed replacement of Annex 1. 2011. [cited 2016 June 28]. Available from: http://www.who.int/biologicals/vaccines/Dengue_DB_5_April_2012_clean.pdf. [Google Scholar]
  • 9.International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use–ICH. ICH-Q6B Test procedures and acceptance criteria for biotechnological/biological products. Geneva: ICH; 1999. [Google Scholar]
  • 10.WHO. Technical Report Series, No. 932. Guidelines for the production and quality control of candidate tetravalent dengue virus vaccines (live). Geneva, 2006. [cited 2016 May 17]. Available from: http://www.who.int/biologicals/publications/trs/areas/vaccines/dengue/TRS932Annex%201_Dengue%20virus%20vacc%20live.pdf. [Google Scholar]
  • 11.Poo J, Galan F, Forrat R, Zambrano B, Lang J, Dayan GH. Live-attenuated tetravalent dengue vaccine in dengue-naive children, adolescents, and adults in Mexico City randomized controlled Phase 1 trial of safety and immunogenicity. Pediatr Infect Dis J. 2011; 30(1): E9–E17. doi: 10.1097/INF.0b013e3181fe05af [DOI] [PubMed] [Google Scholar]
  • 12.Morrison D, Legg TJ, Billings CW, Forrat R, Yoksan S, Lang J. A novel tetravalent dengue vaccine is well tolerated and immunogenic against all 4 serotypes in flavivirus-naive adults. J Infect Dis. 2010; 201:370–377. doi: 10.1086/649916 [DOI] [PubMed] [Google Scholar]
  • 13.Capeding RZ, Luna IA, Bomasang E, Lupisan S, Lang J, Forrat R, et al. Live-attenuated, tetravalent dengue vaccine in children, adolescents and adults in a dengue endemic country: randomized controlled phase I trial in the Philippines. Vaccine. 2011; 29(22):3863–72. doi: 10.1016/j.vaccine.2011.03.057 [DOI] [PubMed] [Google Scholar]
  • 14.Tran NH, Luong CQ, Vu TQH, Forrat R, Lang J, Vu QD, et al. Safety and immunogenicity of recombinant, live attenuated tetravalent dengue vaccine (CYD-TDV) in healthy Vietnamese adults and children. J Vaccines Vaccin. 2012; 3:1–7. [Google Scholar]
  • 15.Lecouturier V, Berry C, Saulnier A, Naville S, Manin C, Girerd-Chambaz Y, et al. Characterization of recombinant yellow fever-dengue vaccine viruses with human monoclonal antibodies targeting key conformational epitopes. Vaccine. 2018; 37(32): 4601–09. doi: 10.1016/j.vaccine.2018.04.065 [DOI] [PubMed] [Google Scholar]
  • 16.Henein S, Swanstrom J, Byers AM, Moser JM, Shaik SF, Bonaparte M, et al. Dissecting antibodies induced by a chimeric yellow fever-dengue, live-attenuated, tetravalent dengue vaccine (CYD-TDV) in naive and dengue exposed individuals. J Infect Dis. 2017; 215(3): 351–358. doi: 10.1093/infdis/jiw576 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Guy B. Immunogenicity of Sanofi Pasteur tetravalent dengue vaccine. J Clin Virol. 2009; 46(Suppl 2):S16–S19. doi: 10.1016/S1386-6532(09)70290-2 [DOI] [PubMed] [Google Scholar]
  • 18.Villar L, Dayan GH, Arredondo-García JL, Rivera DM, Cunha R, Deseda C, et al. Efficacy of a tetravalent dengue vaccine in children in Latin America. N Engl J Med. 2015; 372:113–123. doi: 10.1056/NEJMoa1411037 [DOI] [PubMed] [Google Scholar]
  • 19.Capeding MR, Tran NH, Hadinegoro SR, Ismail HI, Chotpitayasunondh T, Chua MN, et al. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet. 2014; 384(9951):1358–1365. doi: 10.1016/S0140-6736(14)61060-6 [DOI] [PubMed] [Google Scholar]
  • 20.Pascapurnama DN, Labayo HKM, Dapat I, Nagarajegowda DD, Zhao J, Zhang J, et al. Induction of Osteopontin by Dengue Virus-3 Infection in THP-1 Cells: Inhibition of the Synthesis by Brefelamide and Its Derivative. Front. Microbiol. 2017. 8:521. doi: 10.3389/fmicb.2017.00521 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Rungruengphol C, Jaresitthikunchai J, Wikan N, Phaonakrop N, Keadsanti S, Yoksan S, et al. Evidence of plasticity in the dengue virus: Host cell interaction. Microb Pathog. 2015; 86: 18–25. doi: 10.1016/j.micpath.2015.07.003 [DOI] [PubMed] [Google Scholar]
  • 22.Zhou L, Shen LH, Hu LH, Ge H, Pu J, Chai DJ, et al. Retinoid X receptor agonists inhibit phorbol-12-myristate-13-acetate (PMA)-induced differentiation of monocytic THP-1 cells into macrophages. Mol Cell Biochem. 2010; 335(1–2):283–89. doi: 10.1007/s11010-009-0278-z [DOI] [PubMed] [Google Scholar]
  • 23.Osada N, Kohara A, Yamaji T, Hirayama N, Kasai F, Sekizuka T, et al. The genome landscape of the african green monkey kidney-derived vero cell line. DNA Res. 2014; 21(6): 673–683. doi: 10.1093/dnares/dsu029 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Desmyter J, Melnick JL, Rawls WE. Defectiveness of interferon production and of rubella virus interference in a line of African green monkey kidney cells (Vero). J Virol. 1968; 2(10):955–961. doi: 10.1128/JVI.2.10.955-961.1968 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Precioso AR, Palacios R, Thome B, Mondini G, Braga P, Kalil J. Clinical evaluation strategies for a live attenuated tetravalent dengue vaccine. Vaccine. 2015; 33(50):7121–5. doi: 10.1016/j.vaccine.2015.09.105 [DOI] [PubMed] [Google Scholar]
  • 26.Deng SQ, Yang X, Wei Y, Chen JT, Wang XJ, Peng HJ. A Review on Dengue Vaccine Development. Vaccines (Basel). 2020; 8(1):63–76. doi: 10.3390/vaccines8010063 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Almuqrin A, Davidson AD, Williamson MK, Lewis PA, Heesom KJ, Morris S, et al. SARS-Cov-2 vaccine ChAdOx1 nCov19 infection of human cell lines reveals low levels of viral backbone gene transcription alongside very high levels of SARS-Cov-2 S glycoprotein gene transcription. Genome Med. 2021; 13(1):43. doi: 10.1186/s13073-021-00859-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Yang J, Wang W, Chen Z, Lu S, Yang F, Bi Z, et al. A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity. Nature. 2020; 586: 572–7. doi: 10.1038/s41586-020-2599-8 [DOI] [PubMed] [Google Scholar]
  • 29.Milián E, Julien T, Biaggio R, Venereo-Sanchez A, Montes J, Manceur AP, et al. Accelerated mass production of influenza virus seed stocks in HEK-293 suspension cell cultures by reverse genetics. Vaccine. 2017; 35(26):3423–30. doi: 10.1016/j.vaccine.2017.04.065 [DOI] [PubMed] [Google Scholar]
  • 30.Jitoboam K, Phaonakrop N, Libsittikul S, Thepparit C, Roytrakul S, Smith DR. Actin interacts with Dengue Virus 2 and 4 Envelope Proteins. PLOS One. 2016; 11(3): 1–18. doi: 10.1371/journal.pone.0151951 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Whitehead SS. Development of TV003/TV005, a single dose, highly immunogenic live attenuated dengue vaccine; what makes this vaccine different from the Sanofi-Pasteur CYDTM vaccine? Expert Rev Vaccines. 2016; 15:509–17. doi: 10.1586/14760584.2016.1115727 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Osorio JE, Wallace D, Stinchcomb DT. A recombinant, chimeric tetravalent dengue vaccine candidate based on a dengue virus serotype 2 backbone. Expert Rev Vaccines. 2016; 15: 497–508. doi: 10.1586/14760584.2016.1128328 [DOI] [PubMed] [Google Scholar]
  • 33.Weiskopf D, Angelo MA, Bangs DJ, Sidney J, Paul S, Peters B, et al. The human CD8+ T cell responses induced by a live attenuated tetravalent dengue vaccine are directed against highly conserved epitopes. J Virol. 2015;89(1):120–8. doi: 10.1128/JVI.02129-14 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Metz B, Hendriksen CFM, Jiskoot W, Kersten GFA. Reduction of animal use in human vaccine quality control: opportunities and problems. Vaccine. 2002; 20(19–20): 2411–30. doi: 10.1016/s0264-410x(02)00192-5 [DOI] [PubMed] [Google Scholar]
  • 35.WHO. Technical Report Series, No. 979. Guidelines on the quality, safety and efficacy of dengue tetravalent vaccines (live, attenuated). Replacement of Annex 1 of WHO Technical Report Series, No. 932. 2013. Available from: https://www.who.int/biologicals/expert_committee/TRS_979_62nd_report.pdf. [Google Scholar]
  • 36.Paes MV, Lenzi HL, Nogueira ACM, Nuovo GJ, Pinhão AT, Mota EM, et al. Hepatic damage associated with dengue-2 virus replication in liver cells of BALB/c mice. Laboratory investigation. 2009; 89(10): 1140–1151. doi: 10.1038/labinvest.2009.83 [DOI] [PubMed] [Google Scholar]
  • 37.Jiang L, Sun Q. The Role of Autophagy-Mediated Dengue Virus Antibody-Dependent Enhancement Infection of THP-1 Cells. Intervirology. 2020; 63(1–6):57–65. doi: 10.1159/000511420 [DOI] [PubMed] [Google Scholar]
  • 38.Starr T, Bauler TJ, Malik-Kale P, Steele-Mortimer O. The phorbol 12-myristate-13-acetate differentiation protocol is critical to the interaction of THP-1 macrophages with Salmonella Typhimurium. PLoS ONE. 2018; 13(3):e0193601. doi: 10.1371/journal.pone.0193601 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Jin X, Block OT, Rose R, Schlesinger J. Dengue vaccine development and dengue viral neutralization and enhancement assays. Antivir Ther. 2009; 14(6):739–49. doi: 10.3851/IMP1288 [DOI] [PubMed] [Google Scholar]
  • 40.Diamond MS, Edgil D, Roberts TG, Lu B, Harris E. Infection of human cells by dengue virus is modulated by different cell types and viral strains. J Virol. 2000; 74(17):7814–23. doi: 10.1128/jvi.74.17.7814-7823.2000 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Chanput W, Mes JJ, Wichers HJ. THP-1 cell line: An in vitro cell model for immune modulation approach. International Immunopharmacology. 2014; 23(1): 37–45. doi: 10.1016/j.intimp.2014.08.002 [DOI] [PubMed] [Google Scholar]

Decision Letter 0

Henning Ulrich

11 Feb 2022

PONE-D-21-33336Short Communication: Feasibility of dengue vaccine to infect different human cell lines: an alternative potency test using HEK293T cellsPLOS ONE

Dear Dr. Martins de  Almeida Nogueira,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Comments of the Academic Editor:The rationale of this study needs to be clarified and the translational applications of this manuscript discussed in detail. 

Further points:

Stock solution for dilution in Figure 3 needs to be defined in the legend for Figure 3.

Figure needs to be high resolution and  have readable X- and Y- axis units (Figure 1)

Figure legends need to report number of n.

More details are needed regarding statistical analysis.

English writing style may be improved.

Please submit your revised manuscript by Mar 28 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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PLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: I Don't Know

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have submitted a manuscript describing the investigation of alternative human cell lines for dengue fever vaccine testing. This is a pure cell culture experiment without fresh human cells. Unfortunately, characteristics of the cell lines used are presented only incompletely and scattered throughout the paper, making the rationale behind the work very slow to emerge.

The introduction clearly shows that there is a need for tools to study new vaccines. Appropriate literature is used to explain the approach. The importance of the antibody response is hardly presented, here prior knowledge of the reader is required.

Materials and methods and results are well described. In the discussion, the authors conclude that the HEK293T cell is suitable as a model.

Reviewer #2: The present study provided interesting data of the infectivity and potency of the 4 serotypes used to formulate the DengVaxia vaccine in HEK293T cells when compared to Vero cells, though the group also shows the lack of infectivity of these serotypes on THP-1 cells differentiated to macrophages by PMA.

In fact, the more relevant results is related to the potency studies using HEK293T cells when compared to Vero cells that lacks the Type-1 interferon associated responses. As expected, the potency of the 4 serotypes were similar and higher in Vero cells when compared to HEK293T cells. Interestingly, the group could detect differences in HEK293T cells potency within the four serotypes.

However, the group should discuss in deep how this finding could be translated in a more adequate formulation that would be more close to what could be the in the clinical use of the vaccine, a equilibrated and balanced response against all the four serotypes. Based on the potency results, is it possible to predict a better formulation that would result in a more balanced response against the four serotypes? What would be the related amount of each serotype in vaccine formulation based on the HEK293T potency studies? Of course this can be validated by non-human primate immunization studies showing or not a equilibrated response against all the four serotypes in the new formulation based on the the potency studies on HEK293T cells in contrast to a more pronounced antibody production against the serotype 4 as found in the current vaccine formulation that was based on the potency studies based on Vero cells that presents a clear bias, as the result of the lack of Type-1 Interferon responses in these cells.

**********

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Reviewer #2: No

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PLoS One. 2022 May 6;17(5):e0267653. doi: 10.1371/journal.pone.0267653.r002

Author response to Decision Letter 0

21 Mar 2022

Response to the reviewers

PONE- D -21-33336

Comments of the Academic Editor and Reviewers

All the changes are highlighted in yellow in the MS file named “Revised manuscript with Track changes”. The pages and lines referred in the answers below are in that file to find.

1) The rationale of this study needs to be clarified and the translational applications of this manuscript discussed in detail. Answer: We added in the introduction part of the MS more to the rational of the work also regarding the cell lines used in the study (pg 4 and 5 Lines 89-99). In that section 2 new references are cited (refs number 20 and 21). In the discussion section, we considered the translational application of our findings, as suggested by both the Editor and the Reviewer 2. To this matter we added 3 new references (numbers 31, 32 and 33 ) and enhanced so the discussion (pg 18 -19 Lines 411-435).

2) Stock solution for dilution in Figure 3 needs to be defined in the legend for Figure 3. Answer: It is now added to the legend of Figure 3, pg 13.

3) Figure needs to be high resolution and have readable X- and Y- axis units (Figure 1) Answer: Figure 1 resolution is 300 dpi and the X and Y axis of Figure 1 C, F and I was added. That figure normal size is too big to fit in, so we added the scale manually and attached the original graph (Fig 1C ; Fig 1F; Fig 1I) as “Supporting Information Files” .

4) Figure legends need to report number of n. Answer: All legends were revised, and n number was added when missing.

5) More details are needed regarding statistical analysis. Answer: We wrote the Statistical Analysis again (pg 9 lines 203-213).

6) English writing style may be improved. Answer: We revised the MS as to improve the English style. Some examples listed below: Pg 4 Line 80 and 81 was “point to an unbalanced in the viral and antibody titers produced” changed to “point to unbalanced viral and antibody titers”; Pg 16 Lines 361 was “ can super estimate infectivity and therefore give a not necessarily tangible potency value of each serotype” changed to “ may lead to overestimated infectivity and therefore give as an outcome not necessarily tangible potency value of each serotype”; Pg 18 line 397 was “ Thus” changed to “ As a matter of fact” same Pg line 400 was “Moreover, using HEK293T” changed to “ Moreover, by using …”: Pg 18 line 403 was “ vaccine serotype regarding…” changed to “ vaccine serotype with respect to …”; same page line 410 “ VERO cells showing no …” changed to “VERO cells had shown no…”

7) In Acknowledgments we removed the statement “ This work is part of ….” as recommended, since it is already at the Funding Statement section.

Reviewers' comments:

5. Review Comments to the Author

Reviewer #1: The authors have submitted a manuscript describing the investigation of alternative human cell lines for dengue fever vaccine testing. This is a pure cell culture experiment without fresh human cells. Unfortunately, characteristics of the cell lines used are presented only incompletely and scattered throughout the paper, making the rationale behind the work very slow to emerge.

The introduction clearly shows that there is a need for tools to study new vaccines. Appropriate literature is used to explain the approach. The importance of the antibody response is hardly presented, here prior knowledge of the reader is required.

Materials and methods and results are well described. In the discussion, the authors conclude that the HEK293T cell is suitable as a model.

Answer: To address the comment that the “characteristics of the cell lines used are presented only incompletely and scattered throughout the paper, making the rationale behind the work very slow to emerge”, we added in the Introduction a paragraph where we deeper characterized the cells (pg4 and 5 Lines 89-99) and we also included new references (refs number 20 and 21).

Reviewer #2: The present study provided interesting data of the infectivity and potency of the 4 serotypes used to formulate the DengVaxia vaccine in HEK293T cells when compared to Vero cells, though the group also shows the lack of infectivity of these serotypes on THP-1 cells differentiated to macrophages by PMA.

In fact, the more relevant results is related to the potency studies using HEK293T cells when compared to Vero cells that lacks the Type-1 interferon associated responses. As expected, the potency of the 4 serotypes were similar and higher in Vero cells when compared to HEK293T cells. Interestingly, the group could detect differences in HEK293T cells potency within the four serotypes.

However, the group should discuss in deep how this finding could be translated in a more adequate formulation that would be more close to what could be the in the clinical use of the vaccine, a equilibrated and balanced response against all the four serotypes. Based on the potency results, is it possible to predict a better formulation that would result in a more balanced response against the four serotypes? What would be the related amount of each serotype in vaccine formulation based on the HEK293T potency studies? Of course this can be validated by non-human primate immunization studies showing or not a equilibrated response against all the four serotypes in the new formulation based on the the potency studies on HEK293T cells in contrast to a more pronounced antibody production against the serotype 4 as found in the current vaccine formulation that was based on the potency studies based on Vero cells that presents a clear bias, as the result of the lack of Type-1 Interferon responses in these cells.

Answer: In order to properly address the comment “However, the group should discuss in deep how this finding could be translated in a more adequate formulation….” , we added to the discussion 3 new references (Numbers 31, 32 and 33) on the theme and improved our discussion, as described in Pg 18 and 19 Lines 411-435. “Some studies, investigating the virus vaccine viremia of Dengvaxia® in individuals serum negative, showed a predominance for the CYD-4 and highest titer of DENV-4 neutralizing antibodies after vaccination. Regarding the vaccine TV003/TV005 from US NIH/Butantan Institute, consisted of a chimera of DENV-2 attenuated virus (backbone DENV-4) and attenuated DENV-1, DENV-3 and DENV-4, different formulations were tested in an attempt to balance the serotypes antibody titers. Thus, this study demonstrated that balanced infectivity correlated with the production of homotypic antibody [33]. Another vaccine approach developed by Takeda, used DENV-2 as the backbone virus to build a chimeric tetravalent vaccine. Again, differences in seroconversion were detected, but in this case, DENV-2 immunogenicity was higher once compared to the other serotypes. Alteration of concentrations of the vaccine virus like particles lead to a shift on the response. However, the increase of one specific serotype concentration – DENV-4 – with respect to the others resulted in a decrease on the seroconversion of DENV-1 serotype [34]. Yet, though the concentrations play an important role on seroconversion, other factors like cellular response and the virus strategy in the vaccine formulations have also to be explored [35].

Indeed, it might be helpful to use a cell that may better predict infectivity fluctuations and, consequently participate to the development of more uniform vaccines regarding their immunogenicity. Even though our work is a first effort on this token, it already shows that HEK293T might be interesting for testing heterologous DENV vaccines, for their ability to measure differences in serotypes infectivity. Whether this cell could also be a tool for studying cellular responses remains an open subject.”

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Decision Letter 1

Henning Ulrich

13 Apr 2022

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

PONE-D-21-33336R1

Dear Dr. Martins de Almeida Nogueira,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The authors show that HEK293T cell line a promising candidate in the development of potency assay and to identify vaccines against Dengue.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

Acceptance letter

Henning Ulrich

29 Apr 2022

PONE-D-21-33336R1

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

Dear Dr. Martins de Almeida Nogueira:

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on behalf of

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Confocal immunofluorescence graph of VERO cells infected with dengue vaccine.

    Cells were stained with anti-4G2 (pan-flavivirus antibody, pink fluorescence) and PAFI (DNA staining, blue fluorescence). Representative graph of fluorescence intensity (y-axis) against the distance in micrometer (x-axis) of infected VERO cells, obtained from confocal representative image (confocal microscope Leica TCS-SP8 at 200X magnification). n = 2.

    (TIF)

    Click here for additional data file. (126.2KB, tif)
    S2 Fig. Confocal immunofluorescence graph of HEK293T cells infected with dengue vaccine.

    Cells were stained with anti-4G2 (pan-flavivirus antibody, pink fluorescence) and PAFI (DNA staining, blue fluorescence). Representative graph of fluorescence intensity (y-axis) against the distance in micrometer (x-axis) of infected HEK293T cells, obtained from confocal representative image (confocal microscope Leica TCS-SP8 at 200X magnification). n = 2.

    (TIF)

    Click here for additional data file. (141.1KB, tif)
    S3 Fig. Confocal immunofluorescence graph of PMA-treated THP-1 cells infected with dengue vaccine.

    Cells were stained with anti-4G2 (pan-flavivirus antibody, pink fluorescence) and PAFI (DNA staining, blue fluorescence). Representative graph of fluorescence intensity (y-axis) against the distance in micrometer (x-axis) of infected PMA-treated THP-1 cells, obtained from confocal representative image (confocal microscope Leica TCS-SP8 at 200X magnification). n = 2.

    (TIF)

    Click here for additional data file. (114.2KB, tif)
    S1 Data

    (XLSX)

    Click here for additional data file. (64.2KB, xlsx)
    Attachment

    Submitted filename: Response to the reviewers.docx

    Click here for additional data file. (18.4KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

    Articles from PLoS ONE are provided here courtesy of PLOS

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