The influenza virus poses a serious global threat to human health and can cause pandemics associated with high mortality worldwide [1
]. While influenza vaccines have been used in many countries, the seasonal influenza epidemics have not been well controlled. In addition, the outbreak of the swine-origin H1N1 influenza in 2009 resulted in an estimated 300,000 deaths [2
], explicitly demonstrating how the influenza vaccination was insufficient for controlling a potential pandemic. Consequently, there is an urgent need to develop an approach to effectively control influenza. One approach to improve the effectiveness of the influenza vaccine is to include adjuvants. While adjuvants have been used in human vaccines to enhance vaccine efficacy for almost a century, only a few adjuvants are licensed. In particular, there are three main adjuvants, alum, MF59, and AS03, which are included in currently licensed influenza vaccines [4
]. Despite the fact that alum induces humoral immunity, it weakly stimulates cellular immunity [5
]. MF59 has been shown to cause adverse effects, including pain at injection sites and the induction of inflammatory arthritis [6
]. Moreover, cases of rare sleeping disorders and narcolepsy after vaccination with AS03-adjuvanted vaccines were reported [7
]. Therefore, the development of an ideal adjuvant, with regard to potential safety and the ability to enhance both humoral and cellular immune responses that are specific to vaccine antigens, is needed.
Adjuvants targeting pattern-recognition receptors, such as Toll-like receptors (TLRs), are part of a broad approach to stimulate innate immune responses, thereby enhancing antigen-specific immunity and subsequently improving vaccine efficacy [9
]. Recently, numerous studies have shown the effects of TLR4-dependent adjuvants on improving vaccine efficacy [10
]. A TLR4 agonist absorbed on alum in AS04 adjuvant was successfully used in licensed vaccines for human papillomavirus [15
] and the hepatitis B virus [16
]. Thus, developing TLR4 ligands as adjuvants could be beneficial to improving antiviral vaccines such as influenza vaccine. Monophosphoryl lipid A (MPL) is a detoxified derivative of the lipopolysaccharide (LPS) isolated from Salmonella minnesota
R595. MPL has been demonstrated to stimulate innate immunity via TLR4 [17
] and promote Th1-biased immune responses [18
]. Even though MPL has been widely used as an adjuvant in several human vaccines [19
], the complicated purification process and prohibitive cost of MPL have precluded its use as an easy-to-use and cost-effective adjuvant. To address these limitations, Escherichia coli
-produced MPL (named EcML) has been previously produced by direct extraction from genetically engineered E. coli
In this study, we first investigated the action mechanisms of EcML to increase innate immunity and tested its adjuvanticity using the model antigen ovalbumin (OVA). EcML triggered the activation of bone marrow-derived dendritic cells (BMDCs) via TLR4 and increased the antigen processing of cells. Compared to OVA alone, OVA plus EcML-immunized mice showed an enhancement in OVA-specific humoral and cellular immune responses. Finally, we explored the adjuvanticity of EcML in enhancing the pandemic H1N1 (pH1N1, A/California/04/09) influenza vaccine efficacy. EcML improved the protective efficacy of the pH1N1 vaccine antigen in mice with 100% survival after viral challenge by increasing influenza-specific antibody (Ab) titers, hemagglutination inhibition (HI) titers, and cytotoxic T lymphocyte (CTL) activity. Collectively, our results strongly suggest that EcML might be a promising adjuvant candidate for influenza vaccines.
2. Materials and Methods
2.1. Mice and Cells
Six-week-old female C57BL/6 mice were purchased from Orient Bio (Gyeonggi-do, Korea). Six-week-old female wild-type C3H/HeN and congenic TLR4-defective C3H/HeJ mice were purchased from Central Lab. Animal Inc. (Seoul, Korea). All animals were housed in a specific pathogen-free (SPF) facility at the Korea Research Institute of Bioscience and Biotechnology (KRIBB). All experiments employing mice were reviewed and approved by the Institutional Animal Care and Use Committee of the KRIBB. Immature BMDCs were generated using Roswell Park Memorial Institute 1640 media (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 U/mL penicillin, 100 mg/mL streptomycin (Gibco), 20 ng/mL murine granulocyte-macrophage colony-stimulating factor (GM-CSF; Peprotech), and 10 ng/mL murine IL-4 (Peprotech) for 7 days followed by changing with fresh media every 3 days, as previously described [21
2.2. Preparation of Adjuvants
EcML was produced by Eubiologics. Co., Ltd. (Gangwon-do, Korea) as described previously [20
] with slight modifications. Briefly, the EcML was purified from an engineered E. coli
KHSC0055 and formulated as aqueous formulations using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and chloroform, and was then evaporated to remove chloroform. The dried EcML was rehydrated in ultrapure water at 0.45 mg/mL, and suspended by sonication at 60 °C. The MPL from S. minnesota
R595 was purchased from InvivoGen (San Diego, CA, USA) and aqueously formulated using the same processes as for EcML. The resulting EcML and MPL were stored at 4 °C for further use. Alum (Alhydrogel adjuvant 2%) was obtained from InvivoGen.
2.3. Preparation of Influenza Virus
The pH1N1 influenza virus was grown in 9- to 10-day-old SPF embryonated chicken eggs (NamDuck SPF, Sungnam, Korea) for 48 h at 37 °C. The viruses were harvested from the allantoic fluids of the eggs by centrifugation at 3500× g for 10 min at 4 °C, filtrated through 0.45 μm pore size membrane filters (Merck KGaA, Darmstadt, Germany), and then stored at −80 °C for further use. All viral experiments were implemented under conditions of biosafety level 2.
2.4. Cell Viability Assay
Immature BMDCs were stimulated with PBS as a negative control, 0.625, 1.25, 2.5, and 5 μg/mL EcML, 1 μg/mL LPS or 0.5% Triton X-100 (as a positive control) for 24 h at 37 °C. Cell viability was measured using Trypan blue stain 0.4% with the Countess TM automated cell counter (Thermo Fisher Scientific, Waltham, MA, USA).
2.5. In Vitro Activation and Antigen Processing of BMDCs
To investigate the BMDC activation, the cells were stimulated with PBS as a negative control, 0.625, 1.25, 2.5, and 5 μg/mL EcML, or 5 μg/mL MPL for 24 h at 37 °C. The stimulated cells were stained with PE-conjugated monoclonal antibodies (mAbs) against mouse CD40, CD80, CD86, major histocompatibility complex (MHC) class II, and isotype-matched control mAbs (BD Biosciences, San Diego, CA, USA). To examine the antigen processing of BMDCs, we incubated the cells with 5 µg/mL DQ™-OVA, which is OVA conjugated with BODIPY FL dye (a self-quenched dye that emits fluorescence upon proteolytic degradation) (Thermo Fisher Scientific) alone or mixed with either 2.5 μg/mL EcML or MPL for 5 h at 37 °C. The cells were acquired on FACSCalibur flow cytometers (BD Biosciences), and the data were collected and analyzed using FlowJo software version 10 (Tree Star Inc., Ashland, OR, USA).
2.6. Western Blotting
Immature BMDCs generated from C3H/HeN and C3H/HeJ mice were serum-deprived for 3 h and then stimulated with EcML at 0.625, 1.25, and 2.5 μg/mL or 2.5 µg/mL MPL for 30 min at 37 °C. The cells were lysed with a lysis buffer containing protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA) plus a phosphatase inhibitor cocktail (Sigma-Aldrich). The cell lysates were collected by centrifugation at 13,000× g for 15 min at 4 °C, and concentrations of total proteins were determined using a bicinchoninic acid protein assay (Thermo Fisher Scientific). The cell lysates were separated by 12% SDS-PAGE and transferred to PVDF membranes (Merck KGaA). The blots were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 and probed with anti-phospho-IκBα rabbit mAb (Cell Signaling Technology, Beverly, MA, USA) for 16 h at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) Ab (Cell Signaling Technology). The blots were then visualized by chemiluminescence using an enhanced chemiluminescent substrate kit (GE Healthcare, Uppsala, Sweden). The membrane was then subsequently stripped and reprobed with anti-β-actin mAb (Cell Signaling Technology) as a loading control.
2.7. Immunizations and Viral Challenge
C57BL/6 mice were intramuscularly (i.m.) immunized with 10 µg OVA protein (Sigma-Aldrich) alone or in combination with 0.625, 1.25, 2.5, or 5 µg EcML on days 0, 14, and 28. Spleens and sera were collected 2 weeks after the last immunization. In a separate experiment, the mice were i.m. immunized with 10 µg OVA protein alone or mixed with either 2.5 µg EcML or 2.5 μg MPL. The body temperature of the immunized mice was measured at indicated time points using Thermalert TH-5 (Physitemp Instruments Inc., Clifton, NJ, USA). In addition, sera were collected from the mice. In a separate experiment, the mice were i.m. immunized with 10 µg OVA protein alone or mixed with either 25 µg alum, 2.5 µg EcML, or 2.5 µg MPL on days 0, 14, and 28. Spleens and sera were collected on day 14 after the last immunization. In the influenza experiment, the mice were i.m. immunized with pH1N1 split vaccine antigen (A/California/7/2009 NYMC X-179A H1N1; provided by Mogam Biotechnology Research Institute, Gyeonggi-do, Korea), which contained 0.05 μg hemagglutinin (HA), alone or mixed with 25 μg alum, 2.5 μg EcML, or 2.5 μg MPL on days 0 and 14. Fourteen days after the last immunization, spleens and sera were collected from the vaccinated mice. On day 14 after the final vaccination, the mice were intranasally (i.n.) challenged with a lethal dose (50 LD50) of the pH1N1 influenza virus. The body weight and survival of the challenged mice were monitored for 14 days after infection. Mice that lost significantly more than 25% of their body weight were considered to have reached the experimental endpoint and were sacrificed.
2.8. HI Assay
HI titers against the pH1N1 influenza virus were determined using sera from the vaccinated mice, as previously described [12
]. HI titers were calculated as the reciprocals of the highest dilution of sera at which hemagglutination was completely prevented.
2.9. Enzyme-Linked Immunosorbent Assay (ELISA)
Levels of cytokines in the cell culture supernatants were measured using OptEIA kits (BD Biosciences) according to the manufacturer’s instructions. The production of antigen-specific IgG, IgG1, and IgG2b Abs in the sera of the immunized mice were determined by indirect ELISA. Briefly, MaxiSorp 96-well plates (Thermo Fisher Scientific) were coated overnight at 4 °C with 100 µL of 1 µg/mL OVA protein or 0.5 µg/mL pH1N1 split vaccine antigen in carbonate solution, pH 9.5. The plates were blocked with 5% skim milk in PBS for 2 h at 37 °C and were washed with 0.05% Tween-20 in PBS. The plates were then incubated with 100 µL of diluted sera from the vaccinated mice for 2 h at 37 °C, followed by incubation with 100 µL of HRP-conjugated anti-mouse IgG (Cell Signaling Technology), and anti-mouse IgG1 or IgG2b Abs (Southern Biotech, Birmingham, AL, USA) for 1 h at 37 °C. After washing, the reactions were developed with the chromogenic tetramethylbenzidine substrate (BD Biosciences) and then terminated with 2N H2SO4. The optical density was measured at 450 nm using a Versamax microplate reader (Molecular Devices, San Francisco, CA, USA).
2.10. Enzyme-Linked Immunospot (ELISPOT) Assay
The frequencies of antigen-specific IFN-γ-secreting cells were evaluated using a mouse ELISPOT kit (BD Biosciences), as previously described [12
]. Briefly, 14 days after the last vaccination, splenocytes were obtained from the immunized mice and then plated at 5 × 105
cells/well onto purified anti-IFN-γ-coated ELISPOT plates. The cells were treated with 0.5 µg/well of OVA257–264
peptides (Anaspec, San Jose, CA, USA) or the 500 median tissue culture infectious dose (TCID50
)/well of UV-inactivated pH1N1 influenza virus for 3 days at 37 °C. The spot-forming units (SFUs) of antigen-specific IFN-γ-secreting cells were calculated using an ELISPOT plate reader (Cellular Technology Ltd., Cleveland, OH, USA).
2.11. Systemic Inflammatory Responses after Vaccination
To measure the systemic inflammatory responses, sera from the immunized mice were collected. Levels of inflammatory cytokines in the pooled sera (n = 5 per group) were measured using the Lengendplex mouse inflammation panel (13-plex, Biolegend, San Diego, CA, USA) according to the manufacturer’s instructions.
2.12. Statistical Analysis
All of the data were presented as the means ± standard deviations (SDs) and represented three independent experiments. Statistically significant differences between the two and multiple groups were assessed using the two-tailed Student’s t-test and one-way ANOVA, followed by Bonferroni’s correction (ANOVA/Bonferroni), respectively. The p values less than 0.05 (p < 0.05) were considered to be statistically significant. All the analyses were implemented using GraphPad Prism software (GraphPad, San Diego, CA, USA).
Despite the availability of annual vaccinations, influenza remains a significant cause of human infectious disease and, thus, there is a push to improve vaccine effectiveness. An adjuvant capable of enhancing vaccine immunogenicity without compromising safety would be beneficial to improving influenza vaccines. However, the main influenza vaccine adjuvants, including alum, MF59, and AS03, have limitations such as their weak induction of cellular immune responses and safety issues [4
] that need to be addressed. MPL has been used as an adjuvant in several human vaccines [19
] and has reported increasing influenza vaccine efficacy [28
]. However, its disadvantages, including the difficult manufacturing processes and prohibitive cost, have precluded its use as an easy-to-use and cost-effective adjuvant. Thus, an alternative component could be interesting and, in fact, E. coli
-produced MPL (named EcML) has recently been produced by direct extraction from an engineered E. coli
]. The E. coli
strain is a sustainable source and can be easily grown at a very low cost and EcML is directly extracted from the bioengineered E. coli
without hydrolysis, indicating that the manufacture of EcML is more cost-effective than that of MPL [20
]. Here, we revealed that EcML might be safe to use due to no in vitro cell cytotoxicity, unchanged body temperature, and transient systemic inflammatory responses after vaccination in mice. In addition, EcML robustly activated DCs via the TLR4-mediated NF-κB signaling pathway and increased the antigen processing of cells in vitro. Moreover, EcML enhanced antigen-specific humoral and cellular immune responses. Ultimately, EcML effectively improved the protective immunity of the pH1N1 influenza vaccine antigen.
DCs play a major role in innate immunity and serve as a significant link between innate and adaptive immunity [29
]. Thus, targeting antigens to DCs is a crucial strategy in vaccine development. Previously, it was also reported that EcML dose-dependently enhanced the BMDC phagocytic activity against B16F10 melanoma cells [26
]. In this study, we showed that EcML triggered DC activation, including the high production of inflammatory cytokines and the upregulation of the expression of costimulatory and MHC-II molecules. The upregulation of these molecules has been previously seen when using MF59 adjuvants [30
]. In addition, we observed that EcML-treated BMDCs had much higher levels of TNF-α and IL-6 (Figure S1
) than MPL-treated BMDCs. This result suggests that EcML might be better than MPL in enhancing innate immune responses by DCs. TLR4 agonists can activate innate immune responses and consequently augment adaptive immune responses by enhancing Th1-biased responses [31
]. A previous study has demonstrated that MPL stimulated innate immunity via TLR4 [33
]. In this study, we also observed that EcML activated BMDCs via TLR4-mediated NF-κB signaling, conferring the suppressed production of inflammatory cytokines and the low expression of costimulatory molecules (CD40, CD80, and CD86) in TLR4-defective BMDCs. These results are consistent with the previous data showing that EcML-induced IFN-ꞵ production was significantly decreased in DCs by a TLR4-neutralizing antibody [26
]. The antigen processing by antigen-presenting cells is an essential step in presenting antigens to T cells and initiating antigen-specific adaptive immunity [34
]. We showed that EcML more efficiently facilitated the degradation of the DQ-OVA antigen, indicating that EcML enhances antigen processing, thereby enhancing adaptive immune responses.
The safety of a vaccine adjuvant is paramount as vaccines are given prophylactically to healthy individuals. A previous study reported that Lipid A, a toxic domain of LPS, was not detected after the purification of EcML, suggesting that EcML is not contaminated with LPS [20
]. EcML has also been demonstrated to neither significantly increase the spleen weight nor decrease mice body weight after intravenous injection [26
], indicating that EcML may be appropriate for systematic administration. In this study, we further examined the safety of EcML by measuring the cell viability after in vitro treatment and changes in body temperature and systemic inflammatory responses after i.m. immunization in mice. We observed that EcML treatment did not result in any in vitro cytotoxic effects on antigen-presenting cells, including BMDCs and macrophage cell line RAW 264.7 cells (Figure S2
). Previous studies have reported that children vaccinated with the MF59-adjuvanted influenza vaccine showed adverse reactions, including redness and swelling at the injection sites [35
]. We did not observe any such adverse effects in mice after i.m. vaccination with EcML. Mice vaccinated with LPS (an unsafe adjuvant) showed hypothermia which lowered the survival rate after challenge [37
]. In this study, there was no significantly different change in body temperature in the mice vaccinated using EcML. While inflammatory responses are a crucial defense mechanism against viral infection, excessive and persistent inflammation can be detrimental [38
]. Previous studies demonstrated that side effects are mediated through the systemic distribution of TNF-α and IL-6 [39
]. Here, we reported that these cytokines subsided to basal levels in the sera of EcML–OVA-vaccinated mice at 24 h post-injection. Our results indicate that EcML induces transient inflammatory responses after vaccination, suggesting that EcML might be safe to use.
A novel vaccine adjuvant could have the ability to improve both antigen-specific humoral and cellular immunity [41
]. Consistent with a previous study showing that EcML enhanced OVA-specific antibody response in a BALB/c mouse model [20
], our results show that EcML improved OVA-specific Ab production. Moreover, we observed enhanced OVA-specific cellular immune responses by EcML after vaccination using C57BL/6 mice. In particular, EcML increased the number of MHC class I-restricted OVA257–264
peptide-specific IFN-γ-producing T cells and the levels of IFN-γ release, indicating that EcML robustly enhances antigen-specific CTL activity. Additionally, we observed that EcML was better than MPL in improving OVA-specific humoral and cellular immune responses. We speculated that the adjuvanticity of EcML could be with the reason behind the enhanced levels of inflammatory responses observed for EcML compared to MPL. Currently, alum and MF59 are mainly used in influenza vaccines [4
], but these adjuvants primarily induce responses toward the Th2-biased immunity [4
] and may not be sufficiently effective in the protection against influenza virus infections. We then evaluated the efficacy of EcML as an adjuvant for the influenza vaccine, in comparison to alum. We found that EcML robustly enhanced not only humoral immune responses, including Ab production and HI titers, but also cellular immune responses, including CTL activity and IFN-γ release. Notably, EcML-induced adaptive immune responses were higher than those induced by alum. Consequently, EcML fully protected mice (100% survival rate), while alum only partially protected mice (20% survival rate) against pH1N1 influenza virus infection.
MPL has been reported to enhance the protective efficacy of the influenza vaccine [28
]. We observed that EcML provided similarly protective efficacy with MPL, indicating that EcML was as good as MPL in improving the protective immunity of the pH1N1 vaccine. The induction of the cross-protective immunity of the pH1N1 vaccine is beneficial for the protection against different influenza virus subtypes. We observed that the EcML-vaccine group had an enhanced cross-reactive IFN-γ response against H1N1 (A/Puerto Rico/8/34) and reassortant H3N2 (HA and neuraminidase of A/Hong Kong/1/1968 and internal genes of A/Puerto Rico/8/34) by enhancing the release of H1N1- and H3N2- specific-IFN-γ cytokines into the splenocyte culture supernatant after ex vivo stimulation (Figure S3
). Further studies should be performed to clarify this effect of EcML. A combination of different adjuvants in specific formulations can result in the complementary and even synergistic enhancement of immune responses to specific antigens. We also previously reported that a complex of poly-γ-glutamic acid and alum strongly induced the cross-protective efficacy of the pH1N1 vaccine, as compared to each component alone [13
]. The AS04 adjuvant composed of MPL and alum has been used in licensed vaccines for human papillomavirus and hepatitis B virus [43
]. Thus, the combination of EcML with other adjuvants, such as alum, should be further studied to broaden the use of EcML in vaccines against other infectious diseases.