Adjuvant effect of docetaxel on the immune responses to influenza A H1N1 vaccine in mice
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- Chen, J., Yuan, L., Fan, Q. et al. BMC Immunol (2012) 13: 36. doi:10.1186/1471-2172-13-36
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Vaccination remains one of the most effective approaches to prevent the spread of infectious diseases. Immune responses to vaccination can be enhanced by inclusion of adjuvant in a vaccine. Paclitaxel extracted from the bark of the Pacific yew tree Taxus brevifola was previously demonstrated to have adjuvant property. Compared to paclitaxel, docetaxel is another member of taxane family, and is more soluble in water and easier to manipulate in medication. To investigate the adjuvant effect of this compound, we measured the immune responses induced by co-administration of a split inactivated influenza H1N1 vaccine antigen with docetaxel.
When co-administered with docetaxel, lower dose antigen (equivalent to 10 ng HA) induced similar levels of IgG and IgG isotypes as well as HI titers to those induced by higher dose antigen (equivalent to 100 ng HA). Docetaxel promoted splenocyte responses to H1N1 antigen, ConA and LPS, mRNA expressions of cytokines (IFN-gamma, IL-12, IL-4 and IL-10) and T-bet/GATA-3 by splenocytes. The enhanced immunity was associated with up-expressed microRNAs (miR-155, miR-150 and miR-146a) in docetaxel-stimulated RAW264.7 cells. Docetaxel promoted similar IgE level to but alum promoted significantly higher IgE level than the control.
Docetaxel has adjuvant effect on the influenza H1N1 vaccine by up-regulation of Th1/Th2 immune responses. Considering its unique vaccine adjuvant property as well as the safe record as an anti-neoplastic agent clinically used in humans during a long period, docetaxel should be further studied for its use in influenza vaccine production.
- IFN-gamma (IFN-γ)
Foetal calf serum
- Con A
Enzyme linked immunosorbant assay
The current strategy for prevention of annual seasonal influenza is primarily based on the trivalent inactivated vaccine, which consists of split viral envelopes with the protein hemagglutinin (HA) as the main vaccine antigen [1, 2]. Although the method has been utilized for many years, vaccination remains one of the most effective approaches, not only to prevent the spread of the influenza virus but also to mitigate the severity of illness and the impact of the disease  Since the rapid spread of the swine-origin influenza A (H1N1) 2009 pandemic worldwide, the rapid implementation of a vaccine has become a global priority.
A previous investigation found that vaccination with recent seasonal influenza vaccines provided little or no cross-reactive antibody protection against 2009 pandemic influenza A (H1N1) in any age groups . The lack of cross-protective immunity between the pandemic and seasonal influenza virus strains highlighted the urgency of rapid vaccine development. The present global production capacity of trivalent seasonal influenza vaccine is about 876 million doses per year. With a world population of more than 6.5 billion people and the probability that two vaccine doses should be administered in a largely naive population, it should be predicted that about 13 billion doses of pandemic vaccine would be needed for adequate pandemic preparedness. The yield of virus in eggs or cell cultures is another important determinant for the amount of vaccine doses that can be manufactured. In spite of the WHO global pandemic influenza action plan to increase the potential supply of pandemic influenza vaccine , the production of sufficient pandemic vaccine to immunize the world’s population would significantly exceed the existing manufacturing capacities.
To effectively vaccinate the high-risk population against pandemic influenza, two challenges are to produce sufficient quantities of vaccine during a short period and to induce significant immunogenicity and cross-protective immunity after vaccine injections [6–8]. Luckily, both purposes can be achieved by using adjuvant to elicit a strong and broadened immune response. Although many materials have been reported having adjuvant property, alum (a term for aluminum-based mineral salts) is the first adjuvant approved by the U.S. Food and Drug Administration (FDA) in the influenza vaccines for human use. However, highly heterogeneous, difficult to manufacture in a consistent and reproducible manner, and a boost injection required to generate protection limited alum in influenza vaccine use [9, 10]. It is also found that certain antigens do not adsorb well onto alum due to the presence of the same charge on the adjuvant and antigens . MF59 is an oil-in-water emulsion adjuvant, which has been formulated in vaccines licensed for human use . Although adverse effects of MF59 are rare, the events such fever, chills, malaise, myalgia, arthralgia, nausea, headache and rash have been found after immunization of MF59-adjuvanted vaccines [13, 14]. Therefore, searching for new vaccine adjuvants remain an interesting topic.
Paclitaxel, a member in the taxane family, was initially extracted from the bark of the Pacific yew tree Taxus brevifola in early 1960s and its structure was confirmed in 1971 . At the late 1970s, paclitaxel was discovered able to blocks mitosis and cause the death of cancer cells by binding to and stabilizing microtubules [16, 17]. In 1992, the drug was approved for the treatment of advanced ovarian cancer, and then has been successfully used in other solid tumors [18, 19]. The drug has a safe record in humans for almost 20 years. Based on the TLR4 agonist activity of paclitaxel at a low dose for stimulation of proinflammatory mediator release from isolated macrophages, it was previously demonstrated that paclitaxel has an adjuvant effect on the immune responses [20, 21]. When co-administrated with paclitaxel, OVA induced significantly higher IgG, IgG subclass and IgM responses in association with upregulation of mRNA expression of T-bet/GATA-3 than when OVA was immunized alone .
Docetaxel is another member of the taxane family. Compared to paclitaxel, docetaxel is more soluble in water, and easier to manipulate in medication. Docetaxel has also been found to have immunomodulatory properties. Garnett et al. recently reported that intraperitoneal injection of docetaxel after subcutaneous inoculation of a recombinant poxviral vaccine significantly enhanced the immune response in a mouse model . Present study was designed to investigate if co-administration of a split inactivated influenza H1N1 vaccine antigen with docetaxel could enhance the immune responses by measuring serum specific antibody responses, total IgE, hemagglutination inhibition titers (HI), lymphocyte proliferation as well as mRNA of cytokines and transcription factors produced by splenocytes in Balb/c mice. Dose-sparing effect of the influenza antigen was also evaluated when docetaxel was administered with the antigen.
Serum vaccine-specific IgG and IgG isotypes
Lymphocyte proliferative responses
Serum total IgE
Cytokines mRNA expression
Docetaxel and alum induced different patterns of T-bet and GATA-3 mRNA expression as shown in (Figure 6B). Docetaxel + 10 ng HA induced significantly higher both T-bet and GATA-3 mRNA expression than the control without adjuvant (P < 0.05); alum + 10 ng HA induced significantly higher GATA-3 (P < 0.05) than the docetaxel group but similar T-bet mRNA expression to the control (P > 0.05 alone).
MicroRNAs expressed by macrophages stimulated in vitro by docetaxel
Adjuvant properties of docetaxel have been demonstrated for inactivated H1N1 influenza vaccine in a mouse model in the present study. Co-administration of docetaxel with inactivated influenza virus H1N1 induced significantly higher serum specific IgG and the isotype responses, HI titer, splenocyte proliferation in response to ConA, LPS and HA than when influenza vaccine was immunized alone. In addition, significantly increased mRNA expressions of IFN-γ, IL-12, IL-4 and IL-10 by splenocytes in association with up-regulation of mRNA expression of T-bet/GATA-3 were observed in docetaxel-adjuvanted groups. Serum total IgE level in the docetaxel adjuvanted group was significantly lower than the alum-adjuvanted group. MiR-155, miR-150 and miR-146a are up-regulated in Raw 264.7 cells in response to docetaxel.
The mouse model has been used to study the immunity of a host against influenza infection for long time. For example, Cox et al. immunized mice with a split influenza virus vaccine, and observed that the inhibition of viral replication by immunization correlates high influenza specific serum IgG concentrations . Caillet et al. immunized Balb/c mice with a H1N1 influenza vaccine using oil-in-water emulsion AF03 as an adjuvant, and found that the mice receiving AF03-adjuvanted vaccine had antigen-specific antibody titers 3- to 10-fold higher than that in animals administered antigen alone . The antibody response elicited by antigen is dose-dependent. Caillet et al. reported that 0.3 μg influenza vaccine antigen induced lower HI tiers than 3 μg antigen in mice . Similarly, we found that 10 ng HA antigen elicited significantly lower serum IgG and HI titers or the IgG isotypes than 100 ng HA antigen. However, supplement of docetaxel (50 to 200 μg) in 10 ng HA antigen significantly amplified IgG and HI titers, which were similar to the titers elicited by 100 ng HA when docetaxel was added at 100 or 200 μg as indicated in Figures 1 and 3, suggesting that the same level of the immune responses could be induced by smaller dose of antigen if docetaxel is used as an adjuvant in the production of vaccines. Garnett et al. significantly enhanced the immune response to a recombinant poxviral vaccine by injection of docetaxel at 500 μg . In this study, antibody titers had no long significant changes when docetaxel was increased from 100 to 200 μg per dose.
IgG is the most plentiful immunoglobulin in the serum, and provides the considerable protection against most blood infectious agents. During a T-cell dependent immune response, a progressive change takes place in the principal immunoglobulin class of the specific antibodies. This subclass switch is influenced by T-cells and their cytokines. Data in Figure 2 indicated that docetaxel significantly increased the production of all IgG isotypes, which may be associated with simultaneously up-regulated gene expression of T-bet and GATA-3 (Figure 6B), leading to increased production of IFN-γ, IL-12, IL-4 and IL-10 by splenocytes (Figure 6A). While alum enhanced only IgG1 (P < 0.05) but not IgG2a, IgG2b and IgG3 (P > 0.05), which may be related to up-regulated gene expression of GATA-3, resulting in enhanced production of Th2 type cytokines such as IL-4 and IL-10 as shown in (Figure 6A). All these suggest that docetaxel activated both Th1 and Th2 while alum only triggered Th2 type immune responses.
Hemagglutinin has the capacity of binding to erythrocytes resulting in agglutination, which can be visually detected and thus used as assay read-out. The binding of HA to erythrocytes is inhibited by the addition of serum containing anti-HA antibodies. Thus, the concentration of anti-HA antibodies can be defined as HI titer by incubating serial dilutions of sera with HA antigen or whole virus . In humans, HI titer of 1:40 or higher is normally considered protective . Figure 3 showed that docetaxel increased HI titers, indicating that the protection capacity against influenza infection was increased in the immunized animals.
The lymphocyte proliferative response depends on the mitogen used. ConA stimulates T-cell whereas LPS stimulates B cell proliferation. Increased lymphocyte proliferation responses to ConA and LPS were found in docetaxel- and alum-adjuvanted groups (Figure 4), indicating that both T and B cells were activated. In order to induce antibody production, antigen-specific B lymphocytes should be triggered for clonal expansion. Significantly enhanced lymphocyte responses to H1N1 HA antigen, paralleled the increased HA-specific IgG responses in mice immunized with docetaxel- or alum-adjuvanted H1N1 vaccine.
Unlike paclitaxel, docetaxel does not bind to TLR4 nor stimulate proinflammatory cytokine responses . Garnett et al. recently reported that docetaxel modulated CD4+, CD8+, CD19+, natural killer cells, and Treg populations and enhanced CD8+ functions . The adjuvant activity of docetaxel may be related to its immunomodulatory effects. MicroRNAs (miRs) are a broad class of small non-coding RNAs (18–25 nucleotides) with crucial roles in regulation of gene expression. Previous studies have shown that miR-155, miR-150, miR-146a, miR-181a and miR-125b are involved in the innate immune reactions. Stimulation of monocytes with lipopolysaccharide (LPS) induced the expression of miR-146 and miR-155 . MiR-155 and miR-125b were found to be up-regulated and down-regulated, respectively, in Raw 264.7 macrophages in response to LPS. The miR-150 has a dynamic expression profile during lymphocyte development, being highly expressed in mature B cells and T cells but not in their progenitors, its expression is then extinguished after further differentiation of naive T cells into the Th1 and Th2 subsets . MiR-181a has been ascribed functions in hematopoietic differentiation and in T cell differentiation [30, 31]. In this study, miR-181a, miR-155, miR-150, miR-146a and miR-125b were analyzed to identify microRNAs possibly involved in responses to docetaxel stimulation (Figure 7). Only miR-146a showed significant increase 1 hour after stimulation of RAW264.7 cells with docetaxel (Figure 7D). Three hours later, miR-155, miR-150 and miR-146a expressions were enhanced (Figure 7B, C, D), while miR-181a and miR-125b showed no significant change (Figure 7A, E). Increased expression of miR-155 and miR-146a has also been found in our previous study when RAW264.7 cells were stimulated with paclitaxel . These suggested that stimulation manner of docetaxel may be different from that of LPS.
Safety should be taken into account when seeking adjuvant candidates. However, the safety of many drugs largely depends on how the drugs are used. Many drugs are safe at a small dose while becoming toxic when they are administered frequently at a higher dose. Compared to other potential vaccine adjuvant candidates reported in literatures, the toxicity of docetaxel is transparent as it has been clinically used for almost 20 years. When docetaxel was used as an antineoplastic agent, the side-effects such as short-lasting neutropenia and hypersensitive reactions were reported . The other toxicities were hematopoietic (rats, mice, dogs and monkeys), gastrointestinal (dogs, monkeys) and neuromotor (mice), and are either usually mild in severity or easily treated or prevented . In our study, the suggested dose of docetaxel for adjuvant purpose was 100 μg/mice, which was significantly lower than that recommended for cancer treatment (47 mg/kg).
The adjuvant effect of aluminum salts on influenza vaccine has been proven previously . However, frequent use of alum-adjuvanted vaccines could be one of the reasons for IgE-mediated allergy due to activated Th2 immune response [35, 36]. In the present study, alum but not docetaxel promoted the production of IgE significantly higher than that of mice immunized with HA only (Figure 5). The increased serum IgE level may be attributed to higher Th2 (IL-4, IL-10, GATA-3) and lower Th1 (IFN-γ, IL-12, T-bet) responses in mice injected with alum-adjuvated vaccine as indicated in Figure 6.
In summary, docetaxel has an adjuvant effect on a split influenza A H1N1 vaccine by up-regulating Th1 and Th2 immune responses in a mouse model. When co-administered with docetaxel, 10 ng of H1N1 virus antigen (HA) induced similar level of IgG and IgG isotype responses as well as HI titers to those induced by 100 ng of HA. Docetaxel promoted splenocyte proliferative response to H1N1 antigen, ConA and LPS, mRNA expressions of cytokines (IL-4, IL-10, IL-12 and IFN-γ) and T-bet/GATA-3 by splenocytes. The enhanced immune responses may be associated with up-expressed microRNAs (miR-155, miR-150 and miR-146a) as detected in docetaxel-stimulated RAW264.7 cells. Docetaxel promoted similar IgE level to but alum promoted significantly higher IgE level than the control. Considering its unique vaccine adjuvant property as demonstrated here as well as the safe record clinically used in humans during a long period, docetaxel should be further evaluated for its use in vaccines.
Female Balb/c mice were purchased from Shanghai Laboratory Animal Center (SLAC) Co., Ltd. (Shanghai, China), and housed in polypropylene cages with sawdust bedding in hygienically controlled environment. The temperature was controlled at 24 ± 1°C and humidity at 50 ± 10%. Feed and water were supplied ad libitum.
All procedures related to the animals and their care conformed to the internationally accepted principles as found in the Guidelines for Keeping Experimental Animals issued by the government of China. One of the authors (Yu Chen) received license (No. X1003003) for management of experimental animals from the Office in Charge of Experimental Animals of Zhejiang Province. The Department of Veterinary Medicine has the relevant approval to carry out animal study in general and need not additional approval for this study.
Antigen and adjuvant
Split inactivated influenza virus NYMCX-179A (H1N1) was kindly supplied by Zhejiang Provincial Center for Diseases Control and Prevention, which contained 132 μg/ml of hemagglutinin (HA) determined by a quantitative single-radial-immunodiffusion assay essentially as described by Wood et al. . Docetaxel was purchased from Xi’an Hao-xuan Biotechnology Co., Ltd (Xi’an, China). Docetaxel was white powder with purity of 99.5%. Docetaxel was dissolved in absolute ethanol, polysorbate 80, saline (1:1:18) and sterilized by passing through a 0.22 μm filter. The endotoxin level in the solutions was less than 0.5 endotoxin unit (EU)/ml by a gel-clot Limulus amebocyte lysate assay (Bath no., Zhanjiang A & C Biological Ltd., Zhanjiang, China). Aluminum hydroxide Gel (Sigma, A8222) was used as the positive control adjuvant.
Seventy-two Balb/c mice (6 weeks of age) were randomly distributed into nine groups with 8 mice each. All animals were subcutaneously (s.c.) immunized twice with saline, docetaxel (100 μg), Ag (100 or 10 ng HA) or Ag (10 ng HA) + docetaxel (25, 50, 100 or 200 μg) at 3 week intervals, and the alum adjuvanted group as the positive control. Two weeks after the boost, blood samples were collected for measurement of serum HI titers using chicken red blood cells, Ag-specific IgG titers as well as IgG isotypes, and total IgE levels. Splenocytes were prepared for determination of cellular proliferation and production of IFN-γ, IL-12, IL-4, IL-10, T-bet and GATA-3. All the injection solution was in the volume of 0.1 ml per mouse.
Measurement of Vaccine-specific IgG, IgG isotypes and total IgE
Serum samples were analyzed for measurement of vaccine-specific IgG titer and IgG isotype responses by indirect enzyme-linked immunosorbent assay as previously described by Song et al. . Total IgE was measured using a mouse IgE ELISA quantitation kit (Biolegend, Cat. No. 432404) following the manufacturer’s instructions. The sensitivity of the assay was 0.1 ng/ml for mouse IgE. Serum was diluted 1:50 with 1× assay diluent.
Hemagglutination inhibition assay
Serum HI titers were determined according to the protocol adapted from the CDC laboratory-based influenza surveillance manual .
Lymphocyte proliferation assay
Quantification of target genes by real-time PCR
Splenocytes from the H1N1-immunized Balb/c mice prepared as described before were seeded into a 24-well flat-bottom microtiter plate (Nunc) at 5 × 106 in 2 ml complete medium, thereafter 20 μl split virus antigen (equivalent to 2.64 ng HA) was added. The plates were incubated at 37°C in a humid atmosphere with 5% CO2. After 10 h treatment, cells were harvested by centrifugation (380 × g at 4°C for 10 min), and washed with ice-cold PBS, then subjected to RNA extraction. Splenocytes (1 × 107) were lysed in 1 ml of RNAisoTM Plus (Takara, China) reagent and the total RNA was isolated according to the manufacture’s protocol. The concentration of total RNA was quantified by determining the optical density at 260 nm. The total RNA was used and reverse transcription was performed by mixing 1 μg of RNA with 5 μl iScript reagent (Bio-Rad) in a DEPC-treated tube, thereafter nuclease-free water was added to a final volume of 20 μl. The reaction condition for reverse transcription was performed according to the manufacture’s protocol (5 min at 25°C, 30 min at 42°C, 5 min at 85°C, hold at 4°C).
Sequences of primer and probe for quantitative RT-PCR of cytokine and transcription factor
Probe: 5′FAM-CCTGGATTCATCGATAAGCTGCACC-BHQ-1 3′
Probe: 5′FAM-CAGGCAGAGAAGCATGGCCCAGAAA-BHQ-1 3′
IL-12p40 Forward: 5′-TTGCTGGTGTCTCCACTCATG-3′
Probe: 5′FAM-CTGGACTCCCGATGCCCCTGG-BHQ-1 3′
Probe: 5′FAM-CTGTTTCTGGCTGTTACTGCCACGGC-BHQ-1 3′
Probe: 5′FAM-CGCCCGCCTCTGCTGCACG-BHQ-1 3′
Probe: 5′FAM-CTGGGAAGCTGAGAGTCGCGCTCA-BHQ-1 3′
Amplification was carried out in a total volume of 20 μl containing 2 μl of 10 × PCR buffer, 2 μl of MgCl2 (25 mM), 2 μl of dNTPmix (2.5 mM), 0.4 μl of Tag DNA polymerase (Takara, China), 2 μl of cDNA template, 2 μl (5 μM) of each target gene and β-actin specific primers, 1 μl (5 μM) of target gene and β-actin specific probes. Reaction conditions were the standard conditions for the TagMan PCR (15 s denaturation at 95°C, 30 s annealing at 60°C) with 45 PCR cycles. Relative quantification between samples was achieved by the 2-ΔΔCT method  and calculated by software REST 2005 (gifted by Eppendorf company), and is reported as the n-fold difference relative to target gene mRNA expression in the calibrator group (the group of mice immunized with saline) .
MicroRNAs expressed by macrophages stimulated in vitro by docetaxel
Sequences of primer for quantitative RT-PCR of microRNA
Universal reverse primer
Reverse primer: 5′-GCGAGCACAGAATTAATACGACTC-3′
Reverse transcription primer
Poly (T) adapter: 5′-GCGAGCACAGAATTAATACGACTCACTATAGG (T)12VN-3′ (V = A,G,C; N = A,T,G,C) [Virginie Olive 2009]
Data are expressed as mean ± standard deviations (S.D.) in addition to HI titers which are expressed as geometrical mean titer (GMT) ± standard deviations (S.D.) Boniferroni method was used to compare the parameters between groups by SPSS16.0. P-values of less than 0.05 were considered statistically significant.
This study was supported by the National Scientific Foundation of China (30771592). We thank ZY Zhu for providing the split inactivated influenza virus antigen.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.