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 [23
]. 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 [24
]. 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 [24
]. 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 and , 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 [22
]. 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 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 B), leading to increased production of IFN-γ, IL-12, IL-4 and IL-10 by splenocytes (Figure A). 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 A). 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 [25
]. In humans, HI titer of 1:40 or higher is normally considered protective [26
]. Figure 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 ), 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 [27
]. Garnett et al. recently reported that docetaxel modulated CD4+
, natural killer cells, and Treg populations and enhanced CD8+
]. 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 [28
]. 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 [29
]. MiR-181a has been ascribed functions in hematopoietic differentiation and in T cell differentiation [30
]. 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 ). Only miR-146a showed significant increase 1 hour after stimulation of RAW264.7 cells with docetaxel (Figure D). Three hours later, miR-155, miR-150 and miR-146a expressions were enhanced (Figure B, C, D), while miR-181a and miR-125b showed no significant change (Figure A, 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 [21
]. 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 [32
]. 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 [33
]. 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 [34
]. However, frequent use of alum-adjuvanted vaccines could be one of the reasons for IgE-mediated allergy due to activated Th2 immune response [35
]. In the present study, alum but not docetaxel promoted the production of IgE significantly higher than that of mice immunized with HA only (Figure ). 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 .