The combined molecular adjuvant CASAC enhances the CD8+ T cell response to a tumor-associated self-antigen in aged, immunosenescent mice

Ineffective induction of T cell mediated immunity in older individuals remains a persistent challenge for vaccine development. Thus, there is a need for more efficient and sophisticated adjuvants that will complement novel vaccine strategies for the elderly. To this end, we have investigated a previously optimized, combined molecular adjuvant, CASAC (Combined Adjuvant for Synergistic Activation of Cellular immunity), incorporating two complementary Toll-like receptor agonists, CpG and polyI:C, a class-II epitope, and interferon (IFN)-γ in aged mice. In aged mice with typical features of immunosenescence, antigen specific CD8+ T cell responses were stimulated after serial vaccinations with CASAC or Complete/Incomplete Freund’s Adjuvant (CFA/IFA) and a class I epitope, deriving either from ovalbumin (SIINFEKL, SIL) or the melanoma-associated self-antigen, tyrosinase-related protein-2 (SVYDFFVWL, SVL). Pentamer analysis revealed that aged, CASAC/SIL-vaccinated animals had substantially higher frequencies of H-2Kb/SIL-specific CD8+ T cells compared to the CFA/IFA-vaccinated groups. Similarly, higher frequencies of H-2Kb/SVL-pentamer+ and IFN-γ+ CD8+ T cells were detected in the aged, CASAC + SVL-vaccinated mice than in their CFA/IFA-vaccinated counterparts. In both antigen settings, CASAC promoted significantly better functional CD8+ T cell activity. These studies demonstrate that functional CD8+ T cells, specific for both foreign and tumour-associated self-antigens, can be effectively induced in aged immunosenescent mice using the novel multi-factorial adjuvant CASAC.


Introduction
The sequelae of immunosenescence in older individuals, including increased morbidity and mortality from infection and malignancy, represent a critically important public health problem [1]. As a consequence of accumulated dysfunctions in the immune system during ageing, vaccination of older individuals is less efficacious than in the young [2,3]. Previous attempts to increase vaccination efficacy by incorporating various adjuvants, including alum, have shown only modest success [2]. Thus, there is an unmet need for new vaccine strategies for the elderly.
Immunosenescence is characterised by decline in both adaptive and innate immune functions [4,5]. Innate immune responses are activated, mainly, by stimulation of Toll-like receptors (TLRs) [6], the expression and function of which declines with age [7]. Dendritic cells (DCs) from both young and aged individuals exhibit comparable activation in response to most TLR ligands, and are equally capable of direct and cross-presentation of antigens to T cells in vitro [8], underscoring the likely importance of TLR-induced DC activation in promoting adaptive immunity. TLR stimulation is therefore a promising strategy to enhance vaccine efficacy in the elderly. Combinations of TLR agonists may be especially effective, as demonstrated in animal models and clinical trials [6,[9][10][11][12][13].
We previously showed that triggering of multiple TLRs, using a combined adjuvant for synergistic activation of cellular immunity (CASAC), incorporating CpG, polyI:C, interferon (IFN)-γ and MHC-class I and II peptides, results in potent cytotoxic T cell-mediated immunity in young mice [14]. Optimization of the adjuvant formulation and investigation of mechanism of action were also performed [14]. We now report the ability of CASAC to improve vaccination-induced responses in aged mice by promoting induction of antigen-specific cellular immunity to both foreign and self tumourassociated peptide antigens.

Animals and vaccination procedures
Young (6-8 weeks old) and aged (18-22 months old) wild-type C57BL/6 female mice were purchased from Harlan, UK. All animal procedures were performed according to UK Home Office and institutional regulations.

In vivo cytotoxicity assay
The in vivo cytotoxicity assay was performed as previously described [14].

Statistical analysis
The Mann-Whitney U test (GraphPad Prism, USA) was used to compare distributions, with p < 0.05 considered significant.
CASAC enhances responses to a foreign antigenic CD8+ T cell epitope in aged mice CASAC was previously shown to effectively promote T cell immunity to the foreign antigen OVA in young mice [14]. We therefore investigated whether CASAC augments responses to the immunogenic OVA peptide SIL [20] in aged mice, compared to CFA/IFA as a conventional adjuvant control [21]. Using our previously described vaccination protocol [14], young and aged C57BL/6 mice were vaccinated twice at a 10-day interval with SIL, combined either with CFA/IFA or CASAC. The distribution of percentages of H-2K b /SIL-pentamer+ CD8+ T cells was significantly higher (p = 0.0003) in the CASAC-vaccinated group (median 14.55 %), compared to CFA/IFA vaccinations (0.80 %). As shown previously [14], distribution of H-2K b /SIL-pentamer+ CD8+ T cell frequencies was also found to be significantly higher (p = 0.0002) in young mice vaccinated with CASAC (median 13.80 %), compared to their CFA/IFA-treated counterparts (0.29 %). Of importance, the H-2K b /SIL-pentamer+ CD8+ T cell frequencies induced by CASAC vaccinations in both young and aged mice were similar. Although similar percentages were induced in both CASAC-vaccinated cohorts, the absolute numbers of SIL-specific CD8+ T cells (Fig. 2b) showed a significantly lower (p = 0.0033) distribution in aged mice (median 22.30) compared to their young counterparts (396.3). This was due to the lower total numbers of CD8+ T cells in aged mice (Fig. 1a). The function of SIL-specific T cells was assessed by performing in vivo cytolytic assays. Aged mice vaccinated with CASAC showed higher distribution of antigen-specific cytolytic activity (median 88.5 %; Fig. 2c) compared to the CFA/IFA-vaccinated mice (16.8 %; p = 0.0106). Similar outcomes were observed in the young groups (98.0 and 58.1 % for CASAC and CFA/IFA vaccinated groups, respectively; p = 0.0238). These results show that CASAC effectively induced CD8+ T cell mediated immunity to a foreign antigen in both aged and young mice.  The need for efficient vaccine adjuvants applies not only to infectious disease but also to cancer, a disease with increasing age-associated incidence [22]. It is therefore imperative that new adjuvants incorporated in anticancer vaccines are assessed for efficacy in aged immunosenescent individuals.
We have previously shown that immune tolerance to the melanoma-associated self-antigen TRP-2 can be overcome by vaccination of young mice with the TRP-2 peptide SVL combined with CASAC [14]. Functional SVL-specific CD8+ T cells were detected after two rounds of vaccinations; however, responses were of variable magnitude and required vaccination with the higher peptide dose of 400 μg. We therefore performed further optimization studies in young mice and found four rounds of vaccination, using 100 μg SVL peptide with CASAC, consistently induced higher frequencies of SVL-specific CD8+ T cells (data not shown). All studies with aged mice were performed with four rounds of vaccination using 100 μg SVL peptide in combination with either CASAC or CFA/IFA. Distribution of SVL-specific CD8+ T cell frequencies was higher in the SVL + CASAC-vaccinated group (median 2.5 %) compared to the SVL + CFA/IFAvaccinated group (0.7 %), although the difference did not reach statistical significance (p = 0.0673; Fig. 3a). In vivo cytotoxicity studies (Fig. 3b) showed that lysis of SVLloaded splenocytes was significantly higher in SVL + CASAC-vaccinated (median 86.6 %) compared to SVL + CFA/IFA-vaccinated aged mice (median 19.7 %; p = 0.0028). Additionally, distribution of IFN-γ+ CD8+ T cell frequencies after in vitro SVL peptide stimulation was significantly higher in the SVL + CASAC-vaccinated mice (median 8.9 %; Fig. 3c) compared to CFA/IFA-vaccinated mice (1.2 %; p = 0.0028). Thus, vaccination of aged mice with the tumour-associated SVL peptide and CASAC induced antigen-specific CD8+ T cells that had considerably better functional activity than CD8+ T cells induced by SVL + CFA/IFA-vaccination.
In conclusion, we have demonstrated that our combined molecular adjuvant CASAC effectively promotes functional antigen-specific CD8+ T cell responses to vaccination with peptides in aged mice, despite their immunosenescent phenotype. CASAC improved responses in aged mice not only to a highly immunogenic foreign antigen, but also to the tumour-associated selfantigen TRP-2 whose immunogenicity is being evaluated in clinical trials [23]. Restoration of response to vaccination in immunosenescent aged mice by CASAC likely reflects the benefits of multiple TLR triggering on DC function [14,24,25] and provision of IFN-γ could substitute for lack of IFN-γ from CD8+ memory cells during the early phase of immune response. Since CASAC comprises a combination of agents that individually are approved for human use, our findings suggest that a CASAC-based vaccination strategy may be amenable to rapid clinical translation, particularly against chronically experienced antigens such as persistent infections or tumour-associated antigens in older people.

Competing interests
The authors declare that they have no competing interests. Fig. 3 CASAC enhances CD8+ T cell responses to a tumour-associated epitope in aged mice. Aged C57BL/6 mice were vaccinated four times with the SVL peptide from TRP-2 in combination with either CFA/IFA (circles) or CASAC (triangles). a The percentage of H-2K b /SVL-pentamer + CD8+ T cells was assessed after staining with H-2K b /SVL-pentamer. b % Lysis of target cells was assessed (as explained in the legend to Fig. 2c) using CFSE low -SVL-loaded and CFSE high -SIL-loaded splenocytes as antigen-specific and control targets, respectively. c Production and intracellular accumulation of IFN-γ in CD8 + T cells in response to the vaccinated peptide was assessed using flow cytometry, after in vitro SVL peptide stimulation of splenocytes for 5 h. Each symbol represents an individual mouse and the median is indicated by a horizontal line. Data is pooled from 2 independent experiments. The Mann-Whitney U test was used to compare distributions