Efficient induction of tumoricidal cytotoxic T lymphocytes by HLA-A0201 restricted, melanoma associated, L(27)Melan-A/MART-1(26-35) peptide encapsulated into virosomes in vitro

Cancer immunotherapy requires the induction of HLA class I restricted cytotoxic T lymphocytes (CTL) specific for tumor associated antigens (TAA). While a number of TAA have been identified, there is an urgent need for the development of adjuvants capable of stimulating CTL responsiveness. Previously, we reported the capacity of immunopotentiating reconstituted influenza virosomes (IRIV) to enhance CTL responses specific for synthetic peptides simultaneously added to cultures in soluble form. This effect was based on IRIV mediated activation of CD4(+) T cells. Here we investigated the "in vitro" immunogenicity of a novel virosome formulation coupling in a single reagent the adjuvant power of IRIV to the capacity of liposomes to efficiently encapsulate synthetic peptides. As a model epitope we chose L(27)Melan-A/Mart-1(26-35) HLA-A0201 restricted peptide from a melanoma-associated antigen widely used in tumor immunotherapy. The reagent thus developed induced the proliferation of CD4(+) T cells characterized by a T helper 1 cytokine profile and CXCR3 expression. Most importantly, it significantly enhanced the generation of L(27)Melan-A/Mart-1(26-35) specific CTL, as compared to soluble peptides, in particular at low nominal epitope concentrations (<1 microg/ml). These effector cells were able to efficiently kill HBL melanoma cells expressing Melan-A/MART-1 and HLA-A0201. The adjuvant effects observed were also detectable in the absence of CD4(+) T cells. Taken together our results suggest that this highly immunogenic antigenic formulation might qualify for clinical use in active, antigen-specific, melanoma immunotherapy.     

Primary and memory T cell responses induced by hepatitis C virus multiepitope long peptides

To develop a vaccine against hepatitis C virus, we synthesized four long peptides from nonstructural proteins NS3, NS4 and NS5B containing HLA-class I and class II epitopes mainly inducing responses in natural infection. In HLA-A2.1 transgenic mice, the four peptides primed higher CTL responses to 6:7 minimal HLA-A2 epitopes than those induced by the minimal epitopes. HLA-A2.1/HLA-DR1 transgenic mice immunized with one peptide, containing a class II epitope implicated in viral resolution, developed IFNgamma-producing CD4+-T and CD8+-T cells. These peptides recalled HCV-specific IFNgamma-producing cells from HCV-infected patients' PBMC. This support the selection of these domains for inclusion in a vaccine formulation.     

Evaluation of antitumor immunity efficacy of epitope-based vaccine with B16 cell line coexpressing HLA-A2/H-2kb and CTL multiepitope in HLA transgenic mice

Epitope-based vaccination strategies designed to induce tumor-specific CD8 CTL are being widely considered for cancer immunotherapy. HLA-A2-transgenic mouse is a useful tool for measuring the CTL responses in vitro. However, tumor vaccine development is required to address the variables that are not easily evaluated by in vitro assays. With the objective of extending the usage of A2-tansgenic mouse in vaccine efficacy assay, here, we established a B16 tumor cell line coexpressing HLA-A*0201/H-2Kb chimeric gene and a polyepitope construct based on the use of a mammalian expression vector pIRES. The value as a tool for evaluating the antitumor efficacy in vitro as well as in experimental tumor challenge model in vivo has been tested. We found that priming with the polyepitope construct and boosting with the mixture of peptide in A2-transgenic mice resulted in: (1) CTL responses not only against the peptide-sensitized T2 and SW480 cell lines but also the non-sensitized reconstructed B16 cell line; (2) expression of HLA-A*0201/H-2Kb chimeric gene and polyepitopes by B16 led to its rejection by immunized A2-transgenic mice. These data established that the reconstructed B16 cell line stably expressed and efficiently presented the HCC-derived CTL epitopes, making B16 based melanoma suitable for the evaluation of the antitumor efficacy of immune responses to these epitopes. Collectively, these data indicate that the use of this method allows for directly testing of HLA-A2 restricted epitope immunogenicity in the A2-transgenic mice.     

A vector-based minigene vaccine approach results in strong induction of T-cell responses specific of hepatitis C virus

Multiepitope-based vaccines against hepatitis C virus (HCV) were designed in the form of three minigenes encompassing four domains of the NS3, NS4 and NS5B proteins that contain multiple class I/II restricted epitopes. The polyEp-WT minigene encodes all four domains in fusion, the polyEp-C minigene encodes the same fusion but optimised for mammalian translation and the polyEp-E3 minigene has an additional endoplasmic reticulum targeting sequence. Whereas the minigenes vectorised by DNA were poorly immunogenic, adenovirus vectorisation induced strong and broader IFNgamma-ELISpot and CTL responses in HLA-A2 transgenic mice. In addition, polyEp-WT and polyEp-E3 responses were found cross-reactive in a recombinant Listeria-NS3-based surrogate challenge. This study illustrates the potency of vectorised minigenes in the field of HCV vaccine development.     

A novel multivalent human CTL peptide construct elicits robust cellular immune responses in HLA-A*0201 transgenic mice: implications for HTLV-1 vaccine design

Cytotoxic T-lymphocytes are critical in the clearance of chronic viral infections such as HTLV-1. Peptide-based vaccines may have potential application in invoking antiviral CTL responses. In the development of vaccination strategies, it is becoming increasingly important to elicit a broad immune response against several epitopes simultaneously that may provide large population coverage. In the present study, we addressed this issue, namely the processing and presentation of multiple CTL epitopes simultaneously for the generation of multispecific CTL responses. We designed a novel multivalent peptide consisting of three HLA-A(*)0201 restricted CTL epitopes, with intervening double arginine residues in tandem. These epitopes were derived from the HTLV-1 regulatory protein Tax, which is an attractive target for vaccine development against HTLV-1. Arginine residues were included to provide cleavage sites for proteasomes, to generate the intended MHC Class I ligands. Proteasomal digestion studies and mass spectrometry analysis showed cleavage of the multivalent construct to generate the individual epitopes. Immunization of HLA-A(*)0201 transgenic mice with this construct efficiently elicited cellular responses to each intended epitope in vivo, further validating the applicability of this approach. These data may have potential in the development of immunotherapeutic strategies for the treatment of HTLV-1 disease and in the future design of multivalent subunit peptide vaccines.     

Attenuated poxviruses expressing a synthetic HIV protein stimulate HLA-A2-restricted cytotoxic T-cell responses

Efficient HIV vaccines have to trigger cell-mediated immunity directed against various viral antigens. However little is known about the breadth of the response induced by vaccines carrying multiple proteins. Here, we report on the immunogenicity of a construct harbouring a fusion of the HIV-1 IIIB gag, pol and nef genes (gpn) designed for optimal safety and equimolar expression of the HIV proteins. The attenuated poxviruses, MVA and NYVAC, harbouring the gpn construct, induced potent immune responses in conventional mice characterised by stimulation of Gpn-specific IFN-gamma-producing cells and cytotoxic T cells. In HLA-A2 transgenic mice, recombinant MVA elicited cytotoxic responses against epitopes recognised in most HLA-A2+ HIV-1-infected individuals. We also found that the MVA vaccine triggered the in vitro expansion of peripheral blood cells isolated from a HIV-1-seropositive patient and with similar specificity as found in immunised HLA-A2 transgenic mice. In conclusion, the synthetic HIV polyantigen Gpn delivered by MVA is immunogenic, efficiently processed and presented by human MHC class I molecules.     

Induction of a cytotoxic T-cell response to HIV-1 proteins with short synthetic peptides and human compatible adjuvants.

The goal of this study was the induction of a strong CTL response against multiple CTL epitopes present in HIV proteins using short synthetic peptides. Four HLA-A2.1 restricted peptides (RT 476-484, p17 77-85, gp41 814-823, RT 956-964) that showed stable binding to the HLA-A2.1 molecule in an in vitro binding assay were able to elicit a strong specific immune response in HLA-A2.1 transgenic mice when injected with IFA or Montanide. The use of biodegradable microspheres (MS) as adjuvant was also successfully tested for all peptides. When the peptides were injected as a mixture the response was weaker as compared to individual injections of the peptides indicating the occurrence of immunodominance (ID). We are currently investigating whether ID can be overcome by a combined injection of peptide loaded MS with different release patterns. Taken together, it seems feasible to induce a specific CTL response in HLA-A2.1 transgenic mice against several HIV proteins using short synthetic peptides and human compatible adjuvants.     

Definition of MHC-restricted CTL epitopes from non-variable number of tandem repeat sequence of MUC1

Mucin1 (MUC1) is expressed ubiquitously on breast cancer cells and is a potential target for the generation of cytotoxic T cells for vaccination against breast cancer. Thus far studies of the immunogenicity of MUC1 have used peptides from the variable number of tandem repeat (VNTR); mice so immunised can generate strong cellular and antibody responses to the VNTR of human MUC1. We now demonstrate that significant CTL and CTLp can be induced to other regions of MUC1. Using the whole native MUC1 molecule, the human milk fat globule membrane antigen (HMFG) linked to mannan, cytotoxic T cell precursors (CTLp) can be generated in BALB/c, C57BL/6, transgenic HLA-A*0201/K(b) and double transgenic HLA-A*0201/K(b)xhuman MUC1 (A2 K(b)MUC1) mice. By immunising with HMFG and testing selectively on (a) extracellular (non-VNTR); (b) VNTR and (c) intracellular peptides, it was shown that all three regions generated effective CTL. Further, the CTL responses to non-VNTR peptides were as strong as those generated to the VNTR. Epitope prediction algorithms were not particularly helpful to describe CTL epitopes: overlapping peptides had to be synthesised and tested to find the epitopes. Thus, for CTL generation, the whole HMFG molecule is a powerful immunogen when linked to mannan, especially as multiple peptide epitopes for presentation by many Class I molecules are contained within the one molecule. Furthermore, Class I restricted MUC1 CTL were generated in double transgenic A2 K(b)MUC1 mice by immunising with mannan-native mucin (HMFG), suggesting that tolerance to MUC1 can be overcome with mannan-HMFG.     

Induction of protective antiviral cytotoxic T cells by a tubular structure capable of carrying large foreign sequences

Bluetongue virus (BTV) produces large numbers of tubules during infection which are formed by a single virus coded non-structural protein, NS1. The NS1 protein has been fused with full length green fluorescent protein (GFP) and was shown to retain the capacity to form tubules when expressed in heterologous expression systems. Moreover, recombinant purified chimeric tubules were demonstrated to be internalized by macrophages and dendritic cells. The ability of such chimeric tubules to induce protective cytotoxic T lymphocytes (CTL) responses has been assessed by generating chimeric tubules carrying a single CD8(+) T cell epitope from the lymphocytic choriomeningitis virus (LCMV) nucleoprotein. These chimeric tubules were recognized by MHC class I restricted T cell hybridoma in vitro and induced in vivo strong CD8(+) class I-restricted CTL responses in immunized mice. Further, the immunized mice were protected when challenged with a lethal dose of LCMV. This is the first study that demonstrates that the virus derived tubules synthesized by a recombinant non-structural protein carrying a single viral CTL epitope could induce protective immunity against a lethal viral challenge. Since recombinant tubules carrying large inserts can be purified at a large quantity from insect cells, they have potential to develop as safe multi-CTL vaccine delivery systems.     

Quantification of the number of cytotoxic T cells specific for an immunodominant HCV-specific CTL epitope primed by DNA immunization

Priming of strong cellular immune responses to hepatitis C (HCV) is thought to be important for eradication of infection. Although productive infection of HCV occurs only reproducibly in humans and chimpanzees, definition of HCV-specific T cell epitopes in mice is necessary to screen efficiently HCV vaccine strategies for their ability to prime cellular immune responses. Out of seven strains of mice screened for immunodominant CTL epitopes against HCV-1a Core, E2, NS5a and NS5b, only one epitope (p214K9) in only one mouse strain was identified. Enumeration of p214K9-specific CD8+ cells by flow cytometry revealed that the number of epitope specific CTL primed by 'naked' DNA immunization was lower than that reported during viral infection. The p214K9 epitope described here, combined with analysis of CTL responses by flow cytometry, should be instrumental in ranking various HCV vaccine strategies for their ability to prime CTL responses.     

Highly conserved pattern of recognition of influenza A wild-type and variant CD8+ CTL epitopes in HLA-A2+ humans and transgenic HLA-A2+/H2 class I-deficient mice

As an in vivo model for studying human MHC (HLA) class I-restricted CTL responses to viral infection, we established a series of HLA Tg mice expressing HLA-A2, -B7 or -B27 human/mouse hybrid genes on a background deficient for H2 class I (Tg HLA(hyb)/H2 class I DKO). To determine whether CTL recognition of influenza A (flu) infection in Tg HLA-A2(hyb)/H2 DKO mice is similar to HLA-A2+ humans, we compared the HLA-A2-restricted Tg mouse and human CD8+ T-cell responses to an immunodominant flu epitope (wild-type [WT] M1 58-66), as well as a variant of this peptide (var. M1 58-66). Similar to HLA-A2+ humans, our results show WT M1 58-66 is likely the dominant CTL epitope recognized in infected Tg HLA-A2(hyb)/H2 DKO mice. Var. M1 58-66 was also recognized by WT peptide-reactive T cells from both HLA-A2+ humans and Tg mice, although slightly less efficiently than WT M1 58-66 in both cases. Reduced variant recognition was shown to be associated with reduced peptide/A2 binding, as well as a more limited repertoire of utilized TCR Vbeta chains. The similar pattern of recognition and cross reaction observed here for the WT and variant M1 58-66 epitopes with HLA-A2 by human and Tg HLA mouse CTLs indicates that A2-dependent events of Ag processing, presentation and recognition are well-conserved between species. These findings demonstrate that this Tg HLA-A2/H2 DKO model will aid identification and development of epitopes as vaccines for numerous viral and tumor antigens for the HLA-A2 supertype.     

Characterization of immune response to novel HLA-A2-restricted epitopes from zinc transporter 8 in type 1 diabetes

ObjectiveZnT8-specific CD8+ T cells in human type 1 diabetes (T1D) have been reported recently, although the results from different laboratories are inconsistent. We aimed to characterize these ZnT8 specific CD8+ T cells and validate assays to screen peptide libraries.MethodsWe screened HLA-A2-restricted T cell candidate peptides of ZnT8 with different methods including computer algorithms, MHC-peptide binding and dissociation assays in T2 cell line, identification in HLA-A2 transgenic (Tg) mice and in vivo CTL assays. Then ELISpot assay was used to measure peptide-reactive T cell responses in 49 HLA-A2-restricted T1D patients.ResultsWe demonstrated that ZnT8_{107–116(115)}, ZnT8_110–118, and ZnT8_177–186 were novel HLA-A*0201-restricted CTL epitopes in T1D patients. ZnT8_{107–116(115)}, ZnT8_115–123, ZnT8_153–161, ZnT8_177–186 and ZnT8_291–300 represent potentially major biomarkers for T1D. T cell responses against these epitopes showed different distributions between recently diagnosed and long-standing patients. Furthermore, they displayed discriminating performance among different ethnicities. We also compared the performance of the epitope identification strategies used herein. The epitopes which exhibited strong immunogenicity in HLA-A2 Tg mice were also well recognized by T1D patients.ConclusionsThe differences in autoimmune T cell responses among T1D individuals may open new avenues toward T1D prediction and prevention. It also provides efficient strategies for immune intervention.     

Delivery of tumor associated antigens to antigen presenting cells using penetratin induces potent immune responses

Cytoplasmic delivery of proteins or CTL epitopes is crucial for the presentation of antigen for the generation of CTL. We previously described the use of the 16-amino acid peptide penetratin from the Drosophila Antennapedia domain (Int) to transport CTL epitopes into cells. Here we show that, Int, incorporating MUC1 CTL epitopes in tandem is able to facilitate their rapid uptake by macrophages and dendritic cells (DC) in an energy-dependent endocytic pathway. We also demonstrate for the first time that Int conjugated proteins are also able to be efficiently taken up by DC. Furthermore, C57BL/6 and HLA-A2 transgenic mice immunized with the Int-peptides or Int-proteins induce strong IFN-gamma secreting T cells and weak IgG1 antibodies. Immunized C57BL/6 mice were protected against the growth of a MUC1(+) tumor cell line.     

Contrasting Epstein-Barr virus-specific cytotoxic T cell responses to HLA A2-restricted epitopes in humans and HLA transgenic mice: implications for vaccine design

This study investigates the hierarchy of cytotoxic T cell (CTL) responses to twelve HLA A2-restricted epitopes from the latent, lytic and structural proteins of Epstein-Barr virus (EBV) in acute infectious mononucleosis and in healthy seropositive donors and the relative immunogenecity of these epitopes in transgenic mice. Responses to the lytic epitope were uniformly strong in all healthy seropositive individuals and acute infectious mononucleosis donors while moderate or low responses were observed to the latent and structural epitopes, respectively in both groups studied. In contrast, when HLA A2/Kb transgenic mice were immunised with these peptide epitopes, CTL responses were observed to all epitopes with a maximal response to the epitopes within the structural proteins and low to moderate responses to the latent epitopes. This hierarchy of CTL responses in mice was also reflected in an MHC stabilisation analysis. These contrasting CTL responses in humans following natural infection compared to the immunogenicity of these epitopes and their ability to stabilise MHC may need to be considered when designing an EBV vaccine.     

A recombinant live attenuated measles vaccine vector primes effective HLA-A0201-restricted cytotoxic T lymphocytes and broadly neutralizing antibodies against HIV-1 conserved epitopes

Live attenuated measles vaccine (MV) could provide a safe and efficient pediatric vaccination vector to immunize children simultaneously against measles and human immunodeficiency virus type 1 (HIV-1). To evaluate the capacity of a vector derived from the certified Schwarz measles vaccine (MVSchw) to prime effective cytotoxic T cells (CTL) and broad neutralizing antibodies against HIV-1 conserved epitopes, we generated recombinant MVSchw viruses expressing HIV-1 antigens. We demonstrate that a recombinant MVSchw virus expressing an HIV-1-derived CTL polyepitope primes effective HLA-A0201-restricted CTLs against multiple conserved HIV-1 epitopes in mice susceptible to measles and humanized for the major histocompatibility complex (MHC) class-I molecule HLA-A0201. Additionally, we show that a recombinant MVSchw virus expressing an HIV-1(89.6) gp140 glycoprotein whose hyper variable V1, V2 and V3 loops were deleted (DeltaV1V2V3gp140), induces broadly neutralizing antibodies against HIV-1 primary isolates. These results show that the MVSchw pediatric vaccination vector induces efficient cellular and humoral HIV-specific immune responses.     

Identification of novel HLA-A*0201-restricted cytotoxic T lymphocyte epitopes from Zinc Transporter 8

Numerous evidences demonstrated that type 1 diabetes (T1D) is due to a loss of immune tolerance to islet antigens, and CD8⁺ T cells play an important role in the development of T1D. Zinc Transporter 8 (ZnT8) has emerged in recent years as a target of disease-associated autoreactive T cells in human T1D. However, ZnT8-associated CTL specific-peptides have not been identified. In this study, we predicted and identified HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) epitopes derived from ZnT8, and utilized it to immunize HLA-A2.1/Kb transgenic (Tg) mice. The results demonstrated that peptides of ZnT8 containing residues 107–115, 115–123 and 145–153 could elicit specific CTLs in vitro, and induce diabetes in mice. The results suggest that these specific peptides are novel HLA-A*0201-restricted CTL epitopes, and could have therapeutic potential in preventing of T1D disease.     

Comparison of the immune response to a self antigen after DNA immunisation of HLA*A201/H-2Kb and HHD transgenic mice

HHD mice which express human HLA-A2 but no mouse class I, have been shown to have improved cytotoxic lymphocyte (CTL) responses to foreign antigens compared to HLA*A201/Kb transgenic mice. We have previously designed a DNA vaccine to a self antigen. Vaccination of A2.1/H-2Kb mice with this vaccine resulted in CTL in one third of the mice, but the frequency of antigen-specific cells was low. An increased frequency was observed in HHD mice, moreover CTL were obtained from all immunised mice. HHD mice are therefore useful for studying immunisation strategies for inducing and maintaining CD8 responses to over-expressed self antigens.     

Immunotherapy with mannan-MUC1 and IL-12 in MUC1 transgenic mice

Mice immunised with oxidised mannan-MUC1 fusion protein (M-FP) develop MHC restricted CD8(+) cytotoxic T cells. We now demonstrate that in MUC1/HLA-A2 transgenic mice, IL-12 gives enhanced CTL, CTLp and tumor protection. CTLp in MUC1 transgenic mice with M-FP were 1/55,000, and with IL-12, this increased to 1/19,000, with improved tumor protection. Thus, IL-12 is important for effective CTL responses to MUC1 in transgenic mice.     

Novel CD8+ T cell-based vaccine stimulates Gp120-specific CTL responses leading to therapeutic and long-term immunity in transgenic HLA-A2 mice

The limitations of highly active anti-retroviral therapy have necessitated the development of alternative therapeutics for human immunodeficiency virus type-1 (HIV-1)-infected patients with dysfunctional dendritic cells (DCs) and CD4(+) T cell deficiency. We previously demonstrated that HIV-1 Gp120-specific T cell-based Gp120-Texo vaccine by using ConA-stimulated C57BL/6 (B6) mouse CD8(+) T (ConA-T) cells with uptake of pcDNA(Gp120)-transfected B6 mouse DC line DC2.4 (DC2.4(Gp120))-released exosomes (EXO(Gp120)) was capable of stimulating DC and CD4(+) T cell-independent CD8(+) cytotoxic T lymphocyte (CTL) responses detected in wild-type B6 mice using non-specific PE-anti-CD44 and anti-IFN-γ antibody staining by flow cytometry. To assess effectiveness of Gp120-Texo vaccine in transgenic (Tg) HLA-A2 mice mimicking the human situation, we constructed adenoviral vector AdV(Gp120) expressing HIV-1 GP120 by recombinant DNA technology, and generated Gp120-Texo vaccine by using Tg HLA-A2 mouse CD8(+) ConA-T cells with uptake of AdV(Gp120)-transfected HLA-A2 mouse bone marrow DC (DC(Gp120))-released EXO(Gp120). We then performed animal studies to assess Gp120-Texo-induced stimulation of Gp120-specific CTL responses and antitumor immunity in Tg HLA-A2 mice. We demonstrate that Gp120-Texo vaccine stimulates Gp120-specific CTL responses detected in Tg HLA-A2 mice using Gp120-specific PE-HLA-A2/Gp120 peptide (KLTPLCVTL) tetramer staining by flow cytometry. These Gp120-specific CTLs are capable of further differentiating into functional effectors with killing activity to Gp120 peptide-pulsed splenocytes in vivo. In addition, Gp120-Texo vaccine also induces Gp120-specific preventive, therapeutic (for 6 day tumor lung metastasis) and CD4(+) T cell-independent long-term immunity against B16 melanoma BL6-10(Gp120/A2Kb) expressing both Gp120 and A2Kb (α1 and α2 domains of HLA-A2 and α3 domain of H-2K(b)) in Tg HLA-A2 mice. Taken together, the novel CD8(+) Gp120-Texo vaccine capable of stimulating efficient CD4(+) T cell-independent Gp120-specific CD8(+) CTL responses leading to therapeutic and long-term immunity in Tg HLA-A2 mice may represent a new immunotherapeutic vaccine for treatment of HIV-1 patients with CD4(+) T cell deficiency.     

Potent enhancement of cellular and humoral immune responses against recombinant hepatitis B antigens using AS02A adjuvant in healthy adults

Recombinant subunit protein vaccines generally elicit good humoral immune responses, weak helper T cell responses and no cytotoxic T cell responses. Certain adjuvants are known to enhance humoral and cellular immune responses. This study evaluated the humoral, CD4+ T helper and CTL responses induced by the recombinant SL* protein adjuvanted with AS02A in comparison with non-adjuvanted SL* in PBS in two groups of 15 healthy adult volunteers. The AS02A adjuvant contains monophosphoryl lipid A (MPL), QS21 and an oil in water emulsion. The adjuvanted vaccine induced fast and vigorous humoral and helper T cell responses of the Th1 type. Using a pool of overlapping 20mer peptides a cytotoxic response was detected in 6 out of 14 HLA-A2-positive (+) and HLA-A2-negative (-) recipients of the adjuvanted vaccine. All HLA-A2-positive subjects in the adjuvanted group and up to 30% of the subjects in the SL* PBS group displayed a CTL response against selected HLA-A2-restricted CD8+ T cell epitopes. The non-adjuvanted vaccine induced a very weak antibody response and no helper T cell responses. Local and general reactions were more frequently reported by AS02A recipients than in the non-adjuvanted group but the safety profile was considered acceptable. AS02A can be considered as a useful adjuvant that strongly enhances the cellular and humoral responses of subunit protein vaccines.