Establishment of animal models to analyze the kinetics and distribution of human tumor antigen-specific CD8 T cells

Many patients develop tumor antigen-specific T cell responses detectable in peripheral blood mononuclear cells (PBMCs) following cancer vaccine. However, measurable tumor regression is observed in a limited number of patients receiving cancer vaccines. There is a need to re-evaluate systemically the immune responses induced by cancer vaccines. Here, we established animal models targeting two human cancer/testis antigens, NY-ESO-1 and MAGE-A4. Cytotoxic T lymphocyte (CTL) epitopes of these antigens were investigated by immunizing BALB/c mice with plasmids encoding the entire sequences of NY-ESO-1 or MAGE-A4. CD8⁺ T cells specific for NY-ESO-1 or MAGE-A4 were able to be detected by ELISPOT assays using antigen presenting cells pulsed with overlapping peptides covering the whole protein, indicating the high immunogenicity of these antigens in mice. Truncation of these peptides revealed that NY-ESO-1-specific CD8⁺ T cells recognized D^d-restricted 8mer peptides, NY-ESO-1_81-88. MAGE-A4-specific CD8⁺ T cells recognized D^d-restricted 9mer peptides, MAGE-A4_265-273. MHC/peptide tetramers allowed us to analyze the kinetics and distribution of the antigen-specific immune responses, and we found that stronger antigen-specific CD8⁺ T cell responses were required for more effective anti-tumor activity. Taken together, these animal models are valuable for evaluation of immune responses and optimization of the efficacy of cancer vaccines.     

Engineering superior DNA vaccines: MHC class I single chain trimers bypass antigen processing and enhance the immune response to low affinity antigens

It is commonly believed that delivery of antigen into the class I antigen presentation pathway is a limiting factor in the clinical translation of DNA vaccines. This is of particular concern in the context of cancer vaccine development as many immunodominant peptides derived from self tumor antigens are not processed and presented efficiently. To address this limitation, we have engineered completely assembled peptide/MHC class I complexes whereby all three components (class I heavy chain, β₂m, and peptide) are attached by flexible linkers and expressed as a single polypeptide (single chain trimers or SCT). In this study, we tested the efficacy of progressive generations of SCT DNA vaccines engineered to (1) enhance peptide binding, (2) enhance interaction with the CD8 coreceptor, and/or (3) activate CD4⁺ helper T cells. Disulfide trap SCT (dtSCT) have been engineered to improve peptide binding, with mutations designed to create a disulfide bond between the class I heavy chain and the peptide linker. dtSCT DNA vaccines dramatically enhance the immune response to model low affinity antigens as measured by ELISPOT analysis and tumor challenge. SCT engineered to enhance interaction with the CD8 coreceptor have a higher affinity for the TCR/CD8 complex, and are associated with more robust CD8⁺ T cell responses following vaccination. Finally, SCT constructs that coexpress a universal helper epitope PADRE, dramatically enhance CD8⁺ T cell responses. Taken together, our data demonstrate that dtSCT DNA vaccines coexpressing a universal CD4 epitope are highly effective in generating immune responses to poorly processed and presented cancer antigens.     

CD8 T cell adjuvant effects of Salmonella FliCd flagellin in live vaccine vectors or as purified protein

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2^d-restricted CD8⁺ T cell-specific epitope (CS_280–288) derived from the Plasmodium yoelii circumsporozoite (CS) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS_280–288 peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS_280–288 peptide. The results showed that CS_280–288-specific cytotoxic CD8⁺ T cells were primed when BALB/c mice were orally inoculated with the expressing the CS_280–288 epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS_280–288 peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8⁺ T cell responses without the need of a heterologous booster immunization. The CD8⁺ T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c⁺ dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations.     

DNA vaccination as a tool to identify subdominant CD8 T cell epitopes

DNA vaccination is highly efficient at inducing CD8⁺ T cell responses in animal models. Here we investigated whether DNA vaccine technology could be exploited to identify subdominant cytotoxic T lymphocytes (CTL) epitopes. Previous studies have shown that the Sendai virus HN protein does not induce a CD8⁺ T cell response in C57BL/6 mice. Thus, we vaccinated C57BL/6 mice with a DNA vaccine encoding Sendai virus hemagglutinin neuraminidase (HN) protein. The data show that this strategy elicited a potent D^b-restricted CD8⁺ CTL response against at least one subdominant HN-derived epitope. These CTL were able to lyse Sendai virus-infected target cells, demonstrating that the epitope was appropriately processed and present at sufficient levels for T cell recognition. However, these cells did not confer protection against lethal challenge with Sendai virus. These data demonstrate the capacity of DNA vaccine to raise CTL responses to subdominant epitopes, but show that such responses may be limited in their efficacy against non-persistent viruses.     

Prediction and identification of novel IBV S1 protein derived CTL epitopes in chicken

Infectious bronchitis virus (IBV) is a major pathogen common in the poultry industry. Broad cytotoxic T lymphocyte (CTL) response against IBV is one of the crucial factors that help to control viral replication. Spike glycoproteins on the surface of the IBV virion harbor major T cell epitopes. In this study, based on the peptide-binding motifs of chicken MHC I molecules for the BF2*4, BF2*12, BF2*15, and BF2*19 haplotypes, potential CTL epitopes were predicted using S1 proteins from different IBV strains. Twenty-one peptides were predicted to be potential CTL epitopes; they were manually synthesized and the CTL responses to them tested in vitro. Spleen lymphocytes were collected from specific-pathogen free (SPF) chicken that had been immunized with the S1 protein expression plasmid, pV-S1, and were stimulated by the synthesized peptides. IFN-γ secretion and CD8⁺ T cell proliferation in chickens were tested by ELISpot array and flow cytometry, respectively. Four epitopes (P8_SRIQTATDP, P9_SRNATGSQP, P18_GAYAVVNV, and P19_SRIQTATQP) were identified to stimulate CD8⁺ T cell proliferation and IFN-γ secretion, indicating their efficacy as CTL epitopes in chicken. Poly-CTL-epitope DNA vaccine (pV-S1T) was constructed by inserting nucleotide sequences encoding the P8, P9, P18, and P19 CTL epitopes into the pVAX1 vector. Chickens were vaccinated with either pV-S1, pV-S1T, or pVAX1 and the protection efficacy was analyzed, revealing that ninety percent of chickens immunized with pV-S1T were protected after challenge with 10⁶ ELD₅₀ of IBV, demonstrating that these novel CTL epitopes were effective against IBV challenge. This study provides a new method to screen virus CTL epitopes in chicken and to develop poly-CTL-epitope DNA vaccines.     

A novel self-assembled nanoparticle vaccine with HIV-1 Tat₄₉₋₅₇/HPV16 E7₄₉₋₅₇ fusion peptide and GM-CSF DNA elicits potent and prolonged CD8⁺ T cell-dependent anti-tumor immunity in mice

Peptide-based vaccines derived from the E7 protein of human papillomavirus (HPV) type 16 were developed to induce effective T cell responses against established cervical cancer, but have met with limited clinical success. It is necessary to develop novel peptide-based strategies to substantially improve the immune response against HPV16-related cancer. In this study, we aimed to design a novel peptide-based self-assembled nanoparticle HPV16 vaccine by combining the cell-penetrating peptide HIV-1 Tat_49-57 that was fused with the HPV16 E7_49-57 cytotoxic T lymphocyte (CTL) epitope and the granulocyte-macrophage colony stimulating factor (GM-CSF) gene, and to investigate how it improves the immune response and the therapeutic outcome ex vivo and in vivo. Nanoparticles were prepared and identified by transmission electron microscopy (TEM), gel retardation and DNase I protection assays. This type of vaccine formulation formed the 20-80 nm nanoparticles, and greatly improved epitope-specific immunity both ex vivo and in vivo. Importantly, this vaccine type was associated with decreased tumor growth and enhanced long-term survival in the prophylactic and therapeutic mouse models. The underlying mechanisms were determined to involve priming of enhanced frequency of CD8⁺ memory T subtype cells. These results suggest that the nanoparticle Tat-E7/pGM-CSF represents a promising novel approach to enhance the potency of peptide-based cervical cancer vaccines, and this vaccine design strategy may act as a useful reference for research of virus-associated diseases and specific tumor immunotherapies.     

An intact signal peptide on dengue virus E protein enhances immunogenicity for CD8 T cells and antibody when expressed from modified vaccinia Ankara

Dengue is a global public health concern and this is aggravated by a lack of vaccines or antiviral therapies. Despite the well-known role of CD8⁺ T cells in the immunopathogenesis of Dengue virus (DENV), only recent studies have highlighted the importance of this arm of the immune response in protection against the disease. Thus, the majority of DENV vaccine candidates are designed to achieve protective titers of neutralizing antibodies, with less regard for cellular responses. Here, we used a mouse model to investigate CD8⁺ T cell and humoral responses to a set of potential DENV vaccines based on recombinant modified vaccinia virus Ankara (rMVA). To enable this study, we identified two CD8⁺ T cell epitopes in the DENV-3 E protein in C57BL/6 mice. Using these we found that all the rMVA vaccines elicited DENV-specific CD8⁺ T cells that were cytotoxic in vivo and polyfunctional in vitro. Moreover, vaccines expressing the E protein with an intact signal peptide sequence elicited more DENV-specific CD8⁺ T cells than those expressing E proteins in the cytoplasm. Significantly, it was these same ER-targeted E protein vaccines that elicited antibody responses. Our results support the further development of rMVA vaccines expressing DENV E proteins and add to the tools available for dengue vaccine development.     

Narrowed TCR diversity for immunised mice challenged with recombinant influenza A-HIV Env311–320 virus

Understanding CD8+ T cell responses generated by live virus vectors is critical for the rational design of next generation HIV CTL-based vaccines. We used recombinant influenza viruses expressing the HIV Env_311–320 peptide in the neuraminidase stalk to study response magnitude, cytokine production and repertoire diversity for the elicited CD8+ D^dEnv₃₁₁ CTL set. The insertion of the CD8+ D^dEnv₃₁₁ epitope into the NA stalk resulted in a decrease in viral fitness that was reflected in lower lung viral titres. While not affecting the magnitude of endogenous primary influenza-specific responses, the introduction of the D^dEnv₃₁₁ CD8+ T cell epitope altered the hierarchy of responses following secondary challenge. The CD8+ K^dNP₁₄₇ response increased 9-fold in the spleen following secondary infection whereas the CD8+ D^dEnv₃₁₁ response increased 15-fold in the spleen. Moreover, this study is the first to describe narrowing of CD8+ TCR repertoire diversity in the context of an evolving secondary immune response against influenza A virus. Analysis of Vβ bias for CD8+ D^dEnv₃₁₁ T cell responses showed a narrowing of CD8+ Vβ8.1/8.2 D^dEnv₃₁₁ TCR repertoire diversity. This work further emphasizes the importance of understanding vaccine-induced CD8+ T cell responses.     

Towards an immunosense vaccine to prevent toxoplasmosis: protective Toxoplasma gondii epitopes restricted by HLA-A*0201

The ideal vaccine to protect against toxoplasmosis in humans would include antigens that elicit a protective T helper cell type 1 immune response, and generate long-lived IFN-γ-producing CD8⁺ T cells. Herein, we utilized a predictive algorithm to identify candidate HLA-A02 supertype epitopes from Toxoplasma gondii proteins. Thirteen peptides elicited production of IFN-γ from PBMC of HLA-A02 supertype persons seropositive for T. gondii infection but not from seronegative controls. These peptides displayed high-affinity binding to HLA-A02 proteins. Immunization of HLA-A*0201 transgenic mice with these pooled peptides, with a universal CD4⁺ epitope peptide called PADRE, formulated with adjuvant GLA-SE, induced CD8⁺ T cell IFN-γ production and protected against parasite challenge. Peptides identified in this study provide candidates for inclusion in immunosense epitope-based vaccines.     

In silico analysis of MHC-I restricted epitopes of Chikungunya virus proteins: Implication in understanding anti-CHIKV CD8+ T cell response and advancement of epitope based immunotherapy for CHIKV infection

Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus, responsible for acute febrile infection. The high morbidity and socio-economic loss associated with the recent CHIKV epidemics worldwide have raised a great public health concern and emphasize the need to study the immunological basis of CHIKV infection to control the disease. MHC-I restricted CD8⁺ T cell response represent one of the major anti-viral immune responses. Accordingly, it is essential to have a detailed understanding towards CHIKV specific MHC-I restricted immunogenic epitopes for anti-viral CD8⁺ CTL immunogenicity. In the present study, a computational approach was used to predict the conserved MHC-I epitopes for mouse haplotypes (H2-Db and H2-Dd) and some alleles of the major HLA-I supertypes (HLA-A2, -A3, -A24, -B7, -B15) of all CHIKV proteins. Further, an in-depth computational analysis was carried out to validate the selected epitopes for their nature of conservation in different global CHIKV isolates to assess their binding affinities to the appropriate site of respective MHC-I molecules and to predict anti-CHIKV CD8⁺ CTL immunogenicity. Our analyses resulted in fifteen highly conserved epitopes for H2-Db and H2-Dd and fifty epitopes for different HLA-I supertypes. Out of these, the MHC-I epitopes VLLPNVHTL and MTPERVTRL were found to have highest predictable CTL immunogenicities and least binding energies for H2-Db and H2-Dd, whereas, for HLA-I, the epitope FLTLFVNTL was with the highest population coverage, CTL immunogenicity and least binding energy. Hence, our study has identified MHC-I restricted epitopes that may help in the advancement of MHC-I restricted epitope based anti-CHIKV immune responses against this infection and this will be useful towards the development of epitope based anti-CHIKV immunotherapy in the future. However, further experimental investigations for cross validation and evaluation are warranted to establish the ability of epitopes to induce CD8⁺ T cell mediated immune responses.     

Novel vaccines for the treatment of chronic HBV infection based on mycobacterial heat shock protein 70

Immunogenic peptide-based vaccines can raise significant cellular immune responses. Although cytotoxic T lymphocytes (CTL) peptide epitopes are generally poor immunogens, heat shock protein 70 from Mycobacterium tuberculosis (TBhsp70) can overcome this problem since it is a potent adjuvant that links innate and adaptive immune responses. Our goal is to use TBhsp70 as an adjuvant for development of therapeutic vaccines for chronic Hepatitis B virus infection (HBV). To this end, we genetically fused the HBV core 18-27 peptide (HBcAg((18-27))) as a CTL epitope to the C-terminus of TBhsp70 and expressed the resulting protein in methylotropic yeast Pichia pastoris GS115. At the same time, the TBhsp70-HBcAg((18-27)) peptide complex was reconstituted in vitro. We investigated whether TBhsp70-peptide complex and TBhsp70-peptide fusion protein could generate antigen specific CTL responses in vitro. Dendritic cells (DC) from HLA-A2(+) chronic HBV infection and healthy control pulsed with two vaccines were studied phenotypically by FACS analyses and functionally by cytokine release, and HBV-specific CTL response. Our results demonstrate that two vaccines can activate DC of chronic HBV infection and healthy control by upregulation CD40 and CD86, high production of IL-12p70 and TNF-alpha. Furthermore, autologous T cells with DC stimulated by two vaccines can produce IFN-gamma and generate HBV-specific CTL response. However, capacity for CTL response and cytokines production from HBV infections remained inferior to that of healthy controls. Thus, the strategy of utilizing TBhsp70 may provide a novel design for the development of prophylactic and therapeutic vaccines.     

Overlapping synthetic peptides encoding TPD52 as breast cancer vaccine in mice: Prolonged survival

Peptide-based vaccines, one of several anti-tumor immunization strategies currently under investigation, can elicit both MHC Class I-restricted (CD8⁺) and Class II-restricted (CD4⁺) responses. However, the need to identify specific T-cell epitopes in the context of MHC alleles has hampered the application of this approach. We have tested overlapping synthetic peptides (OSP) representing a tumor antigen as a novel approach that bypasses the need for epitope mapping, since OSP contain all possible epitopes for both CD8⁺ and CD4⁺ T cells. Here we report that vaccination of inbred and outbred mice with OSP representing tumor protein D52 (TPD52-OSP), a potential tumor antigen target for immunotherapy against breast, prostate, and ovarian cancer, was safe and induced specific CD8⁺ and CD4⁺ T-cell responses, as demonstrated by development of specific cytotoxic T cell (CTL) activity, proliferative responses, interferon (IFN)-γ production and CD107a/b expression in all mice tested. In addition, TPD52-OSP-vaccinated BALB/c mice were challenged with TS/A breast carcinoma cells expressing endogenous TPD52; significant survival benefits were noted in vaccine recipients compared to unvaccinated controls (p < 0.001). Our proof-of-concept data demonstrate the safety and efficacy of peptide library-based cancer vaccines that obviates the need to identify epitopes or MHC backgrounds of the vaccinees. We show that an OSP vaccination approach can assist in the disruption of self-tolerance and conclude that our approach may hold promise for immunoprevention of early-stage cancers in a general population.     

GP120-specific exosome-targeted T cell-based vaccine capable of stimulating DC- and CD4(+) T-independent CTL responses

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutics. In this study, we generated ovalbumin (OVA)-pulsed and pcDNAgp120-transfected dendritic cell (DC)-released exosomes (EXOova and EXOgp120) and ConA-stimulated C57BL/6 CD8⁺ T cells. OVA- and Gp120-Texo vaccines were generated from CD8⁺ T cells with uptake of EXOova and EXOgp120, respectively. We demonstrate that OVA-Texo stimulates in vitro and in vivo OVA-specific CD4⁺ and CD8⁺ cytotoxic T lymphocyte (CTL) responses leading to long-term immunity against OVA-expressing BL6-10_OVA melanoma. Interestingly, CD8⁺ T cell responses are DC and CD4⁺ T cell independent. Importantly, Gp120-Texo also stimulates Gp120-specific CTL responses and long-term immunity against Gp120-expressing B16 melanoma. Therefore, this novel HIV-1-specific EXO-targeted Gp120-Texo vaccine may be useful in induction of efficient CTL responses in AIDS patients with DC dysfunction and CD4⁺ T cell deficiency.     

Flagellin enhances tumor-specific CD8 T cell immune responses through TLR5 stimulation in a therapeutic cancer vaccine model

Tumor antigen (TA)-specific immunotherapy is an emerging approach for cancer treatment. Potent adjuvants are prerequisites to the immunotherapy for overcoming the low immunogenicity of TAs. We previously demonstrated that a bacterial flagellin, Vibrio vulnificus FlaB, has potent adjuvant activity in various vaccination models. In this study, we investigated whether the FlaB protein could be a potent adjuvant for a human papillomavirus 16 E6 and E7 (E6/E7) peptide-based anticancer immunotherapy. We used an E6/E7-expressing TC-1 carcinoma implantation animal model and tested TA-specific immunomodulation by FlaB. We co-administered the E6/E7 peptide either with or without FlaB into TC-1 tumor-bearing mice and then analyzed the antitumor activity of the peptide. FlaB significantly potentiated specific antitumor immune responses elicited by the peptide immunization, as evidenced by retarded in vivo tumor growth and significantly prolonged survival. We noticed that TC-1 cells do not express Toll-like receptor 5 (TLR5) on their surface and the TLR5 signaling pathway in TC-1 cells was not responsible for the antitumor effect of FlaB. FlaB potentiated the CTL activity and Ag-specific IFN-γ production of CD8⁺ T cells from the draining lymph node and spleen. In addition, this antitumor activity was abrogated following the in vivo depletion of CD8⁺ T cells and in TLR5 knockout (KO) or MyD88 KO mice. These results suggest that flagellin could enhance TA-specific CD8⁺ CTL immune responses through TLR5 stimulation in cancer immunotherapy.     

CD8 T cell response in HLA-A*0201 transgenic mice is elicited by epitopes from SARS-CoV S protein

Cytotoxic CD8⁺ T lymphocytes (CTLs) play an important role in antiviral immunity. Several human HLA-A*0201 restricted CTL epitopes of severe acute respiratory syndrome (SARS) spike (S) protein have been identified in HLA-A*0201 transgenic (Tg) mice, but the mechanisms and properties of immune responses are still not well understood. In this study, HLA-A*0201 Tg mice were primed intramuscularly with SARS S DNA and boosted subcutaneously with HLA-A*0201 restricted peptides. The lymphocytes from draining lymph nodes, spleens and lungs were stimulated with the cognate peptides. Three different methods (ELISA, ELISPOT and FACS) were used to evaluate the immune responses during short and long periods of time after immunization. Results showed that peptide-specific CD8⁺ T cells secreted IFN-γ, TNF-α and IL-2 and expressed CD107a/b on cell surface. IFN-γ⁺CD8⁺ T cells and CD107a/b⁺CD8⁺ T cells distributed throughout the lymphoid and non-lymphoid tissues, but the frequency of peptide-specific CD8⁺ T cells was higher in lungs than in spleens and lymph nodes. The phenotype of the CD8⁺ T cells was characterized based on the expression of IFN-γ. Most of the HLA-A*0201 restricted peptide-specific CD8⁺ T cells represented a memory subset with CD45RB^high and CD62L^low. Taken together, these data demonstrate that immunization with SARS S DNA and HLA-A*0201 restricted peptides can elicit antigen-specific CD8⁺ T cell immune responses which may have a significant implication in the long-term protection. We provide novel information in cellular immune responses of SARS S antigen-specific CD8⁺ T cells, which are important in the development of vaccine against SARS-CoV infection.     

Efficient induction of anti-tumor immunity by a TAT-CEA fusion protein vaccine with poly(I:C) in a murine colorectal tumor model

Protein vaccines may be a useful strategy for cancer immunotherapy because recombinant tumor antigen proteins can be produced on a large scale at relatively low cost and have been shown to be safe for clinical application. However, protein vaccines have historically exhibited poor immunogenicity; thus, an improved strategy is needed for successful induction of immune responses.TAT peptide is a protein transduction domain composed of an 11-amino acid peptide (TAT_47-57: YGRKKRRQRRR). The positive charge of this peptide allows protein antigen fused with it to improve cell penetration. Poly(I:C) is a synthetic double-stranded RNA that is negatively charged and favors interaction with the cationic TAT peptide. Poly(I:C) has been reported on adjuvant role in tumor vaccine through promotion of immune responses. Therefore, we demonstrated that vaccine with a mixture of TAT-CEA fusion protein and poly(I:C) can induce anti-tumor immunity in a murine colorectal tumor model. Splenocytes from mice vaccinated with a mixture of TAT-CEA fusion protein and poly(I:C) effectively induced CEA-specific IFN-γ-producing T cells and showed cytotoxic activity specific for MC-38-cea2 tumor cells expressing CEA. Vaccine with a mixture of TAT-CEA fusion protein and poly(I:C) delayed tumor growth in MC-38-cea-2 tumor-bearing mice. Depletion of CD8⁺ T cells and NK cells reversed the inhibition of tumor growth in an MC-38-cea2-bearing mice, indicating that CD8⁺ T cells and NK cells are responsible for anti-tumor immunity by vaccine with a mixture of TAT-CEA fusion protein and poly(I:C). Taken together, these results suggest that poly(I:C) could be used as a potent adjuvant to induce the anti-tumor immunity of a TAT-CEA fusion protein vaccine in a murine colorectal tumor model.     

Ovalbumin lipid core peptide vaccines and their CD4 and CD8 T cell responses

The lipid core peptide (LCP) system has successfully been used in development of peptide-based vaccines against cancer and infectious diseases (such as group A streptococcal infection). CD8⁺ T cells are important targets for vaccines, however developing a vaccine that activates long-lasting immunity has proven challenging. The ability of LCP vaccines to activate antigen-specific CD8⁺ and/or CD4⁺ T cell responses was tested using compounds that contained two or four copies of OVA_257–264 and/or OVA_323–339 peptides conjugated to LCP, which are recognised by OTI (CD8⁺ specific) and OTII (CD4⁺ specific) T cells, respectively. The LCP–ovalbumin vaccines developed in this study were synthesised in 30% yields and showed no significant haemolytic effect on red blood cells (below 4% haemolysis when tested with compounds at up to 100 μM concentrations). Promising in vivo data in mice suggested that this LCP–ovalbumin vaccine system could act as a novel and potent vehicle for the stimulation of robust antigen-specific CD8⁺ T cell responses.     

Antigenic peptide nanofibers elicit adjuvant-free CD8 T cell responses

Vaccines that elicit robust CD8⁺ T cell responses are desirable for protection against infectious diseases and cancers. However, most vaccine adjuvants fail to elicit robust CD8⁺ T cell responses without inflammation and associated toxicity. We recently reported that self-assembling peptides that form nanofibers in physiological buffers elicited strong adjuvant-free and antigen-specific antibody responses in mice. However, whether or not such nanofibers likewise can elicit strong CD8⁺ T cell responses is unknown. Here, we demonstrate that the self-assembling peptide Q11 conjugated to a CD8⁺ T cell epitope of ovalbumin (Q11-OVA), elicits strong antigen-specific primary and recall responses, and in a vaccination regimen protects against subsequent infection. Importantly, we show that these antigenic peptide nanofibers do not persist as an inflammatory antigen depot at the injection site. Our results demonstrate for the first time that self-assembling peptides may be useful as carriers for vaccines where CD8⁺ T cell-mediated protection is needed.     

Development and preclinical safety evaluation of a new therapeutic HIV-1 vaccine based on 18 T-cell minimal epitope peptides applying a novel cationic adjuvant CAF01

Therapeutic immunization of HIV-1-infected individuals with or without anti-retroviral therapy is a new promising disease prevention. To induce a new cytotoxic T_CD8 lymphocyte (CTL) immunity during chronic HIV-1 infection 15 infrequently targeted but conserved HLA-supertype binding CTL epitopes from Gag, Pol, Nef, Env, Vpu and Vif were identified. The 15 T_CD8 and three T_CD4 helper peptides were GMP synthesised and formulated with a new adjuvant CAF01 which is a synthetic two-component liposomic adjuvant comprising the quaternary ammonium dimethyl-dioctadecyl-ammonium (DDA) and the immune modulator trehalose 6,6′-dibehenate (TDB). Using IFN-γ ELISPOT assay, T-cell immune induction by the vaccine was found to both CD4 and CD8 T-cell restricted peptides in HLA-A2 transgenic mice. Comprehensive toxicity studies of the CAF01 adjuvant-alone and together with different vaccines showed that CAF01 when tested at human dose levels was safe and well tolerated with only local inflammation at the site of injection and no systemic reactions. No pharmacological safety issues were observed in Beagle dogs. The HIV-1 vaccine toxicity study in the Göttingen Minipig^® showed no systemic toxicity from five repetitive i.m. injections, each with a 2-week interval, of either the 18 HIV-1 peptide antigen solution (AFO18) or the AFO18-CAF01, in which the 18 HIV-1 peptides were formulated with the CAF01 adjuvant. Distinct inflammatory responses were observed in the injected muscles of the AFO18-CAF01 vaccine treated animals as a result of the immune stimulating effect of the adjuvant on the vaccine. The results of the toxicity studies provide optimism for phase I clinical trials evaluating the therapeutic HIV-1 T-cell vaccination approach using multiple subdominant minimal epitope peptides applying the novel cationic adjuvant CAF01.