Single chain MHC I trimer-based DNA vaccines for protection against Listeria monocytogenes infection

To circumvent limitations of poor antigen presentation and immunogenicity of DNA vaccines that target induction of CD8(+) T cell immunity, we have generated single chain MHC I trimers (MHC I SCTs) composed of a single polypeptide chain with a linear composition of antigenic peptide, β2-microglobulin, and heavy chain of a MHC class I molecule connected by flexible linkers. Because of its pre-assembled nature, the SCT presents enhanced expression and presentation of the antigenic peptide/MHC complexes at the cell surface. Furthermore, DNA vaccination with a plasmid DNA encoding an SCT incorporating an immunodominant viral epitope elicited protective CD8(+) T cell responses against lethal virus infection. To extend these findings, here we tested the efficacy of SCT DNA vaccines against bacterial infections. In a mouse infection model of Listeria monocytogenes, the SCT DNA vaccine encoding H-2K(d) and the immunodominant peptide LLO 91-99 generated functional primary and memory peptide-specific CD8(+) T cells that confer partial protection against L. monocytogenes infection. DNA immunization of K(d)/LLO(91-99) SCTs generated functional memory CD8(+) T cells independently of CD4(+) T cells, although the expression of cognate or non-cognate CD4(+) helper T cell epitopes further enhanced the protective efficacy of SCTs. Our study further demonstrates that the SCT serves as a potent platform for DNA vaccines against various infectious diseases.     

Strategies to enhance immunogenicity of cDNA vaccine encoded antigens by modulation of antigen processing

Most vaccines are based on protective humoral responses while for intracellular pathogens CD8⁺ T cells are regularly needed to provide protection. However, poor processing efficiency of antigens is often a limiting factor in CD8⁺ T cell priming, hampering vaccine efficacy. The multistage cDNA vaccine H56, encoding three secreted Mycobacterium tuberculosis antigens, was used to test a complete strategy to enhance vaccine’ immunogenicity. Potential CD8⁺ T cell epitopes in H56 were predicted using the NetMHC3.4/ANN program. Mice were immunized with H56 cDNA using dermal DNA tattoo immunization and epitope candidates were tested for recognition by responding CD8⁺ T cells in ex vivo assays. Seven novel CD8⁺ T cell epitopes were identified. H56 immunogenicity could be substantially enhanced by two strategies: (i) fusion of the H56 sequence to cDNA of proteins that modify intracellular antigen processing or provide CD4⁺ T cell help, (ii) by substitution of the epitope’s hydrophobic C-terminal flanking residues for polar glutamic acid, which facilitated their proteasome-mediated generation. We conclude that this whole strategy of in silico prediction of potential CD8⁺ T cell epitopes in novel antigens, followed by fusion to sequences with immunogenicity-enhancing properties or modification of epitope flanking sequences to improve proteasome-mediated processing, may be exploited to design novel vaccines against emerging or ‘hard to treat’ intracellular pathogens.     

Intradermal administration of DNA vaccines combining a strategy to bypass antigen processing with a strategy to prolong dendritic cell survival enhances DNA vaccine potency

Intradermal vaccination via gene gun efficiently delivers DNA vaccines into dendritic cells (DCs) of the skin, resulting in the activation and priming of antigen-specific T cells in vivo. We have previously demonstrated that intradermal delivery of DNA vaccines encoding single-chain trimer (SCT) composed of the most immunogenic epitope of human papillomavirus type 16 (HPV-16) E6 protein (aa49-57), beta2-microglobulin, and MHC class I heavy chain (SCT-E6) can bypass antigen processing and lead to stable cell-surface presentation of E6 peptides. We also showed that co-administration of DNA vaccines with DNA encoding anti-apoptotic proteins can prolong the survival of DNA-transduced DCs, resulting in significant enhancement of antigen-specific CD8(+) T cell immune responses. In the current study, we hypothesized that combining the SCT strategy and antiapoptotic strategy may further enhance DNA vaccine potency by augmenting antigen-specific CD8(+) T cell immune responses and antitumor effects in vaccinated mice. Here, we show that C57BL/6 mice vaccinated with SCT-E6 DNA combined with antiapoptotic protein Bcl-xL DNA generated enhanced E6-specific CD8(+) T cell immune responses compared to mice vaccinated with SCT-E6 DNA and a non-functional mutant Bcl-xL (mtBcl-xL) DNA. Furthermore, we show that mice treated with SCT-E6 and Bcl-xL DNA generated enhanced anti-tumor effects against E6-expressing tumor cells (TC-1/Luciferase) compared to mice treated with SCT-E6 and mtBcl-xL DNA.     

Short peptide sequences containing MHC class I and/or class II epitopes linked to nano-beads induce strong immunity and inhibition of growth of antigen-specific tumour challenge in mice

Peptide based vaccines offer practical advantages, but unmodified peptides usually require an adjuvant or delivery vehicle to promote immunogenicity. When peptides containing ovalbumin (OVA) derived CD4 and CD8 T cell epitopes were conjugated to 0.05 μm nano-beads, they gave strong immune responses and inhibition of growth of tumour cells expressing the CD8 T cell epitope with MHC class I. These responses were inducible with both high (50 μg) and low (5 μg) peptide doses after a single immunisation. The helper CD4 T cell epitope was unnecessary for induction of CD8 T cell or tumour challenge responses. However, the CD4 T cell epitope contained a B cell epitope and triggered strong antibody responses. This simple approach offers a convenient experimental tool and a potentially useful clinical method for peptide immunisation.     

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.     

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.     

Antigen encoded by vaccine vectors derived from human adenovirus serotype 5 is preferentially presented to CD8+ T lymphocytes by the CD8α+ dendritic cell subset

Different subsets of dendritic cells (DC) elicit qualitatively different immune responses. In mice, two lymphoid tissue-resident subsets, CD8α⁺ and CD8α⁻, have been implicated in the induction of T helper 1 (Th1) or Th2 responses, respectively. Moreover, CD8α⁺ DC appear to play a major role in priming CD8⁺ T lymphocyte responses to viral antigens in the course of diverse viral infections. These considerations have been less extensively explored for vaccine vectors derived from viruses. Despite inefficient ex vivo transduction of DC, vectored vaccines derived from human adenoviruses of serotype 5 (Ad5) elicit robust immune responses, predominantly of the Th1 orientation, in humans and mice. At present it is unknown whether Ad5 interacts with DC subsets in a differential manner, thereby influencing the quality of the elicited IR. To address this issue, successive steps (attachment, transgene expression, MHC class I antigen presentation and activation of antigen-specific T lymphocytes) involved in induction of immune responses by Ad5-based vectors have been examined in CD8α⁺ and CD8α⁻ murine DC subsets. Although in both ex vivo and in vivo experiments CD8α⁺ and CD8α⁻ DC subsets captured an Ad5-based vector to a similar extent, transgene expression and subsequent MHC class I display of a transgene-encoded antigen were more efficient in CD8α⁺ DC. Moreover, following in vivo and ex vivo transduction with an Ad5-based vaccine, antigen-specific CD8⁺ T lymphocytes were more efficiently activated by CD8α⁺ DC than by CD8α⁻ DC. Thus, superior antigen expression and MHC class I display in CD8α⁺ DC may contribute to preferred priming of antigen-specific CD8⁺ lymphocytes by Ad5-transduced CD8α⁺ DC.     

Heteroclitic peptides enhance human immunodeficiency virus-specific CD8 T cell responses

The inability of human immunodeficiency virus (HIV)-specific CD8⁺ T cells to durably control HIV replication due to HIV escape mutations and CD8⁺ T cell dysfunction is a key factor in disease progression. A few HIV-infected individuals termed elite controllers (EC) maintain polyfunctional HIV-specific CD8⁺ T cells, minimal HIV replication and normal CD4⁺ T lymphocyte numbers. Thus, therapeutic intervention to sustain or restore CD8⁺ T cell responses similar to those persisting in EC could relieve terminal dependence on antiretrovirals. Vaccination with HIV peptides is one approach to achieve this and our objective in this study was to determine whether certain HIV peptide variants display antigenic superiority over the reference peptides normally included in vaccines. Eight peptide sets were generated, each with a reference peptide and six variants harboring conservative or semi-conservative amino acid substitutions at positions predicted to affect T cell receptor interactions without affecting human class I histocompatibililty-linked antigen (HLA) binding. Recognition across peptide sets was tested with >80 HIV-infected individuals bearing the appropriate HLA alleles. While reference peptides were often the most antigenic, cross-reactivity with variants was common and in many cases, peptide variants were superior at stimulating interferon-γ production or selectively enhanced interleukin-2 production. Although such heteroclitic activity was not generalized for all individuals bearing the HLA class I allele involved, these data suggest that heteroclitic peptide variants could improve the efficacy of therapeutic peptide vaccines in HIV infection.     

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.     

A DNA vaccine encoding a single-chain trimer of HLA-A2 linked to human mesothelin peptide generates anti-tumor effects against human mesothelin-expressing tumors

Mesothelin is highly expressed in a majority of ovarian cancer cells and is expressed at low levels in normal cells. Therefore, mesothelin represents a potential target antigen for ovarian cancer vaccine development. DNA vaccines employing single-chain trimers (SCT) have been shown to bypass antigen processing and presentation and result in significant enhancement of DNA vaccine potency. In the current study, we created a DNA vaccine employing an SCT targeting human mesothelin and characterized the ensuing antigen-specific CD8+ T cell-mediated immune responses and anti-tumor effects against human mesothelin-expressing tumors in HLA-A2 transgenic mice. Our results showed that vaccination with DNA employing an SCT of HLA-A2 linked to human mesothelin epitope aa540-549 (pcDNA3-Hmeso540-beta2m-A2) generated strong human mesothelin peptide (aa540-549)-specific CD8+ T cell immune responses in HLA-A2 transgenic mice. Vaccination with pcDNA3-Hmeso540-beta2m-A2 prevented the growth of HLA-A2 positive human mesothelin-expressing tumor cell lines in HLA-A2 transgenic mice in contrast to vaccination with DNA encoding SCT linked to OVA CTL epitope. Thus, the employment of SCT of HLA-A2 linked to the human mesothelin epitope aa540-549 represents a potential opportunity for the clinical translation of DNA vaccines against human mesothelin-expressing tumors, particularly ovarian cancer cells.     

Dissecting antigen processing and presentation routes in dermal vaccination strategies

The skin is an attractive site for vaccination due to its accessibility and presence of immune cells surveilling this barrier. However, knowledge of antigen processing and presentation upon dermal vaccination is sparse. In this study we determined antigen processing routes that lead to CD8⁺ T cell activation following dermal DNA tattoo immunization, exploiting a model antigen that contains an immunoproteasome-dependent epitope. In agreement with earlier reports, we found that DNA tattoo immunization of wild type (WT) mice triggered vigorous responses to the immunoproteasome-dependent model epitope, whereas gene-deficient mice lacking the immunoproteasome subunits β5i/LMP7 and β2i/MECL1 failed to respond. Unexpectedly, dermal immunization both of irradiated bone marrow (BM) reconstituted mice in which the BM transplant was of WT origin, and of WT mice transplanted with immunoproteasome subunit-deficient BM induced a CD8⁺ T cell response to the immunoproteasome-dependent epitope, implying that both BM and host-derived cells contributed to processing of delivered model antigen. Depletion of radiation-resistant Langerhans cells (LC) from chimeric mice did not diminish tattoo-immunization induced CD8⁺ T cell responses in most mice, illustrating that LC were not responsible for antigen processing and CD8⁺ T cell priming in tattoo-immunized hosts. We conclude that both BM and non-BM-derived cells contribute to processing and cross-presentation of antigens delivered by dermal DNA tattoo immunization.     

A modified epitope identified for generation and monitoring of PSA-specific T cells in patients on early phases of PSA-based immunotherapeutic protocols

Efficacy of vaccination in cancer patients on immunotherapeutic protocols can be difficult to evaluate. The aim of this study was therefore to identify a single natural or modified epitope in prostate-specific antigen (PSA) with the ability to generate high levels of PSA-specific T cells to facilitate monitoring in patients after vaccination against prostate cancer. To the best of our knowledge, this study describes for the first time the peptide specificity of T cells stimulated by endogenously processed PSA antigen. The peptide specificity of HLA-A*0201-restricted CD8⁺ T cells against human and rhesus PSA was investigated both in vivo after DNA vaccination in HLA-A*0201-transgenic mice and in vitro after repetitive stimulation of human T cells with DNA-transfected human dendritic cells (DCs). One of seven native PSA peptides, psa53–61, was able to activate high levels of PSA-specific CD8⁺ T cells in HLA-A*0201-transgenic mice after PSA DNA vaccination. Psa53–61 was also the only peptide that induced human T cells to produce IFNγ after stimulation with PSA transfected DCs, however not in all donors. Therefore, plasmids encoding modified epitopes in predicted HLA-A*0201 sequences were constructed. One of these modified PSA plasmids consistently induced IFNγ producing CD8⁺ T cells to the corresponding modified peptide as well as to the corresponding native peptide, in all murine and human T cell cultures. This study demonstrates a novel concept of introducing a modified epitope within a self-tumor antigen, with the purpose of eliciting a reliable T cell response from the non-tolerized immune repertoire, to facilitate monitoring of vaccine efficacy in cancer patients on immunotherapeutic protocols. The purpose of such a modified epitope is thus not to induce therapeutically relevant T cells but rather to, in case of weak or divergent T cell responses to self antigens/peptides, help answer questions about efficacy of vaccine delivery and about the possibility to induce immune responses in the selected and often immunosuppressed cancer patients.     

Short peptide sequences containing MHC class I and/or class II epitopes linked to nano-beads induce strong immunity and inhibition of growth of antigen-specific tumour challenge in mice

Peptide based vaccines offer practical advantages, but unmodified peptides usually require an adjuvant or delivery vehicle to promote immunogenicity. When peptides containing ovalbumin (OVA) derived CD4 and CD8 T cell epitopes were conjugated to 0.05 microm nano-beads, they gave strong immune responses and inhibition of growth of tumour cells expressing the CD8 T cell epitope with MHC class I. These responses were inducible with both high (50 microg) and low (5 microg) peptide doses after a single immunisation. The helper CD4 T cell epitope was unnecessary for induction of CD8 T cell or tumour challenge responses. However, the CD4 T cell epitope contained a B cell epitope and triggered strong antibody responses. This simple approach offers a convenient experimental tool and a potentially useful clinical method for peptide immunisation.     

Identification of a dominant CD8+ CTL epitope in the SARS-associated coronavirus 2 spike protein

Coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2, has been spreading throughout the world. To date, there are still no approved human vaccines for this disease. To develop an effective vaccine, the establishment of animal models for evaluating post-vaccination immune responses is necessary. In this study, we have identified a CTL epitope in the SARS-CoV-2 spike (S) protein that could be used to measure the cellular immune response against this protein. Potential predicted CTL epitopes of the SARS-CoV-2 S protein were investigated by immunizing BALB/c mice with a recombinant of the receptor-binding domain (RBD) of the S protein. Then, CD8⁺ T cells specific for S-RBD were detected by stimulating with potential epitope peptides and then measuring the interferon-gamma production. Truncation of this peptide revealed that S-RBD-specific CD8⁺ T cells recognized a H2-D^d-restricted S_526–533 peptide. In conclusion, this animal model is suitable for evaluating the immunogenicity of SARS-CoV-2 vaccines.     

Tumor-specific peptide-based vaccines containing the conformationally biased, response-selective C5a agonists EP54 and EP67 protect against aggressive large B cell lymphoma in a syngeneic murine model

Vaccines to large B cell lymphoma were made by the covalent attachment of an epitope from the gp70 glycoprotein (SSWDFITV) to the N-termini of the conformationally biased, response-selective C5a agonists EP54 (YSFKPMPLaR) and EP67 (YSFKDMP(MeL)aR). Syngeneic Balb/c mice were immunized with these EP54/EP67-containing vaccines and challenged with a lethal dose of the highly liver metastatic and gp70-expressing lymphoma cell line RAW117-H10 to evaluate the ability of these vaccines to induce protective immune outcomes. All mice immunized with SSWDFITVRRYSFKPMPLaR (Vaccine 2) and SSWDFITVRRYSFKDMP(MeL)aR (Vaccine 3) were protected to a lethal challenge of RAW117-H10 lymphoma (>170 days survival) and exhibited no lymphoma infiltration or solid tumor nodules in the liver relative to unvaccinated controls (<18 days survival). Vaccines 2 and 3 contained the protease-sensitive double-Arg (RR) linker sequence between the epitope and the EP54/EP67 moieties in order to provide a site for intracellular proteases to separate the epitope from the EP54/EP67 moieties once internalized by the APC and, consequently, enhance epitope presentation in the context of MHC I/II. These protected mice exhibited an immune outcome consistent with increased involvement of CD8⁺ and/or CD4⁺ T lymphocytes relative to controls and mice that did not survive or showed low survival rates as with Vaccines 1 and 4, which lacked the RR linker sequence. CD8⁺ T lymphocytes activated in response to Vaccines 2 and 3 express cytotoxic specificity for gp70-expressing RAW117-H10 lymphoma cells, but not antigen-irrelevant MDA-MB231A human breast cancer cells. Results are discussed against the backdrop of the ability of EP54/EP67 to selectively target antigens to and activate C5a receptor-bearing antigen presenting cells and the prospects of using such vaccines therapeutically against lymphoma and other cancers.     

Aspartate-β-hydroxylase induces epitope-specific T cell responses in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) has a poor prognosis due to high recurrence rate. Aspartate-β-hydroxylase (ASPH) is a highly conserved transmembrane protein, which is over expressed in HCC and promotes a malignant phenotype. The capability of ASPH protein-derived HLA class I and II peptides to generate antigen specific CD4⁺ and CD8⁺ immune responses is unknown. Therefore, these studies aim to define the epitope specific components required for a peptide based candidate vaccine. Monocyte-derived dendritic cells (DCs) generated from the peripheral blood mononuclear cells (PBMCs) of HCC patients were loaded with ASPH protein. Helper CD4⁺ T cells and CD8⁺ cytotoxic T lymphocytes (CTLs) were co-incubated with the DCs; T cell activation was evaluated by flow cytometric analysis. Immunoinformatics tools were used to predict HLA class I- and class II-restricted ASPH sequences, and the corresponding peptides were synthesized. The immunogenicity of each peptide in cultures of human PBMCs was determined by IFN-γ ELISpot assay. ASPH protein-loaded DCs activated both CD4⁺ and CD8⁺ T cells contained within the PBMC population derived from HCC patients. Furthermore, the predicted HLA class I- and class II-restricted ASPH peptides were significantly immunogenic. Both HLA class I- and class II-restricted peptides derived from ASPH induce T cell activation in HCC. We observed that ASPH protein and related peptides were highly immunogenic in patients with HCC and produce the type of cellular immune responses required for generation of anti-tumor activity.     

Optimal induction of HPV DNA vaccine-induced CD8 T cell responses and therapeutic antitumor effect by antigen engineering and electroporation

Since human papillomavirus (HPV) E6 and E7 are promising tumor antigens, we engineered E6 and E7 antigens to generate an optimal HPV DNA vaccine by codon optimization (Co), fusion of E6 and E7, addition of a tissue plasminogen activator (tpa) signal sequence, addition of CD40 ligand (CD40L) or Fms-like tyrosine kinase-3 ligand (Flt3L). The resulting constructs were investigated in terms of their antitumor activity as well as induction of HPV-specific CD8⁺ T cell responses. When E6^Co and E7^Co were fused (E67^Co), CD8⁺ T cell responses specific for E6 or E7 antigen decreased, but the preventive antitumor effect rather improved, demonstrating the importance of broad immunity. Interestingly, Flt3L-fused HPV DNA vaccine exhibited stronger E6- and E7-specific CD8⁺ T cell responses as well as therapeutic antitumor effect than that of CD40L linked HPV DNA vaccine. Finally, the optimal construct, tFE67^Co, was generated by including tpa signal sequence, Flt3L, fusion of E6 and E7, and codon optimization, which induces 23 and 25 times stronger E6- and E7-specific CD8⁺ T cell responses than those of initial E67 fusion construct. In particular, inclusion of electroporation in intramuscular immunization of tFE67^Co further enhances HPV-specific CD8⁺ T cell responses, leading to complete tumor regression in a therapeutic setting. Thus, our results provide valuable insight on effective HPV DNA vaccine design and suggest that tFE67^Co delivered with electroporation may be a promising therapeutic HPV DNA vaccine against cervical cancer.     

Ii-Key/HPV16 E7 hybrid peptide immunotherapy for HPV16+ cancers

Activation of antigen-specific CD4+ T cells is critical for vaccine design. We have advanced a novel technology for enhancing activation of antigen-specific CD4+ T helper cells whereby a fragment of the MHC class II-associated invariant chain (Ii-Key) is linked to an MHC class II epitope. An HLA-DR4-restricted HPV16 E7 epitope, HPV16 E7(8–22), was used to create a homologous series of Ii-Key/HPV16 E7 hybrids testing the influence of spacer length on in vivo enhancement of HPV16 E7(8–22)-specific CD4+ T lymphocyte responses. HLA-DR4-tg mice were immunized with Ii-Key/HPV16 E7(8–22) hybrids or the epitope-only peptide HPV16 E7(8–22). As measured by IFN-γ ELISPOT assay of splenocytes from immunized mice, one of the Ii-Key/HPV16 E7(8–22) hybrids enhanced epitope-specific CD4+ T cell activation 5-fold compared to the HPV16 E7(8–22) epitope-only peptide. We further demonstrated that enhanced CD4+ T cell activation augments the CTL activity of a H-2D^b-restricted HPV16 E7(49–57) epitope in HLA-DR4+ mice using an in vivo CTL assay. Binding assays indicated that the Ii-Key/HPV16 hybrid has increased affinity to HLA-DR4+ cells relative to the epitope-only peptide, which may explain its increased potency. In summary, Ii-Key hybrid modification of the HLA-DR4-restricted HPV16 E7(8–22) MHC class II epitope generates a potent immunotherapeutic peptide vaccine that may have potential for treating HPV16+ cancers in HLA-DR4+ patients.     

DNA/Ad5 vaccination with SIV epitopes induced epitope-specific CD4⁺ T cells, but few subdominant epitope-specific CD8⁺ T cells

The goals of a T cell-based vaccine for HIV are to reduce viral peak and setpoint and prevent transmission. While it has been relatively straightforward to induce CD8⁺ T cell responses against immunodominant T cell epitopes, it has been more difficult to broaden the vaccine-induced CD8⁺ T cell response against subdominant T cell epitopes. Additionally, vaccine regimens to induce CD4⁺ T cell responses have been studied only in limited settings. In this study, we sought to elicit CD8⁺ T cells against subdominant epitopes and CD4⁺ T cells using various novel and well-established vaccine strategies. We vaccinated three Mamu-A*01⁺ animals with five Mamu-A*01-restricted subdominant SIV-specific CD8⁺ T cell epitopes. All three vaccinated animals made high frequency responses against the Mamu-A*01-restricted Env TL9 epitope with one animal making a low frequency CD8⁺ T cell response against the Pol LV10 epitope. We also induced SIV-specific CD4⁺ T cells against several MHC class II DRBw*606-restricted epitopes. Electroporated DNA with pIL-12 followed by a rAd5 boost was the most immunogenic vaccine strategy. We induced responses against all three Mamu-DRB*w606-restricted CD4 epitopes in the vaccine after the DNA prime. Ad5 vaccination further boosted these responses. Although we successfully elicited several robust epitope-specific CD4⁺ T cell responses, vaccination with subdominant MHC class I epitopes elicited few detectable CD8⁺ T cell responses. Broadening the CD8⁺ T cell response against subdominant MHC class I epitopes was, therefore, more difficult than we initially anticipated.     

Role of MHC class Ib molecule,H2-M3 in host immunity against tuberculosis

The MHC class I family comprises both classical (class Ia) and non-classical (class Ib) members. While the prime function of classical MHC class I molecules (MHC class Ia) is to present peptide antigens to pathogen-specific cytotoxic T cells, non-classical MHC-I (MHC class Ib) antigens perform diverse array of functions in both innate and adaptive immunity. Vaccines against intracellular pathogens such as Mycobacterium tuberculosis need to induce strong cellular immune responses. Recent studies have shown that MHC class I molecules play an important role in the protective immune response to M. tuberculosis infection. Both MHC Ia-restricted and MHC class Ib-restricted M. tuberculosis -reactive CD8⁺ T cells have been identified in humans and mice, but their relative contributions to immunity is still uncertain. Unlike MHC class Ia-restricted CD8⁺ T cells, MHC class Ib-restricted CD8⁺ T cells are constitutively activated in naive animals and respond rapidly to infection challenge, hence filling the temporal gap between innate and adaptive immunity. The present review article summarizes the general host immunity against M. tuberculosis infection highlighting the possible role of MHC class Ib molecule, H2-M3 and their ligands (N-formylated peptides) in protection against tuberculosis.