The immune responses of HLA-A*0201 restricted SARS-CoV S peptide-specific CD8⁺ T cells are augmented in varying degrees by CpG ODN, PolyI:C and R848

The induction of antigen specific memory CD8⁺ T cells in vivo is very important to new vaccines against infectious diseases. In the present study, we aimed to evaluate the immune responses of peptide-specific CD8⁺ T cells induced by HLA-A*0201 restricted severe acute respiratory syndrome-associated coronavirus (SARS-CoV) S epitopes plus CpG oligodeoxynucleotide (CpG ODN), PolyI:C and R848 as adjuvants. Furthermore, the generation, distribution and phenotype of long-lasting peptide-specific memory CD8⁺ T cells were assessed by ELISA, ELISPOT and flow cytometry. Our results showed that antigen specific CD8⁺ T cells were elicited by HLA-A*0201 restricted SARS-CoV S epitopes. Furthermore, the frequency of peptide-specific CD8⁺ T cells was dramatically increased after both prime and boost immunization with peptides plus CpG ODN, whereas slight enhancements were induced following boost vaccination with peptides plus PolyI:C or R848. SARS-CoV S peptide-specific IFN-γ⁺CD8⁺ T cells were distributed throughout the lymphoid and non-lymphoid tissues. Results also demonstrated that the HLA-A*0201 restricted peptide-specific CD8⁺ T cells induced by peptides plus CpG ODN carried a memory cell phenotype with CD45RB⁺ and CD62L⁻ and possessed long-term survival ability in vivo. Taken together, our results implied that HLA-A*0201 restricted SARS-CoV S epitopes plus CpG ODN might be the superior candidates for SARS vaccine.     

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.     

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.     

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.     

Immunogenicity of late stage specific peptide antigens of Mycobacterium tuberculosis

Global burden of latent TB infection comprises one-third of the world population. Identifying potential Mycobacterium tuberculosis (Mtb) latency associated antigens that can generate protective immunity against the pathogen is crucial for designing an effective TB vaccine. Usually the immune system responds to a small number of amino acids as MHC Class I or Class II peptides. The precision to trigger epitope specific protective T-cell immune response could therefore be achieved with synthetic peptide-based subunit vaccine.In the present study we have considered an immunoinformatic approach using available softwares (ProPred, IEDB, NETMHC, BIMAS, Vaxijen2.0) and docking and visualizing softwares (CABSDOCK, HEX, Pymol, Discovery Studio) to select 10 peptides as latency antigens from 4 proteins (Rv2626, Rv2627, Rv2628, and Rv2032) of DosR regulon of Mtb. As Intracellular IFN-γ secreted by T cells is the most essential cytokine in Th1 mediated protective immunity, these peptides were verified as potential immunogenic epitopes in Peripheral Blood Mononuclear Cells (PBMCs) of 10 healthy contacts of TB patients (HTB) and 10 Category I Pulmonary TB patients (PTB).The antigen-specific CD4 and CD8 T cells expressing intracellular IFN-γ were analyzed using monoclonal antibodies in all subjects by multi-parameter flow cytometry.Both, PTB and HTB individuals responded to DosR peptides by showing increased frequency of IFN-γ⁺CD4 and IFN-γ⁺CD8 T cells. The T-cell responses were significantly higher in PTB patients in comparision to the HTB individuals. Additionally, our synthetic peptides and pools showed higher frequencies of IFN-γ⁺CD4 and IFN-γ⁺CD8 T cells than the peptides of Ag85B.This pilot study can be taken up further in larger sample size which may support the untapped opportunity of designing Mtb DosR inclusive peptide based post-exposure subunit vaccine.     

Novel HLA-A2-restricted human metapneumovirus epitopes reduce viral titers in mice and are recognized by human T cells

Human metapneumovirus (HMPV) is a major cause of morbidity and mortality from acute lower respiratory tract illness, with most individuals seropositive by age five. Despite the presence of neutralizing antibodies, secondary infections are common and can be severe in young, elderly, and immunocompromised persons. Preclinical vaccine studies for HMPV have suggested a need for a balanced antibody and T cell immune response to enhance protection and avoid lung immunopathology. We infected transgenic mice expressing human HLA-A*0201 with HMPV and used ELISPOT to screen overlapping and predicted epitope peptides. We identified six novel HLA-A2 restricted CD8⁺ T cell (T_CD8) epitopes, with M_39–47 (M39) immunodominant. Tetramer staining detected M39-specific T_CD8 in lungs and spleen of HMPV-immune mice. Immunization with adjuvant-formulated M39 peptide reduced lung virus titers upon challenge. Finally, we show that T_CD8 from HLA-A*0201 positive humans recognize M39 by IFNγ ELISPOT and tetramer staining. These results will facilitate HMPV vaccine development and human studies.     

A liposomal peptide vaccine inducing CD8+ T cells in HLA-A2.1 transgenic mice,which recognise human cells encoding hepatitis C virus (HCV) proteins

Virus specific T cell responses play an important role in resolving acute hepatitis C virus (HCV) infections. Using the HLA-A2.1 transgenic mouse model we investigated the potential of a liposomal peptide vaccine to prime a CD8⁺ T cell response against 10 different HCV epitopes, relevant for human applications. We were able to demonstrate the induction of strong cytotoxic T cell responses and high numbers of IFN-γ-secreting cells, which persisted at high levels for at least 3 months. Co-integrating CpG oligonucleotides into liposomes further increased the number of IFN-γ-secreting cells by 2–10-fold for most epitopes tested. The frequency of specific cells was further analysed with chimeric A2 tetramers bearing the NS31073-1081 epitope and was estimated at 2–23% of the CD8⁺ T cell population. Importantly, mouse effector cells, specific for this epitope, were also capable of lysing a human target cell line expressing HCV proteins. This finding and the specific protection observed in challenge experiments with recombinant vaccinia virus expressing HCV sequences emphasise the biological relevance of the vaccine-induced immune response. In conclusion, such liposome formulations represent a safe and promising strategy to stimulate the CD8⁺ T cell against HCV.     

Identification of non-CSP antigens bearing CD8 epitopes in mice immunized with irradiated sporozoites

Immunization of BALB/c mice with irradiated sporozoites (IrSp) of Plasmodium yoelii can lead to sterile immunity. The circumsporozoite protein (CSP) plays a dominant role in protection. Nevertheless after hyper-immunization with IrSp, complete protection is obtained in CSP-transgenic BALB/c mice that are T-cell tolerant to the CSP and cannot produce antibodies [CSP-Tg/JhT(−/−)]. This protection is mediated exclusively by CD8⁺ T cells [1]. To identify the non-CSP protective T cell antigens, we studied the properties of 34 P. yoelii sporozoite antigens that are predicted to be secreted and to contain strong Kd-restricted CD8⁺ T cell epitopes. The synthetic peptides corresponding to the epitopes were used to screen for the presence of peptide-specific CD8⁺ T cells secreting interferon-γ (IFN-γ) in splenocytes from CSP-Tg/JhT(−/−) BALB/c mice hyper immunized with IrSp. However, the numbers of IFN-γ-secreting splenocytes specific for the non-CSP antigen-derived peptides were 20-100 times lower than those specific for the CSP-specific peptide. When mice were immunized with recombinant adenoviruses expressing selected non-CSP antigens, the animals were not protected against challenge with P. yoelii sporozoites although large numbers of CD8⁺ specific T cells were generated.     

Local innate and adaptive immune responses regulate inflammatory cell influx into the lungs after vaccination with formalin inactivated RSV

Inactivated respiratory syncytial virus (RSV) vaccines tend to predispose for immune mediated enhanced disease, characterized by Th2 responses and airway hypersensitivity reactions. We show in a C57BL/6 mouse model that the early innate response elicited by the challenge virus (RSV versus influenza virus) influences the outcome of the Th1/Th2 balance in the lung after intramuscular priming with inactivated vaccine. Priming of CD4⁺/IFN-γ⁺ T cells by mature dendritic cells administered intravenously and/or priming of a virus specific CD8⁺ T cell response ameliorated the Th2-mediated inflammatory response in the lung, suggesting that vaccination procedures are feasible that prevent vaccine induced immune pathology.     

A T cell epitope-based vaccine protects against chlamydial infection in HLA-DR4 transgenic mice

Vaccination with recombinant chlamydial protease-like activity factor (rCPAF) has been shown to provide robust protection against genital Chlamydia infection. Adoptive transfer of IFN-γ competent CPAF-specific CD4⁺ T cells was sufficient to induce early resolution of chlamydial infection and reduction of subsequent pathology in recipient IFN-γ-deficient mice indicating the importance of IFN-γ secreting CD4⁺ T cells in host defense against Chlamydia. In this study, we identify CD4⁺ T cell reactive CPAF epitopes and characterize the activation of epitope-specific CD4⁺ T cells following antigen immunization or Chlamydia challenge. Using the HLA-DR4 (HLA-DRB1*0401) transgenic mouse for screening overlapping peptides that induced T cell IFN-γ production, we identified at least 5 CPAF T cell epitopes presented by the HLA-DR4 complex. Immunization of HLA-DR4 transgenic mice with a rCPAFep fusion protein containing these 5 epitopes induced a robust cell-mediated immune response and significantly accelerated the resolution of genital and pulmonary Chlamydia infection. rCPAFep vaccination induced CPAF-specific CD4⁺ T cells in the spleen were detected using HLA-DR4/CPAF-epitope tetramers. Additionally, CPAF-specific CD4⁺ clones could be detected in the mouse spleen following Chlamydia muridarum and a human Chlamydia trachomatis strain challenge using these novel tetramers. These results provide the first direct evidence that a novel CPAF epitope vaccine can provide protection and that HLA-DR4/CPAF-epitope tetramers can detect CPAF epitope-specific CD4⁺ T cells in HLA-DR4 mice following C. muridarum or C. trachomatis infection. Such tetramers could be a useful tool for monitoring CD4⁺ T cells in immunity to Chlamydia infection and in developing epitope-based human vaccines using the murine model.     

Identification and characterization of HIV-1-specific CD8+ T cell epitopes presented by HLA-A*2601

Since HLA-A*26 is one of the most common alleles in Asia, where approximately 20% of people have this allele, identification of HIV-1-specific epitopes presented by HLA-A*26 is necessary for studies on the immunopathogenesis of AIDS and vaccine development in Asia. As presented herein, we used the reverse immunogenetics approach to identify HIV-1 epitopes presented by HLA-A*2601, one of the major HLA-A*26 subtypes. We selected 24 HLA-A*2601-binding peptides out of 110 HIV-1 peptides by using a HLA-A*2601 stabilization assay. The ability of these HLA-A*2601-binding peptides to induce peptide-specific CD8(+) T cells was tested by stimulating PBMCs from HIV-1-infected individuals having HLA-A*2601 with these peptides. Four HLA-A*2601-binding peptides induced peptide-specific CD8 T cells. Analysis using HIV-1 recombinant vaccinia-infected C1R-A*2601 cells indicated that these four peptides were HIV-1 epitopes endogenously presented by HLA-A*2601. Two epitope-specific CD8(+) T cells were predominantly detected in HIV-1 infected individuals, suggesting that these epitopes may be useful for vaccine development.     

Memory CD8 T cells elicited by HIV-1 lipopeptide vaccines display similar phenotypic profiles but differences in term of magnitude and multifunctionality compared with FLU- or EBV-specific memory T cells in humans

Differentiation marker, multifunctionality and magnitude analyses of specific-CD8⁺ memory T cells are crucial to improve development of HIV vaccines designed to generate cell-mediated immunity. Therefore, we fully characterized the HIV-specific CD8⁺ T cell responses induced in volunteers vaccinated with HIV lipopeptide vaccines for phenotypic markers, tetramer staining, cytokine secretion, and cytotoxic activities. The frequency of ex vivo CD8⁺ T cells elicited by lipopeptide vaccines is very rare and central-memory phenotype and functions of these cells were been shown to be important in AIDS immunity. So, we expanded them using specific peptides to compare the memory T cell responses induced in volunteers by HIV vaccines with responses to influenza (FLU) or Epstein Barr virus (EBV). By analyzing the differentiation state of IFN-γ-secreting CD8⁺ T cells, we found a CCR7⁻CD45RA⁻CD28^+int/CD28⁻ profile (>85%) belonging to a subset of intermediate-differentiated effector T cells for HIV, FLU, and EBV. We then assessed the quality of the response by measuring various T cell functions. The percentage of single IFN-γ T cell producers in response to HIV was 62% of the total of secreting T cells compared with 35% for FLU and EBV, dual and triple (IFN-γ/IL-2/CD107a) T cell producers could also be detected but at lower levels (8% compared with 37%). Finally, HIV-specific T cells secreted IFN-γ and TNF-α, but not the dual combination like FLU- and EBV-specific T cells. Thus, we found that the functional profile and magnitude of expanded HIV-specific CD8⁺ T precursors were more limited than those of to FLU- and EBV-specific CD8⁺ T cells. These data show that CD8⁺ T cells induced by these HIV vaccines have a similar differentiation profile to FLU and EBV CD8⁺ T cells, but that the vaccine potency to induce multifunctional T cells needs to be increased in order to improve vaccination strategies.     

Peptide-pulsed dendritic cells induce the hepatitis C viral epitope-specific responses of naïve human T cells

Hepatitis C virus (HCV) is a major cause of liver disease. Spontaneous resolution of infection is associated with broad, MHC class I- (CD8⁺) and class II-restricted (CD4⁺) T cell responses to multiple viral epitopes. Only 20% of patients clear infection spontaneously, however, most develop chronic disease. The response to chemotherapy varies; therapeutic vaccination offers an additional treatment strategy. To date, therapeutic vaccines have demonstrated only limited success in clinical trials. Vector-mediated vaccination with multi-epitope-expressing DNA constructs provides an improved approach. Highly-conserved, HLA-A2-restricted HCV epitopes and HLA-DRB1-restricted immunogenic consensus sequences (ICS, each composed of multiple overlapping and highly conserved epitopes) were predicted using bioinformatics tools and synthesized as peptides. HLA binding activity was determined in competitive binding assays. Immunogenicity and the ability of each peptide to stimulate naïve human T cell recognition and IFN-γ production were assessed in cultures of total PBMCs and in co-cultures composed of peptide-pulsed dendritic cells (DCs) and purified T lymphocytes, cell populations derived from normal blood donors. Essentially all predicted HLA-A2-restricted epitopes and HLA-DRB1-restricted ICS exhibited HLA binding activity and the ability to elicit immune recognition and IFN-γ production by naïve human T cells. The ability of DCs pulsed with these highly-conserved HLA-A2- and -DRB1-restricted peptides to induce naïve human T cell reactivity and IFN-γ production ex vivo demonstrates the potential efficacy of a multi-epitope-based HCV vaccine targeted to dendritic cells.     

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.     

Characterization of the cell-mediated immune response to Takeda’s live-attenuated tetravalent dengue vaccine in adolescents participating in a phase 2 randomized controlled trial conducted in a dengue-endemic setting

BackgroundAs robust dengue-specific CD4+ and CD8+ T cell responses are essential for protective immunity, we assessed cell-mediated immune (CMI) responses to a DENV-2-based dengue tetravalent vaccine candidate (TAK-003) in adolescents living in Panama, a dengue-endemic country.MethodsPeripheral blood mononuclear cells were collected from a subset of 67 participants ≥ 10 years old included in a phase 2 clinical trial of TAK-003 (Clinicaltrials.gov: NCT02302066). Following stimulation with dengue peptides, the frequency, magnitude, and cross-reactivity of the CD8+ and CD4+ T cell IFN-γ, TNF-α and IL-2 responses were assessed by flow cytometry.ResultsIntracellular cytokine staining identified NS1, NS3, and NS5 as the most common non-structural (NS) targets of the CD4+ T-cell response (IFN-γ+); NS3 and NS5 were the main NS targets of the CD8+ T cell response (IFN-γ+). Both CD4+ and CD8+ T-cell responses were multi-functional (IFN-γ + TNF-α + IL-2+) and cross-reactive against DENV-1, -3, and -4 serotypes. Similar responses were seen in all CMI assessments irrespective of participant baseline status for dengue neutralizing antibodies and T cells.ConclusionsTAK-003 elicited cross-reactive, multi-functional CD4+ and CD8+ T-cell responses, irrespective of dengue pre-exposure.     

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.     

Immunogenicity and safety of different injection routes and schedules of IC41, a Hepatitis C virus (HCV) peptide vaccine

Background

An effective vaccine would be a significant progress in the management of chronic HCV infections. This study was designed to examine whether different application schedules and injection routes may enhance the immunogenicity of the HCV peptide vaccine IC41.

Methods

In this randomized trial 54 healthy subjects received either subcutaneous (s.c.) or intradermal (i.d.) vaccinations weekly (16 injections) or every other week (8 injections). One group additionally received imiquimod, an activator of the toll-like receptor (TLR) 7. The T cell epitope-specific immune response to IC41 was assessed using [³H]-thymidine CD4+ T cell proliferation, interferon-gamma (IFN-γ) CD8+ and CD4+ ELIspot and HLA-A*0201 fluorescence-activated cell sorting (FACS) tetramer-binding assays.

Results

More than 60% of vaccinees responded in the CD4+ T cell proliferation assay in all groups. An HLA-A*0201 FACS tetramer-binding assay and IFN-γ CD8+ ELIspot class I response of more than 70% was induced in four and three groups, respectively. IC41 induced significant immunological responses in all groups with responder rates of up to 100%. Interestingly, topical imiquimod was not able to enhance immunogenicity but was associated with a lower immune response. Local injection site reactions were mostly transient. Intradermal injections caused more pronounced reactions compared to s.c., especially erythema and edema.

Conclusion

Compared to a previous study intensified dosing and/or i.d. injections enhanced the response rates to the vaccine IC41 in three assays measuring T cell function. Immunization with IC41 was generally safe in this study. These results justify testing IC41 in further clinical trials with HCV-infected individuals.     

Cellular immune responses of pigs induced by vaccination with either a whole cell sonicate or pepsin-digested Brachyspira (Serpulina) hyodysenteriae bacterin

Brachyspira (Serpulina) hyodysenteriae infection of pigs (swine dysentery) causes a mucohemorrhagic diarrhea resulting in significant economic losses for producers. A commercial vaccine consisting of a proteinase-digested bacterin has shown efficacy in the reduction of disease due to B. hyodysenteriae. Vaccines consisting of whole cell bacterins, however, generally fail to protect pigs from disease. In the present study, cellular immune responses induced by a proteinase-digested bacterin were compared to responses induced by a whole cell sonicate antigen preparation. In addition, usage of either squalene or Freund's incomplete adjuvants in combination with each antigen preparation was also compared. Both antigen preparations induced significant cellular immune responses as measured by in vitro (IFN-γ production and T cell proliferation) and in vivo methods (DTH responses). No significant differences were detected in proliferative, interferon-γ (IFN-γ), or delayed type hypersensitivity (DTH) responses by pigs receiving either adjuvant or antigen preparation. T cells (CD3⁺) but not B cells from vaccinated animals proliferated in response to in vitro stimulation with B. hyodysenteriae antigen. CD8⁺ (single positive and CD4/CD8 double positive) and γδ⁺ T cells were particularly responsive. In addition, high percentages of both CD8 single positive and CD4/CD8 double positive cells were detected in antigen-stimulated cultures. These findings demonstrate the unique sensitivity of porcine CD8⁺ T cells to priming for recall response by vaccination with a proteinase-digested B. hyodysenteriae bacterin.     

Delivery of antigenic candidates by a DNA/MVA heterologous approach elicits effector CD8T cell mediated immunity against Trypanosoma cruzi

In this study, we have characterized the immune mechanisms elicited by antigenic candidates, TcG2 and TcG4, delivered by a DNA-prime/MVA-boost approach, and evaluated the host responses to Trypanosoma cruzi infection in C57BL/6 mice. Immunization of mice with antigenic candidates elicited antigen-specific, high-avidity, trypanolytic antibody response (IgG2b > IgG1) and CD8⁺T cells that exhibited type-1 cytolytic effector (CD8⁺CD107a⁺IFN-γ⁺Perforin⁺) phenotype. The extent of TcG2-dependent type 1 B and T cell immunity was higher than that noted in TcG4-immunized mice, and expanded accordingly in response to challenge infection with T. cruzi. The progression of chronic phase in immunized mice was associated with persistence of IgGs, 55–90% reduction in the frequency of proinflammatory (IFN-γ⁺ or TNF-α⁺) CD8⁺T cells, and an increase or emergence of immunoregulatory (IL-10⁺) CD4/CD8 T cells. The tissue parasitism, infiltration of inflammatory infiltrate, parasite persistence, and fibrosis were decreased by 82–92% in heart and skeletal muscle of immunized/chronically infected mice. Control mice exhibited a significantly low antibody response, consistent activation of effector CD8⁺T cells dominated by pro-inflammatory phenotype and mixed cytokine profile (IFN-γ + TNF-α > IL-4 + IL-10), parasite persistence and pathologic damage in chagasic hearts. We conclude that delivery of TcG2 or TcG4 by DNA-rMVA approach elicits effective antibody and CD8⁺T cell mediated immunity against T. cruzi and Chagas disease. The emergence of type 2 cytokine and T cell response in chronic phase was indicative of prevention of clinical disease.