Two Theileria parva CD8 T cell antigen genes are more variable in buffalo than cattle parasites, but differ in pattern of sequence diversity

Background

Theileria parva causes an acute fatal disease in cattle, but infections are asymptomatic in the African buffalo (Syncerus caffer). Cattle can be immunized against the parasite by infection and treatment, but immunity is partially strain specific. Available data indicate that CD8⁺ T lymphocyte responses mediate protection and, recently, several parasite antigens recognised by CD8⁺ T cells have been identified. This study set out to determine the nature and extent of polymorphism in two of these antigens, Tp1 and Tp2, which contain defined CD8⁺ T-cell epitopes, and to analyse the sequences for evidence of selection.

Methodology/Principal Findings

Partial sequencing of the Tp1 gene and the full-length Tp2 gene from 82 T. parva isolates revealed extensive polymorphism in both antigens, including the epitope-containing regions. Single nucleotide polymorphisms were detected at 51 positions (∼12%) in Tp1 and in 320 positions (∼61%) in Tp2. Together with two short indels in Tp1, these resulted in 30 and 42 protein variants of Tp1 and Tp2, respectively. Although evidence of positive selection was found for multiple amino acid residues, there was no preferential involvement of T cell epitope residues. Overall, the extent of diversity was much greater in T. parva isolates originating from buffalo than in isolates known to be transmissible among cattle.

Conclusions/Significance

The results indicate that T. parva parasites maintained in cattle represent a subset of the overall T. parva population, which has become adapted for tick transmission between cattle. The absence of obvious enrichment for positively selected amino acid residues within defined epitopes indicates either that diversity is not predominantly driven by selection exerted by host T cells, or that such selection is not detectable by the methods employed due to unidentified epitopes elsewhere in the antigens. Further functional studies are required to address this latter point.     

Characterization of monoclonal antibodies against waterfowl parvoviruses VP3 protein

Background

Ebola viruses (EBOVs) cause severe hemorrhagic fever with a high mortality rate. At present, there are no licensed vaccines or efficient therapies to combat EBOV infection. Previous studies have shown that both humoral and cellular immune responses are crucial for controlling Ebola infection. CD8⁺ T cells play an important role in mediating vaccine-induced protective immunity. The objective of this study was to identify H-2^d-specific T cell epitopes in EBOV glycoproteins (GPs).

Results

Computer-assisted algorithms were used to predict H-2^d-specific T cell epitopes in two species of EBOV (Sudan and Zaire) GP. The predicted peptides were synthesized and identified in BALB/c mice immunized with replication-deficient adenovirus vectors expressing the EBOV GP. Enzyme-linked immunospot assays and intracellular cytokine staining showed that the peptides RPHTPQFLF (Sudan EBOV), GPCAGDFAF and LYDRLASTV (Zaire EBOV) could stimulate splenoctyes in immunized mice to produce large amounts of interferon-gamma.

Conclusion

Three peptides within the GPs of two EBOV strains were identified as T cell epitopes. The identification of these epitopes should facilitate the evaluation of vaccines based on the Ebola virus glycoprotein in a BALB/c mouse model.     

Polyfunctional CD4+ T cell responses to a set of pathogenic arenaviruses provide broad population coverage

Background

Several arenaviruses cause severe hemorrhagic fever and aseptic meningitis in humans for which no licensed vaccines are available. A major obstacle for vaccine development is pathogen heterogeneity within the Arenaviridae family. Evidence in animal models and humans indicate that T cell and antibody-mediated immunity play important roles in controlling arenavirus infection and replication. Because CD4⁺ T cells are needed for optimal CD8⁺ T cell responses and to provide cognate help for B cells, knowledge of epitopes recognized by CD4⁺ T cells is critical to the development of an effective vaccine strategy against arenaviruses. Thus, the goal of the present study was to define and characterize CD4⁺ T cell responses from a broad repertoire of pathogenic arenaviruses (including lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses) and to provide determinants with the potential to be incorporated into a multivalent vaccine strategy.

Results

By inoculating HLA-DRB1*0101 transgenic mice with a panel of recombinant vaccinia viruses, each expressing a single arenavirus antigen, we identified 37 human HLA-DRB1*0101-restricted CD4⁺ T cell epitopes from the 7 antigenically distinct arenaviruses. We showed that the arenavirus-specific CD4⁺ T cell epitopes are capable of eliciting T cells with a propensity to provide help and protection through CD40L and polyfunctional cytokine expression. Importantly, we demonstrated that the set of identified CD4⁺ T cell epitopes provides broad, non-ethnically biased population coverage of all 7 arenavirus species targeted by our studies.

Conclusions

The identification of CD4⁺ T cell epitopes, with promiscuous binding properties, derived from 7 different arenavirus species will aid in the development of a T cell-based vaccine strategy with the potential to target a broad range of ethnicities within the general population and to protect against both Old and New World arenavirus infection.     

Immunological response induced by abagovomab as a maintenance therapy in patients with epithelial ovarian cancer: relationship with survival—a substudy of the MIMOSA trial

Purpose

To determine whether abagovomab induces protective immune responses in ovarian cancer patients in first clinical remission. The present analysis is a substudy of monoclonal antibody immunotherapy for malignancies of the ovary by subcutaneous abagovomab trial (NCT00418574).

Methods

The study included 129 patients, 91 in the abagovomab arm and 38 in the placebo arm. Circulating CA125-specific cytotoxic T lymphocytes (CTL) were measured by a flow cytometry-based interferon-γ producing assay. Human antimouse antibody and anti-anti-idiotypic (Ab3) were assessed by ELISA. Patients were evaluated before starting the treatment and at different time points during induction and maintenance phases.

Results

A similar percentage of patients in both the placebo and abagovomab arms had CA125-specific CTL (26.3 and 31.8 %, respectively; p = 0.673 by Fisher’s exact test). Patients with CA125-specific CTL in both arms tended to have an increased relapse-free survival (RFS, log-rank test p = 0.095) compared to patients without. Patients (n = 27) in the abagovomab arm without CA125-specific CTL but that developed Ab3 above the cutoff (defined as median Ab3 level at week 22) had a prolonged RFS compared to patients (n = 24) that did not develop Ab3 above the cutoff (log-rank test p = 0.019).

Conclusion

Abagovomab does not induce CA125-specific CTL. However, patients with CA125-specific CTL perform better than patients without, irrespective of abagovomab treatment. Abagovomab-induced Ab3 associate with prolonged RFS in patients without CA125-specific CTL. Further studies are needed to confirm these data and to assess the potential utility of these immunological findings as a tool for patient selection in clinical trial.     

Effect of age and vaccination on extent and spread of Chlamydia pzneumoniae infection in C57BL/6 mice

Background

Chlamydia pneumoniae is an obligate intracellular respiratory pathogen for humans. Infection by C. pneumoniae may be linked etiologically to extra-respiratory diseases of aging, especially atherosclerosis. We have previously shown that age promotes C. pneumoniae respiratory infection and extra-respiratory spread in BALB/c mice.

Findings

Aged C57BL/6 mice had a greater propensity to develop chronic and/or progressive respiratory infections following experimental intranasal infection by Chlamydia pneumoniae when compared to young counterparts. A heptavalent CTL epitope minigene (CpnCTL7) vaccine conferred equal protection in the lungs of both aged and young mice. This vaccine was partially effective in protecting against C. pneumoniae spread to the cardiovascular system of young mice, but failed to provide cardiovascular protection in aged animals.

Conclusions

Our findings suggest that vaccine strategies that target the generation of a C. pneumoniae-specific CTL response can protect the respiratory system of both young and aged animals, but may not be adequate to prevent dissemination of C. pneumoniae to the cardiovascular system or control replication in those tissues in aged animals.     

Decline of influenza-specific CD8+ T cell repertoire in healthy geriatric donors

Background

While influenza vaccination results in protective antibodies against primary infections, clearance of infection is primarily mediated through CD8⁺ T cells. Studying the CD8⁺ T cell response to influenza epitopes is crucial in understanding the disease associated morbidity and mortality especially in at risk populations such as the elderly. We compared the CD8⁺ T cell response to immunodominant and subdominant influenza epitopes in HLA-A2⁺ control, adult donors, aged 21-42, and in geriatric donors, aged 65 and older.

Results

We used a novel artificial Antigen Presenting Cell (aAPC) based stimulation assay to reveal responses that could not be detected by enzyme-linked immunosorbent spot (ELISpot). 14 younger control donors and 12 geriatric donors were enrolled in this study. The mean number of influenza-specific subdominant epitopes per control donor detected by ELISpot was only 1.4 while the mean detected by aAPC assay was 3.3 (p = 0.0096). Using the aAPC assay, 92% of the control donors responded to at least one subdominant epitopes, while 71% of control donors responded to more than one subdominant influenza-specific response. 66% of geriatric donors lacked a subdominant influenza-specific response and 33% of geriatric donors responded to only 1 subdominant epitope. The difference in subdominant response between age groups is statistically significant (p = 0.0003).

Conclusion

Geriatric donors lacked the broad, multi-specific response to subdominant epitopes seen in the control donors. Thus, we conclude that aging leads to a decrease in the subdominant influenza-specific CTL responses which may contribute to the increased morbidity and mortality in older individuals.     

The Effect of HLA Polymorphisms on the Recognition of Gag Epitopes in HIV-1 CRF01_AE Infection

Introduction

The design of a globally effective vaccine rests on the identification of epitopes capable of eliciting effective cytotoxic T lymphocyte (CTL) responses across multiple HIV clades in different populations. This study aims to discern the effect of HLA polymorphisms and the cross-clade reactivity or clade-specificity of epitopes in Thailand where HIV-1 CRF01_AE is circulating.

Materials and Methods

14 peptides based on consensus HIV-1 CRF01_AE amino acid sequences were designed for use in IFN-γ ELISpot assays and ⁵¹Cr release assays among 66 HIV-1 CRF01_AE-infected Thai patients. For ELISpot responders carrying HLA alleles currently unknown to restrict CRF01_AE epitopes, in silico epitope-HLA prediction was performed.

Results

29/66 (43.9%) patients recognized at least one peptide. In total 79 responses were seen against all 14 peptides. 28/79 (35.4%) of the responses were in patients with HLA alleles previously reported to restrict CRF01_AE epitopes, 24/79 (30.4%) responses were in individuals with HLA alleles previously reported to restrict epitopes of HIV clades other than CRF01_AE, and the remaining 27/79 (34.2%) responses were not associated with HLA alleles previously known to restrict HIV epitopes. In silico epitope prediction detected 19 novel, epitope-HLA combinations, and 11/19 (57.9%) were associated with HLA-C alleles. We further confirmed a novel HLA restriction of a previously identified HIV-1 Gag epitope [p24_122–130: PPIPVGDIY (PY9)] by HLA-B*40:01 with a standard ⁵¹Cr release assay.

Discussion

CTL recognition sites in HIV-1 Gag were similar among different clades but the HLA restriction differed in Thai patients. This disparity in HLA restriction along different populations illustrated the importance of clade- and population-specific HLA analysis prior to CTL vaccine design.     

Large-scale validation of methods for cytotoxic T-lymphocyte epitope prediction

Background  

Reliable predictions of Cytotoxic T lymphocyte (CTL) epitopes are essential for rational vaccine design. Most importantly, they can minimize the experimental effort needed to identify epitopes. NetCTL is a web-based tool designed for predicting human CTL epitopes in any given protein. It does so by integrating predictions of proteasomal cleavage, TAP transport efficiency, and MHC class I affinity. At least four other methods have been developed recently that likewise attempt to predict CTL epitopes: EpiJen, MAPPP, MHC-pathway, and WAPP. In order to compare the performance of prediction methods, objective benchmarks and standardized performance measures are needed. Here, we develop such large-scale benchmark and corresponding performance measures and report the performance of an updated version 1.2 of NetCTL in comparison with the four other methods.     

Clinical and immunological responses in metastatic melanoma patients vaccinated with a high-dose poly-epitope vaccine

Background

Safety and cellular immunogenicity of rising doses and varying regimens of a poly-epitope vaccine were evaluated in advanced metastatic melanoma. The vaccine comprised plasmid DNA and recombinant modified vaccinia virus Ankara (MVA) both expressing a string (Mel3) of seven HLA.A2/A1 epitopes from five melanoma antigens.

Methods

Forty-one HLA-A2 positive patients with stage III/IV melanoma were enrolled. Patient groups received one or two doses of DNA.Mel3 followed by escalating doses of MVA.Mel3. Immunisations then continued eight weekly in the absence of disease progression. Epitope-specific CD8+ T cell responses were evaluated using ex-vivo tetramer and IFN-γ ELISPOT assays. Safety and clinical responses were monitored.

Results

Prime-boost DNA/MVA induced Melan-A-specific CD8+ T cell responses in 22/31 (71%) patients detected by tetramer assay. ELISPOT detected a response to at least one epitope in 10/31 (32%) patients. T cell responder rates were <50% with low-dose DNA/MVA, or MVA alone, rising to 91% with high-dose DNA/MVA. Among eight patients showing evidence of clinical benefit—one PR (24 months+), five SD (5 months+) and two mixed responses—seven had associated immune responses. Melan-A-tetramer+ immunity was associated with a median 8-week increase in time-to-progression (P = 0.037) and 71 week increase in survival (P = 0.0002) compared to non-immunity. High-dose vaccine was well tolerated. The only significant toxicities were flu-like symptoms and injection-site reactions.

Conclusions

DNA.Mel3 and MVA.Mel3 in a prime-boost protocol generated high rates of immune response to melanoma antigen epitopes. The treatment was well tolerated and the correlation of immune responses with patient outcomes encourages further investigation.     

Critical components of a DNA fusion vaccine able to induce protective cytotoxic T cells against a single epitope of a tumor antigen

DNA vaccines can activate immunity against tumor Ags expressed as MHC class I-associated peptides. However, priming of CD8(+) CTL against weak tumor Ags may require adjuvant molecules. We have used a pathogen-derived sequence from tetanus toxin (fragment C (FrC)) fused to tumor Ag sequences to promote Ab and CD4(+) T cell responses. For induction of CD8(+) T cell responses, the FrC sequence has been engineered to remove potentially competitive MHC class I-binding epitopes and to improve presentation of tumor epitopes. The colon carcinoma CT26 expresses an endogenous retroviral gene product, gp70, containing a known H2-L(d)-restricted epitope (AH1). A DNA vaccine encoding gp70 alone was a poor inducer of CTL, and performance was not significantly improved by fusion of full-length FrC. However, use of a minimized domain of FrC, with the AH1 sequence fused to the 3' position, led to rapid induction of high levels of CTL. IFN-gamma-producing epitope-specific CTL were detectable ex vivo and these killed CT26 targets in vitro. The single epitope vaccine was more effective than GM-CSF-transfected CT26 tumor cells in inducing an AH1-specific CTL response and equally effective in providing protection against tumor challenge. Levels of AH1-specific CTL in vivo were increased following injection of tumor cells, and CTL expanded in vitro were able to kill CT26 cells in tumor bearers. Pre-existing immunity to tetanus toxoid had no effect on the induction of AH1-specific CTL. These data demonstrate the power of epitope-specific CTL against tumor cells and illustrate a strategy for priming immunity via a dual component DNA vaccine.     

Transcriptomic profile of host response in Japanese encephalitis virus infection

Background

The attenuated Yellow fever (YF) 17D vaccine virus is one of the safest and most effective viral vaccines administered to humans, in which it elicits a polyvalent immune response. Herein, we used the YF 17D backbone to express a Trypanosoma cruzi CD8⁺ T cell epitope from the Amastigote Surface Protein 2 (ASP-2) to provide further evidence for the potential of this virus to express foreign epitopes. The TEWETGQI CD8⁺ T cell epitope was cloned and expressed based on two different genomic insertion sites: in the fg loop of the viral Envelope protein and the protease cleavage site between the NS2B and NS3. We investigated whether the site of expression had any influence on immunogenicity of this model epitope.

Results

Recombinant viruses replicated similarly to vaccine virus YF 17D in cell culture and remained genetically stable after several serial passages in Vero cells. Immunogenicity studies revealed that both recombinant viruses elicited neutralizing antibodies to the YF virus as well as generated an antigen-specific gamma interferon mediated T-cell response in immunized mice. The recombinant viruses displayed a more attenuated phenotype than the YF 17DD vaccine counterpart in mice. Vaccination of a mouse lineage highly susceptible to infection by T. cruzi with a homologous prime-boost regimen of recombinant YF viruses elicited TEWETGQI specific CD8⁺ T cells which might be correlated with a delay in mouse mortality after a challenge with a lethal dose of T. cruzi.

Conclusions

We conclude that the YF 17D platform is useful to express T. cruzi (Protozoan) antigens at different functional regions of its genome with minimal reduction of vector fitness. In addition, the model T. cruzi epitope expressed at different regions of the YF 17D genome elicited a similar T cell-based immune response, suggesting that both expression sites are useful. However, the epitope as such is not protective and it remains to be seen whether expression of larger domains of ASP-2, which include the TEWETGQI epitope, will elicit better T-CD8+ responses to the latter. It is likely that additional antigens and recombinant virus formulations will be necessary to generate a protective response.     

Autologous versus allogeneic peptide-pulsed dendritic cells for anti-tumour vaccination: expression of allogeneic MHC supports activation of antigen specific T cells,but impairs early naïve cytotoxic priming and anti-tumour therapy

Background

Dendritic cells (DC) pulsed with MHC class I-restricted tumour associated antigen (TAA) peptides have been widely tested in pre-clinical models and early clinical studies for their ability to prime cytotoxic T cell (CTL) responses. The effect of co-expression of allogeneic MHC antigens on DC immunogenicity has not been addressed, and has implications for the feasibility of clinical applications.

Objective

This study compared DC from autologous H-2^b or semi-allogeneic F1 H-2^bxk mice pulsed with the H-2^b-restricted model ovalbumin (OVA) peptide SIINFEKL, and compared in vitro and in vivo their ability to (i) activate specific OT1 cells, (ii) prime naïve CTL, and (iii) protect against B16.OVA challenge. Peptide-pulsed autologous and allogeneic DC were also tested in naïve human CTL priming assays.

Results

Semi-allogeneic DC expressed higher levels of co-stimulatory molecules. On pulsing with SIINFEKL they triggered greater proliferation of OT1 cells in vitro and in vivo, but were less effective at naïve CTL priming and tumour protection. Autologous human DC were similarly more potent at naïve CTL priming against the melanoma-associated TAA MART-1 in vitro.

Conclusion

The expression of allogeneic MHC antigens on peptide-pulsed DC impairs naïve CTL priming and anti-tumour effects, despite effective TAA presentation both in vitro and in vivo.

     

Cytotoxic T lymphocyte lysis of HTLV-1 infected cells is limited by weak HBZ protein expression,but non-specifically enhanced on induction of Tax expression

Background

Immunogenetic evidence indicates that cytotoxic T lymphocytes (CTLs) specific for the weak CTL antigen HBZ limit HTLV-1 proviral load in vivo, whereas there is no clear relationship between the proviral load and the frequency of CTLs specific for the immunodominant antigen Tax. In vivo, circulating HTLV-1-infected cells express HBZ mRNA in contrast, Tax expression is typically low or undetectable. To elucidate the virus-suppressing potential of CTLs targeting HBZ, we compared the ability of HBZ- and Tax-specific CTLs to lyse naturally-infected cells, by co-incubating HBZ- and Tax-specific CTL clones with primary CD4⁺ T cells from HLA-matched HTLV-1-infected donors. We quantified lysis of infected cells, and tested whether specific virus-induced host cell surface molecules determine the susceptibility of infected cells to CTL-mediated lysis.

Results

Primary infected cells upregulated HLA-A*02, ICAM-1, Fas and TRAIL-R1/2 in concert with Tax expression, forming efficient targets for both HTLV-1-specific CTLs and CTLs specific for an unrelated virus. We detected expression of HBZ mRNA (spliced isoform) in both Tax-expressing and non-expressing infected cells, and the HBZ_26–34 epitope was processed and presented by cells transfected with an HBZ expression plasmid. However, when coincubated with primary cells, a high-avidity HBZ-specific CTL clone killed significantly fewer infected cells than were killed by a Tax-specific CTL clone. Finally, incubation with Tax- or HBZ-specific CTLs resulted in a significant decrease in the frequency of cells expressing high levels of HLA-A*02.

Conclusions

HTLV-1 gene expression in primary CD4⁺ T cells non-specifically increases susceptibility to CTL lysis. Despite the presence of HBZ spliced-isoform mRNA, HBZ epitope presentation by primary cells is significantly less efficient than that of Tax.

     

Cross-protective peptide vaccine against influenza A viruses developed in HLA-A*2402 human immunity model

Background

The virus-specific cytotoxic T lymphocyte (CTL) induction is an important target for the development of a broadly protective human influenza vaccine, since most CTL epitopes are found on internal viral proteins and relatively conserved. In this study, the possibility of developing a strain/subtype-independent human influenza vaccine was explored by taking a bioinformatics approach to establish an immunogenic HLA-A24 restricted CTL epitope screening system in HLA-transgenic mice.

Methodology/Principal Findings

HLA-A24 restricted CTL epitope peptides derived from internal proteins of the H5N1 highly pathogenic avian influenza A virus were predicted by CTL epitope peptide prediction programs. Of 35 predicted peptides, six peptides exhibited remarkable cytotoxic activity in vivo. More than half of the mice which were subcutaneously vaccinated with the three most immunogenic and highly conserved epitopes among three different influenza A virus subtypes (H1N1, H3N2 and H5N1) survived lethal influenza virus challenge during both effector and memory CTL phases. Furthermore, mice that were intranasally vaccinated with these peptides remained free of clinical signs after lethal virus challenge during the effector phase.

Conclusions/Significance

This CTL epitope peptide selection system can be used as an effective tool for the development of a cross-protective human influenza vaccine. Furthermore this vaccine strategy can be applicable to the development of all intracellular pathogens vaccines to induce epitope-specific CTL that effectively eliminate infected cells.     

Targeting a Polyepitope Protein Incorporating Multiple Class II-Restricted Viral Epitopes to the Secretory/Endocytic Pathway Facilitates Immune Recognition by CD4+ Cytotoxic T Lymphocytes: a Novel Approach to Vaccine Design

The role of CD4⁺ and CD8⁺ cells in the generation of an effective immune response against viral infections is well established. Moreover, there is an increasing realization that subunit vaccines which include both CD4⁺- and CD8⁺-T-cell epitopes are highly effective in controlling viral infections, as opposed to those which are designed to activate a CD8⁺- or CD4⁺-T-cell response alone. One of the major limitations of epitope-based vaccines designed to stimulate virus-specific CD4⁺ T cells is that endogenously expressed class II-restricted minimal cytotoxic-T-lymphocyte (CTL) epitopes are poorly recognized by CD4⁺ CTLs. In the present study we attempted to enhance the efficiency of class II-restricted endogenous presentation of minimal class II-restricted CTL epitopes by specifically targeting a polyepitope protein to class II processing compartments through the endosomal and/or lysosomal pathway. A significantly enhanced stimulation of virus-specific CD4⁺-T-cell clones by antigen-presenting cells (APC) expressing the recombinant polyepitope protein targeted to the endocytic/secretory pathway was readily demonstrated in cytotoxicity assays. In addition, in vitro activation of Epstein-Barr virus- and influenza virus-specific CD4⁺ memory CTLs by the recombinant constructs encoding the polyepitope protein, specifically targeted to the lysosomal compartment, was also demonstrated. The enhanced stimulatory capacity of APC expressing a lysosome-targeted polyepitope protein has important implications for vaccine design.There is now increasing evidence to suggest that both CD4⁺ and CD8⁺ T cells are critical for the generation of an effective immune response against intracellular pathogens. Although both CD4⁺ and CD8⁺ T cells recognize nonnative forms of the antigen in association with major histocompatibility complex (MHC) molecules, the presentation of antigen to these two types of T lymphocytes occurs through distinct pathways (24). In fact, the disparity in antigen presentation to these T cells is not due to processing differences but rather reflects the differences in the capacities of class I and class II molecules to bind antigenic determinants in an intracellular compartment. Indeed, earlier studies have shown that for processing and interaction with MHC class II molecules, antigen expressed de novo needs to be targeted to an endosomal or lysosomal compartment (5). There are two major pathways by which antigens are targeted to these compartments. The traditional pathway involves the phagocytosis or endocytosis of exogenous antigens, followed by degradation by acid proteases in the endosomal or lysosomal compartments (3, 8, 26, 41). On the other hand, class II-restricted presentation of endogenously synthesized proteins mainly involves membrane antigens which are thought to enter the endosomal or lysosomal pathway by internalization from the cell surface (11). Although, in certain experimental systems, cytoplasmic and nuclear proteins may also enter this endogenous pathway, generally these proteins are targets for the class I processing pathway (9, 14, 20, 27).One of the major limitations of the epitope-based vaccines designed to stimulate virus-specific CD4⁺ T cells is that endogenously expressed class II-restricted minimal cytotoxic T-lymphocyte (CTL) epitopes are poorly recognized by CD4⁺ CTLs (2, 35, 38). Based on these observations, we reasoned that a molecular approach that directly routes these epitopes into the MHC class II pathway, such as the endocytic or lysosomal compartments, might facilitate endogenous presentation to CD4⁺ T cells. The lysosome-associated membrane protein (LAMP-1) and the invariant chain (Ii) are transmembrane proteins which are localized predominantly in the lysosomes and endosomes, respectively. The cytoplasmic domains of these proteins contain specific targeting signals that mediate their translocation to the specific compartments. We therefore designed a chimeric polyepitope construct capable of encoding multiple class II-restricted CTL epitopes from Epstein-Barr virus (EBV) and influenza virus linked to the cytoplasmic and/or transmembrane domains of LAMP-1 and the Ii protein, with the aim of targeting the epitopes to the endosomal and lysosomal compartments. The data presented in this study clearly demonstrate that if the endogenously synthesized polyepitope protein is targeted to the endocytic/secretory pathway, processing and presentation of all the epitopes are dramatically enhanced. More importantly, minimal epitope sequences, without any natural flanking sequences, were adequate for efficient stimulation of the virus-specific memory CTL response, a result that has important implications for epitope-based vaccine design.     

Protection against the allergic airway inflammation depends on the modulation of spleen dendritic cell function and induction of regulatory T cells in mice

Background

Allergen-induced imbalance of specific T regulatory (Treg) cells and T helper 2 cells plays a decisive role in the development of immune response against allergens.

Objective

To evaluate effects and potential mechanisms of DNA vaccine containing ovalbumin (OVA) and Fc fusion on allergic airway inflammation.

Methods

Bronchoalveolar lavage (BAL) levels of inflammatory mediators and leukocyte infiltration, expression of CD_11c ⁺CD₈₀ ⁺ and CD_11c ⁺CD₈₆ ⁺ co-stimulatory molecules in spleen dendritic cells (DCs), circulating CD₄ ⁺ and CD₈ ⁺ T cells, Foxp3⁺ in spleen CD₄ ⁺ T cells and spleen CD₄ ⁺ T cells were measured in OVA-sensitized and challenged animals pretreated with pcDNA, OVA-pcDNA, Fc-pcDNA, and OVA-Fc-pcDNA.

Results

OVA-Sensitized and challenged mice developed airway inflammation and Th2 responses, and decreased the proliferation of peripheral CD₄ ⁺and CD₈ ⁺ T cells and the number of spleen Foxp₃ ⁺ Treg. Those changes with increased INF-γ production and reduced OVA-specific IgE production were protected by the pretreatment with OVA-Fc-pcDNA.

Conclusion

DNA vaccine encoding both Fc and OVA showed more effective than DNA vaccine encoding Fc or OVA alone, through the balance of DCs and Treg.     

Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway

Background

Chlamydiae species are of much importance from a clinical viewpoint. Their diversity both in terms of their numbers as well as clinical involvement are presently believed to be significantly underestimated. The obligate intracellular nature of chlamydiae has also limited their genetic and biochemical studies. Thus, it is of importance to develop additional means for their identification and characterization.

Results

We have carried out analyses of available chlamydiae genomes to identify sets of unique proteins that are either specific for all Chlamydiales genomes, or different Chlamydiaceae family members, or members of the Chlamydia and Chlamydophila genera, or those unique to Protochlamydia amoebophila, but which are not found in any other bacteria. In total, 59 Chlamydiales-specific proteins, 79 Chlamydiaceae-specific proteins, 20 proteins each that are specific for both Chlamydia and Chlamydophila and 445 ORFs that are Protochlamydia-specific were identified. Additionally, 33 cases of possible gene loss or lateral gene transfer were also detected.

Conclusion

The identified chlamydiae-lineage specific proteins, many of which are highly conserved, provide novel biomarkers that should prove of much value in the diagnosis of these bacteria and in exploration of their prevalence and diversity. These conserved protein sequences (CPSs) also provide novel therapeutic targets for drugs that are specific for these bacteria. Lastly, functional studies on these chlamydiae or chlamydiae subgroup-specific proteins should lead to important insights into lineage-specific adaptations with regards to development, infectivity and pathogenicity.