Effect of Early Secreted Antigen Target-6 Gene of Mycobacterium Tuberculosis as Genetic Adjuvant for Avian Influenza Virus DNA Vaccine in Chickens

Influenza virus, belongs to the family Orthomyxoviridae and genus Influenza virus A, causes major disease problems and serious economical losses in poultry industry. Highly pathogenic avian influenza H5N1 subtype which is associated with acute infection with high morbidity and mortality in susceptible birds, is still enzootic in poultry in Asia as well as European and African countries. The virus may also possess serious threat to the emergence of influenza pandemic in humans. Vaccination is one of the biosafety measures which has the greatest impact on improving global health and preventing morbidity and mortality due to avian influenza (AI) infection. The explosion of knowledge in molecular immunology has paved radical developments in vaccine technology. Immunization with DNA vaccines and genetic adjuvants as immunostimulators is an attractive approach in the development of future generations of vaccines and adjuvants. The viral envelope proteins, hemagglutinin (HA or H) and neuraminidase (NA or N), have been shown to play key roles in triggering protective immune responses against AI infection. Meanwhile, nucleocapsid protein (NP) may play a central role in cross protection between AI virus serotypes. The Mycobacterium tuberculosis Early Secreted Antigenic Target-6 (ESAT-6) antigen has been shown to elicit both humoral and cellular immunity, thus it has an ability to act as a genetic adjuvant. This study examined the ability of ESAT-6 to modulate antibody response against H5 following vaccination with DNA vaccine in chickens. In order to study the immunological properties of AIV DNA vaccines, several recombinant plasmids pcDNA3.1/H5, pcDNA3.1/N1, pcDNA3.1/NP, pcDNA3.1/H5-ESAT6, pcDNA3.1/N1-ESAT6 and pcDNA3.1/NP-ESAT6 were constructed. The recombinant plasmid constructs were confirmed by restriction enzymes and sequence analyses. The expression of genes of interest in cell culture was confirmed by immunofluorescence test and Western blot analysis. The immunogenicity of the DNA vaccine pcDNA3.1./H5 with and without the presence of ESAT-6 in specific-pathogen-free (SPF) chicks was determined. Sera obtained from the chickens immunized with pcDNA3.1/H5 and pcDNA3.1/H5-ESAT6 demonstrated viral neutralizing activities based on haemagglutination inhibition (HI) test. The sera collected from chicks immunized with pcDNA3.1/H5-ESAT6 have higher HI titer compared to the group which was immunized with pcDNA3.1/H5. However, the increase in HI titer at different post immunization days between these groups was not statistically significant. When the tissue samples from the chest muscle of injection site and spleen from chickens immunized with the DNA vaccine were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR), all the samples were positive for H5 specific transcripts. In summary, the current study delineated that the constructed recombinant plasmids were transcriptionally active in the in vivo chicken model and DNA immunization in SPF chicks with pcDNA3.1/H5 and pcDNA3.1/H5-ESAT6 produced humoral immune response. In conclusion, future studies are required to explore the role of ESAT-6 gene of Mycobacterium tuberculosis as an effective genetic adjuvant for H5 DNA vaccine in chickens

A novel HLA-B18 restricted CD8+ T cell epitope is efficiently cross-presented by dendritic cells from soluble tumor antigen

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8<sup>+</sup>T cell epitope, NY-ESO-1<sub>88–96</sub> (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1<sub>157–165</sub> epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-1<sub>88–96</sub> is much more efficiently cross-presented from the soluble form, than NY-ESO-1<sub>157–165</sub>. On the other hand, NY-ESO-1<sub>157–165</sub> is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A<sub>26–35</sub>; whereas NY-ESO-1<sub>88–96</sub> was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-1<sub>88–96</sub> is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18+ melanoma patients. Surprisingly, all the detectable responses to NY-ESO-1<sub>88–96</sub> from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8<sup>+</sup>T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed

Cross‐priming of TCD8+ specific for cell‐associated antigens

© 2014 Dr. Sara OveissiTCD8+ of the adaptive immune system play critical roles in the host defence against viruses and other pathogens through elimination of infected host cells. After infection, TCD8+ proliferate and adopt effector functions following recognition of specific antigenic‐peptides in the groove of a MHC class I molecule expressed by antigen presenting cells (APC). Professional APCs such as dendritic cells (DCs) are specialized for the priming and activation of naïve TCD8+. DCs are the key cell type responsible for T cell priming. They can either be directly infected and present viral antigens (direct‐priming) or phagocytose infected cell debris and process and present phagocytosed antigens to the specific TCD8+ (cross‐priming). Little is known about the actual contribution of direct versus cross‐priming during an immune response against viral infections. Understanding how DCs regulate TCD8+ responses is central for our ability to favourably manipulate the immune system as well as develop effective targeting strategies for optimal anti‐viral and antitumoral vaccination. To determine the contributions of direct versus cross‐priming to the clearance of an in vivo viral infection, we generated a Cre‐indicator transgenic mouse model utilising cutaneous HSV‐Cre infection to conditionally trigger model antigen expression in infected cells. This was expected to enable us to discriminate between virally infected and hence direct‐presenting DCs from uninfected and cross‐presenting ones in vivo for the first time. Unexpectedly, all generated transgenic mouse lines showed various levels of tolerance towards our model antigen due to undesired protein leakiness at steady state. However, TCD8+ in these neo‐transgene expresser transgenic mice possessed antigen ignorant properties and were non‐specifically activated following acute viral infection and the introduction of other innate immune cell ligands. Therefore, although these generated transgenic mice could not be used for their initial study purpose, their varying expression levels of model antigen make them an excellent model for studying peripheral tolerance induction and its maintenance. Cross‐priming is especially important for anti‐tumour immunity as tumour cells, although carrying tumour associated antigens, do not activate naïve TCD8+ efficiently due to an absence of co‐stimulatory molecules. Remarkably, our group has recently shown that influenza A virus (IAV) infection of allogeneic cells led to tumour protection due to enhanced cross‐priming of TCD8+ specific to cellular antigen. We have previously demonstrated that this enhancement was partially mediated through TLR7 sensing and entirely dependent on MyD88 and IFN signalling pathways, yet independent of the IL‐1β‐inflammasome. To further increase our understanding of cross‐priming enhancement mechanisms found in our system, we have additionally investigated the involvement of other immunological mechanisms in this thesis. Here, we show that IAV enhanced crosspriming is independent from the IL‐18‐inflammasome signalling pathway but that TCD4+ helper play a surprisingly important role for optimal enhancement. Also, through investigations using Batf3‐/‐ mice, we not only confirm the specificity of CD8α+ and CD103+ DC subsets for cross‐presenting, but also demonstrated that there are two types of cross‐priming outcomes: a baseline cross‐priming that is innate signalling independent and an innate immune signal enhanced crosspriming pathway. Interestingly, both types of cross‐priming events were abolished in Batf3‐/‐ mice. This knowledge will be useful to aid future efforts to develop more robust cancer vaccines. Finally, efficient antigen processing and presentation of the TCD8+ epitope is a central factor that determines the extent of specific T‐cell responses. Our group has identified the most immunodominant T‐cell response in the Balb/c mice after IAV infection. Interestingly, this novel epitope is encoded by the intronic region of the non‐structural protein mRNA. To dissect the mechanism of such epitope translation, as being either through spliced mRNA translation or generation using an alternative open reading frame (AltORF), we disabled splicing mechanisms and discovered that generation of the novel epitope occurred through AltORF translation. We will focus on the identification of the exact mechanism(s) underlying the efficient generation of this novel epitope as such mechanisms may provide a great opportunity for developing more efficient IAV T‐cell based vaccine strategies

Resident CD8(+) and migratory CD103(+) dendritic cells control CD8 T cell immunity during acute influenza infection.

The identification of the specific DC subsets providing a critical role in presenting influenza antigens to naïve T cell precursors remains contentious and under considerable debate. Here we show that CD8(+) T lymphocyte (TCD8+) responses are severely hampered in C57BL/6 mice deficient in the transcription factor Batf3 after intranasal challenge with influenza A virus (IAV). This transcription factor is required for the development of lymph node resident CD8(+) and migratory CD103(+)CD11b(-) DCs and we found both of these subtypes could efficiently stimulate anti-IAV TCD8+. Using a similar ex vivo approach, many publications on this subject matter excluded a role for resident, non-migratory CD8(+) DC. We postulate the differences reported can partially be explained by how DC are phenotyped, namely the use of MHC class II to segregate subtypes. Our results show that resident CD8(+) DC upregulate this marker during IAV infection and we advise against its use when isolating DC subtypes

Influenza A virus infection-induced macroautophagy facilitates MHC class II-restricted endogenous presentation of an immunodominant viral epitope

CD4+ T cells recognize peptides presented by major histocompatibility complex class II molecules (MHC-II). These peptides are generally derived from exogenous antigens. Macroautophagy has been reported to promote endogenous antigen presentation in viral infections. However, whether influenza A virus (IAV) infection-induced macroautophagy also leads to endogenous antigen presentation through MHC-II is still debated. In this study, we show that IAV infection leads to endogenous presentation of an immunodominant viral epitope NP311-325 by MHC-II to CD4+ T cells. Mechanistically, such MHC-II-restricted endogenous IAV antigen presentation requires de novo protein synthesis as it is inhibited by the protein synthesis inhibitor cycloheximide, and a functional ER-Golgi network as it is totally blocked by Brefeldin A. These results indicate that MHC-II-restricted endogenous IAV antigen presentation is dependent on de novo antigen and/or MHC-II synthesis, and transportation through the ER-Golgi network. Furthermore, such endogenous IAV antigen presentation by MHC-II is enhanced by TAP deficiency, indicating some antigenic peptides are of cytosolic origin. Most importantly, the bulk of such MHC-II-restricted endogenous IAV antigen presentation is blocked by autophagy inhibitors (3-MA and E64d) and deletion of autophagy-related genes, such as Beclin1 and Atg7. We have further demonstrated that in dendritic cells, IAV infection prevents autophagosome-lysosome fusion and promotes autophagosome fusion with MHC class II compartment (MIIC), which likely promotes endogenous IAV antigen presentation by MHC-II. Our results provide strong evidence that IAV infection-induced autophagosome formation facilitates endogenous IAV antigen presentation by MHC-II to CD4+ T cells. The implication for influenza vaccine design is discussed

LMP2 immunoproteasome promotes lymphocyte survival by degrading apoptotic BH3-only proteins

The role of the immunoproteasome is perceived as confined to adaptive immune responses given its ability to produce peptides ideal for MHC Class‐I binding. Here, we demonstrate that the immunoproteasome subunit, LMP2, has functions beyond its immunomodulatory role. Using LMP2‐deficient mice, we demonstrate that LMP2 is crucial for lymphocyte development and survival in the periphery. Moreover, LMP2‐deficient lymphocytes show impaired degradation of key BH3‐only proteins, resulting in elevated levels of pro‐apoptotic BIM and increased cell death. Interestingly, LMP2 is the sole immunoproteasome subunit required for BIM degradation. Together, our results suggest LMP2 has important housekeeping functions and represents a viable therapeutic target for cancer

Lack of influenza specific T_CD8+ responses in Batf3^o/o mice.

<p>(A) Pre-gating strategy to identify DC (B–C) DC from the inguinal and mediastinal lymph nodes of wildtype or Batf3^o/o mice on a B6 background were analyzed for their expression of either (B) CD8 and CD205 or (C) CD8 and CD103. Representative plots from 3-pooled mice from two independent experiments are shown. (D–E) B6 and Batf3^o/o mice were infected with PR8. On day 10, the absolute number of influenza specific T cells specific for defined peptide sequences were measured in the spleen (D) or BAL (E). The specific T cell response was elucidated following stimulation without peptides (Nil) or the peptides NP_366–374, PA_224–233, PB1-F2_62–70, or PB1_703–711. Shown is the absolute number of IFNγ⁺ CD8⁺ T cells, calculated using the following equation: cell count x%PI⁻ x%CD8⁺ x%IFNγ⁺. Average is taken from between 5–6 mice per group over two independent experiments and the error shows the SEM.</p

Antigen presentation by DC subsets after influenza infection.

<p>B6 mice were inoculated with PR8 and 3 days post infection the lung draining mediastinal lymph nodes were pooled and DC isolated. (A) Gating strategy for isolation of enriched DC subpopulations: CD8⁺ DC were purified on the basis of expression of CD11c and CD8 (upper left; right gate); CD11c⁺CD8⁻ cells (upper left; left gate) were segregated into CD103⁺CD11b⁻ (upper right; top gate) and CD103⁻CD11b⁺ (upper right; lower gate); and finally CD11c⁻ cells were isolated (upper left; bottom gate). (B) The antigen-specific T cell activation for the T cell line specific for the H-2D^b restricted influenza epitope NP_366–374 was assessed using B6 bone-marrow derived DCs pulsed with NP_366–374 peptide at indicated dilutions in a standard ICS assay for IFNγ. (C) Production of IFNγ by NP_366–374 T cells (5×10⁴) co-cultured for 6 hours with serially diluted DC subsets as identified in (A). Data are representative of two independent experiments, which showed a similar trend.</p

PA_224–33 T_CD8+ can be generated in Batf3^o/o mice.

<p>B6 or Batf3^o/o mice were inoculated intraperitoneally with 2.5×10⁶ LPS-treated, PA_224–233 peptide-pulsed B6 bone-marrow-derived DC. 7 days later, the number of PA_224–233 responding T_CD8+ present in the spleen was determined by ICS. Average is taken from 6 mice per group over two independent experiments and the error shows the SEM.</p