Sung-Youl Ko

A Combination of Chemoimmunotherapies Can Efficiently Break Self-Tolerance and Induce Antitumor Immunity in a Tolerogenic Murine Tumor Model

Her-2/neu is a well-characterized tumor-associated antigen overexpressed in human carcinomas such as breast cancer. Because Her-2/neu is a self-antigen with poor immunogenicity due to immunologic tolerance, active immunotherapy targeting Her-2/neu should incorporate methods to overcome immunologic tolerance to self-proteins. In this study, we developed a tolerogenic tumor model in mice using mouse Her-2/neu as self-antigen and investigated whether genetic vaccination with DNA plasmid and/or adenoviral vector expressing the extracellular and transmembrane domain of syngeneic mouse Her-2/neu or xenogenic human Her-2/neu could induce mouse Her-2/neu-specific CTL responses. Interestingly, adenoviral vectors expressing xenogenic human Her-2/neu (AdhHM) proved capable of breaking immune tolerance and of thereby inducing self-reactive CTL and antibodies, but not to the degree required to induce therapeutic antitumor immunity. In attempting to generate therapeutic antitumor immunity against established tumors, we adopted several approaches. Treatment with agonistic anti-glucocorticoid-induced TNFR family-related receptor (GITR) antibody plus AdhHM immunization significantly increased self-reactive CTL responses, and alpha-galactosylceramide (alphaGalCer)-loaded dendritic cells (DC) transduced with AdhHM were shown to break self-tolerance in a tolerogenic murine tumor model. Furthermore, gemcitabine treatment together with either AdhHM plus agonistic anti-GITR antibody administration or alphaGalCer-loaded DC transduced with AdhHM showed potent therapeutic antitumor immunity and perfect protection against preexisting tumors. Gemcitabine treatment attenuated the tumor-suppressive environment by eliminating CD11b(+)/Gr-1(+) myeloid-derived suppressor cells. When combined with immunotherapies, gemcitabine offers a promising strategy for the Ag-specific treatment of human cancer.

Optimization of Codon Usage Enhances the Immunogenicity of a DNA Vaccine Encoding Mycobacterial Antigen Ag85B

ABSTRACT In spite of its many other benefits, DNA vaccine is limited in its application by its insufficient immunogenicity. One promising approach for enhancing its immunogenicity is to maximize its expression in the immunized host. In the current study, we investigated whether codon optimization of the mycobacterial antigen Ag85B gene could enhance the expression and immunogenicity of the Ag85B DNA vaccine. We generated a synthetic humanized Ag85B (hAg85B) gene in which codon usage was optimized for expression in human cells. DNA plasmids with codon-optimized hAg85B increased the level of protein expression in vitro and in vivo. DNA vaccine with hAg85B induced stronger Th1-like and cytotoxic T-cell immune responses in BALB/c mice and generated higher protective immunity in a BALB/c mouse model of Mycobacterium tuberculosis aerosol infection than did the DNA vaccine with wild-type Ag85B. Therefore, our results suggest that codon optimization of mycobacterial antigens (e.g., Ag85B) could improve protein expression and thereby enhance the immunogenicity of DNA vaccines against M. tuberculosis.

Enhanced efficacy of DNA vaccination against Her-2/neu tumor antigen by genetic adjuvants

Certain types of malignant tumors overexpress Her‐2/neu, a transmembrane glycoprotein of the class I receptor tyrosine kinase erbB family. To develop an effective Her‐2/neu vaccine for selective immunotherapy of these malignancies, we prepared Her‐2/neu DNA plasmid encoding the transmembrane and extracellular domain (pHM) and tested the ability of this construct to induce antitumor immunity in animal models. In addition, we investigated the effects of cytokine used as a genetic adjuvant. Modulation by factors that affect T‐cell function or hematopoiesis, including interleukin‐12, interleukin‐15, interleukin‐18, interleukin‐23, Eta‐1, Flt3L and GM‐CSF, was studied in the forms of monocistronic and bicistronic plasmid. Our results demonstrated that vaccination of pHM could induce successful antitumor immunity against Her‐2/neu‐expressing murine tumor cells in BALB/c mice. We also showed that the antitumor activity of pHM was augmented by coadministration and coexpression of different cytokines. Despite the similar levels of gene expression, the antitumor effects of bicistronic plasmids coexpressing Her‐2/neu antigen and cytokine were improved in comparison with coadministration of separate monocistronic plasmid. In particular, coexpression of interleukin‐18 or GM‐CSF with Her‐2/neu increased antitumor activity in both preventive and therapeutic experiments. These findings can help in the decision concerning which of the various cytokine adjuvants should be used for the development of a Her‐2/neu DNA vaccine. In addition, our results from a large panel of cytokine adjuvants in the various tumor models may provide an insight into the important immune components of antitumor immunity.

Comparative analysis of effects of cytokine gene adjuvants on DNA vaccination against Mycobacterium tuberculosis heat shock protein 65

DNA-based vaccines generate potent cellular immunity as well as humoral immunity. It seems evident that cytokines play a crucial role in generation of effector T cell subsets and in determining the magnitude of the response by DNA vaccines. In this study, we compared the effects of several TH1 cytokine genes as adjuvant in DNA vaccination using mycobacterial Hsp65 as a model antigen. Our results demonstrated that although the overall immune response to Hsp65 was enhanced by co-injection of Hsp65 DNA with cytokine genes, each cytokine gene was shown to affect different immune response elements. Co-injection of Hsp65 DNA with IL-12 or GM-CSF led to an increase in IFN-gamma production and represented potent protections against Mycobacterium tuberculosis challenge, while that with Eta-1, IL-12 or IL-18 gene led to an elevated IgG2a/IgG1 ratio. Interestingly, co-administration of Flt3L gene was shown to enhance the Ag-specific CTL response. These results show that the direction and magnitude of immune response in DNA vaccination against Hsp65 of M. tuberculosis could be modulated in different ways by co-injection of an appropriate cytokine gene as adjuvant.

Mediastinal lymph node CD8α− DC initiate antigen presentation following intranasal coadministration of α‐GalCer

Our previous study revealed that α‐galactosylceramide (α‐GalCer) is a potent nasal vaccine adjuvant inducing both potent humoral and cellular immune responses and affording complete protection against viral infections and tumors. However, the antigen‐presenting cells (APC) that are activated by NKT cells and thereby initiate the immune responses following intranasal coadministration of protein antigen and α‐GalCer are poorly understood. We assessed here where antigen presentation occurs and which APC subset mediates the early stages of immune responses when protein antigen and α‐GalCer are intranasally administered. We show that dendritic cells (DC), but not B cells, initiated the mucosal immune responses at mediastinal lymph nodes. Of the DC subsets, the CD8α−B220−CD11c+ DC subset played the most prominent role in the direct and cross‐presentation of protein antigen to naive T cells and in triggering the naive T cells to differentiate into effector T cells. This might be mainly caused by a relatively larger population of CD1dhigh cells of CD8α−B220−CD11c+ DC subset than those of other DC subsets. These results indicate that CD8α−B220−CD11c+ DC is the principal subset becoming immunogenic after interaction with NKT cells and abrogating tolerance to intranasally administered protein antigen when α‐GalCer is coadministered as a nasal vaccine adjuvant.

Split peripheral tolerance: CD40 ligation blocks tolerance induction for CD8 T cells but not for CD4 T cells in response to intestinal antigens

The role of antigen‐presenting cells in the balance between immunity and tolerance to intestinal antigens remains poorly understood. In this study, we examined whether CD40 ligation affects the induction of CD4 and CD8 T cell tolerance in response to intestinal antigens. We show that an agonistic anti‐CD40 mAb treatment did not block the induction of OVA‐specific CD4 T cell tolerance, whereas this approach enabled strong priming of OVA‐specific cytotoxic T cells (CTL), preventing CTL tolerance to intestinal antigen. Such CTL priming was independent of CD4 T cell help but required B7 costimulation. Co‐administration of anti‐CD40 mAb increased the synthesis of IL‐2 and the expression of CD25 by CD8 T cells, but neither IL‐2 production nor CD25 expression by CD4 T cells was enhanced by anti‐CD40 mAb. However, neutralization of TGF‐β together with addition of agonistic anti‐CD40 mAb was able to reverse CD4 T cell tolerance. These findings suggest that the induction of tolerance versus immunity against intestinal antigens is determined by the status of the antigen‐presenting cells and that signals via CD40 differently regulate the outcome of CD4 and CD8 T cells in vivo.

From the Cover: Sublingual vaccination with influenza virus protects mice against lethal viral infection

We assessed whether the sublingual (s.l.) route would be an effective means of delivering vaccines against influenza virus in mice by using either formalin-inactivated or live influenza A/PR/8 virus (H1N1). Sublingual administration of inactivated influenza virus given on two occasions induced both systemic and mucosal antibody responses and conferred protection against a lethal intranasal (i.n.) challenge with influenza virus. Coadministration of a mucosal adjuvant (mCTA-LTB) enhanced these responses and resulted in complete protection against respiratory viral challenge. In addition, s.l. administration of formalin-inactivated A/PR/8 plus mCTA-LTB induced systemic expansion of IFN-γ-secreting T cells and virus-specific cytotoxic T lymphocyte responses. Importantly, a single s.l. administration of live A/PR/8 virus was not pathogenic and induced protection mediated by both acquired and innate immunity. Moreover, s.l. administration of live A/PR/8 virus conferred heterosubtypic protection against respiratory challenge with H3N2 virus. Unlike the i.n. route, the A/PR/8 virus, whether live or inactivated, did not migrate to or replicate in the CNS after s.l. administration. Based on these promising findings, we propose that the s.l. mucosal route offers an attractive alternative to mucosal routes for administering influenza vaccines.

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We assessed whether the sublingual (s.l.) route would be an effective means of delivering vaccines against influenza virus in mice by using either formalin-inactivated or live influenza A/PR/8 virus (H1N1). Sublingual administration of inactivated influenza virus given on two occasions induced both systemic and mucosal antibody responses and conferred protection against a lethal intranasal (i.n.) challenge with influenza virus. Coadministration of a mucosal adjuvant (mCTA-LTB) enhanced these responses and resulted in complete protection against respiratory viral challenge. In addition, s.l. administration of formalin-inactivated A/PR/8 plus mCTA-LTB induced systemic expansion of IFN-γ-secreting T cells and virus-specific cytotoxic T lymphocyte responses. Importantly, a single s.l. administration of live A/PR/8 virus was not pathogenic and induced protection mediated by both acquired and innate immunity. Moreover, s.l. administration of live A/PR/8 virus conferred heterosubtypic protection against respiratory challenge with H3N2 virus. Unlike the i.n. route, the A/PR/8 virus, whether live or inactivated, did not migrate to or replicate in the CNS after s.l. administration. Based on these promising findings, we propose that the s.l. mucosal route offers an attractive alternative to mucosal routes for administering influenza vaccines.

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We assessed whether the sublingual (s.l.) route would be an effective means of delivering vaccines against influenza virus in mice by using either formalin-inactivated or live influenza A/PR/8 virus (H1N1). Sublingual administration of inactivated influenza virus given on two occasions induced both systemic and mucosal antibody responses and conferred protection against a lethal intranasal (i.n.) challenge with influenza virus. Coadministration of a mucosal adjuvant (mCTA-LTB) enhanced these responses and resulted in complete protection against respiratory viral challenge. In addition, s.l. administration of formalin-inactivated A/PR/8 plus mCTA-LTB induced systemic expansion of IFN-γ-secreting T cells and virus-specific cytotoxic T lymphocyte responses. Importantly, a single s.l. administration of live A/PR/8 virus was not pathogenic and induced protection mediated by both acquired and innate immunity. Moreover, s.l. administration of live A/PR/8 virus conferred heterosubtypic protection against respiratory challenge with H3N2 virus. Unlike the i.n. route, the A/PR/8 virus, whether live or inactivated, did not migrate to or replicate in the CNS after s.l. administration. Based on these promising findings, we propose that the s.l. mucosal route offers an attractive alternative to mucosal routes for administering influenza vaccines.