Priming MHC-I-restricted cytotoxic T lymphocyte responses to exogenous hepatitis B surface antigen is CD4+ T cell dependent.

MHC-I (Ld)-restricted, S28-39-specific CTL responses are efficiently primed in H-2d BALB/c mice injected with low doses of native hepatitis B surface Ag (HBsAg) lipoprotein particles without adjuvants. Priming of this CTL response by exogenous HBsAg required CD4+ T cell "help" and IL-12: this CTL response could be neither induced in mice depleted of CD4+ T cells by in vivo Ab treatment, nor in (CD4+ T cell-competent or CD4+ T cell-depleted) IL-12-unresponsive STAT4-/- knockout BALB/c mice. Codelivery of oligonucleotides (ODN) with immunostimulating CpG sequences (ISS) with exogenous HBsAg reconstituted the CTL response to exogenous HBsAg in CD4+ T cell-depleted normal mice and in CD4+ T cell-competent and CD4+ T cell-depleted STAT4-/- BALB/c mice. Injection (by different routes) of "naked" pCI/S plasmid DNA encoding HBsAg into IL-12-responsive or -unresponsive BALB/c mice efficiently primed the MHC-I-restricted, HBsAg-specific CTL response. CTL priming was not detectable when CD4+ T cell-depleted animals were subjected to genetic immunization. In vivo priming of the well-characterized CD8+ CTL response to HBsAg in "high responder" BALB/c mice either by exogenous surface lipoprotein particles or by DNA vaccination is thus CD4+ T cell dependent. CTL priming by exogenous HBsAg, but not by genetic immunization, is IL-12 dependent. The dependence of CTL priming by exogenous HBsAg on CD4+ T cells can be overcome by codelivering ODN with ISS motifs, and this "adjuvants effect" operates efficiently in IL-12-unresponsive mice. The data characterize a feature of the adjuvant effect of ISS-containing ODN on CTL priming that may be of major interest for the design of CTL-stimulating vaccines with efficacy in immunodeficiency conditions.     

Revealing the potential of DNA-based vaccination: lessons learned from the hepatitis B virus surface antigen

DNA-based vaccination is a novel technique to efficiently stimulate humoral (antibody) and cellular (T cell) immune responses to protein antigens. In DNA-based vaccination, immunogenic proteins are expressed in in vivo transfected cells of the vaccine recipients in their native conformation with correct posttranslational modifications from antigen-encoding expression plasmid DNA. This ensures the integrity of antibody-defined epitopes and supports the generation of protective (neutralizing) antibody titers. Plasmid DNA vaccination is furthermore an exceptionally potent strategy to stimulate CD8+ cytotoxic T lymphocyte (CTL) responses because antigenic peptides are efficiently generated by endogenous processing of intracellular protein antigens. These key features make DNA-based immunization an attractive strategy for prophylactic and therapeutic vaccination against extra- and intracellular pathogens. In this brief review, we summarize the current state of expression vector design, DNA delivery strategies, priming immune responses to intracellular or secreted antigens by DNA vaccines and unique advantages of DNA- versus recombinant protein-based vaccines using the hepatitis B surface antigen (HBsAg) as a model antigen.     

Enhancing the immunogenicity of exogenous hepatitis B surface antigen-based vaccines for MHC-I-restricted T cells

Vaccination with either exogenous hepatitis B surface antigen (HBsAg) lipoprotein particles without adjuvants, or plasmid DNA encoding secreted small HBsAg stimulate long-lasting, potent antibody responses in H-2d (BALB/c) and C57Bl/6 (H-2b) mice. Vaccination with exogenous HBsAg primes MHC-I restricted cytotoxic T lymphocyte (CTL) responses to HBsAg in H-2d but not H-2b mice, while DNA vaccination primes HBsAg-specific CTL responses in both mouse strains. We defined vaccination strategies that could elicit CTL responses to exogenous HBsAg in 'low responder' C57Bl/6 mice. We found that the bacterial plasmid DNA itself, synthetic oligodeoxynucleotides containing immunostimulating sequences, or recombinant Th1 cytokines (IL12, IFNgamma) efficiently support priming of CTL responses to exogenous HBsAg in 'low responder' H-2b mice, but have only minor effects on CTL priming in 'high responder' H-2d mice in the high dose range tested. These molecularly well defined adjuvants can thus efficiently support priming of anti-viral T cell responses under 'low responder' conditions.     

Targeting an anti-viral CD8+ T cell response to a growing tumor facilitates its rejection

 We demonstrate in a murine model that targeting an anti-viral T cell response to a growing tumor facilitates priming of a tumor-associated antigen (TAA)-specific, rejecting T cell response. Murine P815 mastocytoma cells grow aggressively in a syngeneic host. Transfected P815/S cells (expressing the hepatitis B surface antigen, HBsAg) also grow as subcutaneous tumors, but occasional ‘spontaneous’ rejections after transient growth are observed. Growth of P815/S tumors (but not of P815 tumors) is efficiently suppressed by a CD8⁺ cytotoxic T lymphocyte (CTL)-dependent immune mechanism in mice primed to HBsAg by DNA–immunization. In hosts immunized against HBsAg by DNA vaccination, HBsAg-specific CTL are generated. This specific CTL reactivity was targeted to s.c.-growing P815 tumors by intra tumor injections of either HBsAg-encoding plasmid DNA or viable P815/S cells; this treatment led to tumor rejection in 70–80% of the tumor-bearing animals. All rejecting animals showed a CD8⁺ CTL-dependent resistance to subsequent challenges by native, non-transfected P815 tumors. Targeting an established anti-viral (‘strong’) CTL response to a growing tumor hence is an efficient strategy to facilitate priming of a rejecting CTL response against (‘weak’) TAA in this system. Received: 18 December 1996 / Accepted: 6 February 1997     

Priming biologically active antibody responses against an isolated,conformational viral epitope by DNA vaccination

The immunodominant, conformational "a" determinant of hepatitis B surface Ag (HBsAg) elicits Ab responses. We selectively expressed the Ab-binding, glycosylated, native a determinant (residue 120-147) of HBsAg in a fusion protein containing C-terminally the HBsAg fragment SII (residue 80-180) fused to a SV40 T-Ag-derived hsp73-binding 77 aa (T(77)) or non-hsp-binding 60 aa (T(60)) N terminus. A DNA vaccine encoding non-hsp-binding secreted T(60)-SII fusion protein-stimulated murine Ab responses with a similar efficacy as a DNA vaccine encoding the secreted, native, small HBsAg. A DNA vaccine encoding hsp73-binding, intracellular T(77)-SII fusion protein-stimulated murine Ab responses less efficiently but comparable to a DNA vaccine encoding the intracellular, native, large HBsAg. HBsAg-specific Abs elicited by either the T(60)-SII-expressing or the T(77)-SII-expressing DNA vaccine suppressed HBsAg antigenemia in transgenic mice that produce HBsAg from a transgene in the liver; hence, a biologically active B cell response cross-reacting with the native, viral envelope epitope was primed by both DNA vaccine constructs. HBsAg-specific Ab and CTL responses were coprimed when an S(20-50) fragment (containing the immunodominant, L(d)-binding epitope S(28-39)) of HBsAg was fused C-terminally to the pCI/T(77)-SII sequence (pCI/T(77)-SII-L(d) DNA vaccine). Chimeric, polyepitope DNA vaccines encoding conformational, Ab-binding epitopes and MHC class I-binding epitopes can thus efficiently deliver antigenic information to different compartments of the immune system in an immunogenic way.     

Th1类细胞因子对pHCV-C重组体诱生免疫应答的增强作用

为了探索Th1类细胞因子IL-2和IL-12对含丙型肝炎病毒(HCV)核心(C)基因重组体诱生的免疫应答的增强作用,本文构建了包含HCVC基因片段的重组质粒pHCV-C,将其单独或与Th1类细胞因子表达质粒pIL-2或pIL-12共免疫BALB/c小鼠,ELISA法检测免疫小鼠血清中的HCVC特异性抗体滴度;以pHCV-C转染SP2/0细胞,经筛选稳定表达HCVC抗原者(SP2/0-HCV-C)为靶细胞,     

Adjuvant activities of novel cytokines, interleukin-23 (IL-23) and IL-27, for induction of hepatitis C virus-specific cytotoxic T lymphocytes in HLA-A*0201 transgenic mice

Searching the sequence databases has revealed two novel cytokines: interleukin-23 (IL-23) and IL-27. These cytokines are quite similar to, but clearly distinct from IL-12 in their structures and T-cell stimulatory fashions. In contrast to IL-12, however, little is known about the roles of IL-23 and IL-27 in the immune regulation. Previously, we evaluated the prime-boost immunization consisting of priming and the first boosting with the hepatitis C virus (HCV)-core expression plasmid, followed by a second boosting with recombinant adenovirus expressing HCV core for induction of HCV core-specific cytotoxic T lymphocytes (CTLs) in BALB/c mice. The present study demonstrates that HCV-specific CTL induction was greatly enhanced by coinoculation of an IL-12 expression plasmid in the prime-boost immunization, indicating the potent adjuvant activity of IL-12. We investigated whether similar adjuvant effects could be exerted by either IL-23 or IL-27 in a prime-boost immunization with HLA-A*0201 transgenic mice. Coadministration of either an IL-23 or an IL-27 expression plasmid, as well as an IL-12 expression plasmid, in a prime-boost immunization enhanced induction of HCV-specific CTLs and led to dramatic increases in the numbers of gamma interferon (IFN-gamma)-producing, HCV-specific CD8+ cells. Further, preinjections of IL-12, IL-23, or IL-27 expression plasmids before immunization resulted in great increases in the number of IFN-gamma-producing, HCV-specific CD8+ cells in response to immunization with recombinant adenovirus. These data revealed that both IL-23 and IL-27, as well as IL-12, are potent adjuvants for epitope-specific CTL induction. The two novel cytokines might offer new prophylactic and therapeutic strategies against infectious pathogens such as HCV.     

Hepatitis B surface antigen vector delivers protective cytotoxic T-lymphocyte responses to disease-relevant foreign epitopes

Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen- and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases.     

Creation of superagonist epitope sequences in the hepatitis B envelope protein using mutagenesis and DNA vaccination

DNA vaccination is a simple and efficient method for the induction of cytotoxic T lymphocytes (CTLs). In the present study, we have examined the effect of the mutations of each of the 12 amino acids of the HBsAg Ld-restricted CTL epitope on the ability of the modified proteins to induce CTLs after DNA-based immunization. Replacement of glutamine or serine by alanine codons in the whole envelope gene created a protein that induced higher CTL activity against cells bearing the wildtype peptide-MHC complex than against the wildtype sequence itself. These results represent the first example of immunogenic mutant sequences (superagonists) that induce higher CTL activity against the wildtype CTL epitope than does the wildtype protein. Because the entire mutant protein is being expressed from the modified plasmid, any of the various steps in epitope processing could be affected by the mutations and lead to increased class I immunogenicity of the peptide sequence.     

IL-27 mediates complete regression of orthotopic primary and metastatic murine neuroblastoma tumors: role for CD8+ T cells

We have shown previously that IFN-gamma-inducing cytokines such as IL-12 can mediate potent antitumor effects against murine solid tumors. IL-27 is a newly described IL-12-related cytokine that potentiates various aspects of T and/or NK cell function. We hypothesized that IL-27 might also mediate potent antitumor activity in vivo. TBJ neuroblastoma cells engineered to overexpress IL-27 demonstrated markedly delayed growth compared with control mice, and complete durable tumor regression was observed in >90% of mice bearing either s.c. or orthotopic intra-adrenal tumors, and 40% of mice bearing induced metastatic disease. The majority of mice cured of their original TBJ-IL-27 tumors were resistant to tumor rechallenge. Furthermore, TBJ-IL-27 tumors were heavily infiltrated by CD8(+) T cells, and draining lymph node-derived lymphocytes from mice bearing s.c. TBJ-IL-27 tumors are primed to proliferate more readily when cultured ex vivo with anti-CD3/anti-CD28 compared with lymphocytes from mice bearing control tumors, and to secrete higher levels of IFN-gamma. In addition, marked enhancement of local IFN-gamma gene expression and potent up-regulation of cell surface MHC class I expression are noted within TBJ-IL-27 tumors compared with control tumors. Functionally, these alterations occur in conjunction with the generation of tumor-specific CTL reactivity in mice bearing TBJ-IL-27 tumors, and the induction of tumor regression via mechanisms that are critically dependent on CD8(+), but not CD4(+) T cells or NK cells. Collectively, these studies suggest that IL-27 could be used therapeutically to potentiate the host antitumor immune response in patients with malignancy.     

Estimating the In Vivo Killing Efficacy of Cytotoxic T Lymphocytes across Different Peptide-MHC Complex Densities

Cytotoxic T lymphocytes (CTLs) are important agents in the control of intracellular pathogens, which specifically recognize and kill infected cells. Recently developed experimental methods allow the estimation of the CTL''s efficacy in detecting and clearing infected host cells. One method, the in vivo killing assay, utilizes the adoptive transfer of antigen displaying target cells into the bloodstream of mice. Surprisingly, killing efficacies measured by this method are often much higher than estimates obtained by other methods based on, for instance, the dynamics of escape mutations. In this study, we investigated what fraction of this variation can be explained by differences in peptide loads employed in in vivo killing assays. We addressed this question in mice immunized with lymphocytic choriomeningitis virus (LCMV). We conducted in vivo killing assays varying the loads of the immunodominant epitope GP33 on target cells. Using a mathematical model, we determined the efficacy of effector and memory CTL, as well as CTL in chronically infected mice. We found that the killing efficacy is substantially reduced at lower peptide loads. For physiological peptide loads, our analysis predicts more than a factor 10 lower CTL efficacies than at maximum peptide loads. Assuming that the efficacy scales linearly with the frequency of CTL, a clear hierarchy emerges among the groups across all peptide antigen concentrations. The group of mice with chronic LCMV infections shows a consistently higher killing efficacy per CTL than the acutely infected mouse group, which in turn has a consistently larger efficacy than the memory mouse group. We conclude that CTL killing efficacy dependence on surface epitope frequencies can only partially explain the variation in in vivo killing efficacy estimates across experimental methods and viral systems, which vary about four orders of magnitude. In contrast, peptide load differences can explain at most two orders of magnitude.     

B subunit of Shiga toxin-based vaccines synergize with alpha-galactosylceramide to break tolerance against self antigen and elicit antiviral immunity

The nontoxic B subunit of Shiga toxin (STxB) targets in vivo Ag to dendritic cells that preferentially express the glycolipid Gb(3) receptor. After administration of STxB chemically coupled to OVA (STxB-OVA) or E7, a polypeptide derived from HPV, in mice, we showed that the addition of alpha-galactosylceramide (alpha-GalCer) resulted in a dramatic improvement of the STxB Ag delivery system, as reflected by the more powerful and longer lasting CD8(+) T cell response observed even at very low dose of immunogen (50 ng). This synergy was not found with other adjuvants (CpG, poly(I:C), IFN-alpha) also known to promote dendritic cell maturation. With respect to the possible mechanism explaining this synergy, mice immunized with alpha-GalCer presented in vivo the OVA(257-264)/K(b) complex more significantly and for longer period than mice vaccinated with STxB alone or mixed with other adjuvants. To test whether this vaccine could break tolerance against self Ag, OVA transgenic mice were immunized with STxB-OVA alone or mixed with alpha-GalCer. Although no CTL induction was observed after immunization of OVA transgenic mice with STxB-OVA, tetramer assay clearly detected specific anti-OVA CD8(+) T cells in 8 of 11 mice immunized with STxB-OVA combined with alpha-GalCer. In addition, vaccination with STxB-OVA and alpha-GalCer conferred strong protection against a challenge with vaccinia virus encoding OVA with virus titers in the ovaries reduced by 5 log compared with nonimmunized mice. STxB combined with alpha-GalCer therefore appears as a promising vaccine strategy to more successfully establish protective CD8(+) T cell memory against intracellular pathogens and tumors.     

IL-12 and NK cells are required for antigen-specific adaptive immunity against malaria initiated by CD8+ T cells in the Plasmodium yoelii model.

CD8+ T cells have been implicated as critical effector cells in protection against preerythrocytic stage malaria, including the potent protective immunity of mice and humans induced by immunization with radiation-attenuated Plasmodium spp. sporozoites. This immunity is directed against the Plasmodium spp. parasite developing within the host hepatocyte and for a number of years has been presumed to be mediated directly by CD8+ CTL or indirectly by IFN-gamma released from CD8+ T cells. In this paper, in BALB/c mice, we establish that after immunization with irradiated sporozoites or DNA vaccines parasite-specific CD8+ T cells trigger a novel mechanism of adaptive immunity that is dependent on T cell- and non-T cell-derived cytokines, in particular IFN-gamma and IL-12, and requires NK cells but not CD4+ T cells. The absolute requirement for CD8+ T cells to initiate such an effector mechanism, and the requirement for IL-12 and NK cells in such vaccine-induced protective immunity, are unique and underscore the complexity of the immune responses that protect against malaria and other intracellular pathogens.     

Construction and preliminary investigation of a plasmid containing a novel immunotoxin DT390-IL-18 gene for the prevention of murine experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a neuropathological animal model for multiple sclerosis. Antigen-presenting cells (APCs) expressing interleukin-18 receptor (IL-18R) were shown to be crucial in the beginning and progress of EAE. In this study we tested the effect of a novel recombinant immunotoxin targeting IL-18R-bearing APC for EAE prevention. The novel eukaryotic plasmid DT390-IL-18-SRalpha, encoding recombinant immunotoxin DT390-IL-18, was constructed. The immunotoxin consisted of IL-18 as the targeting moiety, and a truncated diphtheria toxin (DT) as the toxic moiety. Transfection assay and proliferation inhibition assay proved the immunotoxin could be expressed in vitro and was toxic to the activated mouse T cells. To evaluate the preventive effect of DT390-IL-18-SRalpha on EAE in vivo, cationic liposome-embedded DT390-IL-18-SRalpha was injected into the hind limbs of EAE mice. DT390-IL-18-SRalpha-treated mice showed a delayed manifestation of EAE and decreased symptoms compared to the mice treated with plasmid DT390-SRalpha or phosphate-buffered saline alone. A significant reduction in infiltrating inflammatory cells was detected in the brain tissues from immunotoxin-treated mice as compared with the controls by hematoxylin-eosin staining. This study suggested that the recombinant immunotoxin DT390-IL-18 could be expressed in vitro and in vivo, and prevented murine EAE effectively.     

Immunogene therapy of tumors with vaccine based on xenogeneic epidermal growth factor receptor

The breaking of immune tolerance against self epidermal growth factor receptor (EGFr) should be a useful approach for the treatment of receptor-positive tumors with active immunization. To test this concept, we constructed a plasmid DNA encoding extracellular domain of xenogeneic (human) EGFr (hEe-p) or corresponding control mouse EGFr (mEe-p) and empty vector (c-p). Mice immunized with hEe-p showed both protective and therapeutic antitumor activity against EGFr-positive tumor. Sera isolated from the hEe-p-immunized mice exhibited positive staining for EGFr-positive tumor cells in flow cytometric analysis and recognized a single 170-kDa band in Western blot analysis. Ig subclasses responded to rEGFr proteins were elevated in IgG1, Ig2a, and Ig2b. There was the deposition of IgG on the tumor cells. Adoptive transfer of the purified Igs showed the antitumor activity. The increased killing activity of CTL against EGFr-positive tumor cells could be blocked by anti-CD8 or anti-MHC class I mAb. In vivo depletion of CD4(+) T lymphocytes could completely abrogate the antitumor activity, whereas the depletion of CD8(+) cells showed partial abrogation. The adoptive transfer of CD4-depleted (CD8(+)) or CD8-depleted (CD4(+)) T lymphocytes isolated from mice immunized with hEe-p vaccine showed the antitumor activity. In addition, the increase in level of both IFN-gamma and IL-4 was found. Taken together, these findings may provide a new vaccine strategy for the treatment of EGFr-positive tumors through the induction of the autoimmune response against EGFr in a cross-reaction between the xenogeneic homologous and self EGFr.     

The immunodominant,Ld-restricted T cell response to hepatitis B surface antigen (HBsAg) efficiently suppresses T cell priming to multiple Dd-, Kd-, and Kb-restricted HBsAg epitopes

MHC-I-restricted CTL responses of H-2(d) (L(d+) or L(d-)) and F(1) H-2(dxb) mice to hepatitis B surface Ag (HBsAg) are primed by either DNA vaccines or HBsAg particles. The D(d)/S(201-209) and K(d)/S(199-208) epitopes are generated by processing endogenous HBsAg; the K(b)/S(208-215) epitope is generated by processing exogenous HBsAg; and the L(d)/S(28-39) epitope is generated by exogenous as well as endogenous processing of HBsAg. DNA vaccination primed high numbers of CTL specific for the L(d)/S(28-39) HBsAg epitope, low numbers of CTL specific for the D(d)/S(201-209) or K(d)/S(199-208) HBsAg epitopes in BALB/c mice, and high numbers of D(d)/S(201-209)- and K(d)/S(199-208)-specific CTL in congenic H-2(d)/L(d-) dm2 mice. In F(1)(dxb) mice, the K(d)-, D(d)-, and K(b)-restricted CTL responses to HBsAg were strikingly suppressed in the presence but efficiently elicited in the absence of L(d)/S(28-39)-specific CTL. Once primed, the K(d)- and D(d)-restricted CTL responses to HBsAg were resistant to suppression by immunodominant L(d)/S(28-39)-specific CTL. The L(d)-restricted immunodominant CTL reactivity to HBsAg can thus suppress priming to multiple alternative epitopes of HBsAg, independent of the processing pathway that generates the epitope, of the background of the mouse strain used, and of the presence/absence of different allelic variants of the K and D MHC class I molecules.     

IL-12 gene therapy using an electrically mediated nonviral approach reduces metastatic growth of melanoma

Interleukin-12 (IL-12) has been evaluated in both preclinical and clinical immunotherapy protocols as a potential therapy for melanoma. However, delivery of IL-12 in the form of recombinant protein can result in severe toxicity, and gene therapy has had limited success against B16.F10 murine melanoma. This study investigated the therapeutic effect of delivering a plasmid encoding IL-12 followed by electroporation on primary and secondary tumors. Three treatments of intratumoral (i.t.) plasmid injection and electroporation resulted in 80% of mice with B16.F10 melanoma tumors being tumor free for >100 days (cure). The "cured animals" were resistant to challenge with B16 cells. In a separate experiment, B16 cells were injected on the opposite flank of the treated tumor on the day of treatment. Eighty-seven percent of control mice developed a distant tumor while only 43.8% of mice receiving two or three i.t. electroporation treatments developed a distant tumor. For examination of tumor development in the lungs, mice were injected intravenously with B16.F10 cells then treated with i.m. injections of plasmid with or without electroporation. Only 37.5% of mice receiving i.m. injections and electroporation developed nodules in the lungs compared to 87.5% of mice in the no-treatment group. The results show that administration of a plasmid encoding IL-12 with electroporation has a therapeutic effect on primary tumors as well as distant tumors and metastases.     

High avidity cytotoxic T lymphocytes can be selected into the memory pool but they are exquisitely sensitive to functional impairment

High avidity cytotoxic T lymphocytes (CTL) are important in viral clearance and anti-tumor immunity, however, mechanisms for their optimal generation and maintenance in vivo remain unclear. Immunizing mice with an antibody-DNA vaccine encoding a single CTL epitope, induces a 100 fold higher avidity response than peptide vaccination with the identical epitope. The high avidity response is retained into memory and can be efficiently reactivated with an antibody-DNA boost. In contrast, reactivation of high avidity CTL with peptide, stimulated responses with a significant drop in avidity, suggesting loss or conversion of the high avidity CTL to lower avidity. Similarly, high avidity T cells maintained ex vivo were exquisitely sensitive to signaling with low doses of peptide (1 ng/ml) giving optimal TCR stimulation and resulting in retained avidity, proliferation and ability to kill specific targets. In contrast, high avidity T cells maintained ex vivo with supraoptimal TCR stimulation (10 μg/ml peptide) resulted in reduced avidity and failure to kill tumor cells. They also failed to proliferate, showed a significant increase in apoptosis and expressed high levels of the exhaustion marker programmed death-1 (PD-1) and low levels of the lymphocyte-activation gene 3 (LAG-3). This suggests high avidity T cells are recruited to the memory pool but can be lost by supraoptimal stimulation in vitro and in vivo. This is characterized by loss of function and an increase in cell death. The remaining CTL, exhibit low functional avidity that is reflected in reduced anti-tumor activity. This could contribute to failure of the immune system to control the growth of tumors and has implications for vaccination strategies and adoptive transfer of T cells.     

人白细胞介素12表达质粒与Mtb8.4基因疫苗 联合免疫增强对小鼠结核杆菌感染的免疫保护效果

本课题旨在研究结核分枝杆菌Mtb8.4基因疫苗与人白细胞介素12（hIL-12）联合免疫小鼠所诱导的细胞免疫应答及对小鼠结核杆菌感染的免疫保护效果。40只C57BL/6N小鼠随机分为Mtb8.4基因疫苗＋hIL-12质粒组（联合免疫组）、Mtb8.4基因疫苗组、卡介苗（BCG）组、空载体组和PBS组,基因疫苗、空载体和PBS,经肌内注射法免疫各组小鼠,每隔3周免疫1次,共免疫3次,BCG组经尾部皮下注射1×106 CFU BCG免疫1次。免疫4周后,每组处死3只小鼠,采用酶联免疫吸附法（ELISA）检测脾细胞培养上清中细胞因子水平;乳酸脱氢酶（LDH）释放法检测细胞毒性T细胞（CTL）杀伤活性。每组其余5只小鼠用结核杆菌H37Rv强毒株经尾静脉攻击,4周后,计数肺和脾组织中的结核杆菌菌落数,对小鼠部分肺和脾组织作病理切片,HE染色观察组织病变程度,Z-N染色查抗酸杆菌,观察该疫苗对小鼠结核杆菌感染的免疫保护效果。结果显示,联合免疫组能诱导较强的抗原特异性Th1型细胞免疫应答,免疫小鼠脾细胞培养上清液IFN-γ和IL-2水平（分别为1493.34±8.128pg/mL、747.489±48.676pg/mL）,显著高于Mtb8.4基因疫苗组,与BCG组相当,IL-4分泌减少,特异性CTL杀伤活性增强,对小鼠结核杆菌感染有较好的免疫保护效果,使小鼠肺和脾组织中的结核杆菌菌落数显著减少,组织病变明显减轻,其效果与卡介苗（BCG）组相当,优于Mtb8.4基因疫苗组。表明hIL-12表达质粒与Mtb8.4基因疫苗联合免疫后,能够增强Mtb8.4基因疫苗所诱导的细胞免疫应答,使Mtb8.4基因疫苗的免疫效力得到很大提高。     

Papillomavirus pseudovirus: a novel vaccine to induce mucosal and systemic cytotoxic T-lymphocyte responses

Intestinal mucosa is a portal for many infectious pathogens. Systemic immunization, in general, does not induce a cytotoxic T-lymphocyte (CTL) response at the mucosal surface. Because papillomavirus (PV) naturally infects mucosa and skin, we determined whether PV pseudovirus, i.e., PV-like particles in which unrelated DNA plasmids are packaged, could generate specific mucosal immunity. We found that the pseudovirus that encoded the lymphocytic choriomeningitis virus gp33 epitope induced a stronger CTL response than a DNA vaccine (plasmid) encoding the same epitope given systemically. The virus-like particles that were used to make the pseudoviruses provided an adjuvant effect for induction of CTLs by the DNA vaccine. The PV pseudovirus pseudoinfected mucosal and systemic lymphoid tissues when administered orally. Oral immunization with the pseudovirus encoding human PV type 16 mutant E7 induced mucosal and systemic CTL responses. In comparison, a DNA vaccine encoding E7, when given orally, did not induce a CTL response in intestinal mucosal lymphoid tissue. Further, oral immunization with the human PV pseudovirus encoding E7 protected mice against mucosal challenge with an E7-expressing bovine PV pseudovirus. Thus, PV pseudovirus can be used as a novel vaccine to induce mucosal and systemic CTL responses.