Induction of cytotoxic T lymphocyte responses by cholera toxin-treated bone marrow-derived dendritic cells

Cholera toxin (CT), a powerful mucosal adjuvant, is a potent inducer of Th2-type responses via activation of co-stimulatory molecules for the induction of IgA antibody responses. Less appreciated is the ability of CT to induce and regulate cytotoxic T lymphocyte (CTL) responses. In order to help for clarifying mechanisms underlying the CTL-inducing ability of CT, we have examined the effects of CT on dendritic cells (DCs) that could lead to the induction of cytotoxic CD8(+) T cells. When bone marrow-derived DCs (BM-DCs) were cultured with CT in vitro, B7-1 but not B7-2 molecules were significantly enhanced and allogenic CTL responses were induced. Also, increased numbers of IFN-gamma-secreting CD8(+) T cells were elicited when CT-treated BM-DCs were co-cultured with allogenic CD8(+) CTLs. Antibody blockade of B7-1 on CT-treated BM-DCs suppressed allogenic CTL responses, further indicating the importance of CT-induced B7-1 molecules on DCs for the acquisition of cytolytic function by CTL precursors. CD40 signaling was proven not necessary for the CT-induced CTL response since CT-treated CD40(-/-) BM-DCs developed CTL responses equivalent to those detected in CT-treated BM-DCs derived from normal mice. Our results suggest that CT-treated DCs are effective inducers of CD8(+) CTL, and this induction is mediated through CT's ability to enhance B7-1 expression on DCs.     

Induction of cytotoxic T lymphocyte activity by fusion-active peptide-containing virosomes

Priming of cytotoxic T lymphocyte (CTL) activity with exogenous antigen requires introduction of the antigen into the MHC class I presentation pathway of antigen-presenting cells. In the present study, we used fusogenic reconstituted envelopes (virosomes), derived from influenza virus, as a carrier system for delivery of a synthetic soluble peptide corresponding to a major murine CTL epitope of the influenza virus nucleoprotein (NP). Virosomes containing encapsulated NP-peptide efficiently sensitized target cells for recognition by influenza-specific CTLs generated through priming of mice with infectious virus. Intramuscular immunization of mice with peptide-containing virosomes induced a potent class I MHC-restricted CTL response against influenza-infected target cells. By contrast, an equal dose of NP-peptide encapsulated in fusion-inactivated virosomes did not induce CTL activity, indicating an essential role of the membrane fusion activity of the virosomes in the induction of the response. Likewise, NP-peptide encapsulated in liposomes, NP-peptide mixed with empty virosomes and NP-peptide in IFA failed to induce a CTL response. These results demonstrate that fusion-active virosomes represent a promising delivery system for induction of class I MHC-restricted CTL activity with non-replicating viral antigens.     

Induction of T lymphocyte responses to dengue virus by a candidate tetravalent live attenuated dengue virus vaccine

Development of a safe and immunogenic tetravalent dengue virus (DV) vaccine has been designated as a priority by the World Health Organization. We characterized the T cell response to DV induced by a candidate live attenuated tetravalent DV vaccine as part of a phase I study. Proliferation and cytotoxic T lymphocyte (CTL) responses to multiple DV serotypes were detected in six of six and four of four subjects studied, respectively. Proliferation responses were higher to DV serotypes 1 and 3 than to serotypes 2 and 4. CTL responses were higher to DV serotypes 2 and 3 than to serotype 1, and included serotype cross-reactive responses. Production of interferon-γ, but not IL-4, was observed in response to DV stimulation. This candidate vaccine is immunogenic for both CD4+ and CD8+ T lymphocytes. However, T cell responses to the four DV serotypes were not equivalent, suggesting that the vaccine could be further optimized.     

Induction of cytotoxic T lymphocyte activity by immunization with recombinant Semliki Forest virus: indications for cross-priming

For the rational design of vaccines capable of inducing CD8+ T cell responses knowledge of the identity of the antigen-presenting cell (APC) and the mechanism of antigen presentation is very important. Here, we address these issues for alphavirus-based immunization, in particular immunization with recombinant Semliki Forest virus (rSFV). Studies with dendritic cells (DCs) from various origins revealed that rSFV has a very limited capacity to transfect this cell type in vitro. To further investigate in vivo whether rSFV transfects professional antigen-presenting cells directly or whether the antigens reach APCs via a mechanism of cross-priming we compared the immunological effects of three different SFV-constructs encoding the influenza nucleoprotein (NP). These constructs differ in the amount of NP produced per cell or in the stability of the NP, respectively. Induction of cytotoxic T lymphocytes (CTLs) appeared to benefit from a large amount of stable antigen. In contrast, rapid antigen degradation, and thus availability of antigenic peptides in the transfected cell, was found to be disadvantageous. Based on these in vitro and in vivo results, we hypothesize that antigen presentation after SFV-based immunization proceeds via a mechanism in which APCs are not transfected directly but acquire antigen from other transfected cells and present it to CTLs in a process of cross-priming.     

Combined peptides of human enterovirus 71 protect against virus infection in mice

Human enterovirus 71 (EV71) is a cause of hand, foot and mouth disease (HMFD) in children under 6 years old, and could cause serious neurological complications in some patients. Numerous large outbreaks of EV71 caused HMFD have occurred recently in Asia, especially in China. The cross-reactivity of EV71 with human brain tissue was observed and the cross-reactivity inducing regions were identified in previously study, which suggested that there were two regions in structural proteins of virus should be avoided in the vaccine. Six peptides without cross-reactivity were selected and combined into three vaccine candidates and applied in further evaluation in neonatal mice. The Vac6 comprising the peptides of P_70–159, P_140–249, P_324–443 and P_746–876 of the structural proteins could provide effective protection on pups against virus infection, as shown in viral copies detection and histopathology examination. Immunohistochemical staining results indicated that Vac6 had no cross-reactivity with human brain tissues. Our results suggested that Vac6 could have potential clinical value against EV71 epidemics caused mainly by C4 strains in the mainland of China.     

戊型肝炎病毒重组质粒诱导小鼠产生免疫应答的研究

目的探讨含HEV主要抗原表位基因片段的重组质粒pcHEV23在小鼠体内诱导产生免疫应答的情况,以寻求其作为HEVDNA疫苗的可能性。方法实验分生理盐水对照组(生理盐水)、空质粒对照组(pcDNA3)、免疫组(重组质粒pcHEV23)、交替免疫组(重组质粒pcHEV23+HEVORF23嵌合表达蛋白)4组,分别免疫BALB/c小鼠;ELISA检测血清中HEVIgG抗体的情况;流式细胞仪检测CD4+、CD8+T细胞亚群及CD4+/CD8+比值的变化。结果免疫组、交替免疫组与生理盐水对照组、空质粒对照组相比,IgG抗体明显增高(P<0.01),CD4+及CD4+/CD8+比值也有显著升高(P<0.01);交替免疫组与免疫组相比,IgG抗体明显增高(P<0.01)。结论HEV重组质粒pcHEV23既能较好地刺激细胞免疫,又能产生一定的体液免疫应答,提示是一种有前途的HEV基因疫苗。     

Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by immunization with dendritic cells transduced with replication-defective recombinant adenovirus

We studied the potential of dendritic cells (DCs) in priming hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTLs) in mice. Recombinant adenovirus expressing HCV core (Adex1SR3ST) was employed to express core in DCs. Core-specific CTLs are effectively elicited by injecting Adex1SR3ST-transduced DCs, whereas injection of Adex1SR3ST does not result in effective priming. Further, Adex1SR3ST-transduced DCs more efficiently prime core-specific CTLs than Adex1SR3ST-transduced macrophages, or DCs treated with an anthrax toxin fusion protein reported previously. Upon challenge with recombinant HCV-core-expressing vaccinia virus, vaccinia titers are significantly reduced in mice immunized with Adex1SR3ST-transduced DCs. Thus, adenovirus-transduced DCs may be a promising candidate for a CTL-based vaccine against HCV.     

Inhibition of immune responses against rabies virus by monoclonal antibodies directed against rabies virus antigens.

Treatment of mice with a cocktail of murine anti-rabies monoclonal antibodies (mAb-C) interfered with the ability of these animals to mount a virus-neutralizing antibody response to rabies vaccine. Administered mAb-C did not affect the induction of rabies virus-specific T-helper cells. The magnitude of the inhibition of rabies virus-specific B-cell response was dependent on the concentration of the mAb-C and the duration of the mAb-mediated interference was inversely proportional to the biological half-life of the mAb. As long as the serum titres were above a critical threshold, the suppression could not be overcome even by multiple vaccinations. Since injection of mice with immunocomplexes consisting of inactivated rabies virus and mAb rendered the animals non-responsive to a subsequent vaccination with inactivated rabies virus, it is concluded that the mAb-induced suppression might be caused by the formation of antigen-antibody complexes which exert a negative signalling effect to premature B cells.     

Recombinant proteins of Zaire ebolavirus induce potent humoral and cellular immune responses and protect against live virus infection in mice

Infections with filoviruses in humans are highly virulent, causing hemorrhagic fevers which result in up to 90% mortality. In addition to natural infections, the ability to use these viruses as bioterrorist weapons is of significant concern. Currently, there are no licensed vaccines or therapeutics available to combat these infections. The pathogenesis of disease involves the dysregulation of the host’s immune system, which results in impairment of the innate and adaptive immune responses, with subsequent development of lymphopenia, thrombocytopenia, hemorrhage, and death. Questions remain with regard to the few survivors of infection, who manage to mount an effective adaptive immune response. These questions concern the humoral and cellular components of this response, and whether such a response can be elicited by an appropriate prophylactic vaccine. The data reported herein describe the production and evaluation of a recombinant subunit Ebola virus vaccine candidate consisting of insect cell expressed Zaire ebolavirus (EBOV) surface glycoprotein (GP) and the matrix proteins VP24 and VP40. The recombinant subunit proteins are shown to be highly immunogenic in mice, yielding both humoral and cellular responses, as well as highly efficacious, providing up to 100% protection against a lethal challenge with live virus. These results demonstrate proof of concept for such a recombinant non-replicating vaccine candidate in the mouse model of EBOV which helps to elucidate immune correlates of protection and warrants further development.     

Induction of human gamma delta T cells in myasthenia gravis thymus transplanted SCID mice

BACKGROUND: TCR-gamma delta cells develop by extrathymic differentiation and might play an important role in thymectomy-resistant myasthenia gravis (MG) patients. In this study, we show development of human TCR-gamma delta cells in periphery of SCID mice by transplantation of human MG thymus or thymoma. METHODS: Three pieces of thymoma tissue and four of non-thymoma thymic tissues were obtained from MG patients by thymectomy. Each tissue was transplanted into 5-6 weeks old female SCID mice by intraperitoneal injection or surgical implantation. Rate of human TCR-positive cell development and its subtypes (TCR-alpha beta, TCR-gamma delta) were measured in the mice blood at one, three, and six weeks after the transplantation. RESULTS: Human TCR-positive cells were detected three weeks after transplantation. Rate of TCR-gamma delta T cell development got higher in thymoma transplanted group than in non-thymoma transplanted group. CONCLUSIONS: We could successfully develop human mature T cells in SCID mice by transplantation of human MG thymus or thymoma.     

Tannins protect against skin tumor promotion induced by ultraviolet-B radiation in hairless mice

We recently showed that Tarapod tannic acid (TA), a hydrolyzable tannin extracted from the pods of the Tara tree (Caesalpinia spinosa), was more effective than other tannins tested at inhibiting ultraviolet-B (UV-B)-stimulated hydrogen peroxide activity (an indirect measure of free radicals) in the skin of hairless mice. We also found that Tarapod TA inhibited UV-B-induced ornithine decarboxylase activity and UV-B-stimulated DNA synthesis, two biochemical markers linked to the skin tumor-promoting ability of this physical carcinogen. For this reason, we examined the effect of topical application, force feeding (gavage), and intraperitoneal injections of Tarapod TA on mouse skin chronically treated with UV-B light. Mice were initiated by a single topical application of 7,12-dimethylbenz[a]anthracene (50 nmol) and promoted by two weekly treatments with UV-B light (250 mJ/cm2) for 25 weeks. Topical application of Tarapod TA, 20 minutes before irradiation, resulted in a dose-dependent inhibition of tumor incidence (number of mice with tumors) and tumor yield (number of tumors/mouse), with 8 mg of TA inhibiting tumor yield by 70% at Week 25. Intraperitoneal injections of low doses (10 mg/kg mouse body wt), but not of high doses (25 mg/kg body wt), of TA afforded protection against UV-B-induced papillomas. However, the protection by intraperitoneal injection was lower than that observed by topical application: 10 mg/kg body wt of TA reduced tumor yield by 55%. The force feeding of 10 mg of Tarapod TA before irradiation failed to significantly inhibit the yield of tumors at the end of the experiment but delayed tumor appearance by six weeks. These results suggest that plant tannins administered topically or injected intraperitoneally reduce the tumor-promoting effects of UV-B radiation and thus could be useful photoprotectants.     

Immunogenicity in mice and rabbits of DNA vaccines expressing woodchuck hepatitis virus antigens

The licensed vaccine against hepatitis B virus (HBV) is an effective means to prevent infection, but is not an effective therapeutic strategy to treat established chronic infections when used alone. In an animal model of chronic HBV infection (the woodchuck experimentally infected with woodchuck hepatitis virus (WHV)), the combination of conventional vaccine and potent antiviral drugs has shown promise as a potential therapeutic intervention. This approach might be improved further through the application of newer vaccine technologies. In the present study, we evaluated electroporation (EP)-based intramuscular (i.m.) delivery of a codon-optimized DNA vaccine for the WHV surface antigen (WHsAg) in mice and rabbits. In mice, this immunization procedure compared favorably to vaccination by i.m. injection of the DNA vaccine or i.m. administration of a recombinant WHsAg-alum vaccine, exhibiting characteristics expected to be beneficial for a therapeutic vaccine strategy. These included dose efficiency, consistency, vigorous induction of antibody responses to WHsAg, as well as a Th1 bias. Following scale-up to rabbits, a species that approximates the size of the woodchuck, the EP dosing regimen was markedly more effective than conventional i.m. injection of the DNA vaccine. Taken together, these results provide the foundation for studies of EP-based DNA immunization in the woodchuck in order to further assess its potential as an immunotherapeutic approach for treatment of chronic HBV infection in humans.     

Virosome-mediated delivery of protein antigens in vivo: efficient induction of class I MHC-restricted cytotoxic T lymphocyte activity

Induction of CTL responses against protein antigens is an important aim in vaccine development. In this paper we present fusion-active virosomes as a vaccine delivery system capable of efficient induction of CTL responses in vivo. Virosomes are reconstituted viral membranes, which do not contain the genetic material of the virus they are derived from. Foreign macromolecules, including protein antigens, can be encapsulated in virosomes during the reconstitution process. Functionally reconstituted virosomes retain the cell binding and fusion characteristics of the native virus. Thus, upon uptake by cells through receptor-mediated endocytosis, virosomes will deliver their content to the cell cytosol. In a previous study, we demonstrated that protein antigens delivered in this manner to dendritic cells are efficiently processed for both MHC class I and class II presentation. Here, we studied in vivo induction of cellular immune responses against virosome-encapsulated ovalbumin (OVA) in mice. As little as 0.75 microg OVA delivered by fusion-active virosomes was sufficient to induce a powerful class I MHC-restricted CTL response. All immunization routes that were used (i.m., i.p. and s.c.) resulted in efficient induction of CTL activity. The CTLs induced were cytotoxic in a standard 51Cr-release assay and produced IFNgamma in response to OVA peptide. Thus, virosomes represent an ideal antigen delivery system for induction of cellular immunity against encapsulated protein antigens.     

Antibody, cytokine and cytotoxic T lymphocyte responses in chimpanzees immunized with human papillomavirus virus-like particles

We evaluated antibody, cytokine (IFN-gamma, IL-5, TNF-alpha), and cytotoxic T lymphocyte (CTL) responses in chimpanzees immunized with monovalent or quadrivalent (HPV-6, -11, -16, -18) L1 virus-like particle (VLP) vaccines administered i.m. on aluminum hydroxyphosphate (alum) at weeks 0, 8 and 24. Maximum serum antibody titers to type-specific, neutralizing, conformational epitopes on HPV-11 or -16 L1 VLPs were detected by radioimmunoassay (RIA) four weeks after the second and third immunizations. HPV-11 and -16 neutralizing antibodies were also detected at similar time points with an Human papillomaviruses (HPV) neutralization assay using pseudovirions. Depending on the VLP type used for immunization, HPV type-specific cytokine responses were most frequently seen four weeks after the second or third immunizations and between weeks 44-52. Transient HPV-16 L1-specific CTL activity was observed only between weeks 16-24 in 3 of 22 (13.6%) chimpanzees immunized with HPV-16 L1 VLPs. These findings provide evidence that immunization with multivalent L1 VLPs on alum can evoke both neutralizing antibodies and Th1 and Th2 cytokine responses to several HPV types; however, induction of CTLs is infrequent.     

Induction of hepatitis B virus-specific cytotoxic T lymphocytes response in vivo by filamentous phage display vaccine

The ability of inducing MHC class I restricted cytotoxic T lymphocytes response in vivo via recombinant filamentous phage was investigated. The recombinant filamentous phage particles that displayed the Hepatitis B virus epitope S(28--39) were injected into BALB/c (H-2d) mice without adjuvants. A MHC class I restricted HBs specific CTL response was found 8 days after injection. The potentiality of using the recombinant filamentous phage as anti-virus vaccine was discussed.     

Dendritic cells transfected with Her2 antigen-encoding RNA replicons cross-prime CD8 T cells and protect mice against tumor challenge

Antigen-specific T cells can be induced by direct priming and cross-priming. To investigate cross-priming as a vaccination approach dendritic cells were transfected with cytopathogenic viral RNA-replicons that expressed domains of the tumor-associated Her2-antigen and injected into MHC-discordant mice that did not allow direct priming. Upon tumor challenge 75% of the vaccinated, but none of the mock-vaccinated mice remained tumor-free. The anti-tumor effect required T cells and correlated with the vigor of the cross-primed CD8 T cell response. Her2-specific antibodies were not detected. This study highlights the potential of T cell cross-priming in cancer immunotherapy.     

Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by immunization with dendritic cells treated with an anthrax toxin fusion protein.

As a novel and safe vaccine strategy, the anthrax toxin-mediated antigen delivery system composed of lethal factor (LF) fusion protein and protective antigen (PA) has been studied to prime hepatitis C virus (HCV) core-specific cytotoxic T lymphocytes (CTLs) in vivo. The core epitope fused to LF (LF-core) together with PA induces a negligible core-specific CTL response in mice, whereas core-specific CTL are effectively primed in mice by injecting dendritic cells (DCs) treated in vitro with LF-core and PA. These findings imply that LF fusion protein plus PA in combination with dendritic cells may be useful for a novel T cell vaccine against HCV infection.     

Antibody responses and protection in mice immunized orally against influenza virus

Antibody responses and protection were studied in BALB/c mice immunized orally with formalin-inactivated influenza viruses (A/PR/8/34) combined with cholera toxin B subunit as adjuvant. Influenza virus-specific IgA as well as IgG antibody responses were induced in the mice, depending on the oral dosage frequency. The oral immunization by multiple doses resulted in reduction of viral replication in the nose and prevention of development of infection in the lung after intranasal (i.n.) challenge. The protective effect in the nose was thought to be related to the nasal IgA antibody response. The oral immunization was, however, less efficient for induction of the IgA antibody response and protection in the nose, compared with an i.n. immunization. The oral immunization following subcutaneous priming led to the complete protection in the nose, accompanied by a prompt local IgA antibody response.