Induction by DNA immunization of a protective antitumor cytotoxic T lymphocyte response against a minimal-epitope-expressing tumor

 In order to examine the use of DNA immunization to block tumor growth, we have developed a model system in which a defined 9-amino-acid epitope from the nucleoprotein of influenza virus is used as a surrogate tumor-associated antigen. A mastocytoma cell line of DBA/2 origin (P815) was transfected with a plasmid encoding the minimal H-2K^d-restricted NP(147–155) cytotoxic T lymphocyte (CTL) epitope, pCMV/NPep, to generate the cell line designated P815-NPep. Mice primed and boosted once with a plasmid encoding the full-length NP gene, pCMV/NP, but not with the minigene pCMV/NPep, developed a strong NP(147–155)-specific CTL response within 2 weeks after the boost. When challenged with 10⁴ P815-NPep cells, pCMV/NP-immunized DBA/2 mice were protected from tumor challenge, whereas control mice immunized with the vector backbone rapidly developed lethal tumor. Importantly, the P815-NPep-immune mice were also protected from a subsequent challenge with the untransfected parental tumor P815. By depleting the NP-immune mice of either CD4⁺ or CD8⁺ T cells and then challenging with 10⁴ P815-NPep tumor cells, it was determined that the CD8-depleted mice rapidly developed tumors, whereas the CD4-depleted or non-treated mice were protected. These data clearly indicate that intramuscular (i.m.) plasmid DNA immunization can be used to mobilize an effective CD8⁺ CTL-mediated antitumor response. Received: 8 May 1997 / Accepted: 28 August 1997     

Recognition of naturally processed and ovarian cancer reactive CD8<Superscript>+</Superscript> T cell epitopes within a promiscuous HLA class II T-helper region of NY-ESO-1

NY-ESO-1 is frequently expressed in epithelial ovarian cancer (EOC) and elicits spontaneous humoral and cellular immune responses in a proportion of EOC patients. The identification of NY-ESO-1 peptide epitopes with dual HLA-class I and class II specificities might be useful in vaccination strategies for generating cognate CD4+ T cell help to augment CD8+ T cell responses. Here, we describe two novel NY-ESO-1-derived MHC class I epitopes from EOC patients with spontaneous humoral immune response to NY-ESO-1. CD8+ T cells derived from NY-ESO-1 seropositive EOC patients were presensitized with a recombinant adenovirus encoding NY-ESO-1or pooled overlapping peptides. These epitopes, ESO127-136 presented by HLA-A68 molecule, and ESO127-135 restricted by HLA-Cw15 allele, are located within ESO119-143, a promiscuous HLA-class II region containing epitopes that bind to multiple HLA-DR alleles. The novel epitopes were naturally processed by APC or naturally presented by tumor cell lines. In addition, these epitopes induced NY-ESO-1-specific CTL in NY-ESO-1 seropositive EOC patients. Together, the results indicate that ESO119-143 epitope has dual HLA classes I and II specificities, and represents a potential vaccine candidate in a large number of cancer patients.     

Organismal regulation of XBP-1-mediated unfolded protein response during development and immune activation

The increased demand on protein folding in the endoplasmic reticulum (ER) during bacterial infection activates the unfolded protein response (UPR). OCTR-1-a G protein-coupled catecholamine receptor expressed in neurons-suppresses innate immunity by downregulating a non-canonical UPR pathway and the p38 MAPK pathway. Here, we show that OCTR-1 also regulates the canonical UPR pathway, which is controlled by XBP-1, at the organismal level. Importantly, XBP-1 is not under OCTR-1 control during development, only at the adult stage. Our results indicate that the nervous system temporally controls the UPR pathway to maintain ER homeostasis during development and immune activation.     

Induction of Th1 type response by DNA vaccinations with N, M, and E genes against SARS-CoV in mice

Vaccination against the SARS-CoV infection is an attractive means to control the spread of viruses in public. In this study, we employed a DNA vaccine technology with the levamisole, our newly discovered chemical adjuvant, to generate Th1 type of response. To avoid the enhancement antibody issue, genes encoding the nucleocapsid, membrane, and envelope protein of SARS-CoV were cloned and their expressions in mammalian cells were determined. After the intramuscular introduction into animals, we observed that the constructs of the E, M, and N genes could induce high levels of specific antibodies, T cell proliferations, IFN-gamma, DTH responses, and in vivo cytotoxic T cells activities specifically against SARS-CoV antigens. The highest immune responses were generated by the construct encoding the nucleocapsid protein. The results suggest that the N, M, and E genes could be used as the targets to prevent SARS-CoV infection in the DNA vaccine development.     

Induction of cell-mediated immunity against B16-BL6 melanoma in mice vaccinated with cells modified by hydrostatic pressure and chemical crosslinking

In the preceding paper we have demonstrated an increase in presentation of both major histocompatibility complex antigens (MHC) and a tumor-associated antigen of the weakly immunogenic B16 melanoma by a straight-forward technique. The method consists in modulating the tumor cell membrane by hydrostatic pressure and simultaneous chemical crosslinking of the cell-surface proteins. In B16-BL6 melanoma, the induced antigenic modulation was found to persist for over 48 h, which permitted the evaluation of the ability of modified B16-BL6 cells to induce immunity against unmodified B16-BL6 cells. In the present study, we have shown that a significant systemic immunity was induced only in mice that were immunized with modified B16-BL6 melanoma cells, whereas immunization with unmodified B16-BL6 cells had only a marginal effect when compared to the results in control sham-immunized mice. The induced immunity was specific since a single immunization affected the growth of B16-BL6 tumors but had no effect on MCA 106, an antigenically unrelated tumor. The addition of interleukin-2 to the immunization regimen had no effect on the antitumor responses induced by the modified B16-BL6 cells. The cell-mediated immunity conferred by immunization with treated B16-BL6 cells was confirmed in experiments in vitro where splenocytes from immunized mice could be sensitized to proliferate by the presence of B16-BL6 cells. In addition, the altered antigenicity of these melanoma cells appeared to correlate with their increased susceptibility to specific effectors. Thus,⁵¹Cr-labeled B16-BL6 target cells, modified by pressure and crosslinking, in comparison to control labeled target cells, were lysed in much greater numbers by effectors such as lymphokine-activated killer cells and allogeneic cytotoxic lymphocytes (anti-H-2^b), while such cells remained resistant to lysis by natural killer cells. Our findings indicate that the physical and chemical modifications of the tumor cells that are described here may be considered as a simple yet effective method for the preparation of tumor vaccines, which could be applied in tumor-bearing hosts.     

Induction of protective immune response to intranasal administration of influenza virus-like particles in a mouse model

Human influenza A viruses (IAVs) cause global pandemics and epidemics, which remains a nonignorable serious concern for public health worldwide. To combat the surge of viral outbreaks, new treatments are urgently needed. Here, we design a new vaccine based on virus-like particles (VLPs) and show how intranasal administration of this vaccine triggers protective immunity, which can be exploited for the development of new therapies. H1N1 VLPs were produced in baculovirus vectors and were injected into BALB/c mice by the intramuscular (IM) or intranasal (IN) route. We found that there were significantly higher inflammatory cell and lymphocyte concentrations in bronchoalveolar lavage samples and the lungs of IN immunized mice; however, the IM group had little signs of inflammatory responses. On the basis of our results, immunization with H1N1 influenza VLP elicited a strong T cell immunity in BALB/c mice. Despite T cell immunity amplification after both IN and IM vaccination methods in mice, IN-induced T cell responses were significantly more intense than IM-induced responses, and this was likely related to an increased number of both CD11b^high and CD103⁺ dendritic cells in mice lungs after IN administration of VLP. Furthermore, evaluation of interleukin-4 and interferon gamma cytokines along with several chemokine receptors showed that VLP vaccination via IN and IM routes leads to a greater CD4⁺ Th1 and Th2 response, respectively. Our findings indicated that VLPs represent a potential strategy for the development of an effective influenza vaccine; however, employing relevant routes for vaccination can be another important part of the universal influenza vaccine puzzle.     

Induction of anti-idiotypic humoral and cellular immune responses by a murine monoclonal antibody recognizing the ovarian carcinoma antigen CA125 encapsulated in biodegradable microspheres

The use of biodegradable poly(dl-lactic-co-glycolic acid) microspheres as a cancer vaccine delivery system for induction of anti-idiotypic responses was investigated using a murine monoclonal antibody B43.13 that recognizes the human ovarian cancer antigen CA125. Immunization of mice with mAb B43.13 encapsulated in poly(dl-lactic-co-glycolic acid) microspheres resulted in enhanced humoral and cellular immune responses compared with mAb B43.13 alone or mAb B43.13 mixed with microspheres. The antibody responses could be further enhanced by the co-encapsulation of mAb B43.13 with monophosphoryl lipid A, a non-toxic adjuvant, in microspheres. Anti-idiotypic humoral responses were shown to result in Ab2 antibodies mimicking the nominal antigen CA125 and Ab3 antibodies recognizing CA125. Further, microsphere delivery of mAb B43.13 also resulted in induction of T cell responses involving T2 cells reactive with mAb B43.13 epitopes and T3 cells recognizing CA125. These results indicate that microsphere delivery of Ab1 can induce both humoral and cellular anti-idiotypic responses relevant to cancer antigens. This raises the possibility of the use of such formulations for anti-idiotypic induction immunotherapy for cancer. Received: 27 August 1997 / Accepted: 24 April 1998     

Development of an allogeneic whole-cell tumor vaccine expressing xenogeneic gp100 and its implementation in a phase II clinical trial in canine patients with malignant melanoma

A xenogeneic melanoma-antigen-enhanced allogeneic tumor cell vaccine (ATCV) is an appealing strategy for anti-cancer immunotherapy due to its relative ease of production, and the theoretical possibility that presentation of a multiplex of antigens along with a xenogeneic antigen would result in cross-reaction between the xenogeneic homologs and self-molecules, breaking tolerance and ultimately resulting in a clinically relevant immune response. In this study, we evaluated the efficacy of such a strategy using a xenogeneic melanoma differentiation antigen, human glycoprotein 100 (hgp100) in the context of a phase II clinical trial utilizing spontaneously arising melanoma in pet dogs. Our results demonstrate that the approach was well tolerated and resulted in an overall response rate (complete and partial response) of 17% and a tumor control rate (complete and partial response and stable disease of >6 weeks duration) of 35%. Dogs that had evidence of tumor control had significantly longer survival times than dogs that did not experience control. Delayed type hypersensitivity (DTH) to 17CM98 canine melanoma cells used in the whole cell vaccine was enhanced by ATCV and correlated with clinical response. In vitro cytotoxicity was enhanced by ATCV, but did not correlate with clinical response. Additionally, anti-hgp100 antibodies were elicited in response to ATCV in the majority of patients tested; however, this also did not correlate with clinical response. This approach, along with further elucidation of the mechanisms of tumor protection after xenogeneic immunization, may allow the development of more rational vaccines. This trial also further demonstrates the utility of spontaneous tumors in companion animals as a valid translational model for the evaluation of novel vaccine therapies. E.G. MacEwen deceased     

Advance in treatment strategy and immune reconstruction against HIV-1 infection

HIV-1 can be considered an infection of the immune system, resulting in progressive and ultimately profound immune suppression. The availability of highly active antiretroviral therapy (HAART) has resulted in dramatic changes in the disease course in persons fortunate enough to have access to these medications, but long-term therapy is limited by the development of resistance as well as toxicities of the potent medication regimens. Emerging data indicate that individuals who have non-progressive clinical course control HIV-1 immunologically. This has bolstered hope that the immune response might be effectively augmented in persons with HIV infection. Recent data indicating that immediate treatment of acute infection leads to augmentation of antiviral immune responses have provided evidence that the immune system might be enhanced in certain situations. Therefore, investigation in the reconstitution of anti-HIV immune response in patients under HAART should provide encouragement for continuing to explore methods to obtain meaningful and durable immune enhancement as an adjunct to HAART in HIV-1 infection.     

ROS Production and Apoptosis Induction by Formation of Gts1p-Mediated Protein Aggregates

GTS1 of Saccharomyces cerevisiae is a pleiotropic gene. Its induction leads to a variety of biological phenomena represented by cell aggregation. The C-terminal polyglutamine sequence in Gts1p is indispensable for its pleiotropy and nuclear localization. This sequence is often observed in polyglutamine diseases, such as Huntington disease, and is believed to induce protein aggregation, leading to cell death. In this study, protein aggregates were formed in a polyglutamine-dependent manner in cells inducing GTS1, and heat-shock protein family, translation elongation factor, and mitochondrial proteins were trapped in Gts1p-mediated protein aggregates. Moreover, the polyglutamine sequence of Gts1p was indispensable to the induction of reactive oxygen species (ROS) production and apoptosis. Deletion of the genes encoding Por1p and Yhb1p altered the profiles of ROS production and apoptosis caused by GTS1 induction, suggesting that the trapping of these proteins in Gts1p-mediated protein aggregates inhibits the intrinsic functions of these proteins.     

离心、超滤技术在甲型肝炎减毒活疫苗生产工艺中的应用

采用离心和超滤技术进行甲型肝炎减毒活疫苗生产 ,达到提高病毒的收获率。将含有甲型肝炎病毒的细胞收获液离心 ,上清进行超滤浓缩 ,回收病毒 ,病毒回收效果好。实验证明 ,该方法适合规模化生产。     

Elimination of tumor by CD47/PD-L1 dual-targeting fusion protein that engages innate and adaptive immune responses

The host immune system generally serves as a barrier against tumor formation. Programmed death-ligand 1 (PD-L1) is a critical “don't find me” signal to the adaptive immune system, whereas CD47 transmits an anti-phagocytic signal, known as the “don't eat me” signal, to the innate immune system. These and similar immune checkpoints are often overexpressed on human tumors. Thus, dual targeting both innate and adaptive immune checkpoints would likely maximize anti-tumor therapeutic effect and elicit more durable responses. Herein, based on the variable region of atezolizumab and consensus variant 1 (CV1) monomer, we constructed a dual-targeting fusion protein targeting both CD47 and PD-L1 using “Knobs-into-holes” technology, denoted as IAB. It was effective in inducing phagocytosis of tumor cells, stimulating T-cell activation and mediating antibody-dependent cell-mediated cytotoxicity in vitro. No obvious sign of hematological toxicity was observed in mice administered IAB at a dose of 100 mg/kg, and IAB exhibited potent antitumor activity in an immune-competent mouse model of MC38. Additionally, the anti-tumor effect of IAB was impaired by anti-CD8 antibody or clodronate liposomes, which implied that both CD8+ T cells and macrophages were required for the anti-tumor efficacy of IAB and IAB plays an essential role in the engagement of innate and adaptive immune responses. Collectively, these results demonstrate the capacity of an elicited endogenous immune response against tumors and elucidate essential characteristics of synergistic innate and adaptive immune response, and indicate dual blockade of CD47 and PD-L1 by IAB may be a synergistic therapy that activates both innate and adaptive immune response against tumors.     

Toxoplasma gondii: protective immunity against experimental toxoplasmosis induced by a DNA vaccine encoding the perforin-like protein 1

Toxoplasma gondii is an important zoonotic parasite infecting about one third of the world population, causing congenital infections and eye disease. T. gondii perforin-like protein 1 (TgPLP1) is believed to be involved in the acute virulence of T. gondii in mice, and is therefore of interest as a vaccine candidate. In this study, we constructed a DNA vaccine expressing TgPLP1, and evaluated the immune response in Kunming mice. The gene sequence encoding TgPLP1 was inserted into the eukaryotic expression vector pVAX I, and Kunming mice were immunized intramuscularly with the plasmid. After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally with 1 × 10³ tachyzoites of the virulent T. gondii RH strain. The results showed that pVAX/TgPLP1 alone or with pVAX/IL-18 developed specific anti-TLA (T. gondii lysate antigen) antibodies and specific lymphocyte proliferative responses. Co-injection of pVAX/IL-18 significantly increased the production of IFN-γ and IL-2. Further, challenge experiments showed that co-immunization of pVAX/TgPLP1 with pVAX/IL-18 significantly (P < 0.05) increased survival time (12.7 ± 1.2 days) of immunized mice, compared with pVAX/TgPLP1 alone (11.3 ± 0.9 days). These results demonstrate that TgPLP1 is a potential vaccine candidate against toxoplasmosis, worth further evaluation in other animal hosts. IL-18 could enhance the immune effect of TgPLP1, prolonging the survival time of immunized mice.     

Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund''s adjuvant. Mouse splenocytes were then cultured in Dulbecco''s Modified Eagle''s Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj) ]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.     

Cross‐reactive antibody response to the pandemic A (H1N1) 2009 influenza virus induced by vaccination with a seasonal trivalent influenza vaccine: a longitudinal study of three influenza seasons in Japan

The cross‐reactivity of antibody to the swine‐origin pandemic influenza A (H1N1) 2009 virus induced by vaccination with a seasonal trivalent influenza vaccine was studied. Paired sera from a cohort of adult volunteers vaccinated with a trivalent seasonal influenza vaccine every year from 2006 to 2008 were collected each year and tested by hemagglutination inhibition (HI) for antibody against the pandemic influenza A (H1N1) 2009 virus. There was little increase in the geometric mean titer overall; a slight increase was detected in the sera obtained in the 2007–2008 season but not in the other two seasons. The proportion of individuals with HI antibody titers ≥ 1:40 did not change significantly from year to year. These results indicate that cross‐reactivity of the antibodies induced by a trivalent seasonal vaccine to the pandemic influenza A (H1N1) 2009 virus is marginal.