HER-2/neu mediated down-regulation of MHC class I antigen processing prevents CTL-mediated tumor recognition upon DNA vaccination in HLA-A2 transgenic mice

To study DNA vaccination directed against human HER-2 in the HHD mouse Tg strain, we created a novel HER-2-expressing syngeneic tumor transplantation model. We found that a DNA vaccine encoding the full length HER-2 DNA protected HHD mice from HER-2⁺ tumor challenge by a CTL independent mechanism. A more efficient approach to induce HLA-A2 restricted CTLs, through immunization with a multi-epitope DNA vaccine expressing the HLA-A2 restricted HER-2 369–377, 435–443 and 689–697 epitopes, resulted in high numbers of peptide specific T cells but failed to induce tumor protection. Subsequently we discovered that HER-2 transfected tumor cells down-regulated MHC class I antigen expression and exhibited a series of defects in the antigen processing pathway which impaired the capacity to produce and display MHC class I peptide-ligands to specific CTLs. Our data demonstrate that HER-2 transfection is associated with defects in the MHC class I presentation pathway, which may be the underlying mechanism behind the inability of CTLs to recognize tumors in this HLA-A2 transgenic model. As defective MHC class I presentation may be a common characteristic of HER-2 expressing tumors, vaccines targeting HER-2 should aim at inducing an integrated immune response where also CD4⁺ T cells and antibodies are important components. S. Vertuani and C. Triulzi contributed equally to this work.     

A chimeric multi-human epidermal growth factor receptor-2 B cell epitope peptide vaccine mediates superior antitumor responses

Immunotherapeutic approaches to cancer should focus on novel undertakings that modulate immune responses by synergistic enhancement of antitumor immunological parameters. Cancer vaccines should preferably be composed of multiple defined tumor Ag-specific B and T cell epitopes. To develop a multiepitope vaccine, 12 high ranking B cell epitopes were identified from the extracellular domain of the human epidermal growth factor receptor-2 (HER-2) oncoprotein by computer-aided analysis. Four novel HER-2 B cell epitopes were synthesized as chimeras with a promiscuous T cell epitope (aa 288-302) from the measles virus fusion protein (MVF). Two chimeric peptide vaccines, MVF HER-2(316-339) and MVF HER-2(485-503) induced high levels of Abs in outbred rabbits, which inhibited tumor cell growth. In addition, Abs induced by a combination of two vaccines, MVF HER-2(316-339) and MVF HER-2(628-647) down-modulated receptor expression and activated IFN-gamma release better than the individual vaccines. Furthermore, this multiepitope vaccine in combination with IL-12 caused a significant reduction (p = 0.004) in the number of pulmonary metastases induced by challenge with syngeneic tumor cells overexpressing HER-2. Peptide Abs targeting specific sites in the extracellular domain may be used for exploring the oncoprotein's functions. The multiepitope vaccine may have potential application in the treatment of HER-2-associated cancers.     

In vivo electroporation restores the low effectiveness of DNA vaccination against HER-2/neu in aging

Experimental evidence has been provided that cancer vaccines are less effective at older age than in young adults. In this study, we evaluated the possibility to recover the low effectiveness of DNA immunization against HER-2/neu increasing plasmid uptake by cells from old mice through electroporation with the aim to enhance the activation of specific immune responses. Young and old Balb/c mice received two immunizations with a pCMV-ECDTM DNA plasmid using plasmid intramuscular injection followed by electroporation (IM + E) or plasmid intramuscular injection alone (IM), and successively, they were challenged with syngeneic HER-2/neu overexpressing TUBO cells. Young mice were completely protected whereas less than 60% protection was observed in old mice after IM immunization. IM + E immunization completely protected old mice against a TUBO cell challenge. The protection was associated with increased transgene expression in the site of immunization and with the induction of both humoral and cell-mediated immunity in old mice. We conclude that the effectiveness of anticancer DNA vaccination in old ages may be improved increasing plasmid uptake and transgene expression through electroporation, suggesting the relevant role of the first steps of the immunization process in the success of cancer vaccines at older age.     

Early role of CD4+ Th1 cells and antibodies in HER-2 adenovirus vaccine protection against autochthonous mammary carcinomas

HER-2 is an oncogenic tumor-associated Ag that is overexpressed in several human tumors including breast and ovarian cancer. The efficacy and mechanism of a HER-2-expressing recombinant adenoviral vaccine to protect against tumorigenesis was examined using HER-2 transgenic (BALB-neuT) mice, which develop spontaneous breast tumors in all 10 mammary glands, and also using a transplantable mouse tumor model. Vaccination beginning at 6-8 wk of age (through 19 wk of age) prevented development of spontaneous mammary tumors even after 50 wk, whereas the animals in the control groups had tumors in all mammary glands by 25 wk. Such long-term protection after the last boost has not been achieved previously in this transgenic mouse in which the oncogene is continuously spawning tumorigenesis. Using beta(2)-microglobulin-knockout, IFN-gamma-knockout, and B cell-deficient mice, CD4(+) and CD8(+) cell depletion, and Ab transfer studies, we show that induction of anti-HER-2/neu Abs are both necessary and sufficient for protection, and the IgG2a isotype is most effective. In contrast, CD8(+) T cells are not necessary at all, and CD4(+) T cells are necessary for only 36-48 h after immunization to provide help for B cells but not as effector cells. Equal protection in immunized mice deficient in FcgammaRI/III excluded an FcR-mediated mechanism. Anti-HER-2 serum not only inhibited growth of mammary tumor cell lines expressing HER-2 in vitro but also protected mice from tumors in vivo, suggesting a direct action of Ab on the tumor cells. Such a vaccine may provide Ab-mediated protection against HER-2-expressing breast cancers in humans.     

Peptide vaccines of the HER-2/neu dimerization loop are effective in inhibiting mammary tumor growth in vivo

Human epidermal growth factor receptor-2 (HER-2)/neu (ErbB2), a member of the epidermal growth factor family of receptors, is overexpressed in 20-30% of breast cancers. It is an attractive target for receptor-directed antitumor therapy using mAbs. Unlike other epidermal growth factor receptor family members, HER-2/neu does not bind a high-affinity ligand, but rather functions as the preferred dimerization partner. Pertuzumab (Omnitarg) is a humanized mAb directed against the HER-2/neu dimerization domain that inhibits receptor signaling. The recent definition of the crystal structure of the HER-2/neu-pertuzumab complex demonstrated that the receptor dimerization region encompassed residues 266-333. Based on the three-dimensional structure of the complex, we have designed three conformational peptide constructs (sequences 266-296, 298-333, and 315-333) to mimic regions of the dimerization loop of the receptor and to characterize their in vitro and in vivo antitumor efficacy. All the constructs elicited high-affinity peptide Abs that inhibited multiple signaling pathways including HER-2/neu-specific inhibition of cellular proliferation and cytoplasmic receptor domain phosphorylation. All the peptide Abs showed Ab-dependent cellular cytotoxicity to varying degrees with the 266-296 constructs being equally effective as compared with Herceptin. The 266-296 peptide vaccine had statistically reduced tumor onset in both transplantable tumor models (FVB/n and BALB/c) and significant reduction in tumor development in two transgenic mouse tumor models (BALB-neuT and VEGF(+/-)Neu2-5(+/-)). The 266-296 construct represents the most promising candidate for antitumor vaccination and could also be used to treat a variety of cancers with either normal or elevated expression of HER-2 including breast, lung, ovarian, and prostate.     

Induction of Protective Anti-CTL Epitope Responses against HER-2-Positive Breast Cancer Based on Multivalent T7 Phage Nanoparticles

We report here the development of multivalent T7 bacteriophage nanoparticles displaying an immunodominant H-2k^d-restricted CTL epitope derived from the rat HER2/neu oncoprotein. The immunotherapeutic potential of the chimeric T7 nanoparticles as anti-cancer vaccine was investigated in BALB/c mice in an implantable breast tumor model. The results showed that T7 phage nanoparticles confer a high immunogenicity to the HER-2-derived minimal CTL epitope, as shown by inducing robust CTL responses. Furthermore, the chimeric nanoparticles protected mice against HER-2-positive tumor challenge in both prophylactic and therapeutic setting. In conclusion, these results suggest that CTL epitope-carrying T7 phage nanoparticles might be a promising approach for development of T cell epitope-based cancer vaccines.     

Identification and characterization of the immunodominant rat HER-2/neu MHC class I epitope presented by spontaneous mammary tumors from HER-2/neu-transgenic mice

The HER-2/neu (neu-N)-transgenic mice are a clinically relevant model of breast cancer. They are derived from the parental FVB/N mouse strain and are transgenic for the rat form of the proto-oncogene HER-2/neu (neu). In this study, we report the identification of a MHC class I peptide in the neu protein that is recognized by CD8(+) T cells derived from vaccinated FVB/N mice. This 10-mer was recognized by all tumor-specific FVB/N T cells generated regardless of the TCR Vbeta region expressed by the T cell or the method of vaccination used, establishing it as the immunodominant MHC class I epitope in neu. T cells specific for this epitope were able to cure FVB/N mice of transplanted neu-expressing tumor cells, demonstrating that this is a naturally processed peptide. Altered peptide analogs of the epitope were analyzed for immunogenicity. Vaccination with dendritic cells pulsed with a heteroclitic peptide provided FVB/N and neu-N mice with increased protection against tumor challenge as compared with mice immunized with dendritic cells loaded with either wild-type or irrelevant peptide. Discovery of this epitope allows for better characterization of the CD8(+) T cell responses in the neu-N mouse model in which neu-specific tolerance must be overcome to produce effective antitumor immunity.     

Complementary antitumor immunity induced by plasmid DNA encoding secreted and cytoplasmic human ErbB-2.

A plasmid DNA was constructed to encode the N-terminal 505 aa of human ErbB-2 (E2, HER-2/neu) and designated as secreted ErbB-2 (secE2). Recombinant secE2 protein was detected in the transfected cells and was secreted as an 80-kDa glycoprotein. Vaccination of BALB/c mice with secE2 DNA induced both IgG1 and IgG2a ErbB-2-specific Abs and protected approximately 90% of mice against mouse mammary tumor D2F2, which expressed human ErbB-2 (D2F2/E2). The efficacy of secE2 vaccine was comparable with that of wild-type ErbB-2 DNA, which encodes the entire 1258 aa of ErbB-2 protein, induced only IgG2a E2-specific Abs, and stimulated greater CTL activity. Immune lymphocytes were stimulated in vitro with irradiated 3T3 cells, which expressed ErbB-2, K(d), and B7.1. CTL activity was measured by the lysis of E2-positive target cells and by intracellular IFN-gamma production. To enhance CTL activation, mice were immunized with a combination of secE2 and cytoplasmic E2 (cytE2); the latter encodes the 1258-aa ErbB-2 protein that was released into the cytoplasm upon synthesis. Significant increase in CTL activity was demonstrated after mice were immunized with the combined vaccines and all mice were protected from D2F2/E2 tumor growth. Therefore, secE2, which induced Th2 Ab and weak CTL, conferred similar protection as E2, which induced Th1 Ab and strong CTL. Combined vaccination with secE2 and cytE2 resulted in Th2 Ab, strong CTL, and the most effective protection against tumor growth. The strategy of coimmunization with DNA that direct Ags to different subcellular compartments may be adapted as appropriate to optimize immune outcome.     

HSP110-HER2/neu chaperone complex vaccine induces protective immunity against spontaneous mammary tumors in HER-2/neu transgenic mice

Heat shock proteins (HSPs) are shown to be strong immunoadjuvants, eliciting both innate and adaptive immune responses against cancers. HSP110 is related in sequence to HSP70 and is approximately 4-fold more efficient in binding to and stabilizing denatured protein substrates compared with HSP70. In the present study we evaluated the ability of a heat shock complex of HSP110 with the intracellular domain (ICD) of human HER-2/neu to elicit effective antitumor immune responses and to inhibit spontaneous mammary tumors in FVB-neu (FVBN202) transgenic mice. The HSP110-ICD complex was capable of breaking tolerance against the rat neu protein and inhibiting spontaneous mammary tumor development. This vaccine induced ICD-specific IFN-gamma and IL-4 production. Depletion studies revealed that CD8(+) T cells were involved in protection against challenge with mouse mammary tumors, whereas CD4(+) T cells revealed partial protection. Increased IgG2a Ab titer in the sera of tumor-free animals after vaccination and elevated CD4(+) CD25(+) regulatory T cells in the PBL of tumor-bearing animals suggested that IFN-gamma-producing Th1 cells may be responsible for partial protection of CD4(+) T cells against the mammary tumor challenge, whereas CD4(+)CD25(+) regulatory T cells (Th2 cells) may suppress the antitumor immune responses. Together, these results suggest that HSP110-ICD complex can elicit effective IFN-gamma-producing T cells against spontaneous mammary tumors and that up-regulation of CD4(+) CD25(+) regulatory T cells may prevent complete eradication of the tumor following immunotherapy.     

Novel engineered trastuzumab conformational epitopes demonstrate in vitro and in vivo antitumor properties against HER-2/neu

Trastuzumab is a growth-inhibitory humanized Ab targeting the oncogenic protein HER-2/neu. Although trastuzumab is approved for treatment of advanced breast cancer, a number of concerns exist with passive immunotherapy. Treatment is expensive and has a limited duration of action, necessitating repeated administrations of the mAb. Active immunotherapy with conformational B cell epitopes affords the possibility of generating an enduring immune response, eliciting protein-reactive high-affinity anti-peptide Abs. The three-dimensional structure of human HER-2 in complex with trastuzumab reveals that the Ag-binding region of HER-2 spans residues 563-626 that comprises an extensive disulfide-bonding pattern. To delineate the binding region of HER-2, we have designed four synthetic peptides with different levels of conformational flexibility. Chimeric peptides incorporating the measles virus fusion "promiscuous" T cell epitope via a four-residue linker sequence were synthesized, purified, and characterized. All conformational peptides were recognized by trastuzumab and prevented the function of trastuzumab inhibiting tumor cell proliferation, with 563-598 and 597-626 showing greater reactivity. All epitopes were immunogenic in FVB/N mice with Abs against 597-626 and 613-626 recognizing HER-2. The 597-626 epitope was immunogenic in outbred rabbits eliciting Abs which recognized HER-2, competed with trastuzumab for the same epitope, inhibited proliferation of HER-2-expressing breast cancer cells in vitro and caused their Ab-dependent cell-mediated cytotoxicity. Moreover, immunization with the 597-626 epitope significantly reduced tumor burden in transgenic BALB-neuT mice. These results suggest the peptide B cell immunogen is appropriate as a vaccine for HER-2-overexpressing cancers because the resulting Abs show analogous biological properties to trastuzumab.     

Identification and characterization of a HER-2/neu epitope as a potential target for cancer immunotherapy

Our aim is to develop peptide vaccines that stimulate tumor antigen-specific T-lymphocyte responses against frequently detected cancers. We describe herein a novel HLA-A*0201-restricted epitope, encompassing amino acids 828–836 (residues QIAKGMSYL), which is naturally presented by various HER-2/neu ⁺ tumor cell lines. HER-2/neu(828-836), [HER-2(9₈₂₈)], possesses two anchor residues and stabilized HLA-A*0201 on T2 cells in a concentration-dependent Class I binding assay. This peptide was stable for 3.5 h in an off-kinetic assay. HER-2(9₈₂₈) was found to be immunogenic in HLA-A*0201 transgenic (HHD) mice inducing peptide-specific and functionally potent CTL and long-lasting anti-tumor immunity. Most important, using HLA-A*0201 pentamer analysis we could detect increased ex vivo frequencies of CD8⁺ T-lymphocytes specifically recognizing HER-2(9₈₂₈) in 8 out of 20 HLA-A*0201⁺ HER-2/neu ⁺ breast cancer patients. Moreover, HER-2(9₈₂₈)-specific human CTL recognized the tumor cell line SKOV3.A2 as well as the primary RS.A2.1.DR1 tumor cell line both expressing HER-2/neu and HLA-A*0201. Finally, therapeutic vaccination with HER-2(9₈₂₈) in HHD mice was proven effective against established transplantable ALC.A2.1.HER tumors, inducing complete tumor regression in 50% of mice. Our data encourage further exploitation of HER-2(9₈₂₈) as a promising candidate for peptide-based cancer vaccines.     

Fusion to Listeriolysin O and delivery by Listeria monocytogenes enhances the immunogenicity of HER-2/neu and reveals subdominant epitopes in the FVB/N mouse

Five overlapping fragments of rat HER-2/neu have been expressed in recombinant Listeria monocytogenes. Each fragment of HER-2/neu is secreted as a fusion protein with a truncated, nonhemolytic form of listeriolysin O (LLO). Lm-LLO-EC1, Lm-LLO-EC2, and Lm-LLO-EC3 overlap the extracellular domain of HER-2/neu, whereas Lm-LLO-IC1 and Lm-LLO-IC2 span the intracellular domain. All five strains controlled the growth of established NT-2 tumors, a rat HER-2/neu-expressing tumor line derived from a spontaneously arising mammary tumor in a FVB/N HER-2/neu-transgenic mouse. The antitumor effect of each of these vaccine constructs was abrogated by the in vivo depletion of CD8(+) T cells, although only one known epitope has been defined previously and is present in Lm-LLO-EC2. Anti-HER-2/neu CTL responses were generated by each of the rLm vaccine constructs. With the use of a panel of 3T3 cell lines expressing overlapping fragments of HER-2/neu, regions of HER-2/neu with potential CD8(+) T cell epitopes have been defined. DNA vaccines expressing either a fragment or full-length HER-2/neu were constructed in LLO-fused and non-LLO-fused forms. CTL analysis of the DNA vaccines revealed a broadening in the regions of HER-2/neu recognizable as targets when the target Ag is fused to LLO. These studies show the efficacy of L. monocytogenes-based HER-2/neu vaccines in a murine model of breast cancer and also that the immunogenicity of self-Ags can be increased by fusion to LLO and delivery by L. monocytogenes revealing subdominant epitopes.     

Immunoprevention of mammary carcinoma in HER-2/neu transgenic mice is IFN-gamma and B cell dependent

A vaccine combining IL-12 and allogeneic mammary carcinoma cells expressing p185(neu) completely prevents tumor onset in HER-2/neu transgenic BALB/c mice (NeuT mice). The immune protection elicited was independent from CTL activity. We now formally prove that tumor prevention is mainly based on the production of anti-p185(neu) Abs. In the present studies, NeuT mice were crossed with knockout mice lacking IFN-gamma production (IFN-gamma(-/-)) or with B cell-deficient mice (microMT). Vaccination did not protect NeuT-IFN-gamma(-/-) mice, thus confirming a central role of IFN-gamma. The block of Ab production in NeuT-microMT mice was incomplete. About one third of NeuT-microMT mice failed to produce Abs and displayed a rapid tumor onset. By contrast, those NeuT-microMT mice that responded to the vaccine with a robust production of anti-p185(neu) Ab displayed a markedly delayed tumor onset. In these NeuT-microMT mice, the vaccine induced a lower level of IgG2a and IgG3 and a higher level of IgG2b than in NeuT mice. Moreover, NeuT-microMT mice failed to produce anti-MHC class I Abs in response to allogeneic H-2(q) molecules present in the cell vaccine. These findings show that inhibition of HER-2/neu carcinogenesis depends on cytokines and specific Abs, and that a highly effective vaccine can rescue Ab production even in B cell-deficient mice.     

Application of a gene vaccine targeting HER-2/neu in immunocontraception

HER-2/neu was widely used as a target for tumor prevention and therapy because of its overexpression in many tumors. However, it also plays an important role in proliferation of endometrium, embryo implantation, and development. Here, HER-2/neu was used in immunocontraception. A gene vaccine encoding the extracellular domain of human HER-2/neu was constructed. After immunization, it especially elicited both humoral and cellular responses in mice. Embryo implantation was interfered by intravenous and intraluminal injection of anti-HER-2/neu serum or lymphocytes. Lower fertility was induced after vaccination when compared with the control groups, while injuries to the uterus and ovary were not observed. Our results suggested a new and impactful target for contraceptive vaccines development.     

In the FVB/N HER-2/<Emphasis Type="Italic">neu</Emphasis> transgenic mouse both peripheral and central tolerance limit the immune response targeting HER-2/neu induced by <Emphasis Type="Italic">Listeria monocytogenes</Emphasis>-based vaccines

Listeria monocytogenes-based vaccines for HER-2/neu are capable of breaking tolerance in FVB/N rat HER-2/neu transgenic mice. The growth of implanted NT-2 tumors, derived from a spontaneously occurring tumor in the FVB/N HER-2/neu transgenic mouse, was significantly slower in these mice following vaccination with a series of L. monocytogenes-based vaccines for HER-2/neu. Mechanisms of T cell tolerance that exist in these transgenic mice include the absence of functional high avidity anti-HER-2/neu CD8⁺ T cells and the presence of CD4⁺CD25⁺ regulatory T cells. The in vivo depletion of these regulatory T cells resulted in the slowing in growth of tumors even without the treatment of mice with an anti-HER-2/neu vaccine. The average avidities of responsive CD8⁺ T cells to six of the nine epitopes in HER-2/neu we examined, four of which were identified in this study, are shown here to be of a lower average avidity in the transgenic mice versus wild type FVB/N mice. In contrast, the average avidity of CD8⁺ T cells to three epitopes that showed the lowest avidity in the wild-type mice did not differ between wild type and transgenic mice. This study demonstrates the ability of L. monocytogenes-based vaccines to impact upon tolerance to HER-2/neu in FVB/N HER-2/neu transgenic mice and further defines some of the aspects of tolerance in these mice.     

TCR vaccines against a murine T cell lymphoma: a primary role for antibodies of the IgG2c class in tumor protection

Tumor-associated proteins can act as effective immunotherapeutic targets. Immunization with tumor TCR protein conjugated to the immunogenic protein keyhole limpet hemocyanin (KLH) protects mice from tumor challenge with the murine T cell lymphoma C6VL. The immune mechanisms responsible for this tumor protection are of interest for designing more effective vaccine strategies. Previous studies using depletion experiments had suggested a CD8-mediated component of protection induced by TCR-KLH vaccines. In this study we used CD8alpha knockout, micro MT, and FcgammaR knockout mice to investigate the relative roles of CD8+ T cells and Ab in protective immunity induced by TCR-KLH immunization. We found that CD8+ T cells are not required for tumor protection, although they may contribute to protection. Vaccine-induced Abs are sufficient to mediate protection against this murine T cell lymphoma through an FcR-dependent mechanism. This was confirmed with Ab transfers, which protect challenged mice. Additionally, recombinase-activating gene 1(-/-) splenocytes can mediate Ab-dependent cellular cytotoxicity against this tumor in the presence of bound anti-TCR Abs. IFN-gamma knockout mice demonstrated a requirement for IFN-gamma, probably via generation of IgG2c Abs, in vaccine-induced tumor protection. IFN-gamma knockout mice were not protected by immunization and had a severe impairment in IgG2c Ab production in response to immunization. Although mock-depleted anti-TCR Abs could transfer tumor protection, IgG2c-deficient anti-TCR Abs were unable to transfer tumor protection to wild-type mice. These results suggest that TCR-KLH vaccine-induced tumor protection in the C6VL system is primarily attributable to the induction of IgG2c Abs and humoral immunity.     

HER-2/neu oncogene expression, DNA ploidy and proliferation index in breast cancer.

HER-2/neu gene expression, DNA ploidy and proliferation index were studied in 250 cases of breast cancer. Expression of HER-2/neu was determined by using an antibody to the HER-2/neu receptor. Ki-67 antibody was used to determine the proliferation index of the breast cancers, and the Feulgen method was used to assess DNA amounts in the tumor cells. Histochemical staining was quantitated by image analysis. Of the cancers studied, 72 were positive for overexpression of HER-2/neu protein; of these, 62 (86%) possessed near-tetraploid DNA content, and 47 (65%) had more than one G0G1 stem line (polyploid) of DNA distribution. Cells from the cases negative for HER/2-neu overexpression contained DNA amounts that ranged from diploid to varying degrees of aneuploid. A significant difference in the amounts of cellular proliferation in HER-2/neu overexpressing cancers was found between those that expressed the HER-2/neu receptor on their membranes and those that exhibited mainly cytoplasmic receptors.     

Accelerated HER-2 degradation enhanced ovarian tumor recognition by CTL. Implications for tumor immunogenicity

We investigated the ubiquitination and degradation of a tumor antigen, the HER-2/neu (HER-2) protooncogene product which is overexpressed in epithelial cancers. HER-2 degradation was investigated in the ovarian tumor line, SKOV3.A2, that constitutively overexpressed long-life HER-2. We used as agonist geldanamycin (GA), which initiated downmodulation of HER-2 from the cell surface. HER-2 was polyubiquitinated and degraded faster in the presence than in the absence of GA. GA did not decrease HLA-A2 expression. Presentation of the immunodominant cytotoxic T lymphocyte (CTL) epitope, E75 (369–377) from SKOV.A2 was inhibited by proteasome inhibitors, such as LLnL but was enhanced by cysteine protease inhibitors such as E64, indicating that both the proteasome and cysteine proteases are involved in epitope formation but have different effects. Enhanced tumor recognition was not an immediate or early effect of GA treatment, but was evident after 20 h of GA treatment. In contrast, 20 h GA treatment did not increase tumor sensitivity to LAK cell lysis. Twenty hour GA-treated SKOV3.A2 cells expressed an unstable HER-2 protein synthesized in the presence of GA, of faster electrophoretic mobility than control HER-2. This suggested that the newly synthesized HER-2 in the presence of GA was the main source of epitopes recognized by CTL. Twenty hour GA-treated SKOV3.A2 cells were better inducers of CTL activity directed to a number of HER-2 CTL epitopes, in peripheral blood mononuclear cells compared with control untreated SKOV3.A2 cells. Thus, induction of HER-2 protein instability enhanced the sensitivity of tumor for CTL lysis. Increased HER-2 CTL epitopes presentation may have implications for overcoming the poor immuno-genicity of human tumors, and design of epitope precursors for cancer vaccination.     

Influenza A vaccine based on the extracellular domain of M2: weak protection mediated via antibody-dependent NK cell activity

Vaccination of mice with a peptide corresponding to the extracellular part of M2 protein coupled to the immunodominant domain of hepatitis B core can protect mice from a lethal challenge with influenza A virus. As the extracellular part of M2 protein is highly conserved in all known human influenza A strains, such a vaccine may protect against all human influenza A strains, which would represent a major advantage over current vaccine strategies. The present study demonstrates that protection is mediated exclusively by Abs, a very important feature of a successful preventive vaccine. However, these Abs neither bind efficiently to the free virus nor neutralize virus infection, but bind to M2 protein expressed on the surface of virus-infected cells. The presence of NK cells is important for protection, whereas complement is not, supposing that protection is mediated via Ab-dependent, cell-mediated cytotoxicity. The absence of neutralizing Abs results in much weaker protection than that achieved by vaccination with UV-inactivated influenza virus. Specifically, whereas neutralizing Abs completely eliminate signs of disease even at high viral challenge doses, M2-specific Abs cannot prevent infection, but merely reduce disease at low challenge doses. M2-specific Abs fail to protect from high challenge doses, as vaccinated mice undergo lethal infection under these conditions. In conclusion, protection mediated by M2-hepatitis B core vaccine would be insufficient during the yearly epidemics, for which full protection is desirable, and overall is clearly inferior to protection achieved by immunization with classical inactivated viral preparations.