HPV16 E7抗原CTL表位拟肽设计及活性评价

人乳头瘤病毒(human papillomavirus,HPV)早期基因E7是致癌的关键基因,其表达在宫颈癌细胞癌变进程及维持癌细胞恶性表型方面发挥重要作用,已成为宫颈癌治疗的理想靶标.目前,基于HPV16 E7抗原细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL)表位设计多肽疫苗是抗宫颈癌治疗发展的重要方向,但天然CTL表位肽普遍存在体内半衰期短、激发CTL反应效果不佳等缺点.因此,本研究基于前期HPV16 E7抗原CTL表位鉴定的基础,结合多肽酶解实验结果,进行分子动力学模拟及结合自由能计算,初步筛选了3条表位模拟肽.人工合成相关待测表位肽,并利用T2细胞株测定各肽与HLA-A2分子的结合力.研究结果表明,3条表位模拟肽体外抗酶解能力较天然HPV16 E7抗原CTL表位肽均有提高,以(d)RAHYNIVTF表位模拟肽的效果最为明显.此外,(d)RAHYNIVTF表位模拟肽与HLA-A2分子的结合力也有所提高(荧光系数为2.06).以上结果表明,基于HPV16 E7抗原CTL表位模拟肽进行结构修饰有望为宫颈癌治疗性疫苗的设计奠定基础.     

Cytotoxic T-lymphocyte responses to human papillomavirus type 16 E5 and E7 proteins and HLA-A*0201-restricted T-cell peptides in cervical cancer patients

Previously, we found that human papillomavirus type 16 (HPV-16) E5 protein is a tumor rejection antigen and can induce cytotoxic T-lymphocyte (CTL) activity. Therefore, in this study, human leukocyte antigen A*0201 (HLA-A*0201)-restricted human CTL epitopes of HPV-16 E5 protein were identified using a bioinformatics approach, and the abilities of these predicted peptides to induce an immune response in HLA-A*0201 transgenic mice were confirmed by assaying E5-specific CTLs and in vitro-generated CTLs from normal peripheral blood T lymphocytes of HLA-A2-positive human donors. Second, the CTL responses to HLA-A*0201 CTL epitopes (E5 63-71 and E7 11-20) were examined in HPV-16-infected patients with HLA-A2. Third, the effect of HLA-A-type alleles on CTL activities in response to the entire E5 and E7 proteins was examined in cervical cancer patients. E5 and E7 peptides (but not the whole proteins) stimulated E5- and E7-specific CTL recall responses in HPV-16- and HLA-A2-positive cervical cancer patients, and HPV-16 E5 and E7 proteins stimulated na?ve T cells in HPV-16-negative cervical cancer patients with HLA-A11 and -A24 haplotypes. In summary, this is the first demonstration that E5 63-71 is an HLA-A*0201-restricted T-cell epitope of HPV-16 E5.     

Immunological ignorance of an E7-encoded cytolytic T-lymphocyte epitope in transgenic mice expressing the E7 and E6 oncogenes of human papillomavirus type 16.

Certain human papillomaviruses (HPV) have been implicated in the etiology of cervical malignancies, and the E7 and E6 gene products of HPV type 16 are frequently expressed in these lesions. However, cytolytic T-lymphocyte (CTL)-mediated responses to HPV are rarely detectable in patients with cervical cancer. To examine whether the T-cell response is deficient during the HPV-induced transformation, we produced lines of transgenic (Tg) mice that expressed the E6 and E7 oncogenes in keratinized epithelia. The mice developed severe hypertrophy of all keratinized epithelia, but no malignancies were observed. Although epithelial cells from Tg mice could present at least an E7-encoded CTL epitope (E7 49-57), CTLs from these mice were neither primed to nor made tolerant of this epitope. No quantitative or qualitative differences were seen in the CTL responses of the Tg mice compared to those of their littermates following immunization with the peptide E7 49-57. Immunization of Tg mice with the E7 49-57 peptide protected them against a subcutaneous challenge with tumor cells expressing a transfected E7 gene, yet the skin was unaffected, although the cultured skin epithelial cells from Tg mice expressed E7. Our results suggest that the Tg mice were immunologically ignorant of HPV oncoproteins with respect to a CTL response and that a similar type of ignorance may explain why HPV-associated cervical cancer cells can escape immunological destruction.     

MHC class I antigen processing of an adenovirus CTL epitope is linked to the levels of immunoproteasomes in infected cells

Proteasomes are the major source for the generation of peptides bound by MHC class I molecules. To study the functional relevance of the IFN-gamma-inducible proteasome subunits low molecular mass protein 2 (LMP2), LMP7, and mouse embryonal cell (MEC) ligand 1 in Ag processing and concomitantly that of immunoproteasomes, we established the tetracycline-regulated mouse cell line MEC217, allowing the titrable formation of immunoproteasomes. Infection of MEC217 cells with Adenovirus type 5 (Ad5) and analysis of Ag presentation with Ad5-specific CTL showed that cells containing immunoproteasomes processed the viral early 1B protein (E1B)-derived epitope E1B192-200 with increased efficiency, thus allowing a faster detection of viral entry in induced cells. Importantly, optimal CTL activation was already achieved at submaximal immunosubunit expression. In contrast, digestion of E1B-polypeptide with purified proteasomes in vitro yielded E1B192-200 at quantities that were proportional to the relative contents of immunosubunits. Our data provide evidence that the IFN-gamma-inducible proteasome subunits, when present at relatively low levels as at initial stages of infection, already increase the efficiency of antigenic peptide generation and thereby enhance MHC class I Ag processing in infected cells.     

Use of Major Histocompatibility Complex Class I/Peptide/β2M Tetramers To Quantitate CD8+ Cytotoxic T Lymphocytes Specific for Dominant and Nondominant Viral Epitopes in Simian-Human Immunodeficiency Virus-Infected Rhesus Monkeys

To evaluate the impact of the diversity of antigen recognition by T lymphocytes on disease pathogenesis, we must be able to identify and analyze simultaneously cytotoxic T-lymphocyte (CTL) responses specific for multiple viral epitopes. Many of the studies of the role of CD8(+) CTLs in AIDS pathogenesis have been done with simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys. These studies have frequently made use of the well-defined SIV Gag CTL epitope p11C,C-M presented to CTL by the HLA-A homologue molecule Mamu-A*01. In the present study we identified and fine mapped two novel Mamu-A*01-restricted CTL epitopes: the SIVmac Pol-derived epitope p68A (STPPLVRLV) and the human immunodeficiency virus type 1 (HIV-1) Env-derived p41A epitope (YAPPISGQI). The frequency of CD8(+) CTLs specific for the p11C,C-M, p68A, and p41A epitopes was quantitated in the same animals with a panel of tetrameric Mamu-A*01/peptide/beta2m complexes. All SHIV-infected Mamu-A*01(+) rhesus monkeys tested had a high frequency of SIVmac Gag-specific CTLs to the p11C,C-M epitope. In contrast, only a fraction of the monkeys tested had detectable CTLs specific for the SIVmac Pol p68A and HIV-1 Env p41A epitopes, and these responses were detected at very low frequencies. Thus, the p11C,C-M-specific CD8(+) CTL response is dominant and the p41A- and p68A-specific CD8(+) CTL responses are nondominant. These results indicate that CD8(+) CTL responses to dominant CTL epitopes can be readily quantitated with the tetramer technology; however, CD8(+) CTL responses to nondominant epitopes, due to the low frequency of these epitope-specific cells, may be difficult to detect and quantitate by this approach.     

Human papillomavirus type 16 E7 peptide-directed CD8+ T cells from patients with cervical cancer are cross-reactive with the coronavirus NS2 protein

Human papillomavirus type 16 (HPV16) E6 and E7 oncoproteins are required for cellular transformation and represent candidate targets for HPV-specific and major histocompatibility complex class I-restricted CD8(+)-T-cell responses in patients with cervical cancer. Recent evidence suggests that cross-reactivity represents the inherent nature of the T-cell repertoire. We identified HLA-A2 binding HPV16 E7 variant peptides from human, bacterial, or viral origin which are able to drive CD8(+)-T-cell responses directed against wild-type HPV16 E7 amino acid 11 to 19/20 (E7(11-19/20)) epitope YMLDLQPET(T) in vitro. CD8(+) T cells reacting to the HLA-A2-presented peptide from HPV16 E7(11-19(20)) recognized also the HLA-A2 binding peptide TMLDIQPED (amino acids 52 to 60) from the human coronavirus OC43 NS2 gene product. Establishment of coronavirus NS2-specific, HLA-A2-restricted CD8(+)-T-cell clones and ex vivo analysis of HPV16 E7 specific T cells obtained by HLA-A2 tetramer-guided sorting from PBL or tumor-infiltrating lymphocytes obtained from patients with cervical cancer showed that cross-reactivity with HPV16 E7(11-19(20)) and coronavirus NS2(52-60) represents a common feature of this antiviral immune response defined by cytokine production. Zero of 10 patients with carcinoma in situ neoplasia and 3 of 18 patients with cervical cancer showed > or =0.1% HPV16 E7-reactive T cells in CD8(+) peripheral blood lymphocytes. In vivo priming with HPV16 was confirmed in patients with cervical cancer or preinvasive HPV16-positive lesions using HLA-A2 tetramer complexes loaded with the E6-derived epitope KLPQLCTEL. In contrast, we could not detect E6-reactive T cells in healthy individuals. These data imply that the measurement of the HPV16 E7(11-19(20)) CD8(+)-T-cell response may reflect cross-reactivity with a common pathogen and that variant peptides may be employed to drive an effective cellular immune response against HPV.     

Induction of anti-gp160 cytotoxic T cells cross-reacting with various V3 loop P18 peptides in human immunodeficiency virus type 1 envelope-immunized individuals.

Cytotoxic T lymphocytes (CTL) may be important to prevent cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1), the agent responsible for AIDS. In this study, we investigated the epitope specificity of CTLs induced in individuals immunized against the virus envelope glycoprotein gp160. The determinant of HIV-1 gp160 for the stimulation of CTL is located in a region of high sequence variability among HIV-1 isolates, the so-called V3 loop P18. Using a panel of P18 peptides, we compared the CTL specificities of cells from two individuals immunized with vaccinia virus recombinants expressing the envelope glycoproteins from two different strains of HIV-1, IIIB and SIMI. For this purpose, CTLs specific for the IIIB P18 peptide (RIQRGPGRAFVTIGK) were compared with CTLs for the site from the SIMI isolate (TLHMGPKRAFYATGD). The results indicate that in contrast to CD8+ CTLs induced by the glycoprotein from strain IIIB, CD8+ CTLs induced by strain SIMI strongly cross-reacted with targets presenting P18 peptides as well as envelope proteins from the divergent MN and RF isolates but failed to cross-react with targets that presented the IIIB peptide. These data have implications for the design of an HIV vaccine.     

Oligomerization properties of the viral oncoproteins adenovirus E1A and human papillomavirus E7 and their complexes with the retinoblastoma protein

Human papillomavirus 16 E7 (HPV16 E7) and adenovirus 5 E1A (Ad5 E1A) are encoded by highly divergent viruses yet are functionally similar in their ability to bind the retinoblastoma (pRB) tumor suppressor protein, causing the aberrant displacement of E2F trancription factors. The amino acid residues of HPV16 E7 that are necessary for stability, for inhibition of pRB function, and for cell transformation are also necessary for E7 oligomerization. However, neither the specific oligomerization state of HPV16 E7 nor of Ad5 E1A as a function of pRB-binding has been characterized. To gain insight into HPV16 E7 and Ad5 E1A oligomerization properties, sedimentation equilibrium experiments were performed with recombinant HPV16 E7 and Ad5 E1A proteins. These studies reveal that, despite the overall functional similarities between these proteins, monomers, dimers, and tetramers of HPV16 E7 were detected while only reversible monomer-dimer association was identified for Ad5 E1A. The apparent K(d(monomer)-(dimer)) of HPV16 E7 is approximately 100-fold lower than that of a comparable region of Ad5 E1A, and it is concluded that under physiological protein concentrations HPV16 E7 exists primarily as a dimer. Sedimentation equilibrium experiments of pRB/Ad5 E1A and of pRB/HPV16 E7 complexes demonstrate that the tight association of pRB with the viral oncoproteins does not disturb their inherent oligomerization properties. Taken together, this study demonstrates significant differences between the Ad5 E1A and HPV16 E7 oligomerization states that are potentially related to their distinct structures and specific mechanisms of pRB-inactivation.     

Identification of a novel HLA-A2-restricted cytotoxic T lymphocyte epitope from cancer-testis antigen PLAC1 in breast cancer

Identification of cytotoxic T lymphocyte (CTL) epitopes from tumor antigens is essential for the development of peptide vaccines against tumor immunotherapy. Among all the tumor antigens, the caner-testis (CT) antigens are the most widely studied and promising targets. PLAC1 (placenta-specific 1, CT92) was considered as a novel member of caner-testis antigen, which expressed in a wide range of human malignancies, most frequently in breast cancer. In this study, three native peptides and their analogues derived from PLAC1 were predicted by T cell epitope prediction programs including SYFPEITHI, BIMAS and NetCTL 1.2. Binding affinity and stability assays in T2 cells showed that two native peptides, p28 and p31, and their analogues (p28-1Y9?V, p31-1Y2L) had more potent binding activity towards HLA-A*0201 molecule. In ELISPOT assay, the CTLs induced by these four peptides could release IFN-γ. The CTLs induced by these four peptides from the peripheral blood mononuclear cells (PBMCs) of HLA-A*02⁺ healthy donor could lyse MCF-7 breast cancer cells (HLA-A*0201⁺, PLAC1⁺) in vitro. When immunized in HLA-A2.1/K^b transgenic mice, the peptide p28 could induce the most potent peptide-specific CTLs among these peptides. Therefore, our results indicated that the peptide p28 (VLCSIDWFM) could serve as a novel candidate epitope for the development of peptide vaccines against PLAC1-positive breast cancer.     

Antigen processing defects in cervical carcinomas limit the presentation of a CTL epitope from human papillomavirus 16 E6.

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with the development of cervical cancer. The E6 and E7 proteins of HPV are constitutively expressed in cervical carcinoma cells making them attractive targets for CTL-based immunotherapy. However, few studies have addressed whether cervical carcinomas can process and present HPV E6/E7-derived Ags for recognition by CTL. We generated HLA-A*0201-restricted CTL clones against HPV16 E6(29-38) that recognized HPV16 E6 Ags transfected into B lymphoblastoid cells. These CTL were unable to recognize HLA-A*0201(+) HPV16 E6(+) cervical carcinoma cell lines even when the level of endogenous HPV16 E6 in these cells was increased by transfection. This defect in presentation of HPV16 E6(29-38) correlated with low level expression of HLA class I, proteasome subunits low molecular mass protein 2 and 7, and the transporter proteins TAP1 and TAP2 in the cervical carcinoma cell lines. The expression of all of these proteins could be up-regulated by IFN-gamma, but this was insufficient for CTL recognition unless the level of HPV16 E6 Ag was also increased by transfection. CTL recognition of the HPV16 E6(29-38) epitope in 721.174 B cells was dependent on TAP expression but independent of immunoproteasome expression. Collectively, these findings suggest that presentation of the HPV16 E6(29-38) epitope in cervical carcinoma cell lines is limited both by the level of TAP expression and by the low level or availability of the source HPV E6 oncoprotein. These observations place constraints on the use of this, and potentially other, HPV-derived CTL epitopes for the immunotherapy of cervical cancer.     

Crystal structure of swine major histocompatibility complex class I SLA-1 0401 and identification of 2009 pandemic swine-origin influenza A H1N1 virus cytotoxic T lymphocyte epitope peptides

The presentation of viral epitopes to cytotoxic T lymphocytes (CTLs) by swine leukocyte antigen class I (SLA I) is crucial for swine immunity. To illustrate the structural basis of swine CTL epitope presentation, the first SLA crystal structures, SLA-1 0401, complexed with peptides derived from either 2009 pandemic H1N1 (pH1N1) swine-origin influenza A virus (S-OIV(NW9); NSDTVGWSW) or Ebola virus (Ebola(AY9); ATAAATEAY) were determined in this study. The overall peptide-SLA-1 0401 structures resemble, as expected, the general conformations of other structure-solved peptide major histocompatibility complexes (pMHC). The major distinction of SLA-1 0401 is that Arg(156) has a "one-ballot veto" function in peptide binding, due to its flexible side chain. S-OIV(NW9) and Ebola(AY9) bind SLA-1 0401 with similar conformations but employ different water molecules to stabilize their binding. The side chain of P7 residues in both peptides is exposed, indicating that the epitopes are "featured" peptides presented by this SLA. Further analyses showed that SLA-1 0401 and human leukocyte antigen (HLA) class I HLA-A 0101 can present the same peptides, but in different conformations, demonstrating cross-species epitope presentation. CTL epitope peptides derived from 2009 pandemic S-OIV were screened and evaluated by the in vitro refolding method. Three peptides were identified as potential cross-species influenza virus (IV) CTL epitopes. The binding motif of SLA-1 0401 was proposed, and thermostabilities of key peptide-SLA-1 0401 complexes were analyzed by circular dichroism spectra. Our results not only provide the structural basis of peptide presentation by SLA I but also identify some IV CTL epitope peptides. These results will benefit both vaccine development and swine organ-based xenotransplantation.     

Longevity of antigen presentation and activation status of APC are decisive factors in the balance between CTL immunity versus tolerance

Encounter of Ag by naive T cells can lead to T cell priming as well as tolerance. The balance between immunity and tolerance is controlled by the conditions of Ag encounter and the activation status of the APC. We have investigated the rules that govern this balance in case an environment that normally induces tolerance is reverted into a milieu that promotes T cell priming, using a minimal CTL epitope derived from human adenovirus type 5 E1A. Vaccination of mice s.c. with E1A peptide in IFA readily induces CTL tolerance, resulting in the inability to control E1A-expressing tumors. The present study shows that efficient CTL priming is achieved when this peptide vaccine is combined with systemic administration of APC-activating compounds like agonistic anti-CD40 mAb or polyriboinosinate-polyribocytidylate. Surprisingly, this CTL response is not long-lasting and therefore fails to protect against tumor outgrowth. Disappearance of CTL reactivity was strongly associated with systemic persistence of the peptide for >200 days. In contrast, peptide administered in PBS does not persist and generates long term CTL immunity capable of rejecting Ad5E1A-positive tumors, when combined with CD40 triggering. Thus, presentation of CTL epitopes in an appropriate costimulatory setting by activated APC, although being essential and sufficient for CTL priming, eventually results in tolerance when the Ag persists systemically for prolonged times. These observations are important for the development of immune intervention schemes in autoimmunity and cancer.     

Immunization of mice with vaccinia virus-M2 recombinant induces epitope-specific and cross-reactive Kd-restricted CD8+ cytotoxic T cells.

The M2 protein of respiratory syncytial virus (RSV) is a protective antigen in H-2d, but not H-2b or H-2k mice. None of the other RSV proteins, excluding the surface glycoproteins that induce neutralizing antibodies, is protective in mice bearing these haplotypes. Thus, the M2 protein stands alone as a nonglycoprotein-protective antigen of RSV. The M2 protein is a target for murine Kd-restricted cytotoxic T lymphocytes (CTLs), and the resistance induced by infection with a vaccinia virus-RSV M2 (vac-M2) recombinant is mediated by CD8+ CTLs. Since the nonameric consensus sequence for H-2 Kd-restricted T-cell epitopes and the amino acid sequence of the M2 protein of subgroup A and B strains of RSV are known, the present study sought to identify the specific epitope(s) on the M2 protein recognized by CD8+ CTLs. This was done by examining the ability of four predicted Kd-specific motif peptides present in the M2 amino acid sequence of an RSV subgroup A strain to sensitize target cells for lysis by pulmonary or splenic CTLs obtained from mice infected with RSV or vac-M2. The following observations were made. First, two of the four peptides sensitized target cells for lysis by pulmonary or splenic CTLs induced by infection with either vac-M2 or RSV. Second, one of the two peptides, namely the 82-90 (M2) peptide, sensitized targets at a very low peptide concentration (10(-10) to 10(-12) M). Third, cold-target competition experiments revealed that the predominant CTL population induced by infection with vac-M2 or RSV recognized the 82-90 (M2) peptide, and this CTL population appeared to recognize the 71-79 (M2) peptide in a cross-reactive manner. Fourth, CTL recognition of targets sensitized with either the 71-79 (M2) or the 82-90 (M2) peptide was Kd restricted. Fifth, CTLs induced by infection with RSV subgroup A or B strains recognized the two M2 peptides. The findings suggest that the M2 protein of RSV contains an immunodominant Kd-restricted CTL epitope consisting of amino acid residues 82 to 90 (SYIGSINNI), which are shared by subgroup A and B RSVs.     

Established human papillomavirus type 16-expressing tumors are effectively eradicated following vaccination with long peptides

Peptide-based vaccines aimed at the induction of effective T cell responses against established cancers have so far only met with limited clinical success and clearly need to be improved. In a preclinical model of human papillomavirus (HPV)16-induced cervical cancer we show that prime-boost vaccinations with the HPV16-derived 35 amino-acid long peptide E7(43-77), containing both a CTL epitope and a Th epitope, resulted in the induction of far more robust E7-specific CD8(+) T cell responses than vaccinations with the minimal CTL epitope only. We demonstrate that two distinct mechanisms are responsible for this effect. First, vaccinations with the long peptide lead to the generation of E7-specific CD4(+) Th cells. The level of the induced E7-specific CD8(+) T cell response proved to be dependent on the interactions of these Th cells with professional APC. Second, we demonstrate that vaccination with the long peptide and dendritic cell-activating agents resulted in a superior induction of E7-specific CD8(+) T cells, even when T cell help was excluded. This suggests that, due to its size, the long peptide was preferably endocytosed, processed, and presented by professional APCs. Moreover, the efficacy of this superior HPV-specific T cell induction was demonstrated in therapeutic prime-boost vaccinations in which the long peptide admixed with the dendritic cell-activating adjuvant oligodeoxynucleotide-CpG resulted in the eradication of large, established HPV16-expressing tumors. Because the vaccine types used in this study are easy to prepare under good manufacturing practice conditions and are safe to administer to humans, these data provide important information for future clinical trials.     

Potent Antitumor Activity Generated by a Novel Tumor Specific Cytotoxic T Cell

Hepatocellular carcinoma is one of the most common malignant neoplasms in the world and is the main cause of death in patients with liver cirrhosis. Surgical intervention is not suitable for majority of hepatocellular carcinoma. Investigation of the effective targeting to the tumor cells is essential for both primary tumors and metastases. Tumor specific cytotoxic T lymphocytes (CTL) have been considered to be the attractive vehicles for delivering therapeutic agents toward various tumor diseases. This study was to explore the distribution pattern of CTL carrying the lentiviral vectors with the characteristic of adenoviral E1 gene under the control of the cell activation-dependent CD40 ligand promoter (Lenti/hCD40L/E1AB). Following transduction with adenoviral vectors containing chimeric type 5 and type 35 fiber proteins (Ad5/35-TRAIL), these CTLs produced infectious virus when exposed to HepG2 cells. We assessed the therapeutic ability of CTLs using MTT, Western blot and colony formation assay. The novel CTL harboring Lenti/hCD40L/E1AB and Ad5/35-TRAIL caused proliferation inhibition and significant apoptosis in hepatocellular carcinoma cell lines. Thus, the novel CTL may be useful for the development of gene therapy approaches to hepatocellular carcinoma.     

Selection of Virus Variants and Emergence of Virus Escape Mutants after Immunization with an Epitope Vaccine

In this report, we assessed the evolution of the cytotoxic T-lymphocyte (CTL) response induced by an epitope vaccine. In two macaques immunized with a mixture of lipopeptides derived from simian immunodeficiency virus (SIV) Nef and Gag proteins, CTL responses were directed against the same, single epitope of the Nef protein (amino acids 128 to 137) presenting an alanine at position 136 (Nef 128-137/136A). However, after 5 months of SIV infection, peripheral blood mononuclear cells from both macaques lost their ability to be stimulated by autologous SIV-infected cells while still retaining their capacity to generate cytotoxic responses after specific Nef 128-137/136A peptide stimulation. The sequences of the pathogenic viral isolate used for the challenge showed a mixture of several variants. Within the Nef epitopic sequence from amino acids 128 to 137, 82% of viral variants expressed the epitopic peptide Nef 128-137/136A; the remaining variants presented a threonine at position 136 (Nef 128-137/136T). In contrast, sequence analysis of cloned proviral DNA obtained from both macaques 5 months after SIV challenge showed a different pattern of quasi-species variants; 100% of clones presented a threonine at position 136 (Nef 128-137/136T), suggesting the disappearance of viral variants with an alanine at this position under antiviral pressure exerted by Nef 128-137/136A-specific CTLs. In addition, 12 months after SIV challenge, six of eight clones from one macaque presented a glutamic acid at position 131 (Nef 128-137/131E+136T), which was not found in the infecting isolate. Furthermore, CTLs generated very early after SIV challenge were able to lyse cells sensitized with the Nef 128-137/136A epitope. In contrast, lysis was significantly less effective or even did not occur when either the selected peptide Nef 128-137/136T or the escape variant peptide Nef 128-137/131E+136T was used in a target cell sensitization assay. Dose analysis of peptides used to sensitize target cells as well as a major histocompatibility complex (MHC)-peptide stability assay suggested that the selected peptide Nef 128-137/136T has an altered capacity to bind to the MHC. These data suggest that CTL pressure leads to the selection of viral variants and to the emergence of escape mutants and supports the fact that immunization should elicit broad CTL responses.     

DNA fusion vaccine designed to induce cytotoxic T cell responses against defined peptide motifs: implications for cancer vaccines

DNA vaccination offers a strategy to induce immune attack on cancer cells, but tumor Ags are often weak. Inclusion of a "foreign" protein increases immunogenicity, and we found previously that fusion of the fragment C (FrC) of tetanus toxin to the tumor Ag sequence promotes Ab and CD4(+) responses against B cell tumors. For CTL responses, use of the full two-domain FrC may be less helpful, because known immunogenic MHC class I-binding peptides in the second domain could compete with attached tumor-derived epitopes. Therefore, we removed the second domain, retaining the N-terminal domain, which contains a "universal" helper epitope. We investigated the ability to induce CTL responses of candidate peptides placed at the C terminus of this domain. As test peptides, we repositioned the two known CTL motifs from the second domain to this site. Strong CTL responses to each peptide were induced by the engineered construct, as compared with the native FrC construct. Induced CTLs were able to specifically kill tumor cells transfected with FrC as a surrogate tumor Ag both in vitro and in vivo. Further reduction of the domain to a short helper epitope generated only weak CTL responses against fused peptides, and synthetic peptides mixed with the plasmid containing the first domain were ineffective. The single FrC domain-peptide vaccine design also was able to induce high levels of CTLs against a known epitope from carcinoembryonic Ag. Response to peptide was suppressed if two FrC domains were present, consistent with immunodominance. These principles and designs may have relevance for cancer vaccines delivered via DNA.     

HLA A2.1-restricted cytotoxic T cells recognizing a range of Epstein-Barr virus isolates through a defined epitope in latent membrane protein LMP2.

Cytotoxic T-lymphocyte (CTL) responses induced by persistent Epstein-Barr virus (EBV) infection in normal B-lymphoid tissues could potentially be directed against EBV-positive malignancies if expression of the relevant viral target proteins is maintained in tumor cells. For malignancies such as nasopharyngeal carcinoma and Hodgkin's disease, this will require CTL targeting against the nuclear antigen EBNA1 or the latent membrane proteins LMP1 and LMP2. Here we analyze in detail a B95.8 EBV-reactivated CTL response which is specific for LMP2 and restricted through a common HLA allele, A2.1. We found that in vitro-reactivated CTL preparations from several A2.1-positive virus-immune donors contained detectable reactivity against A2.1-bearing target cells expressing either LMP2A or the smaller LMP2B protein from recombinant vaccinia virus vectors. Peptide sensitization experiments then mapped the A2.1-restricted response to a single epitope, the nonamer CLGGLLTMV (LMP2A residues 426 to 434), whose sequence accords well with the proposed peptide binding motif for A2.1. Most Caucasian and African virus isolates (whether of type 1 or type 2) were identical in sequence to B95.8 across this LMP2 epitope region, although 2 of 12 such isolates encoded a Leu-->Ile change at epitope position 6. In contrast, most Southeast Asian and New Guinean isolates (whether of type 1 or type 2) constituted a different virus group with a Cys-->Ser mutation at epitope position 1. CTLs raised against the B95.8-encoded epitope were nevertheless able to recognize these variant epitope sequences in the context of A2.1 whether they were provided exogenously as synthetic peptides or generated endogenously in B cells transformed with the variant viruses. A CTL response of this kind could have therapeutic potential in that it is directed against a protein expressed in many EBV-positive malignancies, is reactive across a range of virus isolates, and is restricted through a relatively common HLA allele.