甲型流感病毒核壳蛋白在BALB／c小鼠中酶联免疫斑点法表位的筛选及其与CTL表位的相关性研究

目的：利用甲型流感病毒A／Johannesburg／33／94（H3N2）核壳蛋白（NP）全长肽库筛选BAI卫／c（H-2^d）小鼠中NP酶联免疫斑点法（EUSPOT）表位,研究其和细胞毒性T淋巴细胞（CTL）表位的一致性关系,为使用ELlSPOT评价流感病毒NP疫苗的细胞免疫效果提供实验依据。方法：以甲型流感病毒A／PR／8／34（H1N1）（PR8）感染BALB／c（H-2^d）小鼠后,通过检测T细胞分泌γ-干扰素（IFN-1）的ELISPOT法和体内CTL法检测NP所诱发的细胞免疫反应,综合分析ELlSPOT和CTL表位肽之间的关系。结果：Pep36（NP第141-155位氨基酸残基,SNLNDTTYQRTRALV）和Pep37（NP第145-159位氨基酸残基,DTTYQRTRALVRTGM）可以诱发较强的ELlSPOT反应,根据Pep36和Pep37共有序列合成的Pep147-155（NP第147-155位氨基酸残基,TYQRTRALV）可以诱发与这2条多肽相同强度的ELlSPOT反应,表明Pep147-155为NP诱发ELlSPOT反应的最强表位,体内CTL也表明它是最强的CTL表位;Pep95（NP第377-391位氨基酸残基,STLELRSRYWAIRTR）、Pep96（NP第381-395位氨基酸残基,LRSRYWAIRTRSGGN）和其他表位肽诱发的ELISPOT反应较弱,体内CTL反应也较弱。结论：BALB／c（H-2^d）小鼠中,甲型流感病毒NP诱发ELlSPOT反应和CTL反应的表位肽高度相关;实验结果为使用ELlSPOT评价流感病毒NP疫苗的细胞免疫效果提供了实验依据。     

Vaccinia virus-specific human CD4+ cytotoxic T-lymphocyte clones.

Vaccinia virus-specific cytotoxic T-lymphocyte (CTL) clones were established from a healthy donor, who had been immunized with vaccinia virus vaccine, by stimulation of peripheral blood lymphocytes with UV-inactivated vaccinia virus antigen. The phenotype of all of the clones established was CD3+ CD4+ CD8- Leu11-. We used a panel of allogenic vaccinia virus-infected B-lymphoblastoid cell lines and demonstrated that some of the clones recognized vaccinia virus epitopes presented by human leukocyte antigen (HLA) class II molecules. Monoclonal antibodies specific for either HLA-DP or HLA-DR determinant reduced the cytotoxicity of specific clones. The HLA-restricted cytotoxicity of the clones is vaccinia virus specific, because vaccinia virus-infected but not influenza virus-infected autologous target cells were lysed. Using vaccinia virus deletion mutants, we found that some of the CTL clones recognize an epitope(s) that lies within the HindIII KF regions of the vaccinia virus genome. These results indicate that heterogeneous CD4+ CTL clones specific for vaccinia virus are induced in response to infection and may be important in recovery from and protection against poxvirus infections.     

Class I MHC-restricted recognition of cells expressing a gene encoding a 41 amino acid product of the influenza hemagglutinin

Class I H-2Kd-restricted influenza hemagglutinin (HA)-specific CTL recognize two immuno-dominant sites as represented by synthetic peptides spanning epitopes located in the HA1 and hydrophobic transmembrane domains of the influenza HA. Using a vaccinia virus recombinant expression system, we have examined CTL recognition of HA deletion mutants expressed in target cells. We have demonstrated that a truncated influenza HA gene encoding only the transmembrane anchor region containing a class I recognition site and a short segment of the cytoplasmic tail of the HA can be efficiently presented to class I CTL. These results set the stage for detailed analyses of the intracellular events associated with Ag presentation to class I CTL and offer novel possibilities for future vaccine design.     

Efficient processing of the immunodominant, HLA-A*0201-restricted human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitope despite multiple variations in the epitope flanking sequences

Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences.     

The role of the endoplasmic reticulum in antigen processing. N-glycosylation of influenza hemagglutinin abrogates CD4+ cytotoxic T cell recognition of endogenously processed antigen.

Evidence is presented for an endogenous route of Ag processing for CD4+ T cell recognition of influenza hemagglutinin that requires obligatory traffic of de novo synthesized hemagglutinin across the lumen of the endoplasmic reticulum for processing in a cytosolic compartment. I-Ad-restricted T cell clones that recognize synthetic peptides corresponding to two distinct antigenic regions of the HA1 subunit, HA1 56-76 and HA1 177-199, are cytotoxic and, dependent on epitope specificity can recognize endogenously processed Ag and lyse class II+ target cells infected with a recombinant vaccinia-X31 HA virus. HA1 56-76 specific T cell clones fail to recognize (target cells infected with) influenza X31 viruses, containing a single residue change, HA1 63 Asp----Asn that introduces an oligosaccharide attachment site: Asp63Cys64Thr65. Recognition is restored, however, by tunicamycin treatment of mutant virus infected target cells. Inasmuch as N-glycosylation of nascent hemagglutinin polypeptides occurs in the lumen of the endoplasmic reticulum, this indicates a route of endogenous processing for hemagglutinin, requiring transport across the endoplasmic reticulum, which has been confirmed by the failure of CD4+ T cells to recognize a recombinant VACC-hemagglutinin virus in which the same single residue change, HA1 63 Asp----Asn has been introduced by site directed mutagenesis.     

Induction of cytotoxic T cells to a cross-reactive epitope in the hepatitis C virus nonstructural RNA polymerase-like protein.

Cytotoxic T lymphocytes (CTL) have been found to mediate protection in vivo against certain virus infections. CTL also may play an important role in control of infection by hepatitis C virus (HCV), but no CTL epitopes have yet been defined in any HCV protein. The nonstructural protein with homology to RNA polymerase should be a relatively conserved target protein for CTL. To investigate the epitope specificity of CTL specific for this protein, we used 28 peptides from this sequence to study murine CTL. Mice were immunized with a recombinant vaccinia virus expressing the HCV nonstructural region corresponding to the flavivirus NS5 gene (RNA polymerase), and the primed spleen cells were restimulated in vitro with peptides. CTL from H-2d mice responded to a single 16-residue synthetic peptide (HCV 2422 to 2437). This relatively conserved epitope was presented by H-2d class I major histocompatibility complex (MHC) molecules to conventional CD4- CD8+ CTL but was not recognized by CTL restricted by H-2b. Moreover, exon shuffle experiments using several transfectants expressing recombinant Dd/Ld and Kd demonstrated that this peptide is seen in association with alpha 1 and alpha 2 domains of the Dd class I MHC molecule. This peptide differs from the homologous segments of this nonstructural region from three other HCV isolates by one residue each. Variant peptides with single amino acid substitutions were made to test the effect of each residue on the ability to sensitize targets. Neither substitution affected recognition. Therefore, these conservative mutations affected peptide interaction neither with the Dd class I MHC molecule nor with the T-cell receptor. Because these CTL cross-react with all four sequenced isolates of HCV in the United States and Japan, if human CTL display similar cross-reactivity, this peptide may be valuable for studies of HCV diagnosis and vaccine development. Our study provides the first evidence that CD8+ CTL can recognize an epitope from the HCV sequence in association with a class I MHC molecule.     

Analysis of Murine CD8+ T-Cell Clones Specific for the Dengue Virus NS3 Protein: Flavivirus Cross-Reactivity and Influence of Infecting Serotype

Serotype-cross-reactive dengue virus-specific cytotoxic T lymphocytes (CTL) induced during a primary dengue virus infection are thought to play a role in the immunopathogenesis of dengue hemorrhagic fever (DHF) during a secondary dengue virus infection. Although there is no animal model of DHF, we previously reported that murine dengue virus-specific CTL responses are qualitatively similar to human dengue virus-specific CTL responses. We used BALB/c mice to study the specificity of the CTL response to an immunodominant epitope on the dengue virus NS3 protein. We mapped the minimal H-2K^d-restricted CTL epitope to residues 298 to 306 of the dengue type 2 virus NS3 protein. In short-term T-cell lines and clones, the predominant CD8⁺ CTL to this epitope in mice immunized with dengue type 2 virus or vaccinia virus expressing the dengue type 4 virus NS3 protein were cross-reactive with dengue type 2 or type 4 virus, while broadly serotype-cross-reactive CTL were a minority population. In dengue type 3 virus-immunized mice, the predominant CTL response to this epitope was broadly serotype cross-reactive. All of the dengue virus-specific CTL clones studied also recognized the homologous NS3 sequences of one or more closely related flaviviruses, such as Kunjin virus. The critical contact residues for the CTL clones with different specificities were mapped with peptides having single amino acid substitutions. These data demonstrate that primary dengue virus infection induces a complex population of flavivirus-cross-reactive NS3-specific CTL clones in mice and suggest that CTL responses are influenced by the viral serotype. These findings suggest an additional mechanism by which the order of sequential flavivirus infections may influence disease manifestations.     

Identification of amino acids involved in recognition by dengue virus NS3-specific, HLA-DR15-restricted cytotoxic CD4+ T-cell clones.

The majority of T-cell clones derived from a donor who experienced dengue illness following receipt of a live experimental dengue virus type 3 (DEN3) vaccine cross-reacted with all four serotypes of dengue virus, but some were serotype specific or only partially cross-reactive. The nonstructural protein, NS3, was immuno-dominant in the CD4+ T-cell response of this donor. The epitopes of four NS3-specific T-cell clones were analyzed. JK15 and JK13 recognized only DEN3 NS3, while JK44 recognized DEN1, DEN2, and DEN3 NS3 and JK5 recognized DEN1, DEN3, and West Nile virus NS3. The epitopes recognized by these clones on the DEN3 NS3 protein were localized with recombinant vaccinia viruses expressing truncated regions of the NS3 gene, and then the minimal recognition sequence was mapped with synthetic peptides. Amino acids critical for T-cell recognition were assessed by using peptides with amino acid substitutions. One of the serotype-specific clones (JK13) and the subcomplex- and flavivirus-cross-reactive clone (JK5) recognized the same core epitope, WITDFVGKTVW. The amino acid at the sixth position of this epitope is critical for recognition by both clones. Sequence analysis of the T-cell receptors of these two clones showed that they utilize different VP chains. The core epitopes for the four HLA-DR15-restricted CD4+ CTL clones studied do not contain motifs similar to those proposed by previous studies on endogenous peptides eluted from HLA-DR15 molecules. However, the majority of these dengue virus NS3 core epitopes have a positive amino acid (K or R) at position 8 or 9. Our results indicate that a single epitope can induce T cells with different virus specificities despite the restriction of these T cells by the same HLA-DR15 allele. This finding suggests a previously unappreciated level of complexity for interactions between human T-cell receptors and viral epitopes with very similar sequences on infected cells.     

Immediate-Early Transactivator Rta of Epstein-Barr Virus (EBV) Shows Multiple Epitopes Recognized by EBV-Specific Cytotoxic T Lymphocytes

We analyzed the immediate-early transactivator Rta of Epstein-Barr virus (EBV) for its role as a target for specific cytotoxic T lymphocytes (CTL). Panels of overlapping peptides covering the entire amino acid sequence of Rta were synthesized and used to induce and analyze specific CTL responses in EBV-positive donors. Using peptide-pulsed target cells, we found nine different CTL epitopes that are distributed over the entire protein sequence. One epitope restricted by HLA-A24 could be mapped to the decameric sequence DYCNVLNKEF between amino acid positions 28 and 37 of the Rta protein. A second epitope could be assigned to the same region of Rta (residues 25 to 39) and was shown to be restricted by HLA-B18. Another, minimal epitope could be mapped to the nonameric sequence ATIGTAMYK between amino acid positions 134 and 142; this peptide was restricted by HLA-A11. Another four epitopes were proven to be restricted by HLA-A2, -A3, -B61, and -Cw4 and were located between Rta residues 225 and 239, 145 and 159, 529 and 543, and 393 and 407, respectively. For two other epitopes, only the location within the Rta protein is known so far (residues 121 to 135 and 441 to 455); their exact HLA restriction patterns have not yet been identified. Using target cells infected with recombinant vaccinia virus containing the gene for Rta, we showed that six of eight Rta-specific CTL lines recognized the corresponding peptides also after endogenous processing. These data suggest that Rta comprises an important target for EBV-specific cellular cytotoxicity. Together with recent findings of other immediate-early and early proteins also acting as CTL targets, they reveal the role of proteins of the lytic cycle in the immune recognition of EBV-infected cells.     

Overlapping epitopes that are recognized by CD8+ HLA class I-restricted and CD4+ class II-restricted cytotoxic T lymphocytes are contained within an influenza nucleoprotein peptide.

Viral epitopes that are recognized by both HLA class I-restricted and class II-restricted T cells have been defined for a type A influenza virus nucleoprotein (NP) peptide. CD8+ and CD4+ CTL lines have been generated against a synthetic peptide encompassing residues 335 to 349 of NP that are restricted by HLA-B37 and HLA-DQw5, respectively. Both of these CTL populations were capable of specifically lysing influenza A virus-infected targets, indicating that a naturally processed NP peptide(s) was being mimicked by the NP (335-349) peptide. Amino acid residues that are critical for recognition of this NP determinant in the context of HLA-B37 and HLA-DQw5 were investigated by the use of panels of truncated and alanine-substituted NP peptides. The results demonstrate that: 1) truncations in the amino- or carboxy-terminal ends differentially affect CD8+ and CD4+ CTL recognition; 2) the NP (335-349) sequence contains two octapeptide epitopes that share a core of six amino acid residues (NP 338-343); and 3) alanine substitutions at five of these residues abrogated recognition by at least one of the CD8+ and CD4+ CTL lines. Thus, these class I- and class II-restricted CTL lines recognize similar but distinct epitopes, and different structural features of the NP peptide are required for presentation by HLA-B37 and HLA-DQw5. Comparison of the amino acid sequences of the NP peptide presented by HLA-B37 and HLA-DQw5 with other peptides known to be presented by both class I and class II molecules revealed a common motif among these peptides.     

Molecular definition of a major cytotoxic T-lymphocyte epitope in the glycoprotein of lymphocytic choriomeningitis virus.

Analyses with segmental reassortants of lymphocytic choriomeningitis virus (LCMV) RNA have shown that cytotoxic T lymphocytes (CTL) are induced by and recognize proteins encoded by the viral short segment, which specifies two virus structural proteins, glycoprotein (GP) and nucleoprotein (NP). Expression of cDNA copies of these genes in vaccinia virus vectors demonstrates that C57BL/6 (H2bb) mice mount significant CTL responses to both GP and NP. We have used LCMV-specific H2bb-restricted CTL clones and a family of serial C-terminal truncations of the LCMV GP expressed in vaccinia virus to map the precise specificities of the anti-GP clones. Of the 18 CTL clones studied, 1 recognizes NP and the other 17 recognize GP. The reactivities of 14 of the 17 anti-GP CTL clones against the deleted GP molecules have been fully characterized, and two clear patterns of anti-GP activity have emerged, defining at least two CTL epitopes. The first epitope, recognized by only two of the clones, lies within GP residues 1 to 218. The second is recognized by all 12 of the remaining clones and was mapped, by using the GP deletions, to a 22-amino-acid region comprising GP residues 272 to 293. A synthetic peptide representing this area sensitized uninfected syngeneic target cells to lysis both by bulk CTL obtained from the spleen after a primary immunization and by appropriate CTL clones. Two sets of criteria are available which are said to identify potential T-cell epitopes, one based on primary amino acid sequence and the second based on protein secondary structure. Neither of these predictive schemes would have identified region 272 to 293 as a CTL recognition motif, indicating that such programs are of limited usefulness as presently conceived. Analysis of the CTL clones shows clearly that all three families (anti-NP and anti-GP 1 to 218 and 272 to 293) direct efficient cross-reactive killing against a variety of serologically distinct strains of LCMV.     

Identification of a gag-encoded cytotoxic T-lymphocyte epitope from FBL-3 leukemia shared by Friend, Moloney, and Rauscher murine leukemia virus-induced tumors.

FBL-3 is a highly immunogenic murine leukemia of C57BL/6 origin induced by Friend murine leukemia virus (MuLV). Immunization of C57BL/6 mice with FBL-3 readily elicits CD8+ cytotoxic T lymphocytes (CTL) capable of lysing FBL-3 as well as syngeneic leukemias induced by Moloney and Rauscher MuLV. The aim of this current study was to identify the immunogenic epitope(s) recognized by the FBL-3-specific CD8+ CTL. A series of FBL-3-specific CD8+ CTL clones were generated from C57BL/6 mice immunized to FBL-3. The majority of CTL clones (32 of 38) were specific for F-MuLV gag-encoded antigen. By using a series of recombinant vaccinia viruses expressing full-length and truncated F-MuLV gag genes, the antigenic epitope recognized by the FBL-3 gag-specific CTL clones, as well as by bulk-cultured CTL from spleens of mice immune to FBL-3, was localized to the leader sequence of gPr80gag protein. The precise amino acid sequence of the CTL epitope in the leader sequence was identified as CCLCLTVFL (positions 85-93) by examining lysis of targets incubated with a series of synthetic leader sequence peptides. No evidence of other CTL epitopes in the gPr80gag or Pr65gag core virion structural polyproteins was found. The identity of CCLCLTVFL as the target peptide was validated by showing that immunization with the peptide elicited CTL that lysed FBL-3. The CTL elicited by the Gag peptide also specifically lysed syngeneic leukemia cells induced by Moloney and Rauscher MuLV (MBL-2 and RBL-5). The transmembrane peptide was shown to be the major gag-encoded antigenic epitope recognized by bulk-cultured CTL derived from C57BL/6 mice immunized to MBL-2 or RBL-5. Thus, the CTL epitope of FBL-3 is localized to the transmembrane anchor domain of the nonstructural Gag polyprotein and is shared by leukemia/lymphoma cell lines induced by Friend, Moloney, and Rauscher MuLV.     

Overlapping cytotoxic T-lymphocyte and B-cell antigenic sites on the influenza virus H5 hemagglutinin.

To define the recognition site of cytotoxic T lymphocytes (CTLs) on influenza virus H5 hemagglutinin (HA), an H5 HA-specific CTL clone was examined for the ability to recognize monoclonal antibody-selected HA variants of influenza virus A/Turkey/Ontario/7732/66 (H5N9). On the basis of 51Cr release assays with the variants, a CTL epitope was located near residue 168 of H5 HA. To define the epitope more precisely, a series of overlapping peptides corresponding to this region was synthesized and tested for CTL recognition. The minimum peptide recognized by the CTL clone encompassed residues 158 to 169 of H5 HA. Relative to the H3 HA three-dimensional structure, this CTL epitope is located near the distal tip of the HA molecule, also known as a major B-cell epitope on H3 HA. A single mutation at residue 168 (Lys to Glu) in the H5 HA variants abolished CTL recognition; this same amino acid was shown previously to be critical for B-cell recognition (M. Philpott, C. Hioe, M. Sheerar, and V. S. Hinshaw, J. Virol. 64:2941-2947, 1990). Additionally, mutations within this region of the HA molecule were associated with attenuation of the highly virulent A/Turkey/Ontario/7732/66 (H5N9) (M. Philpott, B. C. Easterday, and V.S. Hinshaw, J. Virol. 63:3453-3458, 1989). When tested for recognition of other H5 viruses, the CTL clone recognized the HA of A/Turkey/Ireland/1378/83 (H5N8) but not that of A/Chicken/Pennsylvania/1370/83 (H5N2), even though these viruses contain identical HA amino acid 158-to-169 sequences. These results suggest that differences outside the CTL epitope affected CTL recognition of the intact HA molecule. The H5 HA site defined in these studies is, therefore, important in both CTL and B-cell recognition, as well as the pathogenesis of the virus.     

Crystal structures of murine MHC Class I H-2 D(b) and K(b) molecules in complex with CTL epitopes from influenza A virus: implications for TCR repertoire selection and immunodominance

Cytotoxic T lymphocyte (CTL) responses against influenza A virus in C57BL/6 mice are dominated by a small number of viral peptides among many that are capable of binding to major histocompatibility complex (MHC) class I molecules. The basis of this limited immune recognition is unknown. Here, we present X-ray structures of MHC class I molecules in complex with two immunodominant epitopes (PA(224-233)/D(b) and PB1(703-711)/K(b)) and one non-immunogenic epitope (HA(468-477)/D(b)) of the influenza A virus. The immunodominant peptides are each characterized by a bulge at the C terminus, lifting P6 and P7 residues out of the MHC groove, presenting featured structural elements to T-cell receptors (TCRs). Immune recognition of PA(224-233)/D(b) will focus largely on the exposed P7 arginine residue. In contrast, the non-immunogenic HA(468-477) peptide lacks prominent features in this C-terminal bulge. In the K(b)-bound PB1(703-711) epitope, the bulge results from a non-canonical binding motif, such that the mode of presentation of this peptide strongly resembles that of D(b)-bound peptides. Given that PA(224-233)/D(b), PB1(703-711)/K(b) and the previously defined NP(366-374)/D(b) epitopes dominate the primary response to influenza A virus in C57BL/6 mice, our findings indicate that residues of the C-terminal bulge are important in selection of the immunodominant CTL repertoire.     

Cytotoxic T-lymphocyte clones specific for an immunodominant epitope display discerning antagonistic response to naturally occurring Epstein-Barr virus variants.

In the present study we have identified Epstein-Barr virus isolates which encode variant sequences within an HLA B35-restricted immunodominant cytotoxic T-lymphocyte (CTL) epitope that act as natural antagonists and can inhibit CTL activity on the wild-type epitope. This effect can be demonstrated if the wild-type epitope is presented as a synthetic peptide or when processed from a full-length Epstein-Barr virus protein expressed by recombinant vaccinia constructs. However, this antagonistic effect was only selectively seen with some CTL clones, while a strong agonistic effect was evident for other clones in the presence of the same variant peptide. The data presented in this study strongly suggest that it is unlikely that the variant viruses can completely antagonize a virus-specific CTL response by this mechanism since the host immune response is capable of generating CTLs expressing a diverse array of T-cell receptors. Moreover, many of these CTLs can recognize the variant sequences as efficiently as wild-type epitope.     

Protection against lethal cytomegalovirus infection by a recombinant vaccine containing a single nonameric T-cell epitope.

The regulatory immediate-early (IE) protein pp89 of murine cytomegalovirus induces CD8+ T lymphocytes that protect against lethal murine cytomegalovirus infection. The IE1 epitope is the only epitope of pp89 that is recognized by BALB/c cytolytic T lymphocytes (CTL). Using synthetic peptides, the optimal and minimal antigenic sequences of the IE1 epitope have been defined. To evaluate the predictive value of data obtained with synthetic peptides, recombinant vaccines encoding this single T-cell epitope were constructed using as a vector the hepatitis B virus core antigen encoded in recombinant vaccinia virus. In infected cells expressing the chimeric proteins, only IE1 epitope sequences that were recognized as synthetic peptides at concentrations lower than 10(-6) M were presented to CTL. Vaccination of mice with the recombinant vaccinia virus that encoded a chimeric protein carrying the optimal 9-amino-acid IE1 epitope sequence elicited CD8+ T lymphocytes with antiviral activity and, furthermore, protected against lethal disease. The results thus show for the first time that recombinant vaccines containing a single foreign nonameric CTL epitope can induce T-lymphocyte-mediated protective immunity.     

HA2 subunit of influenza A H1 and H2 subtype viruses induces a protective cross-reactive cytotoxic T lymphocyte response

Influenza H1 subtype-specific CTL can be induced by secondary stimulation of a hybrid protein of the first 81 amino acids of the viral NS1 non-structural protein and the HA2 subunit of A/Puerto Rico/8/34(H1N1) hemagglutinin. In addition, a derivative of this protein with 65 amino acids deleted from the N-terminal end of HA2 can also generate H1 subtype-specific CTL in bulk cultures. CTL clones established by stimulation with the derivative protein demonstrated cross-reactive lysis of target cells infected with virus strains of the H1 and H2 subtypes. Cold target competition experiments with CTL clones as effectors demonstrated that the Ag specificity between these two hybrid proteins is identical. Adoptive transfer of the CTL clone significantly reduced virus titers in the lungs of mice infected with the virus strains of the H1 or H2 subtype but not those infected with the H3 subtype virus in vivo, which reflects the in vitro CTL clone activity. These experiments demonstrate that an epitope on the hemagglutinin that is conserved on virus strains of the H1 and H2 subtypes induces a protective CTL response. These results suggest an alternative approach for developing influenza vaccines by using conserved antigenic sites on the hemagglutinin HA2 subunit to avoid the problem of frequent antigenic mutations of the HA1 subunit antibody binding sites.     

Identification of five MAGE-A1 epitopes recognized by cytolytic T lymphocytes obtained by in vitro stimulation with dendritic cells transduced with MAGE-A1.

MAGE genes are expressed by many human tumors of different histological types but not by normal cells, except for male germline cells. The Ags encoded by MAGE genes and recognized by T cells are therefore strictly tumor-specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes recognized by CTL. Candidate peptides known to bind to a given HLA have been used to stimulate T lymphocytes in vitro. In some instances, CTL clones directed against these synthetic peptides have been obtained, but these clones often failed to recognize tumor cells expressing the relevant gene. Therefore, we designed a method to identify CTL epitopes that selects naturally processed peptides. Monocyte-derived dendritic cells infected with a recombinant canarypoxvirus (ALVAC) containing the entire MAGE-A1 gene were used to stimulate CD8+ T lymphocytes from the blood of individuals without cancer. Responder cell microcultures that specifically lysed autologous cells expressing MAGE-A1 were cloned using autologous stimulator cells either transduced with a retrovirus coding for MAGE-A1 or infected with recombinant Yersinia-MAGE-A1 bacteria. The CTL clones were tested for their ability to lyse autologous cells loaded with each of a set of overlapping MAGE-A1 peptides. This strategy led to the identification of five new MAGE-A1 epitopes recognized by CTL clones on HLA-A3, -A28, -B53, -Cw2, and -Cw3 molecules. All of these CTL clones recognized target cells expressing gene MAGE-A1.     

Poliovirus-specific major histocompatibility complex class I-restricted cytolytic T-cell epitopes in mice localize to neutralizing antigenic regions.

A major histocompatibility complex (MHC) class I-restricted cytotoxic T-lymphocyte (CTL) response is induced in BALB/c mice upon immunization with poliovirus serotype 1 (Mahoney strain). A similar class I-restricted response is also induced upon immunization with purified VP1 capsid proteins. Thus, poliovirus-specific MHC class I CTL responses can be induced independently of viral infection in murine hosts. In experiments using recombinant vaccinia virus vectors expressing different segments of the poliovirus capsid proteins and synthetic peptides, two regions of the VP1 capsid protein appear to contain epitopes recognized by this bulk CTL population. These epitope regions contain a Kd-restricted peptide-binding motif. Interestingly, each of these CTL epitopes is located near previously defined neutralizing antigenic sites.     

Recognition of noninfectious influenza virus by class I-restricted murine cytotoxic T lymphocytes

We have recently shown that murine target cells can be sensitized for lysis by class I-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) using noninfectious influenza virus. Sensitization is dependent on inactivation of viral neuraminidase activity (which can be achieved by heating virus); and requires fusion of viral and cellular membranes. In the present study, we have examined recognition of antigens derived from heat-treated virus by cloned CTL lines induced by immunization with infectious virus. Target cells sensitized with heat-treated virus were recognized by all 11 CTL clones that were specific for internal virion proteins (nucleoprotein and basic polymerase 1), and by one of six clones specific for the major viral glycoprotein (the hemagglutinin). Immunization of mice with heat-treated virus primed their splenocytes for secondary in vitro CTL responses. CTL generated in this manner recognized target cells infected with recombinant vaccinia virus expressing cloned influenza virus gene products. These findings indicate that both integral membrane proteins and internal proteins that comprise virions can be processed by antigen-presenting cells for recognition by class I-restricted CTL. It also appears that not all hemagglutinin determinants recognized on virus-infected cells are presented by cells sensitized with heat-treated virus.