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PubMed1997

Degenerate and promiscuous recognition by CTL of peptides presented by the MHC class I A3-like superfamily: implications for vaccine development.

Recent data demonstrate that HLA class I alleles can be grouped into superfamilies based on similarities of their peptide-binding motifs. In this study, we have tested the immunogenicity and antigenicity of peptides capable of degenerate binding to multiple HLA class I molecules of the A3-like superfamily. The assay systems utilized included both primary in vitro cultures of lymphocytes from healthy donors, as well as in vitro restimulation of lymphocytes from HIV-infected individuals. Several of the peptides capable of binding more than one HLA A3-like class I molecule were also found to be immunogenic in the context of this same group of A3-like molecules (degenerate CTL recognition). Furthermore, some of the CTL lines thus generated demonstrated promiscuous recognition of the cognate epitope in the context of MHC molecules from more than one member of the superfamily. The fine Ag specificity of this phenomenon was further analyzed using two promiscuous CTL clones derived from A3 and A11 individuals, respectively, and specific for an epitope in the HIV-1 reverse transcriptase. By the use of single-amino acid-substitution analogues, it was demonstrated that the fine specificity of the TCR is largely maintained between MHC-matched and MHC-mismatched presentation of peptide within the A3-like superfamily. These results indicate that the similar peptide-binding specificities among different members of the A3-like superfamily can be reflected in a remarkable similarity in the peptide-MHC complex structures engaged by the TCR and responsible for T cell activation.

AIDS Vaccines↗
PubMed1997

Spectratyping of TCRs expressed by CTL-infiltrating minor histocompatibility antigen-disparate allografts.

Minor histocompatibility Ags (HA) play prominent roles in stimulating allograft rejection and are recognized by CTLs that mediate this process. However, there is no information on the diversity of TCRs that are specific for single minor histocompatibility Ag peptides and expressed by CTLs in vivo. We have used the technique of spectratyping to study the diversity of Vbeta usage and beta complementarity-determining region 3 (CDR3) length of TCRs expressed by CTL-infiltrating skin allografts expressing the immunogenic H4 peptide during the process of rejection. Spectratyping revealed overall reduction in diversity of both Vbeta usage and CDR3 length, with sequential application of primary, second-set, and third-set H4-incompatible grafts. This dissection of the array of beta-chains expressed by graft-infiltrating CTLs allowed the direct sequencing of individual beta-chain PCR products. Beta CDR3s were characterized by a net negative charge, as we have observed previously with CDR3s expressed by H4-specific CTL clones selected in vitro. Identical and closely related CDR3 amino acid sequences could be identified that were shared by TCRs that 1) utilized different Vbeta genes, 2) derived from different mice, or 3) derived from different sequential sets of allografts on individual mice. Furthermore, a number of CDR3 sequences expressed by graft-infiltrating CTLs were identical or closely related to sequences we have identified previously in in vitro selected CTL clones that were specific for the H4 peptide.

Amino Acid Sequence↗
PubMed1998

Avoidance of self-reactivity results in skewed CTL responses to rare components of synthetic immunogens.

In studying the CTL recognition of peptide determinants derived from the nuclear Ag La (SS-B), we observed significant skewing of the response toward rare components present within the immunogen. Thus, priming of naive mouse lymphocytes in vitro with a synthetic H-2Kb-binding peptide comprising human La (hLa) residues 51-58 resulted in class I-restricted cytotoxic T cells that failed to recognize naturally presented hLa 51-58 peptide. Instead, the majority of T hybrids recognized a low abundance (< or = 1%) contaminant present at picomolar concentrations in the original synthesis and identified as a peptide adduct containing N,4-t-butyl asparagine at position 6 of the hLa 51-58 sequence. The preferred T cell recognition of the butyl adduct was not due to increased affinity of this peptide for the H-2Kb molecule or to the antagonism of CTL recognizing the unmodified determinant. Rather, the bias in the immune response appeared to be the result of partial self-tolerance to the homologous mouse La 51-58 determinant, which differs from its human counterpart by only a single amino acid at position 1 (T-->I). Accordingly, the CTL response appeared to be focused on "non-self" ligands present within the synthesis, even though they were present at very low concentrations. These observations have significant implications for the use of synthetic peptide vaccines, especially those designed to manipulate responses to self peptides such as tumor Ags in which self-tolerance may result in unexpected reactivity.

Amino Acid Substitution↗
PubMed1998

The efficiency of antigen recognition by CD8+ CTL clones is determined by the frequency of serial TCR engagement.

Using H-2Kd-restricted CTL clones, which are specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS(252-260) (SYIPSAEKI) and permit assessment of TCR-ligand interactions by TCR photoaffinity labeling, we have previously identified several peptide derivative variants for which TCR-ligand binding and the efficiency of Ag recognition deviated by fivefold or more. Here we report that the functional CTL response (cytotoxicity and IFN-gamma production) correlated with the rate of TCR-ligand complex dissociation, but not the avidity of TCR-ligand binding. While peptide antagonists exhibited very rapid TCR-ligand complex dissociation, slightly slower dissociation was observed for strong agonists. Conversely and surprisingly, weak agonists typically displayed slower dissociation than the wild-type agonists. Acceleration of TCR-ligand complex dissociation by blocking CD8 participation in TCR-ligand binding increased the efficiency of Ag recognition in cases where dissociation was slow. In addition, permanent TCR engagement by TCR-ligand photocross-linking completely abolished sustained intracellular calcium mobilization, which is required for T cell activation. These results indicate that the functional CTL response depends on the frequency of serial TCR engagement, which, in turn, is determined by the rate of TCR-ligand complex dissociation.

Animals↗
PubMed1999

A hsp70-2 mutation recognized by CTL on a human renal cell carcinoma.

We performed T cell cloning experiments with a tumor-infiltrating lymphocyte subpopulation derived from a renal cell carcinoma tumor site (RCC-7) in which the TCR clonotypic repertoire had been analyzed in terms of TCRBV complementarity-determining region 3 size distribution. We report in this work the characterization of one of the five RCC-specific MHC class I-restricted CTL clones isolated in RCC-7. This TCRBV6J1S1 CTL recognized only the autologous RCC-7 tumor cell line in the context of HLA-A*0201, and the Ag is encoded by a mutated form of the hsp70-2 gene found in the tumor cells, but not in autologous PBLs nor in 47 other tumors. The identification of this gene was achieved by cotransfecting into COS cells a cDNA library of RCC-7 together with HLA-A*0201. Transfectants expressing the Ag were identified by their ability to stimulate TNF release by the CTL clone. The antigenic peptide is a decamer with a mutated residue at position 8. Half-maximal lysis was obtained with only 5 x 10(-11) M of decapeptide in target sensitization assays compared with 5 x 10(-8) M for the wild-type decapeptide. This difference in recognition was not related to difference in binding HLA-A*0201-presenting molecules, as assessed in an immunofluorescence-based peptide-binding assay using T2 cells. Constitutive hsp70 expression in various tumors suggests that this stress-induced protein may be recognized in situ by tumor-infiltrating lymphocytes. The finding in the tumor of a mutated form of the stress-induced hsp70-2 gene whose product is specifically recognized by TILs with high avidity is discussed in view of the present use of mycobacteria or heterologous heat-shock proteins as immunomodulators or as subunit vaccine candidates.

Animals↗
PubMed1999

Natural variation of the expression of HLA and endogenous antigen modulates CTL recognition in an in vitro melanoma model.

Increasing attention has been devoted to elucidating the mechanism of lost or decreased expression of MHC or melanoma-associated antigens (MAAs), which may lead to tumor escape from immune recognition. Loss of expression of HLA class I or MAA has, as an undisputed consequence, loss of recognition by HLA class I-restricted cytotoxic T cells (CTLs). However, the relevance of down-regulation remains in question in terms of frequency of occurrence. Moreover the functional significance of epitope down-regulation, defining the relationship between MHC/epitope density and CTL interactions, is a matter of controversy, particularly with regard to whether the noted variability of expression of MHC/epitope occurs within a range likely to affect target recognition by CTLs. In this study, bulk metastatic melanoma cell lines originated from 25 HLA-A*0201 patients were analyzed for expression of HLA-A2 and MAAs. HLA-A2 expression was heterogeneous and correlated with lysis by CTLs. Sensitivity to lysis was also independently affected by the amount of ligand available for binding at concentrations of 0.001 to 1 mM. Natural expression of MAA was variable, independent from the expression of HLA-A*0201, and a significant co-factor determining recognition of melanoma targets. Thus, the naturally occurring variation in the expression of MAA and/or HLA documented by our in vitro results modulates recognition of melanoma targets and may (i) partially explain CTL-target interactions in vitro and (ii) elucidate potential mechanisms for progressive escape of tumor cells from immune recognition in vivo.

Antibodies, Monoclonal↗
PubMed2005

Discovery and characterization of highly immunogenic and broadly recognized mimics of the HIV-1 CTL epitope Gag77-85.

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) play an important role in HIV infection. Given the viral genetic diversity, the selection of suitable antigens and epitope variants will be important in the design of an effective vaccine. We have previously shown that combinatorial libraries are useful tools to identify epitope mimics as well as potentially cross-reactive natural sequences in protein databases. We have applied this approach to the HIV Gag p17-derived epitope SL9 (SLYNTVATL) to identify broadly recognized SL9 mimics and to assess the cross-recognition of naturally occurring SL9 variants. Nine nonapeptides were identified that were up to one order of magnitude more effective than SL9 in stimulating CTL responses in PBMC from HIV-infected subjects. Using transgenic mice, we demonstrate that a number of these epitope mimics were able to generate de novo T cell responses that cross-reacted with the original SL9 sequence. Particularly, mimics with changes at the relatively conserved F-pocket anchor residue were frequently cross-recognized. This approach may lead to vaccine candidates with higher in vivo immunogenicity and increased potential for cross-recognition of naturally occurring SL9 variants.

Amino Acid Sequence↗
PubMed2005

Increased rejection of primary tumors in mice lacking B cells: inhibition of anti-tumor CTL and TH1 cytokine responses by B cells.

We investigated the role of B cells in tumor immunity by studying immune responses of mice genetically lacking B cells to primary tumors. IgM(-/-) B cell-deficient mice (BCDM) exhibited enhanced resistance to 3 histologically diverse syngeneic tumors as compared to the wild-type (WT) mice. EL4 thymoma and MC38 colon carcinoma grew progressively in WT mice, but regressed spontaneously in BCDM whereas growth of B16 melanoma was slowed significantly in BCDM as compared to the WT mice. BCDM exhibited increased T cell infiltration of tumors, higher T(H)1 cytokine response and, in the case of MC38, a higher anti-tumor CTL response. The increased tumor resistance of BCDM did not seem to result from intrinsic changes in their non-B immunocytes because adoptive transfer of WT splenic B cells to BCDM abrogated tumor rejection and resulted in diminished anti-tumor T(H)1 cytokine and CTL responses. Studies involving BCR-transgenic mice indicated that B cells may inhibit anti-tumor T cell responses by antigen-nonspecific mechanisms since neither tumor-specific antibodies nor cognate T:B interactions were necessary for inhibition of tumor immunity by B cells. IFN-gamma secretion in splenocyte:tumor co-cultures of tumor-challenged BCDM was inhibited by WT but not CD40(-/-) B cells indicating that B cells may inhibit anti-tumor T(H)1 cytokine responses in a CD40-dependent manner. Adoptive transfer of CD40(-/-) B cells into BCDM resulted in restored growth of MC38 suggesting additional factors other than CD40 are involved in dampening anti-tumor responses. The effects of B cells on anti-tumor response warrant further study.

Animals↗
PubMed1995

Identification of a novel peptide derived from the melanocyte-specific gp100 antigen as the dominant epitope recognized by an HLA-A2.1-restricted anti-melanoma CTL line.

Cytotoxic T lymphocytes (CTL) reactive with human melanoma tumor cells occasionally display cross-reactivity with normal melanocytes. Previously, we identified the melanocyte lineage-specific antigen gp 100 that is expressed by both melanoma cells and normal melanocytes, as a target antigen for tumor-infiltrating lymphocytes derived from a melanoma patient (TIL 1200). Here, we demonstrate that the oligoclonal HLA-A2.1-restricted TIL 1200 line is reactive with 2 distinct peptides derived from the gp 100 protein. Apart from the peptide corresponding to gp 100 amino acids 457-466, we identified the gp 100 peptide 154-162 as a second epitope recognized by TIL 1200. A 100-fold lower concentration of this novel gp 100 peptide was required for target-cell sensitization compared to peptide 457-466, indicating that the 154-162 peptide is the dominant gp 100 epitope for TIL 1200. Together with the recently described gp 100 280-288 epitope, 3 distinct CTL epitopes have now been identified in gp 100, all presented in the context of HLA-A2.1. Therefore, gp 100 is an attractive target antigen in the development of immuno-therapeutic protocols against melanoma.

Amino Acid Sequence↗
PubMed1999

Codon optimization effect on translational efficiency of DNA vaccine in mammalian cells: analysis of plasmid DNA encoding a CTL epitope derived from microorganisms.

Interspecific difference of codon usage is one of the major obstacles for effective induction of specific immune responses against bacteria and protozoa by DNA immunization. Using genes encoding major histocompatibility complex class I-restricted cytotoxic T-lymphocyte (CTL) epitopes, derived from an intracellular bacterium, Listeria monocytogenes and a mouse malaria parasite, Plasmodium yoelii, we report here that the codon optimization level of the genes is not precisely proportional to, but does correlate well with the translational efficiency in mammalian cells, which is concomitantly associated with the induction level of specific CTL response in the mouse. These results suggest that DNA immunization using the gene codon-optimized to mammals through the entire region is very effective.

Animals↗
PubMed1997

Immunosensitization of prostate carcinoma cell lines for lymphocytes (CTL, TIL, LAK)-mediated apoptosis via the Fas-Fas-ligand pathway of cytotoxicity.

Several reports suggest that immunotherapy mediated by cytotoxic lymphocytes is beneficial in the destruction of drug-resistant tumor cells. Cytotoxic T lymphocytes kill target cells by two main mechanisms, namely by the perforin pathway and by the Fas-ligand (Fas-L) pathway. The role of the Fas-L pathway in tumor cell killing is not clear because many Fas(+)-expressing tumor cells are resistant to the Fas-L agonist cytotoxic anti-Fas antibody. The human prostate tumor cell lines (PC-3, DU145, and LnCAP) express Fas on the cell surface but are resistant to killing by anti-Fas antibody. This study examined the sensitivity of prostate tumor cells to Fas-L-mediated cytotoxicity and sensitization of the tumor cells by drugs to Fas-L-mediated killing. All three prostate tumor cell lines are resistant to Fas-L killing as determined by the use of the murine CTL hybridoma PMMI that kills only through the Fas-L pathway. However, the addition of subtoxic concentrations of CDDP or VP-16 significantly sensitized the PC-3 and DU145, but not LnCAP, tumor cells to Fas-L killing and apoptosis by PMMI. The sensitization of tumor cells by drugs was inhibited by neutralizing anti-Fas antibody. These findings demonstrate that immunoresistant Fas(+)-expressing DU145 and PC-3 prostate tumor cells can be sensitized by drugs to Fas-L killing. We then examined the role of Fas-L killing by TIL and LAK cells. All three prostate tumor cell lines were sensitive to killing by TIL and LAK and cell killing was primarily mediated through the Ca(2+)-dependent perforin pathway because it was blocked by the addition of EGTA/MgCl2. Sensitization by CDDP or VP-16 did not significantly augment killing of untreated tumor cells by TIL or LAK cells. However, in the presence of EGTA/MgCl2, the addition of CDDP or VP-16 significantly augmented killing of PC-3 and DU145, but not LnCAP, by TIL and LAK, and killing was blocked by neutralizing anti-Fas antibody. These findings demonstrate that both TIL and LAK exhibit a Fas-L-mediated killing pathway that is revealed once the perforin pathway is blocked by the Ca2+ chelator EGTA/MgCl2. Altogether, these findings show that drug-resistant, Fas(+)-expressing PC-3 and DU145 prostate tumor cells can be sensitized by CDDP and VP-16 to killing by Fas-L-bearing CTL, TIL, and LAK cells. Sensitization of tumor cells by drugs may augment the efficacy of immunotherapy in the eradication of tumor cells that are resistant to Fas-L-mediated killing.

Animals↗
PubMed1999

Novel phenotype associated with in vivo activated CTL precursors.

A previously undefined phenotype of CD8(+) cells that appears to represent in vivo activated CTL precursors (CTLP*) has been identified in the spleens of C57Bl/6 mice responding to a P815 tumor allograft. This population was first evident by the transient expression of very high levels of CD28 and CD44 on day 5 of the allograft response and reached maximal levels on days 7 and 8 before declining on day 9. A transient increase in CD69 expression was also observed on these cells on day 5. In contrast, CTL effectors (CTLE), identified by their CD8(+)CD44(hi)CD62LloCD45RBlo phenotype, were not appreciably detected in the spleen until day 8 and reached maximal levels on day 10. Further characterization of CTLP* on day 7 revealed that they represented blasting cells by increased light scatter and also expressed very high levels of CD54 but not CD122, CD152, or CD154. In addition, the cells had already up-regulated CD49d, asialo GM1, CD11a, and CD95L, and down-regulated their expression of CD62L. A small percentage of these cells also expressed CD25. Day 7 CTLP* sorted on the basis of their CD44(xhi) and CD54(xhi) phenotype did not exhibit cytolytic activity in a standard chromium release assay but became cytotoxic when they were cultured in the presence of exogenous murine IL-2 for 5 days. Granzyme B activity, however, was detected in CTLP* on day 7 at levels equivalent to CTLE on day 10. In order to establish a potential precursor relationship between CTLP* and CTLE, mice were treated with various doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a chemical that has been shown to dose-dependently suppress the in vivo generation of CTLE to P815 tumor cells by altering an early stage of CTLP activation. Results indicated that CTLP* were suppressed by TCDD on day 7 to the same degree that CTLE were suppressed on day 10. Importantly, for controls and for all doses of TCDD, there were approximately 12.5 CTLE on day 10 for every CTLP* detected on day 7. These results suggested that TCDD acted identically across all doses to inhibit the early stages of activation of CTLP but did not affect the final stages of differentiation and expansion to CTLE. This interpretation supports the previous observation that TCDD exposure had to occur within the first 3 days of the allograft response in order to induce suppression of CTLE activity. Taken together, these results support the conclusion that in vivo activated CTLP can be identified by their unique expression of very high levels of CD44, CD28, and/or CD54 prior to their full maturation and clonal expansion to functional CTLE.

Animals↗
PubMed1995

Proteinases are involved in both DNA fragmentation and membrane damage during CTL-mediated target cell killing.

A number of inhibitors with different specificities were used to probe the involvement of proteinases in the mechanism of cytotoxic T lymphocyte (CTL)-mediated lysis. N-Acetyl-L-tyrosine ethyl ester (ATEE) and N-benzoyl-L-arginine ethyl ester (BAEE) are reversible substrate inhibitors of proteinases with trypsin-like and chymotrypsin-like specificities, respectively. BAEE did not prevent either the chromium release or DNA fragmentation induced in mouse tumor target cells by a mixed lymphocyte population. In contrast, ATEE inhibited both processes. The irreversible proteinase inhibitor 3,4-dichloroisocoumarin (DCI) also blocked both chromium release and DNA fragmentation, but at significantly lower concentrations than ATEE. More importantly, chromium release was more susceptible to inhibition by DCI than DNA fragmentation. Addition of a combination of the endonuclease inhibitor aurintricarboxylic acid plus DCI resulted in virtually complete inhibition of both DNA fragmentation and chromium release when the drugs were added at the beginning of the incubation period. In contrast addition of DCI 15 or 30 min following initiation of the lytic cycle abolished the affect of DCI on fragmentation, but not lysis. A model which suggests a dual role for the proteinases in CTL-mediated target cell death is presented. First, proteinases are involved in the initiation of DNA fragmentation. Second, they have an ongoing function in membrane damage.

Animals↗
PubMed2001

Blocking CTL-based cytotoxic pathways reduces apoptosis of transplanted hepatocytes.

BACKGROUND: A major obstacle in allogenic hepatocyte transplantation is increased apoptosis of grafted cells due to CTL-based cytotoxicity. However, whether blockade of Fas- and granzyme-mediated pathways of CTL-based cytotoxicity may provide immune protection to transplanted hepatocytes is poorly defined. Our study aimed to reduce apoptosis of allogenic transplanted hepatocytes by inhibiting granzyme B (GraB) activity and blocking Fas-FasL interaction. MATERIALS AND METHODS: Hepatocyte transplantation was performed by inoculating isolated liver cells from ACI rats (allogenic) or Lewis rats (syngenic) into the spleens of Lewis rats. Recipients were treated with FLIM58, an inhibitory anti-FasL mAb, and GraB inhibitor I alone or a combination of the two drugs for 5 days after transplantation, and were sacrificed at Day 7. Apoptosis of transplanted hepatocytes was detected in situ by TUNEL assay and M30 immunostaining. Glutamate dehydrogenase (GLDH) activity in recipient spleens was examined to evaluate survival of transplanted cells. Recipient spleens were assayed for FasL level with Western blotting and for GraB activity by hydrolysis of GraB substrate. RESULTS: FLIM58 or GraB inhibitor I significantly reduced the percentage of TUNEL-positive and M30-positive hepatocytes and markedly increased GLDH levels in allogenic, but not syngenic, recipient spleens. These effects were more pronounced when the two drugs were used in combination (P < 0.05). Additionally, elevation of FasL and GraB levels in allogenic recipient spleens can be significantly reduced by FLIM58 and GraB inhibitor I, respectively. CONCLUSIONS: Inhibition of GraB activity and blockade of Fas-FasL interaction reduce the apoptosis of allogenic transplanted hepatocytes, and thus improve their survival.

Animals↗
PubMed1997

Identification of a Kb-restricted CTL epitope of beta-galactosidase: potential use in development of immunization protocols for "self" antigens.

The use of recombinant and synthetic vaccines in the treatment of cancer has recently been explored using model tumor associated antigens (TAA), many of which do not model the immunological state of affairs in which the TAA is expressed by normal tissues. One potentially useful model Ag is beta-galactosidase (beta-gal). Because the activity of this enzyme is so easily detectable, this gene has been inserted into a large number of recombinant viruses and tumors useful to the cancer vaccinologist. In addition, numerous transgenic mouse colonies that have tissue-specific expression of beta-gal have been developed, enabling the modeling of tolerance to "self" Ags. Since most of these mice have an H-2b background, we generated cytotoxic T lymphocytes (CTL) capable of recognizing beta-gal-expressing tumor cells of C57BL\6 origin and have determined that their restriction element is the K(b) molecule. Using an allele-specific epitope forecast to generate a panel of candidate peptides, we have determined that the K(b)-restricted sequence is DAPIYTNV and corresponds to amino acids 96-103 of the intact beta-gal molecule. A recombinant vaccinia virus (rVV-ES beta-gal96-103) was constructed that encoded the peptide epitope preceded by an endoplasmic reticulum insertion signal sequence. Tumor cells infected with this rVV were recognized by the original CTL that had been used to identify the epitope. Furthermore, splenocytes of mice immunized with a rVV encoding the full-length beta-gal molecule and restimulated with the DAPIYTNV peptide specifically recognized tumor cells expressing beta-gal. The identification of this immunogenic beta-gal sequence enables the modeling of immunization strategies in animal models of malignant disease in which the target antigen is a "self" protein.

Amino Acid Sequence↗
PubMed1997

Do CTL kill target cells by inducing apoptosis?

Inhibitors of ICE-family proteases (caspases) block many examples of apoptotic cell death in vivo and in vitro, including multiple apoptotic stimuli for T lymphocytes. We have tested whether cell death induced by cytotoxic T lymphocytes was also blocked by caspase inhibitors. We found that the rapid apoptotic target cell death induced by Fas ligand-bearing CTL using the target Fas death pathway was efficiently blocked by caspase inhibitors. In contrast, target lysis induced by the CTL granule exocytosis pathway is not detectably blocked by such inhibitors, although the accompanying apoptotic nuclear damage is efficiently blocked. Thus caspase inhibitors prevent the hallmark phenotype of apoptosis without measurably affecting target cell death as evidenced by lysis.

Animals↗
PubMed1996

Analysis of the contribution of CTL epitopes in the immunobiology of morbillivirus infection.

In Balb/c (H-2d) mice, the nucleoprotein (NP) of measles virus (MV) induces a MHC class I restricted-CTL response to a single 9-amino-acid epitope (aa 281--289). This L(d)-restricted epitope is also present in the NP of the closely related canine distemper virus (CDV). To investigate whether this epitope is immunologically effective when it is present within the primary sequence of a nonviral protein, we have incorporated the 281--289 motif into the human CD36 protein. When cells are infected with vaccinia virus (VV) recombinants expressing this protein, CD36NP, the MV epitope is correctly processed and the cells are lysed by MVNP-specific CTLs. In vivo, VV-CD36NP induced CTLs which protected mice from a lethal dose of CDV, but did not block virus replication. The MVNP contains four other potential L(d)-restricted motifs. To investigate if these could be utilized in the absence of the dominant epitope, a mutant NP was produced in which one of the anchor residues in the aa 281--289 motif was mutated. Cells infected with a VV recombinant expressing this protein (VV-NP F289S) were only poorly lysed by MVNP-specific CTLs. Similarly, immunization of Balb/c mice with VV-NP F289S induced a lower level of CTL activity compared to the VV-NP, but the activity was now directed to three other epitopes. When mice were vaccinated with VV-NP F289S they were only partially protected from a lethal CDV challenge. The significance of these results for MV vaccine development is discussed.

Amino Acid Sequence↗
PubMed1999

High-yield reassortant influenza vaccine production virus has a mutation at an HLA-A 2.1-restricted CD8+ CTL epitope on the NS1 protein.

Current influenza virus vaccines are prepared using high-yield reassortant virus strains obtained from a mixed infection of the new virus strain and a prototype high-yielding virus strain. The high-titered reassortant virus strain used as vaccine seed virus possesses the recent virus HA and NA and contains the internal genes from the high-growing prototype parent. We established a human CD8(+) cytotoxic T cell (CTL) line, 10-2C2, which recognizes an HLA-A2.1-restricted influenza A virus H1, H2, H3 cross-reactive T cell epitope on amino acids 122-130 of the NS1 protein, and unexpectedly we observed that there was decreased lysis of target cells infected with the A/Texas/36/91 (H1N1) vaccine virus strain compared to the lysis of target cells infected with the prototype A/PR/8/34 (H1N1) virus. RT-PCR results showed that the A/Texas vaccine virus strain contained a quasispecies. Approximately 50% of viral RNA of the NS1 gene had a nucleotide substitution that resulted in the N --> K amino acid change at the sixth position of the nonamer peptide. Current influenza vaccines are inactivated and do not contain the NS1 protein; however, future influenza vaccines may include live attenuated vaccines and with this mutation a live virus would fail to induce a CD8(+) CTL response to this epitope in individuals with HLA-A2.1, a very common allele, and potentially have reduced efficacy.

Base Sequence↗