Recombinant Vaccinia Virus Vectors Research Articles

IL-15-independent antiviral function of primary and memory CD8 + T cells

Memory CD8 + T cells are comprised of CD122 hi IL-15-dependent and CD122 lo IL-15-independent subsets. Induction and retention of IL-15-independent memory CD8 + T cells was assessed in IL-15 −/− and wild-type (wt) mice immunized with recombinant vaccinia virus (rVV) or Sindbis virus (rSIN) vectors expressing the identical foreign epitope. Both vectors induced epitope-specific CD8 + T cell expansion and function, independent of IL-15. Similar kinetics of rVV clearance confirmed effective CD8 + T cell function in IL-15 −/− mice. CD44 hi CD122 hi CD8 + T cells, mainly of the CD62L −/lo phenotype, increased more dramatically and declined more rapidly in IL-15 −/− mice, independent of the vector. Rapid IL-15-independent memory CD8 + T cell expansion following challenge of immune mice compensated for the limited memory CD8 + populations in IL-15 −/− mice. However, despite expansion and expression of potent effector function, viral clearance was delayed in the absence of IL-15, coinciding with a rapid loss in cytolytic function.

Use of dual recombinant vaccinia virus vectors to assay viral glycoprotein-mediated fusion with transfection-resistant primary cell targets.

Fusion mediated by the human immunodeficiency virus type-1 (HIV-1) envelope (Env) glycoprotein and the cellular CD4/chemokine receptor complex is the first step in entry and is often analyzed in cell-cell fusion assays that require Env expression by recombinant vaccinia viruses and/or target cell transfection. Primary lymphocytes and macrophages are the principal targets for HIV-1 in vivo, but are poor substrates for transfection, and constructing recombinant vaccinia viruses expressing every novel or mutant env gene is laborious. This chapter describes a fusion assay using two recombinant vaccinia viruses that express distinct RNA polymerases suitable for transfection-resistant targets, such as primary human lymphocytes and macrophages. It also uses env genes contained in plasmid vectors, eliminating the need to construct recombinant vaccinia viruses to analyze each construct. Effector 293T cells are cotransfected with SP6-driven reporter gene and T7-driven env plasmids, then infected with recombinant vaccinia virus expressing T7 polymerase. Primary lymphocyte or macrophage targets are infected with recombinant vaccinia virus expressing SP6 polymerase. Fusion mediated by effector cell Env and endogenous CD4/coreceptors in target lymphocytes or macrophages enables SP6 polymerase-mediated reporter gene transactivation. This approach provides an efficient tool to study fusion mediated by multiple-cloned primary isolate or mutant HIV-1 env genes with the primary target cell types relevant to infection in vivo.

Glycol chitosan improves the efficacy of intranasally administrated replication defective human adenovirus type 5 expressing glycoprotein D of bovine herpesvirus 1

The ability of two soluble formulations, namely chitosan and glycol chitosan, when used as an intranasal adjuvant, to improve the immunogenicity of an intranasal human adenovirus type 5 replication defective expressing bovine herpesvirus 1 (BoHV-1) glycoprotein D based vaccine, was investigated in cattle. Their adjuvant effects on immune response by increasing clinical and especially virological protection against an intranasal BoHV-1 challenge were then evaluated. The best virological protection was obtained in calves immunized with the vaccine vector adjuvanted with glycol chitosan which decreased the challenge BoHV-1 virus excretion titres by 0.5–1.5 log when compared to those obtained in calves immunized with the vaccine vector alone or adjuvanted with chitosan. A slight difference in clinical scores was observed in calves immunized with the adjuvanted vaccine vector compared to calves immunized with the vaccine vector alone. The obtained data suggest that the tested soluble formulation of glycol chitosan has promising potential use as an intranasal adjuvant for recombinant viral vector vaccines in cattle.

Broad human immunodeficiency virus (HIV)-specific T cell responses to conserved HIV proteins in HIV-seronegative women highly exposed to a single HIV-infected partner.

Eighteen highly exposed but persistently seronegative (HEPS) women (HW) and their human immunodeficiency virus (HIV) type 1-seropositive male partners were studied for HIV-specific T cells and other host factors. Circulating HIV-specific T cells were measured by interferon-gamma enzyme-linked immunospot assays, using recombinant vaccinia virus vectors expressing HIV proteins. Nine (50%) of the HW and all HIV-seropositive persons had HIV-specific T cell responses. Only 2 (22%) of the HEPS responders recognized Env, compared with 94% of HIV-seropositive persons. A high percentage (75%) of the HW with HIV-specific T cell responses reported recent HIV exposure. Remarkably, however, long-lived HIV-specific T cells were detected in 2 HW who had an extended period (>3.9 years) of no HIV exposure. These findings have important implications for HIV vaccine design.

Protection in dogs against visceral leishmaniasis caused by Leishmania infantum is achieved by immunization with a heterologous prime-boost regime using DNA and vaccinia recombinant vectors expressing LACK.

A heterologous prime-boost vaccination regime with DNA and recombinant vaccinia virus (rVV) vectors expressing relevant antigens has been shown to enhance specific cellular immune responses and to elicit protection against a variety of pathogens in animal models. In this paper, we describe the effectiveness of the prime-boost strategy by immunizing dogs with a plasmid carrying the gene for the LACK antigen from Leishmania infantum (DNA-LACK) followed by a booster with a rVV containing the same gene (rVV-LACK). Thereafter, animals were challenged with L. infantum to induce visceral leishmaniasis (VL). In the vaccinated dogs as compared with the controls, the outcome of the infection after challenge with a high inoculum (10 8) of L. infantum stationary promastigotes was assessed by tissue parasite load, specific anti- Leishmania antibody production, cytokine level and development of clinical signs of leishmaniasis. We observed a 60% protection against infection in dogs immunized by DNA-LACK prime/rVV/-LACK boost while two doses of DNA-LACK did not elicit protection against the disease. The interleukin 4 (IL-4), interferon gamma (IFNγ) and IL-12 (p40 subunit) cytokine mRNA expression profiles in PBMC as well as lymphocyte proliferative response and the IgG2/IgG1 ratios specific for LACK suggest that in vaccinated animals there is triggering of cellular immune responses. This type of DNA/rVV prime/boost immunization approach may have utility against visceral leishmaniasis in dogs.

Feasibility of using genetic linkage analysis to identify the genes encoding T cell-defined minor histocompatibility antigens.

We have evaluated the utility of genetic linkage analysis to identify genes that encode minor histocompatibility antigens using vaccinia virus vectors as a simple and convenient method for transient expression of class I MHC molecules in lymphoblastoid cell lines. As a test case, we used a CTL clone that recognizes HA-8, a minor histocompatibility antigen encoded by the KIAA0020 gene and presented by HLA-A*0201. EBV-transformed B cell lines from individuals in three large pedigrees from the CEPH reference family collection were infected with a recombinant vaccinia virus vector encoding an HLA-A*0201 transgene, which led to high level expression of the MHC restricting allele HLA-A*0201 on the cell surface. HA-8 expression in the vaccinia-infected target cells was then determined using standard in vitro cytotoxicity assays. Pairwise linkage analysis of the segregation of HA-8 expression in these pedigrees demonstrated that the HA-8 gene was tightly linked with a cluster of marker loci located on the distal portion of chromosome 9p. Analysis of 9p marker haplotypes for individuals in the three families identified several individuals with recombinant haplotypes, and these recombination events were used to refine the precision of the HA-8 gene localization further. The data collectively indicate that the HA-8 gene is localized to a 10.3 cM (corresponding to 3.9 Mb) interval of distal 9p that is thought to encode at least 11 genes, including KIAA0020. These results demonstrate that linkage analysis can be used to map minor histocompatibility genes with high precision and accuracy. Over the next years, refinement and annotation of the human genome sequence will undoubtedly increase the utility of linkage analysis as a tool for identifying minor histocompatibility antigen genes.

Rapid induction of specific cytotoxic T lymphocytes against melanoma-associated antigens by a recombinant vaccinia virus vector expressing multiple immunodominant epitopes and costimulatory molecules in vivo.

A specific cellular immune response directed against a panel of three defined tumor-associated antigen (TAA) epitopes was induced in metastatic melanoma patients by a prime-boost strategy taking advantage of an innovative recombinant vaccinia virus as evaluated by quantitative assessment of cytotoxic T lymphocytes (CTLs) with corresponding specificity. The immunization protocol consisted of the administration of psoralen-UV-treated and replication-incompetent recombinant vaccinia virus encoding the three immunodominant HLA-A*0201-restricted epitopes Melan-A(27-35), gp100(280-288), and tyrosinase(1-9) together with two costimulatory molecules, B7.1 and B7.2, in the context of systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. Boosts were subsequently applied with corresponding synthetic nonapeptides and GM-CSF. Specific CTL induction was assessed by tetramer staining and CTL precursor (CTLp) frequency evaluation. Within 12 days of injection of the recombinant vector, cytotoxic T cell responses specific for engineered epitopes were detectable in three of three patients. During the vaccination treatment, antigen-specific CTLp frequencies exceeding 1:10,000 peripheral CD8(+) T cells could be observed. Tetramer staining also revealed significant increases in specific CD8(+) T cell numbers. We conclude that active specific antitumor vaccination can raise a concurrent and specific cellular immune response against a panel of molecularly defined antigens, thereby increasing the chance of an immune hit against neoplastic cells with heterogeneous antigen expression. Data from this study emphasize the potency of a recombinant vaccinia virus vector encoding multiple minigenes and costimulatory molecules in the context of exogenously administered GM-CSF. Clinical effectiveness of this immunologically active protocol should therefore be explored in appropriately selected groups of patients.

Retroviral vectors produced in the cytoplasmic vaccinia virus system transduce intron-containing genes.

Introns and polyadenylation (pA) sites are known to improve transcript stability and nuclear-cytoplasmic transport and are normally present in efficient gene expression vectors. Standard retroviral vectors, however, do not allow the inclusion of such sequence elements, as mRNA processing at internal splice and pA sites interferes with the production of functional full-length vector genomes. In this report we examined the capability of hybrid vaccinia/retroviral vectors to transduce complex gene cassettes with nuclear RNA processing signals within the retroviral genome. A retroviral vector was constructed that contains a gene of interest (the human coagulation factor IX [FIX] cDNA), including an intron and an internal pA site. The modified proviral vector genome was cloned downstream of a vaccinia virus promoter and was inserted into the vaccinia virus genome. Infection of a packaging cell line with the recombinant vaccinia virus vector resulted in secretion of retroviral particles at average titers of 10(5) CFU per ml of cell culture supernatant. Due to the cytoplasmic transcription and the nonrecognition of nuclear transcription signals in the vaccinia virus system, full-length transcripts were obtained that still contained the intron. In the retrovirally transduced cell lines the FIX transcripts were terminated at the internal pA site. The transcripts were quantitatively spliced, and FIX was secreted. Recombinant cell lines with stable single-copy inserts containing sequence elements necessary for efficient gene function could be generated. Thus, a relatively simple cytoplasmic system for the generation of complex retroviral vectors is described. Retroviral vectors transducing intron-containing gene cassettes may play a further role in gene therapy applications.

Immunization strategies to augment oral vaccination with DNA and viral vectors expressing HIV envelope glycoprotein

Induction of mucosal immunity to the human immunodeficiency virus (HIV) envelope (env; gp160) glycoprotein has been demonstrated with orally administered recombinant vaccinia virus (rVV) vectors and poly( dl-lactide-co-glycolide) (PLG)-encapsulated plasmid DNA expressing gp160. In this study, we investigated the effect of an oral DNA-prime/rVV-boost vaccine regimen in conjunction with adjuvants on the level of gp160-specific cellular and humoral responses in BALB/c mice. We demonstrated that DNA priming followed by a booster with rVV expressing gp160 (vPE16) significantly augmented env-specific immunity in systemic and mucosal tissues of the immunized mice. Association of vPE16 with liposomes and coadministration of liposome-associated β-glucan lentinan or IL-2/Ig-encoded plasmid DNA-encapsulated in PLG microparticles triggered the optimal cell-mediated immune (CMI) responses. Lentinan was found to increase env-specific type 1 cytokine production and cytotoxic T-lymphocyte (CTL) activities but had no effect on humoral responses. On the other hand, IL-2/Ig-mediated increases in both type 1 and 2 activities were associated with higher levels of env-specific CTL and antibody responses. Results of these studies demonstrated the effectiveness of oral vaccines with DNA and rVV vectors in conjunction with immunomodulators in inducing specific immune responses in systemic and mucosal tissues.

Specific immunotherapy by genetically engineered APCs: the "guided missile" strategy.

We tested the hypothesis that APCs genetically engineered to present an Ag and to express Fas ligand (FasL) simultaneously can target and eliminate Ag-specific T cells. Transgenic T cells specific for influenza hemagglutinin (HA) were used as targets. We prepared recombinant vaccinia virus vectors (VVV) to transfer the gene constructs individually or simultaneously into APCs. We prevented unwanted viral replication by attenuating the VVVs with psoralen-UV light treatment. For presentation of the HA Ag, APCs were transduced with cDNA for HA flanked by sequences of the lysosome-associated membrane protein that direct efficient processing and presentation of the Ag by APCs. As a "warhead" for the APCs, we transduced them with the gene for FasL, which induces apoptosis of Fas-expressing activated T cells. To protect the transduced APCs from self-destruction by FasL, we transferred cDNA for a truncated form of Fas-associated death domain, which inhibits Fas-mediated cell death. Our results show that the engineered APCs effectively expressed the genes of interest. APCs transduced with VVV carrying all three gene constructs specifically killed HA-transgenic T cells in culture. Coculture with T cells specific for an unrelated Ag (OVA) had no significant effect. Our in vitro findings show that APCs can be genetically engineered to target and kill Ag-specific T cells and represent a promising novel strategy for the specific treatment of autoimmune diseases.

Adverse effects of MVA-T7 on the transport of Marburg virus glycoprotein

Expression of glycoproteins has been carried out successfully using recombinant vaccinia virus vectors. Especially attractive is the use of recombinant vaccinia viruses which express the DNA-dependent RNA polymerase of the phage T7 (T7-polymerase). The T7-polynerase drives the transcription of plasmid-based genes under the control of the T7 RNA polymerase promoter transfected into the infected cell. Comparison of two different recombinant vaccinia viruses, vTF7-3 and MVA-T7, revealed that post-translational processing of Marburg virus surface glycoprotein (GP) is impaired in the MVA-T7 but not in the vTF7-3 system. Influenza virus hemagglutinin, however, was transported and processed like the authentic protein in both systems. It is shown that transport of GP in the MVA-T7 system is not completely blocked, but the vast majority of molecules remained Endo H-sensitive. Only trace amounts evaded the endoplasmatic reticulum and reached the plasma membrane. Thus, the adverse effects of MVA-T7 on the processing of recombinant glycoproteins cannot be predicted, and correct processing has to be investigated for every expressed glycoprotein.

VP5, the Nonstructural Polypeptide of Infectious Bursal Disease Virus, Accumulates within the Host Plasma Membrane and Induces Cell Lysis

Infectious bursal disease virus (IBDV) encodes a 17-kDa nonstructural polypeptide known as VP5. This polypeptide is not essential for virus replication in vitro but it plays an important role in in vivo dissemination and pathogenesis. We have characterized the expression of VP5 in three eukaryotic systems: (i) IBDV-infected chicken embryo fibroblasts; (ii) BSC-1 cells infected with a recombinant vaccinia virus vector; and (iii) Cos-1 cells transiently transfected with a plasmid vector. Immunofluorescence analyses showed that upon expression VP5 accumulates within the plasma membrane. This finding was consistent with sequence-based topology predictions, indicating that VP5 is a class II membrane protein with a cytoplasmic N-terminus and an extracellular C-terminal domain. Brefeldin A treatment of VP5-expressing cells prevented the accumulation of this polypeptide in the plasma membrane, thus showing the requirement of an active exocytic pathway to reach that compartment. Expression of VP5 was shown to be highly cytotoxic. Induction of VP5 expression resulted in the alteration of cell morphology, the disruption of the plasma membrane, and a drastic reduction of cell viability. VP5-induced cytotoxicity was prevented by blocking its transport to the membrane with Brefeldin A. Our findings suggest that VP5 plays an important role in the release of the IBDV progeny.

Tumor-specific gene delivery using recombinant vaccinia virus in a rabbit model of liver metastases.

Several approaches to gene therapy for cancer have yielded promising results in rodent models. The translation of these results to the clinical realm has been delayed by the lack of tumor models in large animals. We investigated the pattern of transgene (i. e., foreign or introduced gene) expression and virus vector elimination after systemic gene delivery using a thymidine kinase-negative vaccinia virus in a rabbit model of disseminated liver metastases. VX-2 rabbit carcinoma cells were maintained by serial transplantation in the thigh muscles of New Zealand White rabbits, and disseminated liver metastases were established by direct injection of tumor cells into the portal vein of the animals. Different doses of a recombinant thymidine kinase-negative vaccinia virus vector encoding the firefly luciferase reporter gene (i.e., transgene) were injected into tumor-bearing rabbits. Transgene activity in tumors and other organs was measured at multiple time points thereafter. The pattern of development of antibodies against the vaccinia virus vector was also examined. Two-tailed Student's paired t test was used for comparisons of transgene activity. Transgene expression was increased in tumors by at least 16-fold in comparison with expression in other tissues by day 4 after vector injection (all P<. 001) and was maintained for approximately 1 week, providing evidence of tumor-specific gene delivery in this model. Rapid elimination of the circulating vector by the host immune system was observed. Anti-vector antibodies were detectable in serum as early as day 6 and were maintained for more than 3 months. Tumor-specific gene delivery is possible after systemic injection of a thymidine kinase-negative vaccinia virus vector in a model of rabbit liver metastases. Although the period of transgene expression appears limited because of a rapid immune response, the therapeutic window might be sufficient for an enzyme/prodrug gene therapy approach in clinical application.

Immune Modulation by IL-10 Gene Transfer via Viral Vector and Plasmid DNA: Implication for Gene Therapy

In the present report, we have evaluated and compared the modulatory effect of the cytokine interleukin (IL)-10 expressed via viral vector or plasmid DNA on viral antigen-induced cutaneous inflammatory lesions. Our data demonstrate the superior potency of both recombinant vaccinia virus and herpes simplex virus IL-10 expression vectors after single intramuscular administration, but the effects were only short term and only functioned in animals lacking immunity to the viral vectors used for modulation. In contrast, modulatory effects achieved by plasmid DNA expressing IL-10 were delayed in onset and milder in effect but were far more persistent than those achieved by viral vectors. Moreover, plasmid DNA expressing IL-10 provided effective modulation when given repeatedly to animals. Our data also showed that IL-10 gene delivery resulted in a systemic and durable modulatory effect while the effect caused by a single IL-10 protein treatment was transient and confined to the injected site. Our results imply that the viral vector system is superior for obtaining short-term effects, whereas the plasmid DNA approach represents a better strategy to achieve gene therapy to modulate chronic inflammatory lesions.

Protection against establishment of retroviral persistence by vaccination with a live attenuated virus.

Many human viruses not only cause acute diseases but also establish persistent infections. Such persistent viruses can cause chronic diseases or can reactivate to cause acute diseases in AIDS patients or patients receiving immunosuppressive therapies. While the prevention of persistent infections is an important consideration in the design of modern vaccines, surprisingly little is known about this aspect of protection. In the current study, we tested the feasibility of vaccine prevention of retroviral persistence by using a Friend virus model that we recently developed. In this model, persistent virus can be detected at very low levels by immunosuppressing the host to reactivate virus or by transferring persistently infected spleen cells into highly susceptible mice. Two vaccines were analyzed, a recombinant vaccinia virus vector expressing Friend virus envelope protein and a live attenuated Friend virus. Both vaccines reduced pathogenic virus loads to levels undetectable by infectious center assays. However, only the live, attenuated vaccine prevented immunosuppression-induced reactivation of persistent virus. Thus, even very low levels of persistent Friend virus posed a significant threat during immunosuppression. Our results demonstrate that vaccine protection against establishment of retroviral persistence is attainable.

A novel vaccine regimen utilizing DNA, vaccinia virus and protein immunizations for HIV-1 envelope presentation

Recombinant DNA and vaccinia virus (VV) vectors that express envelope (Env) proteins of the human immunodeficiency virus (HIV) have each been prominently utilized in vaccine development. These two vectors (termed DNA-Env and VV-Env) are attractive vaccine candidates due to their abilities to elicit both cytotoxic T-lymphocyte and B-cell responses. Our previous work demonstrated that DNA-Env primed animals, that were relatively unresponsive to DNA-Env boosters, could be immunized with VV-Env to yield more than a 100-fold increase in antibody responses. Here we show: (1) results with an optimized vaccine regimen that primes with DNA-Env, boosts with VV-Env, and re-boosts with purified Env proteins, (2) enhanced responses with 8 rather than 16 week intervals between VV-Env and protein immunizations, and (3) the failure of single Env vaccines to reproducibly elicit responses toward heterologous Env, regardless of the vaccination regimen utilized. Results encourage the use of poly-Env vaccine cocktails administered via DNA/VV/protein regimens in future non-human primate and clinical studies.

Antisense oligonucleotide inhibition of hepatitis C virus (HCV) gene expression in livers of mice infected with an HCV-vaccinia virus recombinant.

Hepatitis C virus (HCV) is the major cause of non-A, non-B hepatitis worldwide. Current treatments are not curative for most infected individuals, and there is an urgent need for both novel therapeutic agents and small-animal models which can be used to evaluate candidate drugs. A small-animal model of HCV gene expression was developed with recombinant vaccinia virus vectors. VHCV-IRES (internal ribosome entry site) is a recombinant vaccinia viral vector containing the HCV 5' nontranslated region (5'-NTR) and a portion of the HCV core coding region fused to the firefly luciferase gene. Intraperitoneal injection of VHCV-IRES produced high levels of luciferase activity in the livers of BALB/c mice. Antisense oligonucleotides complementary to the HCV 5'-NTR and translation initiation codon regions were then evaluated for their effects on the expression of these target HCV sequences in BALB/c mice infected with the vaccinia virus vector. Treatment of VHCV-IRES-infected mice with 20-base phosphorothioate oligonucleotides complementary to the sequence surrounding the HCV initiation codon (nucleotides 330 to 349) specifically reduced luciferase expression in the livers in a dose-dependent manner. Inhibition of HCV reporter gene expression in this small-animal model suggests that antisense oligonucleotides may provide a novel therapy for treatment of chronic HCV infection.

Role of Mucosal Immunity in Herpes Simplex Virus Infection

This study evaluates whether the vaginal mucosal surface of immunized mice can prevent invasion by herpes simplex virus (HSV) and aims to identify immune components that affect immunity after challenge at the vaginal mucosa. Despite the induction of both IgA and IgG vaginal Ab following immunization with recombinant vaccinia virus vectors expressing either glycoproteins B or D, viral infection occurred in most animals even after minimal viral dose challenge. Challenged immune animals, including those genetically unable to generate anti-HSV Ab, survived and showed few if any clinical signs of infection. Experiments with T cell subtype knockout animals and depletion with T cell subset-specific MAb indicated that immunity following vaginal challenge was principally dependent on the function of CD4+ T cells. Our results indicate that anti-HSV vaccines may not provide barrier immunity at the vaginal mucosal site but may be adequate to minimize clinical expression of disease.

General method for the detection and in vitro expansion of equine cytolytic T lymphocytes

Equine immunological research is hindered by the lack of a simple yet reliable general protocol by which to assay CTL activity specific for viral or parasitic antigens. We present here the first comprehensive analysis of the parameters necessary to reliably culture equine T cells and to analyze the antigen specific cytolytic activity of T lymphocytes utilizing the equine infectious anemia virus (EIAV) infection of outbred ponies as a source for in vivo primed T lymphocytes. Effective long-term in vitro culture of equine T cells was determined to require minimally 200 U/ml of recombinant human IL-2. We demonstrated that pokeweed mitogen (PWM) stimulated PBMC generated large quantities of MHC class I and MHC class II expressing autologous lymphoblasts that were used initially to activate and expand antigen specific T lymphocytes and later to serve as a source of target cells in standard chromium release assays. The source of antigen expressed by the PWM lymphoblasts was a recombinant vaccinia virus vector which carried sequences encoding various antigens of interest, but most specifically, the envelope glycoprotein of EIAV. Secondary in vitro stimulation of the T lymphocytes by autologous PWM lymphoblasts expressing EIAV envelope glycoprotein was maximal using a ratio of 10 T cells to one stimulator cell. After antigen stimulation, responding T lymphocytes had antigen specific cytolytic activity and were of both the CD4 and CD8 lineage. The methodology presented here should provide an effective and reliable means by which to analyze the cytolytic activity of equine T lymphocytes to other foreign antigens. Furthermore, we suggest that this method derived for the equine animal model should be applicable to other mammalian and avian model systems that currently lack an effective means by which to analyze antigen specific CTL activity.

Human Melanoma Patients Recognize an HLA-A1-Restricted CTL Epitope from Tyrosinase Containing Two Cysteine Residues: Implications for Tumor Vaccine Development

To identify shared epitopes for melanoma-reactive CTL restricted by MHC molecules other than HLA-A*0201, six human melanoma patient CTL lines expressing HLA-A1 were screened for reactivity against the melanocyte differentiation proteins Pmel-17/gp100, MART-1/Melan-A, and tyrosinase, expressed via recombinant vaccinia virus vectors. CTL from five of the six patients recognized epitopes from tyrosinase, and recognition of HLA-A1+ target cells was strongly correlated with tyrosinase expression. Restriction by HLA-A1 was further demonstrated for two of those tyrosinase-reactive CTL lines. Screening of 119 synthetic tyrosinase peptides with the HLA-A1 binding motif demonstrated that nonamer, decamer, and dodecamer peptides containing the sequence KCDICTDEY (residues 243-251) all reconstituted the CTL epitope in vitro. Epitope reconstitution in vitro required high concentrations of these peptides, which was hypothesized to be a result of spontaneous modification of cysteine residues, interfering with MHC binding. Substitution of serine or alanine for the more N-terminal cysteine prevented modification at that residue and permitted target cell sensitization at peptide concentrations 2 to 3 orders of magnitude lower than that required for the wild-type peptide. Because spontaneous modification of sulfhydryl groups may also occur in vivo, tumor vaccines using this or other cysteine-containing peptides may be improved by amino acid substitutions at cysteine residues.