A plasmid encoding murine granulocyte-macrophage colony-stimulating factor increases protection conferred by a malaria DNA vaccine

Using the murine parasite Plasmodium yoelii (Py) as a model for malaria vaccine development, we have previously shown that a DNA plasmid encoding the Py circumsporozoite protein (PyCSP) can protect mice against sporozoite infection. We now report that mixing a new plasmid PyCSP1012 with a plasmid encoding murine granulocyte-macrophage colony-stimulating factor (GM-CSF) increases protection against malaria, and we have characterized in detail the increased immune responses due to GM-CSF. PyCSP1012 plasmid alone protected 28% of mice, and protection increased to 58% when GM-CSF was added (p < 0.0001). GM-CSF plasmid alone did not protect, and control plasmid expressing inactive GM-CSF did not enhance protection. GM-CSF plasmid increased Abs to PyCSP of IgG1, IgG2a, and IgG2b isotypes, but not IgG3 or IgM. IFN-gamma responses of CD8+ T cells to the PyCSP 280-288 amino acid epitope increased but CTL activity did not change. The most dramatic changes after adding GM-CSF plasmid were increases in Ag-specific IL-2 production and CD4+ T cell proliferation. We hypothesize that GM-CSF may act on dendritic cells to enhance presentation of the PyCSP Ag, with enhanced IL-2 production and CD4+ T cell activation driving the increases in Abs and CD8+ T cell function. Recombinant GM-CSF is already used in humans for medical purposes, and GM-CSF protein or plasmids may be useful as enhancers of DNA vaccines.     

Role of a subdominant H-2Kd-restricted SV40 tumor antigen cytotoxic T lymphocyte epitope in tumor rejection

SV40-transformed mKSA cells (H-2d) readily induce progressively growing tumors in adult syngeneic BALB/c mice while expressing the full complement of H-2d MHC class I antigens. BALB/c mice previously immunized with SV40, soluble SV40 T antigen, or irradiated SV40-transformed syngeneic, allogeneic, or xenogeneic cells reject an mKSA tumor challenge even though these mice have been considered low- or nonresponders to T antigen due to difficulty in demonstrating SV40 T antigen-specific CTL. We have investigated the role of H-2d-restricted CTL in the rejection of SV40 tumors in BALB/c mice. Immunization of BALB/c mice with SV40 induced T antigen-specific CTL which were largely. H-2Ld-restricted. However, following repeated in vitro restimulation with mKSA cells, CTL emerged which recognized a subdominant H-2Kd-restricted epitope corresponding to T antigen residues 499-507. Immunization of BALB/c mice with a recombinant vaccinia virus expressing the T499-507 epitope provided partial protection against a challenge of syngeneic mKSA tumor cells and induced the generation of T499-507-specific CTL. These results indicate that a subdominant H-2Kd-restricted CTL epitope can participate in the rejection of SV40 tumors in BALB/c mice.     

Virus-mediated delivery of antigenic epitopes into dendritic cells as a means to induce CTL

Dendritic cells (DC) are potent inducers of CD8+ T cells and can stimulate protective antitumor immunity when pulsed with an antigenic peptide or protein. We used a replication-deficient adenovirus containing a Kb-restricted antigenic peptide of chicken OVA to study CTL induction in vitro and in vivo after adenovirus-mediated gene transfer into DC. The efficiency of adenovirus-infected DC in eliciting a specific CTL response was compared with immunizations with a recombinant vaccinia virus and DC pulsed with peptide or protein. An immortalized DC line derived from a C57BL/6 mouse and freshly isolated splenic DC from C57BL/6 mice were used in CTL induction. Virus-infected DC elicited the strongest Ag-specific CTL response in vitro and in vivo and induced protective antitumor immunity to a challenge with EG.7 tumors (EL-4 cell line expressing OVA). Direct immunization of mice with recombinant adenovirus resulted in the induction of high titers of neutralizing Abs, which precluded a boost of a CTL response after repeated inoculations. However, repeated injections of virus-infected DC induced only low titers of neutralizing Abs. Furthermore, the presence of neutralizing Abs specific for the virus did not affect the usefulness of infected DC as repeated applications of virus-infected DC boosted the CTL response even in mice previously infected with the recombinant vector. The use of DC infected with a recombinant virus has advantages over other forms of immunization and could provide an alternative approach for designing vaccination therapies.     

Immunization with a single major histocompatibility complex class I-restricted cytotoxic T-lymphocyte recognition epitope of herpes simplex virus type 2 confers protective immunity

We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8(+) cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2Kb-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8(+) T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8(+) CTL in HSV vaccine design.     

Protective efficacy against malaria of a combination sporozoite and erythrocytic stage vaccine

Most malariologists believe that optimal malaria vaccines will induce protective immune responses against different stages of the parasite's life cycle. A multiple antigen peptide (MAP) vaccine based on the Plasmodium yoelii circumsporozoite protein (PyCSP) protects mice against sporozoite challenge by inducing antibodies that prevent sporozoites from invading hepatocytes. A purified recombinant protein vaccine based on the P. yoelii merozoite surface protein-1 (PyMSP-1) protects mice against challenge with infected erythrocytes, presumably by inducing antibodies against the erythrocytic stage of the parasite. We now report studies designed to determine if the PyMSP-1 vaccine protects against challenge with sporozoites, the stage encountered in the field, and if immunization with a combination of the PyCSP and PyMSP-1 vaccines provides additive or synergistic protection against sporozoite challenge. In two experiments, using TiterMax or Ribi R-700 as adjuvant, 3 of 19 mice immunized with the PyMSP-1 vaccine were completely protected against sporozoite challenge. The remaining mice had significantly delayed onset and lower levels of peak parasitemia than did control mice (11.1 +/- 2.8% vs. 36.7 +/- 1.6% in experiment #2, P < 0.01). Immunization with the combination vaccine reduced by approximately 50% the level of antibodies induced to PyCSP and PyMSP-1, as compared to that induced by the individual components. However, in two experiments, there was evidence of additive protection. Six of 19 (31.6%) immunized with the PyCSP vaccine, 3 of 19 (15.8%) immunized with the PyMSP-1 vaccine, and 10 of 19 (52.6%) immunized with the combination were completely protected against sporozoit challenge. This modest increase in protection in the combination group may be a reflection of additive anti-PyCSP and anti-PyMSP-1 immunity, since mice in the combination group had diminished levels of antibodies to each components. These studies indicate that considerable work may be required to optimize the construction, delivery, and assessment of multi-stage malaria vaccines.     

Linear and multiple antigen peptides containing defined T and B epitopes of the Plasmodium yoelii circumsporozoite protein: antibody-mediated protection and boosting by sporozoite infection

We previously reported the identification of a T cell epitope in the N-terminal part of the circumsporozoite protein (CSP) of Plasmodium yoelii yoelii (Pyy). CD4+ T cell clones derived from mice immunized with a 21-mer peptide (amino acids 59-79, referred to as Py1) containing this epitope confer complete protection after passive transfer in mice. These clones proliferate in vitro in the presence of a 13-mer peptide (amino acids 59-71, referred to as Py1T). This shorter peptide was found to behave as a Th epitope in vivo, allowing overcoming of the genetic restriction for production of anti-repeat antibodies in BALB/c mice, when cross-linked to three (QGPGAP) repeats of the Pyy CSP. In this study, we report protection in BALB/c mice, against a challenge with Pyy sporozoites after immunization with linear and multiple antigen peptides containing Py1T as T epitope and three repeats QGPGAP (Py3) as B epitope. Multiple antigen peptide (MAP4-Py1T-Py3)-induced immunity was shown to be more effective than immunity induced by the linear form of the conjugate (Py1T-Py3), protecting against challenges with higher numbers of sporozoites. In both cases, levels of anti-repeat antibodies were strongly correlated with anti-parasite antibodies and protection. When tested in vitro, sera from mice immunized with the protective constructs strongly inhibited Pyy liver stages, while lymph node T cells displayed no cytotoxicity. In vivo, depletion of CD4+ or CD8+ T cells did not affect protection. Furthermore, MAP4-Py1T-Py3-immunized mice were not protected against a challenge with P. yoelii nigeriensis sporozoites, a parasite which has the same Py1T sequence but differs from Pyy in its repeated sequence. These results demonstrate that anti-repeat antibodies raised by immunization with the linear or the MAP form are exclusively responsible for the protection. Furthermore, this antibody response is boosted by a sporozoite challenge, allowing protection against a second challenge.     

Identification of a nonameric H-2Kk-restricted CD8+ cytotoxic T lymphocyte epitope on the Plasmodium falciparum circumsporozoite protein

Class I-restricted CD8+ cytotoxic T lymphocytes (CTL) against the circumsporozoite protein (CSP) protect mice against the rodent malaria parasite, Plasmodium yoelii, and vaccines designed to produce protective CTL against the P. falciparum CSP (PfCSP) are under development. Humans and B10.BR (H-2k) mice have been shown to have CD8+ CTL activity against a 23-amino-acid region of the PfCSP (residues 368 to 390 from the PfCSP 7G8 sequence) that is too long to bind directly to class I major histocompatibility complex molecules. To identify within this 23-amino-acid peptide a shorter peptide that binds to an H-2k class I major histocompatibility molecule, a primarily CD8+ (97.8%) T-cell line (PfCSP TCL.1) was produced by immunizing B10.BR mice with recombinant vaccinia virus expressing the PfCSP and stimulating in vitro spleen cells from these immunized mice with L cells transfected with the PfCSP gene (LPF cells). PfCSP TCL.1 lysed LPF cells and L cells pulsed with peptide PfCSP 7G8 368-390. When 15 overlapping nonamer peptides spanning the 368 to 390 sequence were tested, only one peptide, PfCSP 7G8 375-383 (Y E N D I E K K I), which includes an H-2Kk-binding motif, E at amino acid residue 2, and I at residue 9, sensitized targets for lysis by PfCSP TCL.1. Furthermore, a 10(3)- to 10(4)-fold lower concentration of the nonamer than that of the 23-amino-acid peptide was required to sensitize target cells for lysis by PfCSP TCL.1. Presentation by H-2Kk was demonstrated by using 3T3 fibroblast cells transfected with the murine H-2Kk or H-2Dk genes, and only the H-2Kk transfectants were lysed by PfCSP TCL.1 after incubation with peptide PfCSP 7G8 375-383. Binding to H-2Kk was confirmed by competitive inhibition of binding of labelled peptides to affinity-purified Kk molecules. Substitution of the anchor amino acid residue, E, at position 2 with A dramatically reduced binding to Kk and eliminated the capacity of the peptide to sensitize target cells for killing. Variation of non-anchor residues did not markedly reduce binding to Kk but in some cases eliminated the capacity of the peptide to sensitize targets for cytolysis by PfCSP TCL.1, presumably by eliminating T-cell receptor-binding sites. These data suggest that similar studies with human T cells will be required for optimal development of peptide-based vaccines designed to produce protective class I-restricted CD8+ CTL against the PfCSP in humans.     

Transcriptional and post-transcriptional regulation of interleukin-8

We have previously shown that vaccines expressing virus-derived cytotoxic-T-lymphocyte (CTL) epitopes as short minigenes can confer effective protection against virus challenges, and here we extend these studies to the bacterium Listeria monocytogenes. Host defense against this important human pathogen appears largely T cell mediated, and a nonamer CTL epitope from the listeriolysin O (LLO) protein has been identified in BALB/c mice. We have synthesized this nonamer as a minigene, expressed it in a recombinant vaccinia virus (VV-list), and used this to immunize mice. Memory CTLs cultured from VV-list-immunized mice specifically lyse target cells pulsed with a nonamer peptide identified at LLO amino acid residues 91 to 99. Four weeks postimmunization, mice were challenged with L. monocytogenes. By day 6 following challenge with a sublethal dose of L. monocytogenes, mice immunized with VV-list showed a approximately 2,000- to 6,000-fold reduction in bacteria CFU in the spleen and liver. At this time point, with control mice, bacterial were readily detectable by Gram stain of the liver but were undetectable in the VV-list-immunized animals. Additionally, when a normally lethal dose of bacteria was given, death was delayed in VV-list-immunized animals. This study has demonstrated that a single immunization with a recombinant vaccinia virus bearing only nine amino acids from a bacterial pathogen can induce specific CTLs able to confer partial protection against bacterial challenge.     

Strategy for development of a pre-erythrocytic Plasmodium falciparum DNA vaccine for human use

Data generated in the Plasmodium yoelii rodent model indicated that plasmid DNA vaccines encoding the P.yoelii circumsporozoite protein (PyCSP) or 17 kDa hepatocyte erythrocyte protein (PyHEP17) were potent inducers of protective CD8+ T cell responses directed against infected hepatocytes. Immunization with a mixture of these plasmids circumvented the genetic restriction of protective immunity and induced additive protection. A third DNA vaccine encoding the P. yoelii sporozoite surface protein 2 (PySSP2) also induced protection. The P. falciparum genes encoding the homologues of these three protective P. yoelii antigens as well as another P. falciparum gene encoding a protein that is expressed in infected hepatocytes have been chosen for the development of a human vaccine. The optimal plasmid constructs for human use will be selected on the basis of immunogenicity data generated in mice and nonhuman primates. We anticipate that optimization of multi-gene P. falciparum DNA vaccines designed to protect against malaria by inducing CD8+ T cells that target infected hepatocytes will require extensive clinical trials during the coming years.     

Lysis of CD4+ T cells expressing HIV-1 gag peptides by gag-specific CD8+ cytotoxic T cells

The vast majority of in vitro experiments testing the cytotoxic T lymphocytes (CTL) activity in HIV infection has been performed with target cells consisting of autologous EBV-transformed B lymphoblastoid cell lines (B-LCLs) expressing Human immunodeficiency virus type I (HIV-1) proteins. However data concerning the lysis of primary CD4+ T lymphocytes expressing HIV-1 antigens by CTLs is still lacking. To study the CTL activity against such primary targets, we used a system involving PBMCs of an HIV+ asymptomatic patient (PT) as effector cells and the CD4+ lymphocytes or B-LCLs of his healthy HLA-identical twin brother (HTW) as target cells. These syngeneic targets were either infected with recombinant vaccinia virus containing HIV-1 gag gene (gag-vac), or coated with HIV-1 gag peptides. We demonstrate in this study that PT CTLs (which were CD3+, CD4-, CD8+, TCRalphabeta+, TCRgammadelta-, CD56-) specifically lysed both types of syngeneic target cells expressing gag-vac; however, CD4+ T cells expressing HIV gag proteins were lysed less efficiently than B-LCLs expressing the same HIV epitopes. On the other hand, no specific lysis was detected when the target cells were uninfected or infected by wild-type vaccinia virus.     

Recombinant viruses expressing a human malaria antigen can elicit potentially protective immune CD8+ responses in mice

Extensive studies on protective immunity to rodent malaria provided the basis for the current experiments in which mice were immunized with recombinant (re) influenza and vaccinia viruses expressing selected sequences of the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum. Mice of different H-2 haplotypes immunized with re influenza viruses expressing the immunodominant B cell epitope of this CS protein produced high titers of antibodies to the parasite. A cytotoxic T lymphocyte epitope of the CS protein of P. falciparum, PF3, recognized by CD8+ T cells of H-2(k) mice, was expressed in a re vaccinia virus (VacPf) and a re influenza virus (FluPf). Immunization of mice with either FluPf or VacPf elicited a modest CS-specific CD8+ T cell response detected by interferon gamma secretion of individual immune cells. Priming of mice with FluPf, followed by a booster with VacPf, resulted in a striking enhancement of this T cell response. The reverse protocol, i.e., priming with VacPf followed by a booster with FluPf, failed to enhance the primary response. VacPf also greatly enhanced the primary response of mice injected with P. falciparum sporozoites or with a lipopeptide containing PF3. A booster with FluPf also amplified the response of lipopeptide- or sporozoite-primed mice but less than a VacPf booster did. Although mice are not susceptible to infection by P. falciparum sporozoites, we demonstrated that administration of two distinct immunogens expressing PF3 elicited activated, extravasating CS-specific T cells that protected against an intracerebral VacPf challenge.     

Intraductal papillary mucinous tumors of the pancreas: imaging studies and treatment strategies

Heat shock proteins (hsp's) isolated from murine cancer cells can elicit protective immunity and specific cytotoxic T lymphocytes (CTLs) by channeling tumor-derived peptides bound to hsp's to the major histocompatibility class I antigen presentation pathway. Here we have investigated if hsp70 can be used in a novel peptide vaccine for the induction of protective antiviral immunity and memory CTLs. A CTL epitope from the well-defined lymphocytic choriomeningitis virus (LCMV) system was mixed with recombinant hsp70 in vitro under conditions that optimize peptide binding to hsp70. Mice were immunized with the hsp70-peptide mixture and challenged with LCMV. Virus titers were reduced 10-100-fold in these mice compared to control mice. Immunization with the hsp70-peptide mixture resulted in the development of CTL memory cells that could be reactivated during LCMV infection, and that in a 51Cr-release assay could lyse cells pulsed with the same peptide, but not cells pulsed with another LCMV peptide. These results show that hsp70 can be used with CTL epitopes to induce efficient protective antiviral immunity and the generation of peptide-specific CTLs. The results also demonstrate the usefulness of hsp70 as an alternative to adjuvants and DNA vectors for the delivery of CTL epitopes to antigen-presenting cells.     

Human leukocyte antigen class I- and class II-restricted cytotoxic T lymphocyte responses to measles antigens in immune adults

The study of cytotoxic T cell (CTL) responses to measles polypeptides in persons with different HLA frequencies will provide information for the design of new vaccines. Peripheral blood mononuclear cells derived from African blacks and Caucasians were stimulated with measles virus-infected autologous cells and tested in a standard 51Cr-release assay against autologous B cells infected with vaccinia virus recombinants expressing measles virus antigens. The proportion of subjects who generated CTL to the fusion, hemagglutinin, and nucleoprotein antigens was 43%, 38%, and 28%, respectively. The use of HLA-mismatched targets showed killing to be restricted by both HLA class I and class II antigens, although CD8-mediated class I cytotoxicity predominated. Measles vaccine boosted CTL responses in subjects with low initial activity. These data suggest that the fusion and hemagglutinin proteins are important targets for the measles CTL response.     

Bystander activation of cytotoxic T cells: studies on the mechanism and evaluation of in vivo significance in a transgenic mouse model

Bystander activation, i.e., activation of T cells specific for an antigen X during an immune response against antigen Y may occur during viral infections. However, the low frequency of bystander-activated T cells has rendered it difficult to define the mechanisms and possible in vivo relevance of this nonspecific activation. This study uses transgenic mice expressing a major histocompatibility complex class I-restricted TCR specific for glycoprotein peptide 33-41 of lymphocytic choriomeningitis virus (LCMV) to overcome this limitation. CD8+ T cells from specific pathogen-free maintained, unimmunized "naive" TCR transgenic mice can differentiate into LCMV-specific cytolytic effector CTL during infections with vaccinia virus or Listeria monocytogenes in vivo or mixed lymphocyte culture in vitro. We show that in these model situations (a) nonspecifically activated CTL are able to confer antiviral protection in vivo, (b) bystander activation is largely independent of the expression of a second T cell receptor of different specificity, (c) bystander activation is not mediated by a broadly cross-reactive TCR, but rather by cytokines, (d) bystander activation can be mediated by cytokines such as IL-2, but not alpha/beta-IFN in vitro; (e) bystander activation is, overall, a rare event, occuring in vivo in roughly 1 in 200 of the LCMV-specific CTL during infection of TCR transgenic mice with vaccinia virus; (f) bystander activation does not have a significant functional impact on nontransgenic CTL memory under the conditions tested; and (g) even in the TCR transgenic situation, where unphysiologically high numbers of T cells of a single specificity are present, bystander activation is not sufficient to cause clinically manifest autoimmune disease in a transgenic mouse model of diabetes. We conclude that although bystander activation via cytokines may generate cytolytically active CTL from naive precursors, quantitative considerations suggest that this is usually not of major biological consequence.     

Induction of MHC class I-restricted CTL response by DNA immunization with ubiquitin-influenza virus nucleoprotein fusion antigens

DNA vaccines have been shown to be an effective means of inducing cytotoxic T-lymphocyte (CTL) responses in both young and aged mice. Better understanding of the pathways by which antigens encoded by DNA vaccines are processed and presented to CTL may allow for improvements in CTL responses in older animals. Since CTL recognize short peptides presented by MHC class I molecules, and since ubiquitin-dependent proteolysis is widely believed to be responsible for degradation of endogenously synthesized antigens and generation of these peptide ligands, we sought to use ubiquitin (Ub) conjugation to target influenza virus nucleoprotein (NP) antigen into the Ub-proteasome degradation pathway for MHC class I-restricted antigen processing and presentation. However, the addition of the Ub moiety did not affect the half-life of Ub-NP protein in transiently transfected human rhabdomyosarcoma (RD) cells. Moreover, the modifications of NP DNA vaccine with Ub conjugation did not affect their ability to induce a CTL response specific for the H-2Kd-restricted NP147-155 epitope, as assessed by both percent cytolysis in bulk CTL culture and by CTL precursor (CTLp) frequency in limiting dilution analysis (LDA). In contrast, the anti-NP antibody (Ab) responses were dramatically reduced in mice immunized with low doses (1 microgram) of Ub-NP constructs, compared with mice immunized with wild-type NP DNA. These results demonstrate that Ub conjugation alone does not guarantee targeting of endogenously synthesized antigens for rapid degradation by proteasomes. Furthermore, the ability of ubiquintination to reduce Ab responses to NP without affecting CTL responses suggests that the Ub modifications result in a lower availability of full-length NP from transfected cells in vivo. The implications of these data on antigen presentation and cross-priming are discussed.     

Cross-clade human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte responses in HIV-infected Zambians

We have examined cross-clade HIV-specific cytotoxic T-lymphocyte (CTL) activity in peripheral blood of eight Zambian individuals infected with non-B-clade human immunodeficiency virus type 1 (HIV-1). Heteroduplex mobility assay and partial sequence analysis of env and gag genes strongly suggests that all the HIV-infected subjects were infected with clade C HIV-1. Six of eight C-clade HIV-infected individuals elicited CTL activity specific for recombinant vaccinia virus-infected autologous targets expressing HIV gag-pol-env derived from B-clade HIV-1 (IIIB). Recognition of individual recombinant HIV-1 B-clade vaccinia virus-infected targets expressing gag, pol, or env was variable among the patients tested, indicating that cross-clade CTL activity is not limited to a single HIV protein. These data demonstrate that HIV clade C-infected individuals can mount vigorous HIV clade B-reactive CTL responses.     

Delivery of multiple CD8 cytotoxic T cell epitopes by DNA vaccination

Development of CD8 alphabeta CTL epitope-based vaccines requires an effective strategy capable of co-delivering large numbers of CTL epitopes. Here we describe a DNA plasmid encoding a polyepitope or "polytope" protein, which contained multiple contiguous minimal murine CTL epitopes. Mice vaccinated with this plasmid made MHC-restricted CTL responses to each of the epitopes, and protective CTL were demonstrated in recombinant vaccinia virus, influenza virus, and tumor challenge models. CTL responses generated by polytope DNA plasmid vaccination lasted for 1 yr, could be enhanced by co-delivering a gene for granulocyte-macrophage CSF, and appeared to be induced in the absence of CD4 T cell-mediated help. The ability to deliver large numbers of CTL epitopes using relatively small polytope constructs and DNA vaccination technology should find application in the design of human epitope-based CTL vaccines, in particular in vaccines against EBV, HIV, and certain cancers.     

Identification of Db- and Kb-restricted subdominant cytotoxic T-cell responses in lymphocytic choriomeningitis virus-infected mice

Antiviral cytotoxic T-cells are critical for control of lymphocytic choriomeningitis virus (LCMV) infection in mice. In H-2b mice, the antiviral response is directed against three Db-restricted epitopes in the viral nucleoprotein (NP396-404) and glycoprotein (GP276-286 and GP33-41). Our present data revealed a clear hierarchy among these three epitopes, in which NP396-404 is immunodominant, followed by GP33-41 and GP276-286, respectively. In order to identify additional CTL epitopes in the LCMV nucleoprotein and glycoprotein, we used the motifs for Db2- and Kb-binding peptides, combined with MHC class I-binding assays. Out of 23 Db motif-fitting peptides, we identified 4 Db binders, one of which (GP92-101) turned out to be a new CTL epitope. Among 28 Kb motif-fitting peptides, 12 bound Kb, and one of these (NP205-212) was a CTL epitope. Both newly identified CTL peptides were recognized by LCMV-immune splenocytes after secondary in vitro stimulation. Both peptides bound their MHC class I molecules with intermediate affinity (470 and 170 nM for GP92-101 and NP205-212, respectively). Responses against these peptides were weaker than the responses against the three major epitopes. None of the high affinity binders were new epitopes, suggesting that high affinity binders are either immunodominant epitopes or no epitopes at all. Thus, analysis of 51 Kb and Db motif-fitting peptides yielded 2 new, subdominant epitopes. Immunization of C57BL/6 mice with these peptides, or vaccinia virus recombinants expressing these epitopes as minigenes, protected against chronic LCMV infection, demonstrating that immunization with subdominant epitopes can confer protection against chronic viral infection.     

CD40 ligand/trimer DNA enhances both humoral and cellular immune responses and induces protective immunity to infectious and tumor challenge

CD40/CD40 ligand interactions have a central role in the induction of both humoral and cellular immunity. In this study, we examined whether a plasmid expressing CD40 ligand/trimer (CD40LT) could enhance immune responses in vivo. BALB/c mice were injected with plasmid expressing beta-galactosidase DNA with or without CD40LT DNA or IL-12 DNA, and immune responses were assessed. Mice vaccinated with beta-gal DNA plus CD40LT DNA or IL-12 DNA had a striking increase in Ag-specific production of IFN-gamma, cytolytic T cell activity, and IgG2a Ab. The mechanism by which CD40LT DNA enhanced these responses was further assessed by treating vaccinated mice with anti-IL-12 mAb or CTLA-4 Ig (CTLA4Ig). Production of IFN-gamma and CTL activity was abrogated by these treatments, suggesting that CD40LT DNA was mediating its effects on IFN-gamma and CTL activity through induction of IL-12 and enhancement of B7 expression, respectively. Physiologic relevance for the ability of CD40LT DNA to enhance immune responses by the aforementioned pathways was shown in two in vivo models. First, with regard to CTL activity, mice vaccinated with CD40LT DNA did not develop metastatic tumor following challenge with lethal dose of tumor. Moreover, in a mouse model requiring IL-12-dependent production of IFN-gamma, mice vaccinated with soluble Leishmania Ag and CD40LT DNA were able to control infection with Leishmania major. These data suggest that CD40LT DNA could be a useful vaccine adjuvant for diseases requiring cellular and/or humoral immunity.     

Cultured blood dendritic cells retain HIV-1 antigen-presenting capacity for memory CTL during progressive HIV-1 infection

Dendritic cells (DC) are potent APC that may be involved in the pathogenesis of HIV-1 infection. We studied the APC function of DC from HIV-1-infected subjects that were derived from monocyte-depleted PBMC by culture in human IL-4 and human granulocyte-macrophage CSF. The cultured cells from the HIV-1-infected subjects had similar morphology and phenotype of mature DC (CD80 = 41 +/- 8%, CD86 = 77 +/- 5%, CD40 = 87 +/- 6%, CD1a = 1 +/- 1%) to DC cultured from seronegative subjects. The yield of these DC was lower than from HIV-1-seronegative subjects (4 +/- 0% vs 11 +/- 2%, p < 0.01), and the lower DC yields correlated with lower numbers of blood CD4+ T cells (r = 0.60, p < 0.01) and higher plasma viral load (r = -0.49, p < 0.01). DC from HIV-1-infected subjects were infected with recombinant vaccinia virus vectors expressing Gag, Pol, and Env and were able to stimulate equal or higher levels of MHC class I-restricted, anti-HIV-1 memory CTL (CTLm) than were similarly treated, autologous B lymphocyte cell lines. DC pulsed with peptides representing HIV-1 CTL epitopes stimulated higher levels of anti-HIV-1 CTLm responses than did DC infected with the vaccinia virus-HIV-1 constructs. Allogeneic, MHC class I-matched DC also stimulated anti-HIV-1 CTLm activity in cells from HIV-1-infected subjects. DC from early and late stages of HIV-1 infection had a similar ability to activate CTLm specific for targets expressing either HIV-1 genes via vaccinia virus vectors or HIV-1 immunodominant synthetic peptides. However, DC from either early or late stages of HIV-1 infection could not overcome the defect in anti-HIV-1 CTLm response in advanced infection.