Human CD4+ T cell epitopes from vaccinia virus induced by vaccination or infection

Despite the importance of vaccinia virus in basic and applied immunology, our knowledge of the human immune response directed against this virus is very limited. CD4(+) T cell responses are an important component of immunity induced by current vaccinia-based vaccines, and likely will be required for new subunit vaccine approaches, but to date vaccinia-specific CD4(+) T cell responses have been poorly characterized, and CD4(+) T cell epitopes have been reported only recently. Classical approaches used to identify T cell epitopes are not practical for large genomes like vaccinia. We developed and validated a highly efficient computational approach that combines prediction of class II MHC-peptide binding activity with prediction of antigen processing and presentation. Using this approach and screening only 36 peptides, we identified 25 epitopes recognized by T cells from vaccinia-immune individuals. Although the predictions were made for HLA-DR1, eight of the peptides were recognized by donors of multiple haplotypes. T cell responses were observed in samples of peripheral blood obtained many years after primary vaccination, and were amplified after booster immunization. Peptides recognized by multiple donors are highly conserved across the poxvirus family, including variola, the causative agent of smallpox, and may be useful in development of a new generation of smallpox vaccines and in the analysis of the immune response elicited to vaccinia virus. Moreover, the epitope identification approach developed here should find application to other large-genome pathogens.     

Target Antigen of Vaccinia-Infected Cells Recognized by Virus-Specific Cytotoxic T Lymphocytes

A vaccinia-specific target antigen for recognition of anti-vaccinia cytotoxic T lymphocytes (CTL) was found to be formed on the surface of infected cells through two distinct processes. In the first phase, the expression of the target antigen was dependent on the dose of inoculated virus, without specific protein synthesis. The target antigen seems to be produced by virus-cell fusion. In the second phase, the expression of the target antigen was accompanied by synthesis of an early protein. In spite of the difference in their mode of expression, the first-phase and the second-phase target antigens were cross-reactive in cytotoxicity inhibition assays. Cowpox virus, CPR Cl strain, brought about a lower response than vaccinia virus, IHD-J strain, in both sensitization of CTL and formation of CTL-susceptible cells at both the first and the second phase. The cross-reactive, but inefficient, recognition of anti-vaccinia CTL for cowpox-infected cells suggested a slight difference in the target antigens of the two viruses. Attempts to identify the target antigen were then made by comparing the polypeptide composition of vaccinia virus, cowpox virus, and their recombinants. SDS-PAGE analysis of trypsinactivated viruses revealed 44K (cowpox)/45K (vaccinia) polypeptides which corresponded to the difference in target cell formation. Trypsinization of the viruses also increased the ability of the virus to induce the production of CTL-susceptible target cells.     

Vaccinia virus-specific CD8+ cytotoxic T lymphocytes in humans.

Stimulation of human vaccinia virus immune peripheral blood mononuclear cells in vitro from vaccinia virus-immune donors with live vaccinia virus-infected autologous cells generated vaccinia virus-specific cytotoxic T lymphocytes (CTL) capable of lysing vaccinia virus-infected cells. We generated vaccinia virus-specific CD8+ clones and CD4+ CTL lines by limiting dilution from two donors by using peripheral blood mononuclear cells obtained 2 months or 4 years postrevaccination with vaccinia virus. These results demonstrate that vaccinia virus-specific CTL are generated as a result of immunization of humans with vaccinia virus and that both CD8(+)- and CD4(+)-specific T cells are maintained as memory cells.     

The cytolytic activity of pulmonary CD8+ lymphocytes, induced by infection with a vaccinia virus recombinant expressing the M2 protein of respiratory syncytial virus (RSV), correlates with resistance to RSV infection in mice.

Previous studies demonstrated that the pulmonary resistance to respiratory syncytial virus (RSV) challenge induced by immunization with a recombinant vaccinia virus expressing the M2 protein of RSV (vac-M2) was significantly greater 9 days after immunization than at 28 days and was mediated predominantly by CD8+ T cells. In this study, we have extended these findings and sought to determine whether the level of CD8+ cytotoxic T-lymphocyte (CTL) activity measured in vitro correlates with the resistance to RSV challenge in vivo. Three lines of evidence documented an association between the presence of pulmonary CTL activity and resistance to RSV challenge. First, vac-M2 immunization induced pulmonary CD8+ CTL activity and pulmonary resistance to RSV infection in BALB/c (H-2d) mice, whereas significant levels of pulmonary CTL activity and resistance to RSV infection were not seen in BALB.K (H-2k) or BALB.B (H-2b) mice. Second, pulmonary CD8+ CTL activity was not induced by infection with other vaccinia virus-RSV recombinants that did not induce resistance to RSV challenge. Third, the peak of pulmonary CTL activity correlated with the peak of resistance to RSV replication (day 6), with little resistance being observed 45 days after immunization. An accelerated clearance of virus was not observed when mice were challenged with RSV 45 days after immunization with vac-M2. The results indicate that resistance to RSV induced by immunization with vac-M2 is mainly mediated by primary pulmonary CTLs and that this resistance decreases to very low levels within 2 months following immunization. The implications for inclusion of CTL epitopes into RSV vaccines are discussed in the context of these observations.     

Characterization of mouse hepatitis virus-specific cytotoxic T cells derived from the central nervous system of mice infected with the JHM strain.

The cytotoxic T lymphocyte (CTL) activity of spleen cells from BALB/c (H-2d) mice immunized with the neurotropic JHM strain of mouse hepatitis virus (JHMV) was stimulated in vitro for 7 days. CTL were tested for recognition of target cells infected with either JHMV or vaccinia virus recombinants expressing the four virus structural proteins. Only target cells infected with either JHMV or the vaccinia virus recombinant expressing the JHMV nucleocapsid protein were recognized. Cytotoxic T cell lines were established by limiting dilution from the brains of mice undergoing acute demyelinating encephalomyelitis after infection with JHMV. Twenty of the 22 lines recognized JHMV-infected but not uninfected syngeneic target cells, indicating that they are specific for JHMV. All T-cell lines except one were CD8+. The specificity of the CTL lines was examined by using target cells infected with vaccinia virus recombinants expressing the JHMV nucleocapsid, spike, membrane, and hemagglutinin-esterase structural proteins. Seventeen lines recognized target cells expressing the nucleocapsid protein. Three of the JHMV-specific T-cell lines were unable to recognize target cells expressing any of the JHMV structural proteins, indicating that they are specific for an epitope of a nonstructural protein(s) of JHMV. These data indicate that the nucleocapsid protein induces an immunodominant CTL response. However, no CTL activity specific for the nucleocapsid protein could be detected in either the spleens or cervical lymph nodes of mice 4, 5, 6, or 7 days after intracranial infection, suggesting that the CTL response to JHMV infection within the central nervous system may be induced or expanded locally.     

Increased per cell IFN-γ productivity indicates recent in vivo activation of T cells

Immunization with vaccinia virus causes long-term immunity. Efforts have been made to characterize the T cells responsible for this protection. Recently, T cell subsets were described that not only co-express multiple cytokines, but also show increased per cell cytokine productivity. These highly productive cells are often considered to be the most protective. We used ELISPOT assays to measure per cell IFN-γ productivity of vaccinia-specific T cells in childhood immunized adults immediately before and at different time points after vaccinia re-vaccination. Apart from an increase in frequency, we found a marked increase of IFN-γ productivity following vaccinia re-vaccination. However, these changes were short-lived as both parameters quickly returned to baseline values within 22 days after re-vaccination. Therefore, increased per cell IFN-γ productivity seems to be a sign of recent in vivo T cell activation rather than a stable marker of a distinct T cell subset responsible for long-term immune protection.     

Alveolar macrophages regulate the induction of primary cytotoxic T-lymphocyte responses during influenza virus infection.

Virus-specific cytotoxic T lymphocytes (CTL) are thought to be responsible for the eradication of respiratory influenza virus infections by direct cytolysis of virus-infected epithelial cells. In this study, we provide evidence for a role for alveolar macrophages (AM) in the regulation of pulmonary virus-specific CTL responses. Prior to infection with influenza virus, AM were selectively eliminated in vivo with a liposome-mediated depletion technique, and virus-specific CTL activities of lung and mediastinal lymph node (MLN) cells were assayed ex vivo and compared with those for normal mice. AM depletion resulted in increased primary CTL responses and changed the kinetics of the CTL response. Flow cytometric analysis of lung and MLN cells showed that the percentage of CD8+ cells was not altered after AM depletion and that lung cells from AM-depleted mice had an increased capacity to lyse virus-infected cells. Upon restimulation in vitro, virus-specific CTL activity in lung cells of normal mice was similar to that in lung cells of AM-depleted mice. Furthermore, elimination of AM resulted in increased virus titers in the lung, but virus clearance as a function of time was not affected. Our results show that AM regulate virus-specific CTL responses during respiratory influenza virus infection by removing viral particles, by downregulating the priming and activity of CTL in MLN cells, and by inhibiting the expansion of virus-specific CTL in the lung.     

Requirement for CD4 T cell help in maintenance of memory CD8 T cell responses is epitope dependent

CD4 Th cells play critical roles in stimulating Ab production and in generating primary or maintaining memory CTL. The requirement for CD4 help in generating and maintaining CTL responses has been reported to vary depending on the vector or method used for immunization. In this study, we examined the requirement for CD4 T cell help in generating and maintaining CTL responses to an experimental AIDS vaccine vector based on live recombinant vesicular stomatitis virus (VSV) expressing HIV Env protein. We found that primary CD8 T cell responses and short-term memory to HIV Env and VSV nucleocapsid (VSV N) proteins were largely intact in CD4 T cell-deficient mice. These responses were efficiently recalled at 30 days postinfection by boosting with vaccinia recombinants expressing HIV Env or VSV N. However, by 60 days postinfection, the memory/recall response to VSV N was lost in CD4-deficient mice, while the recall response HIV Env was partially maintained in the same animals for at least 90 days. This result indicates that there are epitope-specific requirements for CD4 help in the maintenance of memory CD8 T cell responses. Our results also suggest that choice of epitopes might be critical in an AIDS vaccine designed to protect against disease in the context of reduced or declining CD4 T cell help.     

Antigen-presenting T cells. II. Clonal responses of alloreactive and virus-specific self-restricted human cytotoxic T cell responses stimulated by T lymphoblasts

The capacity of various stimulator cell types to present alloantigens or viral antigens to resting human CD8+ cytotoxic lymphocyte precursors (CLP) was analyzed in a limiting dilution culture system. Cell sorter-separated T lymphoblasts of both CD4+ and CD8+ phenotypes but not resting T cells were found to efficiently stimulate the clonal development of allogeneic CD8+ CLP. Thus, 5000 CD4+ T lymphoblasts activated as many (one out of 200 to one out of 300) allogeneic CLP as 50,000 peripheral blood mononuclear stimulator cells. This potent stimulator activity was found in CD4+ and CD8+ T lymphoblasts activated by mitogen, anti-T3 monoclonal antibody, or mixed leukocyte reactions. Cytotoxic T cells generated in this system were highly specific for HLA class I antigens. Furthermore, T lymphoblasts infected with mumps virus efficiently induced development of autologous CLP into CTL clones that were virus specific and self-HLA restricted, as shown by split-well analysis. The possible in vivo significance of antigen-presenting T lymphoblasts is discussed.     

CD127+CCR5+CD38+++ CD4+ Th1 effector cells are an early component of the primary immune response to vaccinia virus and precede development of interleukin-2+ memory CD4+ T cells

The stages of development of human antigen-specific CD4+ T cells responding to viral infection and their differentiation into long-term memory cells are not well understood. The inoculation of healthy adults with vaccinia virus presents an opportunity to study these events intensively. Between days 11 and 14 postinoculation, there was a peak of proliferating CCR5+CD38+++ CD4+ effector cells which contained the cytotoxic granule marker T-cell intracellular antigen 1 and included gamma interferon (IFN-gamma)-producing vaccinia virus-specific CD4+ T cells. The majority of these initial vaccinia virus-specific CD4+ T cells were CD127+ and produced interleukin-2 (IL-2) but not CTLA-4 in response to restimulation in vitro. Between days 14 and 21, there was a switch from IFN-gamma and IL-2 coexpression to IL-2 production only, coinciding with a resting phenotype and an increased in vitro proliferation response. The early CCR5+CD38+++ vaccinia virus-specific CD4+ T cells were similar to our previous observations of human immunodeficiency virus (HIV)-specific CD4+ T cells in primary HIV type 1 (HIV-1) infection, but the vaccinia virus-specific cells expressed much more CD127 and IL-2 than we previously found in their HIV-specific counterparts. The current study provides important information on the differentiation of IL-2+ vaccinia virus-specific memory cells, allowing further study of antiviral effector CD4+ T cells in healthy adults and their dysfunction in HIV-1 infection.     

Proliferative and cytotoxic T cells to AIDS virus glycoproteins in chimpanzees immunized with a recombinant vaccinia virus expressing AIDS virus envelope glycoproteins

PBL from chimpanzees immunized with a recombinant vaccinia virus that expresses HIV envelope glycoproteins ("env"), were found to proliferate after stimulation with HIV or with "env". Furthermore, CTL clones lytic for autologous target cells expressing HIV envelope glycoproteins were generated after stimulation of the chimpanzees' PBL with "env", indicating that immunization of these primates with a recombinant vaccinia virus primes HIV-specific CTL and also that HIV envelope glycoproteins serve as target antigens for CTL.     

In vitro studies of the rabbit immune system. VI. Rabbit anti-mouse cytotoxic T effector cells are inhibited by anti-rabbit T cell serum in the absence of complement.

Xenogeneic rabbit anti-mouse cell-mediated cytotoxic activity could be generated by culturing lymphoid cells from mesenteric lymph nodes (MLN), spleen, or peripheral blood of rabbits primed 2 to 8 weeks earlier with mouse tumor or spleen cells. MLN cells, which provided the best source of activity after being cultured with 5 to 10 X 10(6) mitomycin C-treated mouse spleen cells for 4 to 6 days, produced 30 to 90% specific isotope release after 4 to 7 hr incubation with 15Cr-labeled tumor target cells. Xenogeneic cytotoxic activity was primarily H-2 specific and could not be blocked by immune complexes but was abrogated by treatment with goat anti-rabbit thymocyte serum plus complement (ATS + C) before or after culture. Therefore, the activity appeared to be mediated by cytotoxic T lymphocytes (CTL). Furthermore, ATS without C abrogated cytotoxic activity when included in the CTL assay at concentrations of 5 to 15 microliter/10(7) effector cells. The inhibitory activity of ATS was directed to the rabbit effector population and could be absorbed completely by rabbit thymocytes. Antisera to mouse T cells with comparable cytolytic activity in the presence of C did not inhibit murine allogeneic CTL.     

Primary pulmonary cytotoxic T lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza A virus nucleoprotein peptide do not protect mice against challenge.

The nucleoprotein (NP) of influenza A virus is the dominant antigen recognized by influenza virus-specific cytotoxic T lymphocytes (CTLs), and adoptive transfer of NP-specific CTLs protects mice from influenza A virus infection. BALB/c mouse cells (H-2d) recognize a single Kd-restricted CTL epitope of NP consisting of amino acids 147 to 155. In the present study, mice were immunized with various vaccinia virus recombinant viruses to examine the effect of the induction of primary pulmonary CTLs on resistance to challenge with influenza A/Puerto Rico/8/34 virus. The minigene ESNP(147-155)-VAC construct, composed of a signal sequence from the adenovirus E3/19K glycoprotein (designated ES) and expressing the 9-amino-acid NP natural determinant (amino acids 147 to 155) preceded by an alanine residue, a similar minigene NP(Met 147-155)-VAC lacking ES, and a full-length NP-VAC recombinant of influenza virus were analyzed. The two minigene NP-VAC recombinants induced a greater primary pulmonary CTL response than the full-length NP-VAC recombinant. However, NP-specific CTLs induced by immunization with ESNP(147-155)-VAC did not decrease peak virus titer or accelerate clearance of virus in the lungs of mice challenged intranasally with A/PR/8/34. Furthermore, NP-specific CTLs induced by immunization did not protect mice challenged intranasally with a lethal dose of A/PR/8/34. Sequence analysis of the NP CTL epitope of A/PR/8/34 challenge virus obtained from lungs after 8 days of replication in ESNP(147-155)-VAC-immunized mice showed identity with that of the input virus, demonstrating that an escape mutant had not emerged during replication in vivo. Thus, in contrast to adoptively transferred CTLs, pulmonary NP-specific CTLs induced by recombinant vaccinia virus immunization do not have protective in vivo antiviral activity against influenza virus infection.     

Recognition of human T cell leukemia virus type I (HTLV-I) gag and pX gene products by MHC-restricted cytotoxic T lymphocytes induced in rats against syngeneic HTLV-I-infected cells

We established rat T cell lines expressing human T cell leukemia virus type I (HTLV-I) Ag from inbred strains of rats, WKA/H, DA, and F344, to study CTL response against the HTLV-I-infected cells. HTLV-I-specific Ag expressed in these rat cells were HTLV-I gag Ag, p19, p24, and p15, and pX Ag, p40tax and p27rex, but not env Ag, as determined by immunofluorescence and immunoblot assays. By immunization of rats with syngeneic HTLV-I-infected cells, CTL against syngeneic HTLV-I-infected cells and antibodies to HTLV-I Ag were generated in WKA/H and DA rats. The bulk CTL cultures from WKA/H and DA rats lysed specifically syngeneic SV40-transformed kidney cells infected with recombinant vaccinia viruses (RVV) expressing HTLV-I gag and pX Ag, but not those infected with RVV expressing HTLV-I env Ag or a control vaccinia virus. From WKA/H rat CTL cultures, four CTL clones reactive with syngeneic HTLV-I-infected cells were isolated, three of which were specific for p27rex/p21x, but the Ag recognized by the other CTL clone was not defined with any RVV used. These results indicate that HTLV-I gag and pX gene products are recognized by MHC-restricted rat CTL specific for syngeneic HTLV-I-infected cells.     

Immunization with a modified vaccinia virus expressing simian immunodeficiency virus (SIV) Gag-Pol primes for an anamnestic Gag-specific cytotoxic T-lymphocyte response and is associated with reduction of viremia after SIV challenge

The immunogenicity and protective efficacy of a modified vaccinia virus Ankara (MVA) recombinant expressing the simian immunodeficiency virus (SIV) Gag-Pol proteins (MVA-gag-pol) was explored in rhesus monkeys expressing the major histocompatibility complex (MHC) class I allele, MamuA*01. Macaques received four sequential intramuscular immunizations with the MVA-gag-pol recombinant virus or nonrecombinant MVA as a control. Gag-specific cytotoxic T-lymphocyte (CTL) responses were detected in all MVA-gag-pol-immunized macaques by both functional assays and flow cytometric analyses of CD8(+) T cells that bound a specific MHC complex class I-peptide tetramer, with levels peaking after the second immunization. Following challenge with uncloned SIVsmE660, all macaques became infected; however, viral load set points were lower in MVA-gag-pol-immunized macaques than in the MVA-immunized control macaques. MVA-gag-pol-immunized macaques exhibited a rapid and substantial anamnestic CTL response specific for the p11C, C-M Gag epitope. The level at which CTL stabilized after resolution of primary viremia correlated inversely with plasma viral load set point (P = 0.03). Most importantly, the magnitude of reduction in viremia in the vaccinees was predicted by the magnitude of the vaccine-elicited CTL response prior to SIV challenge.     

DNA Immunization with Minigenes: Low Frequency of Memory Cytotoxic T Lymphocytes and Inefficient Antiviral Protection Are Rectified by Ubiquitination

Our previous studies have shown that isolated cytotoxic T lymphocyte (CTL), B-cell, and T-helper epitopes, for which we coined the term minigenes, can be effective vaccines; when expressed from recombinant vaccinia viruses, these short immunogenic sequences confer protection against a variety of viruses and bacteria. In addition, we have previously demonstrated the utility of DNA immunization using plasmids encoding full-length viral proteins. Here we combine the two approaches and evaluate the effectiveness of minigenes in DNA immunization. We find that DNA immunization with isolated minigenes primes virus-specific memory CTL responses which, 4 days following virus challenge, appear similar in magnitude to those induced by vaccines known to be protective. Surprisingly, this vigorous CTL response fails to confer protection against a normally lethal virus challenge, although the CTL appear fully functional because, along with their high lytic activity, they are similar in affinity and cytokine secretion to CTL induced by virus infection. However this DNA immunization with isolated minigenes results in a low CTL precursor frequency; only 1 in ~40,000 T cells is epitope specific. In contrast, a plasmid encoding the same minigene sequences covalently attached to the cellular protein ubiquitin induces protective immunity and a sixfold-higher frequency of CTL precursors. Thus, we show that the most commonly employed criterion to evaluate CTL responses—the presence of lytic activity following secondary stimulation—does not invariably correlate with protection; instead, the better correlate of protection is the CTL precursor frequency. Recent observations indicate that certain effector functions are active in memory CTL and do not require prolonged stimulation. We suggest that these early effector functions of CTL, immediately following infection, are critical in controlling virus dissemination and in determining the outcome of the infection. Finally, we show that improved performance of the ubiquitinated minigenes most probably requires polyubiquitination of the fusion protein, suggesting that the enhancement results from more effective delivery of the minigene to the proteasome.     

Dengue virus-specific cross-reactive CD8+ human cytotoxic T lymphocytes

Stimulation with live dengue virus of peripheral blood mononuclear cells from a dengue virus type 4-immune donor generated virus-specific, serotype-cross-reactive, CD8+, class I-restricted cytotoxic T lymphocytes (CTL) capable of lysing dengue virus-infected cells and cells pulsed with dengue virus antigens of all four serotypes. These CTL lysed autologous fibroblasts infected with vaccinia virus-dengue virus recombinant viruses containing the E gene or several nonstructural dengue virus type 4 genes. These results demonstrate that both dengue virus structural and nonstructural proteins are targets for the cytotoxic T-cell-mediated immune response to dengue virus and suggest that serotype-cross-reactive CD8+ CTL may be important mediators of viral clearance and of virus-induced immunopathology during secondary dengue virus infections.     

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

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

Plasmid DNA vaccine encoding prostatic acid phosphatase is effective in eliciting autologous antigen-specific CD8+ T cells

Prostatic acid phosphatase (PAP) is a prostate cancer tumor antigen and a prostate-specific protein shared by rats and humans. Previous studies indicated that Copenhagen rats immunized with a recombinant vaccinia virus expressing human PAP (hPAP) developed PAP-specific cytotoxic T cells (CTL) with cross reactivity to rat PAP (rPAP) and evidence of prostate inflammation. Viral delivery of vaccine antigens is an active area of clinical investigation. However, a potential difficulty with viral-based immunizations is that immune responses elicited to the viral vector might limit the possibility of multiple immunizations. In this paper, we investigate the ability of another genetic immunization method, a DNA vaccine encoding PAP, to elicit antigen-specific CD8+ T cell immune responses. Specifically, Lewis rats were immunized with either a plasmid DNA-based (pTVG-HP) or vaccinia-based (VV-HP) vaccine each encoding hPAP. We determined that rats immunized with a DNA vaccine encoding hPAP developed a Th1-biased immune response as indicated by proliferating PAP-specific CD4+ and CD8+ cells and IFNγ production. Rats immunized with vaccinia virus encoding PAP did not develop a PAP-specific response unless boosted with a heterologous vaccination scheme. Most importantly, multiple immunizations with a DNA vaccine encoding the rat PAP homologue (pTVG-RP) could overcome peripheral self-tolerance against rPAP and generate a Th1-biased antigen-specific CD4+ and CD8+ T cell response. Overall, DNA vaccines provide a safe and effective method of generating prostate antigen-specific T cell responses. These findings support the investigation of PAP-specific DNA vaccines in human clinical trials.