CD8+ cellular immunity mediates rAd5 vaccine protection against Ebola virus infection of nonhuman primates

Vaccine-induced immunity to Ebola virus infection in nonhuman primates (NHPs) is marked by potent antigen-specific cellular and humoral immune responses; however, the immune mechanism of protection remains unknown. Here we define the immune basis of protection conferred by a highly protective recombinant adenovirus virus serotype 5 (rAd5) encoding Ebola virus glycoprotein (GP) in NHPs. Passive transfer of high-titer polyclonal antibodies from vaccinated Ebola virus-immune cynomolgus macaques to naive macaques failed to confer protection against disease, suggesting a limited role of humoral immunity. In contrast, depletion of CD3(+) T cells in vivo after vaccination and immediately before challenge eliminated immunity in two vaccinated macaques, indicating a crucial requirement for T cells in this setting. The protective effect was mediated largely by CD8(+) cells, as depletion of CD8(+) cells in vivo using the cM-T807 monoclonal antibody (mAb), which does not affect CD4(+) T cell or humoral immune responses, abrogated protection in four out of five subjects. These findings indicate that CD8(+) cells have a major role in rAd5-GP-induced immune protection against Ebola virus infection in NHPs. Understanding the immunologic mechanism of Ebola virus protection will facilitate the development of vaccines for Ebola and related hemorrhagic fever viruses in humans.     

Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity

There is currently a need for vaccines that stimulate cell-mediated immunity-particularly that mediated by CD8+ cytotoxic T lymphocytes (CTLs)-against viral and tumor antigens. The optimal induction of cell-mediated immunity requires the presentation of antigens by specialized cells of the immune system called dendritic cells (DCs). DCs are unique in their ability to process exogenous antigens via the major histocompatibility complex (MHC) class I pathway as well as in their ability to activate naive, antigen-specific CD8+ and CD4+ T cells. Vaccine strategies that target or activate DCs in order to elicit potent CTL-mediated immunity are the subject of intense research. We report here that whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens potently induced antigen-specific, CTL responses, including those mediating tumor protection, in vaccinated animals. Interactions between yeast and DCs led to DC maturation, IL-12 production and the efficient priming of MHC class I- and class II-restricted, antigen-specific T-cell responses. Yeast exerted a strong adjuvant effect, augmenting DC presentation of exogenous whole-protein antigen to MHC class I- and class II-restricted T cells. Recombinant yeast represent a novel vaccine strategy for the induction of broad-based cellular immune responses.     

Multiplex Identification of Antigen-Specific T Cell Receptors Using a Combination of Immune Assays and Immune Receptor Sequencing

Monitoring antigen-specific T cells is critical for the study of immune responses and development of biomarkers and immunotherapeutics. We developed a novel multiplex assay that combines conventional immune monitoring techniques and immune receptor repertoire sequencing to enable identification of T cells specific to large numbers of antigens simultaneously. We multiplexed 30 different antigens and identified 427 antigen-specific clonotypes from 5 individuals with frequencies as low as 1 per million T cells. The clonotypes identified were validated several ways including repeatability, concordance with published clonotypes, and high correlation with ELISPOT. Applying this technology we have shown that the vast majority of shared antigen-specific clonotypes identified in different individuals display the same specificity. We also showed that shared antigen-specific clonotypes are simpler sequences and are present at higher frequencies compared to non-shared clonotypes specific to the same antigen. In conclusion this technology enables sensitive and quantitative monitoring of T cells specific for hundreds or thousands of antigens simultaneously allowing the study of T cell responses with an unprecedented resolution and scale.     

Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans

Protective immunity against Mycobacterium tuberculosis depends on the generation of a T(H)1-type cellular immune response, characterized by the secretion of interferon-gamma (IFN-gamma) from antigen-specific T cells. The induction of potent cellular immune responses by vaccination in humans has proven difficult. Recombinant viral vectors, especially poxviruses and adenoviruses, are particularly effective at boosting previously primed CD4(+) and CD8(+) T-cell responses against a number of intracellular pathogens in animal studies. In the first phase 1 study of any candidate subunit vaccine against tuberculosis, recombinant modified vaccinia virus Ankara (MVA) expressing antigen 85A (MVA85A) was found to induce high levels of antigen-specific IFN-gamma-secreting T cells when used alone in bacille Calmette-Guerin (BCG)-naive healthy volunteers. In volunteers who had been vaccinated 0.5-38 years previously with BCG, substantially higher levels of antigen-specific IFN-gamma-secreting T cells were induced, and at 24 weeks after vaccination these levels were 5-30 times greater than in vaccinees administered a single BCG vaccination. Boosting vaccinations with MVA85A could offer a practical and efficient strategy for enhancing and prolonging antimycobacterial immunity in tuberculosis-endemic areas.     

Enrichment of an antigen-specific T cell response by retrovirally transduced human dendritic cells.

The superior ability of dendritic cells (DC) in triggering antigen-specific T cell responses makes these cells attractive tools for the generation of antitumor or antiviral immunity. We report here an efficient retroviral transduction system for the introduction of antigens into DC. A retroviral vector encoding several CTL epitopes in a string-of-beads fashion in combination with the marker gene green fluorescence protein (GFP) was generated. Polyepitope transduced EBV-LCL could be isolated on the basis of GFP expression and were found to be sensitive to lysis by antigen-specific cytotoxic T cells, demonstrating that antigens encoded by the retroviral construct were stably expressed, processed, and presented in the context of HLA class I molecules. CD34(+) cells isolated from G-CSF mobilized peripheral blood were transduced with high efficiency (40-60%) with this retroviral construct. These cells could be considerably expanded in vitro and differentiated into mature DC without loss of the transduced antigen. DC transduced with the polyepitope constructs were able to mount a CTL response against an influenza epitope in the context of HLA-A2, demonstrating the antigen-specific CTL priming capacity of retrovirally transduced DC. Staining of the T cells with tetramers of HLA-A2 and the influenza virus peptide demonstrated a marked antigen-specific CTL enrichment after 2 in vitro stimulations using DC transduced with the polyepitope. However, additional in vitro stimulations of the T cells with transduced DC did not result in a further enrichment of tetramer staining cells.     

Antigen-specific T-cell induction by vaccination with a recombinant Sendai virus vector even in the presence of vector-specific neutralizing antibodies in rhesus macaques

Recombinant viral vectors are promising vaccine tools for eliciting potent cellular immune responses against immunodeficiency virus infection, but pre-existing anti-vector antibodies can be an obstacle to their clinical use in humans. We have previously vaccinated rhesus macaques with a recombinant Sendai virus (SeV) vector twice at an interval of more than 1 year and have shown efficient antigen-specific T-cell induction by the second as well as the first vaccination. Here, we have established the method for measurement of SeV-specific neutralizing titers and have found efficient SeV-specific neutralizing antibody responses just before the second SeV vaccination in these macaques. This suggests the feasibility of inducing antigen-specific T-cell responses by SeV vaccination even in the host with pre-existing anti-SeV neutralizing antibodies.     

Th-1-type cytotoxic CD8+ T-lymphocyte responses to simian immunodeficiency virus (SIV) are a consistent feature of natural SIV infection in sooty mangabeys

Sooty mangabeys are a natural host of simian immunodeficiency virus (SIV) that remain asymptomatic and do not exhibit increased immune activation or increased T-lymphocyte turnover despite sustained high levels of SIV viremia. In this study we asked whether an altered immune response to SIV contributes to the lack of immunopathology in sooty mangabeys as opposed to species with pathogenic lentivirus infection. SIV-specific cellular immune responses were investigated in a cohort of 25 sooty mangabeys with natural SIV infection. Gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay responses targeting a median of four SIV proteins were detected in all 25 mangabeys and were comparable in magnitude to those of 13 rhesus macaques infected with SIVmac251 for more than 6 months. As with rhesus macaques, Th2 ELISPOT responses to SIV were absent or >10-fold lower than the IFN-gamma ELISPOT response to the same SIV protein. The SIV-specific ELISPOT response was predominantly mediated by CD8+ T lymphocytes; the frequency of circulating SIV-specific CD8+ T lymphocytes ranged between 0.11% and 3.26% in 13 mangabeys. Functionally, the SIV-specific CD8+ T lymphocytes were cytotoxic; secreted IFN-gamma, tumor necrosis factor alpha, and macrophage inflammatory protein 1beta; and had an activated effector phenotype. Although there was a trend toward higher frequencies of SIV-specific CD8+ T lymphocytes in mangabeys with lower viral loads, a significant inverse correlation between SIV viremia and SIV-specific cellular immunity was not detected. The consistent detection of Th1-type SIV-specific cellular immune responses in naturally infected sooty mangabeys suggests that immune attenuation is neither a feature of nor a requirement for maintenance of nonpathogenic SIV infection in its natural host.     

Control of simian/human immunodeficiency virus viremia and disease progression after IL-2-augmented DNA-modified vaccinia virus Ankara nasal vaccination in nonhuman primates

A successful HIV vaccine may need to stimulate antiviral immunity in mucosal and systemic immune compartments, because HIV transmission occurs predominantly at mucosal sites. We report here the results of a combined DNA-modified vaccinia virus Ankara (MVA) vaccine approach that stimulated simian/human immunodeficiency virus (SHIV)-specific immune responses by vaccination at the nasal mucosa. Fifteen male rhesus macaques, divided into three groups, received three nasal vaccinations on day 1, wk 9, and wk 25 with a SHIV DNA plasmid producing noninfectious viral particles (group 1), or SHIV DNA plus IL-2/Ig DNA (group 2), or SHIV DNA plus IL-12 DNA (group 3). On wk 33, all macaques were boosted with rMVA expressing SIV Gag-Pol and HIV Env 89.6P, administered nasally. Humoral responses were evaluated by measuring SHIV-specific IgG and neutralizing Abs in plasma, and SHIV-specific IgA in rectal secretions. Cellular responses were monitored by evaluating blood-derived virus-specific IFN-gamma-secreting cells and TNF-alpha-expressing CD8+ T cells, and blood- and rectally derived p11C tetramer-positive T cells. Many of the vaccinated animals developed both mucosal and systemic humoral and cell-mediated anti-SHIV immune responses, although the responses were not homogenous among animals in the different groups. After rectal challenge of vaccinated and naive animals with SHIV89.6P, all animals became infected. However a subset, including all group 2 animals, were protected from CD4+ T cell loss and AIDS development. Taken together, these data indicate that nasal vaccination with SHIV-DNA plus IL-2/Ig DNA and rMVA can provide significant protection from disease progression.     

Both mucosal and systemic routes of immunization with the live,attenuated NYVAC/simian immunodeficiency virus SIV(gpe) recombinant vaccine result in gag-specific CD8(+) T-cell responses in mucosal tissues of macaques

As most human immunodeficiency virus (HIV) infection occurs via mucosal surfaces, an important goal of vaccination may be the induction of virus-specific immune responses at mucosal sites to contain viral infection early on. Here we designed a study in macaques carrying the major histocompatibility complex class I Mamu-A(*)01 molecule to assess the capacity of the highly attenuated poxvirus NYVAC/simian immunodeficiency virus (SIV) SIV(gpe) vaccine candidate administered by the intranasal, intramuscular, or intrarectal route to induce mucosal immunity. All macaques, including one naive macaque, were exposed to SIV(mac251) by the intrarectal route and sacrificed 48 h after infection. The kinetics of immune response at various time points following immunization with NYVAC/SIV(gpe) and the anamnestic response to SIV(mac251) at 48 h after challenge were assessed in blood, in serial rectal and vaginal biopsy samples, and in tissues at euthanasia with an SIV(mac) Gag-specific tetramer. In addition, at euthanasia, antigen-specific cells producing gamma interferon or tumor necrosis factor alpha from the jejunum lamina propria were quantified in all macaques. Surprisingly, antigen-specific CD8(+) T cells were found in the mucosal tissues of all immunized macaques regardless of whether the vaccine was administered by a mucosal route (intranasal or intrarectal) or systemically. In addition, following mucosal SIV(mac251) challenge, antigen-specific responses were mainly confined to mucosal tissues, again regardless of the route of immunization. We conclude that immunization with a live vector vaccine results in the appearance of CD8(+) T-cell responses at mucosal sites even when the vaccine is delivered by nonmucosal routes.     

Enhancement of anti-tumor immunity specific to murine glioma by vaccination with tumor cell lysate-pulsed dendritic cells engineered to produce interleukin-12

Aim: The aim of this study was to develop an immunotherapy specific to a malignant glioma by examining the efficacy of glioma tumor-specific cytotoxic T lymphocytes (CTL) as well as the anti-tumor immunity by vaccination with dendritic cells (DC) engineered to express murine IL-12 using adenovirus-mediated gene transfer and pulsed with a GL26 glioma cell lysate (AdVIL-12/DC+GL26) was investigated. Experimentl: For measuring CTL activity, splenocytes were harvested from the mice immunized with AdVIL-12/DC+GL26 and restimulated with syngeneic GL26 for 7 days. The frequencies of antigen-specific cytokine-secreting T cell were determined with mIFN-γ ELISPOT. The cytotoxicity of CTL was assessed in a standard 51Cr-release assay. For the protective study in the subcutaneous tumor model, the mice were vaccinated subcutaneously (s.c) with 1×10⁶ AdVIL-12/DC+GL26 in the right flanks on day −21, −14 and −7. On day 7, the mice were challenged with 1×10⁶ GL26 tumor cells in the shaved left flank. For a protective study in the intracranial tumor model, the mice were vaccinated with 1×10⁶ AdVIL-12/DC+GL26 s.c in the right flanks on days −21, −14 and −7. Fresh 1×10⁴ GL26 cells were inoculated into the brain on day 0. To prove a therapeutic benefit in established tumors, subcutaneous or intracranial GL26 tumor-bearing mice were vaccinated s.c with 1×10⁶ AdVIL-12/DC+GL26 on day 5, 12 and 19 after tumor cell inoculation. Results: Splenocytes from the mice vaccinated with the AdVIL-12/DC+GL26 showed enhanced induction of tumor-specific CTL and increased numbers of IFN-γ: secreting T cells by ELISPOT. Moreover, vaccination of AdVIL-12/DC+GL26 enhanced the induction of anti-tumor immunity in both the subcutaneous and intracranial tumor models. Conclusions: These preclinical model results suggest that DC engineered to express IL-12 and pulsed with a tumor lysate could be used in a possible immunotherapeutic strategy for malignant glioma.Korea Research Foundation Grant (KRF-2004-005-E00001).     

Memory immune responses against pandemic (H1N1) 2009 influenza virus induced by a whole particle vaccine in cynomolgus monkeys carrying Mafa-A1*052:02

We made an H1N1 vaccine candidate from a virus library consisting of 144 (?=?16 HA×9 NA) non-pathogenic influenza A viruses and examined its protective effects against a pandemic (2009) H1N1 strain using immunologically na?ve cynomolgus macaques to exclude preexisting immunity and to employ a preclinical study since preexisting immunity in humans previously vaccinated or infected with influenza virus might make comparison of vaccine efficacy difficult. Furthermore, macaques carrying a major histocompatibility complex class I molecule, Mafa-A1*052:02, were used to analyze peptide-specific CD8(+) T cell responses. Sera of macaques immunized with an inactivated whole particle formulation without addition of an adjuvant showed higher neutralization titers against the vaccine strain A/Hokkaido/2/1981 (H1N1) than did sera of macaques immunized with a split formulation. Neutralization activities against the pandemic strain A/Narita/1/2009 (H1N1) in sera of macaques immunized twice with the split vaccine reached levels similar to those in sera of macaques immunized once with the whole particle vaccine. After inoculation with the pandemic virus, the virus was detected in nasal samples of unvaccinated macaques for 6 days after infection and for 2.67 days and 5.33 days on average in macaques vaccinated with the whole particle vaccine and the split vaccine, respectively. After the challenge infection, recall neutralizing antibody responses against the pandemic virus and CD8(+) T cell responses specific for nucleoprotein peptide NP262-270 bound to Mafa-A1*052:02 in macaques vaccinated with the whole particle vaccine were observed more promptly or more vigorously than those in macaques vaccinated with the split vaccine. These findings demonstrated that the vaccine derived from our virus library was effective for pandemic virus infection in macaques and that the whole particle vaccine conferred more effective memory and broader cross-reactive immune responses to macaques against pandemic influenza virus infection than did the split vaccine.     

A replication competent adenovirus 5 host range mutant-simian immunodeficiency virus (SIV) recombinant priming/subunit protein boosting vaccine regimen induces broad,persistent SIV-specific cellular immunity to dominant and subdominant epitopes in Mamu-A*01 rhesus macaques

CTL are important in controlling HIV and SIV infection. To quantify cellular immune responses induced by immunization, CD8(+) T cells specific for the subdominant Env p15m and p54m epitopes and/or the dominant Gag p11C epitope were evaluated by tetramer staining in nine macaques immunized with an adenovirus (Ad) 5 host range mutant (Ad5hr)-SIVenv/rev recombinant and in four of nine which also received an Ad5hr-SIVgag recombinant. Two Ad5hr-SIV recombinant priming immunizations were followed by two boosts with gp120 protein or an envelope polypeptide representing the CD4 binding domain. Two mock-immunized macaques served as controls. IFN-gamma-secreting cells were also assessed by ELISPOT assay using p11C, p15m, and p54m peptide stimuli and overlapping pooled Gag and Env peptides. As shown by tetramer staining, Ad-recombinant priming elicited a high frequency of persistent CD8(+) T cells able to recognize p11C, p15m, and p54m epitopes. The presence of memory cells 38 wk postinitial immunization was confirmed by expansion of tetramer-positive CD8(+) T cells following in vitro stimulation. The SIV-specific CD8(+) T cells elicited were functional and secreted IFN-gamma in response to SIV peptide stimuli. Although the level and frequency of response of peripheral blood CD8(+) T cells to the subdominant Env epitopes were not as great as those to the dominant p11C epitope, elevated responses were observed when lymph node CD8(+) T cells were evaluated. Our data confirm the potency and persistence of functional cellular immune responses elicited by replication competent Ad-recombinant priming. The cellular immunity elicited is broad and extends to subdominant epitopes.     

Adenovirus-transduced dendritic cells injected into skin or lymph node prime potent simian immunodeficiency virus-specific T cell immunity in monkeys

Adenoviral vectors can be used to deliver complex Ag to dendritic cells (DC), and thus may be ideal for stimulating broad T cell responses to viral pathogens and tumors. To test this hypothesis in a relevant primate model, we used recombinant adenovirus serotype 5 vectors expressing SIV Gag Ag to transduce monocyte-derived DC from rhesus macaques, and then immunized donor animals either by intradermal or intranodal injections. T cell responses were evaluated by ELISPOT assay using previously frozen PBMC pulsed with pools of 15-mer peptides representing the Gag sequence. Immunization resulted in rapid and potent induction of T cell responses to multiple regions of Gag, with frequencies approaching 1 Gag-specific T cell per 500 uncultured PBMC. Surprisingly, intradermal and intranodal injections generated a similar intensity and breadth of response, indicating that administration of Ag-expressing DC by either route may be equally effective at inducing immune responses. Detailed analysis of two monkeys revealed CD8(+) T cell responses to several peptide epitopes of Gag not previously described, at least two of which are restricted by MHC class I alleles not currently identified. Repeated vaccination did not induce T cell responses to the adenoviral vector and did not prevent Ag-expressing DC injected under the capsule of the lymph node from migrating to the paracortex and interposing between T cells. However, boost injections of adenovirus-transduced DC were generally limited in efficacy. These findings support the use of adenovirus-transduced DC in the therapy of HIV infection and cancer.     

Antiviral antibodies and T cells are present in the foreskin of simian immunodeficiency virus-infected rhesus macaques

No information exists regarding immune responses to human immunodeficiency virus (HIV) infection in the foreskin or glans of the human penis, although this is a key tissue for HIV transmission. To address this gap, we characterized antiviral immune responses in foreskin of male rhesus macaques (RMs) inoculated with simian immunodeficiency virus (SIV) strain SIVmac251 by penile foreskin exposure. We found a complete population of immune cells in the foreskin and glans of normal RMs, although B cells were less common than CD4(+) and CD8(+) T cells. IgG-secreting cells were detected by enzyme-linked immunospot (ELISPOT) assay in cell suspensions made from the foreskin. In the foreskin and glans of SIV-infected RMs, although B cells were less common than CD4(+) and CD8(+) T cells, SIV-specific IgG antibody was present in foreskin secretions. In addition, cytokine-secreting SIV-specific CD8(+) T cells were readily found in cell suspensions made from the foreskin. Although potential HIV target cells were found in and under the epithelium covering all penile surfaces, the presence of antiviral effector B and T cells in the foreskin suggests that vaccines may be able to elicit immunity in this critical site to protect men from acquiring HIV.     

Improving dendritic cell vaccine immunogenicity by silencing PD-1 ligands using siRNA-lipid nanoparticles combined with antigen mRNA electroporation

Dendritic cell (DC)-based vaccination boosting antigen-specific immunity is being explored for the treatment of cancer and chronic viral infections. Although DC-based immunotherapy can induce immunological responses, its clinical benefit has been limited, indicating that further improvement of DC vaccine potency is essential. In this study, we explored the generation of a clinical-grade applicable DC vaccine with improved immunogenic potential by combining PD-1 ligand siRNA and target antigen mRNA delivery. We demonstrated that PD-L1 and PD-L2 siRNA delivery using DLin-KC2-DMA-containing lipid nanoparticles (LNP) mediated efficient and specific knockdown of PD-L expression on human monocyte-derived DC. The established siRNA-LNP transfection method did not affect DC phenotype or migratory capacity and resulted in acceptable DC viability. Furthermore, we showed that siRNA-LNP transfection can be successfully combined with both target antigen peptide loading and mRNA electroporation. Finally, we demonstrated that these PD-L-silenced DC loaded with antigen mRNA superiorly boost ex vivo antigen-specific CD8⁺ T cell responses from transplanted cancer patients. Together, these findings indicate that our PD-L siRNA-LNP-modified DC are attractive cells for clinical-grade production and in vivo application to induce and boost immune responses not only in transplanted cancer patients, but likely also in other settings.     

Dendritic cells directly trigger NK cell functions: cross-talk relevant in innate anti-tumor immune responses in vivo

Cytotoxic T lymphocytes and natural killer cells are essential effectors of anti-tumor immune responses in vivo. Dendritic cells (DC) 'prime' tumor antigen-specific cytotoxic T lymphocytes; thus, we investigated whether DC might also trigger the innate, NK cell-mediated anti-tumor immunity. In mice with MHC class I-negative tumors, adoptively transferred- or Flt3 ligand-expanded DC promoted NK cell-dependent anti-tumor effects. In vitro studies demonstrated a cell-to-cell contact between DC and resting NK cells that resulted in a substantial increase in both NK cell cytolytic activity and IFN-gamma production. Thus, DC are involved in the interaction between innate and adaptive immune responses.     

Effect of preexisting immunity to adenovirus human serotype 5 antigens on the immune responses of nonhuman primates to vaccine regimens based on human- or chimpanzee-derived adenovirus vectors

In this study we compared a prime-boost regimen with two serologically distinct replication-defective adenovirus (Ad) vectors derived from chimpanzee serotypes C68 and C1 expressing Gag, Pol, gp140, and Nef of human immunodeficiency virus type 1 with a regimen in which replication-defective Ad vectors of the human serotype 5 (AdHu5) were given twice. Experiments were conducted in rhesus macaques that had or had not been preexposed to antigens of AdHu5. There was no significant difference in T-cell responses tested from peripheral blood of the different groups, although responses were overall highest in nonpreexposed animals immunized with the chimpanzee Ad vectors. Preexisting immunity to AdHu5 completely inhibited induction of transgene product-specific antibodies by the AdHu5 vectors without affecting antibody responses to the chimpanzee vectors. Upon euthanasia, T-cell responses were tested from a number of tissues. Preexisting immunity to AdHu5, commonly found in humans, changed the homing pattern of vaccine-induced T cells. In AdHu5-preexposed animals vaccinated with the chimpanzee Ad vectors, frequencies of transgene-specific T cells were higher in spleens than in blood, and in most preexposed animals vaccinated either with AdHu5 vectors or chimpanzee adenovirus vectors, frequencies of such T cells were exceptionally high in livers. The latter results indicate that analysis of T-cell responses solely from blood mononuclear cells of vaccine recipients may not suffice to compare the potencies of different vaccine regimens.     

HLA-A2-restricted T-cell epitopes specific for prostatic acid phosphatase

Prostatic acid phosphatase (PAP) has been investigated as the target of several antigen-specific anti-prostate tumor vaccines. The goal of antigen-specific active immunotherapies targeting PAP would ideally be to elicit PAP-specific CD8+ effector T cells. The identification of PAP-specific CD8+ T-cell epitopes should provide a means of monitoring the immunological efficacy of vaccines targeting PAP, and these epitopes might themselves be developed as vaccine antigens. In the current report, we hypothesized that PAP-specific epitopes might be identified by direct identification of pre-existing CD8+ T cells specific for HLA-A2-restricted peptides derived from PAP in the blood of HLA-A2-expressing individuals. 11 nonamer peptides derived from the amino acid sequence of PAP were used as stimulator antigens in functional ELISPOT assays with peripheral blood mononuclear cells from 20 HLA-A2+ patients with prostate cancer or ten healthy blood donors. Peptide-specific T cells were frequently identified in both groups for three of the peptides, p18–26, p112–120, and p135–143. CD8+ T-cell clones specific for three peptides, p18–26, p112–120, and p299–307, confirmed that these are HLA-A2-restricted T-cell epitopes. Moreover, HLA-A2 transgenic mice immunized with a DNA vaccine encoding PAP developed epitope-specific responses for one or more of these three peptide epitopes. We propose that this method to first identify epitopes for which there are pre-existing epitope-specific T cells could be used to prioritize MHC class I-specific epitopes for other antigens. In addition, we propose that the epitopes identified here could be used to monitor immune responses in HLA-A2+ patients receiving vaccines targeting PAP to identify potentially therapeutic immune responses.     

Eliciting cytotoxic T lymphocytes against acute myeloid leukemia-derived antigens: evaluation of dendritic cell-leukemia cell hybrids and other antigen-loading strategies for dendritic cell-based vaccination

Dendritic cells (DC) have been successfully used in clinical pilot studies to induce tumor-specific immunity as well as clinical response in selected patients. However, DC-based immunotherapy remains a challenge and several parameters need to be examined in order to optimize the induction of anti-tumor immune responses. This study focuses on DC vaccination for leukemia and evaluates the in vitro efficacy of three different strategies for generating antigen-loaded DC-based vaccines for the induction of major histocompatibility complex (MHC) class I-restricted anti-leukemia cytotoxic T lymphocyte (CTL) responses. These included direct fusion of DC with leukemia cells to generate DC-leukemia cell hybrids, and DC pulsed with either apoptotic leukemia cell fragments or whole tumor cell lysates. Using either the U937 cell line or primary human acute myeloid leukemia blasts (AML), DC-leukemia cell hybrids were found to be the most potent in vitro inducers of CTL activity. DC pulsed with apoptotic tumor cell fragments were less efficient, but induced a more potent CTL response compared to tumor lysate-pulsed DC. The CTL responses were both MHC class I-restricted and antigen-specific, as shown by the inability of the CTL to lyse other control targets. The data presented here suggest that the method of antigen loading onto DC may be critical in the design of tumor vaccines.     

Assessing antigen specific HLA-DR+ antibody secreting cell (DR+ASC) responses in whole blood in enteric infections using an ELISPOT technique

Antibody secreting cells (ASCs) generate antibodies in an antigen-specific manner as part of the adaptive immune response to infections, and these cells increase their surface expression of HLA-DR. We have studied this parameter (HLA-DR+ ASC) in patients with recent diarrheal infection using immuno-magnetic cell sorting and an enzyme linked immunospot (ELISPOT) technique that requires only one milliliter of blood. We validated this approach in adult patients with cholera (n = 15) or ETEC diarrhea (n = 30) on days 2, 7 and 30 after showing clinical symptom at the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) hospital in Dhaka, and we compared responses to age-matched healthy controls (n = 7). We found that HLA-DR+ ASC (DR+ASC) responses specific both for T cell-dependent (cholera toxin B subunit), and T cell-independent (lipopolysaccharide) antigens were elevated at day 7 after showing clinical cholera symptom. Similarly, DR+ASCs were elevated against both heat-labile toxin and colonization factors following ETEC infection. We observed significant correlations between antigen-specific DR+ASC responses and antigen-specific, gut homing ASC and plasma antibody responses. This study demonstrates that a simple ELISPOT procedure allows determination of antigen-specific ASC responses using a small volume of whole blood following diarrhea. This technique may be particularly useful in studying DR+ASC responses in young children and infants, either following infection or vaccination.