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.     

Altered helper T lymphocyte function associated with chronic hepatitis B virus infection and its role in response to therapeutic vaccination in humans

Theradigm-hepatitis B virus (HBV) is an experimental lipopeptide vaccine designed to stimulate induction of HBV-specific CTL responses in HLA-A2 individuals. Previous studies had demonstrated high immunogenicity in healthy volunteers, but comparatively weak CTL responses in chronically infected HBV patients. Herein, we examined helper T lymphocyte (HTL) responses in chronically infected patients. Despite normal proliferation and IL-2 secretion, IL-12 and IFN-gamma secretion in vitro in response to the vaccine was reduced compared with healthy volunteers. A similar pattern of cytokine secretion was observed following mitogen stimulation, suggesting a general altered balance of Th1/Th2 responses. Further analysis indicated that HTL recall responses to whole tetanus toxoid protein were reduced in chronically infected subjects, and reduced responsiveness correlated with the outcome of Theradigm-HBV immunization. Finally, experiments in HBV transgenic mice indicated that the nonnatural Pan DR HTL epitope, PADRE, is capable of inducing high levels of IFN-gamma secretion and that its inclusion in a lipopeptide incorporating an immunodominant Ld-restricted CTL epitope resulted in breaking tolerance at the CTL level. Overall, our results demonstrate an alteration in the quality of HTL responses induced in chronically infected HBV patients and suggest that use of a potent HTL epitope may be important to overcome CTL tolerance against specific HBV Ags.     

Direct detection and magnetic isolation of Chlamydia trachomatis major outer membrane protein-specific CD8+ CTLs with HLA class I tetramers

We recently identified HLA class I-presented epitopes in the major outer membrane protein (MOMP) of Chlamydia trachomatis that elicit CTL responses in human genital tract infections. T cells possessing cytolytic activities specific for these epitopes could be detected following in vitro stimulation of peripheral blood CD8(+) T cells with peptides. In the present study we used HLA-A2 tetramers for detailed characterization of MOMP-specific CTL responses. Ex vivo tetramer analysis detected MOMP-specific T cells in the peripheral blood of infected individuals at significant frequencies (0.01-0.20% of CD8(+) T cells). After in vitro stimulation with peptides, the frequencies of MOMP peptide-specific T cells increased up to 2.34% of CD8(+) T cells in bulk cultures. In contrast, HLA-A2/MOMP tetramer-binding T cells were virtually undetectable in the peripheral blood from uninfected individuals, either ex vivo or after 3 wk of in vitro peptide stimulation of their T cells. Magnetically sorted, tetramer-bound T cells specifically lysed peptide-pulsed targets as well as C. trachomatis-infected epithelial cells with nearly 50-fold greater per cell efficiency than that of unsorted populations. This study provides conclusive evidence of in vivo induction of HLA class I-restricted CD8(+) CTL responses to C. trachomatis MOMP. Direct detection of these cells with tetramers will allow their further characterization without prior manipulation and facilitate monitoring of CTL responses during infections and in immunization trials with MOMP-based vaccines.     

HLA-A*0201 T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus nucleocapsid and spike proteins

The immunogenicity of HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) peptide in severe acute respiratory syndrome coronavirus (SARS-CoV) nuclear capsid (N) and spike (S) proteins was determined by testing the proteins' ability to elicit a specific cellular immune response after immunization of HLA-A2.1 transgenic mice and in vitro vaccination of HLA-A2.1 positive human peripheral blood mononuclearcytes (PBMCs). First, we screened SARS N and S amino acid sequences for allele-specific motif matching those in human HLA-A2.1 MHC-I molecules. From HLA peptide binding predictions (http://thr.cit.nih.gov/molbio/hla_bind/), ten each potential N- and S-specific HLA-A2.1-binding peptides were synthesized. The high affinity HLA-A2.1 peptides were validated by T2-cell stabilization assays, with immunogenicity assays revealing peptides N223-231, N227-235, and N317-325 to be the first identified HLA-A*0201-restricted CTL epitopes of SARS-CoV N protein. In addition, previous reports identified three HLA-A*0201-restricted CTL epitopes of S protein (S978-986, S1203-1211, and S1167-1175), here we found two novel peptides S787-795 and S1042-1050 as S-specific CTL epitopes. Moreover, our identified N317-325 and S1042-1050 CTL epitopes could induce recall responses when IFN-gamma stimulation of blood CD8+ T-cells revealed significant difference between normal healthy donors and SARS-recovered patients after those PBMCs were in vitro vaccinated with their cognate antigen. Our results would provide a new insight into the development of therapeutic vaccine in SARS.     

Intranasal delivery of recombinant parvovirus-like particles elicits cytotoxic T-cell and neutralizing antibody responses

We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4(+) and CD8(+) T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8(+) T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in the absence of adjuvant, developed high levels of PPV-specific immunoglobulin G (IgG) and/or IgA in their serum, as well as in mucosal sites such as the bronchoalveolar and intestinal fluids. Antibodies in sera from mice immunized parenterally or intranasally with PPV:VLP were strongly neutralizing in vitro. Intranasal immunization with PPV:VLP carrying the LCMV CD8(+) T-cell epitope also elicited a strong peptide-specific cytotoxic-T-cell (CTL) response. In contrast, mice orally immunized with recombinant PPV:VLP did not develop any antibody or CTL responses. We also showed that mice primed with PPV:VLP are still able to develop strong CTL responses after subsequent immunization with chimeric PPV:VLP carrying a foreign CD8(+) T-cell epitope. These results highlight the attractive potential of PPV:VLP as a safe, nonreplicating antigen carrier to stimulate systemic and mucosal immunity after nasal administration.     

A CD8+ T cell heptaepitope minigene vaccine induces protective immunity against Chlamydia pneumoniae

An intact T cell compartment and IFN-gamma signaling are required for protective immunity against Chlamydia. In the mouse model of Chlamydia pneumoniae (Cpn) infection, this immunity is critically dependent on CD8(+) T cells. Recently we reported that Cpn-infected mice generate an MHC class I-restricted CD8(+) Tc1 response against various Cpn Ags, and that CD8(+) CTL to multiple epitopes inhibit Cpn growth in vitro. Here, we engineered a DNA minigene encoding seven H-2(b)-restricted Cpn CTL epitopes, the universal pan-DR epitope Th epitope, and an endoplasmic reticulum-translocating signal sequence. Immunization of C57BL/6 mice with this construct primed IFN-gamma-producing CD8(+) CTL against all seven CTL epitopes. CD8(+) T cell lines generated to minigene-encoded CTL epitopes secreted IFN-gamma and TNF-alpha and exhibited CTL activity upon recognition of Cpn-infected macrophages. Following intranasal challenge with Cpn, a 3.6 log reduction in mean lung bacterial numbers compared with control animals was obtained. Using a 20-fold increase in the Cpn challenging dose, minigene-vaccinated mice had a 60-fold reduction in lung bacterial loads, compared with controls. Immunization and challenge studies with beta(2)-microglobulin(-/-) mice indicated that the reduction of lung Cpn burdens was mediated by the MHC class I-dependent CD8(+) T cells to minigene-included Cpn CTL epitopes, rather than by pan-DR epitope-specific CD4(+) T cells. This constitutes the first demonstration of significant protection achieved by immunization with a CD8(+) T cell epitope-based DNA construct in a bacterial system and provides the basis for the optimal design of multicomponent anti-Cpn vaccines for humans.     

Identification of a ras oncogene peptide that contains both CD4(+) and CD8(+) T cell epitopes in a nested configuration and elicits both T cell subset responses by peptide or DNA immunization

Mutations in ras proto-oncogenes are commonly found in a diversity of malignancies and may encode unique, non-self epitopes for T cell-mediated antitumor activity. In a BALB/c (H-2(d)) murine model, we have identified a single peptide sequence derived from the ras oncogenes that contained both CD8(+) and CD4(+) T cell epitopes in a nested configuration. This peptide reflected ras sequence 4-16, and contained the substitution of Gly to Val at position 12 ?i.e., 4-16(Val12)?. Mice immunized with this 13-mer peptide induced a strong antigen (Ag)-specific CD4(+) proliferative response in vitro. In contrast, mice inoculated with the wild-type ras sequence failed to generate a peptide-specific T cell response. Additionally, mice immunized with the ras 4-16(Val12) peptide concomitantly displayed an Ag-specific CD8(+) cytotoxic T lymphocyte (CTL) response, as determined by lysis of syngeneic tumor target cells incubated with the nominal 9-mer nested epitope peptide ?i.e., 4-12(Val12)?, as well as lysis of tumor target cells expressing the corresponding ras codon 12 mutation. Analysis of the Valpha- and Vbeta-chains of the T cell receptor (TCR) expressed by these CTL revealed usage of the Valpha1 and Vbeta9 subunits, consistent with the TCR phenotype of anti-ras Val12 CTL lines produced by in vivo immunization with the nominal peptide epitope alone. Moreover, immunization with the nested epitope peptide, as compared to immunization with either the 9-mer CTL peptide alone or an admixture of the 9-mer CTL peptide with an overlapping 13-mer CD4(+) T cell helper peptide ?i.e., 5-17(Val12)? lacking the class I N-terminus anchor site, enhanced the production of the CD8(+) T cell response. Finally, immunization with plasmid DNA encoding the ras 4-16(Val12) sequence led to the induction of both Ag-specific proliferative and cytotoxic responses. Overall, these results suggested that a single peptide immunogen containing nested mutant ras-specific CD4(+) and CD8(+) T cell epitopes: (1) can be processed in vivo to induce both subset-specific T lymphocyte responses; and (2) leads to the generation of a quantitatively enhanced CD8(+) CTL response, likely due to the intimate coexistence of CD4(+) help, which may have implications in peptide- or DNA-based immunotherapies.     

Defining MHC class II T helper epitopes for WT1 tumor antigen

The product of Wilms‘ tumor gene 1 (WT1) is overexpressed in diverse human tumors, including leukemia, lung and breast cancer, and is often recognized by antibodies in the sera of patients with leukemia. Since WT1 encodes MHC class I-restricted peptides recognized by cytotoxic T lymphocytes (CTL), WT1 has been considered as a promising tumor-associated antigen (TAA) for developing anticancer immunotherapy. In order to carry out an effective peptide-based cancer immunotherapy, MHC class II-restricted epitope peptides that elicit anti-tumor CD4⁺ helper T lymphocytes (HTL) will be needed. In this study, we analyzed HTL responses against WT1 antigen using HTL lines elicited by in vitro immunization of human lymphocytes with synthetic peptides predicted to serve as HTL epitopes derived from the sequence of WT1. Two peptides, WT1_124–138 and WT1_247–261, were shown to induce peptide-specific HTL, which were restricted by frequently expressed HLA class II alleles. Here, we also demonstrate that both peptides-reactive HTL lines were capable of recognizing naturally processed antigens presented by dendritic cells pulsed with tumor lysates or directly by WT1+ tumor cells that express MHC class II molecules. Interestingly, the two WT1 HTL epitopes described here are closely situated to known MHC class I-restricted CTL epitopes, raising the possibility of stimulating CTL and HTL responses using a relatively small synthetic peptide vaccine. Because HTL responses to TAA are known to be important for promoting long-lasting anti-tumor CTL responses, the newly described WT1 T-helper epitopes could provide a useful tool for designing powerful vaccines against WT1-expressing tumors.     

Optimized vaccination regimen linked to exhaustive screening approaches identifies 2 novel HLA-B7 restricted epitopes within hepatitis C virus NS3 protein

Broad immune responses, in particular specific for the NS3 protein and mediated by both CD8+ and CD4+T lymphocytes, are thought to play a critical role in the control of hepatitis C virus (HCV) infection. In this study, we searched for novel HLA-B*0702 NS3 restricted epitopes following an optimized NS3NS4 immunization protocol in transgenic mice expressing HLA-B*0702 molecule. Combining predicted and overlapping peptides, we identified two novel epitopes, WPA10 (aa 1111-1120) and LSP10 (aa 1153-1162), which triggered significant IFN-gamma-producing T cell frequencies and high CTL responses. Both epitopes were shown to be immunogenic when used as synthetic peptides to immunize mice. The relevance of these epitopes to humans was demonstrated, as both were able in vitro to recall specific IFN-gamma and IL10-producing cells from peripheral blood mononuclear cells of HCV infected patients. Such epitopes enlarge the pool of NS3-specific CD8+T cell epitopes available to perform immunomonitoring of HCV infection and to develop vaccines.     

Rational design of a multiepitope vaccine encoding T-lymphocyte epitopes for treatment of chronic hepatitis B virus infections

Protein sequences from multiple hepatitis B virus (HBV) isolates were analyzed for the presence of amino acid motifs characteristic of cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes with the goal of identifying conserved epitopes suitable for use in a therapeutic vaccine. Specifically, sequences bearing HLA-A1, -A2, -A3, -A24, -B7, and -DR supertype binding motifs were identified, synthesized as peptides, and tested for binding to soluble HLA. The immunogenicity of peptides that bound with moderate to high affinity subsequently was assessed using HLA transgenic mice (CTL) and HLA cross-reacting H-2^bxd (BALB/c × C57BL/6J) mice (HTL). Through this process, 30 CTL and 16 HTL epitopes were selected as a set that would be the most useful for vaccine design, based on epitope conservation among HBV sequences and HLA-based predicted population coverage in diverse ethnic groups. A plasmid DNA-based vaccine encoding the epitopes as a single gene product, with each epitope separated by spacer residues to enhance appropriate epitope processing, was designed. Immunogenicity testing in mice demonstrated the induction of multiple CTL and HTL responses. Furthermore, as a complementary approach, mass spectrometry allowed the identification of correctly processed and major histocompatibility complex-presented epitopes from human cells transfected with the DNA plasmid. A heterologous prime-boost immunization with the plasmid DNA and a recombinant MVA gave further enhancement of the immune responses. Thus, a multiepitope therapeutic vaccine candidate capable of stimulating those cellular immune responses thought to be essential for controlling and clearing HBV infection was successfully designed and evaluated in vitro and in HLA transgenic mice.     

Diversity of epitope and cytokine profiles for primary and secondary influenza a virus-specific CD8+ T cell responses

Screening with the flow cytometric IFN-gamma assay has led to the identification of a new immunogenic peptide (SSYRRPVGI) [corrected] from the influenza PB1 polymerase (PB1(703--711)) and a mimotope (ISPLMVAYM) from the PB2 polymerase (PB2(198--206)). CD8(+) T cells specific for K(b)PB1(703) make both IFN-gamma and TNF-alpha following stimulation with both peptides. The CD8(+) K(b)PB1(703)(+) population kills PB2(198)-pulsed targets, but cell lines stimulated with PB2(198) neither bind the K(b)PB1(703) tetramer nor become CTL. This CD8(+)K(b)PB1(703)(+) population is prominent in the primary response to an H3N2 virus, although it is much less obvious following secondary challenge of H1N1-primed mice. Even so, we can now account for >40% of the CD8(+) T cells in a primary influenza pneumonia and >85% of those present after H3N2 --> H1N1 challenge. Profiles of IFN-gamma and TNF-alpha staining following in vitro stimulation have been traced for the four most prominent influenza peptides through primary and secondary responses into long-term memory. The D(b)NP(366) epitope that is immunodominant after the H3N2 --> H1N1 challenge shows the lowest frequencies of CD8(+) IFN-gamma(+)TNF-alpha(+) cells for >6 wk, and the intensity of IFN-gamma staining is also low for the first 3 wk. By 11 wk, however, the IFN-gamma/TNF-alpha profiles look to be similar for all four epitopes. At least by the criterion of cytokine production, there is considerable epitope-related functional diversity in the influenza virus-specific CD8(+) T cell response. The results for the K(b)PB1(703) epitope and the PB2(198) mimotope also provide a cautionary tale for those using the cytokine staining approach to identity antigenic peptides.     

Delivery of multiple epitopes by recombinant detoxified adenylate cyclase of Bordetella pertussis induces protective antiviral immunity.

CyaA, the adenylate cyclase toxin from Bordetella pertussis, can deliver its N-terminal catalytic domain into the cytosol of a large number of eukaryotic cells and particularly into professional antigen-presenting cells. We have previously identified within the primary structure of CyaA several permissive sites at which insertion of peptides does not alter the ability of the toxin to enter cells. This property has been exploited to design recombinant CyaA toxoids capable of delivering major histocompatibility complex (MHC) class I-restricted CD8(+) T-cell epitopes into antigen-presenting cells and to induce specific CD8(+) cytotoxic T-lymphocyte (CTL) responses in vivo. Here we have explored the capacity of the CyaA vector carrying several different CD8(+) T-cell epitopes to prime multiple CTL responses. The model vaccine consisted of a polyepitope made of three CTL epitopes from lymphocytic choriomeningitis virus (LCMV), the V3 region of human immunodeficiency virus gp120, and chicken ovalbumin, inserted at three different sites of the catalytic domain of genetically detoxified CyaA. Each of these epitopes was processed on delivery by CyaA and presented in vitro to specific T-cell hybridomas. Immunization of mice by CyaA toxoids carrying the polyepitope lead to the induction of specific CTL responses for each of the three epitopes, as well as to protection against a lethal viral challenge. Moreover, mice primed against the vector by mock CyaA or a recombinant toxoid were still able to develop strong CTL responses after subsequent immunization with a recombinant CyaA carrying a foreign CD8(+) CTL epitope. These results highlight the potency of the adenylate cyclase vector for induction of protective CTL responses with multiple specificity and/or broad MHC restriction.     

Concomitant induction of CD4+ and CD8+ T cell responses in volunteers immunized with Salmonella enterica serovar typhi strain CVD 908-htrA

Type 1 cell-mediated immunity might play an important role in protection from typhoid fever. We evaluated whether immunization with Salmonella enterica serovar Typhi (S. Typhi) strain CVD 908-htrA (a Delta aroC Delta aroD Delta htrA mutant), a leading live oral typhoid vaccine candidate, elicits specific CD4(+) and CD8(+) S. Typhi immune responses. Potent CTL responses and IFN-gamma secretion by CD8(+) T cells were detected following immunization with CVD 908-htrA in high (4.5 x 10(8) CFU) and low (5 x 10(7) CFU) dosages. S. Typhi-specific CTL were observed in six of eight vaccinees (four high and two low dose) after immunization. Mean increases in the frequency of IFN-gamma spot-forming cells (SFC) in the presence of S. Typhi-infected targets were 221 +/- 41 SFC/10(6) PBMC and 233 +/- 87 SFC/10(6) PBMC, in the high and low dose groups, respectively. Strong CD4(+) T cell responses were also observed. Increases in the IFN-gamma production to soluble S. Typhi flagella (STF) occurred in 82 and 38% of the volunteers who received the high and low doses, respectively. Robust correlations were observed between volunteers that responded with IFN-gamma SFC to stimulation with S. Typhi-infected cells and IFN-gamma released in response to stimulation with STF Ags (r = 0.822, p < 0.001) and between CTL and IFN-gamma production to STF (r = 0.818, p = 0.013). These data demonstrating the concomitant induction of both CD4- and CD8-mediated CMI are consistent with a significant role for type 1 immunity in controlling typhoid infection and support the continuing evaluation of CVD 908-htrA as a typhoid vaccine candidate.     

Immunization with dendritic cells loaded with alpha-galactosylceramide at priming phase, but not at boosting phase, enhances cytotoxic T lymphocyte activity against infection by intracellular bacteria

We evaluated the effect of immunization with dendritic cells (DCs) pulsed with alpha-galactosylceramide (alphaGalCer) and listeriolysin O (LLO) 91-99 peptide, a dominant cytotoxic T lymphocyte (CTL) epitope of Listeria monocytogenes by observing the responses of specific CD8(+) T cells and in vivo CTL activity. DCs were pulsed with various combinations of alphaGalCer and LLO91-99 peptide and administered to BALB/c mice. Immunization with DCs pulsed with alphaGalCer and LLO91-99 at priming phase and with DCs pulsed with LLO91-99 alone at boosting phase induced stronger in vivo CTL activity, reduced the bacterial load in spleens of Listeria-challenged mice and augmented CD62L(+) CD8(+) central memory T cells compared with other immunization protocols. The blockade of interferon-gamma (IFN-gamma) at boosting phase reversed the induction of CD8(+) central memory T cells and reduced the bacterial load in spleens of Listeria-challenged mice immunized with DCs pulsed with alphaGalCer and LLO91-99 at both phases, suggesting that alphaGalCer at boosting phase has deleterious effects through IFN-gamma production. These results indicate that immunization with DCs pulsed with CTL epitope peptide together with alphaGalCer at priming phase, but not at boosting phase, is feasible for eliciting a specific CTL activity and protective immunity against infection of intracellular bacteria.     

A rational strategy to design multiepitope immunogens based on multiple Th lymphocyte epitopes

Four HLA-DR-restricted HIV-derived Th lymphocyte (HTL) epitopes cross-reactive with the murine I-A(b) class II molecule were used to evaluate different vaccine design strategies to simultaneously induce multiple HTL responses. All four epitopes were immunogenic in H-2(b) mice, demonstrating the feasibility of murine models to evaluate epitope-based vaccines destined for human use. Immunization with a pool of peptides induced responses against all four epitopes; illustrating immunodominance does not prevent the induction of balanced multispecific responses. When different delivery systems were evaluated, a multiple Ag peptide construct was found to be less efficient than a linear polypeptide encompassing all four epitopes. Further characterization of linear polypeptide revealed that the sequential arrangement of the epitopes created a junctional epitope with high affinity class II binding. Disruption of this junctional epitope through the introduction of a GPGPG spacer restored the immunogenicity against all four epitopes. Finally, we demonstrate that a GPGPG spacer construct can be used to induce HTL responses by either polypeptide or DNA immunization, highlighting the flexibility of the approach.     

Characterization of CD8(+) effector T cell responses in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine

Salmonella enterica serovar Typhi (S. typhi) strain Ty21a remains the only licensed attenuated typhoid vaccine. Despite years of research, the identity of the protective immunological mechanisms elicited by immunization with the Ty21a typhoid vaccine remains elusive. The present study was designed to characterize effector T cell responses in volunteers immunized with S. typhi strain Ty21a typhoid vaccine. We determined whether immunization with Ty21a induced specific CTL able to lyse S. typhi-infected cells and secrete IFN-gamma, a key effector molecule against intracellular pathogens. We measured the functional activity of these CTL by a (51)Cr-release assay using 8-day restimulated PBMC from Ty21a vaccinees as effector cells and S. Typhi-infected autologous PHA-activated PBMC as target cells. Most vaccinees exhibited consistently increased CD8-mediated lysis of targets by postimmunization PBMC when compared with preimmunization levels. We also developed an IFN-gamma ELISPOT assay to quantify the frequency of IFN-gamma spot-forming cells (SFC) in PBMC from Ty21a vaccinees using an ex vivo system. Significant increases in the frequency of IFN-gamma SFC following immunization (mean +/- SD, 393 +/- 172; range 185-548 SFC/10(6) PBMC; p = 0.010), as compared with preimmunization levels, were observed. IFN-gamma was secreted predominantly by CD8(+) T cells. A strong correlation was recorded between the cytolytic activity of CTL lines and the frequency of IFN-gamma SFC (r(2) = 0.910, p < 0.001). In conclusion, this work constitutes the first evidence that immunization of volunteers with Ty21a elicits specific CD8(+) CTL and provides an estimate of the frequency of CD8(+) IFN-gamma-secreting cells induced by vaccination.     

Induction in humans of CD8+ and CD4+ T cell and antibody responses by sequential immunization with malaria DNA and recombinant protein

Vaccine-induced protection against diseases like malaria, AIDS, and cancer may require induction of Ag-specific CD8(+) and CD4(+) T cell and Ab responses in the same individual. In humans, a recombinant Plasmodium falciparum circumsporozoite protein (PfCSP) candidate vaccine, RTS,S/adjuvant system number 2A (AS02A), induces T cells and Abs, but no measurable CD8(+) T cells by CTL or short-term (ex vivo) IFN-gamma ELISPOT assays, and partial short-term protection. P. falciparum DNA vaccines elicit CD8(+) T cells by these assays, but no protection. We report that sequential immunization with a PfCSP DNA vaccine and RTS,S/AS02A induced PfCSP-specific Abs and Th1 CD4(+) T cells, and CD8(+) cytotoxic and Tc1 T cells. Depending upon the immunization regime, CD4(+) T cells were involved in both the induction and production phases of PfCSP-specific IFN-gamma responses, whereas, CD8(+) T cells were involved only in the production phase. IFN-gamma mRNA up-regulation was detected in both CD45RA(-) (CD45RO(+)) and CD45RA(+)CD4(+) and CD8(+) T cell populations after stimulation with PfCSP peptides. This finding suggests CD45RA(+) cells function as effector T cells. The induction in humans of the three primary Ag-specific adaptive immune responses establishes a strategy for developing immunization regimens against diseases in desperate need of vaccines.     

Concomitant helper response rescues otherwise low avidity CD8+ memory CTLs to become efficient effectors in vivo

This report seeks a means of maximizing memory CD8 T cell responses to peptide immunization. Delivery of the CD8 peptide epitope by stress protein, heat shock protein (hsp)70, results in excellent immunogenicity at the acute phase but memory responses were poor both in terms of the number of responding cells as well as their functional avidity. We demonstrate for the first time that hsp70 can also be used as a vehicle to achieve CD4 T cell responses to loaded peptide epitopes and that coimmunization with hsp70 loaded with both CD8 and CD4 peptide epitopes may increase memory up to 3-fold. Furthermore, CD8+ T cell memory responses were of higher avidity measured both by in vitro cytotoxicity assays and a new methodology that measures the avidity of CTL activity in vivo in mice. Our results emphasize that peptide immunization remains a viable approach to induce long-term CD8+ T cell function, providing steps are taken to assure appropriate stimulation of Th cell responses.     

Characterization of a Mycobacterium tuberculosis peptide that is recognized by human CD4+ and CD8+ T cells in the context of multiple HLA alleles

The secreted Mycobacterium tuberculosis 10-kDa culture filtrate protein (CFP)10 is a potent T cell Ag that is recognized by a high percentage of persons infected with M. tuberculosis. We determined the molecular basis for this widespread recognition by identifying and characterizing a 15-mer peptide, CFP10(71-85), that elicited IFN-gamma production and CTL activity by both CD4(+) and CD8(+) T cells from persons expressing multiple MHC class II and class I molecules, respectively. CFP10(71-85) contained at least two epitopes, one of 10 aa (peptide T1) and another of 9 aa (peptide T6). T1 was recognized by CD4(+) cells in the context of DRB1*04, DR5*0101, and DQB1*03, and by CD8(+) cells of A2(+) donors. T6 elicited responses by CD4(+) cells in the context of DRB1*04 and DQB1*03, and by CD8(+) cells of B35(+) donors. Deleting a single amino acid from the amino or carboxy terminus of either peptide markedly reduced IFN-gamma production, suggesting that they are minimal epitopes for both CD4(+) and CD8(+) cells. As far as we are aware, these are the shortest microbial peptides that have been found to elicit responses by both T cell subpopulations. The capacity of CFP10(71-85) to stimulate IFN-gamma production and CTL activity by CD4(+) and CD8(+) cells from persons expressing a spectrum of MHC molecules suggests that this peptide is an excellent candidate for inclusion in a subunit antituberculosis vaccine.     

Utilization of MHC class I transgenic mice for development of minigene DNA vaccines encoding multiple HLA-restricted CTL epitopes

We engineered a multiepitope DNA minigene encoding nine dominant HLA-A2.1- and A11-restricted epitopes from the polymerase, envelope, and core proteins of hepatitis B virus and HIV, together with the PADRE (pan-DR epitope) universal Th cell epitope and an endoplasmic reticulum-translocating signal sequence. Immunization of HLA transgenic mice with this construct resulted in: 1) simultaneous CTL induction against all nine CTL epitopes despite their varying MHC binding affinities; 2) CTL responses that were equivalent in magnitude to those induced against a lipopeptide known be immunogenic in humans; 3) induction of memory CTLs up to 4 mo after a single DNA injection; 4) higher epitope-specific CTL responses than immunization with DNA encoding whole protein; and 5) a correlation between the immunogenicity of DNA-encoded epitopes in vivo and the in vitro responses of specific CTL lines against minigene DNA-transfected target cells. Examination of potential variables in minigene construct design revealed that removal of the PADRE Th cell epitope or the signal sequence, and changing the position of selected epitopes, affected the magnitude and frequency of CTL responses. Our results demonstrate the simultaneous induction of broad CTL responses in vivo against multiple dominant HLA-restricted epitopes using a minigene DNA vaccine and underline the utility of HLA transgenic mice in development and optimization of vaccine constructs for human use.