Identification of a broad coverage HLA-DR degenerate epitope pool derived from carcinoembryonic antigen.

CD4 T cells are important for anti-tumor immune responses. Aside from their role in the activation of CD8 T cells, CD4 T cells also mediate anti-tumor immune responses by recruiting innate immune effectors into the tumor microenvironment. Thus, the search for strategies to boost CD4 T cell immunity is an active area of research. Our goal in this study was to identify HLA-DR epitopes of carcinoembryonic antigen (CEA), a commonly over-expressed tumor antigen. HLA-DR epitopes of CEA were identified using the epitope prediction program, PIC (predicted IC(50)) and tested using in vitro HLA-DR binding assays. Following CEA epitope confirmation, IFN-gamma ELIspot assays were used to detect existing immunity against the HLA-DR epitope panel of CEA in breast and ovarian cancer patients. In vitro generated peptide-specific CD4 T cells were used to determine whether the epitopes are naturally processed from CEA protein. Forty-three epitopes of CEA were predicted, 15 of which had high binding affinity for 8 or more common HLA-DR molecules. A degenerate pool of four, HLA-DR restricted 15 amino acid epitopes (CEA.24, CEA.176/354, CEA.488, and CEA.653) consisting of two novel epitopes (CEA.24 and CEA.488) was identified against which 40% of breast and ovarian cancer patients had pre-existent T cell immunity. All four epitopes are naturally processed by antigen-presenting cells. Hardy-Weinberg analysis showed that the pool is useful in approximately 94% of patients. Patients with breast or ovarian cancer demonstrate pre-existent immune responses to the tumor antigen CEA. The degenerate pool of CEA peptides may be useful for augmenting CD4 T cell immunity.

Formulation and characterization of a ten-peptide single-vial vaccine, EP-2101, designed to induce cytotoxic T-lymphocyte responses for cancer immunotherapy.

Effective vaccines that mediate clinical responses in cancer patients may require generation of broadly specific cytotoxic T lymphocytes (CTL) directed against multiple epitopes and tumor-associated antigens (TAA). Pursuant to this goal we developed a synthetic peptide vaccine, EP-2101, composed of 10 synthetic peptide epitopes and formulated in Montanide ISA 51 adjuvant. Nine of the HLA-A*0201-restricted CTL epitopes were derived from five well-characterized TAA. The universal HLA-DR binding epitope PADRE was also included for T-cell help. Herein we describe studies on the formulation and characterization of the EP-2101 vaccine which supports generation of a sterile single-vial emulsion using standardized processes. The physicochemical properties of the peptides were highly disparate and as such, solubilization studies were required to identify a process which supported sterile filtration of the EP-2101 peptide mix. A homogenization-based formulation process with Montanide ISA 51 and 0.5 mg/ml of each peptide was developed to generate a water-in-oil emulsion. Physical studies indicated the vaccine emulsion to be stable, with little change in visual appearance, viscosity and water droplet size for at least three months. The physical stability of individual peptides in the vaccine emulsion was demonstrated using HPLC and immunogenicity of the vaccine formulation was confirmed in HLA-A*0201/K(b) transgenic mice where T-cell responses could be induced to all epitopes in EP-2101 following vaccination. Our study process is scalable for production of approximately 1.5 liters of potent experimental vaccine for preclinical animal toxicity and phase 1 clinical testing in patients with breast, colon or lung cancer.

Lipopolysaccharide-specific memory B cell responses to an attenuated live cholera vaccine are associated with protection against Vibrio cholerae infection.

BACKGROUND: The single-dose live attenuated vaccine CVD 103-HgR protects against experimental Vibrio cholerae infection in cholera-naïve adults for at least 6 months after vaccination. While vaccine-induced vibriocidal seroconversion is associated with protection, vibriocidal titers decline rapidly from their peak 1-2 weeks after vaccination. Although vaccine-induced memory B cells (MBCs) might mediate sustained protection in individuals without detectable circulating antibodies, it is unknown whether oral cholera vaccination induces a MBC response. METHODS: In a study that enrolled North American adults, we measured lipopolysaccharide (LPS)- and cholera toxin (CtxB)-specific MBC responses to PXVX0200 (derived from the CVD 103-HgR strain) and assessed stool volumes following experimental Vibrio cholerae infection. We then evaluated the association between vaccine-induced MBC responses and protection against cholera. RESULTS: There was a significant increase in % CT-specific IgG, % LPS-specific IgG, and % LPS-specific IgA MBCs which persisted 180 days after vaccination as well as a significant association between vaccine-induced increase in % LPS-specific IgA MBCs and lower post-challenge stool volume (r = -0.56, p < 0.001). DISCUSSION: Oral cholera vaccination induces antigen-specific MBC responses, and the anamnestic LPS-specific responses may contribute to long-term protection and provide correlates of the duration of vaccine-induced protection. CLINICAL TRIALS REGISTRATION: NCT01895855.

Immunization of cancer patients with HER-2/neu-derived peptides demonstrating high-affinity binding to multiple class II alleles.

PURPOSE: The purpose of this study was to immunize patients with HER-2/neu-overexpressing cancer with a multipeptide vaccine comprised of four class II HER-2/neu peptides that had been identified as the most immunogenic in a previous clinical trial. Furthermore, we questioned whether MHC binding affinity could predict the in vivo immunogenicity of the HER-2/neu helper peptides. EXPERIMENTAL DESIGN: Four putative class II HER-2/neu peptides, which were found to generate detectable specific T-cell responses (stimulation index > 2) in a majority of patients in a previous study, were used to formulate a single vaccine. The multipeptide vaccine was administered intradermally with granulocyte macrophage colony-stimulating factor as an adjuvant. Ten patients with HER-2/neu overexpressing breast or lung cancer were enrolled. HER-2/neu peptide-and protein-specific T cell and antibody immune responses were measured. Competitive inhibition assays were used to analyze the class II HER-2/neu peptides for their binding affinity to 14 common HLA-DR alleles. RESULTS: Twenty-five percent of patients developed HER-2/neu peptide-specific T-cell immunity, and 50% developed HER-2/neu peptide-specific antibody immunity. No patient developed HER-2/neu protein-specific T cell or antibody immunity. The majority of peptides exhibited high binding affinity, in vitro, to >/==" BORDER="0">3 of the 14 DR alleles analyzed. CONCLUSION: The group of peptides used in this study demonstrated high binding affinity to multiple DR alleles suggesting that in vitro binding affinity may be able to predict the in vivo immunogenicity of class II peptides. However, only a minority of patients immunized with the multipeptide vaccine developed HER-2/neu peptide-specific T cell or antibody immunity, and none developed HER-2/neu protein-specific immunity.

Oral priming with replicating adenovirus serotype 4 followed by subunit H5N1 vaccine boost promotes antibody affinity maturation and expands H5N1 cross-clade neutralization.

A Phase I trial conducted in 2009-2010 demonstrated that oral vaccination with a replication competent Ad4-H5 (A/Vietnam) vector with dosages ranging from 107-1011 viral particles was well tolerated. HA-specific T-cell responses were efficiently induced, but very limited hemagglutination-inhibiting (HI) humoral responses were measured. However, a single boost of Ad4-H5-Vtn vaccinated individuals with a unadjuvanted licensed H5N1 (A/Vietnam) subunit vaccine resulted in superior HI titers compared with unprimed subjects. In the current study, the impact of Ad4-H5 priming on the quality of the polyclonal humoral immune response was evaluated using a real-time kinetics assay by surface plasmon resonance (SPR). Total binding of serum polyclonal antibodies from the Ad4-H5-Vtn primed groups against both homologous H5N1-A/Vietnam/1194/2004 (clade 1) and heterologous A/Indonesia-5/2005 (clade 2.1) HA1 head domain was significantly higher compared with sera from individuals that received subunit H5N1 vaccination alone. SPR measurements also demonstrated that the antigen-antibody complex dissociation rates (a surrogate for antibody affinity) of serum antibodies against the HA1 of H5N1-A/Vietnam were significantly higher in the Ad4-H5 primed groups compared with those from the unprimed group. Furthermore, strong correlations were observed between the antibody affinities for HA1 (but not HA2) and the virus neutralization titers against the homologous strain and a panel of heterologous clade 2 H5N1 strains. These findings support the concept of oral prime-boost vaccine approaches against pandemic influenza to elicit long-term memory B cells with high affinity capable of rapid response to variant pandemic viruses likely to emerge and adapt to human transmissions.

Epitope identification and vaccine design for cancer immunotherapy.

The development of processes for engineering multi-epitope vaccines based on the identification and selection of epitope packages, along with vaccine design optimization using epitope placements and spacers to optimize processing efficacy, are reviewed. The Epimmune Inc epitope identification process has been applied to numerous cancer types, but also applies to infectious diseases. Epitope-analog efforts in novel vaccine design have also been explored and their uses in prophylactic and therapeutic applications are eagerly anticipated.

Safety and immunogenicity of an oral, replicating adenovirus serotype 4 vector vaccine for H5N1 influenza: a randomised, double-blind, placebo-controlled, phase 1 study.

BACKGROUND: Replication-competent virus vector vaccines might have advantages compared with non-replicating vector vaccines. We tested the safety and immunogenicity of an oral adenovirus serotype 4 vector vaccine candidate (Ad4-H5-Vtn) expressing the haemagglutinin from an avian influenza A H5N1 virus. METHODS: We did this phase 1 study at four sites in the USA. We used a computer-generated randomisation list (block size eight, stratified by site) to assign healthy volunteers aged 18-40 years to receive one of five doses of Ad4-H5-Vtn (10(7) viral particles [VP], 10(8) VP, 10(9) VP, 10(10) VP, 10(11) VP) or placebo (3:1). Vaccine or placebo was given on three occasions, about 56 days apart. Participants, investigators, and study-site personnel were masked to assignment throughout the study. Subsequently, volunteers received a boost dose with 90 µg of an inactivated parenteral H5N1 vaccine. Primary immunogenicity endpoints were seroconversion by haemagglutination-inhibition (HAI), defined as a four-times rise compared with baseline titre, and HAI geometric mean titre (GMT). We solicited symptoms of reactogenicity daily for 7 days after each vaccination and recorded symptoms that persisted beyond 7 days as adverse events. Primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01006798. FINDINGS: We enrolled 166 participants (125 vaccine; 41 placebo) between Oct 19, 2009, and Sept 9, 2010. HAI responses were low: 13 of 123 vaccinees (11%, 95% CI 6-17) and three of 41 placebo recipients (7%, 2-20) seroconverted. HAI GMT was 6 (95% CI 5-7) for vaccinees, and 5 (5-6) for placebo recipients. However, when inactivated H5N1 vaccine became available, one H5N1 boost was offered to all participants. In this substudy, HAI seroconversion occurred in 19 of 19 participants in the 10(11) VP cohort (100%; 95% CI 82-100) and eight of 22 placebo recipients (36%; 17-59); 17 of 19 participants in the 10(11) VP cohort (89%; 67-99) achieved seroprotection compared with four of 22 placebo recipients (18%; 5-40); GMT was 135 (89-205) with 10(11) VP, compared with 13 (7-21) with placebo. The cumulative frequency of abdominal pain, diarrhoea, and nasal congestion after all three vaccinations was significantly higher in vaccinees than placebo recipients (21 [16·8%] of 125 vs one [2·4%] of 41, p=0·017; 24 [19·2%] of 125 vs two [4·9%] of 41, p=0·027; 41 [32·8%] of 125 vs six [14·6%] of 41, p=0·028; respectively). No serious treatment-related adverse events occurred. INTERPRETATION: Oral Ad4 vector priming might enhance the efficacy of poorly immunogenic vaccines such as H5N1. FUNDING: Wellcome Trust Foundation, PaxVax.

Pre-clinical development of a recombinant, replication-competent adenovirus serotype 4 vector vaccine expressing HIV-1 envelope 1086 clade C.

BACKGROUND: There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated. METHODS: The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets. RESULTS: Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization. CONCLUSIONS: The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical trials.

Pre-clinical evaluation of a replication-competent recombinant adenovirus serotype 4 vaccine expressing influenza H5 hemagglutinin.

BACKGROUND: Influenza virus remains a significant health and social concern in part because of newly emerging strains, such as avian H5N1 virus. We have developed a prototype H5N1 vaccine using a recombinant, replication-competent Adenovirus serotype 4 (Ad4) vector, derived from the U.S. military Ad4 vaccine strain, to express the hemagglutinin (HA) gene from A/Vietnam/1194/2004 influenza virus (Ad4-H5-Vtn). Our hypothesis is that a mucosally-delivered replicating Ad4-H5-Vtn recombinant vector will be safe and induce protective immunity against H5N1 influenza virus infection and disease pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: The Ad4-H5-Vtn vaccine was designed with a partial deletion of the E3 region of Ad4 to accommodate the influenza HA gene. Replication and growth kinetics of the vaccine virus in multiple human cell lines indicated that the vaccine virus is attenuated relative to the wild type virus. Expression of the HA transgene in infected cells was documented by flow cytometry, western blot analysis and induction of HA-specific antibody and cellular immune responses in mice. Of particular note, mice immunized intranasally with the Ad4-H5-Vtn vaccine were protected against lethal H5N1 reassortant viral challenge even in the presence of pre-existing immunity to the Ad4 wild type virus. CONCLUSIONS/SIGNIFICANCE: Several non-clinical attributes of this vaccine including safety, induction of HA-specific humoral and cellular immunity, and efficacy were demonstrated using an animal model to support Phase 1 clinical trial evaluation of this new vaccine.

Identification of broad binding class I HLA supertype epitopes to provide universal coverage of influenza A virus.

Influenza virus remains a significant health concern, with current circulating strains that affect millions each year plus the threat of newly emerging strains, such as swine-origin H1N1 and avian H5N1. Our hypothesis is that influenza-derived HLA-class I-restricted epitopes can be identified for use as a reagent to monitor and quantitate human CD8(+) T-cell responses and for vaccine development to induce protective cellular immunity. Protein sequences from influenza A virus strains currently in circulation, agents of past pandemics and zoonotic infections of man were evaluated for sequences predicted to bind to alleles representative of the most frequent HLA-A and -B (class I) types worldwide. Peptides that bound several different HLA molecules and were conserved among diverse influenza subtypes were tested for their capacity to recall influenza-specific immune responses using human donor PBMC. Accordingly, 28 different epitopes antigenic for human donor PBMC were identified and 25 were 100% conserved in the newly emerged swine-origin H1N1 strain. The epitope set defined herein should provide a reagent applicable to quantitate CD8(+) T cell human responses irrespective of influenza subtype and HLA composition of the responding population. In addition, these epitopes may be suitable for vaccine applications directed at the induction of cellular immunity.

Universal influenza DNA vaccine encoding conserved CD4+ T cell epitopes protects against lethal viral challenge in HLA-DR transgenic mice.

The goal of the present study was to design a vaccine that would provide universal protection against infection of humans with diverse influenza A viruses. Accordingly, protein sequences from influenza A virus strains currently in circulation (H1N1, H3N2), agents of past pandemics (H1N1, H2N2, H3N2) and zoonotic infections of man (H1N1, H5N1, H7N2, H7N3, H7N7, H9N2) were evaluated for the presence of amino acid sequences, motifs, that are predicted to mediate peptide epitope binding with high affinity to the most frequent HLA-DR allelic products. Peptides conserved among diverse influenza strains were then synthesized, evaluated for binding to purified HLA-DR molecules and for their capacity to induce influenza-specific immune recall responses using human donor peripheral blood mononuclear cells (PBMC). Accordingly, 20 epitopes were selected for further investigation based on their conservancy among diverse influenza strains, predicted population coverage in diverse ethnic groups and capacity to recall influenza-specific responses. A DNA plasmid encoding the epitopes was constructed using amino acid spacers between epitopes to promote optimum processing and presentation. Immunogenicity of the DNA vaccine was measured using HLA-DR4 transgenic mice and the TriGrid in vivo electroporation device. Vaccination resulted in peptide-specific immune responses, augmented HA-specific antibody responses and protection of HLA-DR4 transgenic mice from lethal PR8 influenza virus challenge. These studies demonstrate the utility of this vaccine format and the contribution of CD4(+) T cell responses to protection against influenza infection.

A degenerate HLA-DR epitope pool of HER-2/neu reveals a novel in vivo immunodominant epitope, HER-2/neu88-102.

PURPOSE: Over the past two decades, there has been significant interest in targeting HER-2/neu in immune-based approaches for the treatment of HER-2/neu+ cancers. For example, peptide vaccination using a CD8 T cell-activating HER-2/neu epitope (amino acids 369-377) is an approach that is being considered in advanced phase clinical trials. Studies have suggested that the persistence of HER-2/neu-specific CD8 T cells could be improved by incorporating human leukocyte antigen (HLA) class II epitopes in the vaccine. Our goal in this study was to identify broad coverage HLA-DR epitopes of HER-2/neu, an antigen that is highly expressed in a variety of carcinomas. EXPERIMENTAL DESIGN: A combination of algorithms and HLA-DR-binding assays was used to identify HLA-DR epitopes of HER-2/neu antigen. Evidence of preexistent immunity in cancer patients against the identified epitopes was determined using IFN-gamma enzyme-linked immunosorbent spot (ELIspot) assay. RESULTS: Eighty-four HLA-DR epitopes of HER-2/neu were predicted, 15 of which had high binding affinity for > or =11 common HLA-DR molecules. A degenerate pool of four HLA-DR-restricted 15-amino acid epitopes (p59, p88, p422, and p885) was identified, against which >58% of breast and ovarian cancer patients had preexistent T-cell immunity. All four epitopes are naturally processed by antigen-presenting cells. Hardy-Weinberg analysis showed that the pool is useful in approximately 84% of population. Lastly, in this degenerate pool, we identified a novel in vivo immunodominant HLA-DR epitope, HER-2/neu(88-102) (p88). CONCLUSION: The broad coverage and natural immunity to this epitope pool suggests potential usefulness in HER-2/neu-targeting, immune-based therapies such as vaccines.

Induction of immune responses and clinical efficacy in a phase II trial of IDM-2101, a 10-epitope cytotoxic T-lymphocyte vaccine, in metastatic non-small-cell lung cancer.

PURPOSE: Generation of broad cytotoxic T-lymphocyte responses against multiple epitopes and tumor-associated antigens (TAAs) may provide effective immunotherapy in patients with cancer. We evaluated a single-vial peptide vaccine consisting of nine HLA-A2 supertype-binding epitopes (two native and seven analog epitopes modified for optimal HLA binding or T-cell receptor stimulation) covering five TAAs and the universal helper pan-DR epitope, formulated as a stable emulsion with incomplete Freund's adjuvant (Montanide ISA 51; Seppic SA, Paris, France). The clinical efficacy, safety, and multiepitope immunogenicity of IDM-2101 was evaluated in patients with stage IIIB or IV non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: A total of 63 patients were enrolled who were positive for HLA-A2. End points included survival, safety, and immune response. IDM-2101 (previously EP-2101) was administered every 3 weeks for the first 15 weeks, then every 2 months through year 1, then quarterly through year 2, for a total of 13 doses. Epitope-specific cytotoxic and helper T-lymphocyte immunogenic responses were measured by the interferon gamma enzyme-linked immunosorbent spot assay. RESULTS: No significant adverse events were noted. Low-grade erythema and pain at the injection site were the most common adverse effects. One-year survival in the treated patients was 60%, and median survival was 17.3 months. One complete and one partial response were identified. Survival was longer in patients demonstrating an immune response to epitope peptides (P < .001). CONCLUSION: IDM-2101 was well tolerated, and evidence of efficacy was suggested.

An HLA-DR-degenerate epitope pool detects insulin-like growth factor binding protein 2-specific immunity in patients with cancer.

Recent studies have shown the importance of helper CD4 T cells in initiating and sustaining tumor-specific CD8 T-cell immunity. This has paved the way for identifying MHC class II epitopes that could be incorporated into class I-based vaccines. In this study, the goal was to identify an HLA-DR-degenerate epitope pool derived from insulin-like growth factor binding protein 2 (IGFBP-2). IGFBP-2, a regulator of insulin-like growth factor action, is overexpressed in the majority of breast and ovarian cancers. Using algorithms, we predicted 29 HLA-DR1-binding epitopes. Binding assays targeting 15 different HLA-DRs revealed that 10 epitopes were degenerate, binding to at least four different HLA-DR variants. An IFN-gamma enzyme-linked immunosorbent spot assay was used to assess immunity to these 10 epitopes in 48 patients with either breast or ovarian cancer and 18 controls. Elevated T-cell immunity in patients was detected in 4 of the 10 epitopes (IGFBP2.17, IGFBP2.22, IGFBP2.249, and IGFBP2.293). The cumulative T-cell frequency of these four epitopes was elevated in patients relative to controls. All four peptides are naturally processed and presented to CD4 T-cells. The degenerate pool of peptides covers nearly 80% of patients and may be useful for augmenting CD4 T-cell immunity in patients undergoing immunization.

Improved immunogenicity of an immunodominant epitope of the HER-2/neu protooncogene by alterations of MHC contact residues.

The HER-2/neu (HER-2) oncogene is expressed in normal epithelial surfaces at low levels and overexpressed in several types of tumors. The low immunogenicity against this self tumor Ag can be improved by developing epitopes with amino acid replacements in their sequences. In this study, three HER-2/neu.369 (HER-2.369) analogue peptides, produced by modifying both anchor positions by introducing L, V, or T at position 2 and V at the C terminus, were analyzed for their capacity to induce CTLs in vitro from human PBMC and in vivo in HLA-A2.1/Kb transgenic mice. One of the analogues (HER-2.369 V2V9) sensitized target cells for HER-2-specific recognition by human CTLs and induced specific CTLs in vitro at 100-fold lower concentrations than the HER-2.369 wild-type epitope. These CTLs were also able to recognize the wild-type epitope and HER-2-expressing tumors in an MHC-restricted manner. Furthermore, a 100-fold lower amount of the HER-2.369 V2V9 analogue compared with the wild-type epitope was required to induce CTLs in HLA-A2.1/Kb transgenic mice. However, the V2V9 analogue demonstrated only marginally better binding to the MHC class I A2 allele compared with wild type. To establish thermodynamic parameters, we developed radiolabeled F3*Y analogues from both the HER-2.369 epitope and the V2V9 analogue. Our results indicate that the high biological activity of the HER-2.369 V2V9 epitope is associated with a slower dissociation kinetic profile, resulting in an epitope with greater HLA-A2 stability.

A decaepitope polypeptide primes for multiple CD8+ IFN-gamma and Th lymphocyte responses: evaluation of multiepitope polypeptides as a mode for vaccine delivery.

Proteins are generally regarded as ineffective immunogens for CTL responses. We synthesized a 100-mer decaepitope polypeptide and tested its capacity to induce multiple CD8(+) IFN-gamma and Th lymphocyte (HTL) responses in HLA transgenic mice. Following a single immunization in the absence of adjuvant, significant IFN-gamma in vitro recall responses were detected for all epitopes included in the construct (six A2.1-, three A11-restricted CTL epitopes, and one universal HTL epitope). Immunization with truncated forms of the decaepitope polypeptide was used to demonstrate that optimal immunogenicity was associated with a size of at least 30-40 residues (3-4 epitopes). Solubility analyses of the truncated constructs were used to identify a correlation between immunogenicity for IFN-gamma responses and the propensity of these constructs to form particulate aggregates. Although the decaepitope polypeptide and a pool of epitopes emulsified in IFA elicited similar levels of CD8(+) responses using fresh splenocytes, we found that the decaepitope polypeptide more effectively primed for in vitro recall CD8(+) T cell responses. Finally, immunogenicity comparisons were also made between the decaepitope polypeptide and a corresponding gene encoding the same polypeptide delivered by naked DNA immunization. Although naked DNA immunization induced somewhat greater direct ex vivo and in vitro recall responses 2 wk after a single immunization, only the polypeptide induced significant in vitro recall responses 6 wk following the priming immunization. These studies support further evaluation of multiepitope polypeptide vaccines for induction of CD8(+) IFN-gamma and HTL responses.