Mark J. Newman

Phase 1 trial of immunological adjuvant QS-21 with a GM2 ganglioside-keyhole limpet haemocyanin conjugate vaccine in patients with malignant melanoma

Increasing doses of saponin fraction QS-21 were administered as immunological adjuvant in a Phase 1 trial with a constant dose of the melanoma ganglioside GM2 covalently attached to keyhole limpet haemocyanin (KLH). Twenty-eight patients with AJCC Stage III or IV melanoma who were free from disease after surgery were treated with six vaccinations administered subcutaneously over a 5-month period. Local and systemic reactions were QS-21 dose-related. Doses of < or = 100 micrograms induced mild local tenderness and inflammation at vaccination sites lasting 2-4 days and occasional brief low-grade fever and malaise, but no significant incapacitation. The 200 micrograms dose induced low-grade fever and malaise after 30% of vaccinations and local reactions as large as 20 cm in diameter were seen in all patients, resulting in discomfort with usage of the injected extremity for 5-10 days. The titres of IgM and IgG antibodies against GM2, and IgG antibodies against KLH, were highest at the 100 and 200 micrograms QS-21 doses. No antibodies against QS-21 were detected. This trial identifies the 100 micrograms dose of QS-21 as the optimal well tolerated dose for induction of antibodies against both the melanoma ganglioside/GM2 and the protein KLH in melanoma patients.

Rationally Engineered Therapeutic Proteins with Reduced Immunogenicity

Chronic administration of protein therapeutics may elicit unacceptable immune responses to the specific protein. Our hypothesis is that the immunogenicity of protein drugs can be ascribed to a few immunodominant helper T lymphocyte (HTL) epitopes, and that reducing the MHC binding affinity of these HTL epitopes contained within these proteins can generate drugs with lower immunogenicity. To test this hypothesis, we studied the protein therapeutic erythropoietin (Epo). Two regions within Epo, designated Epo 91–120 and Epo 126–155, contained HTL epitopes that were recognized by individuals with numerous HLA-DR types, a property common to immunodominant HTL epitopes. We then engineered analog epitopes with reduced HLA binding affinity. These analog epitopes were associated with reduced in vitro immunogenicity. Two modified forms of Epo containing these substitutions were shown to be bioactive and nonimmunogenic in vitro. These findings support our hypothesis and demonstrate that immunogenicity of protein drugs can be reduced in a systematic and predictable manner.

Structure/function studies of QS-21 adjuvant: assessment of triterpene aldehyde and glucuronic acid roles in adjuvant function.

QS-21, a purified Quillaja saponaria saponin immunologic adjuvant, contains two functional groups that we hypothesized to be involved in the adjuvant mechanism of action through charge or Schiff base interaction with a cellular target. Derivatives, prepared by modification of these sites, were prepared and tested for their ability to augment the immunogenicity of the antigen ovalbumin (OVA) in C57BL/6 mice. QS-21 derivatives that were modified at the carboxyl group on an anionic sugar, glucuronic acid, retained adjuvant activity for antibody stimulation, inducing relative increases in antibody titers similar to those induced by QS-21, although the minimum adjuvant dose required for this stimulation was increased several fold relative to the dose of unmodified QS-21. One of these derivatives also retained significant activity for induction of OVA-specific cytotoxic T-lymphocytes. In contrast, QS-21 derivatives modified at an aldehyde on the triterpene did not show adjuvant activity for antibody stimulation or for induction of cytotoxic T-lymphocytes, suggesting that this functional group may be involved in the adjuvant mechanism.

Immunomic Analysis of the Repertoire of T-Cell Specificities for Influenza A Virus in Humans

ABSTRACT Continuing antigenic drift allows influenza viruses to escape antibody-mediated recognition, and as a consequence, the vaccine currently in use needs to be altered annually. Highly conserved epitopes recognized by effector T cells may represent an alternative approach for the generation of a more universal influenza virus vaccine. Relatively few highly conserved epitopes are currently known in humans, and relatively few epitopes have been identified from proteins other than hemagglutinin and nucleoprotein. This prompted us to perform a study aimed at identifying a set of human T-cell epitopes that would provide broad coverage against different virus strains and subtypes. To provide coverage across different ethnicities, seven different HLA supertypes were considered. More than 4,000 peptides were selected from a panel of 23 influenza A virus strains based on predicted high-affinity binding to HLA class I or class II and high conservancy levels. Peripheral blood mononuclear cells from 44 healthy human blood donors were tested for reactivity against HLA-matched peptides by using gamma interferon enzyme-linked immunospot assays. Interestingly, we found that PB1 was the major target for both CD4+ and CD8+ T-cell responses. The 54 nonredundant epitopes (38 class I and 16 class II) identified herein provided high coverage among different ethnicities, were conserved in the majority of the strains analyzed, and were consistently recognized in multiple individuals. These results enable further functional studies of T-cell responses during influenza virus infection and provide a potential base for the development of a universal influenza vaccine.

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-Ab class II molecule were used to evaluate different vaccine design strategies to simultaneously induce multiple HTL responses. All four epitopes were immunogenic in H-2b 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.

Predicting population coverage of T-cell epitope-based diagnostics and vaccines

BackgroundT cells recognize a complex between a specific major histocompatibility complex (MHC) molecule and a particular pathogen-derived epitope. A given epitope will elicit a response only in individuals that express an MHC molecule capable of binding that particular epitope. MHC molecules are extremely polymorphic and over a thousand different human MHC (HLA) alleles are known. A disproportionate amount of MHC polymorphism occurs in positions constituting the peptide-binding region, and as a result, MHC molecules exhibit a widely varying binding specificity. In the design of peptide-based vaccines and diagnostics, the issue of population coverage in relation to MHC polymorphism is further complicated by the fact that different HLA types are expressed at dramatically different frequencies in different ethnicities. Thus, without careful consideration, a vaccine or diagnostic with ethnically biased population coverage could result.ResultsTo address this issue, an algorithm was developed to calculate, on the basis of HLA genotypic frequencies, the fraction of individuals expected to respond to a given epitope set, diagnostic or vaccine. The population coverage estimates are based on MHC binding and/or T cell restriction data, although the tool can be utilized in a more general fashion. The algorithm was implemented as a web-application available at http://epitope.liai.org:8080/tools/population.ConclusionWe have developed a web-based tool to predict population coverage of T-cell epitope-based diagnostics and vaccines based on MHC binding and/or T cell restriction data. Accordingly, epitope-based vaccines or diagnostics can be designed to maximize population coverage, while minimizing complexity (that is, the number of different epitopes included in the diagnostic or vaccine), and also minimizing the variability of coverage obtained or projected in different ethnic groups.

Development of a DNA vaccine designed to induce cytotoxic T lymphocyte responses to multiple conserved epitopes in HIV-1.

Epitope-based vaccines designed to induce CTL responses specific for HIV-1 are being developed as a means for addressing vaccine potency and viral heterogeneity. We identified a set of 21 HLA-A2, HLA-A3, and HLA-B7 restricted supertype epitopes from conserved regions of HIV-1 to develop such a vaccine. Based on peptide-binding studies and phenotypic frequencies of HLA-A2, HLA-A3, and HLA-B7 allelic variants, these epitopes are predicted to be immunogenic in greater than 85% of individuals. Immunological recognition of all but one of the vaccine candidate epitopes was demonstrated by IFN-γ ELISPOT assays in PBMC from HIV-1-infected subjects. The HLA supertypes of the subjects was a very strong predictor of epitope-specific responses, but some subjects responded to epitopes outside of the predicted HLA type. A DNA plasmid vaccine, EP HIV-1090, was designed to express the 21 CTL epitopes as a single Ag and tested for immunogenicity using HLA transgenic mice. Immunization of HLA transgenic mice with this vaccine was sufficient to induce CTL responses to multiple HIV-1 epitopes, comparable in magnitude to those induced by immunization with peptides. The CTL induced by the vaccine recognized target cells pulsed with peptide or cells transfected with HIV-1 env or gag genes. There was no indication of immunodominance, as the vaccine induced CTL responses specific for multiple epitopes in individual mice. These data indicate that the EP HIV-1090 DNA vaccine may be suitable for inducing relevant HIV-1-specific CTL responses in humans.

Identification of Novel HLA-A2-Restricted Human Immunodeficiency Virus Type 1-Specific Cytotoxic T-Lymphocyte Epitopes Predicted by the HLA-A2 Supertype Peptide-Binding Motif

ABSTRACT Virus-specific cytotoxic T-lymphocyte (CTL) responses are critical in the control of human immunodeficiency virus type 1 (HIV-1) infection and will play an important part in therapeutic and prophylactic HIV-1 vaccines. The identification of virus-specific epitopes that are efficiently recognized by CTL is the first step in the development of future vaccines. Here we describe the immunological characterization of a number of novel HIV-1-specific, HLA-A2-restricted CTL epitopes that share a high degree of conservation within HIV-1 and a strong binding to different alleles of the HLA-A2 superfamily. These novel epitopes include the first reported CTL epitope in the Vpr protein. Two of the novel epitopes were immunodominant among the HLA-A2-restricted CTL responses of individuals with acute and chronic HIV-1 infection. The novel CTL epitopes identified here should be included in future vaccines designed to induce HIV-1-specific CTL responses restricted by the HLA-A2 superfamily and will be important to assess in immunogenicity studies in infected persons and in uninfected recipients of candidate HIV-1 vaccines.

Rational Design of a Multiepitope Vaccine Encoding T-Lymphocyte Epitopes for Treatment of Chronic Hepatitis B Virus Infections

ABSTRACT 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-2bxd (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.

Definition of the Mamu A*01 Peptide Binding Specificity: Application to the Identification of Wild-Type and Optimized Ligands from Simian Immunodeficiency Virus Regulatory Proteins

Single amino acid substitution analogs of the known Mamu A*01 binding peptide gag 181-190 and libraries of naturally occurring sequences of viral or bacterial origin were used to rigorously define the peptide binding motif associated with Mamu A*01 molecules. The presence of S or T in position 2, P in position 3, and hydrophobic or aromatic residues at the C terminus is associated with optimal binding capacity. At each of these positions, additional residues are also tolerated but associated with significant decreases in binding capacity. The presence of at least two preferred and one tolerated residues at the three anchor positions is necessary for good Mamu A*01 binding; optimal ligand size is 8–9 residues. This detailed motif has been used to map potential epitopes from SIVmac239 regulatory proteins and to engineer peptides with increased binding capacity. A total of 13 wild type and 17 analog candidate epitopes were identified. Furthermore, our analysis reveals a significantly lower than expected frequency of epitopes in early regulatory proteins, suggesting a possible evolutionary- and/or immunoselection directed against variants of viral products that contain CTL epitopes.