Pamuk Bilsel

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.

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.

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.

An M2 cytoplasmic tail mutant as a live attenuated influenza vaccine against pandemic (H1N1) 2009 influenza virus

The 2009 influenza pandemic brought home the importance of vaccines in infection control. Previously, we demonstrated an M2 cytoplasmic tail mutant H5N1 influenza virus could serve as a live-attenuated vaccine. Here, we adapted that strategy, generating a mutant pandemic (H1N1) 2009 virus that grew well in cell culture, but replicated less well in mice than did wild-type virus. The mutant virus elicited sterile immunity in mice, completely protecting them from challenge with a pandemic (H1N1) 2009 virus. Our results indicate that M2 cytoplasmic tail mutants are suitable for live-attenuated vaccines against pandemic viruses.

M2SR, a novel live single replication influenza virus vaccine, provides effective heterosubtypic protection in mice.

Despite the annual public health burden of seasonal influenza and the continuing threat of a global pandemic posed by the emergence of highly pathogenic/pandemic strains, conventional influenza vaccines do not provide universal protection, and exhibit suboptimal efficacy rates, even when they are well matched to circulating strains. To address the need for a highly effective universal influenza vaccine, we have developed a novel M2-deficient single replication vaccine virus (M2SR) that induces strong cross-protective immunity against multiple influenza strains in mice. M2SR is able to infect cells and expresses all viral proteins except M2, but is unable to generate progeny virus. M2SR generated from influenza A/Puerto Rico/8/34 (H1N1) protected mice against lethal challenge with influenza A/Puerto Rico/8/34 (H1N1, homosubtypic) and influenza A/Aichi/2/1968 (H3N2, heterosubtypic). The vaccine induced strong systemic and mucosal antibody responses of both IgA and IgG classes. Strong virus-specific T cell responses were also induced. Following heterologous challenge, significant numbers of IFN-γ-producing CD8 T cells, with effector or effector/memory phenotypes and specific for conserved viral epitopes, were observed in the lungs of vaccinated mice. A substantial proportion of the CD8 T cells expressed Granzyme B, suggesting that they were capable of killing virus-infected cells. Thus, our data suggest that M2-deficient influenza viruses represent a promising new approach for developing a universal influenza vaccine.

M2SR, a novel live influenza vaccine, protects mice and ferrets against highly pathogenic avian influenza

The emergence of highly pathogenic avian influenza H5N1 viruses has heightened global concern about the threat posed by pandemic influenza. To address the need for a highly effective universal influenza vaccine, we developed a novel M2-deficient single replication (M2SR) influenza vaccine virus and previously reported that it provided strong heterosubtypic protection against seasonal influenza viruses in mice. In the current study, we assessed M2SR induced protection against H5N1 influenza in mice and ferrets. Mice were intranasally inoculated with M2SR viruses containing the HA and NA from A/Vietnam/1203/2004 (M2SR H5N1) or A/California/07/2009 (M2SR H1N1). All M2SR vaccinated mice survived lethal challenge with influenza A/Vietnam/1203/2004 (H5N1), whereas 40% of mice vaccinated with recombinant H5 HA and none of the naïve controls survived. M2SR H5N1 provided sterile immunity, whereas low levels of virus were detected in the lungs of some M2SR H1N1 vaccinated mice. In contrast, recombinant H5 HA vaccinated mice and naïve controls showed systemic infection. M2SR H5N1 induced strong serum and mucosal antibody responses (IgG and IgA classes) against H5 HA, with high hemagglutination inhibition (HAI) titers. In contrast, while M2SR H1N1 elicited cross-reactive antibodies recognizing the H5 HA2 stalk region or the neuraminidase, no HAI activity against H5N1 virus was detected after M2SR H1N1 immunization. Both M2SR H5N1 and H1N1 also protected ferrets against lethal challenge with A/Vietnam/1203/2004. A prime-boost regimen provided optimal protection with no virus detected in the respiratory tract or brain after challenge. As in the mouse model, only the M2SR H5N1 vaccine induced HAI antibodies against the challenge virus in ferrets, while the M2SR H1N1 was able to provide protection without the induction of HAI antibodies. In summary, effective protection against highly pathogenic H5N1 influenza virus was provided by both homologous H5N1 M2SR and heterologous H1N1 M2SR demonstrating the cross-protective attributes of the M2SR platform.

Novel influenza vaccine M2SR protects against drifted H1N1 and H3N2 influenza virus challenge in ferrets with pre-existing immunity

Current influenza vaccines do not provide effective protection against heterologous influenza viruses. The ability of the novel M2SR influenza vaccine to protect against drifted influenza viruses was evaluated in naïve ferrets and in ferrets with pre-existing immunity to influenza. In naïve ferrets, M2SR provided similar protection against drifted challenge viruses as the comparator vaccine, FluMist®. However, in ferrets with pre-existing immunity, M2SR provided superior protection than FluMist in two model systems. In the first model, ferrets were infected with influenza A H1N1pdm and influenza B viruses to mimic the diverse influenza exposure in humans. The pre-infected ferrets, seropositive to H1N1pdm and influenza B but seronegative to H3N2, were then vaccinated with H3N2 M2SR or monovalent H3N2 FluMist virus (A/Brisbane/10/2007, clade 1) and challenged 6 weeks later with a drifted H3N2 virus (clade 3C.2a). Antibody titers to Brisbane/10/2007 were higher in M2SR vaccinated ferrets than in FluMist vaccinated ferrets in the pre-infected ferrets whereas the opposite was observed in naïve ferrets. After challenge with drifted H3N2 virus, M2SR provided superior protection than FluMist monovalent vaccine. In the second model, the impact of homologous pre-existing immunity upon vaccine-induced protection was evaluated. Ferrets, pre-infected with H1N1pdm virus, were vaccinated 90 days later with H1N1pdm M2SR or FluMist monovalent vaccine and challenged 6 weeks later with a pre-pandemic seasonal H1N1 virus, A/Brisbane/59/2007 (Bris59). While cross-reactive serum IgG antibodies against the Bris59 HA were detected after vaccination, anti-Bris59 hemagglutination inhibition antibodies were only detected post-challenge. M2SR provided better protection against Bris59 challenge than FluMist suggesting that homologous pre-existing immunity affected FluMist virus to a greater degree than M2SR. These results suggest that the single replication intranasal M2SR vaccine provides effective protection against drifted influenza A viruses not only in naïve ferrets but also in those with pre-existing immunity in contrast to FluMist viruses.