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.

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 influenzaspecific 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. Additionally, these epitopes may be suitable for vaccine applications directed at the induction of cellular immunity.

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.

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.

The entire phosphoprotein (P) and nucleocapsid (N) protein gene sequences and deduced amino acid sequences for 18 selected vesicular stomatitis virus isolates representative of the natural genetic diversity within the New Jersey serotype are reported. Phylogenetic analysis of the data using maximum parsimony allowed construction of evolutionary trees for the individual genes and the combined N, P, and glycoprotein (G) genes of these viruses. Virtually identical rates of nucleotide substitutions were found for each gene, indicating that evolution of these genes occurs at essentially the same rate. Although up to 19 and 17% sequence differences were evident in the P and N genes, respectively, no variation in gene length or evidence of recombinational rearrangements was found. However, striking evolutionary differences were observed among the amino acid sequences of vesicular stomatitis virus New Jersey N, P, and G proteins. The N protein amino acid sequence was the most highly conserved among the different isolates, indicating strong functional and structural constraints. Conversely, the P protein amino acid sequences were highly variable, indicating considerably fewer constraints or greater evolutionary pressure on the P protein. Much of the remarkable amino acid variability of the P protein resided in a hypervariable domain located between amino acids 153 and 205. The variability within this region would be consistent with it playing a structural role as a spacer to maintain correct conformational presentation of the separate active domains of this multifunctional protein. In marked contrast, the adjacent domain I of the P protein (previously thought to be under little evolutionary constraint) contained a highly conserved region. The colocalization of a short, potentially functional overlapping open reading frame to this region may explain this apparent anomaly.

The significance of the conserved cytoplasmic tail sequence of influenza A virus neuraminidase (NA) was analyzed by the recently developed reverse genetics technique (W.

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.

As the initial step in generating a live attenuated influenza A vaccine, we attempted to substitute an unrelated amino acid sequence (FLAG) for a portion of the neuraminidase (NA) molecule in influenza virus A/WSN/33 (HlNl), using a recently developed technique (reverse genetics [W.