Improve efficacy, especially in the elderly who are at high risk of severe disease and show reduced responses to current flu vaccines. Peptide scanning of T cell responses of healthy human individuals has shown that matrix 1 (M1) and nucleoprotein (NP) are among the prominent targets of CD8+ and CD4+ T cell cross-recognition [3], so they are of interest as vaccine candidates. By sequence homology, NP is .90 conserved among influenza A isolates [4]. Both murine [5] and human [6] cytotoxic T lymphocytes induced by NP of one virus strain have been shown to cross-react with NP from different influenza A strains. The strong immune responses to NP in mice contribute to protection against challenge [7] via CD8+ T cells [5,8], as well as contributions from CD4+ cells [9,10] and antibodies [11?3]. The influenza A matrix (M) gene encodes two highly conserved proteins: an ion channel protein, M2, and the capsid protein, M1. M1 is not a major protective antigen in the mouse and is not well recognized by mouse T cells [14], but has long been known to be recognized byHighly purchase ML 264 immunogenic Simian Adenovirus Vectorhuman T cells [15]. Thus its potential contribution to vaccine protection may be buy CI 1011 underestimated by mouse studies. While epitopes providing targets widely shared among influenza viruses have been identified in multiple viral proteins, not all of them are highly immunogenic when presented by classical vaccines. More potent immunization can be achieved using recombinant vectors to express the influenza antigens and focus immunity on these targets. Recombinant adenovirus vectors are especially effective at eliciting strong T cell responses to transgene products [16?8]. Recombinant adenovirus vectors expressing NP [19] or both NP and M2 [20,21] can protect mice against a range of influenza virus challenges, including highly pathogenic avian H5N1 strains. While potential interference by prior immunity to human adenoviruses has been suggested as a barrier, this issue can be circumvented by use of vectors based on animal adenoviruses [22?5]. Chimpanzee adenoviruses have been shown to be useful vaccine vectors in a variety of animal studies [26?0], and the prevalence of neutralizing antibodies against chimpanzee adenoviruses is low in human populations [31?3], but not all of them are equally immunogenic. In this study, we use a simian adenovirus, PanAd3, isolated from the bonobo Pan paniscus. This novel adenovirus strain was identified in a study of more than 1000 adenoviruses isolated from chimpanzees and bonobos in order to increase the available repertoire of vectors [34]. In the large scale screening experiments, PanAd3 was among the most potently immunogenic in mice and was also among the least frequently recognized by neutralizing antibodies in human sera. We have generated a replication incompetent PanAd3 vector deleted of E1 and E3 regions and expressing a fusion protein of the NP and M1 antigens of influenza A, chosen as targets of broad and cross-reactive T cell immunity in humans [3]. The PanAd3-based vaccine was tested for induction of antibody and T cell responses in the systemic and mucosal compartments in mice, as well as for protection against lethal influenza virus challenge. We demonstrate that PanAd3 expressing conserved influenza virus antigens provided highly effective protection after a single intranasal administration. Thus it shows considerable promise as a vaccine candidate.Materials and Methods Ethics statementAll animal protoc.Improve efficacy, especially in the elderly who are at high risk of severe disease and show reduced responses to current flu vaccines. Peptide scanning of T cell responses of healthy human individuals has shown that matrix 1 (M1) and nucleoprotein (NP) are among the prominent targets of CD8+ and CD4+ T cell cross-recognition [3], so they are of interest as vaccine candidates. By sequence homology, NP is .90 conserved among influenza A isolates [4]. Both murine [5] and human [6] cytotoxic T lymphocytes induced by NP of one virus strain have been shown to cross-react with NP from different influenza A strains. The strong immune responses to NP in mice contribute to protection against challenge [7] via CD8+ T cells [5,8], as well as contributions from CD4+ cells [9,10] and antibodies [11?3]. The influenza A matrix (M) gene encodes two highly conserved proteins: an ion channel protein, M2, and the capsid protein, M1. M1 is not a major protective antigen in the mouse and is not well recognized by mouse T cells [14], but has long been known to be recognized byHighly Immunogenic Simian Adenovirus Vectorhuman T cells [15]. Thus its potential contribution to vaccine protection may be underestimated by mouse studies. While epitopes providing targets widely shared among influenza viruses have been identified in multiple viral proteins, not all of them are highly immunogenic when presented by classical vaccines. More potent immunization can be achieved using recombinant vectors to express the influenza antigens and focus immunity on these targets. Recombinant adenovirus vectors are especially effective at eliciting strong T cell responses to transgene products [16?8]. Recombinant adenovirus vectors expressing NP [19] or both NP and M2 [20,21] can protect mice against a range of influenza virus challenges, including highly pathogenic avian H5N1 strains. While potential interference by prior immunity to human adenoviruses has been suggested as a barrier, this issue can be circumvented by use of vectors based on animal adenoviruses [22?5]. Chimpanzee adenoviruses have been shown to be useful vaccine vectors in a variety of animal studies [26?0], and the prevalence of neutralizing antibodies against chimpanzee adenoviruses is low in human populations [31?3], but not all of them are equally immunogenic. In this study, we use a simian adenovirus, PanAd3, isolated from the bonobo Pan paniscus. This novel adenovirus strain was identified in a study of more than 1000 adenoviruses isolated from chimpanzees and bonobos in order to increase the available repertoire of vectors [34]. In the large scale screening experiments, PanAd3 was among the most potently immunogenic in mice and was also among the least frequently recognized by neutralizing antibodies in human sera. We have generated a replication incompetent PanAd3 vector deleted of E1 and E3 regions and expressing a fusion protein of the NP and M1 antigens of influenza A, chosen as targets of broad and cross-reactive T cell immunity in humans [3]. The PanAd3-based vaccine was tested for induction of antibody and T cell responses in the systemic and mucosal compartments in mice, as well as for protection against lethal influenza virus challenge. We demonstrate that PanAd3 expressing conserved influenza virus antigens provided highly effective protection after a single intranasal administration. Thus it shows considerable promise as a vaccine candidate.Materials and Methods Ethics statementAll animal protoc.