The HAd-neutralizing antibody titer represents the reciprocal of the highest serum dilution that completely inhibited the production of viral cytopathic effect (c

The HAd-neutralizing antibody titer represents the reciprocal of the highest serum dilution that completely inhibited the production of viral cytopathic effect (c.p.e.) and the titers are given as Mean SD. the importance of a heterologous prime-boost approach for achieving an enhanced immune response. The immunization of na?ve or HAd-primed mice with BAd-H5HA bestowed full protection from morbidity and mortality following a potentially lethal challenge with A/Hong Kong/483/97. These results demonstrate the importance of BAd vectors as an alternate or supplement to HAd vectors for influenza pandemic preparedness. INTRODUCTION In recent years, highly pathogenic H5N1 avian influenza viruses, which are highly lethal to domestic poultry and wild birds, Nortadalafil have spread to several countries in Asia, Africa and Europe, primarily due to migrating birds.1 This, coupled with reports of H5N1 influenza infection and mortality in humans particularly in individuals with close contacts with diseased birds,2,3,4 have heightened the need for an effective vaccine against H5N1 viruses to ward off the threat of an impending H5N1-associated influenza pandemic.5 The inherent ability of influenza viruses to mutate and render the strain-specific vaccines ineffective, has underscored the importance of exploring alternative vaccine design, delivery and production strategies to provide protection against antigenically distinct strains of H5N1. As the virus is highly lethal to chickens, the maintenance of a constant supply of embryonated eggs could be difficult in a pandemic, enhancing the need for an egg-independent vaccine strategy to combat a H5N1 pandemic. The currently licensed seasonal influenza vaccines are egg-derived and subtype-specific, thus do not induce protective immune responses against highly pathogenic H5N1 influenza viruses. It is quite obvious that vaccine strategies offering cross protection against antigenically distinct H5N1 viruses is necessary, especially during the period immediately following the emergence of a highly transmissible human H5N1 virus before a strain-matched vaccine could be produced. Adenoviral (Ad) vectors fulfill important criteria of an ideal vaccine vector in terms of efficacy, safety, and stability.6 Ad vectors induce a strong innate immune response7 that enhances the development of high levels of humoral and cellular immune responses by preferential targeting of antigen presenting cells to facilitate antigen presentation.6,8 In addition, we have previously shown that human Ad vector expressing H5N1 antigen offer 100% protection in mice following lethal challenge with distinct strains of H5N1 influenza virus.9 In view of the fact that the levels of Ad antibodies (vector immunity),10,11 vary in the human Nortadalafil population (because of the existence of 50 Ad serotypes that infect Nortadalafil humans), it is believed that vector immunity may adversely impact expression of foreign genes carried by Ad vectors. With the anticipation that vector immunity may not cross-neutralize nonhuman Ad, a number of Ad vectors derived from various Rabbit Polyclonal to Synapsin (phospho-Ser9) animal species are at different stages of development.8,12, 13 We have shown that Ad neutralizing antibodies in human sera do not cross neutralize bovine adenovirus subtype 3 (BAd3).10 This study demonstrates that BAd vectors overcome exceptionally high levels of vector immunity and could therefore serve as an alternate or supplement to HAd vectors for delivering H5HA antigen in a vaccine during a pandemic situation. In addition, our results suggest that BAd vectors could effectively supplement HAd vectors in a heterologous prime-boost approach. RESULTS Characterization of BAd vector (BAd-H5HA) expressing hemagglutinin of H5N1 influenza virus The full coding region of hemagglutinin gene (HA) of H5N1 avian influenza virus, A/Hong Kong/156/97 (HK/156), under the control of cytomegalovirus (CMV) immediate early promoter and bovine growth hormone (BGH) polyadenylation site was inserted in the E1 region of BAd3 genome in the E1-parallel orientation using homologous recombination in bacteria followed by transfection of an infectious clone into an adenoviral E1 expressing cell line (BHH3)14 that supports replication of E1-deleted BAd3 vectors. The recombinant vector, BAd-H5HA (Figure 1a) showed visible cytopathic effect (c.p.e.) on 16 after transfection. In order to determine, whether the HA protein was expressed in BAd-H5HA-infected cells, Western blot analysis was performed on the whole cell extract of virus-infected BHH3 cells. Approximately 47 kDa HA-specific band was clearly visible in the BAd-H5HA-infected cell.