Animals in the vaccine group had lung viral titers slightly lower than na? ve animals at day 6 but the difference was not statistically significant. current strain-matched vaccines and delays vaccine availability. Vaccines inducing immunity to conserved viral proteins could be manufactured and tested in advance and provide cross-protection against novel influenza viruses until strain-matched vaccines became available. Here we test two prototype vaccines for cross-protection against the recent pandemic virus. Methodology/Principal Findings BALB/c and C57BL/6 mice were intranasally immunized with a single dose of cold-adapted (and NP+M2-rAd immunization protected BALB/c and C57BL/6 mice against challenge with a mouse-adapted pH1N1 virus. Conclusion/Significance Cross-protective vaccines such as NP+M2-rAd and virus are effective Povidone iodine against pH1N1 challenge within 3 weeks of immunization. Protection was not dependent on recognition of the highly variable external viral proteins and could be achieved with a single vaccine dose. The rAd vaccine was superior to the vaccine Povidone iodine by certain measures, justifying continued investigation of this experimental vaccine even though vaccine is already available. This study highlights the potential for cross-protective vaccines as a public health option early in an influenza pandemic. Introduction Influenza virus is a significant public health concern, with the average influenza season in the U.S. resulting in millions of cases and tens of thousands of deaths [1]. These deaths occur despite large-scale vaccination efforts, use of multiple antiviral influenza drugs, and in-patient care. Pandemic influenza represents an even greater concern. Current influenza vaccines function by targeting hemagglutinin (HA). Seasonal vaccines are not useful when a major antigenic change occurs in the circulating strain. Due to the time required for manufacture of new strain-matched vaccines, this can result in large proportions of the population being unprotected during the initial pandemic wave. This situation is exemplified by the 2009 2009 H1N1 pandemic. The 2009 2009 pH1N1 is believed to have originated in Mexico during February of 2009 [2]. The virus soon spread to multiple countries, with the first U.S. case identified in mid-April 2009. The WHO officially declared an influenza pandemic in June. The time required for vaccine manufacture, testing and distribution delayed immunization in the U.S. until October, and initially restricted it to high risk individuals due to limited supply. By this time, infection rates were near peak levels. This delay occurred despite rapid identification of the novel strain. The 2009 CSPG4 2009 pH1N1 experience has highlighted the need to develop alternative vaccines operating by mechanisms of protection not dependent on antibodies against HA, the most variable influenza virus antigen. Instead, vaccination can target conserved antigens of influenza virus to generate heterosubtypic immunity protective against diverse influenza A virus strains and subtypes. While heterosubtypic immunity would not prevent infection, studies in animal models have clearly demonstrated that it can reduce the severity of illness and protect against lethal influenza virus challenge. Such cross-protective vaccines could be prepared and stockpiled prior to emergence of a pandemic virus, reducing the time between identification of a novel threat and deployment of the vaccine. Various approaches to heterosubtypic vaccination against influenza have been studied, using one or more of the conserved viral proteins such as NP [3]C[6], matrix protein 1 (M1) or M2 [7]C[9], or the viral Povidone iodine polymerases [5]. Delivery systems for such vaccines have utilized viral vectors [10], plasmid DNA [3], virus-like particles [11], proteins [12], or peptides [13]. Heterosubtypic immunity can be mediated by T cells and/or antibodies directed against such relatively conserved antigens such as NP, M1, M2 and the HA stem [7]. We have previously demonstrated that prime-boost immunization involving boosting with rAd expressing NP and M2 resulted in protection against challenge with divergent influenza strains, including virulent H1N1 and H3N2, and a highly pathogenic H5N1 avian virus [14], [15]. When the rAd vaccine was given intranasally without priming, protection was rapidly induced, with vaccinated animals protected from lethal challenge 2 weeks after a single immunization, and was long-lasting, with protective immunity still present 10 months after immunization [16]. While such vectored vaccines elicit potent immune responses focused against a limited range of viral antigens (such as NP and M2), to date they remain an experimental approach. An alternative may be to utilize live-attenuated, influenza vaccines, which are approved for human use and have been previously demonstrated to induce heterosubtypic immunity [14], [17], [18]. The and rAd-vectored vaccines can each be intranasally administered, thus providing the advantage of immunity at a relevant anatomic site. In this study we have compared the effectiveness of NP+M2-rAd vaccine and a 1977 vaccine against infection with the 2009 2009 pandemic virus. We describe results of testing a single mucosally-administered dose of these vaccines. We measured humoral and cellular immune reactions elicited from the vaccines which were cross-reactive against pH1N1 antigens, as well as the ability of the vaccines.