The full coding region of HA under the control of the cytomegalovirus (CMV) immediate early promoter and bovine growth hormone (BGH) polyadenylation signal (polyA) and full length coding region of NP gene of the A/Vietnam/1203/04 virus under the control of the murine CMV promoter and the simian virus 40 (SV40) polyA were inserted into early region 1 (E1) of the HAd genome using the Cre-recombinase-mediated site-specific recombination system [20]. Both genes in HAd-HA-NP were in the E1-parallel orientation. The recombinant vector, HAd-HA-NP (Figure 1A) showed visible cytopathic effect (c.p.e.) on the ninth day post-transfection. Western blot analysis was done to confirm the expression of HA and NP in 293 cells. Two distinct polypeptide bands of approximate molecular weights 77 kDa and 50 kDa, representing the HA precursor (HA0) and a proteolytic cleavage product (HA1), respectively, (Figure 1B) were observed in the HAd-HA-NP infected 293 cell lysate. A single band at approximate molecular weight of 56 kDa representing NP (Figure 1C) was visible in the HAd-HA-NP infected 293 cell lysate.

To mimic in a mouse model the preexisting immunity against HAd5 observed in the majority of the human population, groups of animals were inoculated i.n. or i.m. with a single dose of 10⁷, 10⁸, or 10⁹ plaque forming units (p.f.u.) HAd-WT. Both i.n. and i.m. primed groups showed a dose-dependent increase in the levels of HAd-specific neutralizing antibody titers (Figure 2). As expected, the highest HAd-specific neutralizing antibody titers in i.n. inoculated HAd-primed groups were observed with a 10⁹ p.f.u. dose of HAd-WT (2240) followed by 10⁸ p.f.u. (1040) and 10⁷ p.f.u. (300) dose (Figure 2). Similarly, the i.m. primed group receiving 10⁹ p.f.u. of HAd-WT developed the highest titer (3040) followed by 10⁸ p.f.u. (1480) and 10⁷ p.f.u. (520) dose groups (Fig. 2). The i.m. primed groups resulted in the development of higher levels of HAd-specific neutralizing antibody titers compared to the i.n. primed groups.

Development of a robust HA-specific antibody response is an important indicator of the immunogenicity and protective efficacy of an influenza vaccine [21]. The i.n. or i.m. immunization of naïve animals with HAd-HA-NP elicited dose-dependent geometric mean (GM) hemagglutination inhibition (HI) titers (Table 1). The 10⁷ i.n. HAd-primed groups developed significantly high serum HI titers compared to the vector control group by the i.n. (120) or i.m. (160) routes of immunization with 10⁸ p.f.u. of HAd-HA-NP. Similarly, 10⁷ i.m. HAd- primed groups developed significantly higher (P≤0.05) serum HI titers compared to the vector control group by both i.m. (105) and i.n. (91) routes of immunization with HAd-HA-NP. The serum HI titers in 10⁷ HAd-primed groups (i.n. or i.m.) immunized with the vaccine were 1.5–2 fold lower than the naïve immunized groups indicating that HAd-preexisting antibodies affected the induction of humoral immune response following immunization with HAd-HA-NP. In the 10⁸ i.n. HAd-primed group, i.n. immunization with HAd-HA-NP induced lower serum HI titers (22) compared to the group immunized i.m. with HAd-HA-NP (80). Interestingly, an increase in the vaccine dose by five-fold resulted in significant (P≤0.05) enhancement of serum HI titers in mice immunized either i.n. (91) or i.m. (105). A similar trend was observed in the case of 10⁸ i.m. HAd-primed groups indicating that alternating the route of priming and immunization or increasing the vaccine dose can partially overcome the vector immunity.

In the 10⁹ i.n. HAd-primed group, i.n. immunization with HAd-HA-NP induced lower serum HI titers (22). Alternating with the i.m. route of immunization resulted in slight improvement in the HI titers (34). Increasing the vaccine dose by five-fold resulted in further improvement in HI titers in mice immunized either i.n. (52) or i.m. (80) indicating that the i.n.-induced (which mimics the natural route of infection in humans) vector immunity can be partially overcome by increasing the vaccine dose (P≤0.05). However, in the 10⁹ i.m. HAd-primed groups immunized with HAd-HA-NP, a HI titer of 30 was induced, and there were no significant changes in the titers by either alternating the route of vaccine inoculation or with an increased vaccine dose. These results indicate that the levels of vector immunity induced by i.m. priming with 10⁹ p.f.u. of HAd5-WT negatively impact the development of a humoral immune response against a HAd vector-based vaccine.

Cell-mediated immunity (CMI) plays an important role in virus clearance and thus contributes to the recovery from an influenza infection [22], [23]. As anticipated, the HAd-HA-NP vaccine elicited significantly higher percentages of NP-147 epitope-specific CD8 T cells in the naïve groups compared to the vector (HAd-ΔE1E3) control group (Figure 3A & 3B). The percentages of NP-147 epitope-specific CD8 T cells in 10⁷ HAd-primed groups (i.n. or i.m.) was significantly higher than in vector control groups following immunization with HAd-HA-NP by either the i.n. or i.m. route. However, the percentages NP-147-specific CD8 T cells were 1.5–2 fold lower in HAd-primed groups (i.n. or i.m.) compared to naïve groups, suggesting that preexisting vector immunity had a modest effect on the induction of CMI following immunization with HAd-HA-NP (Figure 3A & 3B).

The percentages of NP-147 epitope-specific CD8 T cells in 10⁸ HAd-primed groups were two-fold lower compared to the 10⁷ HAd-primed groups (i.n. or i.m.) following immunization with HAd-HA-NP by either route (Figure 3A & 3B). Interestingly, increasing the vaccine dose by five-fold resulted in significantly higher percentages of NP-147 epitope-specific CD8 T cells in the 10⁸ HAd-primed groups compared to both 10⁷ and 10⁸ HAd-primed groups receiving the lower dose (10⁸ p.f.u.) of the vaccine. As expected, a further increase in the level of preexisting vector immunity led to a further decrease in the percentages of NP-147 epitope-specific CD8 T cells. Furthermore, an increase in the vaccine dose by five-fold resulted in significantly higher percentages of NP-147 epitope-specific CD8 T cells in the 10⁹ i.n. primed group compared to the 10⁹ i.n. primed group receiving the lower vaccine dose (10⁸ p.f.u.). However, this increase was not noticeable in the 10⁹ i.m. primed group.

The functionality of HA-518 and NP-147-specific CD8 T cells was assessed by enumerating interferon-γ (IFN-γ) expressing cells by ELISpot assay. Significantly higher numbers of IFN-γ-secreting HA-518- and NP-147-specific CD8 T cells were detected in the spleens from the naïve groups immunized i.n. or i.m. with HAd-HA-NP compared to the vector control groups following stimulation with the HA-518 (P≤0.0001) or NP-147 (P≤0.0001) peptide, respectively (Figure 4A–D). In general, the number of IFN-γ-secreting HA-518- or NP-147-specific CD8 T cells in the spleens of HAd-primed groups immunized with the vaccine were lower compared to the naïve immunized groups, and a five-fold increase in the vaccine dose resulted in an increase in the number of IFN-γ secreting HA-518- or NP-147-specific CD8 T cells in all the primed groups except for the 10⁹ i.m. primed groups (Figure 4A–D). Overall, it seems that with the increase in preexisting HAd5-neutralizing antibodies, there was a titer-dependent decline in the CMI response which significantly improved with an increase in the vaccine dose by five-fold. There was excellent correlation between the ELISpot and NP pentamer staining data.

Unprimed mice immunized with HAd-HA-NP had lung viral titers on day 3 post-challenge below the level of detection (1.5 Log₁₀ EID₅₀/ml). Similarly, the 10⁷ HAd-primed groups (i.n. or i.m.) immunized with HAd-HA-NP had lung viral titers at or below the level of detection (1.5 Log₁₀ EID₅₀/ml) indicating that the preexisting vector immunity did not adversely impact the protective efficacy. However, the 10⁸ HAd-primed groups (i.n. or i.m.) immunized with HAd-HA-NP by the same route that was used for priming exhibited less efficient virus clearance from the lungs. Interestingly, either using a different route of inoculation for priming and vaccination or increasing the vaccine dose by five-fold resulted in lung viral titers at or below the level of detection. In the 10⁹ i.n. HAd-primed groups (i.n. or i.m.) immunized with HAd-HA-NP by the same route that was used for priming, there was only partial (approximately 2–4 logs) virus clearance. By changing the route of priming and immunization, complete protection was observed only in the group where the i.n. priming was followed by a five-fold increase in the vaccine dose administered by either route (i.m. or i.n.). Even a five-fold increase in the vaccine dose did not yield complete protection in the 10⁹ i.m. HAd-primed groups immunized with HAd-HA-NP by either route (i.n. or i.m.) suggesting that the level of preexisting vector-neutralizing antibody titer could serve as an indicator for predicting the efficacy of Ad-based vaccines.

The Purdue University Biosafety Committee and Animal Care and Use Committee approved the protocol for all animal studies at Purdue University, under the auspices of the Institutional Animal Care and Use Committee (IACUC) #A3231-01 which is supported by the American Association for Laboratory Animal Science (AALAS). The 293 cell line was obtained commercially from American Type Culture Collection (ATCC), and the 293Cre cell line was obtained from Merck & Co. The BHH2C cell line which was created in the PI's laboratory used a combination of commercially available cell lines, MDBK and 293. The use of all human cell lines and the construction of BHH2C hybrid cell line were with permission from the Purdue University Institutional Review Boards (IRB) formed in accordance with federal regulations. A Research Exemption was obtained for the use of commercially available human cell lines. The IRBs are a unit of the Human Research Protection Program (HRPP) which is housed within the Office of Research Administration (ORA).

293 (human embryonic kidney cells expressing HAd5 E1 gene products; obtained from ATCC) and 293Cre (293 cells that constitutively expresses Cre-recombinase enzyme (a gift from Merck Inc., Whitehouse Station, NJ) [35] were grown as monolayer cultures in Eagle's minimum essential medium (MEM) (Life Technologies, Gaithersburg, MD) and supplemented with 10% reconstituted bovine serum (Fetal Clone III; Hyclone, Logan, VT) and 50 µg/ml gentamycin. All constructs were purified by cesium chloride density-gradient centrifugation and titrated by plaque assays on a hybrid cell line using MDBK and 293 cell lines (BHH2C) [36] as described previously [37]. The construction and propagation of replication defective HAd-ΔE1E3 (HAd5 vector having deletions in the E1 and E3 regions) has been previously described [11]. HAd5-WT virus was purified by cesium chloride density-gradient centrifugation and titrated by plaque assay on BHH2C cells.

A Cre-recombinase-mediated site-specific recombination technique [20] was used to insert the full-length coding region of the HA gene (with a modified polybasic site) of the A/Vietnam/1203/04 (H5N1) influenza virus under the control of the CMV promoter and BGH polyA. The polybasic cleavage site QRERRRKKR↓G present in the HA gene of A/Vietnam/1203/04 (H5N1) influenza virus was modified to QRETR↓G to reduce the rare possibility of genetic exchange between the HA in HAd-HA-NP vaccine and a circulating strains of influenza A virus. The full-length coding region of NP gene of the A/Vietnam/1203/04 (H5N1) virus under control of the murine CMV promoter and the SV40 polyA was similarly inserted into the E1 region of the HAd genome. Both genes in HAd-HA-NP were in the E1-parallel orientation. The recombinant virus was plaque purified, and its genome was analyzed by restriction enzyme digestions to confirm the presence of HA and NP gene cassettes and the absence of any other major deletion or insertion.

293 cells were mock-infected or infected with an empty vector (HAd-ΔE1E3) or HAd-HA-NP at a multiplicity of infection (m.o.i.) of 20 p.f.u. per cell. Cells were harvested 36 h post-infection, and cell lysates were prepared and analyzed by Western blot [38] with a polyclonal serum against H5 HA (obtained from Immunology and Pathogenesis Branch, Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA, USA) and a monoclonal antibody against NP (obtained from Immunology and Pathogenesis Branch, Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA, USA) to confirm the expression of HA and NP proteins. Mock (PBS) or HAd-ΔE1E3 served as negative controls.

6–8 week old female BALB/c mice (10 animals/group) were inoculated by either the i.m. or i.n. route with a single dose of 10⁷, 10⁸, or 10⁹ p.f.u. of HAd-WT. These groups were referred to as HAd-primed groups. The unprimed (naïve) mice were similarly inoculated with phosphate-buffered saline (PBS). Four weeks after priming, mice were bled by retro-orbital puncture to evaluate the development of HAd-specific neutralizing antibody titers. HAd-primed and naïve (PBS-inoculated) mice were subsequently immunized twice (4 weeks apart) with 1×10⁸ or 5×10⁸ p.f.u. of HAd-HA-NP vaccine by either the i.m. or i.n. route. Additional groups of HAd-primed mice were similarly immunized with 5×10⁸ p.f.u. of HAd-ΔE1E3 (vector control) to serve as negative controls.

Four weeks after final immunization, blood samples were collected through retro-orbital puncture to evaluate the development of HA-specific antibodies. Five animals from each group were euthanized to collect the spleen cells to evaluate the induction of HA- and NP-specific CMI responses. The remaining mice from each group were challenged with 100-fold of 50% mouse infectious dose (MID₅₀) of a reverse genetics derived A/Puerto Rico/8/1934(H1N1) [PR8] containing HA and NA gene fragments of A/Vietnam/1203/04 (H5N1) [VNH5N1-PR8/CDC-RG] [24]. Since this reassortant virus is not lethal and does not produce clinical disease and weight loss in mice, protection efficacy was monitored by viral clearance in the lungs. Three days post-challenge, mice were euthanized, and the lungs were collected to determine viral titers to evaluate protective efficacy. Briefly, thawed lung tissues were homogenized in 1 ml of sterile PBS. These lung homogenates were then titrated in 10-day-old embryonated eggs in a 10-fold dilution, and positive eggs were identified by hemagglutination of horse red blood cells with allantoic fluid. Values were expressed as log10 EID50/ml ± SEM. The limit of virus detection was set as 1.5 log10 EID50/ml [39]. The NP genes are fairly conserved between A/Vietnam/1203-04 (in the Ad vector) and A/PR8/34 (the backbone of the challenge virus); there is 93% identity at the amino acid level and 85% identity at the nucleotide level. One of the major CD8 T cell epitopes (NP147) is 100% conserved in between A/Vietnam/1203-04 and A/PR8/34.

HAd neutralizing serum antibody titers were determined as previously described [13]. The virus neutralization titer was the reciprocal of the highest serum dilution that completely prevented the development of c.p.e. Pre-challenge serum samples were analyzed for the presence of HI antibody titers using horse red blood cells as described previously [40].

Splenocytes were isolated and stained with a murine MHC k^d–specific pentamer for immunodominant NP-147 epitope (Proimmune Inc., Bradenton, FL) conjugated with phycoerythrin (PE) and an anti-CD8 antibody conjugated with Allophycocyanin (APC) (BD PharMingen, San Jose, CA.) as described previously [11]. B cells were removed by staining splenocytes with anti-CD19 flouro-isothiocyanin (FITC) and gating them out in analysis. Flow cytometric analyses were done using BD FACSCantoII, (BD Bioscience, San Jose, CA) to identify the percent NP-147 epitope-specific CD8+ T cells among the total splenic CD8+ T cells.

96-well filter plates (Millipore, Bedford, MA) were coated with an anti-mouse interferon gamma (IFN-γ) antibody (BD Bioscience, San Jose, CA) and incubated at 4°C overnight. Splenocytes (3.3×10⁵ to 1×10⁶ cells per well) from each mice were cultured in the presence of HA-518 or NP-147 peptides in RPMI medium (GIBCO, Grand Island, NY), supplemented with 10% reconstituted fetal bovine serum for 60 h and developed according to an ELISpot protocol [41]. Splenocytes cultured in the presence of phorbol myristate acetate (PMA) and ionomycin (Sigma-Aldrich, Inc., St. Louis, MO) served as positive control within each group.

Log-transformation of titer measurement was assessed by Shapiro-Wilktest, found to be normally distributed and used for the analysis using SAS 9.2. Tukey's multiple comparison was used for calculation of significance. The significance was set at P <0.05.