The priming effect of the H1N1 <i>ca</i> vaccine is induced by the H1 HA.

<p>Ferrets were intranasally inoculated with the H1N1 NC99 <i>ca</i> virus, a reassortant H1N2 <i>ca</i>, H3N2 CA04 <i>ca</i> or H5N1 HK03 <i>ca</i> virus on day 0 and intranasally inoculated with the H5N1 HK03 <i>ca</i> virus five weeks later. The animals were sacrificed 5 days later and B cell ELISpot analysis was performed with lymphocytes isolated from TLN using rH5 HA as antigen. IgG antibody secreting B cells are presented as the number of ASC per 10⁶ lymphocytes.</p

HA-specific antibodies measured by an ELISA assay.

<p>Ferrets were intranasally inoculated with medium or the H1N1 NC99, H3N2 CA04, H5N1 HK03 <i>ca</i> vaccine viruses on day 0 and intranasally inoculated with the HK03 <i>ca</i> vaccine virus six weeks later. Serum samples were collected 6 weeks after the 1st dose (A) and one week after the 2nd dose (B). ELISA was performed with RDE-treated serum using rH5, rH1 or rH3 HA as antigens.</p

Previous exposure with seasonal LAIV or H1N1 <i>ca</i> virus induced a faster H5N1-specific immune response.

<p>(A) Ferrets were intranasally inoculated with either medium or trivalent LAIV on day 0. Six weeks later, ferrets were intranasally inoculated with the H5N1 HK03 <i>ca</i> vaccine and five days later, lymphocytes isolated from TLN were examined for ASC against H2N2 MDV-A, H5N1 HK03 <i>ca</i> viruses and rH5 HA antigens by B cell ELISpot analysis. Ferrets were intranasally inoculated with medium or the monovalent H1N1 NC99, H3N2 CA04, H5N1 HK03 <i>ca</i> vaccine viruses on day 0 and intranasally inoculated with the H5N1 HK03 <i>ca</i> vaccine six weeks later. The B cell ELISpot analysis was performed with lymphocytes isolated from TLN using the indicated <i>ca</i> vaccine virus (B) or rHA (C) as antigens. The IgG antibody secreting B cells are presented as the number of ASC per 10⁶ lymphocytes.</p

Serum antibody response to influenza viruses after one and two doses of intranasal vaccine.

<p>Groups of three ferrets were vaccinated intranasally with the indicated 1^st dose of vaccine and 42 days later were inoculated with a 2^nd dose of vaccine (H5N1 HK03 <i>ca</i> or VN04 <i>ca</i>). Serum samples were collected 6 weeks after the 1^st dose (pre-dose 2), 1 week and 3 weeks after the 2^nd dose, respectively, and antibody titers (geometric mean titers from 3 animals) against the first or second vaccine viruses were determined by microneutralization assay.</p

H5N1-specific B cells were detected in ferrets infected with live attenuated H5N1 <i>ca</i> vaccines.

<p>Ferrets were intranasally administered the H5N1 VN04 <i>ca</i> or HK03 <i>ca</i> viruses on day 0. Five and ten days post-inoculation, ferrets were sacrificed to collect paratracheal lymph nodes (TLN) and a B cell ELISpot assay was performed using lymphocytes isolated from TLN and BPL-inactivated H5N1 HK03 <i>ca</i> virus or rH5 HA antigens. The number of IgM ASC (A) and IgG ASC (B) are presented as per 10⁶ lymphocytes.</p

Effect of the H1N1 and H3N2 <i>ca</i> vaccines on replication of the H5N1 HK03 <i>ca</i> virus in the upper respiratory tract of ferrets.

<p>Groups of ferrets were vaccinated with the indicated virus and four weeks later inoculated with the H5N1 HK03 <i>ca</i> vaccine. Antibody titers against homologous vaccine virus were determined by HAI assay and expressed as geometric mean titer. The H5N1 HK03 <i>ca</i> virus titer in nasal turbinates (NT) on day 3 post-inoculation is expressed as log₁₀EID₅₀ per gram of tissues calculated from the mean of 6 animals.</p

Infection of HAE by avian, but not human, influenza viruses is restricted at temperatures of the proximal airways.

<p>(A) Comparison of multi-cycle virus growth in HAE inoculated with either A/Victoria/3/75 at 32°C (<i>closed triangles</i>) or 37°C (<i>open triangles</i>) and A/Dk/Eng/62 at 32°C (<i>closed circles</i>) or 37°C (<i>open circles</i>) both at MOI∼0.01. Apical viral titers at times shown were determined by standard plaque assay on MDCK cells. Data shown represents the mean titer +/−standard error (SE; n = 3–10 cultures). (B) Adenylate kinase activity released into the apical compartment of HAE over time after inoculation with A/Victoria/3/75 or A/Dk/Eng/62 at 32°C and 37°C as a measure of viral-induced CPE. Data shown represents the mean fold change over adenylate kinase activity derived from mock-inoculated HAE +/−SE (n = 3–8). Significance is noted (*p<0.05) where viral titers or AK levels obtained for A/Dk/Eng/62 at 32°C were statistically different from all other titers/AK measurements (Dk/37°C, Vic/32°C and Vic/37°C) at that particular time point. Significance is noted (^†p<0.05) where AK levels obtained for A/Dk/Eng/62 at 32°C and 37°C were statistically different.</p

Temperature-dependent growth of different serotypes of influenza viruses in HAE.

<p>Multi-step growth kinetics of (A) human influenza virus A/Eng/26/99 or (C) avian influenza virus A/Dk/Sing/97 (MOI∼0.1) at 32°C (<i>open circles</i>, <i>dashed line</i>) or 37°C (<i>closed circles</i>, <i>solid line</i>) in HAE +/−SE (n = 3 cultures). Multi-step growth kinetics in HAE inoculated with an MOI∼0.03 of (B) A/Udorn/307/72 (H3N2) or (D) A/VN/1203/04 (H5N1) at 33°C (<i>open circles</i>, <i>dashed line</i>) or 37°C (<i>closed circles</i>, <i>solid line</i>). Data represents mean titer across two different donors, each performed in duplicate +/−SE. Viral titers were determined by plaque assay in (A) and (B) and by TCID₅₀ assay for (C) and (D). No significant differences in growth between temperatures were found for either A/Eng/26/99 or A/Udorn/307/72. A/VN/1203/04 was significantly restricted for growth at 24, 48 and 72 hrs pi (*p<0.05). (E) Representative histological cross-sections of HAE infected for 72 hrs at 37°C with A/Udorn/307/72 or A/VN/1203/04 and compared to mock-inoculated HAE. H&E counterstain. Scale bar equals 20 µm.</p

Cell tropism of human, avian and avianized viruses in HAE.

<p>Representative cross-sections of inoculated HAE, fixed 24 hrs pi, were probed for viral antigen (NP; green) and α−acetylated tubulin, a marker for ciliated cells (red). Notably, the staining pattern for wild-type A/Victoria/3/75 was identical to that of PR8+Vic HA/NA. Arrows mark ciliated cells infected with either wild-type A/Victoria/3/75 or PR8+Vic HA/NA; arrow-head denotes non-ciliated cells infected by these viruses. These data indicate that viruses with Victoria glycoproteins were able to infect both cell types previously shown to express α2,6 SA <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000424#ppat.1000424-Thompson1" target="_blank">[13]</a>. Viral antigen was detected only in ciliated cells in cultures inoculated with Vic-226-228HA (in the Victoria background with either endogenous N2 or avian N1 or PR8+Chick HA/NA). Scale bar equals 20 µm.</p

Spread and histopathology of avian and human influenza viruses in HAE at temperatures of the proximal and distal airway.

<p>(A) Representative <i>en face</i> photomicrographs of HAE inoculated with either A/Victoria/3/75 or A/Dk/Eng/62 at 32°C or 37°C, fixed at 6, 24, 48 and 72 hrs pi and stained for viral nucleoprotein (<i>green</i>) to determine numbers of cells infected. Scale bar equals 100 µm. (B) Representative histological cross-sections of HAE at 24, 72 and 120 hrs after inoculation with A/Victoria/3/75 or A/Dk/Eng/62 at 32°C or 37°C. H&E counterstain. Scale bar equals 20 µm.</p