Hyperglycosylated DNA design.

<p>Site-directed mutagenesis in five HA1 subunit regions (A, B, C, D, E) was performed following the addition of N-linked glycosylation sites. Nine N-X-S/T motifs were created in the five regions: (1) 83NNT, (2) 86NNT, (3) 94NFT, (4) 127NSS, (5) 138NRT, (6) 156NTT, (7) 161NRS, (8) 182NDT, (9) 252NAT. Triplet amino acids are underlined. Arrows point away from wild-type sequences to amino acid changes resulting in N-linked glycosylation sequences.</p

Total anti-HA IgG titers elicited by DNA-HA and FliC-VLPs.

<p>BALB/c mice were immunized using a prime boost regimen over a three-week interval as shown: (i) FliC-VLP+FliC-VLP, (ii) DNA-HA+DNA-HA, or (iii) DNA-HA+FliC-VLP. Sera were collected two weeks following the booster immunization. Results indicate significantly higher HA-specific IgG titers from DNA-HA vaccine vector priming followed by FliC-VLP boosting. Shown are individual titers (points) and geometric means (lines) for each group. Asterisks (*) indicate statistical significance at p<0.05.</p

Neutralization against H5pp of A/Anhui/1/2005 and A/Hubei/1/2010 strains.

<p>Dose-dependent neutralization curves were plotted against (A) A/Anhui/1/2005 and (B) A/Hubei/1/2010 H5N1 influenza strains. Data indicate that the 83NNT and 127NSS HA mutants elicited higher neutralizing antibodies than the wild-type HA.</p

HI and NT titers against the NIBRG-14 and A/whooper swan/Mongolia/244/2005 viruses.

<p>Neutralizing activity in sera collected from immunized mice was determined by measuring (A) HI and (B) NT titers against the A/Vietnam/1194/2004 (clade 1) H5N1 virus, and (C) HI and (D) NT titers against the A/Whooper Swan/Mongolia/244/2005 (clade 2.2) H5N1 virus. For calculation purposes, an undetectable level was scored as titer = 1. Individual titers (points) and geometric means (lines) are given for each group. Asterisks indicate statistical significance at p<0.05.</p

HI and NT titers against the NIBRG-14 virus.

<p>Neutralizing activities in sera collected from immunized mice were determined by measuring (A) HI and (B) NT titers against the NIBRG-14 (clade 1) H5N1 influenza virus. Results showed that the DNA-HA vector priming/FliC-VLP booster regiment elicited the highest magnitude of neutralizing antibodies. For calculation purposes, undetectable titers were scored as 1. Individual titers (points) and geometric means (lines) are given for each group.</p

Total anti-HA IgG titers elicited by hyperglycosylated DNA vaccine priming followed by FliC-VLP boosting.

<p>Mice were immunized with each immunorefocusing HA vector twice followed with a third boosting dose with FliC-VLPs on a three-week interval. Sera were collected at 2 weeks after final immunization, and determined the anti-HA IgG titers by ELISA. The results showed that no significant differences of the HA-specific total IgG titers of all the immunized groups with the hyperglycosyalted HA DNA vaccines compared to the wild-type control. Individual titers (points) and geometric means (lines) are given for each group.</p

HA sequence alignment.

<p>Results from analyses of amino acid variation in the HA1 of 163 avian influenza virus strains reveal five regions with the highest variability scores (0 to 4 based on Vector NTI Tables). HA1 subunit regions A (HA81–101), B (HA125–144), C (HA150–166), D (HA182–204), and E (HA245–257) were found to have higher scores.</p

Hemadsorption assay.

<p>RBCs were added to 293 cells transfected with mutated DNA-HA vectors to cover cell monolayer. Results for RBC adsorption indicate that cells transfected with the 83NNT, 86NNT, 94NFT, 127NSS, 138NRT and 161NRS vectors retained their hemadsorption function, while cells transfected with the 156NTT, 183NDT and 252NAT vectors did not. Cells transfected with a wild-type DNA-HA vector were used as a positive control (PC); cells tranfected with an empty vector were used as a negative control (NC).</p

Distribution of EV71 genotypes throughout the world from 1997 to 2010.

<p>Bold indicates predominant genotype.</p

Antibody responses elicited by HA proteins in immunized mice.

<p>BALB/c mice were intramuscularly inoculated with two doses of HA protein (15 µg) coupled with one of five adjuvant formulations. Total IgG antibody responses in mice immunized with KAN-1 (A) or Anhui (B) HA were evaluated 2 weeks following the second immunization. HA protein immunization coupled with PELC/CpG adjuvant induced the largest amount of IgG titers. IgG subclasses were detected using ELISA. PELC and PELC/CpG adjuvants coupled with KAN-1 (C) or Anhui (D) HA induced higher levels of IgG1 and IgG2a subtypes compared to the Alum or CpG adjuvants. All values are expressed as geometric mean with a standard error of the mean of five mice per group. Asterisk (*) indicates a statistically significant difference compared to the PELC/CpG group (p<0.05, Student <i>t</i> test).</p