Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011;6(12):e28757.
doi: 10.1371/journal.pone.0028757. Epub 2011 Dec 21.

Polyvalent DNA vaccines expressing HA antigens of H5N1 influenza viruses with an optimized leader sequence elicit cross-protective antibody responses

Shixia Wang  1 , Anthony Hackett, Na Jia, Chunhua Zhang, Lu Zhang, Chris Parker, An Zhou, Jun Li, Wu-Chun Cao, Zuhu Huang, Yan Li, Shan Lu
Affiliations

Polyvalent DNA vaccines expressing HA antigens of H5N1 influenza viruses with an optimized leader sequence elicit cross-protective antibody responses

Shixia Wang et al. PLoS One. 2011.

Abstract

Highly pathogenic avian influenza A (HPAI) H5N1 viruses are circulating among poultry populations in parts of Asia, Africa, and the Middle East, and have caused human infections with a high mortality rate. H5 subtype hemagglutinin (HA) has evolved into phylogenetically distinct clades and subclades based on viruses isolated from various avian species. Since 1997, humans have been infected by HPAI H5N1 viruses from several clades. It is, therefore, important to develop strategies to produce protective antibody responses against H5N1 viruses from multiple clades or antigenic groups. In the current study, we optimized the signal peptide design of DNA vaccines expressing HA antigens from H5N1 viruses. Cross reactivity analysis using sera from immunized rabbits showed that antibody responses elicited by a polyvalent formulation, including HA antigens from different clades, was able to elicit broad protective antibody responses against multiple key representative H5N1 viruses across different clades. Data presented in this report support the development of a polyvalent DNA vaccine strategy against the threat of a potential H5N1 influenza pandemic.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Schematic diagram of influenza A H5 HA gene inserts used in codon optimized DNA vaccines (A/HongKong/156/97 and A/VietNam/1203/2004), including the full length HA antigens with natural leader sequence (H5.wt) or a human tissue plasminogen activator (tPA) leader substituting the natural HA leader sequence (H5.tPA) and the transmembrane/cytoplasmic region truncated HA antigens (H5.dTM) with tPA leader sequence.
The cleavage site between HA1 and HA2 subunits are marked. The numbers above the HA inserts denote the relevant amino acid positions in natural HA proteins.
Figure 2
Figure 2. Antigen expression and immunogenicity of H5-HK HA DNA vaccines.
A. Western blot analysis of the expression, secretion, and susceptibility to cleavage expressed by different H5-HK (A/HongKong/156/97) HA DNA vaccine constructs in transiently transfected 293T cell supernatant (S) and cell lysate (L). B. Temporal serum anti-HA IgG responses in rabbits measured by ELISA at 1∶5000 serum dilution against H5-HK-dTM as coating antigen. The arrows indicate the time of gene gun-mediated DNA immunizations. Each curve represents the OD value of each group of 3 rabbits receiving H5-HK.wt, H5-HK.tPA, H5-HK.dTM DNA vaccine or empty DNA vector as indicated. C. End titration titers of serum anti-H5-HK HA IgG responses at two weeks after the 4th DNA immunization from the same rabbits shown in panel B. D. The hemagglutination inhibition (HI) antibody responses in NZW rabbit sera immunized with different designs of H5-HK HA DNA vaccines collected at 2 weeks after the 4th DNA immunization (DNA-4) or relevant pre-bleed sera as indicated. The HI antibody titers are shown as the geometric means for each group (3 rabbits per group) with standard deviations against H5N1 A/HongKong/483/97 virus. E. Neutralizing antibody (NAb) responses in NZW rabbit sera immunized with different designs of H5-HK HA DNA vaccines collected at 2 weeks after the 4th DNA immunization (DNA-4) or relevant pre-bleed sera as indicated. NAb titers against H5N1 A/HongKong/483/97 virus infection to MDCK cells are shown as the geometric means from each group (3 rabbits per group) with standard deviation. The statistical differences between each testing groups are determined and P values less than 0.05 or 0.01 are indicated.
Figure 3
Figure 3. Antigen expression and immunogenicity of H5-VN HA DNA vaccines.
A. Western blot analysis of the expression, secretion, and susceptibility to cleavage expressed by different H5-VN (A/VietNam/1203/2004) HA DNA vaccine constructs in transiently transfected 293T cell supernatant (S) and cell lysate (L). B. End titration titers of serum anti-H5-VN HA IgG responses at two weeks after the 4th DNA immunization from rabbits immunized with differently designed H5-VN DNA vaccines against H5-VN.dTM as coating. C. The hemagglutination inhibition (HI) antibody responses in NZW rabbit sera immunized with different designs of H5-VN HA DNA vaccines collected at 2 weeks after the 4th DNA immunization (DNA-4) or relevant pre-bleed sera as indicated. The HI antibody titers are shown as the geometric means for each group (3 rabbits per group) with standard deviations against H5N1 A/VietNam/1203/04 virus. D. Neutralizing antibody (NAb) responses in NZW rabbit sera immunized with different designs of H5-VN HA DNA vaccines collected at 2 weeks after the 4th DNA immunization (DNA-4) or relevant pre-bleed sera as indicated. NAb titers against H5N1 A/VietNam/1203/04 virus infection to MDCK cells are shown as the geometric means from each group (3 rabbits per group) with standard deviation. The statistical differences between each testing groups are determined and P values less than 0.05 or 0.01 are indicated.
Figure 4
Figure 4. Neutralizing antibody responses detected by the pseudotyped virus system expressing the H5 VN-HA antigen.
Early time point sera, at 2 weeks after either one (DNA-1) or three (DNA-3) DNA immunizations, were collected from the same groups of rabbits included in Fig. 3. A. Neutralizing antibody titers measured at 50% inhibition (IC50) of virus infection to target cells. B. Neutralizing antibody titers measured at 90% inhibition (IC90) of virus infection to target cells. Data shown are the geometric mean titers of each group with standard deviations. The statistical differences between each testing groups are determined and P values less than 0.05 or 0.01 are indicated.
Figure 5
Figure 5. Levels and specificity of anti-HA IgG (upper panel), hemagglutination inhibition (HI, middle panel), and neutralizing (NAb, lower panel) antibody responses elicited by H5-HK and H5-VN HA DNA vaccines in rabbits immunized with H5-HK-tPA and H5-VN-tPA (3 rabbits/group), respectively, at two weeks after the 4th DNA immunization.
Peak serum level anti-H5 HA IgG responses as measured by ELISA against H5-HK-dTM or H5-VN-dTM antigens. HI antibody titers and NAb were detected against H5N1 A/HongKong/483/97 or A/VietNam/1203/04 virus, respectively. Neutralizations against wild type H5N1 viruses were performed in MDCK cells.
Figure 6
Figure 6. Levels and specificity of anti-HA IgG (upper panel), HI (middle panel), and NAb (lower panel) antibody responses elicited by H5-VN (A/VietNam/1203/2004) and H5-AH (A/Anhui/1/2005) HA DNA vaccines in rabbits immunized with H5-VN-tPA (3 rabbits) and H5-AH-tPA (4 rabbits), respectively, at two weeks after the 4th DNA immunization.
Peak serum level anti-H5 HA IgG responses, as measured by ELISA against the H5-VN-dTM or H5-AH-dTM antigens. HI antibody and NAb titers were detected against H5N1 A/VietNam/1203/04 or A/Anhui/1/2005 virus, respectively. Neutralizations against wild type H5N1 viruses were performed in MDCK cells. Data shown are the geometric mean IgG, HI, or NAb titers of each group with standard deviations. The statistical differences between each testing groups are determined and P values less than 0.05 or 0.01 are indicated.
Figure 7
Figure 7. Levels and specificity of NAb responses against H5 pseudotyped viruses expressing full length H5 HA from A/VietNam/1203/2004 (A/VN/1203/04-pv, Panel A), A/Anhui/1/2005 (A/AH/1/05-pv, Panel B) or A/Indonesia/5/2005 (A/Ind/5/05-pv, Panel C), by different H5-HA DNA vaccines or empty DNA vector in rabbits.
The vaccine group “HK”, “VN”, “AH” or “Ind” represent monovalent H5 DNA vaccines HK.tPA, VN.tPA, AH.tPA, or Ind.tPA, respectively. The “3-valent” group consists of VN-HA.tPA AH-HA.tPA and Ind-HA.tPA H5 DNA vaccines. There were 3 rabbits/group in Vector, HK, VN, AH, and Ind groups, and 4 rabbits/group in 3-valent groups. Rabbit sera tested by the pseudotyped NAb assays were collected at 2 weeks after the 4th DNA immunization. “<” denotes below detection level. The arrow denotes neutralization against the autologous H5 pseudotyped virus. The statistical differences between the testing group and the autologous neutralization group or 3-valent group are indicated by “*” when the p value was less than 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Smith GJ, Fan XH, Wang J, Li KS, Qin K, et al. Emergence and predominance of an H5N1 influenza variant in China. Proc Natl Acad Sci U S A. 2006;103:16936–16941. - PMC - PubMed
    1. Salzberg SL, Kingsford C, Cattoli G, Spiro DJ, Janies DA, et al. Genome analysis linking recent European and African influenza (H5N1) viruses. Emerg Infect Dis. 2007;13:713–718. - PMC - PubMed
    1. Wu WL, Chen Y, Wang P, Song W, Lau SY, et al. Antigenic profile of avian H5N1 viruses in Asia from 2002 to 2007. J Virol. 2008;82:1798–1807. - PMC - PubMed
    1. Chen H, Smith GJ, Zhang SY, Qin K, Wang J, et al. Avian flu: H5N1 virus outbreak in migratory waterfowl. Nature. 2005;436:191–192. - PubMed
    1. Abdel-Ghafar AN, Chotpitayasunondh T, Gao Z, Hayden FG, Nguyen DH, et al. Update on avian influenza A (H5N1) virus infection in humans. N Engl J Med. 2008;358:261–273. - PubMed

Publication types