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. 2019 Sep 25;14(9):e0222201.
doi: 10.1371/journal.pone.0222201. eCollection 2019.

DNA vaccine based on conserved HA-peptides induces strong immune response and rapidly clears influenza virus infection from vaccinated pigs

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DNA vaccine based on conserved HA-peptides induces strong immune response and rapidly clears influenza virus infection from vaccinated pigs

Marta Sisteré-Oró et al. PLoS One. .

Abstract

Swine influenza virus (SIVs) infections cause a significant economic impact to the pork industry. Moreover, pigs may act as mixing vessel favoring genome reassortment of diverse influenza viruses. Such an example is the pandemic H1N1 (pH1N1) virus that appeared in 2009, harboring a combination of gene segments from avian, pig and human lineages, which rapidly reached pandemic proportions. In order to confront and prevent these possible emergences as well as antigenic drift phenomena, vaccination remains of vital importance. The present work aimed to evaluate a new DNA influenza vaccine based on distinct conserved HA-peptides fused with flagellin and applied together with Diluvac Forte as adjuvant using a needle-free device (IntraDermal Application of Liquids, IDAL®). Two experimental pig studies were performed to test DNA-vaccine efficacy against SIVs in pigs. In the first experiment, SIV-seronegative pigs were vaccinated with VC4-flagellin DNA and intranasally challenged with a pH1N1. In the second study, VC4-flagellin DNA vaccine was employed in SIV-seropositive animals and challenged intranasally with an H3N2 SIV-isolate. Both experiments demonstrated a reduction in the viral shedding after challenge, suggesting vaccine efficacy against both the H1 and H3 influenza virus subtypes. In addition, the results proved that maternally derived antibodies (MDA) did not constitute an obstacle to the vaccine approach used. Moreover, elevated titers in antibodies both against H1 and H3 proteins in serum and in bronchoalveolar lavage fluids (BALFs) was detected in the vaccinated animals along with a markedly increased mucosal IgA response. Additionally, vaccinated animals developed stronger neutralizing antibodies in BALFs and higher inhibiting hemagglutination titers in sera against both the pH1N1 and H3N2 influenza viruses compared to unvaccinated, challenged-pigs. It is proposed that the described DNA-vaccine formulation could potentially be used as a multivalent vaccine against SIV infections.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Viral RNA load in nasal swabs by RT-qPCR.
(A) Mean of genomic equivalent copies (GEC) per mL obtained from nasal swabs from seronegative pigs (Experiment I) collected at 0, 5 and 7 dpi (B) and from nasal swabs from seropositive animals (Experiment II) collected at 0, 5, 7, 11 and 14 dpi. Group A (unvaccinated animals) is represented by grey bars and Group B (pCDNA3.1(+)-VC-4-flagellin vaccinated group) by black bars. Dpi, days post-inoculation. Dashed lines indicate the detection limit of the assays: 1.24 log10GEC/mL. Error bars indicate the mean ± SEM.
Fig 2
Fig 2. Serum antibody HA-specific IgG titers detected in sera and BALFs samples by ELISA test.
Mean of serum IgG antibody levels detected at 0, 20 PVD, 35 PVD, and 7 DPI of Groups A and B (A) against HA from A/California/04/09(H1N1)pdm09, and (B) against HA from A/Aichi/2/1968(H3N2) are represented. Mean of BALFs IgG antibody levels detected in pigs sacrificed at 7 and 14 dpi of Groups A and B (C) against HA from A/California/04/09(H1N1)pdm09, and (D) against HA from A/Aichi/2/1968(H3N2). Grey circles/bars refer to group A (unvaccinated group), and black squares/bars refer to group B (pCDNA3.1(+)-VC4-flagellin vaccinated group). OD, optical density. PVD, post-vaccination days and DPI, days post-inoculation. Error bars indicate the mean ± SEM. Statistically significant differences between vaccine treatment groups (P value <0.05) are marked with *: P<0.05, **: P<0.01.
Fig 3
Fig 3. HI activity against pH1N1 from seronegative pigs (Experiment I) and against H3N2 from seropositive pigs (Experiment II).
HI titers obtained with sera from unvaccinated (Group A) and vaccinated (Group B) pigs against (A) the pH1N1 from SIV-seronegative pigs (Experiment I) and (B) the SwH3N2 from SIV-seropositive pigs (Experiment II). Grey circles refer to group A (unvaccinated group) and black squares depict group B (pCDNA3.1(+)-VC-4-flagellin vaccinated group). HI, hemagglutination inhibition. DPI, days post-inoculation. Dashed lines indicate the threshold to obtain a “seroprotective” titer (HI≥40) which has been used as a criterion of immunogenicity in a vaccine. Error bars indicate the mean ± SEM and statistically significant differences between vaccine treatment groups are marked with **: P<0.01.
Fig 4
Fig 4. Antibody HA-specific IgA titers detected in BALFs samples from Experiment II.
Mean of BALFs IgA antibody levels detected in pigs sacrificed at 7 and 14 dpi of Groups A and B (A) against HA from A/California/04/09(H1N1)pdm09, and (B) against HA from A/Aichi/2/1968(H3N2). Grey bars refer to group A (unvaccinated group), and black bars refer to group B (pCDNA3.1(+)-VC4-flagellin vaccinated group). OD, optical density. PVD, post-vaccination days and DPI, days post-inoculation. Error bars indicate the mean ± SEM.
Fig 5
Fig 5. Seroneutralization (SN) titers detected in BALF samples from Experiment II by seroneutralization assay.
Mean of seroneutralization titers detected at 7 and 14 dpi of Groups A and B against the A/swine/Spain/003/2010 H3N2 IV challenged virus. Grey bars refer to group A (unvaccinated group) and black bars depict group B (pCDNA3.1(+)-VC-4-flagellin vaccinated group). SN, seroneutralization. DPI, days post-inoculation. Error bars indicate the mean ± SEM.
Fig 6
Fig 6. Flow cytometry from PBMCs isolated at 35 PVD (Experiment II).
Mean of the percentages of T cells CD4, CD8 and CD4-CD8 DP from unvaccinated (Group A) and vaccinated (Group B). Grey bars refer to group A (unvaccinated group) and black bars depict group B (pCDNA3.1(+)-VC-4-flagellin vaccinated group). Error bars indicate the mean ± SEM and statistically significant differences between vaccine treatment groups are marked with ***: P<0.001.

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Grants and funding

This work was funded in part by the Spanish Government, Ministerio de Economía y Competitividad de España (MINECO), project: AGL2013-48923-C2-2-R, and by the collaborative infrastructure project funded by the European Comission (EC) under Horizon 2020, project Transvac2-730964-INFRAIA-2016-1. IRTA is supported by CERCA Programme/ Generalitat de Catalunya. M.S.O. is supported by MINECO (scholarship n° BES-2014-068506). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.