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. 2011 Nov;18(11):1070-7.
doi: 10.1038/gt.2011.59. Epub 2011 May 5.

Molecular Adjuvant HMGB1 Enhances Anti-Influenza Immunity During DNA Vaccination

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Free PMC article

Molecular Adjuvant HMGB1 Enhances Anti-Influenza Immunity During DNA Vaccination

P Fagone et al. Gene Ther. .
Free PMC article

Abstract

DNA-based vaccines, while highly immunogenic in mice, generate significantly weaker responses in primates. Therefore, current efforts are aimed at increasing their immunogenicity, which include optimizing the plasmid/gene, the vaccine formulation and method of delivery. For example, co-immunization with molecular adjuvants encoding an immunomodulatory protein has been shown to improve the antigen (Ag)-specific immune response. Thus, the incorporation of enhancing elements, such as these, may be particularly important in the influenza model in which high titered antibody (Ab) responses are critical for protection. In this regard, we compared the ability of plasmid-encoded high-mobility group box 1 protein (HMGB1), a novel cytokine in which we have previously mutated in order to increase DNA vaccine immunogenicity, with boost Ag-specific immune responses during DNA vaccination with influenza A/PR/8/34 nucleoprotein or the hemagglutinin of A novel H1N1/09. We show that the HMGB1 adjuvant is capable of enhancing adaptive effector and memory immune responses. Although Ag-specific antibodies were detected in all vaccinated animals, a greater neutralizing Ab response was associated with the HMGB1 adjuvant. Furthermore, these responses improved CD8 T+-cell effector and memory responses and provided protection against a lethal mucosal influenza A/PR/8/34 challenge. Thus, co-immunization with HMGB1 has strong in vivo adjuvant activity during the development of immunity against plasmid-encoded Ag.

Conflict of interest statement

CONFLICT OF INTEREST

The DBW laboratory notes possible commercial conflicts associated with this work, which may include the following: Inovio, BMS, Virxsys, Ichor, Merck, Althea and Aldevron, and possibly others. The funders had no role in the study design, data collection, analysis, decision to publish or preparation of the manuscript.

Figures

Figure 1
Figure 1
HMGB1 enhances APC maturation. (a) Immunohistochemical staining of CD83+ cells (brown color) infiltrating the muscle after pNP vaccination with pHMGB1 adjuvant. Representative pictures of sections from pNP+pVax1 and pNP+pHMGB1 groups are shown. (b) Inguinal lymphonodal cells harvested at 7 days after the final i.m. injection of pNP alone or in combination with pHMGB1 were stained for the markers of activation CD80 and CD86, and flow cytometric analysis was performed. Empty vector pVax1 was used as a negative control and bar graphs. (c) At day 14 post immunization, splenic sections from pHMGB1-adjuvanted mice (side scatter channel (SSC) with pNP or pGag) were stained for CD11c+ (brown color) and negative cells were blue in color. Original magnification is ×100 and boxes (the insert picture at the right-bottom side) are ×400. (d) Quantification of CD11c+ cells. The results are representative of three independent experiments.
Figure 2
Figure 2
Electroporation increase of NP-specific cellular responses by pHMGB1 co-immunization. Effects of adjuvant pHMGB1 on the induction of NP-specific cellular immune responses were measured in the spleens using the standard IFN-γ ELISPOT assay. (a) Immunization schedule for the murine study is shown. (b, c) Immunogenicity of NP. Splenocytes were harvested at 7 days following the third immunizations, 2 weeks apart, administered i.m. with EP (b) or with out EP (c). Negative control immunized animals receiving three injections of empty vector control plasmid (pVax1). Splenocytes were stimulated with multiple pools of overlapping peptides spanning the entire length of the NP and then IFN-γ spot-forming units (SFUs) per million splenocytes were enumerated. Experiments were performed independently at least three times with similar results.
Figure 3
Figure 3
HMGB1-elicited T-cell proliferative responses to NP. After the third immunization, splenocytes were harvested and stimulated with NP to determine NP-specific CD8+ and CD4+ T-cell proliferation capacity. (a) Singlets, lymphocytes and viable CD3+ cells were gated and representative dot plots from each group of mice are shown for NP-specific CD8+ and CD4+ T cells. Average peptide-specific responses are displayed. (b) Proliferative responses for NP are shown as group mean responses ± s.d. with pVax1 values subtracted. Error bars represent s.d. Similar results were observed in two independent experiments. Statistical analysis was performed with Graphpad Prism 5 (Graphpad Software Inc.).
Figure 4
Figure 4
Protective efficacy of the HMGB1-adjuvanted pNP. At 4 weeks after the third immunization, mice were challenged intranasally with 10 lethal dose 50 of A/Puerto Rico/8/34 virus. (a) Kaplan–Meier curve showing survival percentage of each experimental group over the course of 14 days. (b) Average weight loss among survivors of each group tracked over 14 days. Similar results were observed in two independent experiments with at least n=10 per group for each experiment. (c) Histopathological analysis of lung tissue from vaccinated and challenged BALB/c mice. Mice were challenged with the influenza A/Puerto Rico/8/34 strain at 1 month after the final immunization. The mice were killed at 4 days post challenge, and lung tissue was collected for histological review. Representative micrographs from pVax1, pNP and pNP+pHMGB1 groups are shown along with a tissue section of a naive mouse lung for comparison.
Figure 5
Figure 5
Construction and expression of the HS09 DNA vaccine. (a) Schematic representation of the strategy for cloning the human swine flu consensus gene into the pVax1 vector (pH109). Location of the Kozak sequence, the IgE leader peptide, enzyme restriction sites and the pH109 initiation site are displayed. (b) Gel photograph showing fragments of the pH109 plasmid following restriction digestion with Xho1 and BamHI enzymes. (c, d) Western blot analysis of pH109 expression in human 293T cells transfected with 10 μg pVax1 control vector or the pH109 as indicated. Lysates (c) or cell supernatants (d) were extracted at 48 h post transfection and immunoblotting was performed using anti-HA Abs.
Figure 6
Figure 6
Immunogenicity of pH109 with and without pHMGB1 adjuvant. The immunogenicity of pH109 and the effects of HMGB1 adjuvant on the induction of cellular responses were measured. (a) Immunization schedule for the murine study is shown. (b) Splenocytes were stimulated with multiple pools of overlapping peptides spanning the entire length of the HA antigen, standard IFN-γ was performed and IFN-γ spot-forming unit (SFU) were enumerated. The results are representative of three independent experiments. (c) Total anti-HA IgG responses in the blood serum induced with vaccinations using either pH109 or pH109+pHMGB1 DNA vaccine in BALB/c mice as measured by endpoint Ab enzyme-linked immunosorbent assay. Error bars represent±s.d. from the mean and are representative of three independent experiments. (d) To measure type-specific IgG responses, mice were bled and pooled sera were diluted to 1:50 for reaction with HA. The assay was performed in triplicate and values represent mean (n=3) and bars s.d. (e) The nAb titers against an influenza (A/Mexico/InDRE4487/2009) virus infection was measured for Madin–Darby canine kidney cells and data are shown as the geometric means from each group (n=4).

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