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. 2013;8(3):e59081.
doi: 10.1371/journal.pone.0059081. Epub 2013 Mar 18.

M2e-displaying virus-like particles with associated RNA promote T helper 1 type adaptive immunity against influenza A

Lorena Itatí Ibañez  1 , Kenny Roose, Marina De Filette, Michael Schotsaert, Jessica De Sloovere, Stefan Roels, Charlotte Pollard, Bert Schepens, Johan Grooten, Walter Fiers, Xavier Saelens
Affiliations

M2e-displaying virus-like particles with associated RNA promote T helper 1 type adaptive immunity against influenza A

Lorena Itatí Ibañez et al. PLoS One. 2013.

Abstract

The ectodomain of influenza A matrix protein 2 (M2e) is a candidate for a universal influenza A vaccine. We used recombinant Hepatitis B core antigen to produce virus-like particles presenting M2e (M2e-VLPs). We produced the VLPs with and without entrapped nucleic acids and compared their immunogenicity and protective efficacy. Immunization of BALB/c mice with M2e-VLPs containing nucleic acids induced a stronger, Th1-biased antibody response compared to particles lacking nucleic acids. The former also induced a stronger M2e-specific CD4(+) T cell response, as determined by ELISPOT. Mice vaccinated with alum-adjuvanted M2e-VLPs containing the nucleic acid-binding domain were better protected against influenza A virus challenge than mice vaccinated with similar particles lacking this domain, as deduced from the loss in body weight following challenge with X47 (H3N2) or PR/8 virus. Challenge of mice that had been immunized with M2e-VLPs with or without nucleic acids displayed significantly lower mortality, morbidity and lung virus titers than control-immunized groups. We conclude that nucleic acids present in M2e-VLPs correlate with improved immune protection.

Trial registration: ClinicalTrials.gov NCT00819013.

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Conflict of interest statement

Competing Interests: Walter Fiers and Marina De Filette hold patent rights on the use of M2e as a vaccine antigen. Part of the research on M2e in the Saelens group (Ghent University and VIB) is supported by a research collaboration with Sanofi Pasteur. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. The C-terminal arginine-rich domain of recombinant HBc-based VLPs binds nucleic acids.
A. Schematic representation of the recombinant M2e-VLPs and control VLPs used in this study. Light grey boxes on the left in VLP-1818 and VLP-1965 represent three tandem M2e copies; white boxes represent HBc amino acid residues 1–149; dark grey boxes on the right in VLP-1965 and VLP-1632 represent part of the arginine-rich domain of HBc (residues 150–163). In all constructs, a C-terminal cysteine residue (cys) was engineered to improve particle stability. B. Native agarose gel analysis of VLPs, followed by staining with Coomassie Brilliant blue (left) or ethidium bromide (right). C. Purified recombinant VLPs (2 µg each) were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. D. VLP-1632 contains RNA. One µg of nucleic acids purified from VLP-1632, similarly purified plasmid DNA, plasmid DNA and total mammalian cellular RNA were treated with either PBS, RNAse or DNAse and analyzed by agarose gel electrophoresis followed by SYBR Safe staining. Numbers on the left indicate sizes in base pairs of the DNA marker in the left hand lane.
Figure 2
Figure 2. VLPs containing nucleic acids promote a Th1-polarized humoral immune response.
A. Three groups of 16 BALB/c mice were immunized twice intraperitonealy with 10 µg of VLP-1123, VLP-1818 or VLP-1965, without adjuvant. Priming and boosting injections were given at three week intervals. Two weeks after the boost, M2e-specific serum IgG1 and IgG2a titers were determined by M2e peptide ELISA. White and black bars: serum samples after priming and boosting, respectively. IgG1/IgG2a ratios were calculated by dividing the mean of the IgG1 endpoint titers by the mean of the IgG2a endpoint titers, and are shown on the right. (*: P<0.05, **: P<0.01 two-sample t-test). B. The particles were adjuvanted with Alhydrogel and used to immunize BALB/c mice (14 per group, 10 µg of VLP i.p. per animal). Two weeks after priming, M2e-specific (left) and HBc-specific (right) IgG1 and IgG2a titers were determined in sera pooled from the 14 mice. C. Groups of 12 BALB/c mice were immunized twice by i.p. injection of 10 µg of VLP-1123, VLP-1623, VLP-1818 or VLP-1965 formulated with Alhydrogel. Priming and boosting injections were three weeks apart. M2e-specific serum IgG1 and IgG2a titers are shown in the top panels and HBc-specific titers in the bottom panels. White and black bars: serum samples two weeks after priming and boosting, respectively. (*: P<0.05, **: P<0.01, ***: P<0.001, two-sample t-test). Bars in A and C represent averages and error bars represent standard error of the mean. #: titer lower than 100.
Figure 3
Figure 3. VLPs with nucleic acids promote a Th1-polarized cellular immune response.
BALB/c mice (n = 23 per group) were immunized twice by i.p. injection of 10 µg of VLP-1123, VLP-1623, VLP-1818 or VLP-1965 formulated with Alhydrogel. An additional control group received PBS with Alhydrogel (PBS). Priming and boosting injections were two weeks apart. Two weeks after the boost, splenocytes were isolated from five mice in each group and analyzed in triplicate for each mouse. Following 16 h of in vitro stimulation of splenocytes with M2e peptide, IFN-γ secreting cells were quantified by ELISPOT analysis. Results are shown as the average number of spots per 300,000 splenocytes. Bars represent averages with error bars depicting the standard error of the mean (*:P<0.001 two-sample t-test). #: less than 10 spots.
Figure 4
Figure 4. VLP-associated nucleic acids signal through MYD88 and TRIF.
RT-qPCR was used to analyze mRNA expression levels of the pro-inflammatory cytokines IL-1β and IL-6 20 hours after stimulation of WT, MyD88−/− or TRIF−/− DCs with 20 µg of VLP-1123, VLP-1632, VLP-1818, VLP-1965 or 1 µg of ODN 1826. Results represent mean n-fold induction levels compared to unstimulated control cells ± SD from triplicate PCR reactions.
Figure 5
Figure 5. Immunization with VLP-1965 provides better protection against morbidity caused by influenza A virus challenge than VLP-1818.
A. Groups of 12 BALB/c mice were immunized twice with an interval of three weeks with 10 µg of the indicated VLPs, adjuvanted with Alhydrogel. Three weeks after the second immunization, mice were challenged with 4 LD50 of mouse-adapted X47 virus. Survival rate (left; P<0.001, Kaplan Meier test) and body weight (right; P<0.05, two-sample t-test on days 6 and 7 after challenge) were monitored for two weeks starting from the day of challenge. B. Groups of 8 (PBS), 12 (VLP-1818) or 13 (VLP-1965, -1632 and -1123) BALB/c mice were immunized twice with a two-week interval with 10 µg of the indicated VLPs, adjuvanted with Alhydrogel. One group of mice received PBS with Alhydrogel (PBS). Two weeks after the second immunization, mice were challenged with 4 LD50 of mouse-adapted X47 virus. Survival rate (left; P<0.001, Kaplan Meier test) and body weight (right; P<0.05, two-sample t-test on days 6 after challenge) C. Groups of 14 BALB/c mice were immunized three times with three-week intervals by i.p. injection of 10 µg of VLP-1123, VLP-1818 or VLP-1965, adjuvanted with Alhydrogel. Three weeks after the second boost, mice were challenged with 4 LD50 of PR/8 virus. Survival rate (left; P<0.0001) and body weight (right; P<0.05 on days 8, 10 and 12 after challenge).
Figure 6
Figure 6. Immunization with M2e-VLPs reduces lung virus replication.
BALB/c mice were immunized twice two weeks apart with 10 µg of Alhydrogel-adjuvanted PBS, VLP-1123, VLP-1632, VLP-1818 or VLP-1965. Two weeks after the boost, mice were challenged with 4 LD50 of X47 virus. Six days after challenge, four (PBS) or five (all other groups) mice from each group were sacrificed to determine lung virus titers. Virus titers are expressed as mean log10 TCID50/ml and error bars depict the standard error of the mean.
Figure 7
Figure 7. Cellular immune responses and histopathology following challenge.
Mice were immunized as described in the legend of Figure 6. A. Six days after challenging the mice with 4 LD50 of X47 virus, four (PBS) or five (all other groups) mice from each group were sacrificed to prepare splenocytes. IFN-γ responses after M2e (top) or NP (bottom) peptide restimulation were determined by ELISPOT analysis. Bars represent the average number of spots determined for individual mice (analyzed in triplicate) from each group. Error bars represent standard error of the mean. (*:P<0.05). B. Representative hematoxylin-eosin stained lung tissue section prepared on day four after challenge of mice immunized with 10 µg of Alhydrogel-adjuvanted VLP-1632, VLP-1818 or VLP-1965. VLP-1123 (a): diffuse presence of a pronounced interstitial, alveolar (*) and bronchiolar (arrow) mixed inflammatory infiltrate interlaced with necrotic debris. VLP-1818 (b): focal presence of a limited mixed inflammatory infiltrate both in the interstitial, alveolar (*) as well as bronchiolar (arrow) structures. VLP-1965 (c): Some congestion, but no inflammatory reaction.
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L.I.I. was a beneficiary of the Belgian Federal Sciences Administration (Federale Wetenschapsbeleid, BELSPO) and was supported by Ghent University IOF-grant Stepstone IOF08/STEP/001 and Ghent University Special Research Grant BOF12/GOA/014. K.R. was supported by a predoctoral fellowship from Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie (IWT). M.S. was a beneficiary of a “Bijzonder Onderzoeksfonds” research grant from Ghent University and B.S. is postdoctoral fellow with FWO-Vlaanderen. Research related to M2e-based influenza vaccines in the group of X.S. is supported by FWO-Vlaanderen project G.0375.10N, Ghent University IOF-grant Stepstone IOF08/STEP/001, and a research collaboration with Sanofi Pasteur. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.