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. 2024 Aug 8;20(8):e1012393.
doi: 10.1371/journal.ppat.1012393. eCollection 2024 Aug.

Evaluation of a novel intramuscular prime/intranasal boost vaccination strategy against influenza in the pig model

Robin Avanthay  1   2   3 , Obdulio Garcia-Nicolas  1   2 , Nicolas Ruggli  1   2 , Llorenç Grau-Roma  2   4 , Ester Párraga-Ros  5 , Artur Summerfield  1   2 , Gert Zimmer  1   2
Affiliations

Evaluation of a novel intramuscular prime/intranasal boost vaccination strategy against influenza in the pig model

Robin Avanthay et al. PLoS Pathog. .

Abstract

Live-attenuated influenza vaccines (LAIV) offer advantages over the commonly used inactivated split influenza vaccines. However, finding the optimal balance between sufficient attenuation and immunogenicity has remained a challenge. We recently developed an alternative LAIV based on the 2009 pandemic H1N1 virus with a truncated NS1 protein and lacking PA-X protein expression (NS1(1-126)-ΔPAX). This virus showed a blunted replication and elicited a strong innate immune response. In the present study, we evaluated the efficacy of this vaccine candidate in the porcine animal model as a pertinent in vivo system. Immunization of pigs via the nasal route with the novel NS1(1-126)-ΔPAX LAIV did not cause disease and elicited a strong mucosal immune response that completely blocked replication of the homologous challenge virus in the respiratory tract. However, we observed prolonged shedding of our vaccine candidate from the upper respiratory tract. To improve LAIV safety, we developed a novel prime/boost vaccination strategy combining primary intramuscular immunization with a haemagglutinin-encoding propagation-defective vesicular stomatitis virus (VSV) replicon, followed by a secondary immunization with the NS1(1-126)-ΔPAX LAIV via the nasal route. This two-step immunization procedure significantly reduced LAIV shedding, increased the production of specific serum IgG, neutralizing antibodies, and Th1 memory cells, and resulted in sterilizing immunity against homologous virus challenge. In conclusion, our novel intramuscular prime/intranasal boost regimen interferes with virus shedding and transmission, a feature that will help combat influenza epidemics and pandemics.

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Conflict of interest statement

RA, GZ, and AS filed a patent related to the intramuscular prime/intranasal boost vaccine described in this work. All other authors declare no competing interests.

Figures

Fig 1
Fig 1. Replication and immunogenicity of NS1(1–126) LAIV in pigs following intranasal administration.
The indicated animal groups (group size n = 5) were either immunized via the intramuscular route with VSV-Luc (black symbols) or via the intranasal route with pH1N1/09 (orange symbols) or NS1(1–126) LAIV (turquoise symbols). Nasal swabs samples were collected daily and serum samples at weekly time intervals. At day 42, the animals were euthanized and BAL fluid was collected. (A) Viral RNA quantities in nasal swab samples. The dotted lines show the viral genome copies for the animals at each time point. The bold lines represent the mean values per animal group for each time point. (B, C) Detection of pH1N1/09-specific IgG in sera (B) and BAL fluid (C) by ELISA. The bold lines in (B) represent the mean values for each animal group at each time point. (D, E) Detection by ELISA of pH1N1/09-specific IgA in serum (D) and BAL fluid (E). The dotted lines show the optical density at 450 nm (OD450) for all animals at all time points analyzed. The bold lines represent the mean values for each animal group for each time point. (F) Determination of pH1N1/09-neutralizing antibody titres in serum. The neutralizing antibody titres are shown for the individual animal at all time points analyzed (left panel, dotted lines). The mean values are shown as bold lines. The right panel presents the area under the curve (AUC) analysis encompassing all days. (G) Determination of pH1N1/09-neutralizing antibody titres in BAL fluid. Statistical analysis was performed using the two-way ANOVA test (A, B, D) and the one-way ANOVA test (C, E, F right panel, G). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 indicate significant differences.
Fig 2
Fig 2. Intramuscular prime/intranasal boost vaccination protocol reduces LAIV shedding.
(A) Schematic representation of the experimental design. Red points on the timeline indicate the time points of blood sampling. (B-D) Detection of viral RNA copies in nasal swab samples collected after intranasal inoculation of the animals with the indicated LAIV. At day 55 all animals were challenged via the nasal route using 106 ffu of pH1N1/09. Individual animals are represented by dashed lines and group mean values by continuous thick lines. (B) Pigs were first immunized (i.m.) with the VSV-Luc control vaccine followed by intranasal immunisation with NS1(1–126)-ΔPAX LAIV. (C) Pigs were immunized (i.m.) with VSV-H1 and subsequently boosted (i.n.) with NS1(1–126)-ΔPAX LAIV. (D) Animals were primed (i.m.) with VSV-H1 and boosted (i.n.) with NS1(1–126) LAIV. (E) AUC analyses of viral RNA load in nasal swab samples collected between days 0 and 17 after intranasal vaccination with LAIV (calculated with data from B-D). Significant differences of the AUC values were determined with the one-way ANOVA test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Fig 2A was created with Biorender.com.
Fig 3
Fig 3. Antibody responses following intramuscular prime/intranasal boost vaccination of pigs.
SPF pigs (group size n = 5) were immunized according to the intramuscular prime/intranasal boost vaccination regimen depicted in Fig 2A. (A-D) Analysis of pH1N1/09-specific antibodies by ELISA. The IgG concentrations in serum (A, left panel) and saliva (C, left panel) are depicted by dotted lines for individual animals at each time point analyzed. The bold lines represent the mean values for each animal group for each time point analyzed. The specific IgA levels in serum (B, left panel) and saliva (D, left panel) are represented by dotted lines for the individual animals at each time point analyzed. The bold lines represent the mean values per animal group for each time point. Area under curve (AUC) analysis of the respective data (A-D, right panels). (E) Determination of the virus-neutralizing dose 50% (ND50) in the serum of immunized animals. The dotted lines represent the pH1N1/09-neutralizing antibody titres that were detected in the serum of individual animals at the indicated time points. The bold lines represent the mean values for each animal group (E, left panel). AUC analysis of the data (E, right panel). Significant differences were determined using the one-way ANOVA test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
Fig 4
Fig 4. Induction of memory T cell responses in the peripheral blood of pigs following intramuscular prime/intranasal boost vaccination.
PBMCs were isolated from immunized pigs at day 48 and restimulated with live pH1N1/09 (see scheme in Fig 2A). (A-D) Intracellular cytokine staining of CD4+ T cells for TNF (A), IFNγ (B), IL-17 (C), and TNF/IFNγ double-positive cells (D). The frequency of cytokine-positive cells relative to the total number of CD4+ T cells is shown. (E) Frequency of reactivated CD8+ cells that were positive for the indicated cytokines. Significant differences were determined using the Mann-Whitney test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
Fig 5
Fig 5. The prime/boost vaccination strategy induces sterilizing immunity to homologous virus challenge.
Pigs were vaccinated according to the indicated vaccine combinations and challenged at day 55 with pH1N1/09. (A) Determination of viral RNA loads in nasal swab samples collected at days 1 to 5 post infection (left panel). The dotted lines represent the RNA loads in nasal swabs collected from individual animals. The bold lines represent the mean values. The AUC analysis of these data is shown in the right panel. (B, C) Viral RNA loads in lung tissue (B) and BAL fluid (C) collected at day 5 post infection. (D) Histopathological lung lesion scores. Significant differences were determined using the one-way ANOVA test (A-C) and the Kruskal-Wallis test (D) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
Fig 6
Fig 6. Antibody responses of pigs after nasal challenge infection with pH1N1/09.
(A, B) Detection of pH1N1/09-specific IgA in BAL fluid (A) and lung tissue (B) collected 5 days post infection. (C) Detection of pH1N1/09-specific IgG in lung tissue. Significant differences were determined using the one-way ANOVA test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
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References

    1. Uyeki TM, Hui DS, Zambon M, Wentworth DE, Monto AS. Influenza. Lancet. 2022;400(10353): 693–706. doi: 10.1016/S0140-6736(22)00982-5 - DOI - PMC - PubMed
    1. Czubak J, Stolarczyk K, Orzel A, Fraczek M, Zatonski T. Comparison of the clinical differences between COVID-19, SARS, influenza, and the common cold: A systematic literature review. Adv Clin Exp Med. 2021;30(1): 109–14. doi: 10.17219/acem/129573 - DOI - PubMed
    1. Herold S, Becker C, Ridge KM, Budinger GR. Influenza virus-induced lung injury: pathogenesis and implications for treatment. Eur Respir J. 2015;45(5): 1463–78. doi: 10.1183/09031936.00186214 - DOI - PubMed
    1. Kalil AC, Thomas PG. Influenza virus-related critical illness: pathophysiology and epidemiology. Crit Care. 2019;23(1): 258. doi: 10.1186/s13054-019-2539-x - DOI - PMC - PubMed
    1. Rello J, Pop-Vicas A. Clinical review: primary influenza viral pneumonia. Crit Care. 2009;13(6): 235. doi: 10.1186/cc8183 - DOI - PMC - PubMed

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Grants and funding

This work has received funding from the Swiss National Science Foundation (SNSF), grant no. 189903 (A.S., G.Z.; https://data.snf.ch/grants/grant/189903). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.