Immunogenicity and Protective Efficacy of Dose-Sparing Epigraph Vaccine against H3 Swine Influenza A Virus

Abstract

Swine influenza A virus (IAV-S) is a highly prevalent and transmissible pathogen infecting worldwide swine populations. Our previous work has shown that the computationally derived vaccine platform, Epigraph, can induce broadly cross-reactive and durable immunity against H3 IAV-S in mice and swine. Therefore, in this study, we assess the immunogenicity and protective efficacy of the Epigraph vaccine at increasingly lower doses to determine the minimum dose required to maintain protective immunity against three genetically divergent H3 IAV-S. We assessed both antibody and T cell responses and then challenged with three H3N2 IAV-S derived from either Cluster IV(A), Cluster I, or the 2010.1 "human-like" cluster and assessed protection through reduced pathology, reduced viral load in the lungs, and reduced viral shedding from nasal swabs. Overall, we observed a dose-dependent effect where the highest dose of Epigraph protected against all three challenges, the middle dose of Epigraph protected against more genetically similar IAV-S, and the lowest dose of Epigraph only protected against genetically similar IAV-S. The results of these studies can be used to continue developing a broadly protective and low-dose vaccine against H3 IAV-S.

Keywords: Cluster 1; Cluster 2010.1 “human-like”; Cluster IV(A); Epigraph; Swine influenza A virus; dose-sparing.

Figure 1

Antibody responses in dose-sparing. Male and female swine (n = 15/group) were immunized with FluSure XP, 10¹¹vp, 10¹⁰vp, or 10⁹vp of Epigraph. Serum was collected to assess hemagglutination inhibition (HI) antibody responses after (A) prime immunization and (B) boost immunization. Serum from all swine was analyzed for HI antibody responses against representative IAV-S strains isolated from Cluster I-TX/98, Cluster II-CO/99, Cluster IV-OH/09, Cluster IV(A)-WY/13, Cluster IV(B)-MN/12, Cluster IV(C)-IN/11, Cluster IV(E)-KS/11, and the 2010.1 human-like cluster-TX/18. A heatmap of responses is shown on the right. The dotted line indicates an HI titer of 1:40 (5.32 log₂). Data are presented as the mean ± SEM, and statistical analysis was determined with a one-way ANOVA with Tukey’s multiple comparisons follow-up test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

T cell responses in dose-sparing. Male and female swine (n = 15/group) were immunized with FluSure XP, 10¹¹vp, 10¹⁰vp, or 10⁹vp of the Epigraph vaccine. PBMCs were collected to assess cross-reactive T cell responses by interferon-γ ELISpot after a prime and boost immunization against (A) A/Uruguay/716/2007, (B) A/New York/385/2005, and (C) A/Perth/16/2009. The dotted line indicates a limit of detection of 50 spot-forming units (SFU) per million cells analyzed. Data are presented as the mean ± SEM. Statistical analysis was determined with a one-way ANOVA with Tukey’s multiple comparisons follow-up test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Protection against Cluster IV(A) Swine Influenza A Virus. Pigs were immunized twice with FluSure XP, sham vaccine, or 10¹¹vp, 10¹⁰vp, or 10⁹vp of Epigraph vaccine, then challenged with 10⁶ TCID₅₀ of Cluster IV(A) isolate, A/swine/Ohio/11SW87/2011. Lungs (A,B) and tracheas (C,D) were collected for histopathological analysis (A,C) and immunohistochemistry (B,D) five days post-infection. (E) Composite microscopic scores based on an established scoring system. (F) Infectious virus in the lungs was quantified using a TCID₅₀ assay. (G) Daily nasal swabs were collected, and infectious viral shedding was quantified using a TCID₅₀ assay. Data in E, F, and G are presented as the mean ± SEM, and statistical analysis was analyzed by a one-way ANOVA with Tukey’s multiple comparisons follow-up test: ns: no significance, **** p < 0.0001.

Figure 4

Protection against Cluster I Swine Influenza A Virus. Pigs were immunized twice with FluSure XP, sham vaccine, or 10¹¹vp, 10¹⁰vp, or 10⁹vp of Epigraph vaccine, then challenged with 10⁶ TCID₅₀ of Cluster I isolate, A/swine/Texas/4199-2/1998. Lungs (A,B) and tracheas (C,D) were collected for histopathological analysis (A,C) and immunohistochemistry (B,D) five days post-infection. (E) Composite microscopic scores based on an established scoring system. (F) Infectious virus in the lungs was quantified using a TCID₅₀ assay. (G) Daily nasal swabs were collected, and infectious viral shedding was quantified using a TCID₅₀ assay. Data in E, F, and G are presented as the mean ± SEM, and statistical analysis was analyzed by a one-way ANOVA with Tukey’s multiple comparisons follow-up test: ns: no significance, * p < 0.05, ** p < 0.01.

Figure 5

Protection against Cluster 2010.1 Human-like Swine Influenza A Virus. Pigs were immunized twice with FluSure XP, sham vaccine, or 10¹¹vp, 10¹⁰vp, or 10⁹vp of Epigraph vaccine, then challenged with 10^4.5 TCID₅₀ of Cluster 2010.1 human-like isolate, A/swine/Texas/A01785781/2018. Lungs (A,B) and tracheas (C,D) were collected for histopathological analysis (A,C) and immunohistochemistry (B,D) five days post-infection. (E) Composite microscopic scores are based on an established scoring system. (F) Infectious virus in the lungs was quantified using a TCID₅₀ assay. (G) Daily nasal swabs were collected, and infectious viral shedding was quantified using a TCID₅₀ assay. Data in E, F, and G are presented as the mean ± SEM, and statistical analysis was analyzed by a one-way ANOVA with Tukey’s multiple comparisons follow-up test: * p < 0.05.