Heterologous prime-boost H1N1 vaccination exacerbates disease following challenge with a mismatched H1N2 influenza virus in the swine model

Abstract

Influenza A viruses (IAVs) pose a significant threat to both human and animal health. Developing IAV vaccine strategies able to elicit broad heterologous protection against antigenically diverse IAV strains is pivotal in effectively controlling the disease. The goal of this study was to examine the immunogenicity and protective efficacy of diverse H1N1 influenza vaccine strategies including monovalent, bivalent, and heterologous prime-boost vaccination regimens, against a mismatched H1N2 swine influenza virus. Five groups were homologous prime-boost vaccinated with either an oil-adjuvanted whole-inactivated virus (WIV) monovalent A/swine/Georgia/27480/2019 (GA19) H1N2 vaccine, a WIV monovalent A/sw/Minnesota/A02636116/2021 (MN21) H1N1 vaccine, a WIV monovalent A/California/07/2009 (CA09) H1N1, a WIV bivalent vaccine composed of CA09 and MN21, or adjuvant only (mock-vaccinated group). A sixth group was prime-vaccinated with CA09 WIV and boosted with MN21 WIV (heterologous prime-boost group). Four weeks post-boost pigs were intranasally and intratracheally challenged with A/swine/Georgia/27480/2019, an H1N2 swine IAV field isolate. Vaccine-induced protection was evaluated based on five critical parameters: (i) hemagglutination inhibiting (HAI) antibody responses, (ii) clinical scores, (iii) virus titers in nasal swabs and respiratory tissue homogenates, (iv) BALf cytology, and (v) pulmonary pathology. While all vaccination regimens induced seroprotective titers against homologous viruses, heterologous prime-boost vaccination failed to enhance HAI responses against the homologous vaccine strains compared to monovalent vaccine regimens and did not expand the scope of cross-reactive antibody responses against antigenically distinct swine and human IAVs. Mismatched vaccination regimens not only failed to confer clinical and virological protection post-challenge but also exacerbated disease and pathology. In particular, heterologous-boosted pigs showed prolonged clinical disease and increased pulmonary pathology compared to mock-vaccinated pigs. Our results demonstrated that H1-specific heterologous prime-boost vaccination, rather than enhancing cross-protection, worsened the clinical outcome and pathology after challenge with the antigenically distant A/swine/Georgia/27480/2019 strain.

Keywords: hemagglutinin; heterologous prime-boost; influenza A virus; swine; vaccine.

Figure 1

Heterologous prime-boost vaccination did not augment HAI antibody titers against vaccine constituents compared to homologous monovalent and bivalent prime-boost vaccination regimens. Five groups of pigs (n = 6) were vaccinated in a homologous or a heterologous prime-boost regimen 3 weeks apart at day 0 and day 21. Serum samples from the groups were assessed on day 21 at boost vaccination, day 35, and day 49 pp (day −2 pre-challenge) for hemagglutination inhibition titers against (A) A/swine/Georgia/27480/2019 H1N2, (B) A/California/07/2009 H1N1, and (C) A/swine/Minnesota/A02636116/2021 H1N1. Antibody titers are compared between homologous-boosted monovalent GA19 (blue), MN21 (purple), and CA09 (olive) groups, the bivalent CA09+MN21 vaccine group (cyan), and the heterologous prime-boosted vaccine group (crimson red). Bars represent geometric mean HAI titers and error bars represent 95% confidence interval (CI). Statistical analysis was performed by two-way ANOVA test. Statistically significant differences (p < 0.05) between vaccine group geometric means are noted by different lowercase letters.

Figure 2

Bivalent and heterologous prime-boosting vaccination strategies enhanced and prolonged clinical disease following challenge with A/swine/Georgia/27480/2019. Clinical scores including rectal temperature, respiratory rate, and assessment of clinical demeanor (coughing and weakness/depression) were recorded daily following infection with the A/swine/Georgia/27480/2019 H1N2 challenge virus. Specifically, rectal temperature score ranged from 0 to 3 (<39.4°C = 0, 39.4–39.9°C = 1, 40–40.5°C = 2, >40.6°C = 3), respiratory rate per minute score ranged from 0 to 2 (20–40 = 0, 41–59 = 1, >60 = 2), and clinical behavior scores, based on coughing (absent = 0, present = 1) and depression (absent = 0, present = 2), ranged from 0 to 3. Bars represent the geometric mean of clinical scores and error bars denote 95% CI. Statistical analysis was performed by a two-way ANOVA test. Statistically significant differences (p < 0.05) in clinical scores’ means between MV/C and experimental vaccine groups per day are indicated by an asterisk (*).

Figure 3

Mismatched swine influenza vaccination regimens elicited different inflammatory cytology profiles in BALf after influenza infection compared to the MV/C group. Bronchoalveolar lavage fluid (BALf) was harvested from lungs at day −2 pre-challenge for the MV/NC group and at day 5 pc from infected groups. (A) Concentrated cells were added to slides and stained with Wright-Giemsa stain and differential cell counts were determined microscopically. (i) MV/NC group, (ii) MV/C group, (iii) mono GA19 vaccine group, (iv) mono MN21 vaccine group, (v) mono CA09 vaccine group, (vi) bivalent CA09+MN21 bivalent vaccine group, and (vii) mono CA09 → mono MN21 heterologous prime-boosted vaccine group (20 μm scale bar, 40× magnification). (B) Cell counts were expressed as a percentage of total cells. Black bars represent the mean percentage of macrophages, cyan bars denote the mean percentage of neutrophils, and pink bars represent the mean percentage of lymphocytes in each experimental group. Error bars represent the standard error of the mean (SEM). Statistical analysis was performed by two-way ANOVA test. Statistically significant differences (p < 0.05) in population percentages between MV/C and experimental vaccine groups per cellular element are indicated by an asterisk (*).

Figure 4

Mismatched vaccination strategies failed to reduce virus shedding and replication in the respiratory tract. Nasal swabs were collected from pigs daily post-challenge through euthanasia on day 5. Following necropsy, tissue samples from the respiratory tract including nasal turbinates, trachea, right lung lobes including the accessory lung lobe, and left lung lobes were harvested at day 5 post-challenge. Virus load in nasal swab samples was assessed by an RT-PCR assay. Titers were measured as relative equivalent units (REU) of RNA corresponding to a 10-fold dilution series of RNA extracted from infective MDCK culture medium at a 10⁷ TCID₅₀ of the A/swine/Georgia/27480/2019 H1N2 challenge virus. Virus replication in the respiratory tract was determined via TCID₅₀ assay in the respiratory tissue homogenates. (A) Mean viral titers from nasal swabs. (B) Mean viral titers of homogenized nasal turbinates. (C) Mean viral titers of homogenized trachea tissue. (D) Mean viral titers of right lung lobe tissue homogenates. (E) Mean viral titers of left lung lobe tissue homogenates. Black triangles represent MV/C pigs, blue squares represent GA19 vaccinated pigs, purple triangles represent MN21 vaccinated animals, olive rhombuses represent CA09 vaccinated pigs, cyan hexagons represent bivalent CA09+MN21 vaccinated pigs, and crimson red circles represent heterologous-boosted pigs. For A, bars represent mean virus titers and errors bars represent 95% CI. For B to E, horizontal lines represent the mean virus titer per homogenate for each group. Error bars represent 95% CI. Dotted lines indicate the limit of detection. Statistical analysis was performed by two-way ANOVA test (A), one-way ANOVA test (B, E), and Kruskal–Wallis test (C, D). Statistically significant differences (p < 0.05) in mean virus titer between MV/C and experimental vaccine groups are indicated by an asterisk (*).

Figure 5

Heterologous prime-boosting exacerbated pulmonary pathology following challenge with A/swine/Georgia/27480/2019. Lung tissues were collected at necropsy 5 days post-challenge and were fixed in formalin. Five-micrometer sections were stained with H&E and examined by light microscopy. Lung sections were scored by two individuals including a board-certified veterinary pathologist. (A) Composite pulmonary histopathology scores from each experimental group. (B) Right lung mean histopathology scores. (C) Left lung mean histopathology scores. Bars represent mean histopathology scores and errors bars represent 95% CI. Statistical analysis was performed by one-way ANOVA test. Statistically significant differences (p < 0.05) in mean histopathology score with MV/C and experimental vaccine groups are indicated by an asterisk (*). (D) H&E-stained representative lung tissue sections from each experimental group. Parenchyma, 500 μm scale bar (2× magnification); bronchiolar epithelium, 50 μm scale bar (20× magnification); alveolar septa, 20 μm scale bar (40× magnification).