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. 2023 Apr 18:14:1125190.
doi: 10.3389/fimmu.2023.1125190. eCollection 2023.

Hemagglutinin expressed by yeast reshapes immune microenvironment and gut microbiota to trigger diverse anti-infection response in infected birds

Ruyu Xie  1 , Huixia Zhang  1 , Han Zhang  1 , Changyan Li  1 , Daqing Cui  1 , Shujun Li  1 , Zexing Li  1 , Hualei Liu  2 , Jinhai Huang  1
Affiliations

Hemagglutinin expressed by yeast reshapes immune microenvironment and gut microbiota to trigger diverse anti-infection response in infected birds

Ruyu Xie et al. Front Immunol. .

Abstract

Introduction: The H5N8 influenza virus is a highly pathogenic pathogen for poultry and human. Vaccination is the most effective method to control the spread of the virus right now. The traditional inactivated vaccine, though well developed and used widely, is laborious during application and more interests are stimulated in developing alternative approaches.

Methods: In this study, we developed three hemagglutinin (HA) gene-based yeast vaccine. In order to explore the protective efficacy of the vaccines, the gene expression level in the bursa of Fabricius and the structure of intestinal microflora in immunized animals were analyzed by RNA seq and 16SrRNA sequencing, and the regulatory mechanism of yeast vaccine was evaluated.

Results: All of these vaccines elicited the humoral immunity, inhibited viral load in the chicken tissues, and provided partial protective efficacy due to the high dose of the H5N8 virus. Molecular mechanism studies suggested that, compared to the traditional inactivated vaccine, our engineered yeast vaccine reshaped the immune cell microenvironment in bursa of Fabricius to promote the defense and immune responses. Analysis of gut microbiota further suggested that oral administration of engineered ST1814G/H5HA yeast vaccine increased the diversity of gut microbiota and the increasement of Reuteri and Muciniphila might benefit the recovery from influenza virus infection. These results provide strong evidence for further clinical use of these engineered yeast vaccine in poultry.

Keywords: H5N8; Saccharomyces cerevisiae; hemagglutinin (HA); influenza A virus; vaccine.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematic diagram of vaccines preparation, chicken vaccination and virus challenge experiments. (A) Preparation of protein subunit vaccine. The plasmids were constructed as described in Materials and Methods and then transformed into Saccharomyces cerevisiae ST1814G strain to prepare ST1814G/H5HA vaccine. Western blot analysis showed that HA protein could be expressed normally in ST1814G/H5HA vaccine. (M: protein marker; CK: ST1814G yeast lysates, lane 1-2: ST1814G/H5HA yeast lysates. (B) Preparation of DNA-RNA nucleic acid vaccine. The plasmids were constructed as described in Materials and Methods and then transformed into Saccharomyces cerevisiae ST1814G strain or ST1814G/H5HA strain to prepare ST1814G/DNA-RNA vaccine or ST1814G/H5HA/DNA-RNA vaccine. (C) Both the eGFP protein and the mCherry protein could be efficiently expressed in stomach and intestine tissues of chickens. (D) Seven groups of two-week-old chickens were immunized as the schedule indicated and they were challenged intramuscularly with the H5N8 virus on the 6th day after the third immunization.
Figure 2
Figure 2
Protective effects of engineered yeast vaccine. (A–D) The level of both the secreted IgA in anal swabs and the IgG in serum of each group. The significance of differences was determined by two-way analysis of variance. (A, C), samples after the third immunizations; (B, D), samples post challenge), (**p < 0.01; ***p < 0.001; ****p < 0.001). (E) Survival rate of each group at the indicated time post infection. (F) Virus titer determination of each group in the blood and lung. Statistical analysis was analyzed by Student’s t-test. (****p < 0.001 n=3 chickens for each group). (G) H&E-stained section of the liver and spleen from control group showing spot of bleeding, the lung was showing a pattern of necrotizing alveolitis, the alveolar walls are necrotic and alveolar air spaces contained inflammatory cells in the control groups.
Figure 3
Figure 3
Effect of Vaccines on immune related genes of bursa of Fabricius in chickens. (A, B) Biological process analysis of the differentially-expressed genes (DEGs) based on the Gene Ontology databases. Abscissa: biological process; Ordinate: number of genes and –log10 (P value); (A) ST1814G/H5HA group vs ST1814G group; (B) Inactivated vaccine group vs ST1814G group. (C–E) Heatmaps of gene expression levels in BF for indicated gene lists. (C) Genes related to immune system process; (D) Genes related to leukocyte microenvironment of bursa of Fabricius; (E) Genes related to innate immune response.
Figure 4
Figure 4
Effect of vaccines on intestinal microflora diversity post immunization. (A) Venn diagrams (left) and bar charts (right) of common or endemic species. (B) Relative content of bacterial genus in each group. (C) Relative content of bacterial species in each group. (D) Partial least-squares discrimination analysis (PLS-DA) revealed the diversity of chicken gut microbiota among the seven groups. Dots represent individual samples. (E) The analysis of Alpha diversity index, Chao1, Shannon and observed features, showed the diversity of chicken gut microbiota within each group post immunization. Dots represent individual samples.
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