J Vet Sci.  2018 Nov;19(6):817-826. 10.4142/jvs.2018.19.6.817.

Comparison of immunoadjuvant activities of four bursal peptides combined with H9N2 avian influenza virus vaccine

• Zhang C
• Zhou J
• Liu Z
• Liu Y
• Cai K
• Shen T
• Liao C
• Wang C

• KMID: 2427030
• DOI: http://doi.org/10.4142/jvs.2018.19.6.817

Abstract

The bursa of Fabricius (BF) is a central humoral immune organ unique to birds. Four bursal peptides (BP-I, BP-II, BP-III, and BP-IV) have been isolated and identified from the BF. In this study, the immunoadjuvant activities of BPs I to IV were examined in mice immunized with H9N2 avian influenza virus (AIV) vaccine. The results suggested that BP-I effectively enhanced cell-mediated immune responses, increased the secretion of Th1 (interferon gamma)- and Th2 (interleukin-4)-type cytokines, and induced an improved cytotoxic T-lymphocyte (CTL) response to the H9N2 virus. BP-II mainly elevated specific antibody production, especially neutralizing antibodies, and increased Th1- and Th2-type cytokine secretion. BP-III had no significant effect on antibody production or cell-mediated immune responses compared to those in the control group. A strong immune response at both the humoral and cellular levels was induced by BP-IV. Furthermore, a virus challenge experiment followed by H&E staining revealed that BP-I and BP-II promoted removal of the virus and conferred protection in mouse lungs. BP-IV significantly reduced viral titers and histopathological changes and contributed to protection against H9N2 AIV challenge in mouse lungs. This study further elucidated the immunoadjuvant activities of BPs I to IV, providing a novel insight into immunoadjuvants for use in vaccine design.

Keyword

adjuvants; bursal peptides-(I–IV); cellular immune response; humoral immune response; immune protection

MeSH Terms

Adjuvants, Immunologic
Animals
Antibodies, Neutralizing
Antibody Formation
Birds
Bursa of Fabricius
Cytokines
Immunity, Cellular
Immunity, Humoral
Influenza A Virus, H9N2 Subtype
Influenza in Birds*
Lung
Mice
Peptides*
T-Lymphocytes, Cytotoxic
Adjuvants, Immunologic
Antibodies, Neutralizing
Cytokines
Peptides

Figure

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  Fig. 1 Experimental scheme for immunization, sample collection, and challenge.

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  Fig. 2 Effect of bursal peptides (BPs) on antigen-specific immune responses. Mouse sera were collected on days 0, 7, 14, and 21 after the first immunization. Serum hemagglutination inhibition (HI) antibody titers (A) were analyzed by HI assay at 0, 7, 14, and 21 days; antigen-specific antihemagglutinin (anti-HA) (B), antibody subtypes IgG1 (C), and IgG2a (D) titers were analyzed by enzyme-linked immunosorbent assay on days 7 and 21. Data are presented as mean ± SD values from five replicates. AIV, avian influenza virus; PBS, phosphate-buffered saline; HA, hemagglutinin; OD, optical density. *p < 0.05 and **p < 0.01 compared with mice immunized with H9N2 AIV vaccine alone.

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  Fig. 3 Effect of bursal peptides (BPs) on cell-mediated immune responses in mice. Mouse sera (n = 5) were collected on days 7 and 21 post-immunization; Th1 (interferon gamma)-type cytokines (A) and Th2 (interleukin 4)-type cytokines (B) levels were measured by using commercial enzyme-linked immunosorbent assay kits. (C) Mouse splenic lymphocytes were isolated from spleen (n = 5) on day 21 post-immunization for cytotoxic T-lymphocyte (CTL) assay. (D) Flow cytometry analysis of the total CD3+T cells and subsets CD4+ and CD8+T cells from the splenic lymphocytes of immunized mice. Data are presented as mean ± SD values from five replicates. PBS, phosphate-buffered saline; AIV, avian influenza virus; IFN-γ, interferon gamma; IL-4, interleukin-4. *p < 0.05 and **p < 0.01 compared with mice immunized with H9N2 AIV vaccine.

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  Fig. 4 Detection of avian influenza virus (AIV) titers in lungs of H9N2 AIV-challenged mice by quantitative polymerase chain reaction (PCR) and TCID50 assay (TCID50, 50% tissue culture infective dose). Lung samples from individual mice in each group (n = 5) were collected on days 3, 5, and 7 post-challenge with 2.5 × 106 TCID50 AIV A/Chicken/Jiangsu/JS-1/2002 (H9N2). Lung virus copies were determined by quantitative PCR. Each lung sample was diluted to 1 mL with phosphate-buffered saline (PBS), panels A–C in Fig. 4 shows the viral genomes (copies) presented per milliliter of total RNA in lung tissue of individual mice in each group (n = 5). Panels D–F in Fig. 4 shows the viral titers as plaque-forming units (PFU) per milliliter as determined by TCID50 assay. Data are presented as mean ± SD values from five replicates. BP, bursal peptide. *p < 0.05 and **p < 0.01 compared with mice immunized with H9N2 AIV vaccine alone.

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  Fig. 5 Pathology changes in lung sections harvested at days 3, 5, and 7 after challenge and stained with H&E. Lung sections were obtained from mice (n = 5) in each group, fixed in buffered formaldehyde solution, and stained with H&E to assess inflammation and tissue damage after challenge. The arrows indicate representative pathological changes of the lung tissues: inflammation, perivascular and interstitial infiltrates. PBS, phosphate-buffered saline; BP, bursal peptide. 20×.

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  Fig. 6 Pathological scores of lung sections harvested at days 3, 5, and 7 after challenge. Histological scoring for virus-infected mice; mean values for each group are shown (n = 5). Histopathological changes were scored by an investigator “blind” to sample identity Scores were assigned as follows: a score of 1 indicates no pathology, 2 indicates perivascular infiltrates, 3 indicates perivascular and interstitial infiltrates affecting 20% of the lung lobe section, 4 indicates perivascular and interstitial infiltrates affecting 20% to 50% of the lung lobe section, and 5 indicates perivascular and interstitial infiltrates affecting 50% of the lung lobe section. PBS, phosphate-buffered saline; AIV, avian influenza virus; BP, bursal peptide.

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