J Vet Sci.  2014 Jun;15(2):199-208. 10.4142/jvs.2014.15.2.199.

Recombinant Kluyveromyces lactis expressing highly pathogenic porcine reproductive and respiratory syndrome virus GP5 elicits mucosal and cell-mediated immune responses in mice

• Zhao H
• Wang Y
• Ma Z
• Wang Y
• Feng WH

• KMID: 1784640
• DOI: http://doi.org/10.4142/jvs.2014.15.2.199

Abstract

Currently, killed-virus and modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccines are used to control porcine reproductive and respiratory syndrome. However, both types of vaccines have inherent drawbacks; accordingly, the development of novel PRRSV vaccines is urgently needed. Previous studies have suggested that yeast possesses adjuvant activities, and it has been used as an expression vehicle to elicit immune responses to foreign antigens. In this report, recombinant Kluyveromyces lactis expressing GP5 of HP-PRRSV (Yeast-GP5) was generated and immune responses to this construct were analyzed in mice. Intestinal mucosal PRRSV-specific sIgA antibody and higher levels of IFN-gamma in spleen CD4+ and CD8+ T cells were induced by oral administration of Yeast-GP5. Additionally, Yeast-GP5 administered subcutaneously evoked vigorous cell-mediated immunity, and PRRSV-specific lymphocyte proliferation and IFN-gamma secretion were detected in the splenocytes of mice. These results suggest that Yeast-GP5 has the potential for use as a vaccine for PRRSV in the future.

Keyword

GP5; immune responses; Kluyveromyces lactis yeast; PRRSV; vaccine

MeSH Terms

Administration, Oral
Animals
Antibodies, Viral/*immunology
B-Lymphocytes/immunology/virology
Enzyme-Linked Immunosorbent Assay
*Immunity, Cellular
*Immunity, Mucosal
Injections, Subcutaneous
Kluyveromyces/genetics
Mice
Mice, Inbred BALB C
Porcine respiratory and reproductive syndrome virus/*immunology
Recombinant Proteins/genetics/immunology
T-Lymphocytes/immunology/virology
Viral Envelope Proteins/*genetics/*immunology
Viral Vaccines/administration & dosage/*pharmacology
Antibodies, Viral
Recombinant Proteins
Viral Envelope Proteins
Viral Vaccines

Figure

• 

  Fig. 1 Construction and expression of the recombinant Kluyveromyces (K.) lactis. (A) Schematic diagram of Yeast-GP5 vaccine constructs. (B~D) Whole-cell polymerase chain reaction strategies with integration primers were designed to detect targeted integration of pKLAC1 into the K. lactis chromosome. Lane 1, Yeast-GP5; Lane 2, Yeast-vector; Lane M, DNA Marker. (B) 1.9-kb amplicon, single- or tandem-vector integration. (C) 2.3-kb amplicon, multicopy integration. (D) The GP5 gene of porcine reproductive and respiratory syndrome virus (PRRSV). (E) Analysis of GP5 expression in whole recombinant Yeast-GP5 in vitro by Western blotting. Lane 1, Yeast-vector as a negative control; Lane 2, Yeast-GP5-His as a positive control; Lane 3, Yeast-GP5.

• 

  Fig. 2 PRRSV-specific IgG and sIgA responses in immunized mice. (A) PRRSV-specific IgG in serum samples of mice immunized both subcutaneously and orally was analyzed by iELISA using a single dilution (1 : 250) on day 7 after the second immunization (day 14) and the third immunization (day 21). (B) Small-intestinal and vaginal washes of mice immunized orally were collected on day 7 after the third immunization to determine the PRRSV-specific sIgA. Levels of sIgA in secretions were detected by iELISA using a single dilution. The dilution of small-intestinal washes was 1 : 40, while that of vaginal washes was 1 : 20. Sc: subcutaneous. Data represent the mean ± standard deviation (SD). **p < 0.01.

• 

  Fig. 3 Lymphocyte proliferative responses in immunized mice. Splenocytes were isolated from vaccinated mice on day 7 after the final immunization and re-stimulated in vitro with purified HP-PRRSV antigen (10 µg/mL). Following 60 h of stimulation, MTT was added and the OD values were determined after 4 h of incubation. PMA + ionomycin as a positive control; bovine serum albumin (BSA, 2 µg/mL) as an irrelevant antigen control. Data are presented as the mean ± SD. **p < 0.01.

• 

  Fig. 4 PRRSV-specific IL-4 production in T cells. Splenocytes were isolated on day 7 after the final immunization and restimulated in vitro with purified HP-PRRSV antigen (10 µg/mL) for 6 h. The percentages of CD4+IL-4+ T cells were then analyzed by flow cytometry. (A) Scatter plot of one immunized mouse from each group. (B) Statistical assessment of IL-4-secreting cells among total CD4+ T cells in immunized mice. The vertical bars represent the mean ± SD. ns: not significant.

• 

  Fig. 5 PRRSV-specific IFN-γ production in T cells. Splenocytes were isolated and stimulated as in Fig. 4. (A and D) PE Rat IgG1 isotype control. (B and C) Summaries of percentages of IFN-γ+ CD4+ T cells in total CD4+ T cells. (B) Scatter plot of one immunized mouse from each group. (C) Statistical assessment. (E and F) Summaries of percentage of IFN-γ+ CD8+ T cells in total CD8+ T cells. (E) Scatter plot of one immunized mouse from each group. (F) Statistical assessment. Data are presented as the mean ± SD. *p < 0.05.

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