Combined use of ELISA and Western blot with recombinant N protein is a powerful tool for the immunodiagnosis of avian infectious bronchitis.

BACKGROUND: The avian infectious bronchitis virus (IBV) remains a significant source of loss in the poultry industry and early diagnosis is required to prevent the disease from spreading. This study examined the combined use of an ELISA and Western blot (WB) to detect antibodies against the nucleocapsid protein (N) of IBV. The coding sequence for N was amplified by RT-PCR and expressed in Escherichia coli. A soluble recombinant N protein (rN) of approximately 50 kDa was obtained. A total of 389 sera were tested against the rN in ELISA and the results were compared with those of the commercial IDEXX IBV Ab test. ELISA-rN achieved a 90.34% sensitivity and 90.16% specificity. WB confirmed all false negative sera in ELISA-rN or IDEXX test as truly positive. The current study indicate that the combined use of rN in ELISA and WB is a powerful tool for the immunodiagnosis of avian infectious bronchitis. METHODS: Constructed recombinant pAE/n expression vectors were used to transform E. coli BL21(DE3) Star competent cells (Invitrogen). The rN of infectious bronchitis virus was purified by affinity chromatography using HisTrap HP 1 mL columns pre-packed with pre-charged Ni Sepharose in the ÄKTAprime Automated Liquid Chromatography system (GE Healthcare). A total of 389 serum samples from chickens were used to develop and evaluate the ELISA-rN test. To standardize the indirect ELISA development, serum dilutions (1:100, 1:200 and 1:400) and different concentrations of purified rN antigen (50, 100 and 200 ng/well) were tested. Positive and negative sera for IBV were used as controls. The results were compared with those obtained from a commercial kit. Serum samples scored as negative with the commercial kit but as positive with the ELISA-rN were further analysed by Western blot analyses using the rN protein as an antigen. The results of the ELISA-rN were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software GraphPad Prism version 6.0 for Windows (GraphPad Software, USA). RESULTS: The expected cDNA fragment of approximately 1240 bp was successfully amplified by PCR using primers designed to select for the coding region of the N protein. The rN was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of 10 mg/L of rN was obtained. The SDS-PAGE results demonstrated the presence of two distinct bands that had a molecular mass of approximately 45 and 50 KDa. Out of 244 sera that scored positive in the commercial ELISA IDEXX IBV Ab Test, 220 were also positive in the ELISA-rN, yielding an ELISA-rN test sensitivity of 90.16%. Out of 145 sera that scored negative in the IDEXX IBV Ab Test, 131 also scored negative in the ELISA-rN, indicating a specificity of 90.34%. Sera that tested negative in the ELISA-rN and positive in the commercial test also reacted with the rN protein in Western blot. CONCLUSIONS: The association between the ELISA and Western blot techniques developed in this study with a subunit of IBV (rN) were able to detect antibodies that the commercial ELISA did not detect suggesting that the ELISA-rN has greater sensitivity.

Immune responses in bovines to recombinant glycoprotein D of bovine herpesvirus type 5 as vaccine antigen.

Bovine herpesvirus 5 (BoHV-5) is responsible for outbreaks of meningoencephalitis that cause important economic losses in young cattle. BoHV-5 glycoprotein D (gD5) is essential for attachment and penetration into permissive cells and targeting of host immune systems, inducing strong humoral and cellular immune responses. The aim of this study was to evaluate the vaccinal immune response of vaccines formulated with the recombinant BoHV-5 gD (rgD5) in bovines. For the experiment, 72 heifers were randomly allotted into 6 different groups with 12 animals each. Group 1: vaccine formulated using inactivated BoHV-5 (iBoHV-5) adjuvanted with ISA50V2; Group 2: iBoHV-5 associated with 100 µg of rgD5 adjuvanted with ISA50V2; Group 3: 100 µg of rgD5 adjuvanted with ISA50V2; Group 4: 100 µg of rgD5 adjuvanted with Al(OH)3; Group 5: commercial vaccine; and Group 6: control group. Two doses were administered in a 26-day interval and the third after 357 days from primo vaccination. Cattle vaccinated with the vaccines formulated with iBoHV-5 plus rgD5 showed a significant (p < 0.01) five-fold increase in total immunoglobulin G (IgG) for BoHV-5, BoHV-1, and rgD5 as compared with the commercial and control groups. Also, a significant (p < 0.05) increase in IgG1 and IgG2a levels was induced in serum for rgD5. In addition, these same vaccines showed significant (p < 0.01) four-fold higher titers of BoHV-1 and -5 neutralizing antibodies. The results demonstrated that the rgD5 conserved important epitopes that were able to stimulate bovine humoral immunity response capable of viral neutralization of BoHV-1 and -5, suggesting it as a promising vaccine antigen to be used in vaccine for BoHV-1 and -5 endemic areas.

Probiotics Bacillus toyonensis and Saccharomyces boulardii improve the vaccine immune response to Bovine herpesvirus type 5 in sheep.

There have been significant efforts toward the development of more efficient vaccines for animal health. A strategy that may be used to improve vaccine efficacy is the use of probiotics. Bovine herpesvirus 5 (BoHV-5) is an example of an important animal pathogen for which vaccines have provided only limited protection. In this study, we examined the use of the probiotics Bacillus toyonensis and Saccharomyces boulardii as a potential immune modulator to improve vaccine efficiency. Thirty, 5-month-old lambs were randomly grouped in three lots of 10 each and vaccinated at days 0, 21 and 42 of the experiment. They grazed on the same pasture and were fed ad libitum twice a day with commercial sheep feed supplemented with either B. toyonensis (1×106CFU/g of feed) or S. boulardii (1×107CFU/g of feed), or non-supplemented feed. The probiotic supplementation was suspended day 28; thereafter, the next 35days, they were fed with the same commercial feed as control group. Animals supplemented with probiotics showed a significant (p>0.001) increased seroconversions against BoHV-5, and higher neutralizing antibodies titres (p>0.05) to BoHV-5 than non-supplemented animals. At 63days of experiment, splenocytes from the supplemented sheep had higher mRNA transcription levels of cytokines IL-10 and IL-17A. These results suggest that these probiotics could provide a promising means of improving vaccine efficacy.

Immune responses of mice against recombinant bovine herpesvirus 5 glycoprotein D.

Glycoprotein D (gD) is essential for attachment and penetration of Bovine herpesvirus 5 (BoHV-5) into permissive cells, and is a major target of the host immune system, inducing strong humoral and cellular immune responses. The aim of this study was to evaluate in mice the immunogenicity of recombinant BoHV-5 gD (rgD5) expressed in Pichia pastoris. Vaccines formulated with rgD5 alone or adjuvanted with Montanide 50 ISA V2; Emulsigen or Emulsigen-DDA was administered intramuscularly or subcutaneously. Almost all formulations stimulated a humoral immune response after the first inoculation. The only exception was observed when the rgD5 was administered subcutaneously without adjuvant, in this case, the antibodies were observed after three doses. Higher titers of neutralizing antibodies were obtained with the three oil-based adjuvant formulations when compared to non-adjuvanted vaccine formulations. The rgD5 vaccine stimulated high mRNA expression levels of Th1 (INF-γ) and pro-inflammatory cytokines (IL-17, GM-CSF). The results demonstrated that the recombinant gD from BoHV-5 conserved important epitopes for viral neutralization from native BoHV-5 gD and was able to elicit mixed Th1/Th2 immune response in mice.

The immune modulation of Bacillus cereus var. Toyoi in mice immunized with experimental inactivated Bovine Herpesvirus Type 5 vaccine.

In recent years, there been significant progress toward develop more efficient vaccines. Different compounds with adjuvant capacity have been tested; however, no compound has emerged that suitable for universal use. Several efforts have been made to produce effective vaccines against Bovine herpesvirus 5 (BoHV-5), an important cattle pathogen. In this study we examine the use the probiotic Bacillus cereus var. Toyoi as a potential adjuvant to improve BoHV-5 vaccine efficacy. We observed in the supplemented animals a systemic enhanced IgG antibody response toward Th1, and increased IFN-γ, IL-12 and IL-10 cytokines mRNA levels. These results suggest that this probiotic could provide a promising means of improving vaccine efficacy, particularly those vaccines that rely on a cell-mediated immune response.

Cloning and expression of a truncated form of envelope glycoprotein D of Bovine herpesvirus type 5 in methylotrophic yeast Pichia pastoris.

Meningoencephalitis caused by Bovine herpesvirus type 5 (BoHV-5) is responsible for heavy economic losses in the cattle industry. As in other Alphaherpesviruses, the envelope glycoprotein IV (gD), which mediates penetration into host cells, is one of the major candidate antigens for a recombinant vaccine, since it induces a strong and persistent immune response. The DNA coding for a truncated form of BoHV-5 gD (tgD) has been cloned into the Pichia pastoris expression vector pPICZalphaB to allow protein secretion into the medium. After induction with methanol, a approximately 55kDa protein was obtained. Enzyme deglycosylation with Endo H showed a smaller size band in SDS-PGAE, with approximately 50kDa, suggesting that tgD has N-linked oligosaccharides and that it is not hyperglycosylated. The approximately 55kDa protein was recognized by several polyclonal antibodies, including polyclonal antibody anti-tgD and polyclonal antibodies of different animal species immunized with BoHV-5 and BoHV-1. This is the first report of BoHV-5 gD expression in yeast. It was shown that the recombinant truncated form of BoHV-5 gD has antigenic and immunogenic properties similar to the native BoHV-5 gD. Expression of tgD as a secreted protein allows simple and inexpensive purification methods that can be used for further studies to evaluate its immunogenicity in cattle.

Adjuvant effect of green propolis on humoral immune response of bovines immunized with bovine herpesvirus type 5.

Despite recent technological advances in vaccine production, most vaccines depend on the association with adjuvant substances. In this study, propolis, which has been attracting the attention of researchers due to its bioactive properties, was evaluated as an immunological adjuvant. The association of 40mg/dose of an ethanolic extract of green propolis with an inactivated oil vaccine against bovine herpesvirus type 5 (BoHV-5), resulted in a significant increase (P<0.01) in the neutralizing antibody levels, comparing to the bovines that received the same vaccine without propolis. Besides, propolis increased the percentage of animals with high antibody titers (above 32). Phenolic compounds such as artepillin C (3,5-diprenyl-4-hydroxycinnamic acid) and the derivatives of cinnamic acid besides other flavonoid substances were abundant in the propolis extract used, and they could be the main substances with adjuvant action. The effect of the green propolis extract on the humoral immune response can be exploited in the development of new vaccines.

Immunomodulation produced by a green propolis extract on humoral and cellular responses of mice immunized with SuHV-1.

Despite recent technological advances in vaccine production, most the vaccines depend on the association with adjuvant substances. This work evaluated the adjuvant capacity of an ethanol extract of green propolis associated to inactivated Suid herpesvirus type 1 (SuHV-1) vaccine preparations. Mice inoculated with SuHV-1 vaccine plus aluminum hydroxide and 5mg/dose of propolis extract presented higher levels of antibodies when compared to animals that received the same vaccine without propolis. The use of SuHV-1 vaccine with propolis extract alone did not induce significant levels of antibodies, however it was able to increase the cellular immune response, evidenced by the increase in the expression of mRNA to IFN-gamma. Besides, propolis increased the percentage of protected animals against challenge with a lethal dose of SuHV-1. The effect of green propolis extract on the humoral and cellular immune responses may be exploited for the development of effective vaccines.