RESUMO
AIM: The present study was aimed to establish a protocol for the evaluation of the in vitro potency of commercial inactivated Newcastle disease virus (NDV) oil-adjuvanted vaccines using hemagglutination test (HA) and blocking ELISA (B-ELISA) based on polyclonal antibodies. MATERIALS AND METHODS: Aqueous phases from a total of 47 batches of inactivated NDV vaccines manufactured by 20 different companies were extracted with isopropyl myristate. The viral antigen in each sample was detected and quantified by a standard HA test and a B-ELISA assay. To verify the efficiency of the antigen extraction method used in the batches which showed HA and to test the validity of using in vitro antigen quantification by HA and B-ELISA tests, a subset of 13 batches (selected from the total 47 batches) was inoculated in groups of 3-4-week-old specific pathogen-free chickens using the recommended vaccine dose. The immunogenicity of the selected vaccine batches was assessed by the NDV-hemagglutination inhibition antibody titers in individual serum samples collected 4 weeks after vaccination. Further, the efficacy of the vaccines and their protection rates were determined by a challenge test carried out for the vaccinated chickens with the Egyptian 2012 isolate of the virulent NDV genotype VII. RESULTS: A strong correlation was observed between HA titers and B-ELISA mean titers in the tested 47 batches (R2=0.817). This indicated the possibility of using the latter in vitro assays for vaccine potency assessment. The recommended protective NDV antigen titer measured by B-ELISA was determined to be 28 ELISA units per dose. The comparison between the HA titers of the aqueous extracts of test vaccines and the corresponding results of in vivo potency assays (i.e., immunogenicity and efficacy), including antibody titers in the serum of vaccinated birds, indicated that the efficiency of the antigen extraction used may interfere with obtaining a strong correlation between the in vitro and in vivo results. CONCLUSION: HA or B-ELISA tests can be used as rapid and cost-effective alternatives to traditional in vivo potency tests for vaccine potency assessment by quantifying the NDV antigen present in aqueous phase extracts of the tested vaccines. The latter in vitro protocol, however, requires efficient extraction of the antigen to be able to obtain good correlation with the traditional in vivo potency tests.
RESUMO
AIM: The objective of the present study was to prepare a trivalent inactivated vaccine of Newcastle disease virus (NDV), H5N1, and H9N2 viruses. MATERIALS AND METHODS: Three monovalent and a trivalent vaccines were prepared by emulsifying inactivated NDV (LaSota strain), reassortant H5N1, and H9N2 viruses with Montanide ISA 71 oil adjuvant. Parameters used for evaluation of the efficacy of the prepared vaccines in specific pathogen-free chickens were cellular immunity assays (blastogenesis, interferon gamma, interleukin 1 [IL1], and IL6), humoral immunity by hemagglutination inhibition, protection percentage, and shedding. RESULTS: A single immunization with trivalent vaccine-enhanced cell-mediated immunity as well as humoral immune response with 90% protection against challenges with highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic (LP) avian influenza H9N2 viruses with 100% protection after challenge with NDV. CONCLUSION: Development and evaluation of the trivalent vaccine in the study reported the success in preparation of a potent and efficacious trivalent vaccine which is a promising approach for controlling HPAI H5N1, LP H9N2, and ND viral infections.