E

E., B. HPAIV-neutralizing serum antibodies, with the response to HA being greater, thus identifying HA and NA as impartial neutralization antigens. M2 did not induce a detectable neutralizing serum antibody response, and inclusion of M2 with HA or NA reduced the magnitude of the response. Immunization with HA alone or in combination with NA induced total protection against HPAIV challenge. Immunization with NA alone or in combination with M2 did not prevent death following challenge, but extended the time period before death. Immunization with M2 alone experienced no effect on morbidity or mortality. Thus, there was no indication that M2 is usually immunogenic or protective. Furthermore, inclusion of NA in addition to HA in a vaccine OSU-T315 preparation for chickens may not enhance the high level of protection provided by HA. OSU-T315 Avian influenza (AI) is an economically important disease of poultry worldwide. Avian influenza computer virus (AIV) belongs to the genus under the family in the family and (7). However, the role of entire length of the M2 protein of AIV in induction of neutralizing antibodies and protective immunity against highly pathogenic H5N1 influenza computer virus in chickens has not been directly evaluated. The M2 protein is usually conserved among all influenza A viruses and is therefore considered a stylish target for any universal vaccine (8). Antibodies to HA protein alone can protect against lethal AIV difficulties; the inclusion of other surface proteins in the vaccine regimen may improve the protective efficacy. In the present study, we examined the relative contribution of each of the three HPAIV surface proteins (HA, NA, and M2) to induction of neutralizing antibodies and protective immunity in chickens. Recombinant NDV vectors were constructed that individually expressed each of the three HPAIV surface proteins. They were used to immunize chickens either individually or in different possible combinations. Evaluation of the relative neutralization titers of serum antibody, shedding of challenge computer virus, and protection against lethal HPAIV challenge conferred by each of the NDV-vectored HPAIV surface proteins showed that HA glycoprotein was the major contributor to induction of neutralizing antibodies and protective immunity, followed by NA protein, which conferred partial protection. The M2 protein neither induced a detectable level of serum-neutralizing antibodies nor guarded chickens from your HPAIV lethal challenge. MATERIALS AND METHODS Viruses and cells. The HPAIV strain A/Vietnam/1203/2004 (H5N1) was obtained from the Centers for Disease Control and Prevention (CDC; Atlanta, GA). The recombinant live attenuated influenza computer virus (6attWF10:2H5N1) made up of the altered HA gene (deleted polybasic cleavage site) and the NA gene of computer virus strain A/Vietnam/1203/2004 (H5N1) was explained previously (38). OSU-T315 The recombinant version of the avirulent NDV strain OSU-T315 LaSota was generated previously in our laboratory (14, 36). The viruses were propagated in 9-day-old, specific-pathogen-free (SPF) embryonated chicken eggs. The MDCK (Madin-Darby canine kidney), HEp-2 (human epidermoid carcinoma), and DF1 (chicken embryo fibroblast) cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA). MDCK and HEp-2 cells were produced in Eagle’s minimal essential medium (EMEM) made up of 10% fetal bovine serum (FBS) and managed in EMEM with 5% FBS. DF1 cells were produced in Dulbecco’s minimal essential medium (DMEM) with 10% FBS and managed in DMEM with 5% FBS. Computer virus titration. The titers of stock preparations of rNDV were determined by a plaque assay in DF1 cells using a 0.8% methylcellulose overlay and 5% allantoic fluid. The infected cells were incubated at 37C for 3 to 4 4 days until the development of plaques was apparent. The cell monolayers were then fixed with Rabbit Polyclonal to ZP4 methanol and stained with crystal violet for the enumeration of plaques. Titration of rNDVs and AIVs following or growth was performed by limiting dilution in DF1 and MDCK cells, respectively, using the Reed and Muench method as explained previously (13), and the titers were expressed as 50% tissue culture infectious dose (TCID50) models/ml. For both NDVs and AIVs, HA titers were determined using chicken red blood cells (RBC) (29). Fifty percent egg infective dose (EID50) values for rNDVs were determined by infecting five eggs per group for each 10-fold serial dilution. Following 24 h of contamination, eggs were harvested for allantoic fluid, and the presence of computer virus was confirmed by an HA test. For HPAIV challenge viruses, the chicken 50% lethal dose (CLD50) was determined by infecting three.

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