Inactivated vaccine with adjuvants consisting of pattern recognition receptor agonists confers protection against avian influenza viruses in chickens.
ABSTRACT: Use of adjuvant containing pathogen pattern recognition receptor agonists is one of the effective strategies to enhance the efficacy of licensed vaccines. In this study, we investigated the efficacy of avian influenza vaccines containing an adjuvant (CVCVA5) which was composed of polyriboinosinic polyribocytidylic, resiquimod, imiquimod, muramyl dipeptide and levomisole. Avian influenza vaccines adjuvanted with CVCVA5 were found to induce significantly higher titers of hemagglutiniton inhibition antibodies (P?0.01) than those of commercial vaccines at 2-, 3- and 4-week post vaccination in both specific pathogen free (SPF) chickens and field application. Furthermore, virus shedding was reduced in SPF chickens immunized with H9-CVCVA5 vaccine after H9 subtype heterologous virus challenge. The ratios of both CD3(+)CD4(+) and CD3(+)CD8(+) lymphocytes were slowly elevated in chickens immunized with H9-CVCVA5 vaccine. Lymphocytes adoptive transfer study indicates that CD8(+) T lymphocyte subpopulation might have contributed to improved protection against heterologous virus challenge. Results of this study suggest that the adjuvant CVCVA5 was capable of enhancing the potency of existing avian influenza vaccines by increasing humoral and cellular immune response.
Project description:In the present study, virus-like particles (VLPs) were evaluated as a candidate poultry vaccine against avian influenza virus (AIV) subtype H9N2. Specific pathogen-free chickens received a single injection of the VLP vaccine expressing HA and M1 protein of AIV H9N2 (H9 HA VLP) at escalating doses in the presence or absence of ISA70 water-in-oil adjuvant. At 3 weeks post vaccination, we performed hemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) to determine serological immune responses, and challenge studies using SPF chickens. A single dose of H9 HA VLP vaccine induced high levels of HI antibodies and lowered frequencies of virus isolation after the wild-type virus challenge. The addition of ISA70 adjuvant significantly increased the immunogenicity of H9 HA VLP vaccines. Furthermore, it allows differentiation of AIV-infected chickens from vaccinated chickens with an ELISA using nucleocapsid antigen, which offers a promising strategy to differentiate infected from vaccinated animals (DIVA). These results provide support for continued development of the VLP as an animal vaccine against influenza virus.
Project description:Current commercial H9 avian influenza vaccines cannot provide satisfactory protective immunity against antigenic variant influenza viruses in ducks. Poly I:C, when used as an adjuvant, improves humoral and cellular immunity in many animals but has not been tested in ducks. In this study, we investigated the protective efficacy of Poly I:C as an adjuvant for an inactivated H9N2 Avian influenza vaccine in ducks. We found that an H9N2 vaccine administered with poly I:C (H9-PIC vaccine) induced a significantly more rapid response with higher anti-influenza antibody titers than those of the vaccine alone (H9 vaccine). Moreover, virus shedding was reduced in ducks immunized with the H9-PIC vaccine after challenge with an H9 subtype antigenic variant viruses. IFN-?, IFN-?, IL-6 and MHC-II mRNA levels were all elevated in ducks receiving the H9-PIC vaccine. In addition, lower expression level of MHC-I may be a reason for inefficient protective ability against heterologous influenza viruses in H9-PIC vaccination of ducks. In conclusion, poly I:C adjuvant enhanced both humoral and cellular immune responses in ducks induced by immunization of inactivated H9N2 vaccine.
Project description:Avian influenza viruses represent a growing threat of an influenza pandemic. The co-circulation of multiple H9N2 genotypes over the past decade has been replaced by one predominant genotype-G57 genotype, which displays a changed antigenicity and improved adaptability in chickens. Effective H9N2 subtype avian influenza virus vaccines for poultry are urgently needed.In this study, we constructed H9N2 subtype avian influenza virus-like particle (VLP) and evaluated its protective efficacy in specific pathogen-free (SPF) chickens to lay the foundation for developing an effective vaccine against influenza viruses.Expression of influenza proteins in VLPs was confirmed by Western blot, hemagglutination inhibition (HI), and neuraminidase inhibition (NI). The morphology was observed by electron microscopy. A group of 15 three-week-old SPF chickens was divided into three subgroups of five chickens immunized with VLP, commercial vaccine, and PBS. Challenge study was performed to evaluate efficacy of VLP vaccine.The hemagglutinin (HA) and neuraminidase (NA) proteins were co-expressed in the infected cells, self-assembled, and were released into the culture medium in the form of VLPs of diameter ~80 nm. The VLPs exhibited some functional characteristics of a full influenza virus, including hemagglutination and neuraminidase activity. In SPF chickens, the VLPs elicited serum antibodies specific for H9N2 and induced a higher HI titer (as detected by a homologous antigen) than did a commercial H9N2 vaccine (A/chicken/Shanghai/F/1998). Viral shedding from VLP vaccine subgroup was reduced compared with commercial vaccine subgroup and control subgroup.
Project description:BACKGROUND:H9N2 Low pathogenic avian influenza virus (LPAIV) raises public health concerns and its eradication in poultry becomes even more important in preventing influenza. AJSAF is a purified active saponin fraction from the stem bark of Albizzia julibrissin. In this study, AJSAF was evaluated for the adjuvant potentials on immune responses to inactivated H9N2 avian influenza virus vaccine (IH9V) in mice and chicken in comparison with commercially oil-adjuvant. RESULTS:AJSAF significantly induced faster and higher H9 subtype avian influenza virus antigen (H9-Ag)-specific IgG, IgG1, IgG2a and IgG2b antibody titers in mice and haemagglutination inhibition (HI) and IgY antibody levels in chicken immunized with IH9V. AJSAF also markedly promoted Con A-, LPS- and H9-Ag-stimulated splenocyte proliferation and natural killer cell activity. Furthermore, AJSAF significantly induced the production of both Th1 (IL-2 and IFN-?) and Th2 (IL-10) cytokines, and up-regulated the mRNA expression levels of Th1 and Th2 cytokines and transcription factors in splenocytes from the IH9V-immunized mice. Although oil-formulated inactivated H9N2 avian influenza vaccine (CH9V) also elicited higher H9-Ag-specific IgG and IgG1 in mice and HI antibody titer in chicken, this robust humoral response was later produced. Moreover, serum IgG2a and IgG2b antibody titers in CH9V-immunized mice were significantly lower than those of IH9V alone group. CONCLUSIONS:AJSAF could improve antigen-specific humoral and cellular immune responses, and simultaneously trigger a Th1/Th2 response to IH9V. AJSAF might be a safe and efficacious adjuvant candidate for H9N2 avian influenza vaccine.
Project description:In order to produce a dually effective vaccine against H9 and H5 avian influenza viruses that aligns with the DIVA (differentiating infected from vaccinated animals) strategy, we generated a chimeric H9/H5N2 recombinant vaccine that expressed the whole HA1 region of A/CK/Korea/04163/04 (H9N2) and the HA2 region of recent highly pathogenic avian influenza (HPAI) A/MD/Korea/W452/14 (H5N8) viruses. The chimeric H9/H5N2 virus showed in vitro and in vivo growth properties and virulence that were similar to those of the low-pathogenic avian influenza (LPAI) H9 virus. An inactivated vaccine based on this chimeric virus induced serum neutralizing (SN) antibodies against both H9 and H5 viruses but induced cross-reactive hemagglutination inhibition (HI) antibody only against H9 viruses. Thus, this suggests its compatibility for use in the DIVA strategy against H5 strains. Furthermore, the chimeric H9/H5N2 recombinant vaccine protected immunized chickens against lethal challenge by HPAI H5N8 viruses and significantly attenuated virus shedding after infection by both H9N2 and HPAI H5N8 viruses. In mice, serological analyses confirmed that HA1- and HA2 stalk-specific antibody responses were induced by vaccination and that the DIVA principle could be employed through the use of an HI assay against H5 viruses. Furthermore, each HA1- and HA2 stalk-specific antibody response was sufficient to inhibit viral replication and protect the chimeric virus-immunized mice from lethal challenge with both mouse-adapted H9N2 and wild-type HPAI H5N1 viruses, although differences in vaccine efficacy against a homologous H9 virus (HA1 head domain immune-mediated protection) and a heterosubtypic H5 virus (HA2 stalk domain immune-mediated protection) were observed. Taken together, these results demonstrate that the novel chimeric H9/H5N2 recombinant virus is a low-pathogenic virus, and this chimeric vaccine is suitable for a DIVA vaccine with broad-spectrum neutralizing antibody against H5 avian influenza viruses.IMPORTANCE Current influenza virus killed vaccines predominantly induce antihemagglutinin (anti-HA) antibodies that are commonly strain specific in that the antibodies have potent neutralizing activity against homologous strains but do not cross-react with HAs of other influenza virus subtypes. In contrast, the HA2 stalk domain is relatively well conserved among subtypes, and recently, broadly neutralizing antibodies against this domain have been isolated. Therefore, in light of the need for a vaccine strain that applies the DIVA strategy utilizing an HI assay and induces broad cross-protection against H5N1 and H9N2 viruses, we generated a novel chimeric H9/H5N1 virus that expresses the entire HA1 portion from the H9N2 virus and the HA2 region of the heterosubtypic H5N8 virus. The chimeric H9/H5N2 recombinant vaccine protected immunized hosts against lethal challenge with H9N2 and HPAI H5N1 viruses with significantly attenuated virus shedding in immunized hosts. Therefore, this chimeric vaccine is suitable as a DIVA vaccine against H5 avian influenza viruses.
Project description:Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.
Project description:In order to produce an efficient poultry H9 avian influenza vaccine that provides cross-protection against multiple H9 lineages, two Newcastle disease virus (NDV) LaSota vaccine strain recombinant viruses were generated using reverse genetics. The recombinant NDV-H9Con virus expresses a consensus-H9 hemagglutinin (HA) that is designed based on available H9N2 sequences from Chinese and Middle Eastern isolates. The recombinant NDV-H9Chi virus expresses a chimeric-H9 HA in which the H9 ectodomain of A/Guinea Fowl/Hong Kong/WF10/99 was fused with the cytoplasmic and transmembrane domain of the fusion protein (F) of NDV. Both recombinant viruses expressed the inserted HA stably and grew to high titers. An efficacy study in chickens showed that both recombinant viruses were able to provide protection against challenge with a heterologous H9N2 virus. In contrast to the NDV-H9Chi virus, the NDV-H9Con virus induced a higher hemagglutination inhibition titer against both NDV and H9 viruses in immunized birds, and efficiently inhibited virus shedding through the respiratory route. Moreover, sera collected from birds immunized with either NDV-H9Con or NDV-H9Chi were able to cross-neutralize two different lineages of H9N2 viruses, indicating that NDV-H9Con and NDV-H9Chi are promising vaccine candidates that could provide cross-protection among different H9N2 lineage viruses.
Project description:There is an urgent need for identification of a new adjuvant capable of selectively promoting an efficient immune response for use with vaccines and especially subunit vaccines. Our pervious study showed that Bursopentine (BP5) is a novel immunomodulatory peptide and has the ability to significantly stimulate an antigen-specific immune response in mice. In this study, the potential adjuvant activities of BP5 were examined in chickens by coinjection of BP5 and an inactivated avian influenza virus (AIV) (A/Duck/Jiangsu/NJ08/05 [AIV H9N2 subtype]). The results suggested that BP5 markedly elevated serum hemagglutination inhibition (HI) titers and antigen-specific antihemagglutinin (anti-HA) antibody (IgG) levels, induced both Th1 (interleukin 2 [IL-2] and gamma interferon [IFN-?])- and Th2 (IL-4)-type cytokines, promoted the proliferation of peripheral blood lymphocytes, and increased populations of CD3(+) T cells and their subsets CD4(+) (CD3(+) CD4(+)) T cells and CD8(+) (CD3(+) CD8(+)) T cells. Furthermore, a virus challenge experiment revealed that BP5 contributes to protection against homologous avian influenza virus challenge by reducing viral replication in chicken lungs. This study indicates that the combination of inactivated AIVs and BP5 gives a strong immune response at both the humoral and cellular levels and implies that BP5 is a novel immunoadjuvant suitable for vaccine design.
Project description:H5N1 highly pathogenic avian influenza (HPAI) was one of the most important avian diseases in poultry production of China, especially in Guangdong province. In recent years, new H5N1 highly pathogenic avian influenza viruses (HPAIV) still emerged constantly, although all poultry in China were immunized with H5N1 vaccinations compulsorily. To better understand the pathogenicity and transmission of dominant clades of the H5N1 HPAIVs in chicken from Guangdong in 2012, we chose a clade 7.2 avian influenza virus named A/Chicken/China/G2/2012(H5N1) (G2) and a clade 188.8.131.52 avian influenza virus named A/Duck/China/G3/2012(H5N1) (G3) in our study. Our results showed that the chickens inoculated with 10(3) EID50 of G2 or G3 viruses all died, and the titers of virus replication detected in several visceral organs were high but different. In the naive contact groups, virus shedding was not detected in G2 group and all chickens survived, but virus shedding was detected in G3 group and all chickens died. These results showed that the two clades of H5N1 HPAIVs had high pathogenicity in chickens and the contact transmission of them was different in chickens. The results of cross reactive HI assay showed that antigens of G2 and G3 were very different from those of current commercial vaccines isolates (Re-4, Re-6, and D7). And to evaluate the protective efficacy of three vaccines against most isolates form Guangdong belonging to clade 184.108.40.206 in 2012, G3 was chosen to challenge the three vaccines such as Re-4, Re-6, and D7. First, chickens were immunized with 0.3 ml Re-4, Re-6, and D7 inactivated vaccines by intramuscular injection, respectively, and then challenged with 10(6) EID50 of G3 on day 28 post-vaccination. The D7 vaccine had 100% protection against G3 for chickens, the Re-6 vaccine had 88.9%, and the Re-4 vaccine only had 66.7%. Our results suggested that the D7 vaccine could prevent and control H5N1 virus outbreaks more effectively in Guangdong. From the above, it was necessary to conduct continuously epidemiological survey and study the pathogenicity and antigenic variation of avian influenza in Southern China.
Project description:The methods of repeated immunization with inactivated vaccines have been used widely to increase antibody protection against infectious bronchitis virus (IBV). However, compared with DNA vaccines, these methods usually induce poor cellular responses. In the present study, specific pathogen-free (SPF) chickens were immunized intramuscularly with a DNA vaccine carrying the main IBV structural genes (pVAX1-S1, pVAX1-M, and pVAX1-N, respectively) and boosted with the IBV M41 strain inactivated vaccine to assess whether such a new strategy could enhance the immune responses against IBV. The protection efficacy of the DNA vaccine carrying different structural genes for priming was evaluated further. The chickens were immunized primely on day 7 and boosted 2 weeks later. After that, distribution of the DNA vaccine in vivo, the percentage of CD4+CD3+ and CD8+CD3+ subgroups of peripheral blood T-lymphocytes, and the specific IgG and virus neutralizing antibodies were measured. Chickens were then challenged by the nasal-ocular route with the IBV M41 strain 4 weeks after booster immunization. The results demonstrated that priming with a DNA vaccine encoding nucleocapsid protein (pVAX1-N) and boosting with the inactivated IBV vaccine led to the dramatic augmentation of humoral and cellular responses, and provided up to 86.7% rate of immune protection, providing an effective approach to protect chickens from IBV.