Immune Responses Induced by Recombinant Bacillus Subtilis Expressing the Hemagglutinin Protein of H5N1 in chickens.
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ABSTRACT:
SUBMITTER: Mou C
PROVIDER: S-EPMC5159790 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:To develop a safe, effective, and convenient vaccine for the prevention of highly pathogenic avian influenza (HPAI), we have successfully constructed two recombinant lactobacillus strains (LA4356-pH and DLD17-pH) that express the foreign HPAI virus protein hemagglutinin 1 (HA(1)). The mucosal and systemic immune responses triggered by these two recombinant lactobacilli following oral administration to BALB/c mice were evaluated. The results showed that both LA4356-pH and DLD17-pH could significantly increase the specific anti-HA(1) IgA antibody level in the mucosa and the anti-HA(1) IgG level in serum, as well as stimulating the splenic lymphocyte proliferative reaction through increased expression of interleukin-4 (IL-4). Compared with LA4356-pH, DLD17-pH was more effective at inducing systemic and mucosal immune responses, with higher anti-HA(1)-specific IgA and IgG levels. Therefore, DLD17-pH could be a promising oral vaccine candidate against HPAI.
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Project description:Mycoplasma hyopneumoniae (M. hyopneumoniae) is the pathogen of swine enzootic pneumonia, a chronic respiratory disease affecting pigs of all ages. The ciliated epithelial cells of the respiratory tract are the main target invaded and colonized by M. hyopneumoniae. Therefore, the ideal vaccine would be mucosally administered and able to stimulate suitable mucosal immunity and prevent the adherence of pathogens to mucosal cell surfaces. Currently, Bacillus subtilis as a recombinant vaccine carrier has been used for antigen delivery and proved to be effectively enhancing the innate immunity of nasal mucosa. Here, our study attempts to construct recombinant Bacillus subtilis (B.S-P97R1, B.S-P46), which can express the P97R1 or P46 antigen of M. hyopneumoniae, and to evaluate the immune responses in BALB/c mice. Initially, we respectively successfully constructed recombinant B.S-P97R1, B.S-P46 and validated the expression of antigen proteins by Western analysis. Then, recombinant B.S-P97R1 or B.S-P46 were respectively intranasally (i.n.) immunized in mice. Both strong P97R1-specific and P46-specific immunoglobulin G (IgG), secretory immunoglobulin A (SIgA) antibodies were induced in sera, bronchoalveolar lavage fluids (BALs) by ELISA analysis. Moreover, the levels of specific IL-4, IFN-? in the immunized mice were elevated, and the proliferation of lymphocytes was also enhanced. In general, intranasal inoculation of recombinant B.S-P97R1 or B.S-P46 resulted in strong mucosal immunity, cell-mediated and humoral immunity, which was a mixed Th1/Th2-type response. In addition, our results provided a potential novel strategy that may be applied to the development of vaccines against M. hyopneumoniae.
| S-EPMC6822509 | biostudies-literature
Project description:Highly pathogenic avian influenza (HPAI) is a devastating disease of poultry and a serious threat to public health. Vaccination with inactivated virus vaccines has been applied for several years as one of the major policies to control highly pathogenic avian influenza virus (HPAIV) infections in chickens. Viral-vectored HA protein vaccines are a desirable alternative for inactivated vaccines. However, each viral vector possesses its own advantages and disadvantages for the development of a HA-based vaccine against HPAIV. Recombinant Newcastle disease virus (rNDV) strain LaSota expressing HA protein vaccine has shown promising results against HPAIV; however, its replication is restricted only to the respiratory tract. Therefore, we thought to evaluate avian paramyxovirus serotype 3 (APMV-3) strain Netherlands as a safe vaccine vector against HPAIV, which has high efficiency replication in a greater range of host organs. In this study, we generated rAPMV-3 expressing the HA protein of H5N1 HPAIV using reverse genetics and evaluated the induction of neutralizing antibodies and protection by rAPMV3 and rNDV expressing the HA protein against HPAIV challenge in chickens. Our results showed that immunization of chickens with rAPMV-3 or rNDV expressing HA protein provided complete protection against HPAIV challenge. However, immunization of chickens with rAPMV-3 expressing HA protein induced higher level of neutralizing antibodies compared to that of rNDV expressing HA protein. These results suggest that a rAPMV-3 expressing HA protein might be a better vaccine for mass-vaccination of commercial chickens in field conditions.
| S-EPMC7010735 | biostudies-literature
Project description:BackgroundPseudorabies caused by pseudorabies virus (PRV) mainly infects the swine and seriously threatens the biosafety of the other animals, including humans. Since 2011, the outbreaks of PRV mutants have caused enormous economic losses in the swine industry, and traditional vaccines cannot offer enough protection. PRV can transmit by direct contact, aerosol transmission and pollutants. PRV mainly transmit through the nasal mucosa. After infecting the nasal epithelial cells, PRV can quickly infect the olfactory nerve and establish a potential infection of sensory neurons. Therefore, nasal immunity can effectively prevent viral colonization infection. Recombinant Bacillus subtilis has been widely used to deliver antigen and achieve adequate protective immune responses.ResultsThe present study successfully constructed recombinant Bacillus subtilis (B. subtilis) expressing the dominant antigen regions of PRV gC and gD proteins (named B. subtilis-gCa and B. subtilis-gDa). Furtherly, we evaluated the immunogenicity of the two recombinant B. subtilis in mice. The mice intranasal administration with B. subtilis-gCa and B. subtilis-gDa effectively stimulated IgA and IgG immune responses, further regulated specific T lymphocytes proliferative response by IFN-? and IL-10, and ultimately produced high titers of neutralizing antibodies against PRV infection. In particular, B. subtilis-gDa possessed more excellent immune effect than B. subtilis-gCa in mice.ConclusionsThese results suggested that B. subtilis-gCa and B. subtilis-gDa could trigger high levels of mucosal and systemic immune responses and would be potential candidates for developing PRV vaccines.
| S-EPMC6555017 | biostudies-literature
Project description:Bacillus subtilis (B. subtilis) has a variety of proposed beneficial effects for chickens, including growth promotion and disease prevention. In this study, chickens were fed a diet containing B. subtilis for 21 days and growth performance, intestinal morphology, intestinal microbiota, immune responses, and disease resistance were investigated. After 21 days of feeding, chickens fed a diet containing B. subtilis had higher body weights. The concentrations of serum immunoglobulins IgA and IgM were significantly increased by B. subtilis in the diet. Moreover, chickens fed with B. subtilis had greater villus height (VH), shallower crypt depth (CD), and a higher VH/CD ratio in the jejunum than chickens fed a standard control diet. Diet with B. subtilis can balance intestinal microbiota, facilitate an increase in beneficial bacteria, and inhibit the pathogenic bacteria after 21 days of feeding. After an Escherichia coli (E. coli) challenge, the survival rate of chickens fed with B. subtilis was 66.67%, which was significantly higher than the controls. The E. coli contents in spleens and lungs from chickens fed a diet with B. subtilis were lower than those in controls. In addition, B. subtilis can trigger the toll-like receptor 4 and cause induction of proinflammatory cytokine (Il1β, Il6, and Il8) production to develop innate immune responses in chickens. In conclusion, diets containing B. subtilis can improve growth performance, serum immunoglobulin levels, the intestinal villus-crypt system, intestinal homeostasis, immune responses, and disease resistance against E. coli in chickens.
| S-EPMC7396511 | biostudies-literature
Project description:Recombinant baculoviruses with different promoter and regulatory elements were constructed to enhance the expression of target protein and boost the efficacies of avian influenza vaccine. Hemagglutinin gene was cloned into the baculovirus transfer vectors driven by cytomegaloviru (CMV) and White spot syndrome virus immediate-early promoter one (WSSV ie1) promoter respectively, with different regulatory elements. The recombinant baculoviruses were directly used as vaccines to immunize specific pathogen-free chickens. The protein expression levels of recombinant baculoviruses BV-S-HA and BV-S-ITRs-HA were respectively 2.43 and 2.67 times than that of BV-S-con-HA, while the protein expression levels of BV-A-HA and BV-A-ITRs-HA were respectively 2.44 and 2.69 times than that of BV-S-con-HA. Immunoglobulin G (IgG) antibody levels induced by BV-A and BV-S series recombinant baculovirus were significantly higher than the commercialized vaccine group (P < 0.05). Among the groups with same promoter, the IgG antibody levels induced by the baculovirus containing regulatory elements were significantly higher than control group. Additionally, the immune effects induced by BV-A series recombinant baculoviruses with WSSV ie1 promoter were significantly stronger than the BV-S series recombinant baculoviruses with CMV promoter. The avian influenza vaccine prepared based on baculovirus vector can simultaneously stimulate the humoral and cellular immune responses.
| S-EPMC4827022 | biostudies-other
Project description:BACKGROUND: The highly pathogenic avian influenza (HPAI) virus H5N1 causes multi-organ disease and death in poultry, resulting in significant economic losses in the poultry industry. In addition, it poses a major public health threat as it can be transmitted directly from infected poultry to humans with very high (60%) mortality rate. Effective vaccination against HPAI H5N1 would protect commercial poultry and would thus provide an important control measure by reducing the likelihood of bird-to-bird and bird-to-human transmission. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we evaluated the vaccine potential of recombinant soluble trimeric subtype 5 hemagglutinin (sH5(3)) produced in mammalian cells. The secreted, purified sH5(3) was biologically active as demonstrated by its binding to ligands in a sialic acid-dependent manner. It was shown to protect chickens, in a dose-dependent manner, against a lethal challenge with H5N1 after a single vaccination. Protected animals did not shed challenge virus as determined by a quantitative RT-PCR on RNA isolated from trachea and cloaca swabs. Also in mice, vaccination with sH5(3) provided complete protection against challenge with HPAI H5N1. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that sH5(3) constitutes an attractive vaccine antigen for protection of chickens and mammals against HPAI H5N1. As these recombinant soluble hemagglutinin preparations can be produced with high yields and with relatively short lead time, they enable a rapid response to circulating and potentially pandemic influenza viruses.
| S-EPMC2871037 | biostudies-literature
Project description:BACKGROUND:Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome, and is associated with a number of other diseases. PCV2 is widely distributed in most developed swine industries, and is a severe economic burden. With an eye to developing an effective, safe, and convenient vaccine against PCV2-associated diseases, we have constructed a recombinant Bacillus subtilis strain (B. subtilis-Cap) that expresses the PCV2 capsid protein (Cap). METHODS:Electroporation of a plasmid shuttle vector encoding the PCV2 Cap sequence was use to transform Bacillus subtilis. Flow cytometry was used to evaluate in vitro bone marrow derived dendritic cell (BM-DC) maturation and T cell proliferation induced by B. subtilis-Cap. Orally inoculated piglets were used for in vivo experiments; ELISA and western blotting were used to evaluate B. subtilis-Cap induced PCV2-specific IgA and IgG levels, as well as the secretion of cytokines and the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 9 (TLR9). RESULTS:We evaluated the immune response to B. subtilis-Cap in vitro using mouse BM-DCs and in vivo using neonatal piglets orally inoculated with B. subtilis-Cap. Our results showed that the recombinant B. subtilis-Cap activated BM-DCs, significantly increased co-stimulatory molecules (CD40 and CD80) and major histocompatibility complex II, and induced allogenic T cells proliferation. Piglets immunized with B. subtilis-Cap had elevated levels of PCV2-specific IgA in the mucosal tissues of the digestive and respiratory tract, and PCV2-specific IgG in serum (P?<?0.05 or P?<?0.01). Ileal immunocompetent cells, such as the IgA-secreting cells (P <?0.01), intestinal intraepithelial lymphocytes (IELs) (P?<?0.01), CD3+ T lymphocytes (P <?0.01) and CD4+ T lymphocytes (P?<?0.01) increased significantly in the B. subtilis-Cap immunized piglets. Additionally, B. subtilis-Cap inoculation resulted in increased the expression of TLR2 and TLR9 (P <?0.01), and induced the secretion of cytokines IL-1?, IL-6, interferon-?, and ?-defensin 2 (P <?0.01). CONCLUSIONS:We constructed a prototype PCV2 vaccine that can be administered orally and elicits a more robust humoral and cellular immunity than inactivated PCV2. B. subtilis-Cap is a promising vaccine candidate that is safe, convenient, and inexpensive. Further in vivo research is needed to determine its full range of efficacy in pigs.
| S-EPMC7014726 | biostudies-literature
Project description:Previously we demonstrated the versatile utility of the Parapoxvirus Orf virus (ORFV) as a vector platform for the development of potent recombinant vaccines. In this study we present the generation of new ORFV recombinants expressing the hemagglutinin (HA) or nucleoprotein (NP) of the highly pathogenic avian influenza virus (HPAIV) H5N1. Correct foreign gene expression was examined in vitro by immunofluorescence, Western blotting and flow cytometry. The protective potential of both recombinants was evaluated in the mouse challenge model. Despite adequate expression of NP, the recombinant D1701-V-NPh5 completely failed to protect mice from lethal challenge. However, the H5 HA-expressing recombinant D1701-V-HAh5n mediated solid protection in a dose-dependent manner. Two intramuscular (i.m.) injections of the HA-expressing recombinant protected all animals from lethal HPAIV infection without loss of body weight. Notably, the immunized mice resisted cross-clade H5N1 and heterologous H1N1 (strain PR8) influenza virus challenge. In vivo antibody-mediated depletion of CD4-positive and/or CD8-posititve T-cell subpopulations during immunization and/or challenge infection implicated the relevance of CD4-positive T-cells for induction of protective immunity by D1701-V-HAh5n, whereas the absence of CD8-positive T-cells did not significantly influence protection. In summary, this study validates the potential of the ORFV vectored vaccines also to combat HPAIV.
| S-EPMC3869816 | biostudies-literature
Project description:BACKGROUND: Highly pathogenic avian influenza virus (HPAIV) causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans. One effective way to combat avian influenza with pandemic potential is through the vaccination of poultry. Several live vaccines based on attenuated Newcastle disease virus (NDV) that express influenza hemagglutinin (HA) have been developed to protect chickens or mammalian species against HPAIV. However, the zoonotic potential of NDV raises safety concerns regarding the use of live NDV recombinants, as the incorporation of a heterologous attachment protein may result in the generation of NDV with altered tropism and/or pathogenicity. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we generated recombinant NDVs expressing either full length, membrane-anchored HA of the H5 subtype (NDV-H5) or a soluble trimeric form thereof (NDV-sH5(3)). A single intramuscular immunization with NDV-sH5(3) or NDV-H5 fully protected chickens against disease after a lethal challenge with H5N1 and reduced levels of virus shedding in tracheal and cloacal swabs. NDV-sH5(3) was less protective than NDV-H5 (50% vs 80% protection) when administered via the respiratory tract. The NDV-sH5(3) was ineffective in mice, regardless of whether administered oculonasally or intramuscularly. In this species, NDV-H5 induced protective immunity against HPAIV H5N1, but only after oculonasal administration, despite the poor H5-specific serum antibody response it elicited. CONCLUSIONS/SIGNIFICANCE: Although NDV expressing membrane anchored H5 in general provided better protection than its counterpart expressing soluble H5, chickens could be fully protected against a lethal challenge with H5N1 by using the latter NDV vector. This study thus provides proof of concept for the use of recombinant vector vaccines expressing a soluble form of a heterologous viral membrane protein. Such vectors may be advantageous as they preclude the incorporation of heterologous membrane proteins into the viral vector particles.
| S-EPMC3429475 | biostudies-literature