Liposomes containing recombinant gp85 protein vaccine against ALV-J in chickens

To study the potential of liposome vaccines in the clinical prevention of ALV-J, the effect of recombinant gp85 protein of subgroup J avian leukosis virus (ALV-J) entrapped by liposomes in chickens against ALV-J infection was investigated in this paper. A recombinant plasmid (PET28a-gp85) containing the PET28a vector and gp85 gene was constructed and then expressed in Rosetta (DE3) cells with 0.5 mM IPTG to produce recombinant gp85 proteins that could be entrapped by liposomes through reverse-phase evaporation. The chickens were inoculated intramuscularly either once or twice with the liposomes or with Freund's adjuvant emulsion containing recombinant gp85 protein. Sixty chickens were raised to one week old for the first inoculation and to three weeks old for the second inoculation. Chickens raised to five weeks old were challenged with a 10^2.4 50% tissue culture infective dose (TCID₅₀) of ALV-J. Blood samples were collected from each chicken at weekly intervals for serum antibody and viremia analyses. Changes in serum antibodies showed that positive serum antibodies (S/P value >0.6) could be induced in all groups regardless of the frequency of inoculation but improved significantly in the twice-inoculated groups. As well, high levels of antibodies emerged earlier in the Freund's adjuvant groups but persisted longer in the liposome groups. Detection of viremia indicated that the liposomes provide better protection against ALV-J than Freund's adjuvant emulsion and that this protection is directly influenced by serum antibody levels. Overall, this study reveals the potential of liposome vaccines containing recombinant gp85 protein in the clinical prevention of ALV-J.     

Cooperative effects of immune enhancer TPPPS and different adjuvants on antibody responses induced by recombinant ALV-J gp85 subunit vaccines in SPF chickens

To explore the antibody responses and protective effects induced by subgroup J avian leukosis virus (ALV-J) gp85 protein vaccine plus different adjuvants (CpG and white oil adjuvant YF01) combined with the immune enhancer Taishan Pinus massoniana pollen polysaccharide (TPPPS), we immunized SPF chickens with the recombinant ALV-J gp85 protein, along with different adjuvants and immune enhancer, which protected the chickens by inducing different levels of protective antibodies. Results showed that a single injection of gp85 recombinant protein could only produce low-titre antibodies that were maintained over a short time in few chickens. When combined with YF01 or CpG adjuvants, the recombinant protein could induce high-titre antibodies in most of the immunized chickens. Moreover, when the immune enhancer TPPPS was used with the two adjuvants, it further elevated the antibody levels for a longer duration. The eggs from four groups with the highest levels of ALV-J antibodies were collected, hatched, and examined for maternal antibodies. The protection by the maternal antibodies against ALV-J infection in the TPPPS-immunized group was higher than that in the group without TPPPS, which was consistent with the observations in the parents. This study shows that the immune enhancer TPPPS, combined with YF01 or CpG adjuvants, can enhance the immunogenicity of gp85 recombinant proteins, and provide a better immuno-protection. It provides a powerful experimental basis for the development of ALV-J subunit vaccine. Efficient subunit vaccine development will also accelerate the process of purification of ALV-J.     

Recombinant gp90 protein expressed in Pichia pastoris induces a protective immune response against reticuloendotheliosis virus in chickens

Reticuloendotheliosis virus (REV) causes an oncogenic, immunosuppressive and runting syndrome in multiple avian hosts worldwide. In this study, the gp90 protein of REV was secretory expressed in Pichia pastoris with high production level and good antigenicity. To fully utilize the expression potential of the P. pastoris expression system, a panel of Pichia clones carrying increasing copies of the gp90 expression cassette was created using an in vitro multimerization approach and the effects of gene dosage on gp90 expression were investigated. Results demonstrated that an increase in gp90 copy number can significantly improve the yields of gp90 protein. Following expression and scale-up, the gp90 protein production level could reach up to 400mg/L, and the protein could be detected by gp90-specific monoclonal antibody. Investigations of its vaccine efficacy demonstrated that the recombinant gp90 protein was able to induce sustained high levels of antibodies against REV as being detected by ELISA and virus neutralizing test. Furthermore, immunization of chickens with the recombinant gp90 vaccine fully protected the animals from viremia after REV infection. Overall, the yeast-expressed gp90 protein retains good immunogenicity and could be used as a potential subunit vaccine candidate for REV prevention.     

Immunization with oral and parenteral subunit chimeric vaccine candidate confers protection against Necrotic Enteritis in chickens

Following the ban on the use of in-feed antimicrobials, necrotic enteritis (NE) NE is the most important clostridial disease. Vaccination has been considered as a possible approach to prevent NE. Our previous study showed that a chimeric protein product consisting of antigenic epitopes of NetB, Alpha-toxin and Zinc metallopeptidase (Zmp) triggered immune response against C. perfringens. In the current study we optimized the chimeric gene and constructed a fusion protein containing NetB, Alpha-toxin and Metallopeptidase (NAM) for expressing in tobacco plant to use as an edible vaccine for immunizing the chicken against NE. Simultaneously, we expressed and purified a His-tagged recombinant version of the NAM (rNAM) expressed in E. coli BL21 for subcutaneous immunization of chickens. Immunized birds produced strong humoral immune responses against both edible plant-based and parenteral purified rNAM.The responses were determined by the mean titer of antibody in blood samples to be around 9000 and 32,000, for edible and injected rNAM, respectively. Birds immunized subcutaneously showed the most striking responses. However the edible vaccine provided a more long lasting IgY response 14 days after the third vaccination compared to the injected birds. Chickens immunized with either lyophilized leaves expressing rNAM or purified rNAM, subsequently were subjected to the challenge with a virulent C. perfringens strain using an NE disease model. Our results showed that birds immunized both parenterally and orally with recombinant chimeric vaccine were significantly protected against the severity of lesion in the intestinal tract, but the protection provided with the injectable form of the antigen was greater than that of the oral form. Further analysis is needed to check whether these strategies can be used as the potential platform for developing an efficient vaccine against NE.     

The Ag85B protein of the BCG vaccine facilitates macrophage uptake but is dispensable for protection against aerosol Mycobacterium tuberculosis infection

Defining the function and protective capacity of mycobacterial antigens is crucial for progression of tuberculosis (TB) vaccine candidates to clinical trials. The Ag85B protein is expressed by all pathogenic mycobacteria and is a component of multiple TB vaccines under evaluation in humans. In this report we examined the role of the BCG Ag85B protein in host cell interaction and vaccine-induced protection against virulent Mycobacterium tuberculosis infection. Ag85B was required for macrophage infection in vitro, as BCG deficient in Ag85B expression (BCG:^Δ85B) was less able to infect RAW 264.7 macrophages compared to parental BCG, while an Ag85B-overexpressing BCG strain (BCG:^oex85B) demonstrated improved uptake. A similar pattern was observed in vivo after intradermal delivery to mice, with significantly less BCG:^Δ85B present in CD64^hiCD11b^hi macrophages compared to BCG or BCG:^oex85B. After vaccination of mice with BCG:^Δ85B or parental BCG and subsequent aerosol M. tuberculosis challenge, similar numbers of activated CD4⁺ and CD8⁺ T cells were detected in the lungs of infected mice for both groups, suggesting the reduced macrophage uptake observed by BCG:^Δ85B did not alter host immunity. Further, vaccination with both BCG:^Δ85B and parental BCG resulted in a comparable reduction in pulmonary M. tuberculosis load. These data reveal an unappreciated role for Ag85B in the interaction of mycobacteria with host cells and indicates that single protective antigens are dispensable for protective immunity induced by BCG.     

Immunogenicity and therapeutic effects of recombinant Ag85AB fusion protein vaccines in mice infected with Mycobacterium tuberculosis

The immune function of tuberculosis (TB) patients is disordered. By using immune regulators to assist chemotherapy for TB the curative effect might be improved. In this study, a vaccine containing Mycobacterium tuberculosis (M. tuberculosis) recombinant Ag85AB fusion protein (rAg85AB) was constructed and evaluated. The mice were immunized intramuscularly three times at two-week intervals with Ag85AB fusion protein combined with Corynebacterium parvum adjuvant (rAg85AB+CP). In comparison to control mice that received either CP alone or saline, the mice that received rAg85AB+CP had significantly higher number of T cells secreting IFN-γ and higher levels of specific antibodies of IgG, IgG1 and IgG2a isotypes in sera. The specific antibodies also had higher ratios of IgG2a to IgG1, indicating a predominant Th1 immune response. To test for immunotherapy of TB, M. tuberculosis infected mice were given three intramuscular doses of 20 μg, 40 μg or 60 μg of rAg85AB in rAg85AB+CP, or phosphate-buffered saline (PBS), or CP or Mycobacterium phlei (M. Phlei) F.U.36. Compared with the PBS group, 20 µg, 40 µg and 60 µg rAg85AB+CP and M. phlei F.U.36 groups reduced the pulmonary bacterial loads by 0.13, 0.15, 0.42 and 0.40 log₁₀, and the liver bacterial loads by 0.64, 0.64, 0.53 and 0.61 log₁₀, respectively. Pathological changes of lungs were less, and the lesions were limited to a certain extent in 40 µg and 60 µg rAg85AB+CP and M. phlei F.U.36 groups. These results showed that rAg85AB+CP had immunotherapeutic effect on TB, significantly increasing the cellular immune response, and inhibiting the growth of M. tuberculosis.     

重组表达HIV不同亚型gp41蛋白的抗原表位暴露情况分析

目的通过化学发光法检测原核重组HIV I型4种不同亚型gp41膜外区蛋白对阴阳性标本的反应性,间接反映重组表达gp41膜外区蛋白的抗原表位暴露情况,为科研、临床异常标本分析提供分析方法。方法选取HIV I型4个国内主流基因型(BC、AE、B、C)膜外区序列,克隆至pGEX4T-3载体中,优化目标蛋白的纯化工艺,制备出HIV I型gp41膜外区相应蛋白作为包被抗原,与商品化gp41-HRP酶结合物配对后,夹心法进行HIV I型阴阳性标本反应性的测试,从而判断相应蛋白抗原表位暴露情况。结果HIV I型4个亚型的gp41膜外区相同区段在pGEX4T-3原核载体中均能表达出符合预期大小的目的蛋白;纯化后分别作为包被抗原,按相同浓度包被后进行HIV I型阴阳性标本的测试,结果显示不同亚型的gp41表达蛋白与商品化gp41-HRP酶反应性存在较大差异。结论通过原核表达系统高效表达了HIV I型主流亚型gp41膜外区蛋白,化学发光法组建夹心法进行阴阳性标本测试,其反应性不一致,间接反映出不同基因型选取同一表达区域进行体外重组表达其结构折叠方式有区别,此研究为HIV科研、临床标本验证等提供了基础。     

Phosphatidylinositol di-mannoside and derivates modulate the immune response to and efficacy of a tuberculosis protein vaccine against Mycobacterium bovis infection

Mycobacterium bovis infects a wide range of hosts, including domestic livestock, wildlife, and humans. Development of an effective vaccine protecting against bovine tuberculosis would provide a cost-effective tuberculosis control strategy. The objective of this study was to investigate the ability of phosphatidylinositol di-mannoside (PIM₂) and its derivatives to modulate cell-mediated immunity in vivo in a bovine tuberculosis mouse model in response to a relevant antigen, namely a fusion protein of mycobacterial proteins Ag85A and ESAT-6. The addition of synthetic PIM₂ to the vaccine resulted in a significant reduction in lung bacterial counts and a cytokine profile indicating a Th 1 type immune response. The addition of the other PIM₂ derivatives to the vaccine or the fusion protein alone did not result in reduced lung bacterial counts; moreover, the addition of PIM₂ME appeared to negate the induction of an antigen-specific interferon-γ response and protection against tuberculosis. In conclusion, this study provides further evidence that PIMs can function as potent adjuvants for protein or sub-unit vaccines, but subtle structural differences among PIMs can markedly alter the type of immune response induced.     

Matrix-M™ adjuvanted envelope protein vaccine protects against lethal lineage 1 and 2 West Nile virus infection in mice

West Nile virus (WNV) is a mosquito-transmitted flavivirus and an emerging pathogen in many parts of the world. In the elderly and immunosuppressed, infection can progress rapidly to debilitating and sometimes fatal neuroinvasive disease. Currently, no WNV vaccine is approved for use in humans. As there have been several recent outbreaks in the United States and Europe, there is an increasing need for a human WNV vaccine. In this study, we formulated the ectodomain of a recombinant WNV envelope (E) protein with the particulate saponin-based adjuvant Matrix-M™ and studied the antigen-specific immune responses in mice. Animals immunized with Matrix-M™ formulated E protein developed higher serum IgG1 and IgG2a and neutralizing antibody titers at antigen doses ranging from 0.5 to 10 μg compared to those immunized with 3 or 10 μg of E alone, E adjuvanted with 1% Alum, or with the inactivated virion veterinary vaccine, Duvaxyn^® WNV. This phenotype was accompanied by strong cellular recall responses as splenocytes from mice immunized with Matrix-M™ formulated vaccine produced high levels of Th1 and Th2 cytokines. Addition of Matrix-M™ prolonged the duration of the immune response, as elevated humoral and cellular responses were maintained for more than 200 days. Importantly, mice vaccinated with Matrix-M™ formulated E protein were protected from lethal challenge with both lineage 1 and 2 WNV strains. In summary, Matrix-M™ adjuvanted E protein elicited potent and durable immune responses that prevented lethal WNV infection, and thus is a promising vaccine candidate for humans.     

Immunogenicity and protective efficacy of a tuberculosis DNA vaccine expressing a fusion protein of Ag85B-Esat6-HspX in mice

Tuberculosis remains a major infectious disease worldwide due to the low efficacy of available vaccine of the Mycobacterium bovis Bacillus Calmette-Guérin (BCG). DNA vaccines are especially promising candidates; however, the efficacy of DNA vaccine expressing single antigen of Mycobacterium tuberculosis (MTb) is limited. In this study, a plasmid DNA vaccine, pAEH, was constructed and designed to express a fusion protein of the Ag85B, Esat6, and HspX of MTb. Its immunogenicity and protective efficacy as well as therapeutic effect were assessed in a mouse model of tuberculosis. Vaccination with the pAEH significantly increased the frequency of peripheral blood CD4(+) and CD8(+) T cells, but not γδT cells, similar to that of vaccination with the BCG, and induced significantly higher levels of HspX-specific T cell proliferation, as compared with vaccination with BCG or the pHspX. Furthermore, vaccination with the pAEH increased the frequency of Ag85B, Esat6 and HspX-specific IFNγ-secreting T cells, accompanied by significantly higher levels of IFN-γ and IL-2 production ex vivo, as compared with that of the BCG or pHspX-vaccinated mice. Apparently, vaccination with the pAEH induced potent Th1 responses in mice. More importantly, vaccination with the pAEH inhibited the replication of virulent MTb in the lungs and spleens, even after MTb infection, and related lung inflammation in mice. Potentially, the newly developed pAEH vaccine may be used for the prevention and therapeutic intervention of MTb infection.     

Protective immune response against Toxoplasma gondii elicited by a novel yeast-based vaccine with microneme protein 16

Toxoplasma gondii is an obligate intracellular protozoan that can invade all eukaryotic cells and infect all warm-blood animals, causing the important zoonosis toxoplasmosis. Invasion of host cells is the key step necessary for T. gondii to complete its life cycle and microneme proteins play an important role in attachment and invasion of host cells. Microneme protein 16 (TgMIC16) is a new protective protein in T. gondii and belongs to transmembrane microneme proteins (TM-MIC). The TM-MICs are released onto the parasite’s surface as complexes capable of interacting with host cell receptors. In the present study, we expressed the TgMIC16 protein on the surface of Saccharomyce cerevisiae (pCTCON2-TgMIC16/EBY100) and evaluated it as a potential vaccine for BALB/c mice against challenge infection with the RH strain of T. gondii. We immunized BALB/c mice both orally and intraperitoneally. After three immunizations, the immune response was evaluated by measuring antibody levels, lymphocyte proliferative responses, percentages of CD4⁺ and CD8⁺ T lymphocytes, cytokine production, and the survival times of challenged mice. The results showed that the pCTCON2-TgMIC16/EBY100 vaccine stimulated humoral and cellular immune responses. In addition, mice immunized with the pCTCON2-TgMIC16/EBY100 vaccine showed increased survival times compared with non-immunized controls. In summary, TgMIC16 displayed on the cell surface of S. cerevisiae could be used as potential vaccine against toxoplasmosis.     

A novel mitogen fusion protein against CD40 cells with potent vaccine adjuvant properties

A large number of infectious diseases caused by viral or bacterial infections are treatable and/or preventable by vaccination. In addition, ongoing research is aimed at the development of vaccines against other types of diseases, including almost all forms of cancer. The efficacy of a vaccine relies on the antigen-specific response by the entire repertoire of immune competent cells. Here, we have generated a powerful mitogen fusion protein, CD40L-FasL-IgFc, which stimulates CD40⁺ cells robustly. We found that this specific cell activation is accompanied by increased expression of PRDI-BF1 (Blim-1) RNA, an indicator of terminal B-cell differentiation, in cultures stimulated with CD40L-FasL-IgFc. The addition of specific inhibitors of NF-κB and MEK1/2 partially suppressed the observed proliferative effects of CD40L-FasL-IgFc. When tested in vivo, the immune response to influenza HA vaccine was significantly increased by co-administration of CD40L-FasL-IgFc. Moreover, the co-administration of the cDNA expression plasmid encoding CD40L-FasL-IgFc significantly boosted the vaccine response. We now have a unique opportunity to evaluate our novel fusion protein adjuvant, and other similarly constructed fusion proteins, in both protein-based and genetic vaccines.     

Self-assembling protein nanoparticles with built-in flagellin domains increases protective efficacy of a Plasmodium falciparum based vaccine

To eliminate the problems associated with the use of extraneous adjuvants we have designed a Self-Assembling Protein Nanoparticle (SAPN) containing epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) (designated FMP014) and portions of the TLR5 agonist flagellin (designated FMP014_D0D1) as an intrinsic adjuvant. By combining different molar ratios of FMP014 to FMP014_D0D1 monomers before self-assembly, we generated multiple nanoparticles and investigated their biophysical characteristics, immunogenicity and protective efficacy. Immunization with the construct formulated with the ratio 58:2 of FMP014 to FMP014_D0D1 had the highest protective efficacy against a challenge with a transgenic P. berghei sporozoite expressing PfCSP. Increasing the proportion of flagellin per particle resulted in an inverse relationship with levels of both antibody titers and protection. The cytokine profiles of the various immunization groups were evaluated and quantitative amounts of the cytokines IL-2, IFN-γ, IL-12/p70 (Th1); IL4, IL5 (Th2); TNF-α, IL1β, IL-6, KC/GRO (pro-inflammatory), and IL-10 (immunomodulatory) were measured. The relationship of the cytokines to each other revealed a strong immunomodulatory effect depending on the proportion of flagellin in the construct. Our results demonstrate that SAPNs with flagellin may be a promising strategy for the development and delivery of a safe vaccine for infectious diseases.     

Salmonella Enteritidis with double deletion in phoPfliC--a potential live Salmonella vaccine candidate with novel characteristics for use in chickens

Salmonella Enteritidis mutants with deletions in phoP, fliC or phoP fliC were tested for their virulence and their ability to induce parameters of the innate and adaptive immunity in addition to their potential for serological differentiation between vaccinated, non-vaccinated and infected chickens. The double phoP fliC deletion mutant was sufficiently attenuated but not diminished in its capability to inhibit the caecal colonisation and systemic invasion of homologous Salmonella Enteritidis shortly after administration of the vaccine strain to very young chicks. Immunisation with the attenuated ΔphoP fliC mutant resulted in protective effects which were only slightly and insignificantly lower than after “immunisation” with a Salmonella wild-type strain, indicating the capability to induce an intense adaptive immune response and protection against Salmonella exposure in older chickens. The deletion in fliC enabled the effective the differentiation between immunised and infected chickens using a commercially available ELISA kit. The double phoP fliC deletion mutant of Salmonella Enteritidis might be a potential and promising live Salmonella vaccine candidate with novel characteristics for use in poultry.     

Intranasal VLP-RBD vaccine adjuvanted with BECC470 confers immunity against Delta SARS-CoV-2 challenge in K18-hACE2-mice

As the COVID-19 pandemic transitions into endemicity, seasonal boosters are a plausible reality across the globe. We hypothesize that intranasal vaccines can provide better protection against asymptomatic infections and more transmissible variants of SARS-CoV-2. To formulate a protective intranasal vaccine, we utilized a VLP-based platform. Hepatitis B surface antigen-based virus like particles (VLP) linked with receptor binding domain (RBD) antigen were paired with the TLR4-based agonist adjuvant, BECC 470. K18-hACE2 mice were primed and boosted at four-week intervals with either VLP-RBD-BECC or mRNA-1273. Both VLP-RBD-BECC and mRNA-1273 vaccination resulted in production of RBD-specific IgA antibodies in serum. RBD-specific IgA was also detected in the nasal wash and lung supernatants and were highest in VLP-RBD-BECC vaccinated mice. Interestingly, VLP-RBD-BECC vaccinated mice showed slightly lower levels of pre-challenge IgG responses, decreased RBD-ACE2 binding inhibition, and lower neutralizing activity in vitro than mRNA-1273 vaccinated mice. Both VLP-RBD-BECC and mRNA-1273 vaccinated mice were protected against challenge with a lethal dose of Delta variant SARS-CoV-2. Both vaccines limited viral replication and viral RNA burden in the lungs of mice. CXCL10 is a biomarker of severe SARS-CoV-2 infection and we observed both vaccines limited expression of serum and lung CXCL10. Strikingly, VLP-RBD-BECC when administered intranasally, limited lung inflammation at early timepoints that mRNA-1273 vaccination did not. VLP-RBD-BECC immunization elicited antibodies that do recognize SARS-CoV-2 Omicron variant. However, VLP-RBD-BECC immunized mice were protected from Omicron challenge with low viral burden. Conversely, mRNA-1273 immunized mice had low to no detectable virus in the lungs at day 2. Together, these data suggest that VLP-based vaccines paired with BECC adjuvant can be used to induce protective mucosal and systemic responses against SARS-CoV-2.     

Serological comparison of purified antigens 60 and 85A (P32) of Mycobacterium bovis BCG,and purified protein derivative,in active pulmonary tuberculosis

The immunoglobulin G (IgG) response directed against Mycobacterium bovis BCG antigens 60 (A60) and 85A (P32), and purified protein derivative (PPD), was investigated in order to compare the serodiagnostic potentials of these antigens in tuberculosis (TB). The sera of 59 patients with active minimal or moderately advanced pulmonary TB and of 59 healthy control subjects were tested by enzyme-linked immunosorbent assay. The frequencies of positivity were significantly higher (P < 0.001) in patients than in controls and similar with all three antigens. The strongest correlation was found between the responses to A60 and PPD (P < 0.001), the weakest between the responses to A60 and P32 (P < 0.05). Discrepancies were observed in newly diagnosed patients before the institution of specific chemotherapy and in patients with negative direct smears at the time of diagnosis. Untreated patients with negative direct smears presented the lowest sensitivities. P32 was the most effective antigen in diagnosing these cases (50% positivity); A60 was not better than PPD (29% and 21% positivity, respectively). The results presented here emphasize the importance of comparing antigens with the same samples in order to allow their real respective evaluation.     

Protection against bovine respiratory syncytial virus in calves vaccinated with adjuvanted modified live vaccine administered in the face of maternal antibody

Bovine respiratory syncytial virus (BRSV) is major viral contributor to bovine respiratory disease (BRD). BRD is a major cause of morbidity and mortality in all classes of cattle but particularly young beef and dairy calves. Passive antibodies not only help protect the calf against infection, but may interfere with the immune responses following vaccination. The purpose of this study was to evaluate the efficacy of an adjuvanted modified live virus (MLV) vaccine in the presence of well-defined maternal passive immunity. Calves were vaccinated at approximately 1 month of age and challenged ~90 days later when BRSV systemic antibodies were ≤1:4. Body temperature was lower at 6 and 7 days post challenge and other clinical signs were also lower in the vaccinates. Nasal viral shed was 3–4 times lower in the vaccinated animals as measured by virus isolation and polymerase chain reaction (PCR) and peaked 5 days post challenge compared to the controls (who peaked at days 6 and 7). On day 8 following challenge, animals were necropsied, and lung lobes were scored and tested for virus by PCR and indirect fluorescent assay (IFA). There was a 25-fold reduction in PCR virus detection in vaccinates and two of the vaccinated calves’ lungs were PCR negative. Only 29.4% of vaccinated calves were BRSV positive on IFA testing at necropsy, while 87.5% of control calves were BRSV positive. Vaccinated calves developed a mucosal BRSV IgA response with over 50% of the vaccinated calves having IgA prior to challenge and all vaccinated calves were positive following challenge. Additionally, vaccination stimulated the production of Interferon gamma (IFN-γ) in mononuclear cells to prime the immune system. This study established that an adjuvanted MLV vaccine could provide protection against BRSV as measured by clinical, virological, and pathological parameters while also activating both mucosal and systemic immunity.     

Recombinant Ax21 protein is a promising subunit vaccine candidate against Stenotrophomonas maltophilia in a murine infection model

Stenotrophomonas maltophilia is an emerging pathogen that can cause several disease manifestations such as bacteremia, meningitis, respiratory tract infections and others. More seriously, this pathogen has a highly evolving antibiotic resistance profile. Antibiotic misuse is further aggravating the situation by inducing the development of multi- and even pan-resistance. Thus, employing diverse strategies to overcome this increasing antibiotic resistance is of paramount importance. In general, vaccination is one of these strategies that prevents the onset of infection, provides long term protection against infection, and most importantly diminishes the antibiotic consumption, thus, resulting in controlling resistance. Unfortunately, vaccine research concerning S. maltophilia is very scarce in the literature. Ax21 protein is an outer membrane protein implicated in several virulence mechanisms of S. maltophilia such as quorum sensing, biofilm formation, and antibiotic resistance. Our computational analysis of Ax21 revealed its potential immunogenicity. In the current study, Ax21 protein of S. maltophilia was cloned and heterologously expressed in Escherichia coli. Mice were immunized with the purified recombinant antigen using Bacillus Calmette-Guérin (BCG) and incomplete Freund’s adjuvant (IFA) as immune-adjuvants. Enzyme-linked immunosorbent assay (ELISA) revealed significant antigen-specific IgG1, IgG2a and total IgG levels in immunized mice which reflected successful immune stimulation. Immunized mice that were challenged with S. maltophilia showed a substantial reduction in bacterial bioburden in lungs, liver, kidneys, and heart. In addition, liver histological examination demonstrated a remarkable decrease in pathological signs such as necrosis, vacuolation, bile duct fibrosis and necrosis, infiltration of inflammatory cells, and hemorrhage. Whole cell ELISA and opsonophagocytic assay confirmed the ability of serum antibodies from immunized mice to bind and facilitate phagocytosis of S. maltophilia, respectively. To our knowledge, this is the first report to demonstrate the vaccine protective efficacy of Ax21 outer membrane protein against S. maltophilia infection.     

v-src oncogene-specific carboxy-terminal peptide is immunoprotective against Rous sarcoma growth in chickens with MHC class I allele B-F12

B(12) haplotype of the inbred chicken line CB (B12/B12) contains, like the bulk of chicken MHC(B) haplotypes, only a single dominantly expressed class I molecule (B-F). The peptide binding motifs for this major B-F12 molecule in chickens of Rous sarcoma regressor line CB (B12/B12) have been determined. Using stringent and relaxed motifs, several peptides were found in the v-src molecule of the PR-RSV-C, but most of these peptides are identical with that of endogenous c-src. Only the v-src C-tail peptide(517-524) (LPACVLEV) contains critical anchor amino acids (valine at positions 5 and 8) and shows a sequence different from the corresponding c-src peptide. This v-src C-tail peptide up-regulates expression of the B-F12 class I molecule on PBL, as assessed by FACS analysis, and stimulates T cell proliferation in a [3H]thymidine uptake assay. A protective effect of the immune response to LPACVLEV against RSV challenge was demonstrated in CB (B12/B12) chickens immunised with peptides encapsulated in liposomes.     

Immunogenicity and protective efficacy of recombinant fiber-2 protein in protecting SPF chickens against fowl adenovirus 4

Since a novel hyper-virulent fowl adenovirus serotype 4 (FAdV-4) infection occurred in 2015, the novel FAdV-4 has been widely spreading across China, causing significant economic losses to the poultry industry. As the urgency of the issue calls for effective and efficient solutions, the present study investigated the possibility of the fiber-2 protein of the FAdV-4 to serve as a vaccine candidate against the novel FAdV-4. In the research, fiber-2 proteins were expressed in Escherichia coli, and then purified. To evaluate the immunogenicity of the recombinant fiber-2 protein, we investigated both the humoral and cellular immune responses in chickens immunized with fiber-2. The humoral immunity was assessed by detecting IgY antibodies and virus-neutralizing antibodies in chicken serum at 7, 14, 21 days post-immunization (dpi). We examined cellular immune responses by detecting CD3+CD4+ and CD3+CD8+ changes in chickens’ peripheral blood through using flow cytometry at 7, 14, 21 dpi. The cytokine production in the serum of the immunized chickens was detected by ELISA at 7, 14, 21 dpi to further explore the impact of the recombinant protein on the regulation of cytokines. The protective efficacy was determined by the survival rate of the immunized chickens challenged with the novel FAdV-4. The results show that the level of IgY antibodies of the chickens immunized with fiber-2 protein was significantly higher than that of the chickens immunized with an inactivated vaccine against FAdV-4. Moreover, 7 days after immunization, the CD4+ T-cell proliferative response of the chickens immunized with fiber-2 was significantly higher than that of the chickens immunized with the inactivated vaccine. Challenge experiment showed that the fiber-2 protein could provide full protection and the inactivated vaccine could provide 90 percent protection against the FAdV-4. These results suggest that the recombinant fiber-2 protein can be an ideal candidate for subunit vaccines against the disease.