Molecular characterization of a clinical Haemophilus parainfluenzae isolate with cefotaxime resistance and decreased susceptibility to fluoroquinolones

We report an H. parainfluenzae clinical isolate resistant to cefotaxime and with decreased susceptibility to ciprofloxacin recovered from a patient with cystic fibrosis. The isolate had elevated MICs of ampicillin (256 mg/L), amoxicillin-clavulanate (8 mg/L), cefuroxime (8 mg/L) and cefotaxime (4 mg/L), and showed a β-lactamase-producing amoxicillin-clavulanic acid-resistant (BLPACR) phenotype. A bla_TEM-1 plus five amino acid substitutions in the PBP3 were found: Ser385Thr, Val511Ala, Ile519Val, Asn526Lys and Asp551Leu. MIC of ciprofloxacin was 0.5 mg/L, and substitutions in gyrA (Ser84Tyr) and parC (Ser84Phe) genes were detected.     

鼠伤寒沙门菌耐药株拓扑异构酶基因突变分析

目的　探讨鼠伤寒沙门菌对氟喹诺酮类药物的耐药性与拓扑异构酶基因突变之间的关系。方法　三株鼠伤寒沙门菌分离株X2 ,X7,X11对氟喹诺酮类药物环丙沙星的MIC分别为 32、0 38和 0 0 2 3μg ml,X2为耐药株。利用聚合酶链反应 (PCR)对其拓扑异构酶四个耐药基因gyrA ,gyrB ,parC ,和parE进行扩增和序列分析。结果　发现分离株X2的gyrA基因同时在 2 4 8、2 5 9位点发生点突变 (Ser83→Phe ,Asp87→Asn)。分离株X7gyrA基因在 2 4 8位点发生点突变 (Ser83→Tyr)。分离株X11的gyrA基因没有发生突变。X2parC基因QRDR在 2 38位点发生点突变 (Ser80→Arg)。而分离株X7、X11的parC基因没有发生突变。三株鼠伤寒沙门菌分离株的gyrB和parE基因都没有发现突变。结论　gyrA和parC上同时发生突变会导致鼠伤寒沙门菌对环丙沙星的高耐药性     

A novel liposome adjuvant DPC mediates Mycobacterium tuberculosis subunit vaccine well to induce cell-mediated immunity and high protective efficacy in mice

Tuberculosis (TB) is a serious disease around the world, and protein based subunit vaccine is supposed to be a kind of promising novel vaccine against it. However, there is no effective adjuvant available in clinic to activate cell-mediated immune responses which is required for TB subunit vaccine. Therefore, it is imperative to develop new adjuvant. Here we reported an adjuvant composed of dimethyl dioctadecylammonium (DDA), Poly I:C and cholesterol (DPC for short). DDA can form a kind of cationic liposome with the ability to deliver and present antigen and can induce Th1 type cell-mediated immune response. Poly I:C, a ligand of TLR3 receptor, could attenuate the pathologic reaction induced by following Mycobacterium tuberculosis challenge. Cholesterol, which could enhance rigidity of lipid bilayer, is added to DDA and Poly I:C to improve the stability of the adjuvant. The particle size and Zeta-potential of DPC were analyzed in vitro. Furthermore, DPC was mixed with a TB fusion protein ESAT6-Ag85B-MPT64(190-198)-Mtb8.4-Rv2626c (LT70) to construct a subunit vaccine. The subunit vaccine-induced immune responses and protective efficacy against M. tuberculosis H37Rv infection in C57BL/6 mice were investigated. The results showed that the DPC adjuvant with particle size of 400 nm and zeta potential of 40 mV was in good stability. LT70 in the adjuvant of DPC generated strong antigen-specific humoral and cell-mediated immunity, and induced long-term higher protective efficacy against M. tuberculosis infection (5.41 ± 0.38 log₁₀ CFU) than traditional vaccine Bacillus Calmette–Guerin (BCG) (6.01 ± 0.33 log₁₀ CFU) and PBS control (6.53 ± 0.26 log₁₀ CFU) at 30 weeks post-vaccination. In conclusion, DPC would be a promising vaccine adjuvant with the ability to stimulate Th1 type cell-mediated immunity, and could be used in TB subunit vaccine.     

2017-2018年包头地区耐喹诺酮大肠埃希菌临床分离株的耐药特征与机制分析

目的了解内蒙古包头地区临床分离的耐喹诺酮大肠埃希菌的耐药特征以及机制。方法收集2017-2018年内蒙古包头地区11所参与全国细菌耐药监测网医院的大肠埃希菌临床分离株,采用纸片扩散法或自动化仪器法按统一技术方案进行药敏试验,采用酶抑制剂增强试验测定超广谱β-内酰胺酶(ESBLs),分析病原菌耐药性。并且随机从大肠埃希菌喹诺酮类耐药(环丙沙星或/和左氧氟沙星耐药)组中筛选60株作为试验菌株,采用微量肉汤稀释法测定环丙沙星最低抑菌浓度(MIC)值,采用聚合酶链反应检测GyrA、GyrB、ParC、ParE、qnrA、qnrB、qnrS、aac(6′)-Ib以及qepA基因,限制性酶切反应确定aac(6′)-Ib-cr基因型。结果共收集上述医院非重复大肠埃希菌临床分离株4 262株。耐喹诺酮大肠埃希菌分离率居前3位的医院分别为包头市肿瘤医院(79.37%)、内蒙古包钢医院(74.45%)和包头市第八医院(72.52%);前3位科室分别为泌尿科(90.74%)、呼吸内科(87.58%)和肿瘤科(77.87%);前3位标本分别为尿液(73.77%)、痰液等呼吸道标本(65.63%)和引流液(63.27%)。4 262株大肠埃希菌中喹诺酮类耐药组和喹诺酮类非耐药组占比分别为66.31%和33.69%,喹诺酮类耐药组对绝大部分所测抗菌药物的耐药率和产ESBLs的检出率均高于喹诺酮类非耐药组(P<0.05)。60株试验菌株:GyrA、GyrB、ParC和ParE基因的阳性率均为100.00%;GyrA、ParC和ParE亚基突变发生率均为100.00%,GyrB亚基突变发生率为16.67%;发生最多的氨基酸取代种类是Ser83→Leu(100.00%),其次依次为Ser80→Ile(96.67%)和Asp87→Asn(91.67%);质粒介导的喹诺酮类耐药(PMQR)基因阳性率为18.33%,其中检出率由高到低分别为aac(6′)-Ib-cr基因(15.00%)、qnrS基因(6.67%)和qepA基因(3.33%)。在GyrA亚基双突变的基础上,发生ParC亚基单位点(Ser80或Glu84)突变的菌株对环丙沙星的MIC值多在32~64μg/ml范围内;发生ParC亚基双位点(Ser80和Glu84)突变的菌株对环丙沙星的MIC值多为128μg/ml。在所有发生ParE亚基Ser458→Ala突变的菌株中均检测到含有GyrA和ParC亚基突变,与无ParE亚基Ser458→Ala突变的菌株相比,含有ParE亚基Ser458→Ala突变的菌株对环丙沙星的MIC值更高。结论内蒙古包头地区为耐喹诺酮大肠埃希菌高流行地区,且耐药水平高,常呈多药耐药。编码DNA促旋酶和拓扑异构酶Ⅳ这两种药物作用靶蛋白的基因上喹诺酮类耐药决定区(QRDRs)存在多个位点突变是引起本地区大肠埃希菌对喹诺酮类抗菌药物产生高水平耐药的主要原因。     

The APOA4 Thr347->Ser347 polymorphism is not a major risk factor of obesity

OBJECTIVE: The goal of this study was to assess the association between the APOA4 Thr(347)-->Ser(347) polymorphism and BMI and obesity. RESEARCH METHODS AND PROCEDURES: Men and women (n = 3320), randomly recruited in three independent population surveys from the north, east, and south of France, were genotyped for the APOA4 Thr(347)-->Ser(347) polymorphism. RESULTS: There were 1327 overweight (825 men, 502 women) and 611 obese (313 men, 298 women) subjects. The prevalences of subjects carrying at least one Ser(347) allele (*/Ser(347)) were 36.5%, 33.8%, and 34.3% in controls, overweight, and obese subjects, respectively (not significant), and those of the Ser(347)/Ser(347) genotype were 4.5%, 3.0%, and 2.2%, respectively (not significant). In both men and women, mean BMI and body weight were not significantly different among APOA4 genotypes. There was no evidence of heterogeneity among centers, smoking status, alcohol intake, physical activity, and educational level categories. In men, mean waist girth was lower in Ser(347)/Ser(347) (92.2 +/- 9.4 cm) than in Thr(347) carriers (95.9 +/- 10.9 cm; p = 0.01), and plasma triglycerides levels were lower in Ser(347) (1.41 +/- 1.04 mM) than in Thr(347)/Thr(347) carriers (1.55 +/- 1.23 mM; p = 0.01). DISCUSSION: These results suggest that the APOA4 347Ser allele is not a major risk factor for obesity or overweight.     

A novel subunit vaccine co-expressing GM-CSF and PCV2b Cap protein enhances protective immunity against porcine circovirus type 2 in piglets

Porcine circovirus type 2 (PCV2) causes porcine circovirus-associated disease. Capsid (Cap) protein of PCV2 is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. GM-CSF is an immune adjuvant that enhances responses to vaccines. In this study, recombinant baculoviruses Ac-Cap and Ac-Cap-GM-CSF expressing the Cap protein alone and co-expressing the Cap protein and porcine GM-CSF, respectively, were constructed successfully. The target proteins were analyzed by western blotting and IFA. Further, these proteins were confirmed by electron microscopy, which showed that Cap proteins could self-assemble into virus-like particles having diameters of 17–25 nm. Animal experiments showed that pigs immunized with Cap-GM-CSF subunit vaccine showed significantly higher levels of PCV2-specific antibodies and neutralizing antibodies than pigs immunized with the Cap subunit vaccine and a commercial vaccine (Ingelvac CircoFLEX; P < 0.05). After PCV2 wild strain challenged, Pigs receiving the Cap-GM-CSF subunit vaccine showed significantly higher average daily weight gain after wild-type PCV2 challenge than pigs receiving the other three vaccines (P < 0.05). None of PCV2 DNA was detected in all immunized animals, except control animals immunized with phosphate-buffered saline. These results indicated that GM-CSF was a powerful immunoadjuvant for PCV2 subunit vaccines because it enhanced humoral immune response and improved immune protection against PCV2 infection in pigs. Thus, the novel Cap-GM-CSF subunit vaccine has the potential to be used as an effective and safe vaccine candidate against PCV2 infection.     

Protection against infectious bronchitis virus by spike ectodomain subunit vaccine

The avian coronavirus infectious bronchitis virus (IBV) S1 subunit of the spike (S) glycoprotein mediates viral attachment to host cells and the S2 subunit is responsible for membrane fusion. Using IBV Arkansas-type (Ark) S protein histochemistry, we show that extension of S1 with the S2 ectodomain improves binding to chicken tissues. Although the S1 subunit is the major inducer of neutralizing antibodies, vaccination with S1 protein has been shown to confer inadequate protection against challenge. The demonstrated contribution of S2 ectodomain to binding to chicken tissues suggests that vaccination with the ectodomain might improve protection compared to vaccination with S1 alone. Therefore, we immunized chickens with recombinant trimeric soluble IBV Ark-type S1 or S-ectodomain protein produced from codon-optimized constructs in mammalian cells. Chickens were primed at 12 days of age with water-in-oil emulsified S1 or S-ectodomain proteins, and then boosted 21 days later. Challenge was performed with virulent Ark IBV 21 days after boost. Chickens immunized with recombinant S-ectodomain protein showed statistically significantly (P < 0.05) reduced viral loads 5 days post-challenge in both tears and tracheas compared to chickens immunized with recombinant S1 protein. Consistent with viral loads, significantly reduced (P < 0.05) tracheal mucosal thickness and tracheal lesion scores revealed that recombinant S-ectodomain protein provided improved protection of tracheal integrity compared to S1 protein. These results indicate that the S2 domain has an important role in inducing protective immunity. Thus, including the S2 domain with S1 might be promising for better viral vectored and/or subunit vaccine strategies.     

Immunogenicity and immunoprotection of porcine circovirus type 2 (PCV2) Cap protein displayed by Lactococcus lactis

The capsid (Cap) protein, an important immunoprotective protein of porcine circovirus type 2 (PCV2), was expressed on the cell surface of the Gram-positive food-grade bacterium, Lactococcus lactis. Cap protein was fused to the peptidoglycan binding domain (known as the protein anchor domain, PA) of the lactococcal AcmA cell-wall hydrolase. The Cap protein fusion was non-covalently rebound to the surface of non-genetically modified, non-living high-binder L. lactis cells (designated Gram-positive enhancer matrix (GEM) particles). Expression of the recombinant GEM-displaying capsid protein (GEM-PA-Cap) was verified by Western blotting and immunofluorescence and transmission electron microscopy assays. To evaluate the immunogenicity of the recombinant Cap protein (rCap), 20 PCV2-seronegative piglets were immunized with the GEM-PA-Cap subunit vaccine, GEM alone, or phosphate-buffered saline (PBS, challenge control and empty control). Each group consisted of five piglets. The results showed that the level of PCV2-specific antibodies in piglets immunized with the GEM-PA-Cap subunit vaccine was significantly higher than that of the piglets immunized with GEM alone or the control group at all the time points post-vaccination (P < 0.01). After challenge with the PCV2 wild-type strain, piglets that received the GEM-PA-Cap subunit vaccine showed significantly higher average daily weight gain (DWG) and shorter fever duration than the other two groups (P < 0.001). Furthermore, a significant reduction in the gross lung lesion scores and lymph node lesion scores was noted in the GEM-PA-Cap-immunized group compared with the scores of the GEM or PBS-treated group (P < 0.01). The results suggest that recombinant rCap displayed by L. lactis GEM particles provided the piglets with significant immunoprotection from PCV2-associated disease. Thus, the novel GEM-PA-Cap subunit vaccine has potential to be considered an effective and safe candidate vaccine against PCV2 infection in piglets.     

Development of a subunit vaccine containing recombinant Riemerella anatipestifer outer membrane protein A and CpG ODN adjuvant

Riemerella anatipestifer, a Gram-negative bacillus, causes septicemia that can result in high mortality for ducklings. In this study, we evaluated the immune response and protective efficacy provided by a subunit vaccine containing recombinant outer membrane protein A (rOmpA) and plasmid constructs containing CpG oligodeoxynucleotides (ODN). Results showed that CpG ODN enhanced both humoral and cell-mediated immunity elicited by rOmpA as early as two weeks after primary immunization. When compared to ducks immunized with rOmpA, ducks immunized with rOmpA + CpG ODN showed higher levels (p < 0.05) of antibody titer, T cell proliferation, and percentages of CD4⁺ and CD8⁺ T cell in peripheral blood mononuclear cells (PBMCs). The relative fold inductions of mRNA expression of Th1-type (IFN-γ and IL-12), and Th2-type (IL-6) cytokines in PBMCs isolated from ducks immunized with rOmpA + CpG ODN were significantly higher than those of the rOmpA group. Homologous challenge result showed that the rOmpA + CpG ODN vaccine reduced the pathological score by 90% in comparison with the saline control. In conclusion, our study found that CpG ODN can enhance both humoral and cellular immunity elicited by a rOmpA vaccine. The rOmpA + CpG ODN vaccine can be further developed as a subunit vaccine against R. anatipestifer.     

Molecular analysis of high-level ciprofloxacin resistance in Salmonella enterica serovar Typhi and S. Paratyphi A: need to expand the QRDR region?

Fourteen strains of S. Typhi (n=13) and S. Paratyphi A (n=1) resistant to ciprofloxacin were compared with 30 ciprofloxacin decreased-susceptibility strains on the basis of qnr plasmid analysis, and nucleotide substitutions at gyrA, gyrB, parC and parE. In ciprofloxacin-resistant strains, five S. Typhi and a single S. Paratyphi A showed triple mutations in gyrA (Ser83-->Phe, Asp87-->Asn, Glu133-->Gly) and a novel mutation outside the quinolone resistance determining region (QRDR) (Met52-->Leu). Novel mutations were also discovered in an isolate (minimum inhibitory concentration 8 microg/ml) in gyrA gene Asp76-->Asn and outside the QRDR Leu44-->Ile. Out of 30 isolates with reduced susceptibility, single mutation was found in 12 strains only. Genes encoding qnr plasmid (qnr A, qnr B, AAC1-F) were not detected in ciprofloxacin-resistant or decreased-susceptibility strains. Antimicrobial surveillance coupled with molecular analysis of fluoroquinolone resistance is warranted for reconfirming novel and established molecular patterns of resistance, which is quintessential for reappraisal of enteric fever therapeutics.     

Comparison of monovalent glycoprotein B with bivalent gB/pp65 (GP83) vaccine for congenital cytomegalovirus infection in a guinea pig model: Inclusion of GP83 reduces gB antibody response but both vaccine approaches provide equivalent protection against pup mortality

Cytomegalovirus (CMV) subunit vaccine candidates include glycoprotein B (gB), and phosphoprotein ppUL83 (pp65). Using a guinea pig cytomegalovirus (GPCMV) model, this study compared immunogenicity, pregnancy outcome, and congenital viral infection following pre-pregnancy immunization with a three-dose series of modified vaccinia virus Ankara (MVA)-vectored vaccines consisting either of gB administered alone, or simultaneously with a pp65 homolog (GP83)-expressing vaccine. Vaccinated and control dams were challenged at midgestation with salivary gland-adapted GPCMV. Comparisons included ELISA and neutralizing antibody responses, maternal viral load, pup mortality, and congenital infection rates. Strikingly, ELISA and neutralization titers were significantly lower in the gB/GP83 combined vaccine group than in the gB group. However, both vaccines protected against pup mortality (63.2% in controls vs. 11.4% and 13.9% in gB and gB/GP83 combination groups, respectively; p < 0.0001). Reductions in pup viral load were noted for both vaccine groups compared to control, but preconception vaccination resulted in a significant reduction in GPCMV transmission only in the monovalent gB group (26/44, 59% v. 27/34, 79% in controls; p < 0.05). We conclude that, using the MVA platform, the addition of GP83 to a gB subunit vaccine interferes with antibody responses and diminishes protection against congenital GPCMV infection, but does not decrease protection against pup mortality.     

The use of dissolved oxygen-controlled,fed-batch aerobic cultivation for recombinant protein subunit vaccine manufacturing

A simple “off-the-shelf” fed-batch approach to aerobic bacterial cultivation for recombinant protein subunit vaccine manufacturing is presented. In this approach, changes in the dissolved oxygen levels are used to adjust the nutrient feed rate (DO-stat), so that the desired dissolved oxygen level is maintained throughout cultivation. This enables high Escherichia coli cell densities and recombinant protein titers. When coupled to a k_La-matched scale-down model, process performance is shown to be consistent at the 2 L, 20 L, and 200 L scales for two recombinant E. coli strains expressing different protein subunit vaccine candidates. Additionally, by mining historical DO-stat nutrient feeding data, a method to transition from DO-stat to a pre-determined feeding profile suitable for larger manufacturing scales without using feedback control is demonstrated at the 2 L, 20 L, and 200 L scales.     

Immunogenicity and protective efficacy of virus-like particles and recombinant fiber proteins in broiler-breeder vaccination against fowl adenovirus (FAdV)-8b

Inclusion body hepatitis (IBH) is an economically important diseases in broiler chicken industry. Several serotypes of fowl adenovirus (FAdV) can cause IBH, among them, serotype FAdV-8b is associated with the majority of the IBH cases in Canada. Here, we evaluated FAdV-8b virus-like particles (VLPs) and recombinant FAdV-8b fiber proteins (expressed in E. coli) as potential broiler-breeder vaccines against IBH. For assessing the immunogenicity of vaccines, we investigated both humoral and cellular immunity. The humoral immune response was evaluated by determining total IgY and virus-neutralizing antibody in serum at 14, 28, 35 and 60 days post-immunization (dpi). We examined cellular immunity using flow cytometry by determining CD4:CD8 ratio change in peripheral blood after the booster vaccination. The protective effect of vaccines was tested by challenging 14 day-old progeny (n = 30/group) carrying maternal antibodies (MtAb) by challenging with virulent FAdV-8b virus (1 × 10⁷ TCID₅₀, FAdV-8b-SK). Although total IgY levels were comparable in all groups, the neutralizing antibody response in broiler-breeders at 35 and 60 dpi was significantly (p < 0.05) higher those vaccinated with FAdV-8b VLPs followed by FAdV-8b fiber compared to fiber-knob. Moreover, vaccines comprised of FAdV-8b VLPs and FAdV-8b fiber rather than FAdV-8b fiber-knob efficiently elicited the cell-mediated immune response as evidenced by a statistically significant (p < 0.05) CD8⁺ T-cell proliferative response in broiler-breeders four days after the booster vaccination. Unlike FAdV-8b fiber-knob, FAdV-8b VLPs, and FAdV-8b fiber vaccinated broiler-breeders were able to transfer a substantial amount (28.4 ± 9%) of MtAb to their progeny. Challenge revealed that MtAb provided 100% and 82.7% protection in progeny hatched from FAdV-8b VLPs, and FAdV-8b fiber vaccinated broiler-breeders, respectively. Collectively, our data suggest that FAdV-8b subunit vaccine-induced MtAb efficiently protected progeny against clinical IBH and broiler-breeder vaccination with subunit vaccines is a potential approach to protect against IBH.     

A rare non-synonymous c.102C>G SNP in the IFNB1 gene might be a risk factor for cerebral malaria in Indian populations

Interferon beta1 (IFNB1) is a type I interferon that is mainly known for its antiviral activity, but it also regulates a number of anti-inflammatory and immunomodulatory functions. Studies on mouse models of cerebral malaria have established that IFNB1 regulates severe malaria pathogenesis and increases overall survival against malaria. It down-regulates pro-inflammatory cytokines: TNF, IFNG and ICAM-1, resulting in decreased adherence of Plasmodium falciparum parasitized RBC to capillary wall, entry into the brain and delayed onset of death. Therefore, we hypothesized that variations in IFNB1 gene could regulate malarial pathogenesis. We re-sequenced the complete IFNB1 gene along with 900 bp of 5′ up-stream and 500 bp of 3′-UTR in 437 individuals from malaria endemic regions of the Orissa and Chhattisgarh states of India. The subjects comprised of 173 cases of severe malaria, 101 of mild malaria, and 156 ethnically matched asymptomatic controls. Data were statistically compared between cases and controls for their possible association with P. falciparum malarial outcome. Two single nucleotide polymorphisms (SNPs): a synonymous c.153C>T (rs1051922) and a non-synonymous substitution c.102C>G (rs139262191, p.Ser34Arg) were identified. The genotype and allele distribution of c.153C>T did not differ significantly between the study groups [mild, χ²₂ = 4.10, p-value < 0.13 and severe χ²₂ = 0.06, p-value < 0.97]. Interestingly, the rare non-synonymous SNP (rs139262191) was observed only in malaria patients. The differences between all cases and controls did not reach statistical significance, however, a statistically significant difference was observed between the asymptomatic control group and the cerebral malaria group [OR = 20.32, 95% CI = 1.08–382.63, p-value = 0.044]. Moreover, the genotypes between cerebral malaria positive and negative groups were not significantly different [OR = 5.58, 95% CI = 0.61–50.97, p-value = 0.123]. Our findings suggest that the IFNB1 variant, p.Ser34Arg, might be a risk factor for cerebral malaria in Indian populations.     

Fiber knob domain lacking the shaft sequence but fused to a coiled coil is a candidate subunit vaccine against egg-drop syndrome

Egg-drop syndrome (EDS) virus is an avian adenovirus that causes a sudden drop in egg production and in the quality of the eggs when it infects chickens, leading to substantial economic losses in the poultry industry. Inactivated EDS vaccines produced in embryonated duck eggs or cell culture systems are available for the prophylaxis of EDS. However, recombinant subunit vaccines that are efficacious and inexpensive are a desirable alternative. In this study, we engineered chimeric fusion proteins in which the trimeric fiber knob domain lacking the triple β-spiral motif in the fiber shaft region was genetically fused to trimeric coiled coils, such as those of the engineered form of the GCN4 leucine zipper peptide or chicken cartilage matrix protein (CMP). The fusion proteins were expressed predominantly as soluble trimeric proteins in Escherichia coli at levels of 15–80 mg/L of bacterial culture. The single immunization of chickens with the purified fusion proteins, at a dose equivalent to 10 μg of the knob moiety, elicited serum antibodies with high hemagglutination inhibition (HI) activities, similar to those induced by an inactivated EDS vaccine. A dose–response analysis indicated that a single immunization with as little as 1 μg of the knob moiety of the CMP–knob fusion protein was as effective as the inactivated vaccine in inducing antibodies with HI activity. The immunization of laying hens had no apparent adverse effects on egg production and effectively prevented clinical symptoms of EDS when the chickens were challenged with pathogenic EDS virus. This study demonstrates that the knob domain lacking the shaft sequence but fused to a trimeric coiled coil is a promising candidate subunit vaccine for the prophylaxis of EDS in chickens.     

An optimized,highly efficient,self-assembled,subvirus-like particle of infectious bursal disease virus (IBDV)

Infectious bursal disease virus (IBDV) causes immunosuppression in young chickens, leading to increased susceptibility to other diseases and a reduction in the immune response to other vaccines. Thus, IBDV results in great economic losses to the poultry industry. The most effective method of prevention is vaccination. However, medium-virulence vaccines can cause bursal pathological damage and immunosuppression. Here, we describe a safer, self-assembled, subvirus-like particle (sVP) vaccine without a complex purification process. The IBD-VP2 gene was cloned into Pichia pastoris, and the expressed protein self-assembled into T = 1 sVPs (∼23 nm). Immunization experiments showed that the sVP vaccine elicited high IBDV-neutralizing antibodies in each group, and all birds survived challenge with very virulent IBDV (vvIBDV). Additionally, IBDV RNA was not detected, and sterile immunity was achieved. In conclusion, the IBD-sVP is a suitable candidate for a recombinant subunit vaccine against IBDV.     

Fused Mycobacterium tuberculosis multi-stage immunogens with an Fc-delivery system as a promising approach for the development of a tuberculosis vaccine

Tuberculosis (TB) remains a major health problem worldwide. Currently, the Bacilli Calmette-Guérin (BCG) is the only available licensed TB vaccine, which has low efficacy in protection against adult pulmonary TB. Therefore, the development of a safe and effective vaccine against TB needs global attention. In the present study, a novel multi-stage subunit vaccine candidate from culture filtrate protein-10 (CFP-10) and heat shock protein X (HspX) of Mycobacterium tuberculosis fused to the Fc domain of mouse IgG2a as a selective delivery system for antigen-presenting cells (APCs) was produced and its immunogenicity assessed. The optimized gene constructs were introduced into pPICZαA expression vectors, and the resultant plasmids (pPICZαA-CFP-10:Hspx:Fcγ2a and pPICZαA-CFP-10:Hspx:His) were transferred into Pichia pastoris by electroporation. The identification of both purified recombinant fusion proteins was evaluated by SDS-PAGE and immunoblotting. Then the immunogenicity of the recombinant proteins with and without BCG was evaluated in BALB/c mice by assessing the level of IFN-γ, IL-12, IL-4, IL-17 and TGF-β cytokines. Both multi-stage vaccines (CFP-10:HspX:Fcγ2a and CFP-10:HspX:His) induced Th1-type cellular responses by producing high level of IFN-γ (272 pg/mL, p < 0.001) and IL-12 (191 pg/mL, p < 0.001). However, the Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a low level of IL-4 (10 pg/mL) compared to the CFP-10:HspX:His group. The production of IFN-γ to CFP-10:HspX:Fcγ2a was markedly consistent and showed an increasing trend for IL-12 compared with the BCG or CFP-10:HspX:His primed and boosted groups. Findings revealed that CFP-10:Hspx:Fcγ2a fusion protein can elicit strong Th1 antigen-specific immune responses in favor of protective immunity in mice and could provide new insight for introducing an effective multi-stage subunit vaccine against TB.     

Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates

We describe here the preclinical development of a dengue vaccine composed of recombinant subunit carboxy-truncated envelope (E) proteins (DEN-80E) for each of the four dengue serotypes. Immunogenicity and protective efficacy studies in Rhesus monkeys were conducted to evaluate monovalent and tetravalent DEN-80E vaccines formulated with ISCOMATRIX™ adjuvant. Three different doses and two dosing regimens (0, 1, 2 months and 0, 1, 2, and 6 months) were evaluated in these studies. We first evaluated monomeric (DEN4-80E) and dimeric (DEN4-80EZip) versions of DEN4-80E, the latter generated in an attempt to improve immunogenicity. The two antigens, evaluated at 6, 20 and 100 μg/dose formulated with ISCOMATRIX™ adjuvant, were equally immunogenic. A group immunized with 20 μg DEN4-80E and Alhydrogel™ induced much weaker responses. When challenged with wild-type dengue type 4 virus, all animals in the 6 and 20 μg groups and all but one in the DEN4-80EZip 100 μg group were protected from viremia. Two out of three monkeys in the Alhydrogel™ group had breakthrough viremia. A similar study was conducted to evaluate tetravalent formulations at low (3, 3, 3, 6 μg of DEN1-80E, DEN2-80E, DEN3-80E and DEN4-80E respectively), medium (10, 10, 10, 20 μg) and high (50, 50, 50, 100 μg) doses. All doses were comparably immunogenic and induced high titer, balanced neutralizing antibodies against all four DENV. Upon challenge with the four wild-type DENV, all animals in the low and medium dose groups were protected against viremia while two animals in the high-dose group exhibited breakthrough viremia. Our studies also indicated that a 0, 1, 2 and 6 month vaccination schedule is superior to the 0, 1, and 2 month schedule in terms of durability. Overall, the subunit vaccine was demonstrated to induce strong neutralization titers resulting in protection against viremia following challenge even 8-12 months after the last vaccine dose.     

Safety and immunogenicity of a parenterally administered rotavirus VP8 subunit vaccine in healthy adults

BackgroundThe P2-VP8 subunit vaccine for the prevention of rotavirus gastroenteritis is comprised of a truncated VP8 subunit protein from the rotavirus Wa strain (G1[P8]) fused to the tetanus toxin P2 epitope, and adsorbed on aluminum hydroxide for intramuscular administration.MethodsThree groups of 16 adults were randomized to receive three injections of P2-VP8 (12) or placebo (4) at doses of 10, 30 or 60 μg of vaccine. IgG and IgA antibodies to P2-VP8 were assessed by ELISA in serum and lymphocyte supernatant (ALS). Serum samples were tested for neutralizing antibodies to homologous and heterologous strains of rotavirus.ResultsThe vaccine was well-tolerated. All vaccine recipients demonstrated significant IgA responses and all but one demonstrated IgG responses; in the 60 μg cohort, geometric mean titers (GMTs) rose 70- and 80-fold for IgA and IgG, respectively. Homologous neutralizing antibody responses were observed in about half of participants in all three dose cohorts; in the 60 μg cohort, GMTs against Wa rose from 128 to 992. Neutralizing antibody responses were robust to P[8] strains, moderate to P[4] strains and negligible to P[6] strains. ALS IgA responses were dose dependent.ConclusionsThe P2-VP8 subunit vaccine was well tolerated and evoked promising immune responses.Clinical trials registrationNCT01764256     

IEM101, a naturally attenuated Vibrio cholerae strain as carrier for genetically detoxified derivatives of cholera toxin

Two mutants of cholera toxin (CTS106 containing a Pro106-->Ser substitution and CTK63 containing a Ser63-->Lys substitution) with greatly reduced or no toxicity respectively, were expressed in the naturally attenuated IEM101 Vibrio cholerae strain (El Tor, Ogawa) which does not express cholera toxin (CT). Expression was driven by the natural promoter of CT, or by a promoter known to induce strong in vivo expression such as nirB. In the rabbit ileal loop assay, where 10(4) wild type bacteria were sufficient to induce fluid accumulation, 10(9) IEM101 expressing CTS106 bacteria were needed to induce some fluid accumulation, while IEM101 expressing CTK63 was inactive, even when 10(10) cells were used. When used to immunize mice intranasally, all bacteria induced vibriocidal antibodies; however, anti-CT antibodies were not induced by bacteria expressing low levels of CTK63 under the control of the ct promoter. Anti-CT antibodies were successfully induced by bacteria expressing high levels of CTK63 under the control of the nirB promoter, or by bacteria expressing low levels of CTS106. These data show that antibodies against cholera toxin can be induced in vivo by high level expression of a non toxic mutant, or by using a mutant with residual ADP-ribosyltransferase activity.In conclusion, we have shown that IEM101, a naturally attenuated Vibrio strain known to be safe and immunogenic in humans, can be engineered to express immunogenic levels of CTK63, and may represent a good candidate for vaccination against cholera.