Efficacy of an autophagy-targeted DNA vaccine against avian leukosis virus subgroup J

Infection with the avian leukosis virus subgroup J (ALV-J) can lead to neoplastic disease in chickens, inflicting significant economic losses to the poultry industry. Recent reports have identified inhibitory effects of ALV-J on autophagy, a process involving in innate and adaptive immunity. Inspired by this connection between autophagy and immunity, we developed a novel DNA vaccine against ALV-J which includes co-administration of rapamycin to stimulate autophagy. To measure the efficacy of the developed prototype vaccine, five experimental groups of seven-day-old chickens was immunized three times at three-week intervals respectively with vector, pVAX1-gp85, pVAX1-gp85-LC3, pVAX1-gp85 + rapamycin and pVAX1-gp85-LC3 + rapamycin through electroporation. We then tested their antibody titers, cytokine levels and cellular immune responses. The immunoprotective efficacy of the prototype vaccines against the challenge of the ALV-J GD1109 strain was also examined. The results showed that the combination of pVAX1-gp85-LC3 and rapamycin was able to induce the highest antibody titers, and enhance interleukin(IL)-2, IL-10 and interferon (IFN)-γ expression, and the chickens immunized with the combination of pVAX1-gp85-LC3 and rapamycin showed the highest percentage of CD3+ CD8+ T lymphocytes. Based on our results, we suggest that stimulating autophagy can improve the efficacy of DNA vaccines and that our DNA vaccine shows the potential of being a candidate vaccine against ALV-J. This study provides a novel strategy for developing vaccines against ALV-J.     

gp85 protein vaccine adjuvanted with silica nanoparticles against ALV-J in chickens

This study focused on the effect of silica nanoparticles as adjuvant for vaccine applications comprised of gp85, a dominating structural protein of J Subgroup Avian Leukosis Virus (ALV-J), and which was evaluated by comparing with the responsiveness induced by that emulsified in Freund adjuvant. Thirty-six chickens were inoculated twice with gp85 adjuvanted with the silica nanoparticles or Freund’s adjuvant at the 2nd and 3rd week old. Two weeks later, the inoculated chickens were challenged with a 10^2.2 50% tissue culture infective dose (TCID50) of ALV-J. The blood samples were collected weekly to detect the serum antibodies and viremia. Results showed that positive serum antibodies (S/P value > 0.6) against gp85 emerged at the third week in the inoculated chickens, while the antibodies level persisted longer in silica nanoparticles adjuvanted-group to Freund’s adjuvanted-group. Furthermore, viremia in silica nanoparticles adjuvanted-group was recovered more quickly compared with Freund’s adjuvanted-group. Hence our study revealed that silica nanoparticles can effectively improve the protection of gp85 vaccine against ALV-J and present a better performance than Freund’s adjuvant.     

Vaccination with a codon-optimized A27L-containing plasmid decreases virus replication and dissemination after vaccinia virus challenge

Smallpox is a disease caused by Variola virus (VARV). Although eradicated by WHO in 1980, the threat of using VARV on a bioterror attack has increased. The current smallpox vaccine ACAM2000, which consists of live vaccinia virus (VACV), causes complications in individuals with a compromised immune system or with previously reported skin diseases. Thus, a safer and efficacious vaccine needs to be developed. Previously, we reported that our virus-free DNA vaccine formulation, a pVAX1 plasmid encoding codon-optimized VACV A27L gene (pA27LOPT) with and without Imiquimod adjuvant, stimulates A27L-specific production of IFN-γ and increases humoral immunity 7 days post-vaccination. Here, we investigated the immune response of our novel vaccine by measuring the frequency of splenocytes producing IFN-γ by ELISPOT, the TH1 and TH2 cytokine profiles, and humoral immune responses two weeks post-vaccination, when animals were challenged with VACV. In all assays, the A27-based DNA vaccine conferred protective immune responses. Specifically, two weeks after vaccination, mice were challenged intranasally with vaccinia virus, and viral titers in mouse lungs and ovaries were significantly lower in groups immunized with pA27LOPT and pA27LOPT + Imiquimod. These results demonstrate that our vaccine formulation decreases viral replication and dissemination in a virus-free DNA vaccine platform, and provides an alternative towards a safer an efficacious vaccine.     

Expression of H5 hemagglutinin vaccine antigen in common duckweed (Lemna minor) protects against H5N1 high pathogenicity avian influenza virus challenge in immunized chickens

A synthetic hemagglutinin (HA) gene from the highly pathogenic avian influenza (HPAI) virus A/chicken/Indonesia/7/2003 (H5N1) (Indo/03) was expressed in aquatic plant Lemna minor (rLemna-HA). In Experiment 1, efficacy of rLemna-HA was tested on birds immunized with 0.2 μg or 2.3 μg HA and challenged with 10⁶ mean chicken embryo infectious doses (EID₅₀) of homologous virus strain. Both dosages of rLemna-HA conferred clinical protection and dramatically reduced viral shedding. Almost all the birds immunized with either dosage of rLemna-HA elicited HA antibody titers against Indo/03 antigen, suggesting an association between levels of anti-Indo/03 antibodies and protection. In Experiment 2, efficacy of rLemna-HA was tested on birds immunized with 0.9 μg or 2.2 μg HA and challenged with 10⁶ EID₅₀ of heterologous H5N1 virus strains A/chicken/Vietnam/NCVD-421/2010 (VN/10) or A/chicken/West Java/PWT-WIJ/2006 (PWT/06). Birds challenged with VN/10 exhibited 100% survival regardless of immunization dosage, while birds challenged with PWT/06 had 50% and 30% mortality at 0.9 μg HA and 2.2 μg HA, respectively. For each challenge virus, viral shedding titers from 2.2 μg HA vaccinated birds were significantly lower than those from 0.9 μg HA vaccinated birds, and titers from both immunized groups were in turn significantly lower than those from sham vaccinated birds. Even if immunized birds elicited HA titers against the vaccine antigen Indo/03, only the groups challenged with VN/10 developed humoral immunity against the challenge antigen. None (rLemna-HA 0.9 μg HA) and 40% (rLemna-HA 2.2 μg HA) of the immunized birds challenged with PWT/06 elicited pre-challenge antibody titers, respectively. In conclusion, Lemna-expressed HA demonstrated complete protective immunity against homologous challenge and suboptimal protection against heterologous challenge, the latter being similar to results from inactivated whole virus vaccines. Transgenic duckweed-derived HA could be a good alternative for producing high quality antigen for an injectable vaccine against H5N1 HPAI viruses.     

Toll-like receptor 3 adjuvant in combination with virus-like particles elicit a humoral response against HIV

Human Immunodeficiency Virus (HIV) Virus-Like Particles (VLPs) composed of HIV_IIIB Gag and HIV_BaL gp120/gp41 envelope are a pseudovirion vaccine capable of presenting antigens in their native conformations. To enhance the immunogenicity of the HIV Env antigen, VLPs were coupled to VesiVax Conjugatable Adjuvant Lipid Vesicles (CALV) containing one of four toll-like-receptor (TLR) ligands, each activating a receptor with distinct cellular localization and downstream pathways. C57BL/6 mice were vaccinated by intranasal prime followed by two sub-cheek boosts and their sera immunoglobulin and neutralizing potency were measured over a duration of 3 months after vaccination. PBS control, VLPs alone, CALV + VLPs, and VLPs complexed with CALV and ligands for TLR2 (PAM3CAG), TLR3 (dsRNA), TLR4 (MPLA), or TLR7/8 (resiquimod) were evaluated based on antibody titer, IgG1 and IgG2c class switching, germinal center formation, T follicular cells and potency of neutralizing antibodies. Consistently, the TLR3 ligand dsRNA complexed to CALV and in combination with VLPs (CALV(dsRNA) + VLPs) induced the strongest response. CALV(dsRNA) + VLPs induced the highest titers against the recombinant vaccine antigens clade B Bal gp120 and pr55 Gag. Additionally, CALV(dsRNA) + VLPs induced cross-clade antibodies, represented by high titers of antibody to clade c 96ZM651 gp120. CALV(dsRNA) + VLPs induced predominantly IgG2c over IgG1, a response associated with T helper type 1 (Th1)-like cytokines. In turn, CALV(dsRNA) + VLP immunized mice generated the most potent neutralizing antibodies against HIV strain MN.3. Finally, at time of sacrifice, a significant increase in germinal center B cells and T follicular cells was detected in mice which received CALV(dsRNA) + VLPs compared to PBS. Our results indicate that CALV(dsRNA) is a superior adjuvant for HIV VLPs in generating a Th1-like immunoglobulin profile, while prolonging lymph node germinal centers, T follicular cells and generating neutralizing antibodies to a highly sensitive tier 1A variant of HIV.     

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.     

Canine zona pellucida glycoprotein-3: Up-scaled production,immunization strategy and its outcome on fertility

Zona pellucida (ZP) glycoproteins based contraceptive vaccines have been proposed for the management of wildlife population. In the present study, a fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830–844] followed by a dilysine linker and an ectodomain of dog ZP3 (ZP3; aa residues 23–348) without any affinity tag (TT-KK-ZP3) has been expressed in Escherichia coli. The recombinant protein was successfully produced in fed-batch fermentor and purified. The average yield of purified refolded protein was 12.20 ± 0.61 mg/2 g wet cell pellet. Female FvB/J mice immunized with the varying doses of recombinant TT-KK-ZP3 supplemented with alum/PetGel A as adjuvants following a three injection schedule, showed dose dependent increase in serum IgG titer. Antibodies against TT-KK-ZP3 recognized native mouse/dog ZP and significantly inhibited mouse in-vitro fertilization (p = 0.012). Immunized mice showed significant reduction in fertility (p < 0.05). Higher antibody titers were associated with a decrease in the number of pups born to the immunized female mice. To reduce the number of injections, two injection schedule using various dose combinations of TT-KK-ZP3 supplemented with alum revealed lower immunogenicity and contraceptive efficacy as compared to the three injection schedule. To overcome this, CpG motif was included in addition to alum and both intraperitoneal and intranasal route of immunization following the two injection schedule was investigated. Inclusion of CpG significantly enhanced the antibody titer and improved contraceptive efficacy. In the mice immunized following intraperitoneal route, serum/vaginal IgG and in the mice immunized through intranasal route, vaginal IgA seemed to be important for curtailment in fertility. To conclude, the recombinant protein described herein may be a good candidate for developing contraceptive vaccine for the wildlife population management, in particular street dogs.     

A effective DNA vaccine against diverse genotype J infectious hematopoietic necrosis virus strains prevalent in China

Infectious hematopoietic necrosis virus (IHNV) is the most important pathogen threatening the aquaculture of salmonid fish in China. In this study, a DNA vaccine, designated pIHNch-G, was constructed with the glycoprotein (G) gene of a Chinese IHNV isolate SD-12 (also called Sn1203) of genotype J. The minimal dose of vaccine required, the expression of the Mx-1 gene in the muscle (vaccine delivery site) and anterior kidney, and the titers of the neutralizing antibodies produced were used to evaluate the vaccine efficacy. To assess the potential utility of the vaccine in controlling IHNV throughout China, the cross protective efficacy of the vaccine was determined by challenging fish with a broad range of IHNV strains from different geographic locations in China. A single 100 ng dose of the vaccine conferred almost full protection to rainbow trout fry (3 g) against waterborne or intraperitoneal injection challenge with IHNV strain SD-12 as early as 4 days post-vaccination (d.p.v.), and significant protection was still observed at 180 d.p.v. Intragenogroup challenges showed that the DNA vaccine provided similar protection to the fish against all the Chinese IHNV isolates tested, suggesting that the vaccine can be widely used in China. Mx-1 gene expression was significantly upregulated in the muscle tissue (vaccine delivery site) and anterior kidney in the vaccinated rainbow trout at both 4 and 7 d.p.v. Similar levels of neutralizing antibodies were determined with each of the Chinese IHNV strains at 60 and 180 d.p.v. This DNA vaccine should play an important role in the control of IHN in China.     

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.     

The protective effect of a Schistosoma japonicum Chinese strain 23 kDa plasmid DNA vaccine in pigs is enhanced with IL-12

The schistosome integral membrane protein Sm/Sj23 was initially shown to induce protection in mice as a synthetic peptide vaccine and further, as a plasmid DNA vaccine to induce protection in mice, sheep and water buffalo. In this study we asked if we could induce protection against challenge infection in pigs against Schistosoma japonicum by vaccinating them with a plasmid DNA vaccine encoding the S. japonicum Chinese strain 23 kDa membrane protein. Further, we asked if we could enhance protective efficacy of this vaccine by the addition of IL-12. We compared vaccination with SjC23 plasmid DNA alone or with IL-12 plasmid DNA in pigs. Pigs were immunized three times at three weekly intervals. Thirty Chinese Songjang native pigs were divided into three groups. In group A, each pig was immunized with 500 μg of SjC23 plasmid DNA by intramuscular (i.m.) injection in both buttocks. In group B each pig was immunized with 500 μg of SjC23 plasmid DNA, and 500 μg of each of pcDNA3.1-p35 and 500 μg of pcDNA3.1-p40 DNA by i.m. injection. In group C each pig was immunized with 500 μg of pcDNA3.1 as the control. Thirty days post-vaccination, pigs were challenged with S. japonicum cercariae and adult and egg burdens and granuloma size determined 45 days post-challenge. The results showed that worm reduction rates in SjC23 group compared with control group were 29.2% and in the SjC23 + IL-12 group reduced 58.6%. Similarly the female worm reduction rates were 50.8 and 58.8%, the hepatic egg reduction rates were 48.2 and 56.4%, and the mean square measure reduction rates of hepatic egg granulomas were 48.6 and 44.4%, the mean diameter reduction rates of granulomas were 27.6 and 22.8% in pigs vaccinated with SjC23 or SjC23 + IL-12 compared to plasmid vaccinated pigs, respectively. Analysis of sera from pigs vaccinated with SjC23 showed that 4 of 10 pigs had anti-Sj23 antibody responses; with 5 of 10 pigs positive for anti-Sj23 in the SjC23+IL-12 group. These results suggest that vaccination with Sj23 DNA vaccine induces not only a significant reduction in worm and egg burdens, but also significantly reduces the size of egg granulomas, thus is also anti-pathology.     

Differential immune responses to HIV-1 envelope protein induced by liposomal adjuvant formulations containing monophosphoryl lipid A with or without QS21

Liposomes have shown promise as constituents of adjuvant formulations in vaccines to parasitic and viral diseases. A particular type of liposomal construct, referred to as Army Liposome Formulation (ALF), containing neutral and anionic saturated phospholipids, cholesterol, and monophosphoryl lipid A (MPLA), has been used as an adjuvant for many years. Here we investigated the effects of physical and chemical changes of ALF liposomes on adjuvanted immune responses to CN54 gp140, a recombinant HIV-1 envelope protein. While holding the total amounts of liposomal MPLA and the gp140 antigen constant, different liposome sizes and liposomal MPLA:phospholipid molar ratios, and the effect of adding QS21 to the liposomes were compared for inducing immune responses to the gp140. For liposomes lacking QS21, higher titers of IgG binding antibodies to gp140 were induced by small unilamellar vesicle (SUV) rather than by large multilamellar vesicle (MLV) liposomes, and the highest titers were obtained with SUV having the MPLA:phospholipid ratio of 1:5.6. ALF plus QS21 (ALFQ) liposomes induced the same maximal binding antibody titers regardless of the MPLA:phospholipid ratio. ALF MLV liposomes induced mainly IgG1 and very low IgG2a antibodies, while ALF SUV liposomes induced IgG1 ≥ IgG2a > IgG2b antibodies. Liposomes containing QS21 induced IgG1 > IgG2a > IgG2b > IgG3 antibodies. ELISPOT analysis of splenocytes from immunized mice revealed that ALF liposomes induced low levels of IFN-γ, but ALFQ induced high levels. ALF and ALFQ liposomes each induced approximately equivalent high levels of IL-4. Based on antibody subtypes and cytokine secretion, we conclude that ALF liposomes predominantly stimulate Th2, while ALFQ strongly induces both Th1 and Th2 immunity. When CN54 gp140 was adjuvanted with either ALF or ALFQ liposomes, antibodies were induced that neutralized two HIV-1 tier 1 clade C strain pseudoviruses.     

Development of a single-dose recombinant CAMP factor entrapping poly(lactide-co-glycolide) microspheres-based vaccine against Streptococcus agalactiae

Streptococcus agalactiae is an important contagious bovine mastitis pathogen. Although it is well controlled and even eradicated in most Northern European and North American dairy herds, the prevalence of this pathogen remains very high in China. However, research on development of a vaccine against S. agalactiae mastitis is scarce. The aims of the present study were to: (1) develop a single-dose vaccine against S. agalactiae based on poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) encapsulated CAMP factor, a conserved virulent protein encoded by S. agalactiae’s cfb gene; and (2) evaluate its immunogenicity and protective efficacy in a mouse model. The cfb gene was cloned and expressed in a recombinant Escherichia coli strain Trans1-T1. The CAMP factor was tested to determine a safe dose range and then encapsulated in MS of PLGA (50:50) to assess its release pattern in vitro and immune reaction in vivo. Furthermore, a mouse model and a histopathological assay were developed to evaluate bacterial burden and vaccine efficacy. In the low dosage range (<100 μg), CAMP factor had no obvious toxicity in mice. The release pattern in vitro was characterized by an initial burst release (44%), followed by a sustained and slower release over 7 wk. In mice immunized with either pure CAMP factor protein or PLGA-CAMP, increased antibody titers were detected in the first 2 wk, whereas only PLGA-CAMP immunization induced a sustained increase of antibody titers. In mice vaccinated with PLGA-CAMP, mortality and bacteria counts were lower (compared to a control group) after S. agalactiae challenge. Additionally, no pathological lesions were detected in the vaccinated group. Therefore, PLGA-CAMP conferred protective efficacy against S. agalactiae in our mouse model, indicating its potential as a vaccine against S. agalactiae mastitis. Furthermore, the slow-release kinetics of PLGA MS warranted optimism for development of a single-dose vaccine.     

Specific memory B cell response and participation of CD4+ central and effector memory T cells in mice immunized with liposome encapsulated recombinant NE protein based Hepatitis E vaccine candidate

BackgroundLiposome encapsulated neutralizing epitope protein of Hepatitis E virus (HEV), rNEp, our Hepatitis E vaccine candidate, was shown to be immunogenic and safe in pregnant and non-pregnant mice and yielded sterilizing immunity in rhesus monkeys.MethodsThe current study in Balb/c mice assessed the levels and persistence of anti-HEV IgG antibodies by ELISA, frequencies of B, memory B, T and memory T cells by flow cytometry and HEV-specific IgG secreting memory B cells by ELISPOT till 420 days post immunization (PI) with 5 μg rNEp encapsulated in liposome based adjuvant (2 doses, 4 weeks apart). Mice immunized with a lower dose (1 μg) were assessed only for anamnestic response post booster dose.ResultsVaccine candidate immunized mice (5 μg dose) elicited strong anti-HEV IgG response that was estimated to persist for lifetime. At day 120 PI, frequency of memory B cells was higher in immunized mice than those receiving adjuvant alone. Anti-HEV IgG titers were lower in mice immunized with 1 μg dose. A booster dose yielded a heightened antibody response in mice with both high (>800 GMT, 5 μg) and low (⩽100 GMT, 1 μg) anti-HEV IgG titers. At day 6th post booster dose, HEV-specific antibody secreting plasma cells (ASCs) were detected in 100% and 50% of mice with high and low anti-HEV IgG titers, respectively, whereas the frequencies of CD4⁺ central and effector memory T cells were high in mice with high anti-HEV IgG titers only.ConclusionsTaken together, the vaccine candidate effectively generates persistent and anamnestic antibody response, elicits participation of CD4⁺ memory T cells and triggers memory B cells to differentiate into ASCs upon boosting. This approach of assessing the immunogenicity of vaccine candidate could be useful to explore the longevity of HEV-specific memory response in future HEV vaccine trials in human.     

Identification of a variant antigenic neutralizing epitope in hypervariable region 1 of avian leukosis virus subgroup J

Avian leukosis virus subgroup J (ALV-J) is a hypervariable oncogenic retrovirus that causes great economic loss in poultry. Antigenic variations in the variable regions make the development of an effective vaccine a challenging task. In the present study, we identified a variant antigenic neutralizing epitope using reverse vaccinology methods. First, we predicted the B-cell epitopes in gp85 gene of ALV-J strains by DNAman and bioinformatics. Fourteen candidate epitopes were selected and linked in tandem with glycines or serines as a multi-epitope gene. The expressed protein of multi-epitope gene can induce high-titer antibody that can recognize nature ALV-J and neutralize the infectivity of ALV-J strains. Next, we identified a high effective epitope using eight overlapping fragments of gp85 gene reacting with mAb 2D5 and anti-multi-epitope sera. The identified epitope contained one of the predicted epitopes and localized in hyervariable region 1 (hr1), indicating a variant epitope. To better understand if the variants of the epitope have a good antigenicity, we synthesized four variants to react with mAb 2D5 and anti-ALV-J sera. The result showed that all variants could react with the two kinds of antibodies though they showed different antigenicity, while could not react with ALV-J negative sera. Thus, the variant antigenic neutralizing epitope was determined as 137-LRDFIA/E/TKWKS/GDDL/HLIRPYVNQS-158. The result shows a potential use of this variant epitopes as a novel multi-epitope vaccine against ALV-J in poultry.     

Immune response in infants after universal high-dose hepatitis B vaccination: A community-based study in Beijing,China

BackgroundVaccination of infants beginning at birth is recommended to prevent Hepatitis B virus (HBV) infection in China. Compared to 5 μg/dose vaccine administered in other regions in China, a three-dose HB recombinant yeast vaccine at 10 μg/dose has been administered for infants within 24 h after birth, 1 month and 6 months of age in Beijing since 2006. In a community-based retrospective cohort study, factors influencing immunologic vaccine response were evaluated.MethodsA total of 3670 infants who completed a 3-dose 10 μg recombinant HB vaccine regimen and born to hepatitis B antigen negative mothers were included. The effect on anti-HBs titers of maternal nutrient status, infants’ birth condition, growth factors, timeliness of vaccination, dosing interval and the interval until post-vaccination serologic testing (PVST) were evaluated.ResultsA total of 3666 infants with no markers of HBV infection were included in analysis. The mean anti-HB titers were 1767.17 mIU/ml. Only 16.9% of the infants completed their PVST within 30–59 days after the final dose of vaccination. Multivariate linear regression analysis showed that delay in PVST (β = −0.097, p < 0.0001) and maternal folic acid supplementation (β = 0.067, p = 0.002) were associated with log-transformed anti-HB titers. Also a trend toward significant association was observed between the calcium supplementation of infants and log-transformed anti-HBs titers (β = 0.062, p = 0.057). Longer interval between dose 2 and dose 3 was not observed to increase the anti-HB titers after cofactors adjustment.ConclusionsOur findings illustrate the importance of timing of PVST to avoid unnecessary revaccination. Multi-center large cohort studies should verify the effect and magnitude of folate and calcium supplementation on HB vaccine response.     

The role of anti-NHba antibody in bactericidal activity elicited by the meningococcal serogroup B vaccine,MenB-4C

BackgroundMenB-4C (Bexsero®) is a multicomponent serogroup B meningococcal vaccine. For vaccine licensure, efficacy was inferred from serum bactericidal antibody (SBA) against three antigen-specific indicator strains. The bactericidal role of antibody to the fourth vaccine antigen, Neisserial Heparin binding antigen (NHba), is incompletely understood.MethodsWe identified nine adults immunized with two or three doses of MenB-4C who had sufficient volumes of sera and >3-fold increases in SBA titer against a strain with high NHba expression, which was mismatched with the other three MenB-4C antigens that elicit SBA. Using 1 month-post-immunization sera we measured the effect of depletion of anti-NHba and/or anti-Factor H binding protein (FHbp) antibodies on SBA.ResultsAgainst three strains matched with the vaccine only for NHba, depletion of anti-NHba decreased SBA titers by an average of 43–79% compared to mock-adsorbed sera (P < 0.05). Despite expression of sub-family A FHbp (mismatched with the sub-family B vaccine antigen), depletion of anti-FHbp antibodies also decreased SBA by 45–64% (P < 0.05). Depletion of both antibodies decreased SBA by 84–100%. Against a strain with sub-family B FHbp and expression of NHba with 100% identity to the vaccine antigen, depletion of anti-NHba decreased SBA by an average of 26%, compared to mock-adsorbed sera (P < 0.0001), and depletion of anti-FHbp antibody decreased SBA by 92% (P < 0.0001).ConclusionsAnti-NHba antibody can contribute to SBA elicited by MenB-4C, particularly in concert with anti-FHbp antibody. However, some high NHba-expressing strains are resistant, even with an exact match between the amino acid sequence of the vaccine and strain antigens.     

Multiple efficacy studies of an adenovirus-vectored foot-and-mouth disease virus serotype A24 subunit vaccine in cattle using homologous challenge

The safety and efficacy of an experimental, replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was examined in eight independent cattle studies. AdtA24 non-adjuvanted vaccine was administered intramuscularly to a total of 150 steers in doses ranging from approximately 1.0 × 10⁸ to 2.1 × 10¹¹ particle units per animal. No detectable local or systemic reactions were observed after vaccination. At 7 days post-vaccination (dpv), vaccinated and control animals were challenged with FMDV serotype A24 Cruzeiro via the intradermal lingual route. Vaccine efficacy was measured by FMDV A24 serum neutralizing titers and by protection from clinical disease and viremia after challenge. The results of eight studies demonstrated a strong correlation between AdtA24 vaccine dose and protection from clinical disease (R² = 0.97) and viremia (R² = 0.98). There was also a strong correlation between FMDV A24 neutralization titers on day of challenge and protection from clinical disease (R² = 0.99). Vaccination with AdtA24 enabled differentiation of infected from vaccinated animals (DIVA) as demonstrated by the absence of antibodies to the FMDV nonstructural proteins in vaccinates prior to challenge. Lack of AdtA24 vaccine shedding after vaccination was indicated by the absence of neutralizing antibody titers to both the adenovector and FMDV A24 Cruzeiro in control animals after co-mingling with vaccinated cattle for three to four weeks. In summary, a non-adjuvanted AdtA24 experimental vaccine was shown to be safe, immunogenic, consistently protected cattle at 7 dpv against direct, homologous FMDV challenge, and enabled differentiation of infected from vaccinated cattle prior to challenge.     

Simultaneous immunization of mice with Omp31 and TF provides protection against Brucella melitensis infection

Brucella vaccines consisting of live attenuated Brucella strains are currently used in livestock, but safety concerns preclude their application in humans. Subunit vaccines have recently emerged as safe and efficacious alternatives in both humans and animals. In this study, subunit vaccines were developed that consisted of a recombinant outer membrane protein (rOmp31) and the trigger factor chaperone protein (rTF) of Brucella melitensis, either alone or in combination. BALB/c mice that were immunized with rOmp31 + rTF showed comparable but slightly higher TF-specific IgG1 and IgG2a antibodies as compared to mice with rTF alone. Indeed, mice given this combination had titers of rOmp31-specific antibodies similar to those immunized with rOmp31 alone. In lymphocyte reactivation experiments, the splenocytes of immunized mice, whether given either of these antigens alone or as a cocktail, exhibited a strong antigen-specific recall proliferative response and expressed high amounts of IFN-γ, IL-12, IL-10 and IL-6. Both rTF and rTF + rOmp31 vaccinated mice exhibited significantly higher CD4 and CD8 levels compared to the PBS group. The combination of rOmp31 and rTF provided protection against B. melitensis infection comparable to that of vaccine strain Rev.1. In comparison to rTF alone, combination of rTF and rOmp31 caused only a slight increase in protection level. Although combination of rTF and rOmp31 caused a non-significant increase in IFN-γ induction, antibody level, proliferation index and CD4 and CD8 frequencies compared to rTF alone, its cumulative effects on aforesaid parameters may be viewed as a better efficacy.     

Comparison of homologous and heterologous prime-boost immunizations combining MVA-vectored and plant-derived VP2 as a strategy against IBDV

Different immunogens such as subunit, DNA or live viral-vectored vaccines against Infectious Bursal Disease virus (IBDV) have been evaluated in the last years. However, the heterologous prime-boost approach using recombinant modified vaccinia Ankara virus (rMVA), which has shown promising results in both mammals and chickens, has not been tried against this pathogen yet. IBD is a highly contagious and immunosuppressive disease of poultry that affects mainly young chicks. It is caused by IBDV, a double-stranded RNA virus carrying its main antigenic epitopes on the capsid protein VP2. Our objective was to evaluate the immune response elicited by two heterologous prime-boost schemes combining an rMVA carrying the VP2 mature gene (rVP2) and a recombinant VP2 protein produced in Nicotiana benthamiana (pVP2), and to compare them with the performance of the homologous pVP2-pVP2 scheme usually used in our laboratory. The SPF chickens immunized with the three evaluated schemes elicited significantly higher anti-VP2 antibody titers (p < 0.001) and seroneutralizing titers (p < 0.05) and had less T-cell infiltration (p < 0.001), histological damage (p < 0.001) and IBDV particles (p < 0.001) in their bursae of Fabricius when compared with control groups. No significant differences were found between both heterologous schemes and the homologous one. However, the rVP2-pVP2 scheme showed significantly higher anti-VP2 antibody titers than pVP2-rVP2 and a similar tendency was found in the seroneutralization assay. Conversely, pVP2-rVP2 had the best performance when evaluated through bursal parameters despite having a less potent humoral immune response. These findings suggest that the order in which rVP2 and pVP2 are combined can influence the immune response obtained. Besides, the lack of a strong humoral immune response did not lessen the ability to protect from IBDV challenge. Therefore, further research is needed to evaluate the mechanisms by which these immunogens are working in order to define the combination that performs better against IBDV.     

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.