Adjuvant potential of resiquimod with inactivated Newcastle disease vaccine and its mechanism of action in chicken

Resiquimod (R-848), an imidazoquinoline compound, is a potent synthetic Toll-like receptor (TLR) 7 agonist. Although the solitary adjuvant potential of R-848 is well established in mammals, such reports are not available in avian species hitherto. Hence, the adjuvant potential of R-848 was tested in SPF chicken in this study. Two week old chicks were divided into four groups (10 birds/group) viz., control (A), inactivated Newcastle disease virus (NDV) vaccine prepared from velogenic strain (B), commercial oil adjuvanted inactivated NDV vaccine prepared from lentogenic strain (C) and inactivated NDV vaccine prepared from velogenic strain with R-848 (D). Booster was given two weeks post primary vaccination. Humoral immune response was assessed by haemagglutination inhibition (HI) test and ELISA while the cellular immune response was quantified by lymphocyte transformation test (LTT) and flow cytometry post-vaccination. Entire experiment was repeated twice to check the reproducibility. Highest HI titre was observed in group D at post booster weeks 1 and 2 that corresponds to mean log₂ HI titre of 6.4 ± 0.16 and 6.8 ± 0.13, respectively. The response was significantly higher than that of group B or C (P < 0.01). LTT stimulation index (P ≤ 0.01) as well as CD4⁺ and CD8⁺ cells in flow cytometry (P < 0.05) were significantly high and maximum in group D. Group D conferred complete protection against virulent NDV challenge, while it was only 80% in group B and C. To understand the effects of R-848, the kinetics of immune response genes in spleen were analyzed using quantitative real-time PCR after R-848 administration (50 μg/bird, i.m. route). Resiquimod significantly up-regulated the expression of IFN-α, IFN-β, IFN-γ, IL-1β, IL-4, iNOS and MHC-II genes (P < 0.01). In conclusion, the study demonstrated the adjuvant potential of R-848 when co-administered with inactivated NDV vaccine in SPF chicken which is likely due to the up-regulation of immune response genes.     

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.     

Design,synthesis and evaluation of a gonadotropin releasing hormone-based subunit vaccine in rams (Ovis aries)

Immunocastration using gonadotropin-releasing hormone (GnRH)-based vaccines has been investigated in rams to reduce aggressive and sexual behaviour and to control meat quality. Despite considerable efforts, a practical GnRH vaccine has yet to be developed for rams. In the present study, a A GnRH-lipopeptide vaccine (GnRH-LP) including two copies of GnRH, 2-amino-d,l-hexadecanoic acid (C16), and a unique T helper epitope, was examined in rams. Rams received a primary and secondary vaccination of GnRH-LP without additional adjuvant (Group 1) or with the adjuvant AdjuVac™ (Group 2). In both Group 1 and 2 anti-GnRH antibody titres increased after secondary vaccination, however, the antibody titres were higher (p < 0.01) for rams in Group 2. The latter rams showed a marked decrease in testicular size. The marked and sustained reduction in testicular size in rams treated with GnRH-LP + AdjuVac™ provides the basis for an effective immunocastration vaccine in rams.     

Rabies pre-exposure prophylaxis elicits long-lasting immunity in humans

Despite the availability of safe and effective human vaccines, rabies remains a global threat, with an estimated 60,000 human deaths annually attributed to rabies. Pre-exposure prophylaxis against rabies infection is recommended for travelers to countries where rabies is endemic, and also for those with a higher risk of exposure. In this study, the rabies-specific neutralising antibody responses in a cohort of rabies-vaccinated recipients over a period of twenty years have been assessed. In particular, the antibody response to primary vaccinations and boosters, and the waning of antibody post primary vaccination and post booster were investigated. The significance of gender, age at vaccination, vaccine manufacturer and vaccination intervals were also evaluated. These data confirm that rabies vaccination can elicit a neutralising antibody response that can remain at detectable levels for a number of years, without additional booster vaccinations. The antibody response following both primary vaccination and booster was significantly influenced by the gender of the subject (p = 0.002 and 0.03 respectively), with supportive data that suggests an effect by the make of vaccine administered following primary vaccination, with significantly higher VNA titres observed for one vaccine manufactured prior to 2006 (p < 0.001) in a small subset of recipients (n = 5). Additionally, the decay rate was demonstrated through the overall decline in antibody titre for all individuals, which was a 37% and 27% reduction per 2-fold change in time following primary and booster vaccination respectively. Individuals within older age groups demonstrated a significantly faster decline in antibody titre following the primary vaccination course (p = 0.012). Rate of decline in antibody titre was also significantly influenced by the vaccine make following primary course (p < 0.001). The assessment of neutralising antibody titre decline has also provided an insight into the most appropriate timing for booster administration, and enabled the prediction of long term titres from post-vaccination antibody titres.     

A novel live attenuated anthrax spore vaccine based on an acapsular Bacillus anthracis Sterne strain with mutations in the htrA,lef and cya genes

We recently reported the development of a novel, next-generation, live attenuated anthrax spore vaccine based on disruption of the htrA (High Temperature Requirement A) gene in the Bacillus anthracis Sterne veterinary vaccine strain. This vaccine exhibited a highly significant decrease in virulence in murine, guinea pig and rabbit animal models yet preserved the protective value of the parental Sterne strain. Here, we report the evaluation of additional mutations in the lef and cya genes, encoding for the toxin components lethal factor (LF) and edema factor (EF), to further attenuate the SterneΔhtrA strain and improve its compatibility for human use. Accordingly, we constructed seven B. anthracis Sterne-derived strains exhibiting different combinations of mutations in the htrA, cya and lef genes. The various strains were indistinguishable in growth in vitro and in their ability to synthesise the protective antigen (PA, necessary for the elicitation of protection). In the sensitive murine model, we observed a gradual increase (ΔhtrA < ΔhtrAΔcya < ΔhtrAΔlef < ΔhtrAΔlefΔcya) in attenuation – up to 10⁸-fold relative to the parental Sterne vaccine strain. Most importantly, all various SterneΔhtrA derivative strains did not differ in their ability to elicit protective immunity in guinea pigs. Immunisation of guinea pigs with a single dose (10⁹ spores) or double doses (>10⁷ spores) of the most attenuated triple mutant strain SterneΔhtrAlef^MUTΔcya induced a robust immune response, providing complete protection against a subsequent respiratory lethal challenge. Partial protection was observed in animals vaccinated with a double dose of as few as 10⁵ spores. Furthermore, protective immune status was maintained in all vaccinated guinea pigs and rabbits for at least 40 and 30 weeks, respectively.     

Pooled-sera hSBA titres predict individual seroprotection in infants and toddlers vaccinated with 4CMenB

The Serum Bactericidal Antibody assay with human complement (hSBA) using individual immune sera is a surrogate of protection for meningococcal vaccines. Strain coverage of 4CMenB, a licensed vaccine against serogroup B meningococcal (MenB) disease, has been extensively assessed in hSBA using pooled sera, directly or through the Meningococcal Antigen Typing System (MATS). The extent to which pooled-sera hSBA titres reflect individual protection is not yet fully understood.We analysed more than 17000 individual hSBA titres from infants and toddlers vaccinated with 4CMenB, pooled-serum hSBA titres from subsets therein and MATS data from a 40 strain panel representative of invasive MenB disease in England and Wales.Individual hSBA titres segregated in two normal distributions, respectively from responding and non-responding subjects (fit_model-data: r = 0.996, p-values <0.05). No individual subject showed abnormally high titres compared to the distributions. Also, when sera from the same subjects were tested individually and in pool, pooled-sera titre and average of individual titres from the same group were substantially indistinguishable (r = 0.97, p-value <<0.001).We identified a robust mathematical relationship between the mean of individual hSBA titres and the proportion of subjects achieving a protective titre (seroprotection rate, r = 0.95, p-value <<0.001). Using this relation, the seroprotection rate in 15 groups of vaccinees tested against 11 diverse meningococcal isolates was accurately predicted by the hSBA titre of the respective pooled sera (average prediction error 9%).Finally, strains defined covered by MATS had on average 77% predicted seroprotection rate (interquartile range, IQR: 66–100%) and 39% for non-covered strains (IQR: 19–46%).We conclude that seroprotection rates in infants and toddlers vaccinated with 4CMenB can be accurately predicted by pooled-serum hSBA, and that strain coverage defined by MATS is associated with high seroprotection rates.SummaryThe Serum Bactericidal Antibody assay (SBA) from individual sera is a surrogate of protection for meningococcal vaccines. We show that SBA performed on pooled sera predicts individual protection.     

Durability of immunogenicity and strain coverage of MenBvac,a meningococcal vaccine based on outer membrane vesicles: Lessons of the Normandy campaign

ObjectivesMenBvac® is an outer membrane vesicle (OMV)-based meningococcal vaccine. From 2006 to 2012, it was used to control a clonal B outbreak in Normandy (France). We aimed to analyse the durability of the response against the epidemic strain and coverage beyond the vaccine strain. These data should help to optimize the use of OMV-containing vaccines, such as the new 4CMenB/Bexsero® recombinant vaccine.MethodsSerum bactericidal activity (SBA) was measured in two cohorts of children who received their first dose of MenBvac® at 1–5 years of age and accepted to provide a blood sample either one or four years after a 2 + 1 + 1 schedule. All sera were tested against the outbreak strain. Sera from responder subjects were also tested against 12 additional B or C strains which were chosen to entirely, partially, or not at all match the two variable regions (VR1 and VR2) of the PorA vaccine strain.ResultsOnly 47.9% and 31.3% of subjects showed an SBA titre consistent with protection one and four years, respectively, after the last boost. Protective SBA titres were observed in all sera against B or C strains that entirely matched P1.7,16, and was high (75–100%) for all but one strain that partially matched VR1 or VR2. Extrapolating our data to the OMV component of 4CMenB/Bexsero® suggests that 14.5% of the current B strains would be covered based on PorA matching to the OMV component of 4CMenB/Bexsero® (regardless of the coverage of the three other vaccine components).ConclusionsOur data confirm that OMV-based vaccines elicit short-lasting SBA titres and may require repeated booster injections. However, strain coverage may be greater than expected.     

Immune responses following meningococcal serogroups A,C, Y and W polysaccharide vaccination in C2-deficient persons: Evidence for increased levels of serum bactericidal antibodies

Complement C2 deficiency (C2D) is associated with immunological diseases and increased susceptibility to invasive infections caused by encapsulated bacteria such as Neisseria menigitidis. In this study we evaluate the immunogenicity of vaccination against N. menigitidis in C2D.C2D patients (n = 22) and controls (n = 52) were given a tetravalent meningococcal polysaccharide vaccine. Serum bactericidal antibody (SBA) titres (serogroups A, C, Y and W) were analysed using a rabbit complement source. Levels of IgG, IgM, and IgA, factor B, and factor H, polymorphisms of MBL and Fc-gamma receptors were determined.The C2D patients responded with an increased SBA titre to all four serogroups (p < 0.001). The response rates define as SBA titres ≥8 were found to be between 85.7% and 92.5%. The post-vaccination titres for serogroups C, Y and W were equal to healthy controls. C2D patients with a history of invasive infection had a lower post-vaccination SBA titres both compared to healthy C2D persons (p = 0.03) and compared to controls (p < 0.0001). We found that the G2M*n/G2M*n genotype were associated with a higher SBA titres after immunization (p = 0.03). None of the other investigated immunological factors appear to be important in influencing the vaccine responses. Autoimmune diseases in C2D did not affect the vaccine response.In general, vaccination against meningococci gave rise to antibody responses in the C2D patients that equal healthy controls. The response rate was lower to serogroup A and among C2D patients with history of invasive infections. The presence of G2M*n/G2M*n genotype was associated with higher SBA titres after immunization.     

Development of a dual vaccine for prevention of Brucella abortus infection and Escherichia coli O157:H7 intestinal colonization

Zoonoses that affect human and animal health have an important economic impact. In the study now presented, a bivalent vaccine has been developed that has the potential for preventing the transmission from cattle to humans of two bacterial pathogens: Brucella abortus and Shiga toxin-producing Escherichia coli (STEC). A 66 kDa chimeric antigen, composed by EspA, Intimin, Tir, and H7 flagellin (EITH₇) from STEC, was constructed and expressed in B. abortus Δpgm vaccine strain (BabΔpgm). Mice orally immunized with BabΔpgm(EITH₇) elicited an immune response with the induction of anti-EITH₇ antibodies (IgA) that clears an intestinal infection of E. coli O157:H7 three times faster (t = 4 days) than mice immunized with BabΔpgm carrier strain (t = 12 days). As expected, mice immunized with BabΔpgm(EITH₇) strain also elicited a protective immune response against B. abortus infection. A Brucella-based vaccine platform is described capable of eliciting a combined protective immune response against two bacterial pathogens with diverse lifestyles—the intracellular pathogen B. abortus and the intestinal extracellular pathogen STEC.     

Protective efficacy of a DNA vaccine construct encoding the VP2 gene of infectious bursal disease and a truncated HSP70 of Mycobacterium tuberculosis in chickens

Infectious bursal disease (IBD) is an acute, infectious, immunosuppressive disease affecting young chicken worldwide. The etiological agent IBD virus (IBDV) is a double stranded RNA virus with outer capsid protein VP2 of IBDV is the major antigenic determinant capable of inducing neutralizing antibody. DNA vaccines encoding VP2 has been extensively studied achieving only partial protection. However, the efficacy of DNA vaccines against IBDV can be augmented by choosing a potential molecular adjuvant. The goal of the present study is to evaluate the immune response and protective efficacy of a DNA vaccine encoding the C-terminal domain of the heat shock protein 70 (cHSP70) of Mycobacterium tuberculosis gene genetically fused with the full length VP2 gene of IBDV (pCIVP2-cHSP70) in comparison to a ‘DNA prime-protein boost’ approach and a DNA vaccine encoding the VP2 gene (pCIVP2) alone. The results indicate that both pCIVP2-cHSP70 and ‘DNA prime-protein boost’ elicited humoral as well as cellular immune responses. Chickens in the pCIVP2-cHSP70 and ‘DNA prime-protein boost’ groups developed significantly higher levels of ELISA titer to IBDV antigen compared to the group immunized with pCIVP2 alone (p < 0.01). However, significantly higher levels of lymphocyte proliferative response, IL-12 and IFN-γ production were found in the pCIVP2-cHSP70 group compared to ‘DNA prime-protein boost’ group. Additionally, chickens immunized with pCIVP2-cHSP70 and ‘DNA prime-protein boost’ vaccines were completely protected against the vvIBDV whereas pCIVP2 DNA vaccine alone was able to protect only 70%. These findings suggest that the truncated C-terminal HSP70 mediated DNA vaccine genetically fused with the VP2 gene construct stimulated both humoral and cell mediated immune responses and conferred complete protection against IBDV. This novel strategy is perhaps a seminal concept in utilizing HSP70 as an adjuvant molecule to elicit an immune response against IBD affecting chickens.     

Reduced schedules of 4CMenB vaccine in infants and catch-up series in children: Immunogenicity and safety results from a randomised open-label phase 3b trial

BackgroundThis study evaluated the immunogenicity and safety of a licensed meningococcal serogroup B vaccine (4CMenB) administered alone according to reduced schedules in infants or catch-up series in children.MethodsIn this open-label, multicentre, phase 3b study (NCT01339923), infants randomised 1:1:1 received 4CMenB: 2 + 1 doses at 3½–5–11 months or 6–8–11 months of age, 3 + 1 doses at ages 2½–3½–5–11 months. Children aged 2–10 years received 2 catch-up doses administered 2 months apart. Immune responses were measured by hSBA assays against 4 strains specific for vaccine components fHbp, NadA, PorA and NHBA. Sufficiency of immune responses was defined in groups with 2 + 1 doses schedules as a lower limit ≥70% for the 97.5% confidence interval of the percentage of infants with hSBA titres ≥4, 1 month post-dose 2 for fHbp, NadA, PorA. Adverse events were collected for 7 days post-vaccination; serious adverse events (SAEs) throughout the study.Results754 infants and 404 children were enrolled. Post-primary vaccination, 98–100% of infants across all groups developed hSBA titres ≥4 for fHbp, NadA, PorA, and 48–77% for NHBA. Sufficiency of immune responses in infants receiving 2 + 1 schedules was demonstrated for fHbp, NadA, PorA after 2 doses of 4CMenB, as pre-specified criteria were met. Following receipt of 2 catch-up doses, 95–99% of children developed hSBA titres ≥4 for 4CMenB components. Similar safety profiles were observed across groups. A total of 45 SAEs were reported, 3 of which were related to vaccination.ConclusionReduced infant schedules and catch-up series in children were immunogenic and safe, having the potential to widen 4CMenB vaccine coverage.FundingGlaxoSmithKline Biologicals SA.     

Concomitant administration of a fully liquid,ready-to-use DTaP-IPV-HB-PRP-T hexavalent vaccine with a meningococcal serogroup C conjugate vaccine in infants

DTaP-IPV-HB-PRP-T or hexavalent vaccines are indicated for primary and booster vaccination of infants and toddlers against diphtheria, tetanus, pertussis, hepatitis B, poliomyelitis and invasive diseases caused by Haemophilus influenzae type b (Hib). The present study evaluates the safety and immunogenicity of a ready-to-use hexavalent vaccine when co-administered with a meningococcal serogroup C conjugate (MenC) vaccine in infants.This was a phase III, open-label, randomised, multicentre study conducted in Finland. Healthy infants, aged 46–74 days (n = 350), were randomised in a ratio of 1:1 to receive DTaP-IPV-HB-PRP-T vaccine at two, three and four months, either with a MenC vaccine co-administered at two and four months (Group 1; n = 175) or without MenC vaccine (Group 2; n = 175). All infants also received routine rotavirus and 13-valent pneumococcal conjugate vaccines.The proportion of participants with an anti-HBs concentration ⩾10 mIU/mL assessed one month after the third dose of DTaP-IPV-HB-PRP-T vaccine was 97.5% [95%CI: 93.1–99.3] in the coadministration group and 96.1% [95%CI: 91.8–98.6] in the group without MenC vaccine. The proportion of participants with an anti-MenC SBA titre ⩾8 assessed one month after the second dose of MenC vaccine was 100% in the coadministration group. Both primary objectives were achieved.Secondary immunogenicity and safety analyses showed that co-administration of DTaP-IPV-HB-PRP-T and MenC vaccines did not impact the immune response to the antigens of each of the two vaccines. All vaccines were well tolerated and the safety profile of DTaP-IPV-HB-PRP-T vaccine was similar in both groups.ClinicalTrials.gov identifier: NCT01839175; EudraCT number: 2012-005547-24.     

Comparative evaluation of the protective efficacy of two formulations of a recombinant Chlamydia abortus subunit candidate vaccine in a mouse model

Chlamydia abortus (C. abortus) is the causative agent of ovine enzootic abortion (OEA) and poses a zoonotic risk to pregnant women. Current live attenuated 1B vaccines are efficacious but cause disease in vaccinated animals and inactivated vaccines are only marginally protective. We tested the ability of a new C. abortus subunit vaccine candidate based on the conserved and immunogenic polymorphic membrane protein D (Pmp18D) formulated in CpG1826 + FL (Fms-like tyrosine kinase 3 Ligand; Flt3L) or Vibrio cholerae ghosts (VCG) to induce innate and cross protective immunity against genital C. abortus infection. We found that delivery of rPmp18D with VCG was more effective than with CpG + FL in up-regulating the expression of molecules critically involved in T cell activation and differentiation, including MHC II, CD40, CD80, and CD86, activation of TLRs and NLRP3 inflammasome engagement, and secretion of IL-1β and TNF-α but not IL-10 and IL-4. rVCG-Pmp18D-immunized mice elicited more robust antigen-specific IFN-γ, IgA and IgG2c antibody responses compared to CpG + FL-delivered rPmp18D. Based on the number of mice with positive vaginal cultures, length of vaginal shedding, and number of inclusion forming units recovered following challenge with the heterologous C. abortus strain B577, vaccine delivery with VCG induced superior protective immunity than delivery with a combination of CpG1826 and FL, a nasal DC-targeting adjuvant. These results demonstrate that the ability of VCG to enhance protective immunity against genital C. abortus infection is superior to that of CpG + FL adjuvants.     

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.     

Immune responses elicited by the GEN-003 candidate HSV-2 therapeutic vaccine in a randomized controlled dose-ranging phase 1/2a trial

PurposeGEN-003 is a candidate therapeutic HSV-2 vaccine containing a fragment of infected cell protein 4 (ICP4.2), a deletion mutant of glycoprotein D2 (gD2ΔTMR), and Matrix-M2 adjuvant. In a dose-ranging phase 1/2a clinical trial, immunization with GEN-003 reduced viral shedding and the percentage of reported herpetic lesion days. Here we examine the immune responses in the same trial, to characterize vaccine-related changes in antibody and cell-mediated immunity.MethodsParticipants with genital HSV-2 infection were randomized to 1 of 3 doses of GEN-003, antigens without adjuvant, or placebo. Subjects received 3 intramuscular doses, three weeks apart, and were monitored for viral shedding, lesions and immunogenicity. Antibody titers were measured by ELISA and neutralization assay in serum samples collected at baseline and 3 weeks post each dose. T cell responses were assessed pre-immunization and 1 week post each dose by IFN-γ ELISpot and intracellular cytokine staining. Blood was also collected at 6 and 12 months to monitor durability of immune responses.ResultsAntibody and T cell responses increased with vaccination and were potentiated by adjuvant. Among the doses tested, the rank order of reduction in viral shedding follows the ranking of fold change from baseline in T cell responses. Some immune responses persisted up to 12 months.ConclusionAll measures of immunity are increased by vaccination with GEN-003; however, a correlate of protection is yet to be defined.     

Compound 48/80 acts as a potent mucosal adjuvant for vaccination against Streptococcus pneumoniae infection in young mice

Streptococcus pneumoniae, a major respiratory pathogen, is a leading cause of death among children worldwide. Mucosal vaccination is a recommended method to prevent respiratory infection. However, development of mucosal vaccination is usually hindered due to the lack of safe and effective mucosal adjuvants. Mast cell activator compound 48/80 (C48/80) has been used as a mucosal adjuvant in immunization of adult mice, but its adjuvanticity is not clear in the immunization of young mice. In this study, the adjuvanticity of C48/80 was evaluated when intranasally co-administrated with a pneumococcal vaccine candidate strain SPY1 in a young mice model in comparison with a classical mucosal adjuvant cholera toxin (CT) and a relatively safe mucosal adjuvant Pam2CSK4. All three adjuvants enhanced antibody responses, whereas serum IgG titers were maintained at a stable level during the 3 months after the last immunization only in the SPY1 + C48/80 and SPY1 + CT groups. Furthermore, both the SPY1 + CT group and the SPY1 + C48/80 group induced strong Th17 immune response. Notably, C48/80 showed the exceptional ability to promote the clearance of nasal pneumococcal colonization which CT and Pam2CSK4 did not show. We found that C48/80's ability to induce protection against nasal pneumococcal colonization depended on B cells and IL-17A. Additionally, C48/80, as a mucosal adjuvant, showed a greater ability to protect young mice against lethal pneumococcal infection than CT. In comparison with CT, C48/80 also showed a favorable safety. These results reveal a promising perspective for using C48/80 as a mucosal adjuvant to improve protection against pneumococcal diseases early in life.     

Antigenic variation of LaSota and genotype VII Newcastle disease virus (NDV) and their efficacy against challenge with velogenic NDV

Continued monitoring and evaluation of vaccine efficacy against prevalent or newly isolated strains has great importance in advising Newcastle disease (ND) immunization strategy. In this study, we systematically analysed the antigenic variation between genotype VII NDV aSG10 and the commercial vaccine strain LaSota, and assessed their efficacy against challenge with velogenic NDV by serological analysis and animal testing. We show that these two viruses are antigenically distinguishable; anti-NDV aSG10 hyper-immune sera demonstrated higher haemagglutination inhibition (HI) titres (11.13 ± 0.30log₂) against the aSG10 virus, compared with titres against LaSota (9.53 ± 0.50log₂). Conversely, the hyper-immune sera from LaSota showed higher HI titres against LaSota virus (9.73 ± 0.36log₂), but 2-fold lower HI titre against aSG10 (8.87 ± 0.38log₂). Each serum neutralised heterologous virus, but neutralisation titres were always 3- to 6-fold higher against its homologous strain than heterologous virus. The cross-reactivity R value between aSG10 and LaSota was 0.23, indicating that they are loosely related with major antigenic differences within a single serotype. The results of animal tests revealed that the aSG10 vaccine had a significantly higher protection rate than the LaSota vaccine against genotype VII NDV, regardless of intramuscular (IM) or eye drop/intranasal (ED/IN) route of SG10 challenge. Compared with IM administration, chicken flocks needed higher HI antibody levels to obtain sufficient protection when challenged by the natural ED/IN route. These results are highly informative for better control of ND in the poultry industry.     

N-terminal disulfide-bridging of Borrelia outer surface protein C increases its diagnostic and vaccine potentials

OspC is the main target for IgM in early-stage Lyme disease. As such it is employed as its native or recombinant form in routine immunoassays for the determination of Borrelia-specific antibodies. However, recombinant OspC has so far not shown the antigenicity of the native protein. The latter contains an intrinsic signal sequence and an adjacent cysteine residue, the attachment site of the lipid membrane anchor which has been discussed to have an adjuvant effect on the immune reaction. In expression experiments, we have found a recombinant variant, an OspC covalently homodimerized via an N-terminal disulfide bridge, that shows a remarkably enhanced antigenicity without lipid attachment. Three such OspCs derived from different Borrelia strains were subsequently expressed in E. coli and purified under non-reducing conditions. In non-reducing SDS-PAGE, OspC(Δ1–18) exhibited a 48-kDa band of dimeric OspC. When incubated with IgM-OspC-positive human sera, the reaction at 48 kDa was always stronger than at 24 kDa of monomeric OspC(Δ1–18, C19G). A lineblot with OspC(Δ1–18) also showed a higher diagnostic accuracy than that obtained with OspC(Δ1–18, C19G) based on a higher affinity of IgM for the dimeric form. When used for the immunization of mice, dimeric OspC(Δ1–18) induced consistent high-titre antibodies against OspC, whereas OspC(Δ1–18, C19G) failed to provoke significant titres in some animals. We conclude that the disulfide-bridging of 2 OspC molecules via their N termini forms a complex that is more suitable for the determination of IgM-OspC and is a promising candidate for a monovalent vaccine.     

Enhancement of VP6 immunogenicity and protective efficacy against rotavirus by VP2 in a genetic immunization

VP2/VP6 virus like particles (VLPs) are very effective in inducing protection against the rotavirus infection in animal models. Individually, VP6 can also induce protection. However, there is no information about the immunogenicity of VP2. The aim of this work was to evaluate the efficacy of DNA vaccines codifying for VP2 or VP6, alone or combined, to induce protection against the rotavirus infection. Murine rotavirus VP2 and VP6 genes were cloned into the pcDNA3 vector. Adult BALB/c mice were inoculated three times by intramuscular (i.m.) injections with 100 or 200 µg of pcDNA3-VP2 or pcDNA3-VP6 alone or co-administered with 100 µg of pcDNA3-VP2/100 µg of pcDNA3-VP6. Two weeks after the last inoculation, mice were challenged with the wild type murine rotavirus strain epizootic diarrhea of infant mice (EDIM_wt). We found that both plasmids, pcDNA3-VP2 and pcDNA3-VP6, were able to induce rotavirus-specific serum antibodies, but not intestinal rotavirus-specific IgA; only 200 µg of pcDNA3-VP6 induced 35% protection against the infection. A similar level of protection was found when mice were co-administered with 100 µg of pcDNA3-VP2/100 µg of pcDNA3-VP6 (1:1 ratio). However, the best protection (up to 58%) occurred when mice were inoculated with 10 µg of pcDNA3-VP2/100 µg of pcDNA3-VP6 (1:10 ratio). These results indicate that the DNA plasmid expressing VP6 is a better vaccine candidate that the one expressing VP2. However, when co-expressed, VP2 potentiates the immunogenicity and protective efficacy of VP6.     

Safety and immunogenicity of an MF59®-adjuvanted A/H1N1 pandemic influenza vaccine in children from three to seventeen years of age

ObjectivesThis study was designed to identify the optimal dose of an MF59^®-adjuvanted, monovalent, A/H1N1 influenza vaccine in healthy paediatric subjects.MethodsSubjects aged 3–8 years (n = 194) and 9–17 years (n = 160) were randomized to receive two primary doses of A/H1N1 vaccine containing either 3.75 μg antigen with half a standard dose of MF59 adjuvant, 7.5 μg antigen with a full dose of MF59, or (children 3–8 years only), a non-adjuvanted 15 μg formulation. A booster dose of MF59-adjuvanted seasonal influenza vaccine including homologous A/H1N1 strain was given one year after priming. Immunogenicity was assessed by haemagglutination inhibition (HI) and microneutralization assays. Vaccine safety was assessed throughout the study (up to 18 months).ResultsA single priming dose of either MF59-adjuvanted formulation was sufficient to meet the European licensure criteria for pandemic influenza vaccines (HI titres ≥1:40 > 70%; seroconversion > 40%; and GMR > 2.5). Two non-adjuvanted vaccine doses were required to meet the same licensure criteria. After first and second doses, percentage of subjects with HI titres ≥1:40 were between 97% and 100% in the adjuvanted vaccine groups compared with 68% and 91% in the non-adjuvanted group, respectively. Postvaccination seroconversion rates ranged from 91% to 98% in adjuvanted groups and were 68% (first dose) and 98% (second dose) in the non-adjuvanted group. HI titres ≥1:330 after primary doses were achieved in 69% to 90% in adjuvanted groups compared with 41% in the non-adjuvanted group. Long-term antibody persistence after priming and a robust antibody response to booster immunization were observed in all vaccination groups. All A/H1N1 vaccine formulations were generally well tolerated. No vaccine-related serious adverse events occurred, and no subjects were withdrawn from the study due to an adverse event.ConclusionsAn MF59-adjuvanted influenza vaccine containing 3.75 μg of A/H1N1 antigen was well tolerated and sufficiently immunogenic to meet all the European licensure criteria after a single dose in healthy children 3–17 years old.