Effect of delayed anthrax vaccine dose on Bacillus anthracis protective antigen IgG response and lethal toxin neutralization activity

We describe the Bacillus anthracis protective antigen IgG antibody response and the B. anthracis lethal toxin neutralization activity to a delayed dose of anthrax vaccine adsorbed (AVA, BioThrax^®) using validated assays. 373 individuals received 1, 2, or 3 priming doses, 18–24 months afterward, they received a delayed dose of AVA. Overall, 23.6% of subjects showed detectable anti-PA IgG before the boost, compared to 99.2% (P < 0.0001) 28 days after the boost. Geometric mean anti-PA IgG concentration (GMC) was 1.66 μg/mL before and 887.82 μg/mL after the boost (P < 0.0001). The proportion of individuals with four-fold increase in GMC following the boost ranged from 93.8% to 100%. Robust anti-PA IgG levels and B. anthracis lethal toxin neutralization activity are induced when an AVA dose is delayed as long as two years. These data support continuing with the vaccination schedule when a dose is delayed as long as two years rather than restarting the series.     

Comparison of immunogenicity and safety between two paediatric TBE vaccines

TBE vaccination strategies capable of inducing strong paediatric immunogenicity and acceptable reactogenicity are still under evaluation. This single-blind, multi-center, randomized, controlled, phase III clinical study compared the immunogenicity and safety of the two paediatric TBE vaccines available in Europe (FSME-IMMUN^® Junior and Encepur^® Children) following administration of two doses of either vaccine in 303 children aged 1–11 years. Regardless of immunological test or viral antigen used, immunological responses were consistently higher in children vaccinated with FSME-IMMUN^® Junior than those vaccinated with Encepur^® Children. FSME-IMMUN^® Junior is also non-inferior to Encepur^® Children, with respect to NT seropositivity rates (p < 0.0001). Systemic reaction rates were low and similar between the vaccines. However, among children aged 7–11 years, local reactions were significantly higher after the first (p < 0.01) and second (p < 0.001) vaccination with Encepur^® Children than with FSME-IMMUN^® Junior, affecting half the children in the former group: 22.4% and 10.2% with FSME-IMMUN^® Junior vs. 49.0% and 51.0% for Encepur^® Children.     

Anthrax vaccination induced anti-lethal factor IgG: fine specificity and neutralizing capacity

The efficacy biomarker of the currently licensed anthrax vaccine (AVA) is based on quantity and neutralizing capacity of anti-protective antigen (anti-PA) antibodies. However, animal studies have demonstrated that antibodies to lethal factor (LF) can provide protection against in vivo bacterial spore challenges. Improved understanding of the fine specificities of humoral immune responses that provide optimum neutralization capacity may enhance the efficacy of future passive immune globulin preparations to treat and prevent inhalation anthrax morbidity and mortality. This study (n = 1000) was designed to identify AVA vaccinated individuals who generate neutralizing antibodies and to determine what specificities correlate with protection. The number of vaccine doses, years post vaccination, and PA titer were associated with in vitro neutralization, reinforcing previous reports. In addition, African American individuals had lower serologic neutralizing activity than European Americans, suggesting a genetic role in the generation of these neutralizing antibodies. Of the vaccinated individuals, only 69 (6.9%) had moderate levels of anti-LF IgG compared to 244 (24.4%) with low and 687 (68.7%) with extremely low levels of IgG antibodies to LF. Using overlapping decapeptide analysis, we identified six common LF antigenic regions targeted by those individuals with moderate levels of antibodies to LF and high in vitro toxin neutralizing activity. Affinity purified antibodies directed against antigenic epitopes within the PA binding and ADP-ribotransferase-like domains of LF were able to protect mice against lethal toxin challenge. Findings from these studies have important implications for vaccine design and immunotherapeutic development.     

Enhanced early innate and T cell-mediated responses in subjects immunized with Anthrax Vaccine Adsorbed Plus CPG 7909 (AV7909)

NuThrax™ (Anthrax Vaccine Adsorbed with CPG 7909 Adjuvant) (AV7909) is in development. Samples obtained in a phase Ib clinical trial were tested to confirm biomarkers of innate immunity and evaluate effects of CPG 7909 (PF-03512676) on adaptive immunity. Subjects received two intramuscular doses of commercial BioThrax^® (Anthrax Vaccine Adsorbed, AVA), or two intramuscular doses of one of four formulations of AV7909. IP-10, IL-6, and C-reactive protein (CRP) levels were elevated 24–48 h after administration of AV7909 formulations, returning to baseline by Day 7. AVA (no CPG 7909) resulted in elevated IL-6 and CRP, but not IP-10. Another marker of CpG, transiently decreased absolute lymphocyte counts (ALCs), correlated with transiently increased IP-10. Cellular recall responses to anthrax protective antigen (PA) or PA peptides were assessed by IFN-γ ELISpot assay performed on cryopreserved PBMCs obtained from subjects prior to immunization and 7 days following the second immunization (study day 21). One-half of subjects that received AV7909 with low-dose (0.25 mg/dose) CPG 7909 possessed positive Day 21 T cell responses to PA. In contrast, positive T cell responses occurred at an 11% average rate (1/9) for AVA-treated subjects. Differences in cellular responses due to dose level of CPG 7909 were not associated with differences in humoral anti-PA IgG responses, which were elevated for recipients of AV7909 compared to recipients of AVA. Serum markers at 24 or 48 h (i.e. % ALC decrease, or increase in IL-6, IP-10, or CRP) correlated with the humoral (antibody) responses 1 month later, but did not correlate with cellular ELISpot responses. In summary, biomarkers of early responses to CPG 7909 were confirmed, and adding a CpG adjuvant to a vaccine administered twice resulted in increased T cell effects relative to vaccine alone. Changes in early biomarkers correlated with subsequent adaptive humoral immunity but not cellular immunity.     

Evaluation of hepatic changes and local and systemic immune responses in goats immunized with recombinant Peroxiredoxin (Prx) and challenged with Fasciola hepatica

Protection against Fasciola hepatica in goats immunized with Peroxiredoxin (Prx) was assessed. The experimental trial consisted of three groups of seven animals; group 1 were unimmunized and uninfected, group 2 were immunized with adjuvant only and group 3 were immunized with recombinant Prx in adjuvant (immunized and infected). Immunization with Prx in Quil A adjuvant, group 3, induced a reduction in fluke burden of 33.04% when compared to adjuvant control, group 2, although this difference was not significant. The hepatic gross and microscopical morphometric study revealed lower damage in the Prx-immunized compared to group 2 (p < 0.05). Furthermore, immunohistochemical studies revealed that the Prx-immunized group exhibited reduced infiltration of CD4⁺, CD8⁺, IFN-γ⁺ and TCR⁺ (p < 0.05); and CD2⁺ and IL-4⁺ (p < 0.001) in hepatic lesions. Levels of anti-Prx serum IgG in group 3 showed a significant increase at the 4th week after challenge infection compared with group 2 (p < 0.0001). This is the first report of ruminant immunization with recombinant Prx of F. hepatica. The study shows that this vaccine significantly reduces hepatic damage and encourages further studies to improve the vaccine efficacy.     

Phase 3 trial evaluating the immunogenicity and safety of a three-dose BioThrax regimen for post-exposure prophylaxis in healthy adults

Background

This study was conducted to support licensure of a post-exposure prophylaxis indication for BioThrax^® (anthrax vaccine adsorbed) concurrent with antimicrobials for individuals exposed to aerosolized anthrax spores.

Methods

The immunogenicity and safety of a three-dose regimen (0, 2, and 4 weeks) of BioThrax administered subcutaneously (SC) were evaluated in 200 healthy adults 18–65 years of age. Toxin-neutralizing antibody (TNA) was expressed as 50% neutralization factor (NF₅₀) at predetermined time points through Day 100. Safety was assessed by physical examinations, vital signs, solicited local and systemic reactions using web-enabled subject diaries, in-clinic solicited reactions, and unsolicited adverse events (AEs).

Results

The prospectively defined success criteria for the primary and secondary endpoints were met. This required the lower bound of the 95% confidence interval (CI) for the proportion of subjects with a TNA NF₅₀ value to be greater than 40% at Day 63 (primary), Day 70 (secondary) and Days 63–100 (secondary). At Day 63, 71% of subjects achieved a TNA NF₅₀ threshold value ≥0.56, with a lower bound of the 95% CI ≥40% (64%). The percentage of subjects achieving a TNA NF₅₀ threshold value ≥0.56 at Day 70 was 58% (95% CI: 50%, 65%), and the mean value on Days 63–100 (inclusive) was 53% (95% CI: 41%, 55%). The threshold TNA NF₅₀ value of 0.56 was developed from previous rabbit challenge and human immunogenicity studies. No related serious AEs occurred during the study, and no subjects withdrew from the study because of an AE. Tenderness and pain at the injection site were recorded most often in subject diaries following vaccination.

Conclusions

BioThrax, administered as three SC doses at 0, 2, and 4 weeks, was well tolerated. The prospectively defined success criteria for TNA levels on Days 63, 70, and 63–100 were achieved.     

Comparison of two commercial vaccines against visceral leishmaniasis in dogs from endemic areas: IgG,and subclasses,parasitism, and parasite transmission by xenodiagnosis

Background

The incidence of zoonotic canine visceral leishmaniasis (CVL) would decrease if dogs were effectively vaccinated; however, additional data on the efficacy of canine vaccines are required for their approved preventative use.

Purpose

To prospectively evaluate vaccination outcomes using two products commercially available in Brazil, with respect to adverse reactions (reactogenicity), humoral response, disease signs, parasitism, and parasite infectiousness in naturally exposed pet dogs in an endemic area of visceral leishmaniasis (VL).

Methods

From 2010 to 2012, healthy dogs were vaccinated with Leishmune^® (50 animals) or Leish-Tec^® (50 animals). Each dog was examined to identify clinical signs during peri- and post-vaccination procedures every 2 months for 11 months to identify the presence of parasites or parasite DNA in splenic samples using culturing or PCR, respectively. Levels of anti-Leishmania IgG, IgG1, and IgG2 were quantified in sera by ELISA and infectiousness was assessed by xenodiagnosis.

Results

Adverse effects occurred in 2.2% (1/45) and 13.0% (6/46) of the animals in the Leishmune^® and Leish-Tec^® groups, respectively. IgG levels peaked on the 21st day following the first dose of Leishmune^® and on the 21st day after the second dose of Leish-Tec^®. The final seropositivity rate for IgG was 32.5% (13/40) and 30.9% (13/42) in the Leishmune^® and Leish-Tec^® groups, respectively. The Leishmune^® group presented higher levels of IgG1 and IgG2 compared to the Leish-Tec^® group (p < 0.001), and ELISA reactivity in both vaccinated groups was significantly lower (p < 0.001) than in infected positive control dogs. Parasitism was observed in 12.2% (5/41) of the Leishmune^® group, and 7.9% (3/38) of the Leish-Tec^® group, with xenodiagnostic transmission rates of Leishmania to Lutzomyia longipalpis of 5.1% (2/39), and 5.4% (2/37), respectively.

Conclusions

No significant differences were observed in dogs vaccinated with Leishmune^® or Leish-Tec^®, with respect to LVC clinical aspects, parasitism, IgG seropositivity, or dog infectiousness. The Leishmune^®-vaccinated animals presented higher levels of IgG, IgG1, and IgG2. The animals vaccinated with Leish-Tec^® exhibited adverse reactions with greater frequency and severity.     

Serological Correlate of Protection in Guinea Pigs for a Recombinant Protective Antigen Anthrax Vaccine Produced from Bacillus brevis

ObjectiveRecombinant protective antigen (rPA) is the active pharmaceutical ingredient of a second generation anthrax vaccine undergoing clinical trials both in Korea and the USA. By using the rPA produced from Bacillus brevis pNU212 expression system, correlations of serological immune response to anthrax protection efficacy were analyzed in a guinea pig model.MethodsSerological responses of rPA anthrax vaccine were investigated in guinea pigs that were given single or two injections (interval of 4 weeks) of various amounts of rPA combined with aluminum hydroxide adjuvant. Guinea pigs were subsequently challenged by the intramuscular injection with 30 half-lethal doses (30LD₅₀) of virulent Bacillus anthracis spores. Serum antibody titers were determined by anti-PA IgG ELISA and the ability of antibodies to neutralize the cytotoxicity of lethal toxin on J774A.1 cell was measured through the toxin neutralizing antibody (TNA) assay.ResultsTo examine correlations between survival rate and antibody titers, correlation between neutralizing antibody titers and the extent of protection was determined. Toxin neutralization titers of at least 1176 were sufficient to confer protection against a dose of 30LD₅₀ of virulent anthrax spores of the H9401 strain. Such consistency in the correlation was not observed from those antibody titers determined by ELISA.ConclusionNeutralizing-antibody titers can be used as a surrogate marker.     

Clinical evaluation to determine the appropriate paediatric formulation of a tick-borne encephalitis vaccine

Two dose-finding studies and one open label safety study with a paediatric FSME-IMMUN^® formulation were conducted in children and adolescents aged 1–15 years (N = 3697). The 1.2 μg antigen dose was identified as the optimal dose, inducing high seroconversion rates following the primary vaccination series. Adolescents (aged 12–15 years) vaccinated with the optimal paediatric dose (1.2 μg) attained similarly high seroprotective rates to adults (aged 16–35 years) vaccinated with the 2.4 μg formulation of FSME-IMMUN^®. We concluded that the FSME-IMMUN^® paediatric vaccine formulation is safe and highly immunogenic, not only for children <12 years, but also for adolescents <16 years.     

Stochastic humoral immunity to Bacillus anthracis protective antigen: Identification of anti-peptide IgG correlating with seroconversion to Lethal Toxin neutralization

A substantial fraction of individuals vaccinated against anthrax have low to immeasurable levels of serum Lethal Toxin (LeTx)-neutralizing activity. The only known correlate of protection against Bacillus anthracis in the currently licensed vaccine is magnitude of the IgG response to Protective Antigen (PA); however, some individuals producing high serum levels of anti-PA IgG fail to neutralize LeTx in vitro. This suggests that non-protective humoral responses to PA may be immunodominant in some individuals. Therefore, to better understand why anthrax vaccination elicits heterogeneous levels of protection, this study was designed to elucidate the relationship between anti-PA fine specificity and LeTx neutralization in response to PA vaccination. Inbred mice immunized with recombinant PA produced high levels of anti-PA IgG and neutralized LeTx in vitro and in vivo. Decapeptide binding studies using pooled sera reproducibly identified the same 9 epitopes. Unexpectedly, sera from individual mice revealed substantial heterogeneity in the anti-PA IgG and LeTx neutralization responses, despite relative genetic homogeneity, shared environment and exposure to the same immunogen. This heterogeneity permitted the identification of specificities that correlate with LeTx-neutralizing activity. IgG binding to six decapeptides comprising two PA epitopes, located in domains I and IV, significantly correlate with seroconversion to LeTx neutralization. These results indicate that stochastic variation in humoral immunity is likely to be a major contributor to the general problem of heterogeneity in vaccine responsiveness and suggest that vaccine effectiveness could be improved by approaches that focus the humoral response toward protective epitopes in a greater fraction of vaccinees.     

Antigen-specific B memory cell responses to lipopolysaccharide (LPS) and invasion plasmid antigen (Ipa) B elicited in volunteers vaccinated with live-attenuated Shigella flexneri 2a vaccine candidates

We evaluated B memory responses in healthy adult volunteers who received one oral dose of live-attenuated Shigella flexneri 2a vaccine. LPS-specific B_M cells increased from a median of 0 at baseline to 20 spot forming cells (SFC)/10⁶ expanded cells following vaccination (p = 0.008). A strong correlation was found between post-vaccination anti-LPS B_M cell counts and peak serum anti-LPS IgG titers (rs = 0.95, p = 0.0003). Increases in B_M specific for IpaB approaching significance were also observed. In sum, oral vaccination with live-attenuated S. flexneri 2a elicits B_M cells to LPS and IpaB, suggesting that B_M responses to Shigella antigens should be further studied as a suitable surrogate of protection in shigellosis.     

Enhanced humoral and mucosal immune responses after intranasal immunization with chimeric multiple antigen peptide of LcrV antigen epitopes of Yersinia pestis coupled to palmitate in mice

Yersinia pestis is the causative agent of the most deadly disease plague. F1 and V antigens are the major vaccine candidates. Six protective epitopes of V antigen of varying length (15-25aa) were assembled on a lysine backbone as multiple antigen peptide (MAP) using standard Fmoc chemistry. Palmitate was coupled at amino terminus end. Amino acid analysis, SDS-PAGE, immunoblot and immunoreactivity proved the authenticity of MAP. MAP was immunized intranasally encapsulated in PLGA (polylactide-co-glycolide) microspheres and with/without/adjuvants murabutide and CpG ODN 1826 (CpG), in three strains of mice. Humoral and mucosal immune responses were studied till day 120 and memory response was checked after immunization with native V antigen on day 120. Epitope specific serum and mucosal washes IgG, IgA, IgG subclasses and specific activity were measured by indirect ELISA and sandwich ELISA, respectively. IgG and IgA peak antibody titers of all the MAP construct formulations in sera were ranging from 71,944 to 360,578 and 4493 to 28,644, respectively. MAP with CpG showed significantly high (p < 0.0001) antibody titers ranging from 101,690 to 360,578 for IgG and 28,644 for IgA. Mucosal peak IgG and IgA titers were ranging from 1425 to 8072 and 1425 to 7183, respectively in intestinal washes and 799-4528 and 566-4027, respectively in lung washes. MAP with CpG showed significantly high (p < 0.001) SIgA titers of 8000 in lung and 16,000 in intestinal washes. IgG isotyping revealed IgG2a/IgG1 ratio > 1 with CpG. Serum and mucosal antipeptide IgG and IgA specific activities correlated well with antibody titers. All the constituent peptides contributed towards immune response. Structural analysis of MAP revealed little or no interaction between the peptides. Present study showed MAP to be highly immunogenic with high and long lasting antibody titers in serum and mucosal washes with good recall response with/without CpG as an adjuvant which can be used for vaccine development for plague.     

Age-dependent association between IgG2 and IgG3 subclasses to Pf332-C231 antigen and protection from malaria,and induction of protective antibodies by sub-patent malaria infections,in Daraweesh

The certainty of the protective role of acquired immunity in malaria is the major drive for malaria vaccine development. In this study, we measured the levels of total IgG and IgG subclasses to four candidate malaria vaccine antigens; MSP2-3D7, MSP2-FC27, AMA-1 and Pf332-C231, in plasma obtained from a cohort of 136 donors from Daraweesh in Sudan. The cohort was followed for malaria infection for 9 years. After an initial analysis, the immune response to Pf332-C231 antigen was the only one found associated with protection, thus taken for further analysis. The number of previous clinical malaria episodes experienced by the donors was used as an index for relative protection. The number of these episodes was found to be negatively correlated with the levels of pre-existing total IgG, IgG2 and IgG3 to Pf332-C231 (correlation coefficient, CC – 0.215, p = 0.012; CC – 0.195, p = 0.023 and CC – 0.211, p = 0.014, respectively), and also with age (CC – 0.311, p < 0.001). Unexpectedly, equal levels of Pf332-C231 antibodies were induced by both patent and sub-patent infections regardless of the number of previous malaria episodes (1–7). Combining the correlation analysis with a multi-linear regression, three variable markers for protection were emerged, two age-dependent, the antibody response to Pf332-C231 and an unidentified marker (likely immune response to other antigens), and the third was an age-independent unidentified marker (possibly gene polymorphisms). In conclusion, this report suggests a protective effect for IgG subclasses to Pf332-C231 antigen against malaria.     

Induction of protection in mice against a respiratory challenge by a vaccine formulated with the Chlamydia major outer membrane protein adjuvanted with IC31®

IC31^®, a novel adjuvant, has been shown to be effective by increasing the levels of IFN-γ in animal models when delivered with several antigens. Here, we tested the ability of IC31^®, to enhance the protective ability of the Chlamydia trachomatis native major outer membrane protein (nMOMP). BALB/c mice were immunized by the intramuscular (i.m.) and subcutaneous (s.c.) routes with nMOMP + IC31^®. Another group of animals was immunized with nMOMP + Alum and as a negative control mice were immunized with ovalbumin (Ova) + IC31^®. Animals immunized with nMOMP + IC31^® developed high Chlamydia-specific IgG titers. The serum levels of IgG1 were higher than those of the IgG2a. T cells, from the spleens of mice immunized with IC31^®-adjuvanted nMOMP demonstrated a strong lymphoproliferative reaction to Chlamydia elementary bodies (EB) compared with the groups immunized with nMOMP + Alum or Ova + IC31^®. A similar comparison between these groups of mice revealed that the levels of IFN-γ in the supernatants from stimulated T-cells were significantly higher in animals immunized with nMOMP + IC31^®. Following an intranasal challenge with C. trachomatis, the mice immunized with IC31^®-adjuvanted nMOMP showed significant protection. The change in body weight, an indication of the severity of the infection, was significantly less reduced in mice immunized with nMOMP + IC31^®. Furthermore, the weight of the lungs, as well as the pulmonary Chlamydia burden, was significantly lower in the animals immunized with nMOMP + IC31^® when compared with the groups immunized with nMOMP + Alum or with Ova + IC31^®. In conclusion, these results provide the rationale for further preclinical testing of IC31^® using other chlamydial antigens, and support the potential evaluation of this adjuvant in human vaccines against Chlamydia.     

Surface display of Clonorchis sinensis enolase on Bacillus subtilis spores potentializes an oral vaccine candidate

Clonorchis sinensis (C. sinensis) infections remain the common public health problem in freshwater fish consumption areas. New effective prevention strategies are still the urgent challenges to control this kind of foodborne infectious disease. The biochemical importance and biological relevance render C. sinensis enolase (Csenolase) as a potential vaccine candidate. In the present study, we constructed Escherichia coli/Bacillus subtilis shuttle genetic engineering system and investigated the potential of Csenolase as an oral vaccine candidate for C. sinensis prevention in different immunization routes. Our results showed that, compared with control groups, both recombinant Csenolase protein and nucleic acid could induce a mixed IgG1/IgG2a immune response when administrated subcutaneously (P < 0.001), intraperitoneally (P < 0.01) and intramuscularly (P < 0.001) with worm reduction rate of 56.29%, 15.38% and 37.42%, respectively. More importantly, Csenolase could be successfully expressed as a fusion protein (55 kDa) on B. subtilis spore indicated by immunoblot and immunofluorescence assays. Killed spores triggered reactive Th1/Th2 immune response and exhibited protective efficacy against C. sinensis infection. Csenolase derived oral vaccine conferred worm reduction rate and egg reduction rate at 60.07% (P < 0.001) and 80.67% (P < 0.001), respectively. The shuttle genetic engineering system facilitated the development of oral vaccine with B. subtilis stably overexpressing target protein. Comparably vaccinal trails with Csenolase in different immunization routes potentialize Csenolase an oral vaccine candidate in C. sinensis prevention.     

Correlation between anthrax lethal toxin neutralizing antibody levels and survival in guinea pigs and nonhuman primates vaccinated with the AV7909 anthrax vaccine candidate

The anthrax vaccine candidate AV7909 is being developed as a next generation vaccine for a post-exposure prophylaxis (PEP) indication against anthrax. AV7909 consists of the Anthrax Vaccine Adsorbed (AVA, BioThrax®) bulk drug substance adjuvanted with the immunostimulatory oligodeoxynucleotide (ODN) compound, CPG 7909. The addition of CPG 7909 to AVA enhances both the magnitude and the kinetics of antibody responses in animals and human subjects, making AV7909 a suitable next-generation vaccine for use in a PEP setting. The studies described here provide initial information on AV7909-induced toxin-neutralizing antibody (TNA) levels associated with the protection of animals from lethal Bacillus anthracis challenge. Guinea pigs or nonhuman primates (NHPs) were immunized on Days 0 and 28 with various dilutions of AV7909, AVA or a saline or Alhydrogel + CPG 7909 control. Animals were challenged via the inhalational route with a lethal dose of aerosolized B. anthracis (Ames strain) spores and observed for clinical signs of disease and mortality. The relationship between pre-challenge serum TNA levels and survival following challenge was determined in order to calculate a threshold TNA level associated with protection. Immunisation with AV7909 induced a rapid, highly protective TNA response in guinea pigs and NHPs. Surprisingly, the TNA threshold associated with a 70% probability of survival for AV7909 immunized animals was substantially lower than the threshold which has been established for the licensed AVA vaccine. The results of this study suggest that the TNA threshold of protection against anthrax could be modified by the addition of an immune stimulant such as CPG 7909 and that the TNA levels associated with protection may be vaccine-specific.     

Evaluation of the protective efficacy of recombinant protective antigen vaccine (GC1109)-immunized human sera using passive immunization in a mouse model

The protective efficacy of human sera from vaccinated individuals with a new recombinant protective antigen anthrax vaccine (GC1109) against lethal spore challenge was evaluated in a mouse model. Eighteen human sera were selected from the vaccinated individuals based on their toxin neutralizing assay (TNA) titer (ED₅₀ of 55 to 668). The selected sera were diluted and passively transferred to A/J mice and the mice were subsequently challenged with 100 × LD₅₀ of Bacillus anthracis Sterne spores. The correlation between the survival rate of passively immunized mice and the TNA ED₅₀ of transferred sera was presented (r = 0.873, P-value < 0.001). The estimated TNA titer for 50% survival rate against lethal challenge was 197 (95% confidence interval of 149 and 260). The result suggest that GC1109 is protective against exposure to B. anthracis and the TNA titer of vaccinated serum can be an indicator for protective efficacy.     

Serum antibody response to three non-typeable Haemophilus influenzae outer membrane proteins during acute otitis media and nasopharyngeal colonization in otitis prone and non-otitis prone children

Non-typeable Haemophilus influenzae (NTHi) is the most common bacteria responsible for episodic acute otitis media (AOM; non-otitis prone), recurrent AOM (rAOM; otitis prone) and AOM treatment failure (AOMTF) in children. In this 3.5 years of prospective study, we measured the serum antibody response to outer membrane proteins D, P6 and OMP26 of NTHi in children with AOM (n = 26), rAOM (n = 32), AOMTF (n = 27). The geometric mean titers (GMTs) of IgG at their acute AOM visit against Protein D in otitis prone children were significantly lower compared to AOMTF (p value < 0.01) and non-otitis prone (p value < 0.03) children; otitis prone children had significantly lower IgG levels to P6 compared to AOMTF children (p value < 0.02); otitis prone children had significantly lower IgG levels to OMP26 compared to AOMTF children (p value < 0.04). Comparing acute to convalescent titers after AOM, otitis prone and AOMTF children had no significant change in total IgG against all the three proteins, while non-otitis prone children had significant increases to Protein D. Anti-protein D, P6 and OMP26 antibody levels measured longitudinally during NP colonization between age 6 and 24 months in 10 otitis prone children and 150 non-otitis prone children showed <2-fold increases over time in otitis prone children compared to >4 fold increases in the non-otitis prone children (p value < 0.001). We conclude that otitis prone children mount less of an IgG serum antibody response toward Protein D, P6 and OMP26 after AOM which may account for recurrent infections. The data on acute sera of otitis prone vs non-otitis prone children and the acute-to-convalescence response in non-otitis prone children point to a possible link of anti-PD to protection. Moreover, the data suggest that otitis prone children should be evaluated for their responses to Protein D, P6 and OMP26 vaccine antigens of NTHi.     

Enhancement of the immune responses to vaccination against foot-and-mouth disease in mice by oral administration of an extract made from Rhizoma Atractylodis Macrocephalae (RAM)

This study was designed to evaluate the effects of oral administration of a water extract made from the Rhizoma Atractylodis Macrocephalae (RAM) on the immune responses in mice immunized with FMDV type O vaccine. Thirty-five ICR mice were randomly divided into five groups with seven animals in each group, and orally administered daily for 4 days at a dose equivalent to 0, 0.0625, 0.125, 0.25 or 0.5 g of dried RAM, respectively. After that, the animals were subcutaneously immunized twice with FMDV vaccine at 2-week intervals. Blood samples were collected 3 weeks after boosting for measurement of FMDV-specific IgG titers and the IgG subclasses, lymphocyte proliferation as well as production IL-5 and IFN-γ. Results indicated that serum FMDV-specific IgG titers and the IgG subclass responses were significantly enhanced in mice orally administered RAM at the dose of 0.25 or 0.5 g when compared with the control group (P < 0.05). Splenocyte proliferation in response to Con A and LPS and production of IL-5 and IFN-γ by splenocytes were also significantly enhanced (P < 0.05). Considering the immunomodulatory effect and safety of RAM demonstrated in this study, this herb deserves further investigation to evaluate its potential improvement of FMD vaccination in other animals such as pigs, goats and cattle.     

Immunotherapy with the saponin enriched-Leishmune vaccine versus immunochemotherapy in dogs with natural canine visceral leishmaniasis

Leishmune^®, the first licensed vaccine for prophylaxis against canine visceral leishmaniasis (CVL) and is also immunotherapeutic when used with double saponin adjuvant concentration. The Leishmune^® therapeutic vaccine was assessed for immunotherapy (IT) in 31 infected dogs and for immunochemotherapy (ICT) in combination with allopurinol or amphotericinB/allopurinol, in 35 dogs. Compared to infected untreated control dogs, at month 3, both treatments increased the proportion of dogs showing intradermal response to Leishmania antigen to a similar extent (from 8 to 67%, in the IT and to 76%, in the ICT groups), and conversely reduced from 100 to 38% (IT) and to 18% (ICT) the proportion of symptomatic cases, from 54 to 12% (IT) and to 15% (ICT) the proportion of parasite evidence in lymph nodes and from 48 to 19% (IT) and 12% (ICT) the proportion of deaths, indicating that the immunotherapy with enriched-Leishmune^® vaccine promotes the control of the clinical and parasitological signs of CVL rendering most dogs asymptomatic although PCR positive. By month 8, negative lymph node PCR results were obtained in 80% of the ICT-treated dogs, but only in 33% of the IT group (p = 0.0253), suggesting that the combination of additional chemotherapy with Leishmune^®-enriched saponin vaccination abolished, not only the symptoms but also the latent infection condition, curing the dogs. The animals were followed up until 4.5 years after the beginning of the experiment and, compared to the untreated control group at month 3 (12/25 dogs; 48%), a decrease in the rate of CVL deaths was only seen after ICT treatment (7/35 dogs; 20%; 0.0273) but not after IT treatment (10/31 dogs; 32%; p = 0.278), pointing out an additional advantage of the ICT treatment with the enriched-Leishmune^® in the control and cure of CVL.