Decrease of the incidence of human and canine visceral leishmaniasis after dog vaccination with Leishmune in Brazilian endemic areas

Leishmune^®, the first prophylactic vaccine licensed against canine visceral leishmaniasis (CVL), has been used in Brazil since 2004, where seropositive dogs are sacrificed in order to control human visceral leishmaniasis (VL). We demonstrate here that vaccination with Leishmune^® does not interfere with the serological control campaign (110,000 dogs). Only 1.3% of positivity (76 among 5860) was detected among Leishmune^® uninfected vaccinees. We also analyzed the possible additive effect of Leishmune^® vaccination over dog culling, on the decrease of the incidence of CVL and VL in two Brazilian endemic areas, from 2004 to 2006. In Araçatuba, a 25% of decline was seen in CVL with a 61% decline in human cases, indicating the additive effect of Leishmune^® vaccination of 5.7% of the healthy dogs (1419 dogs), on regular dog culling. In Belo Horizonte (BH), rising curves of canine and human incidence were observed in the districts of Barreiro, Venda Nova and Noroeste, while the canine and human incidence of Centro Sul, Leste, Nordeste, Norte, Pampulha and Oeste, started to decrease or maintained a stabilized plateau after Leishmune^® vaccination. Among the districts showing a percent decrease of human incidence (−36.5%), Centro Sul and Pampulha showed the highest dog vaccination percents (63.27% and 27.27%, respectively) and the lowest dog incidence (−3.36% and 1.89%, respectively). They were followed by Oeste, that vaccinated 25.30% of the animals and experienced an increase of only 12.86% of dog incidence and by Leste and Nordeste, with lower proportions of vaccinees (11.72% and 10.76%, respectively) and probably because of that, slightly higher canine incidences (42.77% and 35.73%). The only exception was found in Norte district where the reduced human and canine incidence were not correlated to Leishmune^® vaccination. Much lower proportions of dogs were vaccinated in Venda Nova (4.35%), Noroeste (10.27%) and Barreiro (0.09%) districts, which according to that exhibited very increased canine incidences (24.48%, 21.85% and 328.57%, respectively), and pronounced increases in human incidence (14%, 4% and 17%, respectively). The decrease of canine (p = −0.008) and human incidences (p = −0.048) is directly correlated to the increase of the number of vaccinated dogs, confirming the additive control effect of Leishmune^® vaccination over dog culling, reducing the parasite reservoir, protecting dogs and, in this way, reducing the risk of transmission of VL to humans and becoming a new effective control tool.     

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.     

T-cell-derived cytokines,nitric oxide production by peripheral blood monocytes and seric anti-Leishmania (Leishmania) chagasi IgG subclass patterns following immunization against canine visceral leishmaniasis using Leishvaccine and Leishmune

It is generally accepted that distinct cytokine expression by the cellular immune response plays a critical role during the outcome of experimental as well as natural canine visceral Leishmaniasis (CVL). Despite the fact that immunoprophylaxis of CVL has become an important control strategy and protective immunity has been reported upon immunization with whole as well as purified Leishmania antigens, the cytokine profile of T-cells triggered by anti-CVL vaccines still remain to be determined. Herein, we have developed a cross-sectional analysis of German Shepherd dogs submitted to vaccination protocols with Leishvaccine (n = 6) and Leishmune^® (n = 6). Our data identified distinct immunological profiles elicited by Leishvaccine and Leishmune^®, with the Leishvaccine triggering a mixed, IFN-γ and IL-4, cytokine pattern in addition to high levels of anti-Leishmania IgG1, whereas the Leishmune^® induced an immunological pattern characterized by enhanced levels of IFN-γ, NO and anti-Leishmania chagasi IgG2. It was important to notice that despite the distinct immunological patterns triggered by Leishvaccine and Leishmune^®, the ability of both immunobiologicals to activate T-cell-derived IFN-γ synthesis further suggesting their immunogenic potential against CVL. These findings added support to our hypothesis that both antigenic composition (whole antigen in Leishvaccine versus purified antigen in Leishmune^®) as well as the adjuvant nature (BGC and saponin) used for the vaccine formulation may count for the distinct activation pattern observed.     

Treatment of canine visceral leishmaniasis by the vaccine Leish-111f + MPL-SE

Immunotherapy of canine visceral leishmaniasis (CVL) may provide an alternative to both marginally effective chemotherapy and undesired euthanasia of infected dogs and could have a great impact not only on animal welfare, but also on control of human disease. Therefore, we examined the potential immunotherapeutic efficacy of the subunit vaccine Leish-111f + MPL-SE, which has undergone rigorous preclinical testing and been demonstrated safe in human clinical trials. Two separate trials were performed in Salvador, Brazil, to evaluate the vaccine for therapeutic efficacy against CVL caused by natural infection: an Open Trial and a Blinded Trial. In the Open Trial 59 dogs with clinically active CVL were sequentially allocated to four groups: group 1 received Leish-111f + MPL-SE; group 2 was treated with Glucantime; group 3 received a combination of the vaccine and Glucantime; and group 4 was given no treatment. At the 6-month assessment, the 13 non-treated dogs had either died or showed no clinical improvement. In contrast, most dogs in groups 1–3 showed initial improvement (100%, 80%, and 92%, respectively). Upon evaluation for a mean of 36 months after therapy, the following cure rates were observed: 75% for group 1 dogs (exact 95% confidence interval [CI] 43–95%), 64% for group 2 dogs (exact 95% CI 31–89%), and 50% for group 3 dogs (exact 95% CI 19–81%). Therapeutic efficacy of the Leish-111f + MPL-SE vaccine was reconfirmed in a subsequent Blinded Trial. The vaccine was effective for mild cases of CVL and was compromised in dogs with severe disease. Although further studies are required to understand mechanisms of action, the Leish-111f + MPL-SE vaccine is a promising tool to control VL in both dogs and humans.     

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.     

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.     

Factors associated with herpes zoster vaccination status and acceptance of vaccine recommendation in community pharmacies

Objectives

1. Identify patient characteristics, awareness and knowledge associated with herpes zoster (HZ) vaccination status. 2. Identify self-reported reasons for not receiving Zostavax^®. 3. Assess the impact of a patient education program by measuring post-intervention interest in obtaining the Zostavax^® vaccine across reasons for being unvaccinated.

Methods

A cross-sectional design with patients aged 60 years or older in 51 community pharmacies in Alabama and Florida was utilized. During the Introductory Pharmacy Practice Experience in summer 2013, 137 immunization-certified student pharmacists provided patient education on HZ and Zostavax^® to unvaccinated patients using the Shingles Vaccine Information Statement. An interviewer-administered questionnaire assessed patient awareness of HZ, receipt of recommendations to receive Zostavax^®, and patient characteristics as well as vaccination status, reasons for being unvaccinated and interest in obtaining Zostavax^® after the educational session.

Results

A total of 681 patients participated in a conversation with a student pharmacist regarding their HZ vaccination status. The majority were female (57.6%), white (84.6%), and unvaccinated (73.6%). Results from logistic regression suggest that participants were more likely to be vaccinated if they received a recommendation from a healthcare provider (OR = 5.15), received the influenza vaccine during the previous year (OR = 3.56), or knew that Zostavax^® was recommended for individuals over 60 years of age (OR = 3.55). The most frequently provided reasons for being unvaccinated were “haven’t gotten around to it/forgot” (27.2%) and “didn’t know it was needed” (27.1%). After the educational session, the majority (72.5%) of unvaccinated patients were interested in speaking with their pharmacist or physician about receiving Zostavax^®. Analysis suggests that interest differed across initial reason for being unvaccinated (χ² = 64.44; p < 0.01).

Implications/conclusions

Recommendations from healthcare providers are valued by patients and can improve vaccination rates. The patient education program increased interest in receiving Zostavax^® and this interest differed depending on the reason provided for being unvaccinated.     

Anthrax vaccine adsorbed: Further evidence supporting continuing the vaccination series rather than restarting the series when doses are delayed

Whether to restart or continue the series when anthrax vaccine doses are missed is a frequent medical management problem. We applied the noninferiority analysis model to this prospective study comparing the Bacillus anthracis protective antigen (PA) IgG antibody response and lethal toxin neutralization activity at day 28 to the anthrax vaccine adsorbed (AVA) (Biothrax^®) administered on schedule or delayed. A total of 600 volunteers were enrolled: 354 in the on-schedule cohort; 246 in the delayed cohort. Differences were noted in immune responses between cohorts (p < 0.0001) and among the racial categories (p < 0.0001). Controlling for covariates, the delayed cohort was non-inferior to the on-schedule cohort for the rate of 4-fold rise in both anti-PA IgG concentration (p < 0.0001) and TNA ED₅₀ titers (p < 0.0001); as well as the mean log₁₀-transformed anti-PA IgG concentration (p < 0.0001) and the mean log₁₀-transformed TNA ED₅₀ titers (p < 0.0001). Providing a missed AVA dose after a delay as long as 5–7 years, elicits anti-PA IgG antibody and TNA ED₅₀ responses that are robust and non-inferior to the responses observed when the 6-month dose is given on-schedule. These important data suggest it is not necessary to restart the series when doses of the anthrax vaccine are delayed as long as 5 or more years.     

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.     

The immune enhancement of a novel soy lecithin/β-glucans based adjuvant on native Neospora caninum tachyzoite extract vaccine in mice

Efficient, cost-effective and safe Th1-immunity-inducing vaccine formulations are paramount for achieving protection against Neospora caninum. In this study, a new adjuvant (Providean-AVEC^®) was used in the development of a N. caninum vaccine and evaluated in a mouse model. Soluble N. caninum tachyzoite native protein extract (sNcAg) was selected as vaccine antigen based on its capacity to activate production of pro-inflammatory cytokines on dendritic cells. Vaccines containing 4 and 0.4 μg of sNcAg, and Providean-AVEC^®, ISCOM-Matrix or aluminum hydroxide (Alum) were tested in BALB/c mice. While mice vaccinated with 4 μg of sNcAg + Providean-AVEC^® developed specific antibodies shortly after the first dose, the rest of the high antigen payload formulations only induced seroconversion after the booster. Mice immunized with the high payload ISCOM vaccine (4 μg sNcAg) or with either low or high payload Providean-AVEC^® formulations (0.4 μg and 4 μg sNcAg, respectively) elicited higher IgG2a than IgG1 serum levels, and IFN-γ anamnestic responses with a Th1-cytokine biased profile. These animals had no histological signs of cerebral lesions and parasite burden assessed by quantitative real-time PCR was not detected. Vaccine preparations including Providean-AVEC^® as adjuvant limited N. canimum multiplication even with only a tenth of antigen payload compared to vaccines containing other adjuvants. Using adjuvants to specifically activate dendritic cells, combined with a careful antigen selection can enhance cellular responses to inert N. caninum vaccines.     

Probiotics reduce symptoms of antibiotic use in a hospital setting: A randomized dose response study

Probiotics are known to reduce antibiotic associated diarrhea (AAD) and Clostridium difficile associated diarrhea (CDAD) risk in a strain-specific manner. The aim of this study was to determine the dose-response effect of a four strain probiotic combination (HOWARU^® Restore) on the incidence of AAD and CDAD and severity of gastrointestinal symptoms in adult in-patients requiring antibiotic therapy. Patients (n = 503) were randomized among three study groups: HOWARU^® Restore probiotic 1.70 × 10¹⁰ CFU (high-dose, n = 168), HOWARU^® Restore probiotic 4.17 × 10⁹ CFU (low-dose, n = 168), or placebo (n = 167). Subjects were stratified by gender, age, and duration of antibiotic treatment. Study products were administered daily up to 7 days after the final antibiotic dose. The primary endpoint of the study was the incidence of AAD. Secondary endpoints included incidence of CDAD, diarrhea duration, stools per day, bloody stools, fever, abdominal cramping, and bloating. A significant dose-response effect on AAD was observed with incidences of 12.5, 19.6, and 24.6% with high-dose, low-dose, and placebo, respectively (p = 0.02). CDAD was the same in both probiotic groups (1.8%) but different from the placebo group (4.8%; p = 0.04). Incidences of fever, abdominal pain, and bloating were lower with increasing probiotic dose. The number of daily liquid stools and average duration of diarrhea decreased with higher probiotic dosage. The tested four strain probiotic combination appears to lower the risk of AAD, CDAD, and gastrointestinal symptoms in a dose-dependent manner in adult in-patients.     

Evaluation of recombinant Mycoplasma hyopneumoniae P97/P102 paralogs formulated with selected adjuvants as vaccines against mycoplasmal pneumonia in pigs

Pig responses to recombinant subunit vaccines containing fragments of eight multifunctional adhesins of the Mycoplasma hyopneumoniae (Mhp) P97/P102 paralog family formulated with Alhydrogel^® or Montanide™ Gel01 were compared with a commercial bacterin following experimental challenge. Pigs, vaccinated intramuscularly at 9, 12 and 15 weeks of age with either of the recombinant formulations (n = 10 per group) or Suvaxyn^® M. hyo (n = 12), were challenged with Mhp strain Hillcrest at 17 weeks of age. Unvaccinated, challenged pigs (n = 12) served as a control group. Coughing was assessed daily. Antigen-specific antibody responses were monitored by ELISA in serum and tracheobronchial lavage fluid (TBLF), while TBLF was also assayed for cytokine responses (ELISA) and bacterial load (qPCR). At slaughter, gross and histopathology of lungs were quantified and damage to epithelial cilia in the porcine trachea was evaluated by scanning electron microscopy. Suvaxyn^® M. hyo administration induced significant serological responses against Mhp strain 232 whole cell lysates (wcl) and recombinant antigen F3_P216, but not against the remaining vaccine subunit antigens. Alhydrogel^® and Montanide™ Gel01-adjuvanted antigen induced significant antigen-specific IgG responses, with the latter adjuvant eliciting comparable Mhp strain 232 wcl specific IgG responses to Suvaxyn^® M. hyo. No significant post-vaccination antigen-specific mucosal responses were detected with the recombinant vaccinates. Suvaxyn^® M. hyo was superior in reducing clinical signs, lung lesion severity and bacterial load but the recombinant formulations offered comparable protection against cilial damage. Lower IL-1β, TNF-α and IL-6 responses after challenge were associated with reduced lung lesion severity in Suvaxyn^® M. hyo vaccinates, while elevated pathology scores in recombinant vaccinates corresponded to cytokine levels that were similarly elevated as in unvaccinated pigs. This study highlights the need for continued research into protective antigens and vaccination strategies that will prevent Mhp colonisation and establishment of infection.     

Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB,an adenovirus-rabies recombinant vaccine

Twenty-seven red foxes (Vulpes vulpes) were each offered a bait containing ONRAB^®, a recombinant oral rabies vaccine that uses a human adenovirus vector to express the immunogenic rabies virus glycoprotein; 10 controls received no vaccine baits. Serum samples collected from all foxes before treatment, and each week post-treatment for 16 weeks, were tested for the presence of rabies virus neutralizing antibody (RVNA). In the bait group, a fox was considered a responder to vaccination if serum samples from 3 or more consecutive weeks had RVNA ≥0.5 IU/ml. Using this criterion, 79% of adult foxes (11/14) and 46% of juveniles (6/13) responded to vaccination with ONRAB^®. Serum RVNA of adults first tested positive (≥0.5 IU/ml) between weeks 1 and 3, about 4 weeks earlier than in juveniles. Adults also responded with higher levels of RVNA and these levels were maintained longer. Serum samples from juveniles tested positive for 1–4 consecutive weeks; in adults the range was 2–15 weeks, with almost half of adults maintaining titres above 0.5 IU/ml for 9 or more consecutive weeks. Based on the kinetics of the antibody response to ONRAB^®, the best time to sample sera of wild adult foxes for evidence of vaccination is 7–11 weeks following bait distribution. Thirty-four foxes (25 ONRAB^®, 9 controls) were challenged with vulpine street virus 547 days post-vaccination. All controls developed rabies whereas eight of 13 adult vaccinates (62%) and four of 12 juvenile vaccinates (33%) survived. All foxes classed as non-responders to vaccination developed rabies. Of foxes considered responders to vaccination, 80% of adults (8/10) and 67% of juveniles (4/6) survived challenge. The duration of immunity conferred to foxes would appear adequate for bi-annual and annual bait distribution schedules as vaccinates were challenged 1.5 years post-vaccination.     

Oral vaccination and protection of striped skunks (Mephitis mephitis) against rabies using ONRAB

Skunks are one of the most important rabies vector species in North America due to their wide geographic distribution, high susceptibility to the rabies virus, and tendency to inhabit areas around human dwellings and domestic animals. Oral vaccination is a cost-effective, socially acceptable technique often used to control rabies in terrestrial wildlife; however, control of rabies in skunks has proven especially challenging due to the lack of a vaccine effective by the oral route in this species. In this study, we examined the antibody response of captive striped skunks (Mephitis mephitis) to ONRAB^® and tested the protection afforded by the vaccine against rabies virus. Thirty-one skunks were each offered one ONRAB^® vaccine bait, 25 skunks were administered ONRAB^® via direct instillation into the oral cavity (DIOC) and ten controls received no vaccine. A blood sample was collected from controls and vaccinates 6 weeks prior to treatment, and then 5 and 7 weeks post-vaccination (PV). A competitive ELISA was used to detect rabies antibody (RAb). Pre-vaccination sera for all skunks, and sera for all controls throughout the serology study, were negative for RAb. Fifty-eight percent (18/31) of skunks in the bait group and 100% (25/25) of skunks that received ONRAB^® DIOC had detectable RAb by 7 week PV. All 10 controls succumbed to experimental rabies infection. In the group of skunks administered ONRAB^® DIOC, 100% (23/23) survived challenge 247 days PV. Survival of skunks presented ONRAB^® baits was 81% (25/31). In the bait group, all 18 skunks that had detectable RAb by 7 week PV survived challenge. Seven additional skunks without detectable RAb prior to week 7 PV also survived. Lack of any remarkable pathology in study animals, together with positive serology and challenge results, supports that ONRAB^® is a safe and effective oral rabies vaccine for use in skunks.     

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.     

CD4 T-cell responses among adults and young children in response to Streptococcus pneumoniae and Haemophilus influenzae vaccine candidate protein antigens

We characterized cytokine profiles of CD4⁺ T-helper (h) cells in adults and young children to ascertain if responses occur to next-generation candidate vaccine antigens PspA, PcpA, PhtD, PhtE, Ply, LytB of Streptococcus pneumonia (Spn) and protein D and OMP26 of non-typeable Haemophilus influenzae (NTHi). Adults had vaccine antigen-specific Th1 and Th2 cells responsive to all antigens evaluated whereas young children had significant numbers of vaccine antigen-specific CD4⁺ T cells producing IL-2, (p = 0.004). Vaccine antigen-specific CD4⁺ T-cell populations in adults were largely of effector (T_EM) and/or central memory (T_CM) phenotypes as defined by CD45RA⁻CCR7⁺ or CD45RA⁻CCR7⁻ respectively; however among young children antigen-specific IL-2 producing CD4⁺ T cells demonstrated CD45RA⁺ expression (non-memory cells). We conclude that adults have circulating memory CD4⁺ T cells (CD45RA⁻) that can be stimulated by all the tested Spn and NTHi protein vaccine candidate antigens, whereas young children have a more limited response.     

Genetically modified Bifidobacterium displaying Salmonella-antigen protects mice from lethal challenge of Salmonella Typhimurium in a murine typhoid fever model

We developed a novel vaccine platform utilizing Bifidobacterium as an antigen delivery vehicle for mucosal immunization. Genetically modified Bifidobacterium longum displaying Salmonella-flagellin on the cell surface was constructed for the oral typhoid vaccine. The efficiency of this vaccine was evaluated in a murine model of typhoid fever. We then orally administered 2.5 × 10⁷ CFU of the recombinant Bifidobacterium longum (vaccine) or parental Bifidobacterium longum, or PBS to BALB/C mice every other day for 2 weeks. After the administration, a total of 42 mice (14 mice in each group) were challenged with Salmonella Typhimurium (1.0 × 10⁷ CFU/mouse). While 12 mice in the PBS group, and 9 in the parental Bifidobacterium longum group died (median survival: 14 and 25 days), only two in the vaccine group died. These data support that our genetically modified Bifidobacterium antigen delivery system offers a promising vaccine platform for inducing efficient mucosal immunity.     

Humoral immune response to oral rabies vaccination in raccoon kits: Problems and implications

Little is known about the immunogenicity of RABORAL V-RG^® (V-RG), an oral rabies vaccine, in raccoon kits (Procyon lotor). The objectives of this study were to characterize the immunogenicity of V-RG in young kits and investigate the potential impact of maternal antibodies on response to vaccination of nursing raccoon kits. Raccoon kits (n = 30) were vaccinated at either 3 weeks of age, 7 weeks of age, or assigned as contact controls. Nineteen kits (73%) that were whelped by unvaccinated mothers responded to V-RG exposure (orally or indirect contact) by production of detectable rabies virus neutralizing antibodies (RVNA) while 7 (27%) kits did not respond to V-RG exposure. Four kits were whelped by a mother with high levels of RVNA and all four kits acquired maternal rabies antibodies. At approximately 9 months of age, all kits were inoculated with a killed rabies vaccine, IMRAB3^®. The kits which initially responded to V-RG oral vaccination or contact with vaccinated littermates demonstrated a rapid anamnestic response. In contrast, the V-RG non-responders and those with acquired maternal antibodies exhibited a primary immune response to IMRAB3^®, where RVNA levels were substantially lower on days 5 and 7 than the levels in the animals with an anamnestic response. These findings suggest that the naïve contact kits and the nonresponsive kits most likely remained susceptible to rabies virus infection whereas the ones demonstrating response to V-RG would not have been susceptible to a rabies virus infection.     

Induction of immunogenicity by live attenuated Leishmania donovani centrin deleted parasites in dogs

Zoonotic visceral leishmaniasis, caused by the intracellular protozoan parasite Leishmania infantum, is a neglected tropical disease that is often fatal when untreated. Dogs are considered the main reservoir of L. infantum in zoonotic VL as the presence of infected dogs may increase the risk for human infection. Canine visceral leishmaniasis (CVL) is a major veterinary and public health problem in Southern Europe, Middle East and South America. Control of animal reservoirs relies on elimination of seropositive dogs in endemic areas. However, treatment of infected dogs is not considered a favorable approach as this can lead to emergence of drug resistance since the same drugs are used to treat human infections. Therefore, vaccination against CVL remains the best alternative in control of the animal reservoirs. In this study, we present data on the immunogenicity profile of a live attenuated parasite LdCen^−/− in a canine infection model and compared it to that of Leishmune^®, a commercially available recombinant vaccine. The immunogenicity of the LdCen^−/− parasites was evaluated by antibody secretion, production of intracytoplasmic and secreted cytokines, activation and proliferation of T cells. Vaccination with LdCen^−/− resulted in high immunogenicity as revealed by the higher IgGTotal, IgG1, and IgG2 production and higher lymphoproliferative response. Further, LdCen^−/− vaccinated dogs showed higher frequencies of activated CD4+ and CD8+ T cells, IFN-γ production by CD8+ T cells, increased secretion of TNF-α and IL-12/IL-23p40 and decreased secretion of IL-4. These results contribute to the understanding of immunogenicity elicited by live attenuated L. donovani parasites and, consequently, to the development of effective vaccines against visceral leishmaniasis.     

Local and systemic immune responses in pigs intramuscularly injected with an inactivated Mycoplasma hyopneumoniae vaccine

The immune response induced by intramuscular administration of a commercial inactivated Mycoplasma hyopneumonie whole-cell vaccine (Suvaxyn^®MH One) was investigated in conventional M. hyopneumoniae-free pigs. The animals were assigned randomly to two groups: non-vaccinated and vaccinated. Pigs in the vaccinated group were injected intramuscularly with the vaccine at 7 days of age, whereas non-vaccinated pigs received physiological saline solution (PBS). Pigs were euthanized and necropsied at 30, 36 and 58 days of age. Blood, bronchoalveolar lavage (BAL) fluid, spleen, lung and bronchial lymph nodes (BLN) were collected. Serum and BAL fluid were tested for the presence of antibodies by ELISA. Monomorphonuclear cells from the peripheral blood and tissues were isolated to quantify the T cell subsets by flow cytometry, and cytokine production by ELIspot and ELISA. Antibodies against M. hyopneumoniae were detected in serum of most vaccinated pigs at 30 days of age. M. hyopneumoniae specific IgG, IgM and IgA were detected in BAL fluid from vaccinated animals, but not from control animals. Significantly higher numbers of IL-12 secreting cells were observed in the lung at day 58 in the vaccinated than in the non-vaccinated group (p < 0.05). The number of IL-10 secreting cells from BLN was also higher in the vaccinated group at day 58 (p < 0.05). After restimulation in vitro, lymphocytes from BLN and lungs secreted significantly higher levels of IL-12 in the vaccinated group at day 58. These results show that the vaccine induced both systemic and mucosal cellular and humoral immune responses.