Immune response and protective efficacy of two new adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02, administered with a Streptococcus agalactiae ghost vaccine in Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae is one of the most important pathogens infecting tilapia worldwide and causes meningoencephalitis, septicemia and high mortalities with considerable losses. Various types of vaccines have been developed against S. agalactiae infection, such as inactivated vaccines, live attenuated vaccines and subunit vaccines. Bacterial ghosts (BGs) are nonliving, empty cell envelopes and have been reported as novel vaccine candidates. Therefore, the main aims of this study were to develop an S. agalactiae ghost vaccine (SAGV) and to evaluate the immune response and protective effect of SAGV against S. agalactiae with two novel adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02. Nile tilapia, mean weight 50 g, were divided into four groups as follows; 1) fish injected with PBS as control, 2) fish injected with the SAGV alone; 3) fish injected with the SAGV+Montanide™ ISA 763B VG; and 4) fish injected with SAGV+Montanide™ GEL02. Following vaccination, innate immunity parameters including serum lysozyme, myeloperoxidase, catalase, and bactericidal activity were all significantly enhanced. Moreover, specific serum IgM antibodies were induced and reached their highest level 2-8 weeks post vaccination. Importantly, the relative percent survival of tilapia vaccinated against the SAGV formulated with both adjuvants was 80-93%. Furthermore, the transcription of immune-related genes (IgM, TCRß, IL-1ß, IL-8 and TNFα) were up-regulated in tilapia after vaccination, indicating that both cellular and humoral immune responses were induced by these adjuvanted vaccines. In summary, Montanide™ ISA 763B VG and Montanide™ GEL02 can enhance immunoprotection induced by the SAGV vaccine against streptococcosis, demonstrating that both have value as potential adjuvants of fish vaccines.

Immunization with recombinant protein Ag43::UpaH with alum and 1,25(OH)2D3 adjuvants significantly protects Balb/C mice against urinary tract infection caused by uropathogenic Escherichia coli.

The majority of urinary tract infections (UTIs) are caused by uropathogenic Escherichia coli (UPEC). Designing a vaccine will certainly reduce the occurrence of infection and antibiotic resistance of the isolates. Antigen 43 (Ag43) and autotransporter H (UpaH) have been associated with the virulence of UPEC. In the present study, the efficacy of different formulations of a hybrid protein composed of Ag43 and UpaH with and without alum and 1,25(OH)2D3 (Vitamin D3) adjuvants were evaluated in mice model. A significant increase in IgG and cellular responses was developed against Ag43::UpaH as compared to the control mice. The addition of alum or a mixture of alum and Vitamin D3 to the protein significantly enhanced the serum IgG responses and tended to remain in a steady state until 6 months. In addition, the mentioned formulations produced significant amounts of IgG1, IL-4, and IL-17 as compared to the fusion protein alone. In addition to the mentioned formulations, the combination of protein with Vitamin D3 also resulted in significantly higher serum IgA and IFN-γ levels as compared to the fusion protein alone. Mice immunized with fusion plus alum and formulation protein admixed with both alum and Vitamin D3 significantly reduced the bacterial load in the bladders and kidneys of mice as compared to the control. In this study, for the first time, the ability of a novel hybrid protein in combination with adjuvants alum and Vitamin D3 was evaluated against UPEC. Our results indicated that fusion Ag43::UpaH admixed with alum and Vitamin D3 could be a promising candidate against UTIs.

Unusual case of polyarthritis and hepatorenal syndrome associated with Leptospira interrogans infection in a dog: A case report.

Leptospirosis is a zoonotic disease caused by spirochetal bacterial of the genus Leptospira affecting virtually all mammals. The infection has a broad range of effects, from mild clinical manifestation to multiple organ failure, and ultimately death. A 5-months-old male unvaccinated dog was admitted to the University Veterinary Teaching Hospital presenting dullness, dehydration, jaundiced mucous, bloody diarrhea, vomiting, and hyporexia. Microscopic agglutination test (MAT) detected serological titers of 1:1.600 for serogroup Canicola. After five days of monitoring by the medical team he developed fever and swelling of carpal and tarsal joints, accompanied by functional limitation. Initial antimicrobial treatment was instituted for leptospirosis. Polyarthritis responsiveness to glucocorticoid therapy was observed through decreasing signs of inflammation of the affected joints. The diagnosis of leptospirosis was further confirmed by molecular investigation for Leptospira spp. on blood and synovial fluid samples. Amplification and sequencing of the secY partial gene characterized the infective bacterial as Leptospira interrogans. From the 7th day the respiratory condition worsened and on Day 14 the patient evolved to death, when necropsy and histological evaluation were performed. Prominent anatomopathological findings included: fibrinous polyarthritis, bronchointerstitial pneumonia, intense hepatocyte dissociation, cholestasis, and periportal multifocal hepatitis, diffuse acute tubular necrosis, and significant dystrophic mineralization in the renal parenchyma, lungs, and atrial endocardium. Here, we present a case report of systemic clinical manifestations polyarthritis associated with the presence of leptospiras in the synovial fluid. We highlight the need for richer knowledge about the different clinical manifestations of leptospirosis.

Hospital-based evidence on cost-effectiveness of brucellosis diagnostic tests and treatment in Kenyan hospitals.

Hospitals in Kenya continue to use the Febrile Antigen Brucella Agglutination Test (FBAT) to diagnose brucellosis, despite reports showing its inadequacy. This study generated hospital-based evidence on the performance and cost-effectiveness of the FBAT, compared to the Rose Bengal Test (RBT).Twelve hospitals in western Kenya stored patient serum samples that were tested for brucellosis using the FBAT, and these were later re-tested using the RBT. Data on the running time and cost of the FBAT, and the treatment prescribed for brucellosis, were collected. The cost-effectiveness of the two tests, defined as the cost in US Dollars ($) per Disability Adjusted Life Year (DALY) averted, was determined, and a basic sensitivity analysis was run to identify the most influential parameters. Over a 6-month period, 180 patient serum samples that were tested with FBAT at the hospitals were later re-tested with RBT at the field laboratory. Of these 24 (13.3%) and 3 (1.7%) tested positive with FBAT and RBT, respectively. The agreement between the FBAT and RBT was slight (Kappa = 0.12). Treatment prescribed following FBAT positivity varied between hospitals, and only one hospital prescribed a standardized therapy regimen. The mean $/DALY averted when using the FBAT and RBT were $2,065 (95% CI $481-$6,736) and $304 (95% CI $126-$604), respectively. Brucellosis prevalence was the most influential parameter in the cost-effectiveness of both tests. Extrapolation to the national level suggested that an estimated $338,891 (95% CI $47,000-$1,149,000) per year is currently spent unnecessarily treating those falsely testing positive by FBAT. These findings highlight the potential for misdiagnosis using the FBAT. Furthermore, the RBT is cost-effective, and could be considered as the mainstay screening test for human brucellosis in this setting. Lastly, the treatment regimens must be harmonized to ensure the appropriate use of antibiotics for treatment.

Isotype-specific Antibody Responses to Mycobacterium avium paratuberculosis Antigens Are Associated With the Use of Biologic Therapy in Inflammatory Bowel Disease.

BACKGROUND: The role of Mycobacterium avium paratuberculosis [MAP] in inflammatory bowel disease [IBD], especially Crohn's disease [CD] is controversial due conflicting results and lack of reproducibility and standardised tests. The current study focuses on the role of MAP in disease progression and genetic susceptibility, as MAP is likely one of many factors involved in the complex pathogenesis of IBD, potentially affecting a subgroup depending on genetic susceptibility. METHODS: Serum from 812 patients was evaluated with seven immunoglobulin [Ig] isotype-specific serology tests assessing humoral response to three different MAP antigens. For each of these in total 21 tests, the intra-assay and inter-assay coefficients were used to evaluate test accuracy. Reliable assays were subsequently analysed in relation to disease characteristics and need for biologic therapy/surgery. Genome-wide genotyping was available for all participants. Genetic determinants of humoral response to MAP antigens were evaluated using genome-wide association analysis and polygenic risk scores [PRS]. RESULTS: High IgA or IgM response to MAP2609 was associated with increased use of biologic therapy in CD and ulcerative colitis [UC] [odds ratios 2.69; 95% confidence interval 1.44-5.01; and 2.60, 1.46-4.64, respectively]. No associations were seen for risk of surgery [p-values > 0.29]. We could not identify genetic determinants nor polygenic risk scores for MAP response with genome-wide significance. CONCLUSIONS: Extensive assays for serological response to MAP were evaluated using stringent criteria for reliability. Increased IgA and IgM response to MAP antigens was seen in patients exposed to biologic therapy, but no genetic determinants underlying this humoral response were found.

Iron Deficiency Anemia at Time of Vaccination Predicts Decreased Vaccine Response and Iron Supplementation at Time of Vaccination Increases Humoral Vaccine Response: A Birth Cohort Study and a Randomized Trial Follow-Up Study in Kenyan Infants.

Background: Iron deficiency may impair adaptive immunity and is common among African infants at time of vaccination. Whether iron deficiency impairs vaccine response and whether iron supplementation improves humoral vaccine response is uncertain. Methods: We performed two studies in southern coastal Kenya. In a birth cohort study, we followed infants to age 18 mo and assessed whether anemia or iron deficiency at time of vaccination predicted vaccine response to three-valent oral polio, diphtheria-tetanus-whole cell pertussis-Haemophilus influenzae type b vaccine, ten-valent pneumococcal-conjugate vaccine and measles vaccine. Primary outcomes were anti-vaccine-IgG and seroconversion at age 24 wk and 18 mo. In a randomized trial cohort follow-up, children received a micronutrient powder (MNP) with 5 mg iron daily or a MNP without iron for 4 mo starting at age 7.5 mo and received measles vaccine at 9 and 18 mo; primary outcomes were anti-measles IgG, seroconversion and avidity at age 11.5 mo and 4.5 y. Findings: In the birth cohort study, 573 infants were enrolled and 303 completed the study. Controlling for sex, birthweight, anthropometric indices and maternal antibodies, hemoglobin at time of vaccination was the strongest positive predictor of: (A) anti-diphtheria and anti-pertussis-IgG at 24 wk (p = 0.0071, p = 0.0339) and 18 mo (p = 0.0182, p = 0.0360); (B) anti-pertussis filamentous hemagglutinin-IgG at 24 wk (p = 0.0423); and (C) anti-pneumococcus 19 IgG at 18 mo (p = 0.0129). Anemia and serum transferrin receptor at time of vaccination were the strongest predictors of seroconversion against diphtheria (p = 0.0484, p = 0.0439) and pneumococcus 19 at 18 mo (p = 0.0199, p = 0.0327). In the randomized trial, 155 infants were recruited, 127 and 88 were assessed at age 11.5 mo and 4.5 y. Compared to infants that did not receive iron, those who received iron at time of vaccination had higher anti-measles-IgG (p = 0.0415), seroconversion (p = 0.0531) and IgG avidity (p = 0.0425) at 11.5 mo. Interpretation: In Kenyan infants, anemia and iron deficiency at time of vaccination predict decreased response to diphtheria, pertussis and pneumococcal vaccines. Primary response to measles vaccine may be increased by iron supplementation at time of vaccination. These findings argue that correction of iron deficiency during early infancy may improve vaccine response.

Gastroprotective effect of phytoncide extract from Pinus koraiensis pinecone in Helicobacter pylori infection.

For centuries, herbs have been used by traditional therapists around the world to treat gastrointestinal tract disorders, such as gastritis. We hypothesized that the anti-Helicobacter pylori properties of phytoncide, which is extracted from pinecone waste, would facilitate use as a natural gastroprotective product to treat gastrointestinal tract disorders. Thus, we investigated in vitro antibacterial efficacy against H. pylori by agar diffusion assay. To determine the gastroprotective properties of phytoncide, we conducted hematoxylin and eosin staining, performed assays for the detection of the cytotoxin gene, and evaluated pro-inflammatory cytokine expression in H. pylori-infected C57BL/6 mice. Phytoncide significantly inhibited the survival of H. pylori in the gastrointestinal system of C57BL/6 mice. Reduction of gastric severity in H. pylori-infected mice was associated with reductions in the expression levels of pro-inflammatory cytokines in the gastric mucosa, and of the cytotoxin CagA gene in phytoncide treated groups (P < 0.05 and P < 0.01). In conclusion, phytoncide significantly inhibited the growth of H. pylori in gastro tissue, possibly due to the abundant α-pinene present in the phytoncide as detected by HPLC analysis. Further studies are needed to validate our findings, but we suggest that phytoncide has the potential to be used as a natural ingredient in anti-H. pylori products.

Leptospiral antibody prevalence and surgical treatment outcome in horses with Equine Recurrent Uveitis (ERU) in Switzerland.

OBJECTIVE: To evaluate leptospiral antibody prevalence in 65 horses with ERU and compare outcome in 36 surgically treated eyes (2010-2015). PROCEDURES: Retrospective data analysis of horses with ERU (n = 65). C-value calculation with microagglutination assay titer (MAT) results for Leptospira spp. Evaluation of follow-up data after pars plana vitrectomy (PPV, n = 21 eyes) and suprachoroidal cyclosporine device implantation (SCDI, n = 15 eyes). Differences between groups were statistically analyzed using Fishers exact test, significance set at P < .05. RESULTS: Positive leptospiral titers were found in 28/65 blood, 31/65 aqueous humor (AH), and 19/20 vitreal (post-PPV) samples. The most common intraocular serovars were Leptospira interrogans grippotyphosa, pomona, and bratislava. Intraocular antibody production was suspected in samples of 22 horses (c-values > 1). Mean follow-up of surgical cases was 3.8 years (PPV) and 3.4 years (SCDI). PPV was performed in 21 eyes with positive, SCDI in 15 eyes with negative leptospiral test results. Uveitis recurred less often after PPV (2/21) compared to SCDI (6/15, P = .04). Retinal detachment occurred after PPV only (5/21, SCDI 0/15, P = .06), whereas only SCDI-treated eyes were enucleated (PPV 0/21, SCDI 3/15, P = .06). Blindness or visual impairment was equally likely to occur in both treatment groups after surgery (PPV 7/21, SCDI 7/15, P = .5). CONCLUSIONS: Leptospiral antibody prevalence is high in horses with ERU in Switzerland. Recurrence of uveitis is uncommon following PPV in the present study; an increased risk of retinal detachment exists. Enucleation is more often warranted in horses after SCDI in this study due to a higher uveitis recurrence.

Optimization of an Experimental Vaccine To Prevent Escherichia coli Urinary Tract Infection.

Urinary tract infections (UTI) affect half of all women at least once during their lifetime. The rise in the numbers of extended-spectrum beta-lactamase-producing strains and the potential for carbapenem resistance within uropathogenic Escherichia coli (UPEC), the most common causative agent of UTI, create an urgent need for vaccine development. Intranasal immunization of mice with UPEC outer membrane iron receptors FyuA, Hma, IreA, and IutA, conjugated to cholera toxin, provides protection in the bladder or kidneys under conditions of challenge with UPEC strain CFT073 or strain 536. On the basis of these data, we sought to optimize the vaccination route (intramuscular, intranasal, or subcutaneous) in combination with adjuvants suitable for human use, including aluminum hydroxide gel (alum), monophosphoryl lipid A (MPLA), unmethylated CpG synthetic oligodeoxynucleotides (CpG), polyinosinic:polycytidylic acid (polyIC), and mutated heat-labile E. coli enterotoxin (dmLT). Mice intranasally vaccinated with dmLT-IutA and dmLT-Hma displayed significant reductions in bladder colonization (86-fold and 32-fold, respectively), with 40% to 42% of mice having no detectable CFU. Intranasal vaccination of mice with CpG-IutA and polyIC-IutA significantly reduced kidney colonization (131-fold) and urine CFU (22-fold), respectively. dmLT generated the most consistently robust antibody response in intranasally immunized mice, while MPLA and alum produced greater concentrations of antigen-specific serum IgG with intramuscular immunization. On the basis of these results, we conclude that intranasal administration of Hma or IutA formulated with dmLT adjuvant provides the greatest protection from UPEC UTI. This report advances our progress toward a vaccine against uncomplicated UTI, which will significantly improve the quality of life for women burdened by recurrent UTI and enable better antibiotic stewardship.IMPORTANCE Urinary tract infections (UTI) are among the most common bacterial infection in humans, affecting half of all women at least once during their lifetimes. The rise in antibiotic resistance and health care costs emphasizes the need to develop a vaccine against the most common UTI pathogen, Escherichia coli Vaccinating mice intranasally with a detoxified heat-labile enterotoxin and two surface-exposed receptors, Hma or IutA, significantly reduced bacterial burden in the bladder. This work highlights progress in the development of a UTI vaccine formulated with adjuvants suitable for human use and antigens that encode outer membrane iron receptors required for infection in the iron-limited urinary tract.

High efficacy and economical procedure of oral vaccination against Lactococcus garvieae/Streptococcus iniae in rainbow trout (Oncorhynchus mykiss).

The present study was aimed to examine the efficacy of chitosan-alginate coated vaccines against pathogenicity of Lactococcus garvieae and Streptococcus iniae in rainbow trout. Fish were divided into four groups including: Group A: fish immunized by chitosan-alginate coated vaccine, Group B: fish immunized by non-coated vaccine, Group C: fish feed by chitosan-alginate coated pellets without vaccine and Group D: fish feed by basic diet (non-coated and without vaccine). In groups A and B, the vaccination was carried out for 14 days and after that supplemented with fundamental diet (control diet). Comparable to groups A and B, fish of group C were also fed 14 days with test diets and after that fed control food. On day 0, 20, 40 and 60 of the experiment, serum samples were given. Fish have been challenged with live L. garvieae and S. iniae after 60 days. The levels of bactericidal activity and complement activity among innate immunity components extended on day 20 of the research and after that decreased in group A and B (P < 0.05) all through the examination. The relative expression of IL-6 and IgM in groups A and B extended on examination day 20. The expression of these genes illustrated no advancements in different groups in during the examination (P > 0.05). In group A, the serum antibody titer against L. garvieae and S. iniae broadly raised on day 40 and 60 of examination, whereas in group B, the immune response titer against S. iniae and L. garvieae illustrated a significant elevation on day 60 of the trial (P < 0.05). After challenge with live bacteria, survival rate of 83 ± 9.1%(challenged with S. iniae) and 72.18 ± 9.8% (challenged with L. garvieae) were gotten independently in group A, which were higher than survival of other exploratory groups (P < 0.05). In conclusion, the results of the present examination appear that the orally vaccination of rainbow trout with chitosan-alginate covered vaccine stimulates immunity system and also efficiently protects rainbow trout against Lactococcus garvieae and Streptococcus iniae.

Selenium Nanoparticles Induce Potent Protective Immune Responses against Vibrio cholerae WC Vaccine in a Mouse Model.

The aim of this study was to evaluate the efficacy of selenium nanoparticle (an immune booster) and naloxone (an opioid receptor antagonist) as a new adjuvant in increasing immune responses against killed whole-cell Vibrio cholerae in a mouse cholera model. The Se NPs were synthesized and characterized by UV-visible, DLS, and zeta potential analysis. The SEM image confirmed the uniformity of spherical morphology of nanoparticle shape with 34 nm in size. The concentration of the Se NPs was calculated as 0.654 µg/ml in the ICP method. The cytotoxic activity of Se NPs on Caco-2 cells was assessed by the MTT assay and revealed 82.05% viability of cells after 24 h exposure with 100 µg/ml of Se NPs. Female BALB/C mice were orally immunized three times on days 0, 14, and 28, and challenge experiments were performed on immunized neonates with toxigenic V. cholerae. Administration of Se NP diet led to significant increase in V. cholerae-specific IgG and IgA responses in serum and saliva and caused protective immunity and 83.3% survival in challenge experiment against 1 LD50 V. cholerae in a group receiving diet of Se NPs compared with other groups including Dukoral vaccine. The IL-4 and IL-5 were significantly increased in response to WC+daily diet of Se NPs with or without naloxone. Naloxone proved no effect on IL-4 and IL-5 increase and is proposed as null in the cytokine and antibody production process. These results reveal that daily diet of Se NPs could efficiently induce immune cell effectors in both humoral and mucosal levels.

Phoenix dactylifera, mentha piperita and montanide™ ISA-201 as immunological adjuvants in a chicken model.

This study evaluated plant-based immune-adjuvants from crude extracts of Phoenix dactylifera and Mentha piperita as promising adjuvants for vaccines because of the limited side effects associated with plant extracts. In addition, Montanide™ ISA 201 previously used in vaccines in cattle. Eight different infectious coryza (IC) vaccines were prepared from three serovars [A (W strain and local strain), C (Modesto strain) and B (0222 strain)] with eight Avibacterium paragallinarum vaccines adjuvants formulae using liquid paraffin, Montanide™ ISA 71, Montanide™ ISA 201, and Montanide™ Gel adjuvants, P. dactylifera and M. piperita as immune-stimulants at a concentration of 1 mg and 2 mg incorporated with or without liquid paraffin oil as an adjuvant. These vaccines were applied in a chicken model. After a single immunization, the eight vaccine formulations were evaluated using the ELISA and Microplate agglutination test. Evidence of protection in the immunized birds was based on the results after challenge and bacterial isolation. The incorporation of the crude aqueous extract of P. dactylifera or M. piperita at a concentration of 2 mg in a liquid paraffin oil adjuvanted IC vaccine could be employed as an efficient adjuvant for chicken to IC vaccine to enhance immune responses. Also,Montanide™ ISA 201 may be the best adjuvant to be used to enhance the protective response against Av. paragallinarum. Our results confirm that aqueous extracts of M. piperita leaves and P. dactylifera fruit have immunomodulatory potentials in vivo and elevated serum antibodies against Av. Paragallinarum.

Immunodominance of LipL3293-272 peptides revealed by leptospirosis sera and therapeutic monoclonal antibodies.

BACKGROUND/PURPOSE: Leptospirosis is a neglected zoonosis, imposing significant human and veterinary public health burdens. In this study, recombinant LipL3293-147 and LipL32148-184 middle domain of LipL3293-184, and LipL32171-214, and LipL32215-272 of c-terminal LipL32171-272 truncations were defined for immunodominance of the molecule during Leptospira infections revealed by leptospirosis sera. RESULTS: IgM-dominant was directed to highly surface accessible LipL32148-184 and Lipl32171-214. IgG dominance of LipL32148-184 revealed by rabbit anti-Leptospira sera and convalescent leptospirosis paired sera were mapped to highly accessible surface of middle LipL32148-184 truncation whereas two LipL32148-184 and LipL32215-272 truncations were IgG-dominant when revealed by single leptospirosis sera. The IgM-dominant of LipL32148-214 and IgG-dominant LipL32148-184 peptides have highly conserved amino acids of 70% identity among pathogenic and intermediate Leptospira species and were mapped to the highly surface accessible area of LipL32 molecule that mediated interaction of host components. IgG dominance of two therapeutic epitopes located at LipL32243-253 and LipL32122-130 of mAbLPF1 and mAbLPF2, respectively has been shown less IgG-dominant (<30%), located outside IgG-dominant regions characterized by leptospirosis paired sera. CONCLUSION: The IgM- and IgG-dominant LipL32 could be further perspectives for immunodominant LipL32-based serodiagnosis and LipL32 epitope-based vaccine.

Protec™ improves innate immune response and specific antibody response against Lactococcus garvieae in rainbow trout (Oncorhynchus mykiss).

Protec™ is a commercial aquafeed (Skretting Italia) containing a combination of glucans, vitamin C, vitamin E and zinc (immune support pack). No research information concerning its capability to improve fish immune response is available, so in this study the potential immunomodulatory effects of Protec™ were investigated in rainbow trout (Oncorhynchus mykiss). Head kidney (HK) leukocytes from adult fish (100 g, n = 6) were in vitro incubated with Protec™ immune support pack resulting in significantly higher respiratory burst activity and proliferation. Specifically, sonicated Protec™ immune support pack (160 µg/ml) induced a respiratory burst response similar to that promoted by zymosan and lipopolysaccharide (LPS), while non-sonicated Protec™ immune support pack induced a response comparable to that of cells stimulated with phorbol myristate acetate (PMA). Moreover, the proliferation of leukocytes exposed to sonicated Protec™ immune support pack (20 µg/ml) was significantly higher than that of cells stimulated with zymosan, and it was comparable to the proliferation of cells stimulated with phytohaemagglutinin (PHA) and LPS. Afterwards, a feeding trial was performed in a rainbow trout farm. Two groups of juvenile rainbow trout (10 g) were acclimated for 7 weeks before the experiment and fed daily with a commercial control diet (Optiline HE, Skretting Italia) at 2% BW/day. At the end of acclimation, one group of fish was fed with Protec™ diet (Skretting Italia) at 2% BW/day whereas the other group continued to feed the control diet at the same level for further 4 weeks. Then, fish were sampled (HK leukocytes from n = 6 fish/group, serum from n = 12 fish/group) or intraperitoneally vaccinated against lactococcosis (n = 160/dietary group/time point). Fish fed the same diets for further 4 weeks after vaccination, then feeding returned to the control diet in both groups until the end of the trial. The specific antibody response was recorded at 4 and 8 weeks after vaccination (n = 12 fish/group). The administration of Protec™ significantly enhanced the respiratory burst activity of leukocytes and the synthesis of specific IgM against Lactococcus garvieae, whereas the serum lysozyme activity was unaffected. The present research suggests that the administration of Protec™ can improve both innate and adaptive immune response of rainbow trout, proving to be an interesting strategy for enhancing the immune reactivity of fish to vaccines.

Immunization with the ferric iron-binding periplasmic protein HitA provides protection against Pseudomonas aeruginosa in the murine infection model.

Pseudomonas aeruginosa is a notorious pathogen with increasing multi-drug resistance. This situation makes it urgent to develop a prophylactic vaccine against this pathogen. Different virulence factors play a crucial role in P. aeruginosa infection. This study focused on evaluation of the iron acquisition protein HitA as a potential vaccine candidate against P. aeruginosa in a murine infection model. The recombinant ferric iron-binding periplasmic protein HitA was overexpressed in Escherichia coli and was purified using metal affinity chromatography. The purified antigen was administered to mice in combination with Bacillus Calmette-Guérin (BCG) as an adjuvant using different vaccination regimens. Serum samples were tested for IgG1, IgG2a and total IgG antibody responses which were extremely significant. Following challenge of mice with P. aeruginosa, there was a significant reduction in bacterial load in lungs of immunized mice compared to negative control mice. Opsonophagocytic assay supported the previous results. In addition, histopathological examination of livers of challenged mice showed a significant improvement difference between immunized mice and negative control mice in various histopathological parameters. Up to our knowledge, this is the first report that investigates HitA as a potential vaccine antigen. Overall, the results of this study demonstrate the protective effect of HitA recombinant protein and highlight its importance as a promising vaccine candidate against P. aeruginosa infection.

Effect of atorvastatin on humoral immune response to 23-valent pneumococcal polysaccharide vaccination in healthy volunteers: The StatVax randomized clinical trial.

BACKGROUND: The immunomodulatory effects of statins on vaccine response remain uncertain. Therefore, the objective of this study was to determine if atorvastatin enhances pneumococcal-specific antibody titer following 23-valent pneumococcal polysaccharide vaccination. METHODS: Double-blind, placebo-controlled, single-center randomized clinical trial entitled StatVax. Subjects were enrolled between June and July 2014 and followed up through September 2014. 33 healthy volunteers signed informed consent after volunteer sampling. 11 participants were excluded; 22 healthy volunteers without prior pneumococcal vaccination were enrolled and completed the study. Participants were randomized to receive a 28-day course of 40 mg atorvastatin (n = 12) or matching lactose placebo (n = 10). On day 7 of treatment, Pneumovax 23 was administered intramuscularly. The primary outcome was fold change in total pneumococcal-specific antibody titer determined by a ratio of post-vaccination titer over baseline titer. Secondary outcomes included serotype-specific pneumococcal antibody titer, seroconversion, complete blood counts (CBC), erythrocyte sedimentation rate (ESR), and serum cytokine analysis. RESULTS: Of the 22 randomized patients (mean age, 23.86; SD, 4.121; 11 women [50%]), 22 completed the trial. Total anti-pneumococcal antibody titer in the atorvastatin group went from a baseline mean of 32.58 (SD, 15.96) to 147.7 (SD, 71.52) µg/mL at 21 days post-vaccination while titer in the placebo group went from a mean of 30.81 (SD, 13.04) to 104.4 (SD, 45) µg/mL. When comparing fold change between treatment groups, there was a significant increase in fold change of total anti-pneumococcal antibody titer in the atorvastatin group compared to the placebo group (2-way ANOVA, p = .0177). CONCLUSIONS: Atorvastatin enhances antigen-specific primary humoral immune response to a T cell-independent pneumonia vaccination. Pending confirmation by larger cohort studies of target populations, peri-vaccination conventional doses of statins can become a novel adjuvant for poorly-immunogenic polysaccharide-based vaccines. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT02097589.

Effect of immunostimulatory feed supplements on the development of acquired immunity in rainbow trout (Oncorhynchus mykiss).

Immunostimulatory feed supplements are an increasingly common feature of aquaculture management and their benefit has been confirmed for a wide area of products. However, these investigations have often focused on the benefit of these supplements on the innate immune system. In the current project, we investigated a mixture of two commercial feed supplements (Biotronic® Top 3 and Levabon® Aquagrow E) with a known protective effect against bacterial infections. The effect of the supplemented diet on antibody titters of Oncorhynchus mykiss vaccinated against Yersinia ruckeri was determined by ELISA. Furthermore, an infection trial was performed to confirm the effect of the supplements on the survival of the fish. Finally, their effects on the growth parameters of the fish were also determined. The results from this study found no significant effect on the general antibody titters. However, when considering only the titters of specific anti-Y ruckeri antibodies, the supplemented feed was associated with an improved response to the vaccine, significantly better than in the fish that had received the control feed.

Melatonin enhances responsiveness to Dichelobacter nodosus vaccine in sheep and increases peripheral blood CD4 T lymphocytes and IgG-expressing B lymphocytes.

The immunomodulatory functions mediated by melatonin support its use as vaccine adjuvant. Previously, we have demonstrated that melatonin enhances antibody responses in sheep vaccinated against Dichelobacter nodosus. Here, we analyze the effect of melatonin on T and B lymphocyte subsets in peripheral blood of sheep vaccinated against D. nodosus. We also compare the use of melatonin in implants and in injections. Melatonin administration either as implants or by injection produced higher antibody titers against A1 and C serotypes compared to those animals that received only the vaccine. These results support the use of melatonin as an adjuvant in vaccination against D. nodosus. Firstly, melatonin induces higher antibody titer than the vaccine alone, secondly, melatonin increase IgG+ B lymphocytes and CD4+ T lymphocytes in vaccinated sheep. These results suggest that melatonin enhances T CD4 cell activation and subsequently secondary humoral immune responses. Further studies are required to determine the mechanism underlining the immunomodulatory role of melatonin in the context of vaccination.

A Bacteriophage T4 Nanoparticle-Based Dual Vaccine against Anthrax and Plague.

Bacillus anthracis and Yersinia pestis, the causative agents of anthrax and plague, respectively, are two of the deadliest pathogenic bacteria that have been used as biological warfare agents. Although Biothrax is a licensed vaccine against anthrax, no Food and Drug Administration-approved vaccine exists for plague. Here, we report the development of a dual anthrax-plague nanoparticle vaccine employing bacteriophage (phage) T4 as a platform. Using an in vitro assembly system, the 120- by 86-nm heads (capsids) of phage T4 were arrayed with anthrax and plague antigens fused to the small outer capsid protein Soc (9 kDa). The antigens included the anthrax protective antigen (PA) (83 kDa) and the mutated (mut) capsular antigen F1 and the low-calcium-response V antigen of the type 3 secretion system from Y. pestis (F1mutV) (56 kDa). These viral nanoparticles elicited robust anthrax- and plague-specific immune responses and provided complete protection against inhalational anthrax and/or pneumonic plague in three animal challenge models, namely, mice, rats, and rabbits. Protection was demonstrated even when the animals were simultaneously challenged with lethal doses of both anthrax lethal toxin and Y. pestis CO92 bacteria. Unlike the traditional subunit vaccines, the phage T4 vaccine uses a highly stable nanoparticle scaffold, provides multivalency, requires no adjuvant, and elicits broad T-helper 1 and 2 immune responses that are essential for complete clearance of bacteria during infection. Therefore, phage T4 is a unique nanoparticle platform to formulate multivalent vaccines against high-risk pathogens for national preparedness against potential bioterror attacks and emerging infections.IMPORTANCE Following the deadly anthrax attacks of 2001, the Centers for Disease Control and Prevention (CDC) determined that Bacillus anthracis and Yersinia pestis that cause anthrax and plague, respectively, are two Tier 1 select agents that pose the greatest threat to the national security of the United States. Both cause rapid death, in 3 to 6 days, of exposed individuals. We engineered a virus nanoparticle vaccine using bacteriophage T4 by incorporating key antigens of both B. anthracis and Y. pestis into one formulation. Two doses of this vaccine provided complete protection against both inhalational anthrax and pneumonic plague in animal models. This dual anthrax-plague vaccine is a strong candidate for stockpiling against a potential bioterror attack involving either one or both of these biothreat agents. Further, our results establish the T4 nanoparticle as a novel platform to develop multivalent vaccines against pathogens of high public health significance.

Protective immune mechanisms of Yifei Tongluo, a Chinese herb formulation, in the treatment of mycobacterial infection.

Yifei Tongluo (YFTL) is a traditional Chinese medicine (TCM) formulation which has been shown clinical efficacy in treatment of patients with multidrug-resistant tuberculosis in China. However, the underlying mechanisms of the effects of YFTL are lacking. This study investigated the effects of YFTL on immune regulation with a mouse lung infection model with Bacille Calmette-Guérin (BCG). We found that compared with untreated mice, the lung mycobacterial load in YFTL-treated mice was significantly reduced, accompanied by alleviated pulmonary inflammation with reduction of pro-inflammatory cytokines and increase of prostaglandin E2 (PGE2). Flow cytometry analyses showed that Th1 cells were significantly higher in the lungs of YFTL-treated mice at early infection time. The results suggest that YFTL-treatment down-regulates pulmonary inflammation, which facilitates a rapid infiltration of Th1 cells into the lungs. Moreover, the Th1 cells in the lungs were resolved faster at later time concomitant with increased the regulatory T cells (Tregs). The reduction of mycobacterial burden associated with improved tissue pathology, faster Th1 cell trafficking, and accelerated resolution of Th1 cells in the lungs of YFTL-treated mice indicates that YFTL improves mycobacterial clearance by maintaining lung homeostasis and dynamically regulating T cells in the lung parenchyma, and suggests that YFTL can be used as host-directed therapies that target immune responses to mycobacterial infection.