Cellular Basis for the Enhanced Efficacy of the Fms-Like Tyrosine Kinase 3 Ligand (FL) Adjuvanted VCG-Based Chlamydia abortus Vaccine.

Efficacious vaccines are needed to control genital chlamydial diseases in humans and the veterinary industry. We previously reported a C. abortus (Cab) vaccine comprising recombinant Vibrio cholerae ghosts (rVCG) expressing the conserved and immunogenic N-terminal region of the Cab polymorphic membrane protein D (rVCG-Pmp18.1) protein that protected mice against intravaginal challenge. In this study, we investigated the immunomodulatory effect of the hematopoietic progenitor activator cytokine, Fms-like tyrosine kinase 3-ligand (FL) when co-administered with the rVCG-Pmp18.1 vaccine as a strategy to enhance the protective efficacy and the potential mechanism of immunomodulation. Groups of female C57BL/6J mice were immunized and boosted twice intranasally (IN) with rVCG-PmpD18.1 with and without FL or purified rPmp18.1 or rVCG-gD2 (antigen control) or PBS (medium) per mouse. The results revealed that co-administration of the vaccine with FL enhanced antigen-specific cellular and humoral immune responses and protected against live Cab genital infection. Comparative analysis of immune cell phenotypes infiltrating mucosal and systemic immune inductive tissue sites following immunization revealed that co-administration of rVCG-Pmp18.1 with FL significantly enhanced the number of macrophages, dendritic and NK cells, γδ and NK T cells in the spleen (systemic) and iliac lymph nodes (ILN) draining the genital tract (mucosal) tissues compared to rVCG-Pmp18.1 alone. Furthermore, FL enhanced monocyte infiltration in the ILN, while CD19+ B cells and CD4+ T cells were enhanced in the spleen. These results indicate that the immunomodulatory effect of FL is associated with its ability to mobilize innate immune cells and subsequent activation of robust antigen-specific immune effectors in mucosal and systemic lymphoid tissues.

Epidemiology, Treatments, and Vaccine Development for Antimicrobial-Resistant Neisseria gonorrhoeae: Current Strategies and Future Directions.

Neisseria gonorrhoeae is the second most common bacterial sexually transmitted infection in the world after Chlamydia trachomatis. The pathogen has developed resistance to every antibiotic currently approved for treatment, and multidrug-resistant strains have been identified globally. The current treatment recommended by the World Health Organization is ceftriaxone and azithromycin dual therapy. However, resistance to azithromycin and ceftriaxone are increasing and treatment failures have been reported. As a result, there is a critical need to develop novel strategies for mitigating the spread of antimicrobial-resistant N. gonorrhoeae through improved diagnosis and treatment of resistant infections. Strategies that are currently being pursued include developing molecular assays to predict resistance, utilizing higher doses of ceftriaxone, repurposing older antibiotics, and developing newer agents. In addition, efforts to discover a vaccine for N. gonorrhoeae have been reignited in recent years with the cross-protectivity provided by the N. meningitidis vaccine, with several new strategies and targets. Despite the significant progress that has been made, there is still much work ahead to combat antimicrobial-resistant N. gonorrhoeae globally.

Efficacy and safety of a non-mineral oil adjuvanted injectable vaccine for the protection of Atlantic salmon (Salmo salar L.) against Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the causative agent of Rainbow Trout Fry Syndrome which has had a major impact on global salmonid aquaculture. Recent outbreaks in Atlantic salmon in Scotland and Chile have added to the need for a vaccine to protect both salmon and trout. At present no licensed vaccines are available in Europe, leaving antibiotics as the only course of action to contain disease outbreaks. Outbreaks generally occur in fry at temperatures between 10 and 15 °C. Recently outbreaks in larger fish have given added impetus to the development of a vaccine which can provide long term protection from this highly heterogeneous pathogen. Most fish injectable vaccines are formulated with oil emulsion adjuvants to induce strong and long lasting immunity, but which are known to cause side effects. Alternative adjuvants are currently sought to minimise these adverse effects. The current study was performed to assess the efficacy of a polyvalent, whole cell vaccine containing formalin-inactivated F. psychrophilum to induce protective immunity in Atlantic salmon. The vaccine was formulated with an adjuvant containing squalene and aluminium hydroxide, and was compared to a vaccine formulated with a traditional oil adjuvant, Montanide ISA 760VG, and a non-adjuvanted vaccine. Duplicate groups of salmon (23.5 ± 6.8 g) were vaccinated with each of the vaccine formulations or phosphate buffered saline by intraperitoneal injection. Fish were challenged by intramuscular injection with F. psychrophilum six weeks post-vaccination to test the efficacy of the vaccines. Cumulative mortality reached 70% in the control salmon, while the groups of salmon that received vaccine had significantly lower mortality than the controls (p = 0.0001), with no significant difference in survival between vaccinated groups. The squalene/alum adjuvant was safe, more readily metabolised by the fish and induced less histopathological changes than the traditional oil adjuvant.

Nonpyrogenic Molecular Adjuvants Based on norAbu-Muramyldipeptide and norAbu-Glucosaminyl Muramyldipeptide: Synthesis, Molecular Mechanisms of Action, and Biological Activities in Vitro and in Vivo.

Fatty acyl analogues of muramyldipeptide (MDP) (abbreviated N-L18 norAbuGMDP, N-B30 norAbuGMDP, norAbuMDP-Lys(L18), norAbuMDP-Lys(B30), norAbuGMDP-Lys(L18), norAbuGMDP-Lys(B30), B30 norAbuMDP, L18 norAbuMDP) are designed and synthesized comprising the normuramyl-l-α-aminobutanoyl (norAbu) structural moiety. All new analogues show depressed pyrogenicity in both free (micellar) state and in liposomal formulations when tested in rabbits in vivo (sc and iv application). New analogues are also shown to be selective activators of NOD2 and NLRP3 (inflammasome) in vitro but not NOD1. Potencies of NOD2 and NLRP3 stimulation are found comparable with free MDP and other positive controls. Analogues are also demonstrated to be effective in stimulating cellular proliferation when the sera from mice are injected sc with individual liposome-loaded analogues, causing proliferation of bone marrow-derived GM-progenitors cells. Importantly, vaccination nanoparticles prepared from metallochelation liposomes, His-tagged antigen rOspA from Borrelia burgdorferi, and lipophilic analogue norAbuMDP-Lys(B30) as adjuvant, are shown to provoke OspA-specific antibody responses with a strong Th1-bias (dominance of IgG2a response). In contrast, the adjuvant effects of Alum or parent MDP show a strong Th2-bias (dominance of IgG1 response).

Structure-activity relationship of lipid core peptide-based Group A Streptococcus vaccine candidates.

Infection with Group A Streptococcus (GAS) can result in a range of different illnesses, some of which are fatal. Currently, our efforts to develop a vaccine against GAS focuses on the lipid core peptide (LCP) system, a subunit vaccine containing a lipoamino acid (LAA) moiety which allows the stimulation of systemic antibody activity. In the present study, a peptide (J14) representing the B-cell epitope from the GAS M protein was incorporated alongside a universal T-helper epitope (P25) in four LCP constructs of different spatial orientation or LAA lengths. Through structure-activity studies, it was discovered that while the alteration of the LCP orientation had a weaker effect on immunostimulation, increasing the LAA side chain length within the construct increased antibody responses in murine models. Furthermore, the mice immunised with the lead LCP construct were also able to maintain antibody activity throughout the course of five months. These findings highlight the importance of LAA moieties in the development of intranasal peptide vaccines and confirmed that its side chain length has an effect on the immunogenicity of the structure.

Adverse and long-term protective effects following oil-adjuvanted vaccination against Aeromonas salmonicida in rainbow trout.

Prophylactic measures against Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis, have been an active field of research for decades, with studies mainly focused on Atlantic salmon (Salmo salar). In the present study we have examined the protective and adverse effects of mineral oil-adjuvanted injection vaccines on rainbow trout (Oncorhynchus mykiss). A commercial vaccine and an experimental auto vaccine, as well as their respective adjuvant formulations alone were used to evaluate their individual effects, both prior to and during an experimental waterborne infection challenge. Macro- and microscopic examination revealed signs of vaccine-induced adverse effects from 10 weeks to 14 months post vaccination. Both vaccines induced statistically significant protection during the experimental challenge (P=0.018 for both vaccines), as well as significantly elevated levels of specific circulating antibodies prior to and during the experimental challenge when compared to an unvaccinated control group. During the early, critical time points of the infection, both vaccines appeared to protect against pathological changes to the liver and spleen, which provides a probable explanation for the reduced mortality seen in the vaccinated groups. A significant correlation was found between the level of A. salmonicida-specific antibodies measured prior to challenge and the endpoint survival of each group after the experimental infection, and furthermore, the levels of these antibodies remained elevated for at least 14 months post vaccination.

Anti-adhesion methods as novel therapeutics for bacterial infections.

Anti-adhesion therapies for bacterial infections offer an alternative to antibiotics, with those therapies bacteria are not killed but are prevented from causing harm to a host by inhibiting adherence to host cells and tissues, a prerequisite for the majority of infectious diseases. The mechanisms of these potential therapeutic agents include inhibition of adhesins and their host receptors, vaccination with adhesins or analogs, use of probiotics and dietary supplements that interfere with receptor-adhesin interactions, subminimal inhibitory concentrations of antibiotics and manipulation of hydrophobic interactions. Once developed, these drugs will contribute to the arsenal for fighting infectious disease in the future, potentially subverting antibiotic resistance.

Evaluation of two outer membrane proteins, Aha1 and OmpW of Aeromonas hydrophila as vaccine candidate for common carp.

Aeromonas hydrophila is an important fish pathogen responsible for huge economic losses in aquaculture sector. The bacterial outer membrane proteins (OMPs), especially adhesins play a key role in the virulence of the bacteria and are considered potential vaccine candidates. We evaluated the immunogenicity of two important outer membrane proteins namely Aha1 and OmpW of A. hydrophila. These proteins were over-expressed in Escherichia coli, purified and used for the vaccination of common carp. Sequence analysis predicted that, Aha1 and OmpW are adhesins and antigenic. Common carp immunized with recombinant Aha1 and OmpW proteins showed significant antibody production and a relative percentage survival of 52 and 71 respectively indicating their protective efficacy against A. hydrophila infection.

Explicit hypoxia targeting with tumor suppression by creating an "obligate" anaerobic Salmonella Typhimurium strain.

Using bacteria as therapeutic agents against solid tumors is emerging as an area of great potential in the treatment of cancer. Obligate and facultative anaerobic bacteria have been shown to infiltrate the hypoxic regions of solid tumors, thereby reducing their growth rate or causing regression. However, a major challenge for bacterial therapy of cancer with facultative anaerobes is avoiding damage to normal tissues. Consequently the virulence of bacteria must be adequately attenuated for therapeutic use. By placing an essential gene under a hypoxia conditioned promoter, SalmonellaTyphimurium strain SL7207 was engineered to survive only in anaerobic conditions (strain YB1) without otherwise affecting its functions. In breast tumor bearing nude mice, YB1 grew within the tumor, retarding its growth, while being rapidly eliminated from normal tissues. YB1 provides a safe bacterial vector for anti-tumor therapies without compromising the other functions or tumor fitness of the bacterium as attenuation methods normally do.

Transcriptional profiles of multiple genes in the anterior kidney of channel catfish vaccinated with an attenuated Aeromonas hydrophila.

A total of 22 uniquely expressed sequence tags (ESTs) were identified from channel catfish anterior kidney subtractive cDNA library at 12 h post vaccination with an attenuated Aeromonas hydrophila (AL09-71 N+R). Of the 22 ESTs, six were confirmed to be significantly (P < 0.05) induced by the vaccination. Of 88 channel catfish genes selected from literature, 14 were found to be significantly (P < 0.05) upregulated by the vaccination. The transcriptional levels of the total 20 genes induced by the vaccination were then compared to that induced by the virulent parent A. hydrophila (AL09-71) at different time points. At 3 h post vaccination (hpv) or infection (hpi), Na(+)/K(+) ATPase α subunit was upregulated the most. At 6 and 12 hpv or hpi, hepcidin and interleukin-1ß were induced the highest. At 24 hpv or hpi, hepcidin was upregulated the most, followed by lysozyme c. At 48 hpi, lysozyme c and hepcidin were significantly induced. When vaccinated fish were challenged by AL09-71, relative percent of survival of vaccinated fish were 100% at 14 days post vaccination (dpv). Transcriptional levels of toll-like receptor 5 and hepcidin were significantly upregulated in vaccinated fish at 14 dpv. Taken together, our results suggest that vaccination with attenuated A. hydrophila mimics infection by live bacteria, inducing multiple immune genes in channel catfish.

Dental caries: from infection to prevention.

Dental caries is one of the most prevalent diseases in humans, second only to the common cold. It causes irreversible damage to the grinding machinery involved in the intake of food and hence causes great distress. The changes in the homeostasis of the oral cavity with an overgrowth of Streptococcus mutans is recognized as the primary cause of the disease. Most treatments are now aimed at either elimination of this bacterium or suppression of its virulence. S. mutans strongly adheres and releases acids by the fermentation of carbohydrates, leading to the demineralization of the tooth. This attachment is mediated mostly by the interaction of surface proteins and bacterial polysaccharides. Ambiguities in the basic treatment of dental caries, such as the use of fluoride and antibiotics, vitalize the deployment of probiotic therapies for its cure. The growing research in herbal treatments has led to the discovery of various phytochemicals to limit the virulence of S. mutans. This review focuses on the properties of S. mutans in cariogenicity and outlines ways to combat dental caries.

Comparison of the impact of pro- and antiinflammatory immune processes at the two immune-deviated sites, eye and solid tumor and possible consequences for the antitumoral therapy with fever inducers.

Findings from various epidemiological studies suggest that acute inflammation and fever may decrease the risk of developing certain types of cancer. In an established tumor situation acute inflammation and fever resulted in tumor regression in some cases, however, treatment was sometimes ineffective or even deleterious. It has been suggested that chronic inflammation contributes to carcinogenesis and in an established tumor situation to malignancy. In order to better understand the role of acute inflammation and fever in the treatment of cancer, we compare some of the current knowledge about the effects of pro- and antiinflammatory immunological processes in the two immune-deviated sites, eye and cancer. Striking similarities between these two sites have been demonstrated. The knowledge gained in experimental studies abrogating immune privilege in the eye could provide insights into the inconsistent results of proinflammatory cancer therapy. The eye could be used as a model to develop hypotheses on how to abolish the immunological tolerance state of a solid tumor and make it susceptible to immunological destruction. The immune privilege of the eye is abrogated, at least transiently, in the initial stages of tissue- or organ-specific cellular autoimmune responses and this effect is possibly supported by complement- fixing/proinflammatory antibodies. An overview of possible consequences for the tumor therapy especially in connection with inflammation and fever inducers such as bacterial vaccines or mistletoe preparations, which are therapeutically used to interfere with the privileged immune state of the tumor, is given.

The concept of "tailor-made", protein-based, outer membrane vesicle vaccines against meningococcal disease.

Protein-based, outer membrane vesicle (OMV) vaccines have previously proven to be efficacious against serogroup B meningococcal disease in Norway and Cuba. Currently, a public health intervention is going on in order to control a serogroup B epidemic in New Zealand. The scale-up and standardization of vaccine production required for controlling the New Zealand epidemic has allowed the establishment of large-scale GMP manufacturing for OMV vaccines. The outcome of this will be licensing of the vaccine in New Zealand and possibly other countries. The availability of licensed OMV vaccines raises the question of whether such vaccines may provide the opportunity to control other outbreaks and epidemics. For instance, such a vaccine could control a localised outbreak of group B meningococci in Normandy, France. "Tailor-made" vaccines, focusing on the sub-capsular antigens may also be considered for use in sub-Saharan Africa for the prevention of the recurrent outbreaks by serogroups A and W135 meningococci. This assumption is based on the epidemiological observation that meningococcal outbreaks in Africa are clonal and are strikingly stable regarding their phenotypic characteristics.

Evaluating the post-licensure effectiveness of a group B meningococcal vaccine in New Zealand: a multi-faceted strategy.

A nationwide strategy to control a group B meningococcal disease epidemic in New Zealand using an epidemic strain-specific vaccine (MeNZB ) commenced in 2004. In the absence of randomised controlled trials investigating the efficacy of this particular vaccine, a complement of observational methods are planned to evaluate the post-licensure effectiveness of this vaccine strategy. The two main approaches involve a Poisson regression model investigating the overall impact of the MeNZB programme on disease rates over time capitalising on detailed population-based disease surveillance data and the staged roll-out of the vaccine campaign, and a case-control study that aims to estimate vaccine effectiveness in pre-school children. The studies are designed to minimise the potential biases inherent in all observational methods and provide critical data on the effectiveness of a major public health intervention.

Infection prophylaxis of gunshot wounds using probiotics.

OBJECTIVE: To report a new method of surgical infection prophylaxis for postoperative gunshot wounds to the extremities. METHOD: Gunshot wounded animals were divided into three groups: treatment (probiotic Sporobacterin), antibiotic (cephalosporin cefamezin) and control (no treatment). Histological studies of wound-bed tissue were taken on days 1, 3, 5 and 10 of the study. RESULTS: The probiotic administered per os was more effective than antibiotics for prophylaxis of surgical infection. CONCLUSION: The probiotic's effect is based on the natural defence mechanism activated after injury--the bacterial translocation of saprophytic bacteria from the gut to the wound. DECLARATION OF INTEREST: None.

Humoral antibody response of Atlantic salmon, Salmo salar L., vaccinated against Moritella viscosa.

Immunization of Atlantic salmon, Salmo salar L., with experimental vaccine for protection against M. viscosa was tested. The antigen preparation used for the comparative studies was sonicate formalin killed M. viscose unwashed bacteria. Vaccination was carried out by intraperitoneal injection of bacterin in Biojec mineral oil as an adjuvant. When accumulated mortality in the control group with PBS of fish challenged by i.p. injection of M. viscose reached 100% with a challenge dose of 2.3 x 10(6) CFU, mortality in the experimental group was 71.23%. A positive correlation between the survival rates and corresponding median antibody levels of each group was found at the challenge time.

Adjuvant regulation of cytokine profile and antibody isotype of immune responses to Mycoplasma agalactiae in mice.

Traditionally, adjuvants have been administered with antigens to enhance immunity. We studied the effect of several adjuvants such as Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), lipopolysaccharide (LPS), homopolymers of polyinosinic-polycytidylic acid (poly I:C) and polyadenylic-polyuridylic acid (poly A:U), lithium chloride (LiCl), saponin Quil A and calcium phosphate gel (CaHPO(4)) on the immune response of mice to formalin-inactivated Mycoplasma agalactiae. The specific antibody or cytokine producing splenocytes were detected by ELISAspot and immunocytochemistry, respectively. Depending on the adjuvant given, the number of M. agalactiae-specific antibody producing cells was increased 2.5-6-fold. IgG was the major class of M. agalactiae-specific antibodies followed by IgM, IgA and IgE. Among IgG isotypes, FCA, FIA, Quil A and CaHPO(4) induced an IgG1 response with substantial increase of the IgG2a, IgG2b and IgG3 isotypes while poly I:C shifted the response toward an IgG2a/IgG3 production. Finally, poly A:U induced an IgG2b response while LPS and LiCl augmented the IgG3/IgG1/IgG2a secretion. FCA augmented IL-4, IL-5 and IL-10 production suggesting a strong Th2 response, while IFN-gamma and IL-12 remained low; poly I:C enhanced IFN-gamma, IL-12 and TNF-alpha eliciting a Th1 response; poly A:U resulted in a IL-10, IL-5, IL-6 and IL-12 secretion; and LPS enhanced the IL-10, IL-6 and TNF-alpha production. Our data show that adjuvants augment M. agalactiae-specific antibody production and lead to B cell isotype-switching via the appropriate cytokine milieu. Certain adjuvants, such as poly I:C, therefore, appear as promising immune enhancers for vaccination against M. agalactiae infections.

Antigenicity of Mycobacterium paratuberculosis superoxide dismutase in mice.

Mycobacterium paratuberculosis (MPT) is the etiologic agent of paratuberculosis. The disease is prevalent in cattle worldwide, and exacts a heavy financial toll. Effective control requires the development of acellular vaccines offering a better protection than the current available vaccines without side effects and allowing the discrimination between infected and vaccinated animals. We studied the immune response of mice to the MPT superoxide dismutase (SOD) alone or adjuvanted by Ribi. We cloned, overexpressed and purified this antigen in Escherichia coli. Spleen cells from immunized mice, after exposure to recombinant MPT SOD (MPT rSOD), produced significant levels of IFNgamma, TNFalpha and IL-6. IFNgamma and TNFalpha production was increased by the addition of Ribi. In contrast, low levels of NO, IL-4 and IL-10 were secreted by spleen cells culture from immunized mice. The immunoglobulin isotype distribution analysis showed that Ribi adjuvant clearly induced a significantly higher anti-MPT rSOD antibody production of all classes tested and decreased the IgG1/IgG2a ratio thus improving the Th1 response. Delayed-type hypersensitivity responses in mice footpads were observed only in mice immunized with MPT rSOD emulsified in Ribi. Vaccination of MPT rSOD emulsified with Ribi induced both a Th2 and Th1 type of immune response with the later slightly more pronounced. The results presented here on the immunogenicity of MPT SOD suggest that this antigen should be further tested as a candidate antigen for a future acellular vaccine against paratuberculosis.

Staphylococcus aureus capsular polysaccharide type 5 conjugate and whole cell vaccines stimulate antibody responses in cattle.

Dairy heifers were immunized subcutaneously with one of four different vaccines which contained preparations of Staphylococcus aureus capsular polysaccharide type 5 (CP5) and a mineral oil adjuvant, or received a placebo containing saline and adjuvant. The vaccine containing a CP5-human serum albumin conjugate (CP5-HSA) and the vaccine with formaldehyde inactivated whole cells expressing CP5, both elicited strong anti-CP5 antibody responses. After two injections three weeks apart and a third injection 10 months later, the mean level and duration of the anti-CP5 antibody response was significantly higher in the whole cell group. No differences were found between the two groups with regard to the relative proportion of IgG subclasses, and the antibody responses to the polysaccharide were composed of both the IgG1 and IgG2. Vaccines containing only free CP5 or CP5 mixed with HSA produced weak and transient humoral immune responses. Only animals vaccinated with the whole cell vaccine or the conjugate vaccine showed responses to CP5 in a lymphocyte proliferation assay conducted one year after the third vaccination. This study indicates that CP5 expressed on the surface of formaldehyde inactivated whole cells, emulsified in an oil adjuvant, gives a strong and long lasting immune response in cattle. The use of conjugation technology, although effective, might not be necessary in order to achieve an immune response against S. aureus CP5 in cattle.

Salmonella. typhi OMPs induced immunomodulation in peritoneal macrophages.

The immunomodulatory properties of outer membrane proteins (OMPs) from S. typhi Ty2 were studied in mouse model at 72 hr and 20 days post-infection. Inspite of reduction in the number of macrophages and their protein content observed in the immunized group vis-à-vis infected group, OMPs activated macrophages showed significant upregulation of NO. At 20 days post infection, the level remained almost the same suggesting the prolonged cytotoxic and cytostatic activity due to the long lasting effects of OMPs activated macrophages. Higher activity of SOD in these aged cells pointed out towards the protective efficacy of OMPs to keep the macrophages themselves away from the noxious effects of O2-. Lower level of acid phosphatase in the macrophages from immunized mice group indicated the involvement of oxygen dependent rather than oxygen independent killing process. The enhanced uptake of organisms and their killing could be related to the production of oxygen and nitrogen radicals in the OMPs immunized group.