Alginate-chitosan microcapsules improve vaccine potential of gamma-irradiated Listeria monocytogenes against listeriosis in murine model.

Listeria monocytogenes is a cause of infectious food-borne disease in humans, characterized by neurological manifestations, abortion, and neonatal septicemia. It is intracellular bacterium, which limits the development of protective inactivated vacines. Adjuvants capable of stimulating cellular immune response are important tools for developing novel vaccines against intracellular bacteria. The aim of this study was to evaluate the vaccine potential of L. monocytogenes inactivated by gamma irradiation (KLM-γ) encapsulated in alginate microcapsules associated or not with chitosan against listeriosis in the murine model. At the fourth day after challenge there was a reduction in bacterial recovery in mice vaccinated with KLM-γ encapsulated with alginate or alginate-chitosan, with lower bacterial loads in the spleen (10 fold) and liver (100 fold) when compared to non-vaccinated mice. In vitro stimulation of splenocytes from mice vaccinated with alginate-chitosan-encapsulated KLM-γ resulted in lymphocyte proliferation, increase of proportion of memory CD4+ and CD8+ T cell and production of IL-10 and IFN-γ. Interestingly, the group vaccinated with alginate-chitosan-encapsulated KLM-γ had increased survival to lethal infection with lower L. monocytogenes-induced hepatic inflammation and necrosis. Therefore, KLM-γ encapsulation with alginate-chitosan proved to have potential for development of novel and safe inactivated vaccine formulations against listeriosis.

Revisiting the Development of Vaccines Against Pathogenic Leptospira: Innovative Approaches, Present Challenges, and Future Perspectives.

Human vaccination against leptospirosis has been relatively unsuccessful in clinical applications despite an expressive amount of vaccine candidates has been tested over years of research. Pathogenic Leptospira encompass a great number of serovars, most of which do not cross-react, and there has been a lack of genetic tools for many years. These obstacles have hampered the understanding of the bacteria's biology and, consequently, the identification of an effective antigen. Thus far, many approaches have been used in an attempt to find a cost-effective and broad-spectrum protective antigen(s) against the disease. In this extensive review, we discuss several strategies that have been used to develop an effective vaccine against leptospirosis, starting with Leptospira-inactivated bacterin, proteins identified in the genome sequences of pathogenic Leptospira, including reverse vaccinology, plasmid DNA, live vaccines, chimeric multi-epitope, and toll- and nod-like receptors agonists. This overview should be able to guide scientists working in the field to select potential antigens and to choose the appropriate formulation to administer the candidates.

Immunoprotective Activity Induced by Leptospiral Outer Membrane Proteins in Hamster Model of Acute Leptospirosis.

Leptospirosis is a zoonotic disease of worldwide distribution, affecting both humans and animals. The development of an effective vaccine against leptospirosis has long been pursued but without success. Humans are contaminated after direct contact with the urine of infected animals or indirectly by contaminated water or soil. The vaccines available consist of inactivated whole-bacterial cells, and the active immunoprotective antigen is the lipopolysaccharide moiety, which is also the basis for serovar classification. However, these vaccines are short-lasting, and protection is only against serovars contained in the preparation. The search for prevalent antigens, present in pathogenic species of Leptospira, represents the most cost-effective strategy for prevention of leptospirosis. Thus, the identification of these antigens is a priority. In this study, we examined the immunoprotective effect of eight leptospiral recombinant proteins using hamster as the challenge model. Animals received subcutaneously two doses of vaccine containing 50 µg of each recombinant protein adsorbed on alum adjuvant. Two weeks after the booster, animals were challenged with virulent leptospires and monitored for 21 days. All proteins were able to induce a specific immune response, although significant protective effects on survival rate were observed only for the proteins Lsa14, rLIC13259, and rLIC11711. Of these, only rLIC13259 and rLIC11711 were found to be highly prospective in promoting renal clearance. The sterilizing potential of both proteins will be further investigated to elucidate the immunoprotective mechanisms involved in leptospirosis control. These are the first proteins involved with human complement components with the capacity to protect against virulent challenge and to eliminate the bacteria from the host.

Variations in the immune and metabolic response of proactive and reactive Sparus aurata under stimulation with Vibrio anguillarum vaccine.

Environmental insults, such as exposure to pathogens, modulate the behavioural coping style of animals to stressors, and repeated exposure to stressful environments may lead to species-specific infection phenotypes. To analyse the influence of stress behavioural phenotypes on immune and metabolic performance, gilthead sea bream (Sparus aurata L.) were first screened for proactive and reactive coping styles. Once characterized, both behavioural phenotypes fish groups were bath vaccinated with bacterin from Vibrio anguillarum, an opportunistic widespread pathogen of fish. Gills and liver were sampled at 0 (control group), 1, 3 and 7 days post-vaccination. Immune-, oxidative stress- and metabolic-related transcripts (il1ß, tnfα, igm, gpx1, sod, cat, lpl, ghr1 and ghr2), metabolic endpoints (glucose, cholesterol and triglycerides), hepatic health indicators (aspartate aminotransferase, alanine transaminase and alkaline phosphatase), oxidative stress status (esterase activity, total antioxidant capacity and total oxidative status) and stress biomarkers (cortisol) were determined. Present results indicate that screening for coping styles in the gilthead sea bream segregated the two distinct phenotypes as expected: proactive and reactive. Results also indicate that under bath vaccination proactive fish show high immune response and lower metabolism, whereas reactive fish show low immune and higher metabolic responses.

Immunogenicity of a Bivalent Non-Purified Recombinant Vaccine against Botulism in Cattle.

Botulism is a potentially fatal intoxication caused by botulinum neurotoxins (BoNTs) produced mainly by Clostridium botulinum. Vaccination against BoNT serotypes C and D is the main procedure to control cattle botulism. Current vaccines contain formaldehyde-inactivated native BoNTs, which have a time-consuming production process and pose safety risks. The development of non-toxic recombinant vaccines has helped to overcome these limitations. This study aims to evaluate the humoral immune response generated by cattle immunized with non-purified recombinant fragments of BoNTs C and D. Cattle were vaccinated in a two-dose scheme with 100, 200 and 400 µg of each antigen, with serum sampling on days 0, 56, 120, and 180 after vaccination. Animals who received either 200 or 400 µg of both antigens induced titers higher than the minimum required by the Brazilian ministry of Agriculture, Livestock and Food Supply and achieved 100% (8/8) seroconversion rate. Animals vaccinated with commercial toxoid vaccine had only a 75% (6/8) seroconversion rate for both toxins. Animals that received doses containing 400 µg of recombinant protein were the only ones to maintain titers above the required level up until day 120 post-vaccination, and to achieve 100% (8/8) seroconversion for both toxins. In conclusion, 400 µg the recombinant Escherichia coli cell lysates supernatant was demonstrated to be an affordable means of producing an effective and safe botulism vaccine for cattle.

LemA and Erp Y-like recombinant proteins from Leptospira interrogans protect hamsters from challenge using AddaVax™ as adjuvant.

BACKGROUND: Recombinant subunit vaccines have been extensively evaluated as promising alternatives against leptospirosis. Here, we evaluated two proteins in formulations containing the adjuvant AddaVax™ as vaccine candidates for prevention and control of leptospirosis. METHODS: Recombinant proteins rErp Y-like and rLemA were characterized by ELISA to assess their ability to bind extracellular matrix (ECM) components and fibrinogen. Groups of eight hamsters were immunized intramuscularly with rErp Y-like or rLemA mixed with a squalene-based adjuvant (AddaVax), and then vaccine efficacy was determined in terms of protection against a lethal challenge. The humoral immune response was determined by ELISA, and the evidence of sub-lethal infection was evaluated by histopathology and kidney culture. RESULTS: rLemA protein binds laminin, fibrinogen, and collagen type IV, while rErp Y-like interacts with fibrinogen. Significant protection was achieved for rLemA and rErp Y-like vaccines, which showed 87.5% and 62.5% survivals, respectively. On day 28, the humoral immune response was significantly greater in the vaccine groups as compared to that in the control group, and the response was predominantly based on IgG2/3. The surviving animals showed negative results in culture isolation but presented with tissue lesions in the lungs and kidneys. CONCLUSION: Cumulatively, our findings suggest that LemA and Erp Y-like proteins act as adhesins and are able to protect against mortality, but not against tissue lesions. Moreover, AddaVax is a novel adjuvant with potential for improving the immunogenicity of leptospiral vaccines.

Recombinant Hsp33 and OmpC protein can serve as promising divalent vaccine with protection against Vibrio anguillarum and Edwardsiella tarda in flounder (Paralichthys olivaceus).

Vibrio anguillarum and Edwardsiella tarda are severe aquaculture pathogens shared similar epidemiological characteristics and susceptible to flounder (Paralichthys olivaceus). In our previous studies, recombinant(r) protein heat shock protein 33 (rHsp33) from V. anguillarum and outer membrane protein C (rOmpC) from E. tarda were proved to have protection against V. anguillarum and E. tarda, respectively. In this paper, the cross protection of rHsp33 against E. tarda and rOmpC against V. anguillarum, and the protection of divalent vaccine candidate (rHsp33 + rOmpC, rHC) against both V. anguillarum and E. tarda were evaluated. RHC, rHsp33, and rOmpC were vaccinated to flounder, respectively, and the percentages of surface immunoglobulin-positive (sIg+) cells in peripheral blood lymphocytes (PBLs), serum IgM, specific antibodies against V. anguillarum or E. tarda, specific antibodies against rHsp33, rOmpC or rHC, the expression of immune-related genes and relative percent survival (RPS) against V. anguillarum or E. tarda were measured. The results showed that: RHC could induced the enhancement of sIg + cells and high levels of specific antibodies against both V. anguillarm and E. tarda; Also a significant increase of specific antibodies against rHsp33, rOmpC or rHC, and up-regulation of gene expression of CD3, CD4-1, CD4-2, CD8α, CD8ß and IgM in spleen, head-kidney, and hindgut, RPS of 70 ±â€¯3.45% against V. anguillarum and 60 ±â€¯1.48% against E. tarda, respectively. In addition, rHsp33 induced specific antibodies against E. tarda and rOmpC, and had a RPS of 43.3 ±â€¯3.73% against E. tarda; rOmpC could evoke specific antibodies against V. anguillarum and rHsp33, and had a RPS of 44 ±â€¯1.27% against V. anguillarm; The results demonstrated that there was cross protection of rHsp33 against E. tarda and rOmpC against V. anguillarum, rHC as a divalent vaccine can induce significant immune response and efficient protection against both E. tarda and V. anguillarum in flounder.

An integrative in-silico approach for therapeutic target identification in the human pathogen Corynebacterium diphtheriae.

Corynebacterium diphtheriae (Cd) is a Gram-positive human pathogen responsible for diphtheria infection and once regarded for high mortalities worldwide. The fatality gradually decreased with improved living standards and further alleviated when many immunization programs were introduced. However, numerous drug-resistant strains emerged recently that consequently decreased the efficacy of current therapeutics and vaccines, thereby obliging the scientific community to start investigating new therapeutic targets in pathogenic microorganisms. In this study, our contributions include the prediction of modelome of 13 C. diphtheriae strains, using the MHOLline workflow. A set of 463 conserved proteins were identified by combining the results of pangenomics based core-genome and core-modelome analyses. Further, using subtractive proteomics and modelomics approaches for target identification, a set of 23 proteins was selected as essential for the bacteria. Considering human as a host, eight of these proteins (glpX, nusB, rpsH, hisE, smpB, bioB, DIP1084, and DIP0983) were considered as essential and non-host homologs, and have been subjected to virtual screening using four different compound libraries (extracted from the ZINC database, plant-derived natural compounds and Di-terpenoid Iso-steviol derivatives). The proposed ligand molecules showed favorable interactions, lowered energy values and high complementarity with the predicted targets. Our proposed approach expedites the selection of C. diphtheriae putative proteins for broad-spectrum development of novel drugs and vaccines, owing to the fact that some of these targets have already been identified and validated in other organisms.

Egg quality in laying hens exposed to Mycoplasma gallisepticum F-strain attenuated vaccine.

Mycoplasma gallisepticum causes coughing, ocular and nasal discharge, reduction in feed intake, lower and uneven growth, decline in egg production and quality, and increase in mortality. Among the attenuated vaccination strains, MGF can reduce clinical signs and lesions in layer hens, stimulate immune responses of cellular and humoral basis, act as an instrument of competitive exclusion in relation to field strains, and reduce the use of antimicrobials. This study aimed to investigate the effects of attenuated MG F-strain vaccination on egg quality in 3 groups of 30 hens each, being one control and 2 vaccinated. Vaccination was applied by ocular route at 8 and 12 wk of age. Comparisons were made among unvaccinated hens; vaccinated at 8 wk of age; and vaccinated at 8 and 12 wk of age. There were no statistical differences in eggshell thickness and weight among groups. Eggs from twice vaccinated birds yielded a Haugh unit significantly lower than the other groups without affecting egg classification. There was no significant difference in ELISA results between the vaccinated groups.

Vaccination with recombinant Clostridium perfringens toxoids α and ß promotes elevated antepartum and passive humoral immunity in swine.

Due to the increasingly restricted use of antimicrobials in animal production systems, the prevention and control of Clostridium perfringens type A- and C-induced diarrhea in piglets should be based on passive immunization via the prepartum vaccination of sows. Given the current obstacles in the production of conventional clostridial vaccines, the use of recombinant proteins has been considered to represent a promising alternative. In the present study, the neutralizing antibody response of immunized sows and their litters to a bivalent vaccine containing the C. perfringens recombinant toxoids alpha (rTA) and beta (rTB) produced in Escherichia coli was assessed. Rabbits (n=8) and pregnant sows (n=7) were immunized with 200µg of each recombinant antigen using Al(OH)3 as adjuvant. The alpha and beta antitoxin titer detected in the rabbits' serum pool was 9.6 and 20.4IU/mL, respectively. The mean alpha and beta antitoxin titers in the sows' sera were 6.0±0.9IU/mL and 14.5±2.2IU/mL, and the corresponding individual coefficients of variation (CV) were 16.04% and 14.91%, respectively. The mean alpha and beta antitoxin titers in the litters' serum pools were 4.2±0.4IU/mL and 10.9±1.7IU/mL, and the CV between litters was 9.23% and 9.85%, respectively. The results showed that the rTA and rTB proteins produced and tested in the present study induced an immune response and can be regarded as candidates for the development of a commercial vaccine against C. perfringens type A- and C-induced diarrhea in pigs.

Study on the efficacy of Leptospira vaccines developed from serovars isolated from Trinidad and comparison with commercial vaccines using a hamster model.

A hamster model was used to determine the efficacy of commercially prepared canine vaccines against Leptospira serovars circulating in Trinidad and to assess the effectiveness of killed whole-cell vaccines prepared from local isolates. The local isolates used for vaccine preparation and challenge were isolates of serovars Copenhageni and Mankarso obtained from a local dog and rodent. Their estimated lethal dose-50 (LD(50)) were 5 and 10 organisms, respectively and clinical signs observed on infection were consistent with leptospirosis. An unvaccinated control group of hamsters and other groups of hamsters that had been vaccinated with 3 doses of (i) in-house whole-cell Copenhageni vaccine, (ii) in-house whole-cell Mankarso vaccine, (iii) commercial vaccine Brand A or (iv) commercial vaccines Brand B were challenged with 1000 times the LD(50) of the respective challenge serovar. The most commonly used commercial vaccine (Brand A) did not offer protection to challenged hamsters, whereas Brand B facilitated the renal carrier state of the Leptospira organism. In contrast the whole-cell vaccines developed from local strains of serovars Copenhageni and Mankarso, protected all hamsters tested from both clinical disease and renal carrier states.

Intranasal immunisation with recombinant Lactococcus lactis displaying either anchored or secreted forms of Proteus mirabilis MrpA fimbrial protein confers specific immune response and induces a significant reduction of kidney bacterial colonisation in mice.

Proteus mirabilis, a common cause of urinary tract infections in humans, can express different fimbriae. MR/P fimbriae may contribute to bacterial colonisation, and its structural protein MrpA represents a promising candidate antigen for mucosal vaccination. Commercial complex vaccines have limited, short-lived protection and are incapable of eliciting mucosal responses against putative antigens related to virulence. The development of mucosal live vaccines using food-grade lactic acid bacterium Lactococcus lactis as antigen vehicle is an attractive alternative and a safe vaccination strategy against P. mirabilis infection. Here, we report the construction of L. lactis strains modified to produce MrpA via two cellular locations, cell wall-anchored and secreted. Protection assays against P. mirabilis infection and evaluation of the immune response generated after immunisation were conducted in a mouse model. MrpA protein was efficiently expressed by L. lactis strain and caused a significant induction of specific serum IgG and IgA in the animals immunised with L. lactis pSEC:mrpA and L. lactis pCWA:mrpA respectively. A significant reduction of renal bacterial colonisation was observed in both groups of mice (P<0.05) after P. mirabilis challenge. This is the first example of a P. mirabilis fimbrial antigen expressed in a food-grade live strain with promising applications in vaccine design.

A DNA vaccine encoding Cu,Zn superoxide dismutase of Brucella abortus induces protective immunity in BALB/c mice.

This study was conducted to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). Intramuscular injection of plasmid DNA carrying the SOD gene (pcDNA-SOD) into BALB/c mice elicited both humoral and cellular immune responses. Animals injected with pcDNA-SOD developed SOD-specific antibodies which exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the DNA vaccine elicited a T-cell-proliferative response and also induced the production of gamma interferon, but not interleukin-10 (IL-10) or IL-4, upon restimulation with either recombinant SOD or crude Brucella protein, suggesting the induction of a typical T-helper-1-dominated immune response in mice. The pcDNA-SOD (but not the control vector) induced a strong, significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308; the level of protection was similar to the one induced by B. abortus vaccine strain RB51. Altogether, these data suggest that pcDNA-SOD is a good candidate for use in future studies of vaccination against brucellosis.

Identification and characterization of a Brucella abortus ATP-binding cassette transporter homolog to Rhizobium meliloti ExsA and its role in virulence and protection in mice.

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not fully understood yet. Furthermore, genes that allow Brucella to reach the intracellular niche and to interact with host cells need to be identified. Using the genomic survey sequence (GSS) approach, we identified the gene encoding an ATP-binding cassette (ABC) transporter of B. abortus strain S2308. The deduced amino acid sequence encoded by this gene exhibited 69 and 67% identity with the sequences of the ABC transporters encoded by the exsA genes of Rhizobium meliloti and Mesorhizobium loti, respectively. Additionally, B. abortus ExsA, like R. meliloti and M. loti ExsA, possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Furthermore, ortholog group analysis placed B. abortus ExsA in ortholog group 6 of ABC transporters more likely to be involved in bacterial pathogenesis. In R. meliloti, ExsA is an exopolysaccharide transporter essential for alfalfa root nodule invasion and establishment of infection. To test the role of ExsA in Brucella pathogenesis, an exsA deletion mutant was constructed. Replacement of the wild-type exsA by recombination was demonstrated by Southern blot analysis of Brucella genomic DNA. Decreased survival in mice of the Brucella DeltaexsA mutant compared to the survival of parental strain S2308 demonstrated that ExsA is critical for full bacterial virulence. Additionally, the B. abortus exsA deletion mutant was used as a live vaccine. Challenge experiments revealed that the exsA mutant strain induced superior protective immunity in BALB/c mice compared to the protective immunity induced by strain S19 or RB51.

DNA immunisation against the CFA/I fimbriae of enterotoxigenic Escherichia coli (ETEC).

The CFA/I fimbria promotes the attachment of enterotoxigenic Escherichia coli (ETEC) to the surface of human enterocytes. The generation of a protective immune response requires the induction of antibodies able to block the CFA/I-mediated binding of ETEC to receptors located on the small intestine epithelium or on the surface of human red blood cells, in hemagglutination tests. An eukaryotic expression plasmid, pBLCFA, encoding the CFA/I gene under the control of the human cytomegalovirus major immediate-early promoter was constructed as a prototype DNA vaccine against ETEC. pBLCFA-tranfected BHK-21 cells secreted a peptide cross-reacting with a monoclonal antibody raised against CFA/I subunits. BALB/c mice immunized intramuscularly with one or two doses of purified pBLCFA developed CFA/I-specific serum antibodies for at least 52 weeks, composed predominantly of the IgG1 subclass. pBLCFA-induced antibodies bind mainly to epitopes exposed on the surface of intact CFA/I fimbriae and do not react with immune recessive epitopes found in other ETEC fimbra sharing amino acid homologies with CFA/I. Furthermore, pBLCFA-induced antibodies were able to block the adhesive properties of the CFA/I fimbriae, as evaluated by the ability to inhibit the hemagglutination promoted by CFA/I-expressing ETEC cells. These results suggest that secretion of CFA/I encoded by pBLCFA preserves important conformational epitopes required for the generation of protective antibodies against the adhesive properties of the CFA/I fimbriae and open new perspectives for the development of DNA vaccines against enteric bacterial pathogens.

Immunization with bacterial antigens: piscirickettsiosis.

Piscirickettsiosis is a septicaemic disease of salmonid fish caused by the obligated intracellular rickettsia, Piscirickettsia salmonis. This disease was first reported in 1989 in salmon cultured in sea water netpens in southern Chile where it is still a major problem causing high mortality among cultured salmonids. In recent years related agents have been reported in farmed salmonids from Ireland, Canada and Norway. Mortality, however, at these locations has been reported to be low. Because of the recent description of piscirickettsiosis and its aetiological agent, knowledge about the immune response of fish against this organism is limited. At present, there is only one paper in the literature dealing with this subject. To standardise challenge methods for testing the efficacy of vaccination, lethal dose 50% and infectivity dose 50% were determined for coho salmon (Oncorhynchus kisutch) and rainbow trout (O. mykiss) using intraperitoneal (i.p.) injections of P. salmonis. Experiments using bath challenge methods failed to reproduce the disease using rainbow trout although low levels of infection in their tissues were found. In a field trial, using formalin killed bacterins injected i.p. into pre-smolt coho salmon, the fish were naturally challenged by placing them in sea water where endemic piscirickettsiosis occurred. The results showed that some of the vaccinated fish groups experienced lower cumulative mortality than the non-vaccinated control group (X < 0.05), suggesting an immunoprotective response in these animals. A trial was also conducted with formalin-killed bacterins in rainbow trout using different antigen concentrations with and without booster injections. Fish were challenged by IP injection of P. salmonis. Vaccinated fish showed less mortality than their respective infected control. Unfortunately the challenge was not strong enough because mortality in the infected control fish was low (20%). Antibody levels measured by radio-immuno-assay increased until day 40 post vaccination. The highest levels of antibody were obtained in the sera of fish vaccinated with concentrated antigen using booster injections.

Interference of immune globulin with measles and rubella immunization.

Passively acquired antibody may interfere with the active antibody response to live viral vaccines such as measles and rubella. To evaluate the duration of this inhibitory effect, we measured the measles and rubella antibody responses of Apache children immunized with measles, mumps, and rubella vaccine at varying intervals after administration of an immune globulin termed bacterial polysaccharide immune globulin (BPIG). This specific immune globulin contained measles and rubella antibody titers similar to those in standard intramuscularly and intravenously administered immune globulins. Antibody responses to measles vaccine were inhibited for up to 5 months after a BPIG dose of 80 mg IgG per kilogram of body weight, but responses to rubella vaccine were inhibited for only 2 months. Most children who had a decreased measles antibody response to primary measles, mumps, and rubella immunization given 1 1/2 to 4 months after BPIG administration responded to a booster immunization given 6 months after their last BPIG dose. We conclude that high doses of immune globulin (> 10 mg/kg) may inhibit the antibody response to measles for more than 3 months. We propose that the interval between administration of immune globulin and measles and rubella immunization be adjusted on the basis of the dose of immune globulin.