The HSP70 and IL-1ß of Nile tilapia as molecular adjuvants can enhance the immune protection of DNA vaccine against Streptococcus agalactiae infection.

Globally, streptococcal disease caused by Streptococcus agalactiae is known for its high mortality rate, which severely limits the development of the tilapia breeding industry. As a third-generation vaccine, DNA vaccines have shown great application prospects in the prevention and control of aquatic diseases, but their low immunogenicity limits their development. The combination of DNA vaccines and molecular adjuvants proved to be an effective method for inducing protective immunity. This study constructed recombinant plasmids encoding tilapia HSP70 and IL-1ß genes (pcHSP70 and pcIL-1ß) to verify their effectiveness as molecular adjuvants for S. agalactiae DNA vaccine (pcSIP) in the immunized tilapia model. The results revealed that serum-specific IgM production, enzyme activities, and immune-related gene expression in tilapia immunized with pcSIP plus pcHSP70 or pcIL-1ß were significantly higher than those in tilapia immunized with pcSIP alone. It is worth noting that combination with molecular adjuvants improved the immune protection of DNA vaccines, with a relative percentage survival (RPS) of 51.72% (pcSIP plus pcHSP70) and 44.83% (pcSIP plus pcIL-1ß), respectively, compared with that of pcSIP alone (24.14%). Thus, our study indicated that HSP70 and IL-1ß in tilapia are promising molecular adjuvants of the DNA vaccine in controlling S. agalactiae infection.

Using Surface Immunogenic Protein as a Carrier Protein to Elicit Protective Antibody to Multiple Serotypes for Candidate Group B Streptococcal Glycan Conjugate Vaccines.

Group B Streptococcus (GBS) is a life-threatening opportunistic pathogen, particularly in pregnant women, infants, and the elderly. Currently, maternal vaccination is considered the most viable long-term option for preventing GBS mother-to-infant infection, and two polysaccharide conjugate vaccines utilizing CRM197 as a carrier protein have undergone clinical phase II trials. Surface immunogenic protein (Sip), present in all identified serotypes of GBS strains so far, is a protective surface protein of GBS. In this study, the type Ia capsular polysaccharide (CPS) of GBS was utilized as a model to develop candidate antigens for a polysaccharide conjugate vaccine by coupling it with the Sip of GBS and the traditional carrier protein CRM197. Serum analysis from immunized New Zealand rabbits and CD1 mice revealed that there was no significant difference in antibody titers between the Ia-Sip group and Ia-CRM197 group; however, both were significantly higher than those observed in the Ia polysaccharide group. Opsonophagocytosis and passive immune protection results using rabbit serum indicated no significant difference between the Ia-Sip and Ia-CRM197 groups, both outperforming the Ia polysaccharide group. Furthermore, serum from the Ia-Sip group had a cross-protective effect on multiple types of GBS strains. The challenge test results in CD1 mice demonstrated that the Ia-Sip group provided complete protection against lethal doses of bacteria and also showed cross-protection against type III strain. Our study demonstrates for the first time that Ia-Sip is immunogenic and provides serotype-independent protection in glycan conjugate vaccines, which also indicates Sip may serve as an excellent carrier protein for GBS glycan conjugate vaccines and provide cross-protection against multiple GBS strains.

BacScan: a novel genome-wide strategy for uncovering broadly immunogenic proteins in bacteria.

In response to the global threat posed by bacterial pathogens, which are the second leading cause of death worldwide, vaccine development is challenged by the diversity of bacterial serotypes and the lack of immunoprotection across serotypes. To address this, we introduce BacScan, a novel genome-wide technology for the rapid discovery of conserved highly immunogenic proteins (HIPs) across serotypes. Using bacterial-specific serum, BacScan combines phage display, immunoprecipitation, and next-generation sequencing to comprehensively identify all the HIPs in a single assay, thereby paving the way for the development of universally protective vaccines. Our validation of this technique with Streptococcus suis, a major pathogenic threat, led to the identification of 19 HIPs, eight of which conferred 20-100% protection against S. suis challenge in animal models. Remarkably, HIP 8455 induced complete immunity, making it an exemplary vaccine target. BacScan's adaptability to any bacterial pathogen positions it as a revolutionary tool that can expedite the development of vaccines with broad efficacy, thus playing a critical role in curbing bacterial transmission and slowing the march of antimicrobial resistance.

Recombinant lactococcal-based oral vaccine for protection against Streptococcus agalactiae infections in tilapia (Oreochromis niloticus).

Streptococcosis outbreaks caused by Streptococcus agalactiae infection in tilapia aquaculture have been consistently reported and associated with high mortality and morbidity leading to significant economic losses. Existing vaccine candidates against Streptococcus spp. are designed for intraperitoneal injections that are not practical and labor-intensive which have prompted farmers to protect aquatic animals with antibiotics, thus encouraging the emergence of multidrug resistant bacteria. In this study, a live recombinant L. lactis vaccine expressing a 1403 bp surface immunogenic protein (SIP) and a 1100 bp truncated SIP (tSIP) gene was developed and evaluated against S. agalactiae infection in tilapia. Both SIP and tSIP sequences were cloned and transformed into L. lactis. The recombinant L.lactis vaccine was orally administered to juvenile tilapia for a month. Detection of SIP-specific serum IgM in vaccinated groups compared to control groups indicated that recombinant proteins expressed from L. lactis could elicit immunogenic reactions in tilapia. Fish immunized with the tSIP vaccine also showed the highest level of protection compared to other test groups, and the mortality rate was significantly reduced compared to both control groups. The relative percentage of survival (RPS) against S. agalactiae for both SIP and tSIP-vaccinated groups was 50 % and 89 %, respectively, at 14 days post-challenge. Significant up-regulation of IgM, IL-1ß, IL-10, TNF-α and IFN-γ were observed at day 34 between the vaccinated and control groups. These results indicated that the recombinant lactococcal tSIP vaccine can elicit both cell-mediated and humoral responses and is recommended as a potential oral vaccine against S. agalactiae infection. Future work will include further in vivo challenge assessments of this vaccine candidate fused with adjuvants to boost immunogenicity levels in tilapia.

Whole proteome screening to develop a potent epitope-based vaccine against Coxiella burnetii: a reverse vaccinology approach.

Coxiellosis is known as a threat to human health. This study aimed to develop an epitope-based vaccine against coxiellosis using a whole proteome investigation. In this case, the whole proteome of Coxiella burnetii was collected from the database, then different assessments were performed to select immunogenic proteins. The selected proteins were used for epitopes prediction. The epitope-based vaccine was made using the best-selected epitopes and HBHA protein. The physical and chemical features, as well as secondary and tertiary structures of the developed vaccine were analyzed. The interaction between the developed vaccine and TLR4/MD2 receptor was examined using molecular docking and molecular dynamic simulation. Finally, in silico cloning, codon optimization, and immune response simulation for the developed vaccine were performed. The findings supported a stable, hydrophilic, antigenic and non-allergenic vaccine with a molecular weight equal to 59.261 kDa and 542 amino acid residues in length. The findings showed that the developed vaccine not only could dock to TRL4/MD2 receptor with an affinity of -20.9 kcal/mol and 15 hydrogen bonds, but also the protein-protein complex was stable during molecular dynamic simulation with the binding free energy of -57.9 ± 6.9 kcal/mol. Furthermore, the optimized sequence of the developed vaccine with a CAI value of 0.97, could be cloned into the pET-21a (+) vector. Finally, The results confirmed that the developed vaccine could strongly trigger primary and secondary immune responses. Evidently, the developed vaccine can be an interesting candidate to apply.Communicated by Ramaswamy H. Sarma.

A comprehensive review on immunogen and immune-response proteins of SARS-CoV-2 and their applications in prevention, diagnosis, and treatment of COVID-19.

Exposure to severe acute respiratory syndrome-corona virus-2 (SARS-CoV-2) prompts humoral immune responses in the human body. As the auxiliary diagnosis of a current infection, the existence of viral proteins can be checked from specific antibodies (Abs) induced by immunogenic viral proteins. For people with a weakened immune system, Ab treatment can help neutralize viral antigens to resist and treat the disease. On the other hand, highly immunogenic viral proteins can serve as effective markers for detecting prior infections. Additionally, the identification of viral particles or the presence of antibodies may help establish an immune defense against the virus. These immunogenic proteins rather than SARS-CoV-2 can be given to uninfected people as a vaccination to improve their coping ability against COVID-19 through the generation of memory plasma cells. In this work, we review immunogenic and immune-response proteins derived from SARS-CoV-2 with regard to their classification, origin, and diverse applications (e.g., prevention (vaccine development), diagnostic testing, and treatment (via neutralizing Abs)). Finally, advanced immunization strategies against COVID-19 are discussed along with the contemporary circumstances and future challenges.

A nanocarrier immersion vaccine encoding surface immunogenic protein confers cross-immunoprotection against Streptococcus agalactiae and Streptococcus iniae infection in tilapia.

Streptococcosis is a highly contagious aquatic bacterial disease that poses a significant threat to tilapia. Vaccination is a well-known effective measure to prevent and control fish bacterial diseases. Among the various immunization methods, immersion vaccination is simple and can be widely used in aquaculture. Besides, nanocarrier delivery technology has been reported as an effective solution to improve the immune effect of immersion vaccine. In this study, the surface immunogenic protein (Sip) was proved to be conserved and potential to provide cross-immunoprotection for both Streptococcus agalactiae (S. agalactiae) and Streptococcus iniae (S. iniae) by multiple sequences alignment and Western blotting analysis. On this basis, we expressed and obtained the recombinant protein rSip and connected it with functionalized carbon nanotubes (CNT) to construct the nanocarrier vaccine system CNT-rSip. After immersion immunization, the immune effect of CNT-rSip against above two streptococcus infections was evaluated in tilapia based on some aspects including the serum specific antibody level, non-specific enzyme activities, immune-related genes expression and relative percent survival (RPS) after bacteria challenge. The results showed that compared with control group, CNT-rSip significantly (P < 0.05) increased the serum antibody levels, related enzyme activities including acid phosphatase, alkaline phosphatase, lysozyme and total antioxidant capacity activities, as well as the expression levels of immune-related genes from 2 to 4 weeks post immunization (wpi), and all these indexes peaked at 3 wpi. Besides, the above indexes of CNT-rSip were higher than those of rSip group with different extend during the experiment. Furthermore, the challenge test indicated that CNT-rSip provided cross-immunoprotection against S. agalactiae and S. iniae infection with RPS of 75 % and 72.41 %, respectively, which were much higher than those of other groups. Our study indicated that the nanocarrier immersion vaccine CNT-rSip could significantly improve the antibody titer and confer cross-immuneprotection against S. agalactiae and S. iniae infection in tilapia.

Exploring the Immunoprotective Potential of a Nanocarrier Immersion Vaccine Encoding Sip against Streptococcus Infection in Tilapia (Oreochromis niloticus).

Tilapia, as one of the fish widely cultured around the world, is suffering severe impact from the streptococcus disease with the deterioration of the breeding environment and the increasing of breeding density, which brings serious economic loss to tilapia farming. In this study, the surface immunogenic protein (Sip) of Streptococcus agalactiae (S. agalactiae) was selected as the potential candidate antigen and connected with bacterial nano cellulose (BNC) to construct the nanocarrier subunit vaccine (BNC-rSip), and the immersion immune effects against S. agalactiae and Streptococcus iniae (S. iniae) in Nile tilapia were evaluated on the basis of the serum antibody level, non-specific enzyme activity, the immune-related gene expression and relative percent survival (RPS). The results indicated that Sip possessed the expected immunogenicity according to the immunoinformatic analysis. Compared with the rSip group, BNC-rSip significantly induced serum antibody production and improved the innate immunity level of tilapia. After challenge, the RPS of BNC-rSip groups were 78.95% (S. agalactiae) and 67.86% (S. iniae), which were both higher than those of rSip groups,31.58% (S. agalactiae) and 35.71% (S. iniae), respectively. Our study indicated that BNC-rSip can induce protective immunity for tilapia through immersion immunization and may be an ideal candidate vaccine for controlling tilapia streptococcal disease.

Computational design of a novel multi-epitope vaccine against Coxiella burnetii.

Query fever is a zoonotic disease caused by Coxiella burnetii. There is no universal method for the prevention of this disease. Recombinant vaccine is a potent strategy that can be utilized for this purpose. The current study was conducted to develop a multi-epitope vaccine against Coxiella burnetii. Hence, OmpA, Tuf2, GroEL, Mip and sucB antigens were used for the prediction of epitopes. Then, a multi-epitope vaccine was developed based on a molecular adjuvant and fragments that contained the best MHCI, B cell, MHCII and IFN-γ epitopes. The features of the developed vaccine including physicochemical parameters, antigenicity and protein structures were assessed. Also, interaction between the developed vaccine and TLR4/MD2 receptor along with molecular dynamics of the ligand-receptor complex were investigated. Finally, the codon adaptation and cloning were conducted for the developed vaccine. According to the results, molecular weight, instability index, antigenicity and random coil percentage of the developed vaccine were 54.4 kDa, 32.84, 1.1936 and 34.92%, respectively. Besides, residues distribution in core region of the refined model was 85%. The results demonstrated that the developed vaccine could dock to its receptor with the lowest energy of -976.7 as well as RMSD value of the complex was between 0.15 and 0.22 nm. Also, the results showed that CIA index of the codon adapted sequence was 0.95. Finally, cloning results revealed that nucleotide sequence of the developed vaccine could be successfully cloned into pET-21a (+). Based on these results, it seems that the developed vaccine can be a suitable candidate to prevent Coxiella burnetii.

Differential antibody responses to Giardia lamblia strain variants expressing dissimilar levels of an immunogenic protein.

AIMS: Giardia lamblia is a protozoan parasite that causes giardiasis, one of the most common worldwide gastrointestinal diseases. For rational development of a Giardia vaccine, increasing our understanding of the host-Giardia interaction is crucial. In this study, we analysed the immunogenicity and antigenicity of two G lamblia strain variants [GS and GS-5G8 (+)], which express different levels of the variant-specific surface protein (VSP) 5G8 and also analysed the intestinal histological changes associated with Giardia infection. METHODS AND RESULTS: We evaluated the antibody responses induced by G lamblia strains in infected, reinfected and immunized C3H/HeJ mice using ELISA, flow cytometry, Western blotting and histological analysis. Our results showed that G lamblia GS-5G8 (+) was more immunogenic and antigenic than the GS strain. The antibody response against the GS-5G8 (+) strain primarily recognized 5G8 protein. Serum antibody from infected and reinfected mice exhibited specific agglutination of trophozoites in vitro. GS-5G8 (+)-infected mice showed higher CD19+ infiltrating cell levels compared to GS-infected animals. CONCLUSION: G lamblia strains with different expression levels of an immunogenic antigen (VSP 5G8) induce differential antibody responses. A better understanding of the immunogenic proteins of G lamblia will contribute to the rational development of an effective vaccine against this parasitic disease.

Development and analytical validation of real-time PCR for the detection of Streptococcus agalactiae in pregnant women.

BACKGROUND: Group B Streptococcus (GBS) is the leading cause of invasive neonatal infection. In this study, we aimed to evaluate the analytical validation of qualitative real-time polymerase chain reaction (qPCR) as a means to detect GBS. METHODS: Genomic DNA (gDNA) was purified from 12 ATCC bacterial strains, two belonging to GBS and the remainder acting as negative controls. Additionally, gDNA was isolated from 21 strains of GBS from various serotypes (Ia, Ib and II-VIII). All gDNA was used to evaluate the analytical validation of the qPCR method employing a specific Taqman probe. Inclusivity, exclusivity, anticipated reportable range, the limit of detection and robustness were evaluated. The methods used are described in international guidelines and other existing reports. The performance of this qPCR method for detecting GBS was compared to other microbiological methods used with vaginal-rectal samples from pregnant women. RESULTS: Our qPCR method for detecting GBS was analytically validated. It has a limit of detection of 0.7 GE/µL and 100% analytical specificity. It detects all strains of GBS with the same level of performance as microbiological methods. CONCLUSION: Data suggest that this qPCR method performs adequately as a means to detect GBS in vaginal-rectal swabs from pregnant women.

Mucosal Vaccination with Lactococcus lactis-Secreting Surface Immunological Protein Induces Humoral and Cellular Immune Protection against Group B Streptococcus in a Murine Model.

Group B Streptococcus (GBS) is the primary etiological agent of sepsis and meningitis in newborns and is associated with premature birth and stillbirth. The development of a licensed vaccine is one of the pending challenges for the World Health Organization. Previously, we showed that oral immunization with surface immune protein (SIP) decreases vaginal colonization of GBS and generates functional opsonizing antibodies, which was determined by opsonophagocytic assays (OPA) in vitro. We also showed that the protein has an adjuvant vaccine profile. Therefore, an oral vaccine based on SIP may be an attractive alternative to employ in the development of new vaccines against GBS. Lactococcus lactis is a highlighted oral vaccine probiotic inducer of the mucosal immune response. This bacterium could serve as an antigen-delivering vehicle for the development of an edible vaccine and has been used in clinical trials. In this study, we showed that an oral vaccine with a recombinant L. lactis strain secreting SIP from GBS (rL. lactis-SIP) can induce protective humoral and cellular immunity in an experimental model of GBS vaginal colonization in C57BL/6 mice. Mice immunized with rL. lactis-SIP were protected against clinical symptoms and bacterial colonization after GBS vaginal colonization. Our rL. lactis-SIP vaccine also induces an increase of immunoglobulin G (IgG) and immunoglobulin A (IgA) specifically against SIP. The adoptive transfer of serum from vaccinated mice to naïve mice generated protection against GBS vaginal colonization. Moreover, the rL. lactis-SIP strain induces the activation of SIP-specific T cells, which could decrease GBS vaginal colonization and generate protective antibodies when transferred to other mice. Our experimental observations strongly support the notion that rL. lactis-SIP induces protective humoral and cellular immunity and could be considered as a novel alternative in the development of vaccines for GBS.

Characterization of the main immunogenic proteins in Brucella infection for their application in diagnosis of brucellosis.

Brucellosis is an important zoonotic bacterial disease widespread in the world. The key step of control this disease is accurate diagnosis and elimination of diseased animals. The classic diagnostic methods, such as tube agglutination test, are inaccurate and nonspecific, because of cross-reaction with Yersinia enterocolitica serotype O:9. Previously, several proteins were reported as Brucella main immunogens. In this study, we used animal infection model to evaluate antibody production against OMP16, BP26, BLS, BCSP31, VirB12, SodC and GroEL proteins and investigated their application in diagnosis of brucellosis. The results showed that the BP26 and BLS are two best immunogenic proteins. In further study, we detected 44 clinical bovine sera using western blot, showing that the BP26 and BLS reacted with 30 Brucella-positive sera, but false-positive results were also shown in 14 Brucella-free sera. In an indirect ELISA assay, compared to lipopolysaccharide-based ELISA, the conformance of the BP26-based ELISA was 92.68 % in Brucella-positive sera, but only 52.94 % in Brucella-free sera. The BLS-based ELISA can hardly differentiate positive sera from negative sera. Besides, truncated fragments of the BP26 protein cannot exclude false-positive results in detection of Brucella-free sera. Altogether, although Brucella main immunogenic proteins have good reaction with Brucella-positive sera, false-positive reaction with Brucella-free sera may lead to misdiagnosis of brucellosis, suggesting that it should be more careful to use these immunogenic proteins as antigen targets to diagnosis of brucellosis.

Surface Immunogenic Protein of Streptococcus Group B is an Agonist of Toll-Like Receptors 2 and 4 and a Potential Immune Adjuvant.

Vaccine-induced protection against pathogens, especially subunit-based vaccines, are related to antigen properties but mainly in their ability to stimulate the immune system by the use of an adjuvant. Modern vaccines are formulated with a high level of antigen purity, where an efficient adjuvant is necessary. In this context, the use of protein Toll-Like Receptor (TLR) agonists as vaccine adjuvants has been highlighted because of their optimal immunogenicity and minimal toxicity. The Surface Immunogenic Protein (SIP) from Group B Streptococcus (GBS) has gained importance as a new potential protein-based vaccine. Recently, we reported that recombinant SIP (rSIP) expressed by E. coli and purified by High Performance Liquid Chromatography (HPLC) alone induces a protective humoral immune response. In this study, we present the immunomodulatory properties of rSIP as a protein-based adjuvant, as an agonist of TLR. To this end, we showed that C57BL/6 bone marrow-derived dendritic cells pulsed by rSIP resulted in enhanced CD40, CD80, CD86, and Major Histocompatibility Complex (MHC) class II as well as increased secretion proinflammatory cytokines Interleukin (IL)-6, Interferon (IFN)-γ, Tumor Necrosis Factor (TNF)-α, and IL-10. Next, we investigated the in vivo effect of rSIP in the absence or presence of ovalbumin (OVA) on antigen-specific antibody secretion in C57BL/6 mice. Immunization with rSIP plus OVA showed that anti-OVA IgG2a and IgG1a increased significantly compared with OVA alone in C57BL/6 mice. Also, the immunization of rSIP plus OVA generates increased serum cytokines levels characterized by IL-12p70, IL-10, IL-4, and IFN-γ. Interestingly, we observed that rSIP stimulate Toll Like Receptor (TLR)2 and TLR4, individually expressed by Human embryonic kidney (HEK) 293-derived TLR reporter cells. These findings suggest that rSIP is a new potential protein TLR agonist adjuvant and may be employed in the development of new vaccines.

In silico design and in vitro analysis of a recombinant trivalent fusion protein candidate vaccine targeting virulence factor of Clostridium perfringens.

Necrotic enteritis (NE) is a multifactorial disease in broiler that is caused by colonization of Clostridium perfringens in their gastrointestinal tract. Recently several immunogenic proteins from virulent C. perfringens have been considered as vaccines to provide protection against NE. In this study, a novel trivalent fusion protein including immunogenic epitopes of three virulence factors of, NetB, alpha toxin and a metallopeptidase protein (NAM) was designed using in silico studies. Circular dichroism spectra was applied for determination of secondary structure and folding properties of the purified recombinant NAM (rNAM) expressed in E. coli. The antigenicity of rNAM was confirmed by induction of immune response in rabbit and neutralization experiments of the toxins in cell culture studies. To this end, anti-rNAM antisera neutralized the crude toxins produced by a wild type virulent C. perfringens strain using chicken hepatocellular carcinoma (LMH) cell lines. The cells were exposed to a mixture of anti-rNAM antisera and 2 × LD50 doses of the toxins. The result showed 94% viability of the cells against the crude toxins, in the presence of anti-rNAM antisera. Our study suggests that combination of metallopeptidase protein along with alpha toxin and NetB toxins is a potent immunogen which is able to neutralize the toxicity of crude extracellular toxins. The recombinant chimeric NAM could be a suitable and effective subunit vaccine candidate to prevent NE disease caused by C. perfringens.

Determination of virulence associated immunogenic proteins in some of Lactococcus garvieae strains.

Lactococcosis disease incident caused by Lactococcus garvieae has been increased with increasing aquaculture productions and outbreaks of the disease have become a threat on farmed species. To prevent lactococcosis, inactivated vaccine has been used, however, it only provides protection when given by injection. Other than inactivated vaccine, various vaccines such as subunit vaccines can be developed. In the present study, total protein profile of 43 strains of L. garvieae isolated from fish, milk and cheese by SDS-PAGE and virulence associated immunogenic proteins of L. garvieae strains using western blot with hyper-immune rabbit sera were determined. After analyzing whole-cell lysate protein of L. garvieae strains with SDS-PAGE, protein bands were ranged between 8.00 and 140.00 kDA. Among strains, variable protein bands were ranged between 17.00 and 48.00 kDa with some variability in the staining intensity of the protein bands and formed in 6 clusters. The immunogenic protein bands were ranged between 25.00 - 75.00 kDa. Only a variable and highly immunogenic protein band was observed between 40.00 and 45.00 kDa. Most of the strain including Lgper had 44.00 kDa immunogenic protein while nonvirulent ATCC strain had 42.50 kDA immunogenic protein. Predominant immuno-reactive proteins encoded by genes can be used as a subunit vaccine.

Oral vaccine based on a surface immunogenic protein mixed with alum promotes a decrease in Streptococcus agalactiae vaginal colonization in a mouse model.

The Surface Immunogenic Protein (SIP) of Group B Streptococcus (GBS) had been described as a good target for vaccine development. To date, SIP has been reported as a highly conserved protein, and in a mouse model it induces protection against lethal GBS challenge. Also, similar effects have been described by intranasal immunization with a SIP-based vaccine. In this study, we show the immune response induced by an oral SIP-based vaccine formulated on alum in a mouse model. Our vaccine can reduce vaginal GBS colonization and induce specific SIP-antibodies with opsonophagocytosis activities against GBS. Moreover, we observed the activation of T-cells producing IFN-γ, TNF-α, IL-10, IL-2, and increased expression of the transcription factor T-bet, suggesting a Th1-type humoral response. The oral SIP-based vaccine is a novel alternative in the development of a vaccine against GBS.

Bovine intramammary infection associated immunogenic surface proteins of Streptococcus uberis.

In spite of the increasing prevalence of Streptococcus uberis mastitis, its pathogenesis and associated virulence factors are not clearly defined. The aim of this study was to identify virulence associated genes and their products that can be used to develop effective vaccine to control bovine S. uberis mastitis. S. uberis was co-cultured with primary bovine mammary epithelial cells (PBMEC) or infused into mammary gland of dairy cows. The messenger RNA (mRNA) from S. uberis associated with PBMEC after 2 h or 4 h of co-culture was purified and sequenced. Results showed that virulence-associated genes such as surface lipoprotein (slp), infection induced histidine kinase (iihK), infection induced response regulator (iirR) and extracellular sugar binding protein 1 and 2 (exsbP1 and exsbP2) were among the top-up-regulated genes. To verify this observation in vivo, quantitative real time PCR (qRT - PCR) was conducted on mRNA of S. uberis recovered from milk of infected mammary glands 24 h post infection. Results revealed that in vitro up-regulated virulence-associated genes were also significantly up regulated under in vivo conditions. The iihK and iirR are flanked by exsbP1 and exsbP2 genes and this entire operon seems to be involved in adaptation to glands micro-environment, survival and colonization of the bovine mammary glands. Based on immunogenic epitope prediction of proteins encoded by these up-regulated genes during early stages of host-bacterial interactions slp, exsbP1 and exsbP2 genes were selected, cloned and expressed in E. coli. The purified recombinant proteins (rSlP, rExsbP1 & rExsbP2) reacted strongly with convalescent serum from cows experimentally infected with S. uberis confirming that they are immunogenic. These proteins may serve as potential targets to develop an effective vaccine against S. uberis mastitis.

Characterization of BIP protein of G. lamblia as a potential immunogen in a mouse infection model.

Giardia lamblia is a protozoan parasite that causes one of the most common gastrointestinal diseases worldwide. To eliminate the parasite from the host intestine, it is necessary the activation of B-cell and T-cell dependent mechanisms. The knowledge about Giardia antigens that can stimulate the host immune response is limited. Recently, it has been described the Binding Immunoglobulin Protein (BIP) of G. lamblia (71kDa) as a potential immunogen. Additionally, our group has identified a highly immunogenic antigen (5G8 protein) of G. lamblia with a relative molecular mass of approximately 70kDa. There is some evidence suggesting that the 5G8 protein may activate both humoral and cellular immune responses. Based on these observations and preliminary mass spectrometry analyses, we hypothesized that the antigen 5G8 could be the BIP protein. In the present study, we characterize immunochemically the BIP protein of Giardia. Flow cytometric assays and western blotting were used to determine the expression profile of BIP and 5G8 antigens in Giardia trophozoites. The differences in expression profile indicated that BIP and 5G8 are not the same molecule. ELISA and Western blotting assays revealed that BIP protein was recognized by antibodies produced during G. lamblia infection in C3H/HeN mice. MTT assays did not reveal the activation of cellular immune response induced by BIP protein in vitro. In addition, we identified the potential B-cell and T-cell epitopes of G. lamblia BIP protein. This molecule is a conserved protein among Giardia strains and other pathogens. The complete immunological characterization of this antigen will contribute to a better understanding of the host-parasite interactions in Giardia infection.

Immunoproteomics Approach for Screening of Vaccine Candidates against Intestinal Botulism.

Intestinal botulism is an infectious form of botulism in which disease results from ingesting spores, which is followed by spore germination and intraluminal production of botulinum neurotoxins over an extended period. Botulinum neurotoxin is produced by endospore forming bacteria called C. botulinum. Immunoproteomic study was used to screen the cross reactive immunogenic proteins of Clostridium botulinum type B using C. botulinum type B live spore antiserum. The whole cell proteins were separated by two dimensional gel electrophoresis and transferred to polyvinylidene difluoride membranes. Further, the Western blotting was performed with mouse pups immune serum against C. botulinum type B live spores. Eight predominant cross immunoreactive proteins were identified by mass spectrometry. These immunogenic proteins might be used to develop novel subunit vaccine candidates against the intestinal botulism.