Unveiling promising immunogenic targets in Coxiella burnetii through in silico analysis: paving the way for novel vaccine strategies.

BACKGROUND: Coxiella burnetii, an intracellular pathogen, serves as the causative agent of zoonotic Q fever. This pathogen presents a significant threat due to its potential for airborne transmission, environmental persistence, and pathogenicity. The current whole-cell vaccine (WCV) utilized in Australia to combat Q fever exhibits notable limitations, including severe adverse reactions and limited regulatory approval for human use. This research employed the reverse vaccinology (RV) approach to uncover antigenic proteins and epitopes of C. burnetii, facilitating the development of more potent vaccine candidates. METHODS: The potential immunogenic proteins derived from C. burnetii RSA493/Nine Mile phase I (NMI) were extracted through manual, automated RV, and virulence factor database (VFDB) methods. Web tools and bioinformatics were used to evaluate physiochemical attributes, subcellular localization, antigenicity, allergenicity, human homology, B-cell epitopes, MHC I and II binding ratios, functional class scores, adhesion probabilities, protein-protein interactions, and molecular docking. RESULTS: Out of the 1850 proteins encoded by RSA493/NMI, a subset of 178 demonstrated the potential for surface or membrane localization. Following a series of analytical iterations, 14 putative immunogenic proteins emerged. This collection included nine proteins (57.1%) intricately involved in cell wall/membrane/envelope biogenesis processes (CBU_0197 (Q83EW1), CBU_0311 (Q83EK8), CBU_0489 (Q83E43), CBU_0939 (Q83D08), CBU_1190 (P39917), CBU_1829 (Q83AQ2), CBU_1412 (Q83BU0), CBU_1414 (Q83BT8), and CBU_1600 (Q83BB2)). The CBU_1627 (Q83B86 ) (7.1%) implicated in intracellular trafficking, secretion, and vesicular transport, and CBU_0092 (Q83F57) (7.1%) contributing to cell division. Additionally, three proteins (21.4%) displayed uncharacterized functions (CBU_0736 (Q83DJ4), CBU_1095 (Q83CL9), and CBU_2079 (Q83A32)). The congruent results obtained from molecular docking and immune response stimulation lend support to the inclusion of all 14 putative proteins as potential vaccine candidates. Notably, seven proteins with well-defined functions stand out among these candidates. CONCLUSIONS: The outcomes of this study introduce promising proteins and epitopes for the forthcoming formulation of subunit vaccines against Q fever, with a primary emphasis on cellular processes and the virulence factors of C. burnetii.

Evaluating the immunogenicity of gold nanoparticles conjugated RBD with Freund's adjuvant as a potential vaccine against SARS-CoV-2.

BACKGROUND: and Introduction: SARS-CoV-2 is currently considered as the most challenging issue in the field of health and medicine by causing a global pandemic. Vaccines are counted as a promising candidate to terminate this deadly pandemic. Various structural proteins in SARS-CoV-2 have recently drawn attention to be utilized as candidate vaccines to stimulate immune responses against COVID-19. MATERIALS AND METHODS: In current study, the RBD protein was cloned and expressed in E. coli host. Then, the expressed RBD protein was purified and its characterizations were evaluated through various methods. Gold nanoparticles, which were utilized as a carrier for candidate Nano-vaccine, were synthesized via oxidation-reduction reaction. While Gold NPs-conjugated RBD was injected into the second treatment group, in the first candidate vaccine, RBD was injected into the first treatment group solely. Complete and Incomplete Freud's Adjuvant were also utilized for both treatment groups to enhance the immune responses against RBD antigen. Immunizations were repeated 2 times in 14-day intervals to boost the immune system of BALB/c mice. The humoral and cell-mediated immune responses were examined through immune and cytokine assays. RESULTS: Our outcomes demonstrate that strong short-term humoral immunity (IgM) was induced in both the first and second treatment group, while long-term humoral responses (IgG) were only observed in the second treatment group. While stronger short- and long-term humoral (IgM and IgG, respectively) were observed in the second treatment group, particular cytokines production (TNF-ɑ and IFN-γ) as a marker of cell-mediated responses were significantly higher in the first treatment group. DISCUSSION AND CONCLUSION: Our study results show the high potentiality of RBD protein as an appropriate stimulating antigen in vaccine synthesis and testifies RBD-based candidate vaccines to control the COVID-19 pandemic. Our outcomes also recommend that Nano-vaccines can be more suitable candidates when stronger long-term immune responses matter.

Intramuscular Immunization of Streptococcus pyogenes SF370 protein extract and identification of multiple virulence factors through proteomic profiling in RHD induced Balb/c mice.

Group A streptococcus (GAS) and autoimmunity are associated with heart related mitral valve damage, in adults. In this study Balb/c mice were intramuscularly immunized with S. pyogenes SF370 for 4 weeks. Prior to euthanization, physiological parameters like body weight and electrical signalling of the heart were recorded. After euthanization, the heart tissue homogenate was prepared and proteomic alterations were studied using SDS-PAGE and 2D electrophoresis. The expression levels of inflammatory genes like TNFα, IFNγ and TGF-ß were quantified using real time PCR. Insilico analysis was performed to identify the functions of hypothetical proteins and virulence factors involved in the induction of rheumatic carditis. The results showed a reduction in body weight, ulceration, inflammation, cardiac lesions and prolonged PR interval in mice immunized with S. pyogenes SF370, as a result of RHD. The heart related proteins like α-actinin, fatty acid binding protein-heart, myosin light chain 3, hemoglobin subunit alpha, myoglobin regulatory light chain 2, (ventricular/cardiac muscle isoform), myosin-6, troponin-1 were found to be up-regulated when compared with the control. The functional annotation of S. pyogenes (SF370) was carried out by retrieving 1696 identified proteins and 653 hypothetical protein sequences in NCBI genome database. The conserved domain was identified for 505 proteins. The pfam database documented that the super families of 279 sequences and 40 signal peptides enabled the classification of proteins in different categories like biological (20%), cellular (22%) and molecular functions (36%). Putative transcription repair coupling factor and putative lysine aminopeptidase N terminal are the two virulence factors identified by VICMPRED in S. pyogenes SF370. The two identified virulence factors are involved in altering the mice heart proteome and thereby controlling the streptococcus pyogenes infection. Thus, the results of the present study reveals the role of immunogenic proteins in induction of rheumatic carditis and to elucidate the molecular mechanisms leading to autoimmune reactions in Balb/c mice.

Purified Splenic amastigotes of Leishmania donovani-Immunoproteomic approach for exploring Th1 stimulatory polyproteins.

Visceral leishmaniasis (VL) represents one of the most challenging infectious diseases worldwide. The reason that once infected, patient develops immunity against Leishmania parasite has paved way to develop prophylactic vaccines against disease, but only some of these have moved ahead for clinical trials. Herein, the study to explore novel and potential vaccine candidates was extended to pathogenic form of parasite, that is, amastigote form which is less explored due to complexity of its purification process. Methods and results. Classical protocol of purification of splenic amastigotes was modified to obtain highly pure amastigotes which was confirmed by Western blotting in support with proteomics studies. Fractionation and sub-fractionation of purified splenic amastigotes revealed four sub-fractions, belonging to 97 to 68 kDa and 68 to 43 kDa ranges, which showed long-lasting protection with remarkable Th1-type cellular responses in hamsters vaccinated with these sub-fractions (LTT, NO, QRT-PCR). Further proteomics analysis, to identify and understand the precise nature and function of these protective protein sub-fractions, identified a total of 47 proteins including twenty-five hypothetical proteins/unknowns. Amastigote stage has potential Th1-stimulatory vaccine candidates, notably, among identified proteins, major were uncharacterized proteins/hypothetical proteins, which once characterized may serve as novel and potential vaccine candidates/drug targets.

Characterization of novel antigenic vaccine candidates for nile tilapia (Oreochromis niloticus) against Streptococcus agalactiae infection.

Streptococcus agalactiae is one of the important pathogens responsible for high mortality and economic losses of the tilapia industry worldwide. Based on ten serovars of S. agalactiae infection, subunit vaccine with conserved antigens is promising strategy corresponding stimulated long-term immunity and provides protection for animals against different serotypes of S. agalactiae. In the present study, eight proteins (AP, AL, LivK, ESAT6, essA, essB, essC and esaA) were selected from the S. agalactiae serotype Ia genome as immunogenic antigens with bioinformation and immune experiment assays. These recombinant proteins were successfully obtained through expression in Escherichia coli and the immunogenicity was assessed in tilapia challenge model. The results showed that the recombinant proteins caused high-level-specific antibodies production and high lysozyme activities, suggesting that the recombinant proteins induced specific humoral immune response and innate immune response of tilapia. The signficant increase were observed in the cytokines levels of TNF-α, IL-1ß, IFN-γ, cc1, cc2 and immune-related genes levels of CD8α and MHC factors in the spleen and head kidney tissues, suggesting that the recombinant proteins induced immune response of tilapia through cytokines signal pathway and activated high cytotoxic T-lymphocyte (CTL) activity of tilapia. Furthermore, vaccinated tilapia conferred high levels of protection against challenge with a lethal dose of highly virulent serovar Ⅰa (highest RPS was 91.60% in AL and essC protein groups). Our results indicated that the eight recombinant proteins induced high level of immune responses and offered protection against S. agalactiae infection, could be potential subunit vaccine candidates.

The intestinal milieu influences the immunoproteome of male and female Heligmosomoides polygyrus bakeri L4 stage.

The gastrointestinal nematode Heligmosomoides polygyrus bakeri shows enhanced survival in mice with colitis. As the antibody response plays an important role in antiparasitic immunity, antibodies against male and female L4 H. polygyrus were examined in mice with and without colitis. Levels of specific antibodies in the mucosa and serum were determined by enzyme-linked immunosorbent assay and immunogenic proteins of male and female parasites were identified using 2D electrophoresis and mass spectrometry. The function of identified proteins was explored with Blast2Go. Nematodes in mice with colitis induced higher levels of specific immunoglobulin G (IgG1) and IgA, a lower level of IgE in the small intestine and a higher level of IgE in serum against female L4. Infected mice with colitis recognized 12 proteins in male L4 and 10 in female L4. Most of the recognized proteins from male L4 were intermediate filament proteins, whereas the proteins from female L4 were primarily actins and galectins. Nematodes from mice with colitis were immunogenically different from nematodes from control mice. This phenomenon gives new insights into helminth therapy as well as host-parasite interactions.

Immunization with Larrea divaricata Cav. proteins elicits opsonic antibodies against Pseudomonas aeruginosa and induces phagocytic activity of murine macrophages.

Pseudomonas aeruginosa is an opportunistic pathogen implicated in nosocomial infections for which no vaccines have been approved. Larrea divaricata Cav. (Jarilla) is a widely spread plant in America and it is used in folk medicine to treat several pathologies. It has also been shown that antibodies elicited against Jarilla proteins of crude extract (JPCE) cross-react with proteins from gram-negative bacteria. In this study we aim to assess the contribution of anti-JPCE antibodies in the opsonophagocytosis of P. aeruginosa by murine macrophages. Levels of reactivity of anti-JPCE IgG and IgA antibodies against cell and membrane proteins suggest that these proteins induce a response that could favor opsonic bacterial recognition, which is important for the elimination of bacteria on mucous membranes, useful in the early stages of infection. Opsonophagocytosis assays also show that these antibodies could favor bacteria intake. These results together with previous observations that indicate that anti-JPCE antibodies are able to neutralize P. aeruginosa enzymes point L. divaricata proteins as candidates for vaccine development.

Immuno-proteomic analysis of Trichinella spiralis, T. pseudospiralis, and T. papuae extracts recognized by human T. spiralis-infected sera.

The present study explored potentially immunogenic proteins of the encapsulated (Trichinella spiralis) and non-encapsulated (T. pseudospiralis, T. papuae) species within the genus Trichinella. The somatic muscle larval extracts of each species were subjected to immunoblotting analysis using human T. spiralis-infected serum samples. Fifteen reactive bands of all three species were selected for further protein identification by liquid chromatography-tandem mass spectrometry, and their possible functions were ascertained using the gene ontology. Our findings showed immunogenic protein patterns with molecular mass in the range of 33-67 kDa. Proteomic and bioinformatic analysis revealed a wide variety of functions of 17 identified proteins, which are associated with catalytic, binding, and structural activities. Most proteins were involved in cellular and metabolic processes that contribute in the invasion of host tissues and the larval molting processes. The parasite proteins were identified as actin-5C, serine protease, deoxyribonuclease-2, and intermediate filament protein ifa-1. This information may lead to alternative tools for selection of potential diagnostic protein markers or aid in the design of vaccine candidates for prevention and control of Trichinella infection.

Protective efficacy of six immunogenic recombinant proteins of Vibrio anguillarum and evaluation them as vaccine candidate for flounder (Paralichthys olivaceus).

Vibrio anguillarum is a severe bacterium that causes terminal haemorrhagic septicaemia in freshwater and marine fish. Virulence-associated proteins play an important role in bacterial pathogenicity and could be applied for immunoprophylaxis. In this study, six antigenic proteins from V. anguillarum were selected and the immune protective efficacy of their recombinant proteins was investigated. VirA, CheR, FlaC, OmpK, OmpR and Hsp33 were recombinantly produced and the reactions of recombinant proteins to flounder-anti-V. anguillarum antibodies (fV-ab) were detected, respectively. Then the recombinant proteins were injected to fish, after immunization, the percentages of surface membrane immunoglobulin-positive (sIg+) cell in lymphocytes, total antibodies, antibodies against V. anguillarum, antibodies against recombinant proteins and relative percent survival (RPS) were analyzed, respectively. The results showed that all the recombinant proteins could react to fV-ab, proliferate sIg + cells in lymphocytes and induce production of total antibodies, specific antibodies against V. anguillarum or the recombinant proteins; the RPS of rVirA, rCheR, rFlaC, rOmpK, rOmpR and rHsp33 against V. anguillarum was 70.27%, 27.03%, 16.22%, 62.16%, 45.95% and 81.08%, respectively. The results revealed that rHsp33, rVirA and rOmpK have good protections against V. anguillarum and could be vaccine candidates against V. anguillarum.

Antibody formation towards porcine tissue in patients implanted with crosslinked heart valves is directed to antigenic tissue proteins and αGal epitopes and is reduced in healthy vegetarian subjects.

BACKGROUND: Glutaraldehyde-fixed porcine heart valves (ga-pV) are one of the most frequently used substitutes for insufficient aortic and pulmonary heart valves which, however, degenerate after 10-15 years. Yet, xeno-immunogenicity of ga-pV in humans including identification of immunogens still needs to be investigated. We here determined the immunogenicity of ga-pV in patients with respect to antibody formation, identity of immunogens and potential options to reduce antibody levels. METHODS: Levels of tissue-specific and anti-αGal antibodies were determined retrospectively in patients who received ga-pV for 51 months (n=4), 25 months (n=6) or 5 months (n=4) and compared to age-matched untreated subjects (n=10) or younger subjects with or without vegetarian diet (n=12/15). Immunogenic proteins were investigated by Western blot approaches. RESULTS: Tissue-specific antibodies in patients were elevated after 5 (1.73-fold) and 25 (1.46-fold, both P<.0001) months but not after 51 months, whereas anti-Gal antibodies were induced 4.75-fold and 3.66-fold after 5 and 25 months (both P<.0001) and still were significantly elevated after 51 months (2.85-fold, P<.05). Western blots of porcine valve extracts with and without enzymatic deglycosylation revealed strong specific staining at ≈65 and ≈140 kDa by patient sera in either group which were identified by 2D Western blots and mass spectrometry as serum albumin and collagen 6A1. Vegetarian diet reduced significantly (0.63-fold, P<.01) the level of pre-formed αGal but not of tissue-specific antibodies. CONCLUSION: Immune response in patients towards ga-pV is induced by the porcine proteins albumin and collagen 6A1 as well as αGal epitopes, which seemed to be more sustained. In contrast, in healthy young subjects pre-formed anti-Gal antibodies were reduced by a meat-free nutrition.

How does temperature influences the development of lactococcosis? Transcriptomic and immunoproteomic in vitro approaches.

Lactococcus garvieae is the aetiological agent of lactococcosis, a haemorrhagic septicaemia that affects marine and freshwater fish, with special incidence and economic relevance in farmed rainbow trout. Water temperature is one of the most important predisposing factors in the development of lactococcosis outbreaks. Lactococcosis in trout usually occur when water temperatures rise to about 18 °C, while fish carriers remain asymptomatic at temperatures below 13 °C. The aim of this work was to analyse the differences in the complete transcriptome response of L. garvieae grown at 18 °C and at 13 °C and to identify the immunogenic proteins expressed by this bacterium at 18 °C. Our results show that water temperature influences the expression of L. garvieae genes involved in the lysis of part of the bacterial cell population and in the cold response bacterial adaptation. Moreover, the surface immunogenic protein profile at 18 °C suggests an important role of the lysozyme-like enzyme, WxL surface proteins and some putative moonlighting proteins (proteins with more than one function, usually associated with different cellular locations) as virulence factors in L. garvieae. The results of this study could provide insights into the understanding of the virulence mechanisms of L. garvieae in fish.

Oxetane Grafts Installed Site-Selectively on Native Disulfides to Enhance Protein Stability and Activity In†Vivo.

A four-membered oxygen ring (oxetane) can be readily grafted into native peptides and proteins through site-selective bis-alkylation of cysteine residues present as disulfides under mild and biocompatible conditions. The selective installation of the oxetane graft enhances stability and activity, as demonstrated for a range of biologically relevant cyclic peptides, including somatostatin, proteins, and antibodies, such as a Fab arm of the antibody Herceptin and a designed antibody DesAb-Aß against the human Amyloid-ß peptide. Oxetane grafting of the genetically detoxified diphtheria toxin CRM197 improves significantly the immunogenicity of this protein in mice, which illustrates the general utility of this strategy to modulate the stability and biological activity of therapeutic proteins containing disulfides in their structures.

Host defences against Giardia lamblia.

Giardia spp. is a protozoan parasite that inhabits the upper small intestine of mammals and other species and is the aetiological agent of giardiasis. It has been demonstrated that nitric oxide, mast cells and dendritic cells are the first line of defence against Giardia. IL-6 and IL-17 play an important role during infection. Several cytokines possess overlapping functions in regulating innate and adaptive immune responses. IgA and CD4(+) T cells are fundamental to the process of Giardia clearance. It has been suggested that CD4(+) T cells play a double role during the anti-Giardia immune response. First, they activate and stimulate the differentiation of B cells to generate Giardia-specific antibodies. Second, they act through a B-cell-independent mechanism that is probably mediated by Th17 cells. Several Giardia proteins that stimulate humoral and cellular immune responses have been described. Variant surface proteins, α-1 giardin, and cyst wall protein 2 can induce host protective responses to future Giardia challenges. The characterization and evaluation of the protective potential of the immunogenic proteins that are associated with Giardia will offer new insights into host-parasite interactions and may aid in the development of an effective vaccine against the parasite.

De novo identification of bacterial antigens of a clinical isolate by combining use of proteosurfaceomics, secretomics, and BacScan technologies.

Background: Emerging infectious diseases pose a significant threat to both human and animal populations. Rapid de novo identification of protective antigens from a clinical isolate and development of an antigen-matched vaccine is a golden strategy to prevent the spread of emerging novel pathogens. Methods: Here, we focused on Actinobacillus pleuropneumoniae, which poses a serious threat to the pig industry, and developed a general workflow by integrating proteosurfaceomics, secretomics, and BacScan technologies for the rapid de novo identification of bacterial protective proteins from a clinical isolate. Results: As a proof of concept, we identified 3 novel protective proteins of A. pleuropneumoniae. Using the protective protein HBS1_14 and toxin proteins, we have developed a promising multivalent subunit vaccine against A. pleuropneumoniae. Discussion: We believe that our strategy can be applied to any bacterial pathogen and has the potential to significantly accelerate the development of antigen-matched vaccines to prevent the spread of an emerging novel bacterial pathogen.

Does sourdough bread provide clinically relevant health benefits?

During the last decade, scientific interest in and consumer attention to sourdough fermentation in bread making has increased. On the one hand, this technology may favorably impact product quality, including flavor and shelf-life of bakery products; on the other hand, some cereal components, especially in wheat and rye, which are known to cause adverse reactions in a small subset of the population, can be partially modified or degraded. The latter potentially reduces their harmful effects, but depends strongly on the composition of sourdough microbiota, processing conditions and the resulting acidification. Tolerability, nutritional composition, potential health effects and consumer acceptance of sourdough bread are often suggested to be superior compared to yeast-leavened bread. However, the advantages of sourdough fermentation claimed in many publications rely mostly on data from chemical and in vitro analyzes, which raises questions about the actual impact on human nutrition. This review focuses on grain components, which may cause adverse effects in humans and the effect of sourdough microbiota on their structure, quantity and biological properties. Furthermore, presumed benefits of secondary metabolites and reduction of contaminants are discussed. The benefits claimed deriving from in vitro and in vivo experiments will be evaluated across a broader spectrum in terms of clinically relevant effects on human health. Accordingly, this critical review aims to contribute to a better understanding of the extent to which sourdough bread may result in measurable health benefits in humans.

Detection of immunoreactive proteins of Escherichia coli, Streptococcus uberis, and Streptococcus agalactiae isolated from cows with diagnosed mastitis.

Introduction: Mastitis is a widespread mammary gland disease of dairy cows that causes severe economic losses to dairy farms. Mastitis can be caused by bacteria, fungi, and algae. The most common species isolated from infected milk are, among others, Streptococcus spp., and Escherichia coli. The aim of our study was protein detection based on both in silico and in vitro methods, which allowed the identification of immunoreactive proteins representative of the following species: Streptococcus uberis, Streptococcus agalactiae, and Escherichia coli. Methods: The study group included 22 milk samples and 13 serum samples obtained from cows with diagnosed mastitis, whereas the control group constituted 12 milk samples and 12 serum samples isolated from healthy animals. Detection of immunoreactive proteins was done by immunoblotting, while amino acid sequences from investigated proteins were determined by MALDI-TOF. Then, bioinformatic analyses were performed on detected species specific proteins in order to investigate their immunoreactivity. Results: As a result, we identified 13 proteins: 3 (molybdenum cofactor biosynthesis protein B, aldehyde reductase YahK, outer membrane protein A) for E. coli, 4 (elongation factor Tu, tRNA uridine 5-carboxymethylaminomethyl modification enzyme MnmG, GTPase Obg, glyceraldehyde-3-phosphate dehydrogenase) for S. uberis, and 6 (aspartate carbamoyltransferase, elongation factor Tu, 60 kDa chaperonin, elongation factor G, galactose-6-phosphate isomerase subunit LacA, adenosine deaminase) for S. agalactiae, which demonstrated immunoreactivity to antibodies present in serum from cows with diagnosed mastitis. Discussion: Due to the confirmed immunoreactivity, specificity and localization in the bacterial cell, these proteins can be considered considered potential targets in innovative rapid immunodiagnostic assays for bovine mastitis, however due to the limited number of examined samples, further examination is needed.

Expression of heterologous heparan sulphate binding protein of Helicobacter pylori on the surface of Lactobacillus rhamnosus GG.

Helicobacter pylori (H. pylori) is one of most commonly found pathogen in the stomach. In spite of emergence of different treatment strategies, H. pylori infection remains difficult to treat. The bioengineered probiotic lactobacilli that could displace H. pylori and simultaneously present immunogenic peptides such as heparan sulphate binding protein (Hsbp) to elicit immune response could emerge as a potential therapeutic agent. The aim of this study was to discover the anti-H. pylori activities and faster exclusion of H. pylori from host cells by the recombinant strain of Lactobacillus expressing the immunogenic Hsbp protein. The results were promising and showed a 65% reduction in H. pylori adhesion after two hours of pre-incubation with recombinant-LGG and HeLa S3 cells, followed by the adhesion of H. pylori pathogen (P < 0.002). Additionally, 36% and 39% reduction were examined in co-incubation and post-incubation with recombinant-LGG, respectively. When challenged with H. pylori, the proinflammatory cytokine expression was also down regulated in recombinant-LGG treated HeLa S3 cells. This promising result provides a new insight of bioengineered probiotic lactobacilli which could displace H. pylori and simultaneously has immunogenic properties thereby may be useful to prevent H. pylori infection. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03428-4.

Biomarker Discovery by ORFeome Phage Display.

Phage display is an efficient and robust method for protein-protein interaction studies. Although it is mostly used for antibody generation, it can be also utilized for the discovery of immunogenic proteins that could be used as biomarkers. Through this technique, a genome or metagenome is fragmented and cloned into a phagemid vector. The resulting protein fragments from this genetic material are displayed on M13 phage surface, while the corresponding gene fragments are packaged. This packaging process uses the pIII deficient helperphage, called Hyperphage (M13KO7 ΔpIII), so open reading frames (ORFs) are enriched in these libraries, giving the name to this method: ORFeome phage display. After conducting a selection procedure, called "bio-panning," relevant immunogenic peptides or protein fragments are selected using purified antibodies or serum samples, and can be used as potential biomarkers. As ORFeome phage display is an in vitro method, only the DNA or cDNA of the species of interest is needed. Therefore, this approach is also suitable for organisms that are hard to cultivate, or metagenomic samples, for example. An additional advantage is that the biomarker discovery is not limited to surface proteins due to the presentation of virtually every kind of peptide or protein fragment encoded by the ORFeome on the phage surface. At last, the selected biomarkers can be the start for the development of diagnostic assays, vaccines, or protein interaction studies.

Development of a Novel Multi-Epitope Vaccine Candidate against Streptococcus Iniae Infection in Fish: An Immunoinformatics Study.

Streptococcus Iniae infection is recognized as a disease with substantial economic losses, infecting a wide range of fish species. The limitations of current vaccines and strategies have led to the identification of new methods to control this disease. Multi-epitope vaccines which employ various immunogenic proteins can be promising. The current research project aimed to design an efficient multi-epitope vaccine against Streptococcus Iniae infection in fish. To this end, six immunogenic proteins of Streptococcus Iniae, including FBA, ENO, Sip11, GAPDH, MtsB, and SCPI proteins, were applied for epitope prediction. The best B cell, T cell, and IFNγ epitopes of the immunogenic proteins, as well as interleukin-8, were used to construct a multi-epitope vaccine. Thereafter, different parameters of the designed vaccine, including physicochemical features, antigenicity, secondary structure, and tertiary structure, were evaluated. Moreover, the interaction of the interleukin-8 domain of the designed vaccine and its receptor was investigated by molecular docking strategy. Finally, nucleotide sequence of the vaccine was adapted to express in Escherichia coli. The results of the present study pointed out that the designed vaccine was a stable vaccine with molecular weight and antigenicity score of 45 kDa and 0.936, respectively. Furthermore, the structure analysis results revealed that the designed vaccine contained 23.49% alpha helix, with 90.5% residues in favored region. Finally, it was demonstrated that the interleukin-8 domain of the designed vaccine could be successfully docked to its receptor with the lowest energy of -1020.9. Based on the obtained results, it seems that the designed vaccine can be an efficient candidate to prevent Streptococcus Iniae infection in fish.

Three novel immunogenic proteins determined through 2-Dimensional electrophoresis and mass spectrometry with immune serum confer protection against challenge with porcine Pasteurella multocida in mouse models.

Pasteurella multocida is an important zoonotic pathogen that causes multiple diseases in both animals and humans. Test of good immunogenic proteins is beneficial for vaccine development and disease control. In the present study, we determined four novel immunogenic proteins of P. multocida by using 2-DE MALDI-TOF MS with immune serum. These four proteins included a trimethylamine-N-oxide reductase TorA, a translation elongation factor Ts, a phosphoglyceromutase PGAM, and a peroxiredoxin PrX. Among these proteins, TorA, Prx, and PGAM were successfully expressed by using E. coli. Western-blotting assays showed that recombinant TorA, Prx, and/or PGAM displayed good reactions with infectious sera of P. multocida serogroups A, B, D and F. Immunization of either rTorA, rPrx, and/or rPGAM induced significantly high levels of antibodies as well as IFN-γ, IL-4 and IL-10 in mice (P < 0.01). Protective efficacy tests revealed that vaccination of either rTorA, rPrx, and/or rPGAM protected 60% ~ 80% of the tested mice against the challenge with P. multocida field isolate. Our results obtained from the present study suggest that these three proteins could be tested as good vaccine candidates against P. multocida infections.