Vaccine Candidate Antigens Research Articles

Nanoparticle formulation enhanced protective immunity provoked by PYGPI8p-transamidase related protein (PyTAM) DNA vaccine in Plasmodium yoelii malaria model

We have previously reported the new formulation of polyethylimine (PEI) with gamma polyglutamic acid (γ-PGA) nanoparticle (NP) to have provided Plasmodium yoelii merozoite surface protein-1 (PyMSP-1) plasmid DNA vaccine with enhanced protective cellular and humoral immunity in the lethal mouse malaria model. PyGPI8p-transamidase-related protein (PyTAM) was selected as a possible candidate vaccine antigen by using DNA vaccination screening from 29 GPI anchor and signal sequence motif positive genes picked up using web-based bioinformatics tools; though the observed protection was not complete. Here, we observed augmented protective effect of PyTAM DNA vaccine by using PEI and γ-PGA complex as delivery system. NP-coated PyTAM plasmid DNA immunized mice showed a significant survival rate from lethal P. yoelii challenge infection compared with naked PyTAM plasmid or with NP-coated empty plasmid DNA group. Antigen-specific IgG1 and IgG2b subclass antibody levels, proportion of CD4 and CD8T cells producing IFN-γ in the splenocytes and IL-4, IFN-γ, IL-12 and TNF-α levels in the sera and in the supernatants from ex vivo splenocytes culture were all enhanced by the NP-coated PyTAM DNA vaccine. These data indicates that NP augments PyTAM protective immune response, and this enhancement was associated with increased DC activation and concomitant IL-12 production.

Identification and bioinformatic analysis of a putative calcium-dependent protein kinase (CDPK6) from Toxoplasma gondii.

Toxoplasma gondii is recognized as an opportunistic human pathogen with a worldwide distribution. Development of effective vaccines is considered the only ideal way to control T. gondii infection. However, only one live vaccine is commercially available for use in sheep and goats. Therefore, the identification of more effective antigenic proteins is very important. In this study, we identified a novel putative calcium-dependent protein kinase of T. gondii, TgCDPK6, and further analyzed its potential antigenicity using a bioinformatic approach. The physical and chemical characteristics, transmembrane domain, epitopes, advanced structure, and functional sites of TgCDPK6 were predicted by multiple bioinformatic approaches. Twenty-six post-translational modification sites were identified in the protein. The secondary structure showed that 58.35% amino acids of TgCDPK6 are exposed to the solvent interface, and the high hydrophilic domains were distributed in amino acid positions 21-59, 68-81, 156-205, 245-271, 280-294, 297-324, 334-356, 367-393, 474-498, and 543-553. The advanced structure of TgCDPK6 was developed by a homology modeling method and was validated by PROCHECK, which showed that most amino acid residues were in the most favored regions. Using these analyses, 10 potential epitopes were predicted. The results indicated that TgCDPK6 could be a vaccine candidate antigen against T. gondii.

Genetic Analysis of Erythrocyte Binding Antigen 175 (EBA-175), Apical Membrane Antigen (AMA-1) and Merozoite Surface Protein 3 (MSP-3) Allelic Types in Plasmodium Falciparum Isolates From Rural Area in Senegal

Background: Several of the intended P. falciparum vaccine candidate antigens are highly polymorphic and could render a vaccine ineffective if their antigenic sites were not represented in the vaccine. This study aimed to characterize genetic diversity of vaccine candidate antigens merozoite surface protein-3 (MSP-3), apical membrane antigen-1 (AMA-1) and erythrocyte binding antigen (EBA-175) in P. falciparum isolates from Senegal. Methods: DNA analysis was completed on 170 isolates of P. falciparum collected from Keur Soce in Senegal between 2006 and 2008. Genetic diversity was determined in the three P. falciparum genes by, PCR followed by restriction fragment length polymorphism (RFLP). Results: From 170 samples collected, successful, PCR products were obtained from 135 (79%), 140 (82%) and 128 (75%) for AMA-1, MSP-3 and EBA-175, respectively. The results showed that the EBA-175 gene presented 4 different alleles [EBA-175F_loop (62.3%), EBA-175C_loop (46.1%), EBA-175~400bp (17.6%), EBA-175~360bp (8.4%)]. Regarding the MSP-3 patterns, the analysis revealed the presence of three alleles MSP-3_K1 (49.2%), MSP-3_3D7 (54.2%) and MSP-3~350bp (15%). For AMA-1, the results showed three different alleles AMA-1_K1 (39%), AMA-1_HB3 (33%), AMA-1_3D7 (32%). Conclusion: Characterization of the genetic diversity in Plasmodium isolates from Keur Soce in Senegal in the three genes investigated showed a high degree of polymorphism. These findings are helpful in the formulation of a vaccine considering restricted repertoire populations.

Allelic Diversity of Polymorphic AMA-1 (Apical Membrane Antigen 1) Vaccine Candidate Antigen of Plasmodium falciparum in Two Population of Imported and Indigenous Cases in South-East of Iran using Nested-PCR and RFLP

The Plasmodium falciparum Apical Membrane Antigen 1 (AMA1) is a leading Malaria Vaccine Candidate Antigen. Antigenic variation is one of the main obstacles in the development of a universal effective malaria vaccine. Antibodies against AMA1 have been shown to block parasite invasion of human erythrocytes. Therefore, detailed studies on the molecular polymorphism PFAMA1 a Geographic area before each experiment or design the vaccine is administered. This study was designed to determine the distribution of AMA1 allele class of Plasmodium falciparum in two population of imported cases and indigenous cases in South-East of Iran .We used the Nested PCR and RFLPs methods with specific primers and restriction enzymes, which improves the three AMA1 allele class(K1-3D7-HB3).Overall the 94 confirmed P. falciparum samples obtained from four different districts, in two population of imported cases(46) and indigenous cases(48) in the south East of Iran. There are three classes of allelic AMA1 were compared in two populations of Indigenous and Imported, 3D7 in Indigenous Population was the higher prevalence than Imported Population. Considering on these results, 3D7 alleles suitable candidates for malaria vaccine design can be. The data reported here will be valuable for the development of AMA1 based malaria vaccine.

Plastid transformation in lettuce (Lactuca sativa L.) by biolistic DNA delivery.

The interest in producing pharmaceutical proteins in a nontoxic plant host has led to the development of an approach to express such proteins in transplastomic lettuce (Lactuca sativa L.). A number of therapeutic proteins and vaccine antigen candidates have been stably integrated into the lettuce plastid genome using biolistic DNA delivery. High levels of accumulation and retention of biological activity suggest that lettuce may provide an ideal platform for the production of biopharmaceuticals.

Differing Patterns of Selection and Geospatial Genetic Diversity within Two Leading Plasmodium vivax Candidate Vaccine Antigens

Differing Patterns of Selection and Geospatial Genetic Diversity within Two Leading Plasmodium vivax Candidate Vaccine Antigens

Evaluation and comparison of the potential of two ferritins as anti-tick vaccines against Haemaphysalis longicornis

BackgroundTick control is an essential aspect of controlling the spread of tick-borne diseases affecting humans and animals, but it presently faces several challenges. Development of an anti-tick vaccine is aimed at designing cost-effective and environmentally friendly protection against ticks and tick-borne diseases as an alternative to the use of chemical acaricides. A single vaccine from the tick midgut protein Bm86 is currently available for field applications, but its efficacy is limited to only some tick species. Identification of candidate vaccine antigens that can affect multiple tick species is highly desirable. The hard tick Haemaphysalis longicornis has two kinds of the iron-binding protein ferritin (HlFER), an intracellular HlFER1 and a secretory HlFER2, and RNA interference experiments showed that these are physiologically important in blood feeding and reproduction and in protection against oxidative stress. Here we investigated the potential of targeting HlFERs for tick control by immunizing the host with recombinant HlFERs (rHlFER1 and rHlFER2).MethodsRabbits were immunized with rHlFERs three times subcutaneously at two-week intervals. Antisera were collected before the first immunization and a week after each immunization to confirm the antigen-specific serum antibody titer by serum ELISA. Two weeks after the final immunization, the rabbits were challenged with tick infestation. After dropping, tick feeding and reproduction parameters were evaluated to determine vaccine efficacy. To demonstrate the effects of antibodies, oxidative stress was detected in the eggs and larvae.ResultsThe antibody titer of rHlFER-immunized rabbits greatly increased after the second immunization. Antibodies exhibited cross-reactivity with rHlFERs and reacted with tick native HlFERs in Western blot analysis. Significantly lower bodyweight was observed in the ticks infested from the rHlFER2-immunized rabbit compared to those from the control rabbit. Reduced oviposition and hatching rate were observed in both rHlFER-immunized groups. rHlFER2 showed a higher vaccine efficacy. The antibodies against rHlFERs were detected in the eggs, and higher levels of oxidative stress biomarkers in the eggs and larvae, of ticks from rHlFER vaccinated rabbits.ConclusionCollectively, these results showed that HlFER2 has a good potential as an anti-tick vaccine antigen that may affect multiple tick species.

Population genetics and natural selection in the gene encoding the Duffy binding protein II in Iranian Plasmodium vivax wild isolates

Region II of Duffy binding protein (PvDBP-II) is one of the most promising blood-stage vaccine candidate antigens against Plasmodium vivax and having knowledge of the nature and genetic polymorphism of PvDBP-II among global P. vivax isolates is important for developing a DBP-based vaccine. By using PCR and sequencing, the present molecular population genetic approach was carried out to investigate sequence diversity and natural selection of dbp-II gene in 63 P. vivax isolates collected from unstable and low transmission malaria-endemic areas of Iran during 2008–2012. Also, phylogenetic analysis, the diversifying natural selection, and recombination across the pvdbp-II gene, including regions containing B-cell epitopes were analyzed using the DnaSP and MEGA4 programs. Twenty two single nucleotide polymorphisms (SNPs, including 20 non-synonymous and 2 synonymous) were identified in PvDBP-II, resulting in 16 different PvDBP-II haplotypes among the Iranian P. vivax isolates. High binding inhibitory B-cell epitope (H3) overlapping with intrinsically unstructured/disordered region (aa: 384–392) appeared to be highly polymorphic (D384G/E385K/ K386N/Q/R390H), and positive selective pressure acted on this region. Most of the polymorphic amino acids, which are located on the surface of the protein, are under selective pressure that implies increased recombination events and exposure to the human immune system.In summary, PvDBP-II gene displays genetic polymorphism among Iranian P. vivax isolates and it is under selective pressure. Mutations, recombination, and positive selection seem to play a role in the resulting genetic diversity, and phylogenetic analysis of DNA sequences demonstrates that Iranian isolates represent a sample of the global population. These results are useful for understanding the nature of the P. vivax population in Iran and also for development of PvDBP-II-based malaria vaccine.

Use of In Vivo-Induced Antigen Technology to Identify In Vivo-Expressed Genes of Campylobacter jejuni During Human Infection

Campylobacter jejuni is a prevalent foodborne pathogen worldwide. Human infection by C. jejuni primarily arises from contaminated poultry meats. Genes expressed in vivo may play an important role in the pathogenicity of C. jejuni. We applied an immunoscreening method, in vivo-induced antigen technology (IVIAT), to identify in vivo-induced genes during human infection by C. jejuni. An inducible expression library of genomic proteins was constructed from sequenced C. jejuni NCTC 11168 and was then screened using adsorbed, pooled human sera obtained from clinical patients. We successfully identified 24 unique genes expressed in vivo. These genes were implicated in metabolism, molecular biosynthesis, genetic information processing, transport, and other processes. We selected six genes with different functions to compare their expression levels in vivo and in vitro using real-time RT-PCR. The results showed that the selected six genes were significantly upregulated in vivo but not in vitro. In short, these identified in vivo-induced genes may contribute to human infection of C. jejuni, some of which may be meaningful vaccine candidate antigens or diagnosis serologic markers for campylobacteriosis. IVIAT may present a significant and efficient method for understanding the pathogenicity mechanism of Campylobacter and for finding targets for its prevention and control.

Co-colonization by Haemophilus influenzae with Streptococcus pneumoniae enhances pneumococcal-specific antibody response in young children

BackgroundStreptococcus pneumoniae (Spn), Haemophilus influenzae (Hi) and Moraxella catarrhalis (Mcat) are common bacterial pathogens of respiratory infections and common commensal microbes in the human nasopharynx (NP). The effect of interactions among theses bacteria during co-colonization of the NP on the host immune response has not been evaluated. The objective of this study was to assess the impact of co-colonization by Hi or Mcat on the systemic antibody response to vaccine protein candidate antigens of Spn and similarly the impact of co-colonization by Spn and Mcat on antibody responses to Hi vaccine protein candidate antigens. MethodsSerum samples were collected from healthy children at 6, 9, 15, 18, and 24 months of age when they were colonized with Spn, Hi, Mcat or their combinations. Quantitative ELISA was used to determine serum IgA and IgG against three Spn antigens and three Hi antigens, and as well as whole cells of non-typeable (NT) Spn and Hi. ResultsNP colonization by Spn increased serum IgA and IgG titers against Spn antigens PhtD, PcpA and PlyD and whole cells of NTSpn, and co-colonization of Hi or Mcat with Spn resulted in further increases of serum pneumococcal-specific antibody levels. NP colonization by Hi increased serum IgA and IgG titers against Hi antigens P6, Protein D and OMP26 and whole cells of NTHi, but co-colonization of Spn or Mcat with Hi did not result in further increase of serum NTHi-specific antibody levels. ConclusionCo-colonization of Hi or Mcat with Spn enhances serum antibody response to NTSpn whole cells and Spn vaccine candidate antigens PhtD, PcPA and PlyD1. Co-colonization appears to variably modulate pathogen species-specific host adaptive immune response.

Oral Immunization with Recombinant Lactobacillus acidophilus Expressing the Adhesin Hp0410 of Helicobacter pylori Induces Mucosal and Systemic Immune Responses

Helicobacter pylori infection is relatively common worldwide and is closely related to gastric mucosa-associated lymphoid tissue (MALT) lymphoma, chronic gastritis, and stomach ulcers. Therefore, a safe and effective method for preventing H. pylori infection is urgently needed. Given that developing an effective vaccine against H. pylori is one of the best alternatives, H. pylori adhesin Hp0410 was expressed in the food-grade bacterium Lactobacillus acidophilus. The recombinant live bacterial vaccine was then used to orally vaccinate mice, and the immunoprotective effects of Hp0410-producing strains were investigated. H. pylori colonization in the stomach of mice immunized with the recombinant L. acidophilus was significantly reduced, in comparison with that in control groups. Furthermore, mucosal secretory IgA antibodies were elicited in the mucosal tissue of mice immunized with the recombinant bacteria, and specific anti-Hp0410 IgG responses were also detected in mouse serum. There was a significant increase in the level of protection against gastric Helicobacter infection following a challenge with H. pylori Sydney strain 1 (SS1). Our results collectively indicate that adhesin Hp0410 is a promising candidate vaccine antigen, and recombinant L. acidophilus expressing Hp0410 is likely to constitute an effective, low-cost, live bacterial vaccine against H. pylori.

Identification of the immunoproteome of the meningococcus by cell surface immunoprecipitation and MS

Most healthy adults are protected from meningococcal disease by the presence of naturally acquired anti-meningococcal antibodies; however, the identity of the target antigens of this protective immunity remains unclear, particularly for protection against serogroup B disease. To identify the protein targets of natural protective immunity we developed an immunoprecipitation and proteomics approach to define the immunoproteome of the meningococcus. Sera from 10 healthy individuals showing serum bactericidal activity against both a meningococcal C strain (L91543) and the B strain MC58, together with commercially available pooled human sera, were used as probe antisera. Immunoprecipitation was performed with each serum sample and live cells from both meningococcal strains. Immunoprecipitated proteins were identified by MS. Analysis of the immunoproteome from each serum demonstrated both pan-reactive antigens that were recognized by most sera as well as subject-specific antigens. Most antigens were found in both meningococcal strains, but a few were strain-specific. Many of the immunoprecipitated proteins have been characterized previously as surface antigens, including adhesins and proteases, several of which have been recognized as vaccine candidate antigens, e.g. factor H-binding protein, NadA and neisserial heparin-binding antigen. The data demonstrate clearly the presence of meningococcal antibodies in healthy individuals with no history of meningococcal infection and a wide diversity of immune responses. The identification of the immunoreactive proteins of the meningococcus provides a basis for understanding the role of each antigen in the natural immunity associated with carriage and may help to design vaccination strategies.

Alterations in immunodominance of Streptococcus mutans AgI/II: Lessons learned from immunomodulatory antibodies

Streptococcus mutans antigen I/II (AgI/II) has been widely studied as a candidate vaccine antigen against human dental caries. In this report we follow up on prior studies that indicated that anti-AgI/II immunomodulatory monoclonal antibodies (MAbs) exerted their effects by destabilizing the native protein structure and exposing cryptic epitopes. We show here that similar results can be obtained by immunizing mice with truncated polypeptides out of the context of an intra-molecular interaction that occurs within the full-length molecule and that appears to dampen the functional response against at least two important target epitopes. Putative T cell epitopes that influenced antibody specificity were identified immediately upstream of the alanine-rich repeat domain. Adherence inhibiting antibodies could be induced against two discrete domains of the protein, one corresponding to the central portion of the molecule and the other corresponding to the C-terminus.

Screening of Genes Expressed In Vivo During Interaction Between Chicken and Campylobacter jejuni

Chicken are considered as the most important source of human infection by Campylobacter jejuni, which primarily arises from contaminated poultry meats. However, the genes expressed in vivo of the interaction between chicken and C. jejuni have not been screened. In this regard, in vivo-induced antigen technology (IVIAT) was applied to identify expressed genes in vivo during interaction between chicken and C. jejuni, a prevalent foodborne pathogen worldwide. Chicken sera were obtained by inoculating C. jejuni NCTC 11168 into Leghorn chickens through oral and intramuscular administration. Pooled chicken sera, adsorbed against in vitrogrown cultures of C. jejuni, were used to screen the inducible expression library of genomic proteins from sequenced C. jejuni NCTC 11168. Finally, 28 unique genes expressed in vivo were successfully identified after secondary and tertiary screenings with IVIAT. The genes were implicated in metabolism, molecular biosynthesis, genetic information processing, transport, regulation and other processes, in addition to Cj0092, with unknown function. Several potential virulence-associated genes were found to be expressed in vivo, including chuA, flgS, cheA, rplA, and Cj0190c. We selected four genes with different functions to compare their expression levels in vivo and in vitro using real-time RT-PCR. The results indicated that these selected genes were significantly upregulated in vivo but not in vitro. In short, the expressed genes in vivo may act as potential virulence-associated genes, the protein encoded by which may be meaningful vaccine candidate antigens for campylobacteriosis. IVIAT provides an important and efficient strategy for understanding the interaction mechanisms between Campylobacter and hosts.

Elongation Factor-1α Is a Novel Protein Associated with Host Cell Invasion and a Potential Protective Antigen of Cryptosporidium parvum

The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-β- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis.

Vaccinomics, the new road to tick vaccines

Ticks are a threat to human and animal health worldwide. Ticks are considered to be second worldwide to mosquitoes as vectors of human diseases, the most important vectors of diseases that affect cattle industry worldwide and important vectors of diseases affecting pets. Tick vaccines are a cost-effective and environmentally friendly alternative to protect against tick-borne diseases through the control of vector infestations and reducing pathogen infection and transmission. These premises stress the need for developing improved tick vaccines in a more efficient way. In this context, development of improved vaccines for tick-borne diseases will be greatly enhanced by vaccinomics approaches starting from the study of tick–host–pathogen molecular interactions and ending in the characterization and validation of vaccine formulations. The discovery of new candidate vaccine antigens for the control of tick infestations and pathogen infection and transmission requires the development of effective screening platforms and algorithms that allow the analysis and validation of data produced by systems biology approaches to tick research. Tick vaccines that affect both tick infestations and pathogen transmission could be used to vaccinate human and animal populations at risk and reservoir species to reduce host exposure to ticks while reducing the number of infected ticks and their vectorial capacity for pathogens that affect human and animal health worldwide.

Assessment of antibody-dependent respiratory burst activity from mouse neutrophils on Plasmodium yoelii malaria challenge outcome.

New tools are required to expedite the development of an effective vaccine against the blood-stage infection with the human malaria parasite Plasmodium falciparum. This work describes the assessment of the ADRB assay in a mouse model, characterizing the functional interaction between antimalarial serum antibodies and FcRs upon neutrophils. We describe a reproducible, antigen-specific assay, dependent on functional FcR signaling, and show that ADRB activity is induced equally by IgG1 and IgG2a isotypes and is modulated by blocking FcR function. However, following immunization of mice with the blood-stage vaccine candidate antigen MSP142, no measurable ADRB activity was induced against PEMS and neither was vaccine efficacy modulated against Plasmodium yoelii blood-stage challenge in γ(-/-) mice compared with WT mice. In contrast, following a primary, nonlethal P. yoelii parasite challenge, serum from vaccinated mice and nonimmunized controls showed anti-PEMS ADRB activity. Upon secondary challenge, nonimmunized γ(-/-) mice showed a reduced ability to control blood-stage parasitemia compared with immunized γ(-/-) mice; however, WT mice, depleted of their neutrophils, did not lose their ability to control infection. Thus, whereas neutrophil-induced ADRB against PEMS does not appear to play a role in protection against P. yoelii rodent malaria, induction of ADRB activity after challenge suggests that antigen targets of anti-PEMS ADRB activity remain to be established, as well as further supporting the observation that ADRB activity to P. falciparum arises following repeated natural exposure.

Plasmodium falciparum: Genetic diversity and complexity of infections in an isolated village in western Thailand

Genetic diversity of Plasmodium falciparum is intimately associated with morbidity, mortality and malaria control strategies. It is therefore imperative to study genetic makeup and population structure of this parasite in endemic areas. In Kong Mong Tha, an isolated village in western Thailand, the majority of P. falciparum infections are asymptomatic. In this study we investigated complexity of infections and single nucleotide polymorphisms (SNPs) in the P. falciparum population of Kong Mong Tha, and compared results with those previously obtained from Mae Sod, in northwestern Thailand, where the majority of infections were symptomatic. Using PCR-based determination of the 5′ merozoite surface protein 1 gene (msp1) recombinant types, we found that 39% of 59 P. falciparum isolates from Kong Mong Tha had multiple 5′ recombinant types with a mean number of 1.54. These values were much lower than those obtained from Mae Sod: 96% for multiple infections and with a mean number of 3.61. Analysis of full-length sequences of two housekeeping genes, the P-type Ca2+-transporting ATPase gene (n=33) plus adenylosuccinate lyase gene (n=33), and three vaccine candidate antigen genes, msp1 (n=26), the circumsporozoite protein gene, csp (n=30) and the apical membrane antigen 1 gene, ama 1 (n=32), revealed that in all of these genes within-population SNP diversity was at similar levels between Kong Mong Tha and Mae Sod, suggesting that the extent of MOI and clinical manifestations of malaria are not strongly associated with genetic diversity. Additionally, we did not detect significant genetic differentiation between the two parasite populations, as estimated by the Wright's fixation index of inter-population variance in allele frequencies, suggesting that gene flow prevented the formation of population structuring. Thus, this study highlights unique features of P. falciparum populations in Thailand. The implications of these finding are discussed.

Identification of immunoreactive proteins ofStreptococcus agalactiaeisolated from cultured tilapia in China

Streptococcus agalactiae (Group B streptococcus, GBS) is an important zoonotic pathogen that can cause lethal infections in humans and animals, including aquatic species. Immunoreactive proteins of the S.agalactiae strain, GD201008-001, isolated from cultured tilapia in China, were screened by immunoproteomics using hyperimmune sera, convalescent guinea pig sera and GD201008-001-infected tilapia antisera as primary detection antibodies. A total of 16 different proteins were identified including 13 novel immunoreactive proteins of S.agalactiae. Four proteins, serine-rich repeat glycoprotein 1, branched-chain alpha-keto acid dehydrogenase (BKD) subunit E2, 5'-nucleotidase family protein and ornithine carbamoyltransferase, were shown to react with the three types of sera and thus were considered to represent novel S.agalactiae vaccine candidate antigens. Our findings represent the basis for vaccine development for piscine S.agalactiae and are necessary for understanding virulence factors and immunogenicity of S.agalactiae with different hosts.

A novel iron transporter inStreptococcus iniae

Streptococcus iniae is a major pathogen that results in considerable economic loss to fish farms. Restricted availability of iron is a huge obstacle to survival for pathogenic bacteria during infection, and iron acquisition is important in bacterial virulence. In this study, S. iniae HD-1 was shown not to produce siderophores (low-molecular-weight compounds) but rather to require iron-containing proteins for growth under iron-restricted conditions. The adenosine triphosphate (ATP)-binding-cassette (ABC) transporter system (ftsABCD), which is cotranscribed by four downstream genes, namely, ftsA, ftsB, ftsC and ftsD, was identified as responsible for haem utilization of S.iniae. Analysis of the corresponding recombinant protein, FtsB, indicated that it is a putative lipoprotein which plays a role in haem utilization and is produced in vivo during infection with S.iniae HD-1, and therefore may be a potential candidate antigen for a streptococcal vaccine.