Screening and Molecular Cloning of a Protective Antigen from the Midgut of Haemaphysalis longicornis

Vaccination is considered a promising alternative for controlling tick infestations. Haemaphysalis longicornis midgut proteins separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane were screened for protective value against bites. The western blot demonstrated the immunogenicity of 92 kDa protein (P92). The analysis of the P92 amino acid sequence by LC-MS/MS indicated that it was a H. longicornis paramyosin (Hl-Pmy). The full lenghth cDNA of Hl-Pmy was obtained by rapid amplification of cDNA ends (RACE) which consisted of 2,783 bp with a 161 bp 3'' untranslated region. Sequence alignment of tick paramyosin (Pmy) showed that Hl-Pmy shared a high level of conservation among ticks. Comparison with the protective epitope sequence of other invertebrate Pmy, it was calculated that the protective epitope of Hl-Pmy was a peptide (LEEAEGSSETVVEMNKKRDTE) named LEE, which was close to the N-terminal of Hl-Pmy protein. The secondary structure analysis suggested that LEE had non-helical segments within an α-helical structure. These results provide the basis for developing a vaccine against biting H. longicornis ticks.     

Immunization effect of recombinant P27/30 protein expressed in Escherichia coli against the hard tick Haemaphysalis longicornis (Acari: Ixodidae) in rabbits

We investigated the induction of resistance to Haemaphysalis longicornis infestation in rabbits that had been immunized with recombinant H. longicornis P27/30 protein. The success of immunological control methods is dependent upon the use of potential key antigens as tick vaccine candidates. Previously, we cloned a gene encoding 27 kDa and 30 kDa proteins (P27/30) of H. longicornis, and identified P27/30 as a troponin I-like protein. In this study, rabbits that were immunized with recombinant P27/30 expressed in Escherichia coli showed the statistically significant longer feeding duration for larval and adult ticks (P<0.05), low engorgement rates in larval ticks (64.4%), and an apparent reduction in egg weights, which suggest that H. longicornis P27/30 protein is a potential candidate antigen for a tick vaccine. These results demonstrated that the recombinant P27/30 protein might be a useful vaccine candidate antigen for biological control of H. longicornis.     

A Novel Recombinant BCG Vaccine Encoding Eimeria tenella Rhomboid and Chicken IL-2 Induces Protective Immunity Against Coccidiosis

A novel recombinant Bacille Calmette-Guerin (rBCG) vaccine co-expressed Eimeria tenella rhomboid and cytokine chicken IL-2 (chIL-2) was constructed, and its efficacy against E. tenella challenge was observed. The rhomboid gene of E. tenella and chIL-2 gene were subcloned into integrative expression vector pMV361, producing vaccines rBCG pMV361-rho and pMV361-rho-IL2. Animal experiment via intranasal and subcutaneous route in chickens was carried out to evaluate the immune efficacy of the vaccines. The results indicated that these rBCG vaccines could obviously alleviate cacal lesions and oocyst output. Intranasal immunization with pMV361-rho and pMV361-rho-IL2 elicited better protective immunity against E. tenella than subcutaneous immunization. Splenocytes from chickens immunized with either rBCG pMV361-rho and pMV361-rho-IL2 had increased CD4⁺ and CD8⁺ cell production. Our data indicate recombinant BCG is able to impart partial protection against E. tenella challenge and co-expression of cytokine with antigen was an effective strategy to improve vaccine immunity.     

Cloning,expression and immunogenicity of ferric enterobactin binding protein Fep B from <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7

The gene coding for ferric enterobactin binding protein from E. coli O157:H7 was amplifi ed. This gene was cloned and expressed as C-terminal His (6)-tagged protein. The SDS-PAGE analysis of the total protein revealed only two distinct bands, with molecular masses of 31kDa and 34kDa. The Ni-NTA chromatography purifi ed FepB and the osmotically shocked periplasmic fraction of IPTG induced cells showed only a single band of 31 kDa. Polyclonal mouse antibody was raised against the recombinant protein during 4 weeks after immunization. Western blot analysis of the recombinant FepB with mouse antiserum revealeda single band of 31 kDa. Identification and purification of FepB helped reveal its appropriate molecular mass. Polyclonal antibody raised against the recombinant protein reacted with bacterial FepB. The recombinant protein FepB could have a protective effect against E. coli O157:H7 and might be useful as an effective vaccine.     

Application of a Chimeric Protein Construct having Enterotoxin B and Toxic Shock Syndrome Toxin Domains of S. aureus in Immunodiagnostics

Staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 are the super antigens responsible for diseases such as staphylococcal food poisoning and toxic shock syndrome. At low serum concentrations, SEB can trigger toxic shock, profound hypotension and multi organ failure and hence is recognized as biowarfare molecule. In this study, a multidomain fusion protein (r-TE) was generated with specificity for SEB and toxic shock syndrome toxin (Tsst-1). The fusion gene comprising the conserved regions of seb and the tsst genes was codon-optimized for expression in Escherichia coli and encoded a 26 kDa recombinant multidomain chimeric protein (r-TE). Hyperimmune antiserum raised against r-TE specifically reacted with SEB (~28 kDa) and Tsst-1 (~22 kDa) components during Western blot analysis and by plate ELISA in confirmed toxin producing strains of S. aureus. The antigenicity of the SEB component of the r-TE protein was also confirmed using TECRA kit. The described procedure of creating a single protein molecule carrying components of two different toxins whilst still retaining the original antigenic determinants of individual toxins proved highly advantageous in the development of rapid, reliable and cost effective immunoassays and may also have the potential to serve as candidate molecule for vaccine studies.     

Plasmodium vivax: Comparison of the immune responses between oral and parenteral immunization of rPv54 in BALB/c mice

The merozoite surface protein-1 (MSP-1) from Plasmodium vivax was evaluated as an oral vaccine candidate by cloning and expressing the interspecies conserved block 10 (ICB10) of the MSP-1 from a Korean isolate in Escherichia coli. The expressed fusion protein contained ICB10 and a maltose-binding protein (MBP), rPv54, has a molecular weight of approximately 54 kDa as determined by SDS-PAGE analysis. IgG against rPv54 was successfully produced in BALB/c mice by oral immunization and sustained for more than 4 months. IgG2b was dominantly produced in both oral and parenteral immunizations. The rPv54 increased the frequency of NK, NKT, CD4+ T, CD8+ T, and B cells in both immunizations. IL-5 and TNF-α were increased in both significantly. In conclusion, rPv54 might be a valuable potential vaccine candidate for the oral and parenteral immunization against vivax malaria.     

Identification of a cysteine-rich antimicrobial peptide from salivary glands of the tick Rhipicephalus haemaphysaloides

The presence of an effective immune response in the hemocoel of ticks is crucial for survival, as it prevents the invasion of pathogens throughout the animal's body. Antimicrobial peptides (AMPs) play an important role in this response by rapidly killing invading microorganisms. In this study, a subtraction hybridization cDNA library was constructed from the salivary glands of the unfed and fed female tick Rhipicephalus haemaphysaloides, and a novel cysteine-rich AMP designated Rhamp (R. haemaphysaloides antimicrobial peptide) was isolated and identified. The Rhamp was encoded by a gene with an open reading frame of 303 bp which encoded a mature peptide with 8 kDa molecular weight. No identity was found by BLAST search to any database entries. The sequence encoding the Rhamp was subcloned into the pGEX-4T vector and expressed in Escherichia coli. The recombinant protein of Rhamp showed chymotrypsin and elastase-inhibitory activity and markedly inhibited the growth of Gram-negative bacteria, including Pseudomonas aeruginosa, Salmonella typhimurium, and E. coli. Moreover, the recombinant protein also exerted low hemolytic activity. These results indicate the Rhamp is a novel antimicrobial peptide with proteinase activity from the tick R. haemaphysaloides.     

Recombinant Yellow Fever Viruses Elicit CD8+ T Cell Responses and Protective Immunity against Trypanosoma cruzi

Chagas’ disease is a major public health problem affecting nearly 10 million in Latin America. Despite several experimental vaccines have shown to be immunogenic and protective in mouse models, there is not a current vaccine being licensed for humans or in clinical trial against T. cruzi infection. Towards this goal, we used the backbone of Yellow Fever (YF) 17D virus, one of the most effective and well-established human vaccines, to express an immunogenic fragment derived from T. cruzi Amastigote Surface Protein 2 (ASP-2). The cDNA sequence of an ASP-2 fragment was inserted between E and NS1 genes of YF 17D virus through the construction of a recombinant heterologous cassette. The replication ability and genetic stability of recombinant YF virus (YF17D/ENS1/Tc) was confirmed for at least six passages in Vero cells. Immunogenicity studies showed that YF17D/ENS1/Tc virus elicited neutralizing antibodies and gamma interferon (IFN-γ) producing-cells against the YF virus. Also, it was able to prime a CD8⁺ T cell directed against the transgenic T. cruzi epitope (TEWETGQI) which expanded significantly as measured by T cell-specific production of IFN-γ before and after T. cruzi challenge. However, most important for the purposes of vaccine development was the fact that a more efficient protective response could be seen in mice challenged after vaccination with the YF viral formulation consisting of YF17D/ENS1/Tc and a YF17D recombinant virus expressing the TEWETGQI epitope at the NS2B-3 junction. The superior protective immunity observed might be due to an earlier priming of epitope-specific IFN-γ-producing T CD8⁺ cells induced by vaccination with this viral formulation. Our results suggest that the use of viral formulations consisting of a mixture of recombinant YF 17D viruses may be a promising strategy to elicit protective immune responses against pathogens, in general.     

Rickettsia raoultii,the predominant Rickettsia found in Dermacentor silvarum ticks in China–Russia border areas

Since the year 2000, clinical patterns resembling tick-borne rickettsioses have been noticed in China–Russia border areas. Epidemiological data regarding species of the aetiological agent, tick vector prevalence and distribution as well as incidence of human cases in the areas are still sparse to date. In order to identify Rickettsia species occurring in the areas, we investigated Dermacentor silvarum collected in the selected areas. Rickettsia raoultii was the predominant Rickettsia found in D. silvarum evident with ompA, ompB, gltA and 17 kDa protein genes. The Rickettsia prevalence in D. silvarum appeared to be 32.25 % with no sex difference. The results extend the common knowledge about the geographic distribution of R. raoultii and its candidate vector tick species, which suggest an emerged potential threat of human health in the areas.     

Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle

Bacillus thuringiensis (Bt) Cry8D insecticidal proteins are unique among Cry8 family proteins in terms of its insecticidal activity against adult Scarab beetles, such as Japanese beetle (Popillia japonica Newman). From the sequence homology with other Bt Cry proteins especially those active against beetles, such as Cry3Aa whose 3D structure is available, the structure of the Cry8D protein has been predicted to be a typical three-domain Cry protein type. In addition, the activation process of Cry8D in gut juice of susceptible insects is presumed to be similar to that of Cry3A (Yamaguchi et al., 2008). In this study, the activation process of Cry8Da in insect gut juice was closely examined. Japanese beetle gut juice proteases digested the 130 kDa Cry8Da protein to produce a 64 kDa protein. This 64 kDa protein was active against both adult and larval Japanese beetle and considered to be an activated toxin. N-terminal sequencing of this 64 kDa protein revealed that the Cry8Da leader sequence consisting of 63 amino acid residues from M¹ to F⁶³ was removed. As in the case of Cry3Aa, the proteases further digested the 64 kDa protein to two 8 kDa and 54 kDa fragments. N-terminal amino acid analysis of these smaller fragments indicated that the proteases digested the loop between Alpha Helix (Alpha for short) 3 and Alpha 4. This means that the 8 kDa fragment consists of Alpha 1-3 of Domain I and that the 54 kDa fragment contains the remaining Domain I and full Domain II and Domain III. Size exclusion chromatography and anion exchange chromatography could not separate these 64, 54 and 8 kDa proteins suggesting that the 54 kDa and 8 kDa fragments are still forming the toxin complex equivalent to the 64 kDa protein by size and ionic charge. The sequencing and chromatography results suggest that the gut juice proteases merely nicked the loop between Alpha 3 and Alpha 4. This nicking process appeared to be essential for receptor binding of the Cry8Da toxin. BBMV binding assay revealed that the Cry8Da toxin bound to BBMV preparations from both adult and larval Japanese beetle only after the loop was nicked. Only the 54 kDa fragment bound to the BBMV preparations but not the 64 kDa protein. Ligand blot showed that the protease activated Cry8Da toxin, presumably the 54 kDa fragment, bound to specific BBMV proteins, one or more of those would be receptor(s). The sizes and binding affinities of these Cry8Da-bound proteins of Japanese beetle BBMV differed between larvae and adults.     

Toxoplasma gondii: The immunogenic and protective efficacy of recombinant ROP2 and ROP4 rhoptry proteins in murine experimental toxoplasmosis

Toxoplasmosis is a one of the most world-wide spread zoonosis representing a very serious clinical and veterinary problem. In the presented study, we evaluated the protective efficacy of a combined recombinant ROP2 and ROP4 subunit vaccine in a chronic Toxoplasma gondii infection in mice. The recombinant ROP2 (rROP2) and ROP4 (rROP4) proteins were cloned and expressed in Escherichia coli and then used for the immunization of C3H/HeJ mice. Both antigens generated a strong systemic mixed Th1/Th2 response polarized towards IgG1 antibody isotype. In contrast to rROP2 stimulating only the specific IL-2 release, rROP4 and crude toxoplasma lysate antigen (TLA) used as a source of native forms of the parasite proteins induced significant proliferation of splenocytes and specific production of IFN-γ as well as IL-2, the Th1-type cytokines. Challenge of rROP2 and rROP4-vaccinated mice with cysts of low virulent T. gondii DX strain resulted in a partial protection effect with a significantly lower brain parasites load when compared with control animals. In the immunized group of mice the brain cysts number was reduced by nearly 46% as was determined in two independent experiments. These results suggest that, similar to ROP2, rhoptry protein ROP4 could be a very good candidate for future anti-T. gondii multicomponent vaccine based on the recombinant forms of different parasite proteins.     

Leishmania donovani: identification of novel vaccine candidates using human reactive sera and cell lines

Leishmaniasis is a complex of diseases caused by protozoan parasites belonging to the genus Leishmania. The development of specific resistance against re-infection after cure suggests that a vaccine approach is feasible. Various studies in humans and experimental animals strongly suggest that Th1 type of cell-mediated immune response is important for protection against the disease. A defined antigen that could elicit a specific T-cell-mediated immune response in the host would be an ideal candidate for the vaccine against this parasite. In order to select a candidate antigen, we established a screening system to identify the recombinant clone, expressing antigen having T-cell epitopes from a cDNA library. We screened the library using an established Leishmania specific cell line (LSCL) from a naive healthy human subject. The cell line with predominantly CD4+ cells behaved in a Leishmania specific manner. Fifty-two immuno-reactive clones were screened against the LSCL in vitro and we identified three cDNA clones expressing recombinant antigens that could induce proliferation of these cells to produce INFgamma. The protective efficacy of one of these recombinant proteins was investigated in a hamster model of experimental visceral leishmaniasis and showed protection against a virulent challenge. The identified antigens might be potential candidates for vaccine against Leishmania.     

Psoroptes ovis: identification of vaccine candidates by immunoscreening

Serum from successful vaccine trials against the sheep scab mite, Psoroptes ovis, was used to immunoscreen a cDNA library constructed from mixed-stage and gender P. ovis to identify potential recombinant vaccine candidates. Immunodominant recombinant proteins recognised by IgG in these sera were selected for further analysis. Two candidates were identified in this way; a catchin-like protein (CLP) and a novel mu class glutathione S-transferase (GST). Both candidates were expressed in bacteria as recombinant proteins, the GST as an active enzyme, and combined with four other recombinant allergens in a multi-component recombinant vaccine. Strong serum IgG responses were induced in sheep against each of the components of the recombinant vaccine, however, the protective efficacy of the vaccine could not be determined because of variability in the establishment of a challenge infection.     

Recombinant expression of in silico identified Bcell epitope of epsilon toxin of Clostridium perfringens in translational fusion with a carrier protein

Epsilon toxin secreted by Clostridium perfringens types B and D has been directly implicated as the causative agent of fatal enterotoxemia in domestic animals. The aim of the present study is to use in silico approach for identification of B-cell epitope(s) of epsilon toxin, and its expression in fusion with a carrier protein to analyze its potential as vaccine candidate(s). Using different computational analyses and bioinformatics tools, a number of antigenic determinant regions of epsilon toxin were identified. One of the B cell epitopes of epsilon toxin comprising the region (amino acids 40-62) was identified as a promising antigenic determinant. This Etx epitope (Etx_40-62) was cloned and expressed as a translational fusion with B-subunit of heat labile enterotoxin (LTB) of E. coli in a secretory expression system. Similar to the native LTB, the recombinant fusion protein retained the ability to pentamerize and bind to GM₁ ganglioside receptor of LTB. The rLTB.Etx_40-62 could be detected both with anti-Etx and anti-LTB antisera. The rLTB.Etx_40-62 fusion protein thus can be evaluated as a potential vaccine candidate against C. perfringens.

Abbreviations

aa - amino acid(s), Etx - epsilon toxin of Clostridium perfringens, LTB - B-subunit of heat labile enterotoxin of E. coli.     

Immunization with the basic membrane protein (BMP) family ABC transporter elicits protection against Enterococcus faecium in a murine infection model

Enterococcus faecium is evolving as a multi-resistant pathogen causing infections with high morbidity and mortality. A protective vaccine against E. faecium is lacking up till now. ATP-binding cassette (ABC) transporter proteins have important functions in bacteria to maintain survival and homeostasis. In the present study, we evaluated the basic membrane protein (BMP) family ABC transporter substrate-binding protein, designated herein as BMP, as a potential vaccine candidate against E. faecium. Recombinant BMP of E. faecium was expressed in Escherichia coli, and purified by metal affinity chromatography. Swiss albino mice were immunized with the recombinant BMP combined with Bacillus Calmette–Guérin (BCG) and/or alum as adjuvants. Mice immunized with BMP combined with alternating BCG and alum developed BMP-specific IgG and were protected against E. faecium challenge as evidenced from organ bioburden and histopathological examination. Furthermore, serum from immunized mice showed enhanced opsonophagocytic activity and protected mice against E. faecium challenge by passive immunization. Bioinformatic analysis revealed appreciable degrees of homology between E. faecium BMP and proteins from other pathogens which suggests BMP could be a useful vaccine against multiple pathogens. To our knowledge, this is the first report of in-vivo evaluation of BMP as a potential vaccine candidate against E. faecium.     

Vaccination against Streptococcus pneumoniae Using Truncated Derivatives of Polyhistidine Triad Protein D

Polyhistidine triad protein D (PhtD) has been described as a promising vaccine candidate for use against Streptococcus pneumoniae, but there has been a lack of examination of its structure and of which region(s) of the protein are targeted by protective immune responses. In this study, we purified recombinant truncated derivatives of PhtD and examined their secondary structural composition, as well as their capacity to bind antibodies from polyclonal murine serum generated against the full length protein. This allowed the identification of a particularly immunogenic fragment of PhtD, which was also purified and characterised. The truncated derivatives were tested as vaccine antigens in mouse models of pneumococcal sepsis and colonisation, using alum and E. coli heat labile toxin B subunit respectively as adjuvants. These experiments revealed that whilst the immunogenic region identified may be a promising candidate to protect against sepsis, the full length PhtD was ineffective at conferring significant protective immunity. These results are significant for the potential for PhtD to be used in novel vaccines, which are currently being tested in clinical trials.