WO2011071404A1 - Imunogénios suas composições e processo para a sua preparação e suas aplicações - Google Patents

Imunogénios suas composições e processo para a sua preparação e suas aplicações Download PDF

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Publication number
WO2011071404A1
WO2011071404A1 PCT/PT2009/000075 PT2009000075W WO2011071404A1 WO 2011071404 A1 WO2011071404 A1 WO 2011071404A1 PT 2009000075 W PT2009000075 W PT 2009000075W WO 2011071404 A1 WO2011071404 A1 WO 2011071404A1
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Prior art keywords
protein
fragment
antigen
immunogens
immune response
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PCT/PT2009/000075
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English (en)
French (fr)
Portuguese (pt)
Inventor
Maria Antónia PEREIRA DA CONCEIÇÃO
Sofia Judite Marques Da Costa
António Manuel OLIVEIRA CASTRO
André Augusto DA SILVA ALMEIDA
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Escola Superior Agrária De Coimbra
Hitag - Biotechnology, Lda.
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Priority to CA2783968A priority Critical patent/CA2783968A1/en
Priority to SG2012042651A priority patent/SG181618A1/en
Priority to BR112012013997A priority patent/BR112012013997A2/pt
Priority to EP09803934A priority patent/EP2510945A1/en
Priority to PCT/PT2009/000075 priority patent/WO2011071404A1/pt
Priority to US13/514,987 priority patent/US9610335B2/en
Application filed by Escola Superior Agrária De Coimbra, Hitag - Biotechnology, Lda. filed Critical Escola Superior Agrária De Coimbra
Priority to JP2012543039A priority patent/JP5832445B2/ja
Priority to CN200980163333.3A priority patent/CN102811732B/zh
Priority to AU2009356279A priority patent/AU2009356279A1/en
Publication of WO2011071404A1 publication Critical patent/WO2011071404A1/pt
Priority to IL220278A priority patent/IL220278A0/en
Priority to HK13106661.0A priority patent/HK1179519A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0003—Invertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the preparation of immunogens, a process for their preparation and their use in recombinant protein expression systems.
  • the present invention provides a novel adjuvant which will application can utter *
  • immunogens namely recombinant proteins containing the peptide sequence
  • the unrelated fragment acquires (in the case of poorly immunogenic anigens) sufficient immunological characteristics to lead to development only by its administration to the host of a host response, characterized in particular by the production of specific immunoglobulins.
  • immunogens resulting from the addition of the peptide with the sequence: M fragment, called fragment H, derived from a Fasciola hepatic calcium binding protein, to an unrelated antigen.
  • the resulting construct has immunogenic activity triggering an immune response when administered to an individual characterized by the production of specific antibodies against the unrelated antigen.
  • the present invention is useful for any application which is intended for the production of an immune response against an antigen by an individual to whom the H fragment immunogen and the unrelated antigen is administered.
  • the present invention describes an adjuvant type application which may have
  • the present invention is an alternative to the present adjuvants and, when used as recombinant protein expression systems, allows the production of proteins with immunogenic characteristics which, without. any other additive causes the development of an immune response, i.e. in an individual likely to develop an immune response.
  • One of the major challenges in antibody development today is obtaining a sufficiently immunogenic antigen to develop the immune response.
  • the antigen is not immunogenic
  • parallel injection of adjuvants is used to enhance the immune response or antibody development.
  • These adjuvants are toxic, cause pain in the injected host and therefore their use is highly discouraged or, in many potential adjuvants, even prohibited.
  • the advantage of the present technique lies precisely in this state of the art, as it describes a methodology for obtaining modified antigens sufficiently immunogenic to develop an immune response without the use of adjuvants.
  • fragment H excreted / secreted by the adult Fasciola hepatic worm with the sequence identical or at least 90% structurally similar to SEQ ID NO 2. designated fragment H;
  • the protein or protein fragment of interest is a pathogenic protein such as: a viral protein, a bacterial protein or a protozoan protein.
  • a pathogenic protein such as: a viral protein, a bacterial protein or a protozoan protein.
  • the protein or protein fragment of interest may be CWG. CD4, 1L5, Pfsp and Ent. PAL, CPI 2 and LEC, BG or Toxo.
  • the immunogens described above may be used as medicaments. While still more preferably they may be used as vaccines or adjuvants. Please note that in some even more preferential cases, a vaccine may be used that only comprises:
  • fragment H excreted / secreted by the adult Fasciola hepatic worm with the sequence identical or at least 90% structurally similar to SEQ ID NO 2. designated fragment H;
  • compositions containing the immunogens described above may contain the immunogens in therapeutically efficient amounts and with a pharmacologically suitable carrier such as excipients, adjuvants, among others,
  • compositions may contain only 100% of one of the previously described immunogens.
  • compositions may comprise the following elements: one of the above described immunogens with concentrations between 1 and 100 mg in a volume between 100 and 1000 m 1 diluted in phosphate buffered solution - 0.01 M phosphate, 0.1 M NaCl, pH 7.2.
  • Another embodiment of the present invention is the description of an adjuvant which
  • CWG and Cp 12 between 1 and 100 mg in a volume between 100 and 1000 m 1 diluted in buffered phosphate solution - 0.01 M phosphate, 0.1 M NaCl, pH 7.2 administered to mice, this induced an increase in intensity and speed with which a specific immune response against the CWG and CPI 2 fragments is developed.
  • Still another embodiment of the present invention is the description of a vaccine which
  • immunogen preparation which comprises the addition of fragment H to an unrelated polypeptide at any position in the sequence corresponding to the polypeptide to be used as immunogen, that is to say at the beginning, end or any fragment of the polypeptide. Even more preferably they may use various fragments and / or proteins such as CWG. CD4, IL5, Pfsp and Ent, PAL. CP 12. LEC, BG or Toxo.
  • the present invention is useful for producing an immune response by increasing specific antibody titers in serum against proteins or other antigens and may be applied in particular for the production of specific polyclonal antibodies, immunotherapy and immunoprophylaxis. in the production of vaccines, adjuvants, diagnostic methods and other applications directly obtained by developing a specific immune response.
  • Antisera are usually produced by the injection of an immunogen from
  • the amount of immunogen that must be administered to produce the desired response varies greatly depending on the animal species and / or subspecies used, the adjuvant used, the route of administration, the frequency of injections, and the immunogenicity of the antigen itself.
  • the quality and quantity of antibodies obtained depend on the size and condition of the immunogen. Small polypeptides and non-protein molecules may require conjugation to larger proteins to elicit an immune response.
  • Adjuvants may be used for a variety of purposes, including: enhancing the immunogenicity of purified or recombinant antigens; reduce the amount of antigen or number of immunizations needed to induce immunity protective; improve vaccine efficacy in newborns, the elderly or immunocompromised individuals: as a system of antigen release or antigen uptake by the mucosae.
  • the benefits of incorporating adjuvant into any formulation must be outweighed by the risk of adverse reactions.
  • One of the biggest challenges in adjuvant research is to increase potency and minimize toxicity.
  • each adjuvant Due to the effects of size, electrical charge and hydrophobicity, which regulate protein incorporation into the adjuvant formulation, it is difficult to predict which adjuvant will be most effective or suitable for a given protein or peptide.
  • epitope changes may occur during formulation or conjugation.
  • carrier proteins the existence of an immunity against it is a major limitation.
  • each adjuvant generates a characteristic immune response profile.
  • FIG. 1 A-Deduced amino acid sequence for FhS polypeptide (SEQ ID NO 1); Deduced amino acid sequence for polypeptide called FhS fragment H (SEQ ID NO 2).
  • FIG. 3 Results of the demonstrations performed with the constructions containing the CWG fragment.
  • Wells F 7 F g - FCWG fractions 1,2 collected from Ni-NTA column.
  • CDl were inoculated periodically and harvested periodically according to the protocol described in table 2; (a) Results obtained with plates containing the recombinant CWG antigen; b) Results obtained with plates containing the recombinant HCWG antigen; C - ELISA optical density results performed on sera from C 1 mice collected 83 days after the last inoculation with CWG (CWG group), HCWG (HCWG group), FCWG (FCWG group) and CD1 without any treatment (Neg Group) . The values represent the average optical densities of the 3 CD1 used in each group. D - Immunoblottings performed with a nitrocellulose membrane containing the recombinant FCWG antigen. FG- Nitrocellulose membrane containing Schwartz starch stained FCWG antigen.
  • Figure 4 Results of the demonstrations performed with the constructions containing the CPI fragment.
  • A Coomassie Blue Stained Tris-Tricine SDS-PAGE Gels.
  • PM Prestaincd SDS-PAGE Standards Marker (BioRad).
  • B Results of optical densities of ELISAs performed with sera from CD12 (CP12 group), HCP12 (group HCP12) and CD1 mice untreated (NEG group). The values represent the average optical densities of the 3 CD1 used in each group.
  • CD1 were periodically inoculated and harvested periodically according to the protocol described in table 2; (a) Results obtained with plates containing the recombinant CPI 2 antigen; b) Results obtained with plates containing the recombinant antigen HCP12; C - Immunoblottings performed with a nitrocellulose membrane containing the recombinant antigen FCP12. FC- Nitrocellulose membrane containing Schwartz starch stained FCP12 antigen. PM - molecular weights. Sera from the post 8 to IP collection of the negative group (g, hei), CP12 (d, eef) and HCP12 (a, bec) inoculated group diluted 1/1000 were incubated with a NC strip containing FCP12 ON antigen at 4 ° C.
  • FIG. 5 Results of the demonstrations performed with the construction containing the BG fragment.
  • Wells Fi F 2 - BG fractions 1, 2 collected from Ni-NTA column;
  • Wells F 4 F 5 - HBG fractions 1, 2 collected from Ni-NTA column;
  • B - EUS Optical Density Results Those performed with sera from HBG-inoculated CD1 mice (HBG group) and untreated CD1 mice (NEG group). The values represent the average optical densities of the 3 CD1 used.
  • CD1 were periodically inoculated and harvested periodically according to the protocol described in Table 2.
  • Figure 6 Results of the demonstrations performed with the construct containing the Ent fragment.
  • A. Results of EL1SA optical densities performed with sera from HEnt-inoculated CD1 mice (HEnt group) and untreated CD1 mice (NEG group). The values represent the average optical densities of the 3 CD1 used. CD1 were inoculated periodically and harvested periodically according to the protocol described in Table 2.
  • Figure 7 Results of the demonstrations performed with the construction containing the Pfsp fragment.
  • A- Results of EL1SA optical densities performed with sera from HPsp-inoculated C1 mice (HPfsp group) and untreated CD1 mice (NEG group). The values represent the average optical densities of the 3 CD1 used. CD1 were inoculated periodically and harvested periodically according to the protocol described in Table 2.
  • B - Immunoblottings performed with a nitrocellulose membrane containing the recombinant FPfsp antigen.
  • Serum pools from the negative (c), HPfsp (def) inoculated group, post 6 to IP (d) and 14 day post 7 to IP (f) diluted 1/200 cultures were incubated with a NC containing the FPfsp ON antigen at 4 ° C. a and b) immunoblotings performed with negative rabbit sera (a) and immunized against F (b) antigen diluted 1/100. As a conjugate G-HRP protein diluted 1/1000 was used and developed with 4-chloro-naphthol.
  • FIG. 8 Results of the demonstrations performed with the construct containing the IL5 fragment.
  • A- Results of optical densities of ELISAs performed on sera from HIL5 (HIL5 group) inoculated CD1 and untreated CD1 mice (NEG group). The values represent the average optical densities of the 3 CD1 used.
  • CD1 were periodically inoculated and harvested periodically according to the protocol described in Table 2.
  • B-Immunoblottings performed with a nitrocellulose membrane containing recombinant FIL5 antigen.
  • PM molecular weights.
  • CDl were inoculated periodically and harvested periodically according to the protocol described in Table 2.
  • B Immunoblottings performed with a nitrocellulose membrane containing the recombinant CD4 antigen.
  • PM molecular weights.
  • Serum pools of the negative (a) and HCD4 (b) inoculated group from day 14 post 7 to IP diluted 1/500 were incubated with a NC strip containing the 4 ° CD4 ON antigen ⁇ .
  • As a conjugate G-HRP protein diluted 1/1000 was used and stained with
  • FIG. 11 Results of the demonstrations performed with the construction containing the PAL fragment.
  • A- Results of the optical densities of ELISAs performed with sera from HPAL (HPAL group) inoculated CD1 and untreated CD1 mice (NEG group). The values represent the average optical densities of the 3 CDl used. CDl were inoculated periodically and harvested periodically according to the protocol described in Table 2.
  • B - immunoblottings performed with a nitrocellulose membrane containing the recombinant HPAL antigen.
  • FIG. 12 Results of the demonstrations performed with the construction containing the LEC fragment.
  • A- Result of the optical densities of ELISAs performed with sera from HLEC-inoculated CD1 mice (HLEC group) and CD1 if any treatment (NEG group). The values represent the average optical densities of the 3 CDl used. CDl were inoculated periodically and harvested periodically according to the protocol described in Table 2.
  • B - Immunoblottings performed with a nitrocellulose membrane containing the recombinant HLEC antigen.
  • PM molecular weights.
  • the present invention relates to fusion proteins comprising in their
  • H Hepatic fasciola - hereinafter referred to as H (Helminth) fragments (SEQ ID NO 2) or similar sequences preferably having at least 90 to 95% homology. preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% similarity; Protein fragments or unrelated proteins allow a significant increase in the immunogenicity levels of these polypeptides.
  • a first aspect of the present invention relates to a fusion protein comprising an amino acid sequence of the H fragment structure followed by an amino acid sequence structurally similar to an unrelated protein fragment or protein.
  • Another aspect of the invention relates to an expression vector comprising a polynucleotide sequence encoding said fusion protein.
  • Still another aspect of the invention relates to the preparation of antigens for injection into an animal likely to develop an immune response.
  • the present invention also relates to a method of administering the
  • the invention further relates to the use of said fusion protein for the production of polyclonal antibodies specific to the added protein or protein fragments.
  • the present invention relates to antigens fused to amino acid sequences in secreted calcium-binding proteins secreted by the adult Fasciola hepatic worm, namely the protein designated Fh8 or fasciolin (Genbank number AF213970).
  • This strategy allows for increased immunogenicity levels of antigens that are fused to H fragments, and such increased immunogenicity allows for a gain in inducing an immune response by individuals to whom it is administered.
  • This system significantly increases the immunogenicity levels of the antigen of interest by allowing the individual to whom it is administered to develop a more intense immune response, namely in the production of specific antibody titers against the antigen of interest. This process enables the use of low antigen
  • the immunological characteristics of the antigen resulting from the addition of the H fragment with the antigen of interest allow the development of a specific response against the antigen of interest without the presence of a significant response against the H fragment.
  • the immune response occurs following injection of the immunogen without another one. additive, namely the presence of another adjuvant, the antigen administered may be denatured or not.
  • Results supporting this invention relate to demonstrations using the 11 amino acid amino acid fragment of the Fh8 protein to increase the immunogenicity of unrelated proteins or protein fragments that are used as an example. These procedures are. however, potentially extendable to any polypeptide.
  • Results refer to examples using Fh8 fragment H (SEQ ID NOs 1 and 2).
  • H fragments corresponding to the sequence of the Fh8 N-terminal fragment specifically the addition of SEQ ID NO 2 polypeptide by molecular biology procedures prior to the sequence corresponding to the polypeptide intended for use as an immunogen.
  • This construct can be accomplished by including this sequence by molecular biology techniques, namely by using suitable restriction enzymes and by adding before and after the fragment sequence such restriction sites, methodology used in the demonstrations performed, or by other processes such as the addition of DNA fragments with the sequence of interest (linkers) to a PCR product, or other applicable strategies.
  • the small amplitude of the H fragment allows the use of a wide variety of strategies for fusion with the polypeptide of interest.
  • F8 sequence insertion process can be performed using molecular biology techniques, notably through the use of restriction enzymes, which is used in the demonstration processes.
  • the invention has been applied to various fragments and proteins with different immunogenic characteristics, from proteins or fragments described as poorly immunogenic such as CWG, CD4, or fragments which by their characteristics, notably their molecular mass, would be poorly immunogenic such as IL5, Pfsp and Ent. as well as proteins described as being very immunogenic such as P ⁇ L, moderately immunogenic antigens such as CP12 and ECF, and other targets with unknown characteristics such as BG and Toxo.
  • Several different protocols were also applied throughout the experiments varying in the protein concentration of the administrations given to the mice and the time periods between them. These various protocols have demonstrated the versatility of the invention. The possibility of administering the antigen under conditions
  • mice were used as experimental models and the antigens were intraperitoneally administered.
  • Antibody production for CWP, IL5, Ent, Toxo, BG and CPI 2 targets was also evaluated in rabbits using subcutaneous administrations with similar results (not shown).
  • Antigens were produced and isolated under denaturing conditions under the same conditions using NiNTA agarose resin (QIAGEN). Anligens were prepared for immunization after PBS dialysis and 0.22 ⁇ filter filtration sterilization.
  • CWP Cyst wall protein protein of the Giardia lamblia cyst.
  • the original sequence is 1089 bp and the region to be amplified is from 527 bp to 931 bp (GenBank Accession No. XM__001710190).
  • the fragment was PCR amplified and subcloned into the pQE (CWP) vector. Constructs containing fragment H followed by CWP (HCWP), and containing the Fh8 sequence fused to the CWP sequence (FhSCWP) were also prepared.
  • Inocula with the same amount of protein (50 pg) were administered at the periodicity as shown in Table 2.
  • Blottings performed using the FhSCWG antigen confirm the results of EUS As and demonstrate the specificity of the antibodies produced.
  • the tests of Immunofluorescence with the parasite demonstrates that the antibodies produced recognize the native protein in the wall of this structure.
  • CP12 is a surface protein of Cryptosporidhon parvum.
  • the CP12 fragment (GenBank No. XM_625821) used in this work is 213 bp and corresponds to the nucleotide sequence of the CP 12 protein without its transmembrane domain.
  • the fragment was PCR amplified and subcloned into the pQE vector (CP 12), constructs containing fragment H followed by CPI 2 (HCP12) were also prepared.
  • the antigens were produced and isolated under
  • Immunofluorescence with the parasite demonstrates that the antibodies produced recognize the native protein in the wall of this structure.
  • the fragment amplified during the work has a size of 163 bp encoding a 5 kDa protein and is comprised of the 291 bp-453 bp region of a gene with a size of 456 bp (GenBank Accession No. XM_645825) of Entamoeba histolytica cyst wall specifw glycoprotein Jacob.
  • Inocula with the same amount of protein (50 ⁇ g) were administered with the Table 2. Results showed a significant increase after 4 to D ?.
  • Blottings performed with FhSEnt protein confirm the specificity of the antibodies produced. Antibody titers are maintained even 90 days after the last inoculation performed. Immunofluorescence assays with the parasite demonstrate that the antibodies produced recognize the native protein in the wall of this structure.
  • Human interleukin 5 is a hematopoietic growth factor. having an 816 bp nucleotide sequence encoding 134 amino acids (GenBank No. BC069137.1).
  • the IL5 fragment used for evaluation corresponds to a small part of 1L5. consisting of 144 bp corresponding to a 5 'end exon of IL5 encoding 48 amino acids.
  • Inocula with the same amount of protein (20 pg) were administered with the periocity described in Table 2. Results showed a significant increase after 4 to PI. Blottings performed with FhSILS protein confirm the specificity of the antibodies produced.
  • the Toxo protein is an oocyst wall protein of Toxoplasma gondii with 1846 bp encoding 499 amino acids (GenBank Accession No to EU851867.1).
  • the Toxo fragment corresponds to the 2nd. comprised between bp 2875 and 3238.
  • Inocula were administered with the same amount of protein (20 pg) with periodicity is described in Table 2. The results showed a significant increase after 4 to IP. Toxo protein blottings confirm the specificity of the antibodies produced.
  • the CD4 protein is a Dicentrarchus labrax lymphocyte wall receptor (GenBank No. AMB849812.1).
  • the CD4 fragment corresponds to two domains of this receptor comprised between 193 and 734 bp.
  • Inocula were administered with 30 pg with the periodicity described in Table 2. The results showed a significant increase after 4 to IP.
  • CD4 protein blottings confirm the specificity of the antibodies produced.
  • PAL protein is a wall protein of this bacterium (GenBank No. YP001250824). THE PAL fragment corresponds to the complete protein. They were administered with 30 pg inocula with the periocity described in Table 2. Results showed a significant increase after 2 ⁇ . PAL protein blottings confirm the specificity of the antibodies produced.
  • Escherichia coli XL1 5 / 21 ⁇ 2 (Stratagene) and Escherichia coli Ml 5 [pREP4 (QIAGEN) strains were used for the cloning of pGEM-T Easy plasmids (Promega) and pQE30 (QIAGEN) plasmids, respectively.
  • Plasmid DNA was isolated and purified by the Wizard® Plus SV Kit.
  • PCR polymerase chain
  • the pQE30 vector containing the Fh8 polypeptide coding gene was used as the PCR reaction template (Castro, 2001; Silva et al, 2004).
  • the PCR reaction began with a 1 minute denaturation step at 95 ° C, followed by 30 cycles of amplification, 45 seconds of denaturation at 94 ° C, 30 seconds of pairing at 50 ° C and 45 seconds of polymerization at 100 ° C. 72 ° C.
  • GTGTTCAA-3 respondent * Toxo_KpnI H fragment cycles: 5-TG amplification (30 sec digested with Sac I and ATGCGCGGT 95 ° C, 30 sec 55 ° C and PCR product ACCCTAGGG 45 sec 72 T) .7 min. corresponding to A ACGAC-3 'at 72 ° C
  • the thermal cycler used for all PCR reactions was My Cycler TM Thermal Cycler (BioRad).
  • the PCR reaction mix was composed of 1 ⁇ L of sample.
  • PCR pellets obtained from PCR were eluted in the pGEM vector and after digestion with restriction enzymes Saci and Kpnl were subcloned into vector pQE30, pQE30 containing fragment H (pQEH) or pQE30 containing fragment Fh8 (pQEF), digested with Saci and Kpnl,
  • Binding reaction to pGEM-T Easy vector was by mixing 3 ⁇ L of DNA sample (PCR product or restriction enzyme digestions) with .1 ⁇ L of pGEM-T Easy vector (Promega ), 5 ⁇ L DNA ligase 2X enzyme buffer (Promega) and 1 ⁇ L DNA T4 Ligase enzyme (Promega), making a final volume of 10 ⁇ L. This reaction occurred at room temperature overnight or overnight. 1 hour and 30 minutes at 37 ° C.
  • E. coli XL1 Blue was transformed with the binding product.
  • the cells were then spread on LB /
  • the inserts resulting from restriction enzyme digestions performed in this work were inserted into the pQE vector.
  • pQEH or pQEFhS by mixing 6 ⁇ L of insert with 2 ⁇ L of pQE vector, 1 ⁇ L of 10X ligase buffer (Promega) and 1L of DNA T4 Ligase enzyme (Promega). This reaction occurred at room temperature overnight or for 1 hour and 30 minutes at 37 ° C.
  • Kpnl digestion was performed by mixing 26 L DNA, 3 ⁇ L J buffer (Promega) and 1 ⁇ L Kpnl (Promega), making a final volume of 30 ⁇ L.
  • L was digested in agarose gel and the resulting 20 ⁇ L was digested with BamHI, in which 2 ⁇ L of 10X K buffer (Promega) and 1 ⁇ L of BamHI (Promega) were mixed.
  • the result of digestion was visualized on appropriate percentage (w / v) agarose gel.
  • a 200 ml preculture was prepared and grown overnight at 37 ° C with stirring and 2 liters of culture induced by placing 100 ml saturated culture and 900 ml LB medium containing 100 pg. / ml Ampicillin, 50 pg / ml Kanamycin and 1 ⁇ M IPTG. After 5 hours incubation cells were harvested by centrifuging 20 minutes at 4000 rpm at 4 ° C. Cell lysis was performed by incubating the cells with 40 mL of 8 M urea buffer, pH 8.0, and stirring overnight. The extract was centrifuged at 13,000 rpm for 15 minutes at room temperature and the supernatant was collected. After recovery of the supernatant. It was filtered through a glass wool column and applied to the NIMTA column (Amersham Biosciences), pre-equilibrated with 8M urea, pH 8.0.
  • Protein quantification was performed by the Bradford method, with the Protein Assay reagent (BioRad) diluted 1: 5 and the optical density read at a wavelength of 595 nm.
  • the calibration curve was obtained by reading the optical density at 595 nm of known bovine serum albumin (BSA) concentration solutions with this reagent.
  • BSA bovine serum albumin
  • the amount of protein administered varied between the various examples, from 10 to 50 pg as described above for each case.
  • Recombinant HLEC protein was prepared in 8M urea and recombinant antigen was concentrated and buffer replaced with 50mM Urea prepared PBS containing pyrogen-free water using centricon ( ⁇ micon) with 3 Kda cut-off membrane. Inocula were prepared extemporaneously by diluting the concentrated protein in the appropriate volume of sterile, pyrogen-free PBS to ensure that the urea concentration was less than 10 mM, and the inoculum was filtered through a 0.2 ⁇ pyrogen-free filter. .
  • Each group is made up of 3 mice and inoculations were periodically administered according to the protocols described in table 2 via the mtraperitoneal route and blood was collected periodically at the tail according to the protocols described in table 2.
  • Tris-Tricine gels used to analyze the collected fractions were based on Schagger's Tris-Tricine systems. H. and Jagow. G. (1987) and Laemmli's SDS-PAGE (1970). Thus, the adopted system consisted of two gels: a 15% resolvent gel and a 4% packaging gel.
  • the resolving gel contained 3.3 mL of 30% acrylamide, 2,205 mL of gel buffer, 705 ⁇ L glycerol. 367.5 ⁇ L of water. 150 ⁇ L 10% PSA4 and 9 ⁇ L TEMED.
  • the packaging gel contained 700 ⁇ L 30% acrylamide, 1.25 mL gel buffer, 3 mL water, 200 ⁇ L 10% PSA and 5 ⁇ L TEMED.
  • transfer buffer 25 mM Tris, 0.2 M glycine, 100 ml methanol
  • 2 filter papers 25 mM Tris, 0.2 M glycine, 100 ml methanol
  • nitrocellulose membrane nitrocellulose membrane
  • SDS-PAGE gel SDS-PAGE gel on which proteins were run
  • sponges needed to assemble the sandwich '.
  • the nitrocellulose membrane (0.45 mm, Schleicher & Schell) was saturated with 5% PBS-milk for 1h at room temperature.
  • the 2X membrane was washed with 0.3% PBS-Tween (PBS-T).
  • the membrane was incubated with the assay serum diluted in PBS-milk at the appropriate concentration overnight at 4 ° C.
  • the 3X membrane was washed with PBS-T.
  • Protein G-peroxidase (Bio Rad) diluted 1/1000 in PBS-milk was added. Incubated at room temperature 2h.
  • the membrane was washed 3X in PBS-T and revealed with 15 mg 4-chloro-l-naphthol dissolved in 5 ml cold methanol, 20 ml PBS and 1 m 25 H, O to 30%.
  • Optical density was read at 490 nm on a model 680 ELISA plate reader (Biorad).
  • the parasite sample was placed in each well of the immunofluorescence slide and allowed to dry in the oven until the pellet crystallized.
  • Immunological reactions in the presence of adjuvants had been previously evaluated, such as CWG and CD4 fragments which had been shown to be poorly immunogenic, CPI 2 and LEC fragments which had been shown to have intermediate immunogenic characteristics, or PAL which had been shown to be a very antigen.
  • immunogenic, or further fragments such as the Ent, IL5 and Pfsp fragments, whose biochemical characteristics, namely protein family, molecular mass and amino acid sequence determined that they would be poorly immunogenic.
  • fragments like Toxo and BG were also used, whose immunological characteristics were totally unknown,
  • FCWG was blotted as antigen. Localization of the recombinant FCWG protein as well as possible polymers was performed with the anti-Fh8 specific immunosorbent (Figure 3D.i) diluted 1/100 which allows the visualization of FCWG polymers. Ensure that. using serum pools diluted 1/200 and, the negative groups, and CWG HCWG crop nine days after IP 5 and IP 6 after the pre-emergence corresponding to the FCWG. Immunoblottings at the same dilutions were performed using the Fh8 antigen (for evaluation of anti-H fragment antibody production) and no precipitate appeared. These results show that antibodies raised by the HCWG group are specific to the CWG fragment with no significant cross reactions with E, coli antigens and no presence of anti-H fragment Ig.
  • group HCP12 being visible in group CP12 the appearance of anti-CP12 Ig from 6 to IP.
  • anti-CP12 Ig titers evolve throughout the experiment.
  • the increased immunogenicity can be observed by the existence of an earlier immune response with higher titers.
  • BG protein The immunological characteristics of this fragment were unknown. After obtaining the pQEHBG constructs, the respective denaturing recombinant antigens were produced and analyzed ( Figure 5A).
  • BG was blotted. There is, using the sera from day 7 post harvest IP diluted to 1/1000, and the negative groups HBG the appearance of precipitates corresponding BG not observed the presence of significant cross-react with E. coli antigens. [132] In order to assess whether the produced Ig were capable of recognizing the native protein in the Giardia trophozoite wall,
  • Protein fragment Ent Due to the low molecular weight of the polypeptide (7Kda) and because it represents only part of a protein, this fragment had characteristics associated with low immunogenicity.
  • Pfsp protein fragment Due to the low molecular weight of the polypeptide (7 Kda) and because it represents only part of a protein, this fragment had characteristics associated with low immunogenicity.
  • IL5 protein fragment Due to the low molecular weight of the polypeptide (7 Kda) and the fact that it represents only a part of a protein with high homology to mouse IL 5 and notoriously low immunogen, this fragment had characteristics associated with low immunogenicity.
  • CD1 mouse inoculations with about 20 pg of HIL5 (Table 2).
  • Assessment of the presence of specific immune response was performed by ELISA with plates containing the HIL5 antigen ( Figure 8A).
  • Antibody titres increase with inoculations over the period evaluated.
  • Toxo Protein The immunological characteristics of this fragment were unknown. After obtaining the pQEHToxo constructions, we proceeded to the production and analysis of the respective recombinant antigens.
  • Toxo was blotted as antigen. It can be seen using sera, day 4 post harvest and IP diluted 1/1000 HToxo group of the appearance of precipitates corresponding Toxo recombinant protein was not observed the presence of significant cross-react with E. coli antigens.
  • CD4 fragment This fragment had been shown to be poorly immunogenic. After obtaining the pQEHCD4 constructs, the respective recombinant antigens were produced and analyzed under denaturing conditions. a recombinant antigen of the expected molecular weight has been produced,
  • CD 1 mouse inoculations with 30 ⁇ g of HCD4 antigens (Table 2). Evaluation of the presence of specific immune response was performed by ELISA with plates containing the HCD4 antigen ( Figure 10A). There is the appearance of anti-CD4 antibodies from 4 to inoculation. Antibody titers reach a plateau after 4 IP which remains 82 days after last inoculation.
  • PAL protein This protein had been shown to be very immunogenic.
  • pQEHPAL constructs were obtained by producing and analyzing the respective recombinant antigens.
  • LEC Protein This protein was considered moderately immunogenic yet Due to its haemagglutinating activity, when in its native form, it presented as an added difficulty the need to develop specific antibodies against a denatured structure
  • Liver Fascia ⁇ a heterologous expression and functional characterization of a thioredoxin peroxidase.
  • Cryptosporidium pan tm Identification of a new surface adhesion preparation on sporozoite and oocyst by screening of a phage-display cDNA library. Experimental Parasitology. Doi: 10,101 / j.exp for.2006.09.018,

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