WO2014200325A2 - Consortium of the recombinant influenza a viruses flu-ns1-124- l7/l12-h5n1, flu-ns1-124-omp16-h5n1, flu-ns1-124-l7/l12-h1n1 and flu-ns1- 124-omp16-h1n1, family ortomyxoviridae, genus influenzavirus, expressing brucella immunodominant proteins destined to generate a vaccine against brucellosis - Google Patents

Consortium of the recombinant influenza a viruses flu-ns1-124- l7/l12-h5n1, flu-ns1-124-omp16-h5n1, flu-ns1-124-l7/l12-h1n1 and flu-ns1- 124-omp16-h1n1, family ortomyxoviridae, genus influenzavirus, expressing brucella immunodominant proteins destined to generate a vaccine against brucellosis Download PDF

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WO2014200325A2
WO2014200325A2 PCT/KZ2014/000006 KZ2014000006W WO2014200325A2 WO 2014200325 A2 WO2014200325 A2 WO 2014200325A2 KZ 2014000006 W KZ2014000006 W KZ 2014000006W WO 2014200325 A2 WO2014200325 A2 WO 2014200325A2
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flu
vaccine
brucella
nsl
omp16
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PCT/KZ2014/000006
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WO2014200325A3 (en
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Abylay Rysbayuly SANSYZBAY
Kaisar Kazybayevich TABYNOV
Bolat Amanbayevich YESPEMBETOV
Kulyaisan Turlybayevna Sultankulova
Berik Mikhitovich KHAIRULLIN
Nurlan Tamambayevich Sandybayev
Andrei Yur'yevich YEGOROV
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The Republican Government Enterprise On The Basis Of Economic Control Rights "Research Institute For Biological Safety Problems" Of The Sience Committee Of The Ministery Of Education And Science Of The Republic Of Kazakhstan
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Publication of WO2014200325A2 publication Critical patent/WO2014200325A2/en
Publication of WO2014200325A3 publication Critical patent/WO2014200325A3/en

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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
    • A61K39/02—Bacterial antigens
    • A61K39/098—Brucella
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins

Definitions

  • the present invention relates to the fields of biotechnology, veterinary and public health, and describes a recombinant influenza viral vector vaccine for the prevention of brucellosis.
  • B. abortus is an intracellular pathogen
  • the main criterion for new candidate vaccines is their ability to elicit a cellular immune response in animals. It is well recognized that the two key components of the protective reaction in infected animals are the formation of Thl CD4 + lymphocytes secreting interferon-gamma (IFN- ⁇ ), a critical cytokine which is required to regulate the anti-brucellosis activity of macrophages [Jiang X. et al., Infect Immun. (61) 1993, 124-134], and CD8 + T lymphocytes that lyse Bruce lla-infected cells [Oliveira S.C. et al., Eur J Immunol. (25) 1995, 2551-2557].
  • IFN- ⁇ interferon-gamma
  • the closest vaccine to the claimed invention is a recombinant Semliki Forest virus (SFV) which can induce expression of the B. abortus proteins Cu-Zn superoxide dismutase (Cu-Zn SOD) or translation initiation factor 3 (TIF3) in vivo in mice after immunization via the intraperitoneal route.
  • SFV Semliki Forest virus
  • Cu-Zn SOD Cu-Zn superoxide dismutase
  • TIF3 translation initiation factor 3
  • the essence of this invention is that reverse genetics methods have been employed to construct novel, genetically-stable, safe and protective vaccines against Brucella abortus.
  • the recombinant influenza viral vectors Flu-NSl-124-L7/L12-H5Nl, Flu-NSl-124-Om l6-H5Nl, Flu-NS l -124-L7/L12-HlNl and Flu-NS l-124-Ompl6-HlNl , which are based on the influenza A viral strain subtypes H5N1 and H1N1, were modified by inserting the genetic sequences of the Brucella protein L7/L12 (ribosomal) or Om l6 (outer membrane) into the viral NS1 gene, enabling the recombinant influenza viral vectors to express these proteins in vivo in the cells of organisms that they infect.
  • L7/L12 ribosomal protein and Ompl6 are immunodominant B. abortus proteins that elicit a cellular immune response (Thl and CD8 + T cells) [Oliveira S.C. and Splitter G.A. Vaccine 14 (1996), 959-962; Pasquevich K.A. et al., Infect. Immun. 77 (2009), 436-445; Mallick A.I. et al., Vaccine 25 (2007), 3692-3704; Luo D. et al, Infect. Immun. 74 (2006), 2734-2741], A large body of data [Campbell C.H. et al., J. Infect. Dis.
  • influenza A virus contains a segmented genome consisting of eight negative-strand RNA fragments.
  • NS viral nonstructural protein
  • Nep nuclear export protein
  • H1N1 The A/Puerto Rico/8/34 (H1N1) strain was used as the backbone for obtaining influenza A virus vectors expressing Brucella L7/L12 or Ompl6 sequences in a form of fusion proteins with N- terminal 124 amino acid residues of NS1.
  • influenza viral vectors were constructed for the purpose of specific prophylaxis of brucellosis (Flu-NS 1-124-L7/L12-H5N1, Flu-NS l-124-Ompl 6-H5Nl, Flu-NS 1 - 124-L7/L 12-H 1 N 1 and Flu-NS 1 - 124-Omp 16-H IN 1 ; influenza A viral vectors of the subtypes H5N1 and H1N1) containing the genetic sequence of the Brucella proteins L7/L12 or Om l6 inserted into the viral NS1 gene.
  • the recombinant influenza viral vectors display good reproductive characteristics in chicken embryos despite possessing a truncated NS1 gene, are genetically stable over five consecutive passages in chicken embryos, and can express the fusion proteins NS1-L7/L12 or NSl-Om l6 in both infected chicken embryos and in vivo in the cells of organisms which they infect.
  • these vaccines in cattle induced a strong antigen-specific T-cell immune response, as indicated by a high number of CD4 + and CD8 + cells, as well as the concentration of IFN- ⁇ , and most importantly provided a high level of protectiveness comparable to the commercial B. abortus SI 9 vaccine and superior to the B. abortus S19 vaccine in combination with Montanide GelOl adjuvant [Tabynov K. et al., Vaccine (2014) DOI:10.1016/j.vaccine.2014.02.058]. Furthermore it is shown that the developed vaccines can effectively differentiate vaccinated animals from infected animals.
  • FIG. 1 Schematic representation of the influenza virus N57 gene (A) and recombinant chimeric NS1 genes of recombinant influenza A viral vectors of the subtypes ⁇ 5 ⁇ 1 and ⁇ 1 ⁇ 1 containing the genetic sequences of the Brucella proteins L7/L12 or Ompl6 (B)
  • Figure 2 Genetic stability of the viral constructs after five passages in chicken embryos (CE), as determined by RT-PCR (A) and confirmation of expression of the Brucella proteins L7/L12 or Ompl6 by the viral constructs using Western blotting (B).
  • Figure 1A 1) Flu-NSl- 124-Ompl6-H5Nl ; 2) pHW plasmid encoding the NSl-124-Ompl6 genes; 3) Flu-NSl-124- Ompl 6-HlNl ; 4) Plasmid NSl-124-Ompl6; 5) Flu-NSl-124-L7/L12-H5Nl ; 6) Plasmid NS1- 124- L7/L12; 7) Flu-NSl-124-L7/L12-HlNl ; 8) Plasmid NS 1-124- L7/L12.
  • Figure IB 1) lysed allantoic fluid (AF) of uninfected CE; 2) lysed AF of CE infected with Flu-NSl-124-Ompl6- H5N1 ; 3) lysed AF of CE infected with Flu-NSl-124-Ompl6-HlNl; 4) lysed AF of uninfected CE; 5) lysed AF of CE infected with Flu-NS l-124- L7/L12-H5N1 ; 6) lysed AF of CE infected with Flu-NSl-124- L7/L12-H5N1
  • FIG. 4 Rectal temperature in cattle after immunization. Vaccination of cattle was carried out twice with an interval of 28 days with viral constructs vaccine formulation only (L7/L12- Ompl6) or a combination thereof with adjuvants (L7/L12-Om l6-Montanide GelOl or L7/L12- Ompl6-chitosan) or a single with commercial vaccine B. abortus SI 9. The animals of the negative control group as an inoculum were administered with PBS.
  • FIG. 5 Titers of IgG, IgGl and IgG2a antibodies against Brucella L7 L12 and Ompl6 proteins in cattle on day 28 (A) and 56 (B) after the initial immunization. Antibodies were determined in ELISA. Vaccination of cattle was carried out twice with an interval of 28 days with viral constructs vaccine formulation only (L7/L12-Ompl6) or a combination thereof with adjuvants (L7/L12-Ompl 6-Montanide GelOl or L7/L12-Ompl6-chitosan) or a single with commercial vaccine B abortus S19. The animals of the negative control group as an inoculum were administered with PBS.
  • Figure 6 Number of CD4 + , CD8 + and the concentration of IFN- ⁇ (in the cell supernatant) in samples of isolated lymphocytes wich were stimulated with Brucella L7/L7 and Ompl6 proteins on a day 28 (A) and 56 (B) after the initial vaccination of cattle.
  • Vaccination of cattle was carried out twice with an interval of 28 days with viral constructs vaccine formulation only (L7/L12-Ompl6) or a combination thereof with adjuvants (L7/L12-Ompl6-Montanide GelOl or L7/L12-Ompl6-chitosan) or a single with commercial vaccine B.
  • abortus SI 9 9.
  • FIG. 7 Protectiveness of vaccine samples in cattle, estimated by the effectiveness of vaccination (A), the index of infection (B), the amount allocated Brucella from lymph nodes (C), as well as rectal temperature (D) after challenge.
  • Vaccination of cattle was carried out twice with an interval of 28 days with viral constructs vaccine formulation only (L7/L12-Ompl6) or a combination thereof with adjuvants (L7/L12-Om l6-Montanide GelOl or L7/L12-Ompl6- chitosan) or a single with commercial vaccine B.
  • abortus SI 9 The animals of the negative control group as an inoculum were administered with PBS. The challenge of cattle (5 animals in each group) was performed with virulent B.
  • Vero cells were co-transfected by LonzaNucleofectorTM (Cologne, Germany) technique with 0.5 ⁇ g ⁇ l of plasmids encoding the PB1, PB2, PA, NP, M genes and NS (chimeric) gene of A/Puerto Rico/8/34 (H1N1) virus; and the HA and NA genes of A/chicken/Astana/6/05 (H5N1) or A/New Caledonia/20/99 (H1N1) strains.
  • the HA protein sequence of the H5 virus was attenuated by means of exchanging its polybasic cleavage site to one containing a trypsin-dependent sequence.
  • the NS genes were modified to express NS1 fusion proteins containing a sequence of 124 N-terminal amino acids from the NS1 protein coupled with a sequences of B. abortus derived proteins: L7/L12 (GenBank: AAA19863.1) or Om l6 (GenBank: AAA59360.1), ended with double stop codon. Brucella sequences were obtained synthetically.
  • the supernatants of transfected cells were used for inoculation into 10- day-old embryonated chicken eggs (CE; Lohmann Tierzucht GmbH, Cuxhaven, Germany]) which was incubated at 34 °C for 48 hours.
  • Vaccine batches were produced in CE after three egg passages of viral constructs.
  • FIG. 1 A and B Schematic representations of the influenza virus NSl gene and the chimeric recombinant NS7 gene in the recombinant influenza A viral vectors of the subtypes H5N1 and H1N1 containing the genetic sequences of the Brucella L7/L12 or Ompl6 antigens are shown in Fig. 1 A and B.
  • Example 2 The genetic stability of recombinant strains of influenza A virus subtypes H5N1 and
  • the NS -fusion protein encoding genes of the viral constructs were sequenced at passages 1, 3 and 5 using the Sanger method with the commercial kit Prism BigDyeTM Terminator v3.1 (ABI, Foster City, CA, USA) on a automatic 16-capillary sequencer Genetic Analyser 3130 xl (ABI). All of the viral constructs replicated well in CE. It should be noted that during the initial passages in CE, the viral constructs had low infection and hemagglutination titers; however, as the number of passages increased, the titers also increased (Table 1). By the fifth passage, the infectious titers of the viral constructs ranged from 7.95 ⁇ 0.22 to 9.2 ⁇ 0.14 lg EID 50 /ml.
  • Vaccine samples were prepared from the viral constructs Flu-NS 1 - 124-L7/L 12-H5N 1 , Flu- NSl-124-Ompl6-H5Nl, Flu-NSl-124-L7/L12-HlNl and Flu-NS l-124-Ompl6-HlNl, which accumulated in 10-day-old CE at 34 °C for 48 h.
  • the obtained allantoic suspensions of viral constructs with the same antigenic structure (H5N1 or H1N1) were combined in a single pool in a 1 :1 ratio to obtain the bivalent vaccine formulation.
  • the resulting mixtures of viral constructs (L7/L12-Ompl6) were combined with adjuvants such as Montanide GelOl (L7/L12- Ompl 6-MontanideGel01 ; Seppic, France) in a 80:20 ratio by volume (according to the manufacturer's recommendations) or chitosan oligosaccharide lactate (L7/L12-Ompl6-chitosan; Sigma- Aldrich) in a final concentration of 0.05%, and the mixtures were stirred using a magnetic stirrer for 5-7 min.
  • the prepared vaccine samples were stored at 2-8 °C until use.
  • Cattle in the positive control group were immunized once subcutaneously in the neck region (right side) with a commercial vaccine B. abortus SI 9 (Shchelkovsky Biokombinat, Russia) at a dose of 80 x 10 9 CFU/animal (according to the manufacturer's instructions).
  • Cattle in the negative control group were administered subcutaneously with 2.0 ml of PBS.
  • blood samples were taken on days 0, 7, 14, 28, 35, 42, 56 post-IV from the jugular vein (serum and whole blood using Vacutainer tubes; Becton Dickinson, USA) for hematological (hemoglobin concentration, hematocrit, red blood cells, white blood cells, platelets, stab and segmented neutrophils, eosinophils, lymphocytes, and monocytes) and biochemical (total bilirubin, direct bilirubin, creatinine, cholesterol, total protein, urea, glucose, aspartate aminotransferase, and alanine aminotransferase) studies.
  • hematological hemoglobin concentration, hematocrit, red blood cells, white blood cells, platelets, stab and segmented neutrophils, eosinophils, lymphocytes, and monocytes
  • biochemical total bilirubin, direct bilirubin, creatinine, cholesterol, total protein, urea, glucose, aspart
  • the immunogenicity of the experimental and control vaccines was evaluated by assessing the presence of a humoral (IgG, IgGl, IgG2a) and T cell immune response (CD4 + , CD8 + , IFN- ⁇ ) in the vaccinated cattle at 28 and 56 days after IV; blood serum (10 ml per Becton Dickinson Vacutainer tube) and whole blood (heparinized tubes [100 U/ml] in a volume of 50-70 ml) samples were collected from the vaccinated cattle. 6.1 Antibody response to the Brucella L7/L12 and Ompl6 proteins in vaccinated cattle
  • the concentration of IFN- ⁇ was also determined. As shown in Fig. 6, significant (P ⁇ 0.0001) accumulation of IFN- ⁇ after stimulation with Brucella L7/L12 and Ompl 6 proteins was observed in the samples from the animals vaccinated with the viral constructs vaccine formulation only, as well as its combination with Montanide GelOl, or the B. abortus SI 9 vaccine as compared to the control samples (without stimulation). Significant accumulation of IFN- ⁇ was not observed in the samples from the group of animals vaccinated with L7/L12-Om l6-chitosan.
  • lymph nodes submandibular, retropharyngeal, right subscapular, left subscapular, right inguinal, left inguinal, mediastinal, bronchial, portal, para-aortic, pelvic, udder, mesenteric
  • parenchymal organs liver, kidney, spleen and bone marrow.
  • 17 organs were sampled from each animal. The organs were plated onto TSA plates and incubated at 37 °C for 4 weeks, during which time the growth of bacterial colonies was periodically counted.
  • An animal was considered to be infected if a Brucella colony was detected from the culture of one or more organs.
  • the results of the bacteriological examination were evaluated as the number of animals from which no colonies were isolated (effectiveness of vaccination) and by the index of infection (the number of organs and lymph nodes from which were isolated Brucella).
  • Determination of the number of virulent Brucella in the lymph nodes of the challenged animals was used as an additional indicator to evaluate protective efficacy.
  • the collected retropharyngeal or right subscapular lymph nodes were homogenized in 4 ml of 0.1% Triton-PBS, and 100 ⁇ aliquots of 10-fold serial dilutions were plated in triplicate onto TSA plates, incubated for 14 days at 37 °C, and the number of CFU were counted.
  • the recombinant influenza viral vectors described herein may be ideal vaccine candidates to prevent brucellosis in pigs and humans. It has been experimentally confirmed that recombinant influenza viral vectors in which the viral NS1 gene has been modified are capable of replicating and can subsequently induce immune responses in pigs and humans [Vincent A., et al., Vaccine 25(47) (2007), 7999-8009; Wressnigg ⁇ ., et al., Vaccine 27(21) (2014), 2851-2857; Stukova M., et al., Tuberculosis 86 (3-4) (2006), 236-246]. Moreover, these recombinant influenza viral vectors are completely safe in pigs and humans.
  • influenza viral vectors could be offered for prophylactic immunization of cattle against brucellosis as bivalent vaccine formulations or mixtures of recombinant influenza viral vectors expressing the Brucella L7/L12 and Om l6 proteins.
  • Bivalent vaccine formulations could be prepared by combining allantoic suspensions of two recombinant influenza viral vectors (Flu-NSl-124-L7/L12-H5Nl + Flu-NS l-124-Ompl6- H5N1 or Flu-NSl-124-L7/L12-HlNl + Flu-NSl-124-Ompl6-HlNl) with the same antigenic structure (H5N1 or HlNl) in a l : l ratio.
  • the resulting mixtures of recombinant influenza viral vectors could be mixed with a stabilizing medium (for example, peptone with sucrose), dispensed into ampoules and freeze-dried.
  • a stabilizing medium for example, peptone with sucrose
  • Vaccination of animals via the conjunctival or subcutaneous routes using a cross-immunization scheme is recommended, whereby the animals would receive a prime vaccination with a mixture of recombinant influenza viral vectors of the H5N1 subtype, and then receive a booster vaccination 21 -28 days later with a mixture of recombinant influenza viral vectors of the HlNl subtype.
  • a commercial polymeric adjuvant such as Montanide GelOl (final concentration of 10-20%; Seppic, France) as the solvent.
  • Other immunization schemes could also be tested and employed in different animals to increase the effectiveness of the candidate vaccine.

Abstract

This invention relates to the fields of biotechnology, veterinary science and public health, and describes a vector vaccine which can be used to prevent brucellosis. The essence of the invention is that reverse genetics methods have been used to construct novel, genetically-stable recombinant influenza viral vectors based on the influenza A viral vectors Flu-NSl-124-L7/L12- H5N1, Flu-NSl-124-Omp16-H5Nl, Flu-NSl-124-L7/L12-HlNl and Flu-NSl-124-Omp16- H1N1 (of the subtypes H5N1 and H1N1) that contain the genetic sequences of the Brucella proteins L7/L12 (ribosomal) or Omp16 (outer membrane) inserted into the viral NS1 gene, which express these Brucella proteins in vivo and are safe in cattle. Our studies showed that this vaccine candidate in cattle induced a strong antigen-specific T-cell immune response, and most importantly provided a high level of protectiveness comparable to the commercial B. abortus S19 vaccine and superior to the B. abortus S19 vaccine in combination with Montanide Gel01 adjuvant. Furthermore it is shown that the developed vaccines can effectively differentiate vaccinated animals from infected animals.

Description

DESCRIPTION OF THE INVENTION
CONSORTIUM OF THE RECOMBINANT INFLUENZA A VIRUSES FLU-NS1-124- L7 L12-H5N1, FLU-NS1-124-OMP16-H5N1, FLU-NS1-124-L7 L12-H1N1 AND FLU-NS1- 124-OMP16-H1N1, FAMILY ORTOMYXOVIRIDAE, GENUS INFLUENZAVIRUS, EXPRESSING BRUCELLA IMMUNODOMINANT PROTEINS DESTINED TO GENERATE A VACCINE AGAINST BRUCELLOSIS
FIELD OF THE INVENTION
The present invention relates to the fields of biotechnology, veterinary and public health, and describes a recombinant influenza viral vector vaccine for the prevention of brucellosis. BACKGROUND OF INVENTION
At present, brucellosis among animals (cattle, sheep and goats) is prevented using live attenuated vaccines from the strains B. abortus 19, RB51 or Rev 1. These vaccines possess a high immunogenic effectiveness, but have a number of serious disadvantages, primarily related to their ability to induce abortion in pregnant animals, secretion of the vaccine strain into the milk of vaccinated animals and the difficulty of differentiating between vaccinated animals and infected animals (only a concern for the B. abortus 19 and Rev 1) [Schurig G.G. et al., Vet Microbiol. 90 (2002), 479-496]. Furthermore, B. abortus 19 and RB 51 strains can cause systemic brucellosis in humans [Ashford D.A. et al., Vaccine. 22 (2004), 3435-3439].
Given that B. abortus is an intracellular pathogen, the main criterion for new candidate vaccines is their ability to elicit a cellular immune response in animals. It is well recognized that the two key components of the protective reaction in infected animals are the formation of Thl CD4+ lymphocytes secreting interferon-gamma (IFN-γ), a critical cytokine which is required to regulate the anti-brucellosis activity of macrophages [Jiang X. et al., Infect Immun. (61) 1993, 124-134], and CD8+ T lymphocytes that lyse Bruce lla-infected cells [Oliveira S.C. et al., Eur J Immunol. (25) 1995, 2551-2557].
Attempts by various research groups to elicit effective Thl CD4+ and CD8+ T cell anti- brucellosis immune responses have resulted in the development of subunit (recombinant protein) vaccines [Al-Mariri A. et al., Infect Immun (69) 2001, 4816-4822; Tabatabai L.B. et al., Vaccine (12) 1994, 919-924; Olive a S.C. et al., Vaccine (14) 1996, 959-962; Oliveira S.C. et al., Gene (140) 1994, 137-138; Cassataro J. et al, Infect Immun. (73) 2005, 8079-8088; Pasquevich K.A. et al., Infect Immun. (77) 2009, 436-445; Mallick A.I. et al, Vaccine (25) 2007, 3692-3704] and DNA vaccines [Leclercq S. et al., Vaccine (15) 1997, 1851-1857; Onate A.A. et al., Infect Immun (71) 2003, 4857-4861; Mayfield J.E. et al., Gene (63) 1988, 1-9; Cassataro J. et al, Infect Immun. (73) 2005, 6537-6546; Luo D. et al., Infect Immun. (74) 2006, 2734-2741], however in terms of protective efficacy, subunit and DNA vaccines are still inferior to commercial live attenuated vaccines.
An alternative strategy for the development of safe and effective brucellosis vaccines is the use of live genetically-modified vectors, i.e., non-pathogenic microorganisms (bacteria and viruses) that express a Brucella antigen. To date, Escherichia coli [Harms J.S. et al., J Immune Based The Vaccines. (7) 2009, 1], Salmonella enterica [Zhao Z. et al., Vaccine (27) 2009, 5214- 5219], Ochrobactrum anthropi [He Y. et al, Infect Immun. 70(5) 2002, 2535-2543] and Semliki Forest virus (SFV) [Cabrera A. et al., Immunobiology 214(6) 2009, 467-474] have been used as vectors for expressing Brucella proteins in vivo. It has been shown that the tested bacterial (intracellular) and viral vectors are capable of infecting a wide range of cell types and expressing Brucella antigens within the infected cells. Furthermore, in all cases, Thl CD4+ and CD8+ T-cell anti-brucellosis immune responses were elicited in immunized animals.
The closest vaccine to the claimed invention, total essential indications (a prototype), is a recombinant Semliki Forest virus (SFV) which can induce expression of the B. abortus proteins Cu-Zn superoxide dismutase (Cu-Zn SOD) or translation initiation factor 3 (TIF3) in vivo in mice after immunization via the intraperitoneal route. Despite being replication-defective, this vector can infect a wide range of hosts, and forms pronounced antigen-specific Thl CD4+ and CD8+ T-cell immune responses in immunized mice. Use of this vector with the Cu-Zn SOD and TIF3 Brucella inserts provides comparable protection against infection with B. abortus S2308 in mice compared to a live vaccine based on strain RB51 [Onate A.A. et al., Infection and Immunity. 73 (6) (2005), 3294-3300; Cabrera A. et al., Immunobiology. 214 (6) (2009), 467- 474] . The disadvantage of SFV vector is that there is insufficient information on the safety of this virus in farm animals, which may be grounds for limiting the use of this vector as a vaccine against brucellosis.
BRIEF SUMMARY OF INVENTION
The essence of this invention is that reverse genetics methods have been employed to construct novel, genetically-stable, safe and protective vaccines against Brucella abortus. The recombinant influenza viral vectors Flu-NSl-124-L7/L12-H5Nl, Flu-NSl-124-Om l6-H5Nl, Flu-NS l -124-L7/L12-HlNl and Flu-NS l-124-Ompl6-HlNl , which are based on the influenza A viral strain subtypes H5N1 and H1N1, were modified by inserting the genetic sequences of the Brucella protein L7/L12 (ribosomal) or Om l6 (outer membrane) into the viral NS1 gene, enabling the recombinant influenza viral vectors to express these proteins in vivo in the cells of organisms that they infect. The abovementioned (Background of invention) shortcomings of commercial vaccines explain why vaccination restrictions exist in many countries prone to brucellosis, which has further aggravated the epizootic situation of this disease worldwide. Based on the above, the basis of the present invention is to produce a novel anti-brucellar vaccine candidate with a similar immunogenicity and protectiveness to existing commercial vaccines but lacking their disadvantages.
This problem was solved by employing a novel strategy to develop safe and effective vaccines - the use of live genetically-modified vectors (nonpathogenic microorganisms) expressing Brucella antigens, In view of the positive results obtained using live viral vectors and the practical advantages of the reverse genetics method, which enables genetic manipulation of RNA-containing viruses [Neumann G. et al., Proc. Natl. Acad. Sci. USA 96 (1999), 9345-9350; Pleschka S. et al., J. Virol. 70 (1996), 4188-4192], we propose that recombinant influenza A viruses expressing the Brucella L7/L12 or Ompl6 proteins may potentially represent a novel candidate vector vaccine against brucellosis. According to published data, L7/L12 ribosomal protein and Ompl6 are immunodominant B. abortus proteins that elicit a cellular immune response (Thl and CD8+ T cells) [Oliveira S.C. and Splitter G.A. Vaccine 14 (1996), 959-962; Pasquevich K.A. et al., Infect. Immun. 77 (2009), 436-445; Mallick A.I. et al., Vaccine 25 (2007), 3692-3704; Luo D. et al, Infect. Immun. 74 (2006), 2734-2741], A large body of data [Campbell C.H. et al., J. Infect. Dis. 135 (1977), 678-680; Brown I.H. et al., Vet. Rec. 143 (1998), 637-638; Gunning R.F. et al., Vet. Rec. 145 (1999), 556-557; Graham D.A. et al., Vet. Rec. 150 (2002), 201-204] has confirmed the ability of influenza viruses to infect cattle and elicit a serological reaction and, in some cases clinical disease, which provided the basis for choosing influenza A viruses as the vaccine vector in this study. Thus, the attenuated influenza A viruses selected as the vector should be able to infect cattle and express the recombinant Brucella proteins. The influenza A virus contains a segmented genome consisting of eight negative-strand RNA fragments. Of these, the smallest fragment (NS), encoding two proteins: viral nonstructural protein (NS 1) and nuclear export protein (Nep), is convenient target for genetic manipulation since NS1 is able to tolerate foreign sequences exceeding its own length [Kittel C. et al., Virology 324 (2004), 67-73]. Thus, the ORF of NS 1 was used for inserting Brucella sequences in this study. The A/Puerto Rico/8/34 (H1N1) strain was used as the backbone for obtaining influenza A virus vectors expressing Brucella L7/L12 or Ompl6 sequences in a form of fusion proteins with N- terminal 124 amino acid residues of NS1.
In total, four recombinant influenza viral vectors were constructed for the purpose of specific prophylaxis of brucellosis (Flu-NS 1-124-L7/L12-H5N1, Flu-NS l-124-Ompl 6-H5Nl, Flu-NS 1 - 124-L7/L 12-H 1 N 1 and Flu-NS 1 - 124-Omp 16-H IN 1 ; influenza A viral vectors of the subtypes H5N1 and H1N1) containing the genetic sequence of the Brucella proteins L7/L12 or Om l6 inserted into the viral NS1 gene. Technically, the recombinant influenza viral vectors display good reproductive characteristics in chicken embryos despite possessing a truncated NS1 gene, are genetically stable over five consecutive passages in chicken embryos, and can express the fusion proteins NS1-L7/L12 or NSl-Om l6 in both infected chicken embryos and in vivo in the cells of organisms which they infect. Comprehensive studies showed that all of the viral constructs vaccine formulation, as well as their combination with commercial adjuvants (Montanide GelOl or chitosan), via the conjunctival method using cross prime (influenza virus subtype H5N1) and booster (influenza virus subtype H1N1) vaccination schedules at an interval of 28 days compared to the commercial bacterial vaccine B. abortus SI 9 were completely safe in cattle. The viral constructs vaccine formulations with Montanide GelOl adjuvant promoted formation of IgG antibodies (with a predominance of antibodies of isotype IgG2a) against Brucella L7/L12 and Ompl6 proteins in ELISA. Moreover, these vaccines in cattle induced a strong antigen-specific T-cell immune response, as indicated by a high number of CD4+ and CD8+ cells, as well as the concentration of IFN-γ, and most importantly provided a high level of protectiveness comparable to the commercial B. abortus SI 9 vaccine and superior to the B. abortus S19 vaccine in combination with Montanide GelOl adjuvant [Tabynov K. et al., Vaccine (2014) DOI:10.1016/j.vaccine.2014.02.058]. Furthermore it is shown that the developed vaccines can effectively differentiate vaccinated animals from infected animals.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Schematic representation of the influenza virus N57 gene (A) and recombinant chimeric NS1 genes of recombinant influenza A viral vectors of the subtypes Η5Ν1 and Η1Ν1 containing the genetic sequences of the Brucella proteins L7/L12 or Ompl6 (B)
Figure 2. Genetic stability of the viral constructs after five passages in chicken embryos (CE), as determined by RT-PCR (A) and confirmation of expression of the Brucella proteins L7/L12 or Ompl6 by the viral constructs using Western blotting (B). Figure 1A: 1) Flu-NSl- 124-Ompl6-H5Nl ; 2) pHW plasmid encoding the NSl-124-Ompl6 genes; 3) Flu-NSl-124- Ompl 6-HlNl ; 4) Plasmid NSl-124-Ompl6; 5) Flu-NSl-124-L7/L12-H5Nl ; 6) Plasmid NS1- 124- L7/L12; 7) Flu-NSl-124-L7/L12-HlNl ; 8) Plasmid NS 1-124- L7/L12. Figure IB: 1) lysed allantoic fluid (AF) of uninfected CE; 2) lysed AF of CE infected with Flu-NSl-124-Ompl6- H5N1 ; 3) lysed AF of CE infected with Flu-NSl-124-Ompl6-HlNl; 4) lysed AF of uninfected CE; 5) lysed AF of CE infected with Flu-NS l-124- L7/L12-H5N1 ; 6) lysed AF of CE infected with Flu-NSl-124- L7/L12-H5N1
Figure 3. Amino acid sequences of the recombinant fusion proteins NSl-Om l6 (A) and NS1-L7/L12 (B) expressed by the recombinant influenza A viral vectors
Figure 4. Rectal temperature in cattle after immunization. Vaccination of cattle was carried out twice with an interval of 28 days with viral constructs vaccine formulation only (L7/L12- Ompl6) or a combination thereof with adjuvants (L7/L12-Om l6-Montanide GelOl or L7/L12- Ompl6-chitosan) or a single with commercial vaccine B. abortus SI 9. The animals of the negative control group as an inoculum were administered with PBS.
Figure 5. Titers of IgG, IgGl and IgG2a antibodies against Brucella L7 L12 and Ompl6 proteins in cattle on day 28 (A) and 56 (B) after the initial immunization. Antibodies were determined in ELISA. Vaccination of cattle was carried out twice with an interval of 28 days with viral constructs vaccine formulation only (L7/L12-Ompl6) or a combination thereof with adjuvants (L7/L12-Ompl 6-Montanide GelOl or L7/L12-Ompl6-chitosan) or a single with commercial vaccine B abortus S19. The animals of the negative control group as an inoculum were administered with PBS. The data are presented as geometric mean titer (GMT) ± standard error (SE); * P = 0.04; ** from P = 0.004 to P = 0.005; *** P = 0.0005; **** P < 0.0001; Statistical analysis was performed using a one way ANOVA (Tukey's multiple comparisons test).
Figure 6. Number of CD4+, CD8+ and the concentration of IFN-γ (in the cell supernatant) in samples of isolated lymphocytes wich were stimulated with Brucella L7/L7 and Ompl6 proteins on a day 28 (A) and 56 (B) after the initial vaccination of cattle. Vaccination of cattle was carried out twice with an interval of 28 days with viral constructs vaccine formulation only (L7/L12-Ompl6) or a combination thereof with adjuvants (L7/L12-Ompl6-Montanide GelOl or L7/L12-Ompl6-chitosan) or a single with commercial vaccine B. abortus SI 9. * from P = 0.01 to P = 0.04; ** from P = 0.001 to P = 0.006; *** from P = 0.0002 to P = 0.0006; **** P < 0.0001; c from P = 0.0007 to P = 0.02. Statistical analysis was performed using a one way ANOVA (Tukey's multiple comparisons test or Sidak's multiple comparisons test).
Figure 7. Protectiveness of vaccine samples in cattle, estimated by the effectiveness of vaccination (A), the index of infection (B), the amount allocated Brucella from lymph nodes (C), as well as rectal temperature (D) after challenge. Vaccination of cattle was carried out twice with an interval of 28 days with viral constructs vaccine formulation only (L7/L12-Ompl6) or a combination thereof with adjuvants (L7/L12-Om l6-Montanide GelOl or L7/L12-Ompl6- chitosan) or a single with commercial vaccine B. abortus SI 9. The animals of the negative control group as an inoculum were administered with PBS. The challenge of cattle (5 animals in each group) was performed with virulent B. abortus 544 using a subcutaneous route of administration at a dose of 5 x 10s CFU/animal. * P = 0.02; ** from P = 0.004 to P = 0.008; *** from P = 0.0001 to P = 0.0006. Statistical analysis was performed using a one way ANOVA (Tukey's multiple comparisons test).
DETAILED DESCRIPTION OF THE INVENTION
The following are the most preferred embodiments of the present invention. These embodiments are described for a better understanding of the invention and it is understood that the present invention is not limited to the following description. Therefore, when considering the description presented herein, it is obvious that individuals skilled in this field could modify any component to generate appropriate variants within the scope of the present invention. For a better understanding of the invention, the following examples of its specific execution are provided.
EXAMPLES Example 1
Construction of recombinant strains of influenza A virus subtypes H5N1 and H1N1 expressing Brucella L7/L12 or Ompl6 proteins.
All viruses were generated by a standard reverse genetics method using 8 bidirectional plasmids pHW2000. Briefly, Vero cells were co-transfected by LonzaNucleofector™ (Cologne, Germany) technique with 0.5 μg μl of plasmids encoding the PB1, PB2, PA, NP, M gens and NS (chimeric) gene of A/Puerto Rico/8/34 (H1N1) virus; and the HA and NA genes of A/chicken/Astana/6/05 (H5N1) or A/New Caledonia/20/99 (H1N1) strains. The HA protein sequence of the H5 virus was attenuated by means of exchanging its polybasic cleavage site to one containing a trypsin-dependent sequence. The NS genes were modified to express NS1 fusion proteins containing a sequence of 124 N-terminal amino acids from the NS1 protein coupled with a sequences of B. abortus derived proteins: L7/L12 (GenBank: AAA19863.1) or Om l6 (GenBank: AAA59360.1), ended with double stop codon. Brucella sequences were obtained synthetically. The supernatants of transfected cells were used for inoculation into 10- day-old embryonated chicken eggs (CE; Lohmann Tierzucht GmbH, Cuxhaven, Germany]) which was incubated at 34 °C for 48 hours. Vaccine batches were produced in CE after three egg passages of viral constructs. Four recombinant influenza A viral constructs of the subtypes H5N1 or H1N1 expressing the Brucella proteins L7/L12 or Ompl6 were obtained by a reverse genetics method: Flu-NSl-124-L7/L12-H5Nl, Flu-NSl-124-Ompl6-H5Nl, Flu-NSl-124-L7/L12-HlNl and Flu-NSl-124-Ompl6-HlNl. Schematic representations of the influenza virus NSl gene and the chimeric recombinant NS7 gene in the recombinant influenza A viral vectors of the subtypes H5N1 and H1N1 containing the genetic sequences of the Brucella L7/L12 or Ompl6 antigens are shown in Fig. 1 A and B.
Example 2 The genetic stability of recombinant strains of influenza A virus subtypes H5N1 and
H1N1, and evaluation of the expression of Brucella proteins in vitro.
Five consecutive passages of four of the viral constructs were accumulated in 10-day-old CE for genetic stability testing. The allantoic cavity of the CE were infected using 10'4 dilutions of inoculate. The genetic stability of the viral constructs was confirmed by reverse transcription- polymerase chain reaction (RT-PCR) using NS segment specific primers (sense 5'- ACTACTTCTAGAGAAGACAAAGCAAAAGCAGGGTGACA-3' and antisense 5'- ACTACTCTGCAGATTAACCC TCACTAAAAGTAGAAACAAG-3'). At passages 1, 3 and 5; the size of the NS amplicons was compared to those of pHW plasmids encoding the corresponding genes. The NS -fusion protein encoding genes of the viral constructs were sequenced at passages 1, 3 and 5 using the Sanger method with the commercial kit Prism BigDye™ Terminator v3.1 (ABI, Foster City, CA, USA) on a automatic 16-capillary sequencer Genetic Analyser 3130 xl (ABI). All of the viral constructs replicated well in CE. It should be noted that during the initial passages in CE, the viral constructs had low infection and hemagglutination titers; however, as the number of passages increased, the titers also increased (Table 1). By the fifth passage, the infectious titers of the viral constructs ranged from 7.95 ± 0.22 to 9.2 ± 0.14 lg EID50/ml. Examination of the NSl gene by RT-PCR confirmed that all of the viral constructs were genetically stable in CE over 5 passages (Fig. 2A). The sizes of the NSl genes of the viral constructs containing Brucella proteins L7/L12 or O pl6 corresponded to the size of that amplified from the control pHW plasmids (1 110 and 1242 bp, respectively). These results were confirmed by the sequencing data, which showed that the nucleotide sequences of the NS 1 gene of all of the viral constructs corresponded to the Brucella proteins L7/L12 or Ompl6 (data not shown). Western blotting (Fig. 2B) demonstrated that the fusion proteins NS1- L7/L12 and NSl-Ompl 6, with molecular masses of approximately 27 kDa and 33 kDa, respectively, were correctly expressed in CE infected with the viral constructs. The amino acid sequences of the recombinant fusion proteins NSl-Ompl6 and NS 1 -L7/L12 are shown in Fig. 3 A and B.
Table 1. Infection and hemagglutination titers for the viral constructs during passage in chicken embryos (CE)
Passage Infection (log 10 EIDso/ml) /hemagglutination titer
level/biological Flu-NSl-124- Flu-NS 1-124- Flu-NS 1-124- Flu-NS 1-124- system L7/L12-H5N1 Ompl6-H5Nl L7/L12-H1N1 Ompl 6-HlNl
1/CE 7.28±0.3/l : 128 6.82±0.14/1 : 16 4.45±0.14/1 :128 4.2±0.08/1 :4
3/CE 8.78±0.14/1 :256 7.95±0.14/1 :256 7.95±0.22/l :128 6.95±0.14/1 :256
5/CE 8.95±0.22/l :512 8.45±0.08/l :512 9.2±0.14/1 :512 7.95±0.22/l :256
Example 3
Preparation of vaccines.
Vaccine samples were prepared from the viral constructs Flu-NS 1 - 124-L7/L 12-H5N 1 , Flu- NSl-124-Ompl6-H5Nl, Flu-NSl-124-L7/L12-HlNl and Flu-NS l-124-Ompl6-HlNl, which accumulated in 10-day-old CE at 34 °C for 48 h. The obtained allantoic suspensions of viral constructs with the same antigenic structure (H5N1 or H1N1) were combined in a single pool in a 1 :1 ratio to obtain the bivalent vaccine formulation. Furthermore, the resulting mixtures of viral constructs (L7/L12-Ompl6) were combined with adjuvants such as Montanide GelOl (L7/L12- Ompl 6-MontanideGel01 ; Seppic, France) in a 80:20 ratio by volume (according to the manufacturer's recommendations) or chitosan oligosaccharide lactate (L7/L12-Ompl6-chitosan; Sigma- Aldrich) in a final concentration of 0.05%, and the mixtures were stirred using a magnetic stirrer for 5-7 min. The prepared vaccine samples were stored at 2-8 °C until use.
Example 4 Cattle and vaccination.
Used 25 head of cattle (heifers), Kazakh white breed (meat direction) aged 1-1.5 years-old. All animals were seronegative for B. abortus, which was confirmed by analysis of blood serum using the Rose Bengal test (RBT; Antigen, Almaty, Kazakhstan), serum agglutination test (SAT; Microgen, Moscow, Russia), complement fixation test (CFT; Microgen) and enzyme-linked immunosorbent assays (ELISA; Brucella-Ab C-ELISA, Svanova Biotech AB, Sweden) according to the manufacturers' instructions. Heifers were divided into five groups (5 animals per group): three experimental groups vaccinated with L7/L12-Ompl6, L7/L12-Ompl6- MontanideGelOl or L7/L12-Ompl6-chitosan, one negative control group (PBS), and one positive control (B. abortus SI 9) group. Each group of animals was kept in a separate room and had free access to water and feed throughout the experiment. Cattle in the experimental groups were immunized twice via the conjunctival route of administration at an interval of 28 days with vaccines generated from the viral vector subtypes H5N1 (prime vaccination) and H1N1 (booster vaccination). The detailed animal immunization scheme is shown in Table 2. Cattle in the positive control group were immunized once subcutaneously in the neck region (right side) with a commercial vaccine B. abortus SI 9 (Shchelkovsky Biokombinat, Russia) at a dose of 80 x 109 CFU/animal (according to the manufacturer's instructions). Cattle in the negative control group were administered subcutaneously with 2.0 ml of PBS.
Table 2. The immunization schedule of cattle (heifers) with viral constructs vaccine formulations
Vaccine* Number of Dose prime Dose booster animals vaccination vaccination
(H5N1), log10 (mm), iog1() EID50 / animal EID50 / animal
L7/L12-Ompl6 5 8.74+8.74 8.5+8.25
L7/L12-Ompl6-Montanide GelOl 5 8.64+8.64 8.4+8.15
L7/L 12-Omp 16-chitosan 5 8.43+8.43 8.2+7.95
* Volume vaccine for cattle by conjunctival method of administration was 1 ml (0.5 ml to each eye) Example 5
Safety assessment of vaccines. The safety of the vaccines generated from the viral constructs was determined and compared with the negative (PBS) and positive (5. abortus SI 9) control groups. The vaccinated cattle were clinically observed daily by thermometry for 56 days after the initial vaccination (IV). Furthermore, blood samples were taken on days 0, 7, 14, 28, 35, 42, 56 post-IV from the jugular vein (serum and whole blood using Vacutainer tubes; Becton Dickinson, USA) for hematological (hemoglobin concentration, hematocrit, red blood cells, white blood cells, platelets, stab and segmented neutrophils, eosinophils, lymphocytes, and monocytes) and biochemical (total bilirubin, direct bilirubin, creatinine, cholesterol, total protein, urea, glucose, aspartate aminotransferase, and alanine aminotransferase) studies.
This study showed that immunization of animals with viral constructs vaccine formulation only, or its combination with adjuvants did not have any negative impact on the overall clinical status (behavior, appetite, etc.) of the animals throughout the observation period. The body temperature of the animals in the experimental groups was within normal limits during the observation period (Fig. 4). No side effects (expiration, conjunctivitis, etc.) were observed at the site of conjunctival administration.
In the positive control group vaccinated with B. abortus SI 9, no animals showed any signs of any disease or changes in behavior or appetite during the period of clinical observation, similarly to the animals in the negative control group (PBS). However, the animals in the positive control group displayed an increase of body temperature up to 40.9°C for 1-3 days after vaccination (Fig. 4). Furthermore, infiltrates up to 7 cm in diameter formed at the site of subcutaneous vaccination, which were completely resorbed by 14 days after vaccination.
Hematological and biochemical analysis revealed that all of the studied parameters remained consistent with normal physiological ranges [Hussain S.A. et al., Turk. J. Vet. Anim. Sci. 37 (2013), 329-336] during the entire period of observation in all groups, and in spite of dynamic changes, did not exceed the limits of the normal ranges (data not shown). The only exception occurred in the group of animals vaccinated with the B. abortus SI 9 vaccine, in which band neutrophils were detected at 7 days after vaccination; according to Gromyko [Ecological Bulletin of Northern Kaukaz. 2 (2005), 80-94], band neutrophils are associated with a slight infectious process (in this case, with vaccination). Example 6 Determination of the immunogenicity of the vaccines.
The immunogenicity of the experimental and control vaccines was evaluated by assessing the presence of a humoral (IgG, IgGl, IgG2a) and T cell immune response (CD4+, CD8+, IFN-γ) in the vaccinated cattle at 28 and 56 days after IV; blood serum (10 ml per Becton Dickinson Vacutainer tube) and whole blood (heparinized tubes [100 U/ml] in a volume of 50-70 ml) samples were collected from the vaccinated cattle. 6.1 Antibody response to the Brucella L7/L12 and Ompl6 proteins in vaccinated cattle
ELISAs (Fig. 5A) demonstrated that single immunization with the viral construct vaccine formulations did not significantly (P = 0.4-0.9 versus negative control group) increase the GMT of IgG antibodies against the brucellosis Ompl6 and L7/L12 proteins. In contrast, a significant (P <0.0001) increase in the GMT of IgG antibodies against brucellosis antigens was observed in the positive control group (B. abortus SI 9) compared to the experimental groups during the period of observation.
After booster vaccination of the experimental groups of cattle (Fig. 5B) significant accumulation of IgG antibodies against brucella proteins was only observed in animals vaccinated with L7/L12-Om l6-MontanideGel01 (P = 0.005 and P = 0.0008 compared to L7/L12-Ompl6 and L7/L12-Ompl6-chitosan, respectively). Despite this, the accumulated IgG antibody titers in the group vaccinated with L7/L12-Ompl 6-MontanideGel01 were still significantly lower (P < 0.0001) than the positive control group.
It should be noted that the ratios of IgG antibody isotypes in the experimental groups were significantly different to the positive control (B. abortus SI 9) group. IgG2a antibodies predominated in the cattle from the experimental groups, IgGl antibodies predominated in the positive control group.
6.2 T-cell immune response in vaccinated cattle 6.2.1 Flow cytometry
Immunophenotypic analysis of lymphocytes isolated from whole blood of the cattle and subsequently stimulated with Brucella L7/L12 and Ompl6 proteins demonstrated that the animals vaccinated with the viral construct vaccine formulations formed a strongly pronounced T cell-mediated immune response, even after the prime vaccination. Antigen specific cellular immune responses were formed, due to the fact that in the samples collected from the animals vaccinated with the viral construct vaccine formulations, the numbers of CD4+ and CD8+ (Fig. 6) cells after stimulation with Brucella L7/L12 and OMP16 proteins were significantly higher (from P = 0.01 to P < 0.0001) than that of the control samples (without stimulation); the only exception was the L7/L12-Ompl6-chitosan vaccine, in which the number of CD4+ cells after stimulation with Brucella proteins was not significantly different to the control samples after both prime (P = 0.07) and booster (P = 0.27) vaccination.
Among the adjuvants tested, only Montanide GelOl contributed significantly to stimulation of the T-cell immune response. After stimulation with Brucella antigens in vitro, the number of CD4+ and CD8+ cells in the samples from the animals vaccinated with vaccines containing Montanide GelOl was significantly higher (from P = 0.01 to P = 0.0006) than the other' experimental groups, and did not differ significantly to that of the positive control group vaccinated with B. abortus SI 9 (from P = 0.2 to P = 0.6). Moreover, among the experimental groups, only the samples from the animals vaccinated with the L7/L12-Ompl6-MontanideGel01 had a significantly higher number of CD4+ (P = 0.009) and CD8+ (P = 0.02) cells after booster vaccination than after prime vaccination.
6.2.2 IFN-γ
The concentration of IFN-γ, a cytokine which is one of the main indicators of the formation of Thl and a cytotoxic cellular immune response, was also determined. As shown in Fig. 6, significant (P <0.0001) accumulation of IFN-γ after stimulation with Brucella L7/L12 and Ompl 6 proteins was observed in the samples from the animals vaccinated with the viral constructs vaccine formulation only, as well as its combination with Montanide GelOl, or the B. abortus SI 9 vaccine as compared to the control samples (without stimulation). Significant accumulation of IFN-γ was not observed in the samples from the group of animals vaccinated with L7/L12-Om l6-chitosan. It should be noted that the highest levels of IFN-γ accumulation after stimulation with Brucella antigens was observed in the samples from animals vaccinated with L7/L12-Ompl6-MontanideGel01 ; the lFN-γ levels for this group were significantly higher (P = 0.01 or P = 0.0003) than the other experimental groups (28 days after the prime vaccination) and even slightly superior (P = 0.12 or P = 0.22) to that of the positive control group vaccinated with B. abortus SI 9. Booster immunization did not significantly (P = 0.09 to P = 0.99) increase the concentration of IFN-γ in the samples from the animals in the experimental and control groups. Example 7 Assessment protectiveness of vaccines.
On day 60 post-IV, cattle from the experimental, negative (PBS) and positive (B. abortus SJ9) control groups were subcutaneously challenged with a virulent strain of B. abortus 544 at a dose of 5 x 108 CFU/animal. On day 30 after challenge, all animals after euthanized by intravenous administration of sodium pentobarbital and slaughtered aseptically for sampling of the lymph nodes (submandibular, retropharyngeal, right subscapular, left subscapular, right inguinal, left inguinal, mediastinal, bronchial, portal, para-aortic, pelvic, udder, mesenteric) and parenchymal organs (liver, kidney, spleen and bone marrow). In total, 17 organs were sampled from each animal. The organs were plated onto TSA plates and incubated at 37 °C for 4 weeks, during which time the growth of bacterial colonies was periodically counted. An animal was considered to be infected if a Brucella colony was detected from the culture of one or more organs. The results of the bacteriological examination were evaluated as the number of animals from which no colonies were isolated (effectiveness of vaccination) and by the index of infection (the number of organs and lymph nodes from which were isolated Brucella).
Determination of the number of virulent Brucella in the lymph nodes of the challenged animals was used as an additional indicator to evaluate protective efficacy. For this purpose, the collected retropharyngeal or right subscapular lymph nodes were homogenized in 4 ml of 0.1% Triton-PBS, and 100 μΐ aliquots of 10-fold serial dilutions were plated in triplicate onto TSA plates, incubated for 14 days at 37 °C, and the number of CFU were counted.
As shown in Fig. 7, the highest level of protection was achieved with Flu-L7/L12-Ompl6- MontanideGelOl ; the effectiveness of vaccination and index of infection for this group were 100% and 0, respectively. Good results were also obtained with Flu-L7/L12-Ompl6, which had a similar effectiveness of vaccination (60%), index of infection and number of cultured Brucella (P = 0.99 or P> 0.99) to the group vaccinated with the B. abortus S19 vaccine. The lowest effectiveness of vaccination (40%) was observed for Flu-L7/L12-Om l6-chitosan. Despite this, the number of Brucella cultured from the lymph nodes and index of infection in this group was significantly lower (P = 0.02 or P = 0.007) than that of the negative control group (PBS), and not significantly different to the other experimental groups (from P = 0.29 to P - 0.98) or the positive control group (P = 0.62 or P = 0.92) groups. After challenge with B. abortus 544, the body temperature of the animals in the experimental groups remained within the normal range (37.5-39.5 °C) during the entire period of observation (30 days), while the body temperature of the animals in the negative and positive control groups increased to 40.0 °C on days 1-3 and day 2 post-challenge, respectively. Example 8
Differentiation of infected from vaccinated animals.
Blood samples were collected from the cattle on days 0, 7, 14, 30, 37, 44, 60 post-IV and also 7, 14, 21 and 30 days after challenge with B. abortus 544 for serologic screening tests, such as the RBT, SAT and CFT. Additionally, the serum of cattle vaccinated with B. abortus 19 was analyzed at the same time points after vaccination. No antibodies were detected in cattle vaccinated with Flu-L7/L12-Om l6, Flu-L7/L12-Ompl6-MontanideGel01 or Flu-L7/L12- Ompl6-chitosan in the test periods after prime and boost vaccination using the RBT, SAT or CFT. However, as expected, in the group of cattle vaccinated with B. abortus 19, antibodies were detected using the RBT, SAT and CFT at 7 and 30 days after vaccination, respectively (data not shown). After challenge of the cattle vaccinated with Flu-L7/L12-Om l 6, Flu-L7/L12- Ompl6-MontanideGel01 or Flu-L7/L12-Ompl6-chitosan (data not shown), in most animals antibodies were initially detected using the RBT (primarily) at 14 days, and were detected using the SAT and CFT by 30 days.
INDUSTRIAL APPLICABILITY
Industrial application of the invention is possible in the fields of veterinary and public health as a vector vaccine for the prevention of brucellosis in farm animals (cattle, sheep, goats and pigs) and humans. The embodiments of the invention described herein have clearly demonstrated the ability of vector vaccines based on recombinant influenza viral vectors expressing the Brucella immunodominant proteins L7/L12 or Ompl6 to protect cattle from brucellosis. Theoretically, these recombinant influenza viral vector vaccines may also have the ability to protect against brucellosis in other species of farm animals, as well as humans. This assumption is based on the fact that the Brucella proteins L7/L12 and Ompl6 expressed by the recombinant influenza viral vectors are conserved within all species and biovars of Brucella {Brucella abortus, Brucella melitensis, Brucella suis, and others). Our recent results [Tabynov K. et al., Vaccine 32 (2014), 2034-2041] demonstrate that these recombinant influenza viral vectors can effectively replicate and express foreign Brucella proteins in the body such natural immunity to influenza animal as cattle. This gives us confidence that the obtained recombinant influenza viruses could be used as vectors to express Brucella proteins and generate a protective immune response against brucellosis in other species such as sheep, goats and pigs. Indeed, the recombinant influenza viral vectors described herein may be ideal vaccine candidates to prevent brucellosis in pigs and humans. It has been experimentally confirmed that recombinant influenza viral vectors in which the viral NS1 gene has been modified are capable of replicating and can subsequently induce immune responses in pigs and humans [Vincent A., et al., Vaccine 25(47) (2007), 7999-8009; Wressnigg Ν., et al., Vaccine 27(21) (2014), 2851-2857; Stukova M., et al., Tuberculosis 86 (3-4) (2006), 236-246]. Moreover, these recombinant influenza viral vectors are completely safe in pigs and humans.
Industrially, we propose the recombinant influenza viral vectors could be offered for prophylactic immunization of cattle against brucellosis as bivalent vaccine formulations or mixtures of recombinant influenza viral vectors expressing the Brucella L7/L12 and Om l6 proteins. Bivalent vaccine formulations could be prepared by combining allantoic suspensions of two recombinant influenza viral vectors (Flu-NSl-124-L7/L12-H5Nl + Flu-NS l-124-Ompl6- H5N1 or Flu-NSl-124-L7/L12-HlNl + Flu-NSl-124-Ompl6-HlNl) with the same antigenic structure (H5N1 or HlNl) in a l : l ratio. The resulting mixtures of recombinant influenza viral vectors could be mixed with a stabilizing medium (for example, peptone with sucrose), dispensed into ampoules and freeze-dried. Vaccination of animals via the conjunctival or subcutaneous routes using a cross-immunization scheme is recommended, whereby the animals would receive a prime vaccination with a mixture of recombinant influenza viral vectors of the H5N1 subtype, and then receive a booster vaccination 21 -28 days later with a mixture of recombinant influenza viral vectors of the HlNl subtype. To enhance the protective efficacy of the vaccine, it could be administered in combination with a commercial polymeric adjuvant such as Montanide GelOl (final concentration of 10-20%; Seppic, France) as the solvent. Other immunization schemes could also be tested and employed in different animals to increase the effectiveness of the candidate vaccine.

Claims

Consortium of the recombinant influenza A viruses Flu-NSl-124-L7/L12-H5Nl (6:2 reassortant of influenza A viruses A/Puerto' Rico/8/34 HlNl and A/chicken/Astana/6/05 H5N1), Flu-NSl-124-Ompl6-H5Nl (6:2 reassortant of influenza A viruses A/Puerto Rico/8/34 HlNl and A chicken/Astana/6/05 H5N1), Flu-NSl-124-L7/L12-HlNl (6:2 reassortant of influenza A viruses A Puerto Rico/8/34 HlNl and A/New Caledonia/20/99 HlNl) and Flu-NSl-124- Ompl6-HlNl (6:2 reassortant of influenza A viruses A/Puerto Rico/8/34 HlNl and A/New Caledonia/20/99 HlNl), family Ortom xoviridae, genus Influenzavirus, that stably expressing the Brucella immunodominant ribosomal L7 L12 or outer membrane proteins Ompl6 from open reading frame NS1 gene, and destined to generate a vaccine against brucellosis.
PCT/KZ2014/000006 2013-06-14 2014-05-23 Consortium of the recombinant influenza a viruses flu-ns1-124- l7/l12-h5n1, flu-ns1-124-omp16-h5n1, flu-ns1-124-l7/l12-h1n1 and flu-ns1- 124-omp16-h1n1, family ortomyxoviridae, genus influenzavirus, expressing brucella immunodominant proteins destined to generate a vaccine against brucellosis WO2014200325A2 (en)

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CN109116035A (en) * 2018-09-14 2019-01-01 重庆理工大学 A kind of Brucella antibody competition AlphaLISA detection kit and its detection method
CN111638329A (en) * 2020-06-09 2020-09-08 南方医科大学 ELISPOT detection kit for detecting brucellosis and application thereof
CN111638329B (en) * 2020-06-09 2021-06-01 南方医科大学 ELISPOT detection kit for detecting brucellosis and application thereof
CN113637703A (en) * 2021-08-06 2021-11-12 河北科技师范学院 Construction method and application of Brucella L7/L12 and GroES eukaryotic expression vector
CN113637703B (en) * 2021-08-06 2023-08-25 河北科技师范学院 Construction method and application of Brucella L7/L12 and GroES eukaryotic expression vector

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