Authors:
Kaissar Tabynov*, Zhailaubay Kydyrbayev, Sholpan Ryskeldinova, Bolat Yespembetov, Nazym Syrymkyzy, Indira Akzhunusova and Abylai Sansyzbay
Affiliations:
The Research Institute for Biological Safety Problems, Zhambulskaya oblast, Kordaiskiy rayon, Gvardeisky, Republic of Kazakhstan
Dates:
Received: 20 February, 2014; Accepted: 19 April, 2014; Published: 21 April, 2014
*Corresponding author:
Kaissar Tabynov, The Research Institute for Biological Safety Problems, Zhambulskaya oblast, Kordaiskiy rayon, Gvardeisky, Republic of Kazakhstan, Tel/fax: + 7 (72636) 72228; Email: tabynov_81@mail.ru
Citation:
Tabynov K, Kydyrbayev Z, Ryskeldinova S, Yespembetov B, Syrymkyzy N, et al. (2015) Safety of the Novel Vector Vaccine Against Brucella Abortus Based on Recombinant Influenza Viruses Expressing Brucella L7/L12 and OMP16 Proteins, in Cattle. J Vaccines Immun 1: 001-007.
Copyright:
© 2015 Tabynov K. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Keywords:
Influenza virus; vector vaccine; Brucella abortus; Safety; Cattle

This paper presents the results of a study of the safety of new vector vaccine against B. abortus based on recombinant influenza A subtype H5N1 or H1N1 (viral constructs vaccine formulation) viruses expressing Brucella ribosomal protein L7/L12 and Omp16, in cattle. To increase the effectiveness of the vaccine, adjuvants such as Montanide Gel01 or chitosan were included in its composition. Immunization of cattle (5 animals per group) with the viral constructs vaccine formulation only, or its combination with adjuvants Montanide Gel01 or chitosan, were conducted via the conjunctival method using cross prime (influenza virus subtype H5N1) and booster (influenza virus subtype H1N1) vaccination schedules. Vaccine candidates were evaluated in comparison with the positive (Brucella abortus) and negative (PBS) controls. Comprehensive studies involving thermometry and clinical examination, hematology and biochemical blood analysis, showed that all of the viral constructs vaccine formulation, as well as their combination with adjuvants, compared to the commercial bacterial vaccine Brucella abortus were completely safe in cattle. Furthermore it is shown that the developed vaccines can effectively differentiate vaccinated animals from infected animals.

Introduction

Brucella abortus is a facultative intracellular pathogen capable of infecting and causing disease in both domestic animals and humans [1]. At present, brucellosis among cattle is prevented using live attenuated vaccines from the strains B. abortus 19 or RB51. These vaccines have a high immunogenic effectiveness, but have a number of serious disadvantages, primarily related to their ability to induce abortion in pregnant cows, secretion of the vaccine strain into the milk of vaccinated animals when they are used in adult cattle and the difficulty of differentiating between vaccinated animals and infected animals (only a concern for the B. abortus 19) [2]. Furthermore, both strains are pathogenic to humans [3]. Therefore, the development of an effective - and at the same time safe - vaccine against B. abortus is currently a problem.

In an effort to create an effective and safe vaccine against B. abortus, several research groups have developed subunit (recombinant proteins) [4-12], a DNA [13-18], or live vector vaccines (based on bacteria and viruses) [19-22]. All of these vaccines were safe when tested in animal models (laboratory mice), and some when tested in cattle. However, these vaccines remain inferior to commercial live attenuated vaccines in terms of protectiveness.

To solve this very significant problem we first proposed vector vaccine based on recombinant influenza viruses expressing the Brucella L7/L12 or Omp16 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 (NS1) and nuclear export protein (Nep) - is a convenient target for genetic manipulation as NS1 is able to tolerate foreign sequences exceeding its own length [25]. Thus, the ORF of NS1 was used for inserting Brucella sequences in this study. The А/Puerto Rico/8/34 (H1N1) strain was used as the backbone for obtaining influenza A virus vectors expressing Brucella L7/L12 or Omp16 sequences in the form of fusion proteins with the N-terminal 124 amino acid residues of NS1.

Our previous studies have shown that a bivalent vaccine formulation comprising a mixture of recombinant influenza A virus subtype H5N1 or H1N1 expressing the ribosomal L7/L12 or Omp16 proteins in prime and booster immunization mode (via conjunctival injection) in cattle induced a strong antigen-specific T-cell immune response, and most importantly provided a high level of protectiveness comparable to the commercial Brucella abortus vaccine and superior to the Brucella abortus vaccine in combination with Montanide Gel01 adjuvant [24]. Based on this, the next stage of our study was to evaluate the safety of the proposed new live vector vaccine in cattle. Additionally, we evaluated the possibility of differentiating infected animals from vaccinated animals using the developed vaccine.

Materials and Methods

Bacterial strains

The vaccine strain B. abortus 19 (Shchelkovsky Biokombinat, Moscow oblast, Russia) and the virulent strain B. abortus 544 (obtained from our institute’s collection of microorganisms) were used in this study. The bacterial cells were cultured under aerobic conditions in tryptone soy agar (TSA; Sigma, St. Louis, MO, USA) at 37°C. All experiments with live Brucella were performed in biosafety level 3 facilities.

Generation of viruses

All viruses were generated as described previously [24]. Vaccine batches were produced in 10-day-old embryonated chicken eggs (CE; Lohmann Tierzucht GmbH, Cuxhaven, Germany) after three egg passages of viral constructs (Flu-NS1-124-L7/L12-H5N1, Flu-NS1-124-Omp16-H5N1, Flu-NS1-124-L7/L12-H1N1 и Flu-NS1-124-Omp16-H1N1).

Preparation of vaccines

Vaccine samples were prepared from the viral constructs Flu-NS1-124-L7/L12-H5N1, Flu-NS1-124-Omp16-H5N1, Flu-NS1-124-L7/L12-H1N1 and Flu-NS1-124-Omp16-H1N1, which accumulated in 10-day-old CE (Lohmann Tierzucht GmbH) 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 (Flu-L7/L12-Omp16) were combined with adjuvants such as Montanide Gel01 (Flu-L7/L12-Omp16-MontanideGel01; Seppic, France) in a 80:20 ratio by volume (according to the manufacturer’s recommendations) or chitosan oligosaccharide lactate (Flu-L7/L12-Omp16-chitosan; Sigma-Aldrich) in a final concentration of 0.05%, and the mixtures were stirred using a magnetic stirrer for 5-7 min.

Cattle and bioethics

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 Flu-L7/L12-Omp16, Flu-L7/L12-Omp16-MontanideGel01 or Flu-L7/L12-Omp16-chitosan, one negative control group (PBS), and one positive control (Brucella abortus) group. Each group of animals was kept in a separate room and had free access to water and feed throughout the experiment.

This study was carried out in compliance with national and international laws and guidelines on animal handling. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Research Institute for Biological Safety Problems of the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan (Permit Number: 0513/107).

Vaccination

Cattle in the experimental groups were immunized twice via the conjunctival route of administration at an interval of 30 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 1. Cattle in the positive control group were immunized once subcutaneously in the neck region (right side) with a commercial vaccine Brucella abortus (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.

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    Table 1:

    The immunization schedule of cattle (heifers) with viral constructs vaccine formulations.

Safety assessment of vaccines

The safety of the vaccines generated from the viral constructs was determined and compared with the negative (PBS) and positive (Brucella abortus) control groups. The vaccinated cattle were clinically observed daily by thermometry for 60 days after the initial vaccination (IV). Furthermore, blood samples were taken on days 0, 7, 14, 30, 37, 44, 60 post-IV from the jugular vein (serum and whole blood using Vacutainer tubes; Becton Dickinson, USA) for hematological and biochemical studies.

Hematological and biochemical blood tests

For hematological analysis, whole blood samples were assayed using the 540 T-Coulter Counter (Coulter Electronics, Hialeah, FL, USA) and ADVIA120 Hematology System (Bayer Healthcare LLC, Tarrytown, NY, USA) automatic blood analyzers; the following parameters were determined: hemoglobin concentration, hematocrit, red blood cells, white blood cells, platelets, stab and segmented neutrophils, eosinophils, lymphocytes, and monocytes.

Biochemical studies of the serum samples were performed on a VITALAB Selectra 2 automated analyzer (Merck, Germany) using commercial kits from DiaSys Diagnostic Systems GmbH (Germany); the following parameters were determined: total bilirubin, direct bilirubin, creatinine, cholesterol, total protein, urea, glucose, aspartate aminotransferase, and alanine aminotransferase.

Differentiation of infected from vaccinated animals

In order to differentiate of infected from vaccinated animals, blood samples were collected from cattle on days 0, 7, 14, 30, 37, 44, 60 post-IV, and 7, 14, 21 and 30 days after post-challenge for analysis by the RBT (Antigen), SAT (Microgen) and CFT (Microgen) according to the instructions included with the kits. On day 60 post-IV, cattle from the experimental, negative (PBS) and positive (Brucella abortus) control groups were subcutaneously challenged with a virulent strain of B. abortus 544 at a dose of 5 x 108 CFU/animal.

Statistical analysis

We counted the mean and standard deviation of rectal temperature, hematological and biochemical parameters in groups of cattle. These values ​​were compared with the normal physiological values.

Results

1 Safety assessment of the vaccines in cattle

Clinical observations with thermometry: 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 (Figure 1). No side effects (expiration, conjunctivitis, etc.) were observed at the site of conjunctival administration.

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    Figure 1

    Rectal temperature in cattle after vaccination. Vaccination of cattle was carried out twice with an interval of 30 days with viral constructs vaccine formulation only (Flu-L7/L12-Omp16) or a combination thereof with adjuvants (Flu-L7/L12-Omp16-Montanide Gel01 or Flu-L7/L12-Omp16-chitosan) or a single with commercial vaccine Brucella abortus. The animals of the negative control group as an inoculum were administered with PBS. The data are presented as mean ± standard deviation (SD).


In the positive control group vaccinated with Brucella abortus, 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 (Figure 1). Furthermore, infiltrates up to 7 cm in diameter formed at the site of subcutaneous vaccination, which were completely resorbed by 14 days after vaccination.

Hematologic and biochemical studies: Hematological and biochemical analysis revealed that all of the studied parameters remained consistent with normal physiological ranges [25, 26] during the entire period of observation in all groups, and in spite of dynamic changes, did not exceed the limits of the normal ranges (Figures 2 and 3). The only exception occurred in the group of animals vaccinated with the Flu-L7/L12-Omp16-MontanideGel01 and Brucella abortus vaccines, in which band neutrophils were detected at 7 days after vaccination; according to Gromyko [27], band neutrophils are associated with a slight infectious process (in this case, with vaccination).

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    Figure 2

    Hematological studies of cattle whole blood samples on days 0, 7, 14, 30, 37, 44, 60 after the initial vaccination. Vaccination of cattle was carried out twice with an interval of 30 days with viral constructs vaccine formulation only (Flu-L7/L12-Omp16) or a combination thereof with adjuvants (Flu-L7/L12-Omp16-Montanide Gel01 or Flu-L7/L12-Omp16-chitosan) or a single with commercial vaccine Brucella abortus. The animals of the negative control group as an inoculum were administered with PBS. The data are presented as mean ± standard deviation (SD).

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    Figure 3

    Biochemical studies of cattle blood serum samples on days 0, 7, 14, 30, 37, 44, 60 after the initial vaccination. Vaccination of cattle was carried out twice with an interval of 30 days with viral constructs vaccine formulation only (Flu-L7/L12-Omp16) or a combination thereof with adjuvants (Flu-L7/L12-Omp16-Montanide Gel01 or Flu-L7/L12-Omp16-chitosan) or a single with commercial vaccine Brucella abortus. The animals of the negative control group as an inoculum were administered with PBS. The data are presented as mean ± standard deviation (SD).


Differentiation of vaccinated and infected 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-Omp16, Flu-L7/L12-Omp16-MontanideGel01 or Flu-L7/L12-Omp16-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-Omp16, Flu-L7/L12-Omp16-MontanideGel01 or Flu-L7/L12-Omp16-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.

Discussion

The present work is a continuation of a series of studies aimed at developing a safe and effective vaccine against B. abortus. As already pointed out to solve the problem of specific prophylaxis B. abortus we was first proposed vector vaccine based on recombinant influenza viruses expressing Brucella L7/L12 or Omp16 proteins. Our previous studies have shown that this vaccine in cattle induced a strong antigen-specific T-cell immune response, and most importantly provided a high level of protectiveness comparable to the commercial Brucella abortus vaccine and superior to the Brucella abortus vaccine in combination with Montanide Gel01 adjuvant [24]. Safety data of developed vaccines, as well as its ability to differentiate infected animals from vaccinated animals have never been described and presented for the first time in this paper.

Inclusion of adjuvants in the vaccine was due to the need to enhance its effectiveness. In view of the conjunctival route of vaccine administration, we focused on commercial adjuvants such as Montanide Gel01 and chitosan, which according to the manufacturer’s recommendations and in some publications [28-31] can be incorporated into vaccines with a mucosal route of administration.

In previous studies, it was shown that as the size of the NS1 gene decreased in viral vectors, the degree of attenuation of the influenza A viruses increased [32]; however, it is well known that attenuation of influenza viruses may be dependent on the properties of the foreign insert in the C-terminal part of the trancated NS1 protein [33]. Therefore, we considered it necessary to study the safety or degree of attenuation of the constructed recombinant influenza A viruses in cattle. It should be noted that in these studies, along with the traditional methods used for assessing the safety of veterinary vaccines, we also employed more sensitive methods such as hematological and biochemical blood analysis, which enable the early detection of any disease with unclear clinical symptoms in the body. Thus, as a result of our comprehensive studies involving clinical examination with thermometry, hematology and biochemical blood analysis, it was found that all of the viral constructs vaccine formulation, alone or in combination with adjuvants, were completely safe for cattle in prime and booster immunization mode compared to the commercial Brucella abortus vaccine. In our previous studies we demonstrated the replication-deficient properties of the viral vectors and confirmed the absence of viral transmission from vaccinated to non-vaccinated animals [34]. Interestingly, one vaccine vector is based on the prepandemic flu A/H5N1 delNS1 vaccine. It was previously shown that this vaccine is completely safe and immunogenic when tested in a variety of laboratory models (chickens, ferrets and rhesus macaques) [35] and humans [36]. Therefore, we assume that this vaccine could not only be used for cattle but also for humans in the future.

The final stage of this study investigated whether it was possible to distinguish between vaccinated and infected animals when the recombinant influenza A viruses expressing the Brucella proteins L7/L12 and Omp16 were used as vaccines. The use of traditional brucellosis vaccines is significantly complicated by the difficulty of differentiating between vaccinated and infected animals due to the presence of an O chain in the Brucella lipopolysaccharide responsible for the agglutinogenic properties of serum [2]. The recombinant influenza A viruses we constructed do not contain this factor; therefore, as expected, and in contrast to the positive control group of animals (B. abortus 19), no antibodies were detected using the RBT, SAT or CFT in cattle serum during the entire observation period after prime and boost vaccination with Flu-L7/L12-Omp16, Flu-L7/L12-Omp16-MontanideGel01 or Flu-L7/L12-Omp16-chitosan. After challenge of the cattle vaccinated with viral construct vaccine formulations, 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. In the groups of animals immunized with B. abortus 19 antibodies could be detected using the RBT, SAT and CFT at 7 days post-challenge. These data show that after immunization with Flu-L7/L12-Omp16, Flu-L7/L12-Omp16-MontanideGel01 or Flu-L7/L12-Omp16-chitosan, it is possible to effectively differentiate between vaccinated animals and infected animals.

Thus, we can conclude that our proposed new candidate vaccine against B. abortus - bivalent vaccine formulation consisting of a mixture of recombinant influenza A viruses subtypes H5N1 or H1N1 expressing Brucella ribosomal protein L7/L12 or Omp16 in prime and booster immunization mode (with conjunctival injection) completely safe for cattle, furthermore it is can effectively differentiate infected from vaccinated animals. Based on the data, as well as previously published results [24], for practical use in cattle we recommended bivalent vaccine formulation containing the adjuvant Montanide Gel01.

Acknowledgements

The authors express their gratitude to Professor Egorov A. (HSC Development GmbH, Tulln, Austria) for his help in the production of recombinant influenza viruses expressing Brucella Omp16 or L7/L12 proteins. We also would like to express our gratitude to employees of the Research Institute for Biological Safety Problems, E.M. Kozhamkulov, D.A. Inkarbekov, T.M. Gotskina, N.N. Assanzhanova and N.N. Zinina for their assistance in studying the safety of the vaccines in cattle. The work was carried out under the project “Development of Products for Preventing Bovine Brucellosis” as part of the research program “Bovine Brucellosis: Monitoring the Epizoological Situation and Developing Means of Diagnosis and Prevention” for 2012-2014 funded by the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan.

  1. Godfroid J, Cloeckaert A, Liautard JP, Kohler S, Fretin D, et al. (2005) From the discovery of the Malta fever’s agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis. Vet Res 36: 313-326.
  2. Schurig GG, Sriranganathan N, Corbel MJ (2002) Brucellosis vaccines: past, present and future. Vet Microbiol 90: 479-496.
  3. Ashford DA, di Pietra J, Lingappa J, Woods C, Noll H, et al. (2004) Adverse events in humans associated with accidental exposure to the livestock brucellosis vaccine RB51. Vaccine 22: 3435-3439.
  4. Al-Mariri A, Tibor A, Mertens P, De Bolle X, Michel P, et al. (2001) Protection of BALB/c mice against  Brucella abortus 544 challenge by vaccination with bacterioferritin or P39 recombinant proteins with CpGoligodeoxynucleotides as adjuvant. Infect Immun 69: 4816-4822.
  5. Tabatabai LB, Pugh GW Jr. (1994) Modulation of immune responses in Balb/c mice vaccinated with Brucellaabortus Cu-Zn superoxide dismutase synthetic peptide vaccine. Vaccine 12: 919-924.
  6. Oliveira SC, Splitter GA (1996) Immunization of mice with recombinant L7/L12 ribosomal protein confers protection against Brucella abortus infection. Vaccine 14: 959-962.
  7. Oliveira SC, Zhu Y, Splitter G (1994) Sequences of the rplJL operon containing the L10 and L7/L12 genes from Brucella abortus. Gene 140: 137-138.
  8. Oliveira SC, Zhu Y, Splitter GA (1994) Recombinant L7/L12 ribosomal protein and gamma-irradiated Brucella abortus induce a T-helper 1 subset response from murine CD4+ T cells. Immunology 83: 659-664.
  9. Oliveira SC, Harms JS, Banai M, Splitter GA (1996) Recombinant Brucella abortus proteins that induce proliferation and gamma-interferon secretion by CD4+ T cells from Brucella-vaccinated mice and delayed-type hypersensitivity in sensitized guinea pigs. Cell Immunol 172: 262-268.
  10. Cassataro J, Estein SM, Pasquevich KA, Velikovsky CA, de la Barrera S, et al. (2005) Vaccination with the recombinant Brucella outer membrane protein 31 or a derived 27-amino-acid synthetic peptide elicits a CD4+ T helper 1 response that protects against Brucella melitensis infection. Infect. Immun 73: 8079-8088.
  11. Pasquevich KA, Estein SM, García Samartino C, Zwerdling A, Coria LM, et al. (2009) Immunization with recombinant Brucella species outer membrane protein Omp16 or Omp19 in adjuvant induces specific CD4+ and CD8+ T cells as well as systemic and oral protection against Brucella abortus infection. Infect Immun 77: 436-445.
  12. Mallick AI, Singha H, Chaudhuri P, Nadeem A, Khan SA, et al. (2007) Liposomised recombinant ribosomal L7/L12 protein protects BALB/c mice against Brucella abortus 544 infection. Vaccine 25: 3692-3704.
  13. Leclercq S, Harms JS, Oliveira SC (2003) Enhanced efficacy of DNA vaccines against an intracellular bacterial pathogen by genetic adjuvants. Curr Pharm Biotechnol 4: 99-107.
  14. Kurar E, Splitter GA (1997) Nucleic acid vaccination of Brucella abortus ribosomal L7/L12 gene elicits immune response. Vaccine 15: 1851-1857.
  15. Oñate AA, Céspedes S, Cabrera A, Rivers R, González A, et al. (2003) A DNA vaccine encoding Cu, Zn superoxide dismutase of Brucella abortus induces protective immunity in BALB/c mice. Infect. Immun 71: 4857-4861.
  16. Mayfield JE, Bricker BJ, Godfrey H, Crosby RM, Knight DJ, et al. (1988) The cloning, expression, and nucleotide sequence of a gene coding for an immunogenic Brucella abortus protein. Gene 63: 1-9.
  17. Cassataro J, Velikovsky CA, de la Barrera S, Estein SM, Bruno L, et al. (2005) A DNA vaccine coding for the Brucella outer membrane protein 31 confers protection against B. melitensis and B. ovis infection by eliciting a specific cytotoxic response. Infect Immun 73: 6537-6546.
  18. Luo D, Ni B, Li P, Shi W, Zhang S, et al. (2006) Protective immunity elicited by a divalent DNA vaccine encoding both the L7/L12 and Omp16 genes of Brucella abortus in BALB/c mice. Infect Immun 74: 2734-2741.
  19. Harms JS, Durward MA, Magnani DM, Splitter GA (2009) Evaluation of recombinant invasive, non-pathogenic Eschericia coli as a vaccine vector against the intracellular pathogen, Brucella. J. Immune Based The Vaccines. 7: 1.
  20. Zhao Z, Li M, Luo D, Xing L, Wu S, et al. (2009) Protection of mice from Brucella infection by immunization with attenuated Salmonella enteric serovar typhimurium expressing A L7/L12 and BLS fusion antigen of Brucella. Vaccine 27: 5214-5219.
  21. He Y, Vemulapalli R, Schurig GG (2002) Recombinant Ochrobactrum anthropi Expressing Brucella abortus Cu,Zn Superoxide Dismutase Protects Mice against B. abortus Infection Only after Switching of Immune Responses to Th1 Type. Infect Immun 70: 2535-2543.  
  22. Cabrera A, Sáez D, Céspedes S, Andrews E, Oñate A (2009) Vaccination with recombinant Semliki Forest virus particles expressing translation initiation factor 3 of Brucella abortus induces protective immunity in BALB/c mice. Immunobiology 214: 467-474.
  23. Kittel C, Sereinig S, Ferko B, Stasakova J, Romanova J, et al. (2004) Rescue of influenza virus expressing GFP from the NS1 reading frame. Virology 324: 67-73.
  24. Tabynov K , Kydyrbayev Zh, Ryskeldinova Sh, Yespembetov B, Zinina N, et al. (2014) Novel influenza virus vectors expressing Brucella L7/L12 or Omp16 proteins in cattle induce a strong T-cell immune response, as well as high protectiveness against B. abortus infection. Vaccine 32: 2034-41.
  25. Veterinary Medicine: A Textbook of the Diseases of Cattle, Horses, Sheep, Pigs, and Goats. 10th edn., Saunders Elsevier, Philadelphia. 2007.
  26. Hussain SA, Uppal SK, Randhawa C, Sood NK, Mahajan SK (2013) Clinical characteristics, hematology, and biochemical analytes of primary omasal impaction in bovines. Turk J Vet Anim Sci 37: 329-336.
  27. Gromyko EB (2005) Assessment of the body of cows by methods of biochemistry. Ecological Bulletin of Northern Kazkaz 2: 80-94.
  28. Deville S, Arous JB, Bertrand F, Borisov V, Dupuis L (2012) Efficacy of intranasal and spray delivery of adjuvanted live vaccine against infectious bronchitis virus in experimentally infected poultry. Procedia in Vaccinology 6: 85-92.
  29. Read RC, Naylor SC, Potter CW, Bond J, Jabbal-Gill I, et al. (2005) Effective nasal influenza vaccine delivery using chitosan. Vaccine 23: 4367-74.
  30. Mills KH, Cosgrove C, McNeela EA, Sexton A, Giemza R, et al. (2003) Protective levels of diphtheria-neutralizing antibody induced in healthy volunteers by unilateral priming-boosting intranasal immunization associated with restricted ipsilateral mucosal secretory immunoglobulin a. Infect Immun 71: 726-32.
  31. McNeela EA, Jabbal-Gill I, Illum L, Pizza M, Rappuoli R, et al. (2004) Intranasal immunization with genetically detoxified diphtheria toxin induces T cell responses in humans: enhancement of Th2 responses and toxin-neutralizing antibodies by formulation with chitosan. Vaccine 22: 909-14.
  32. Egorov A, Brandt S, Sereinig S, Romanova J, Ferko B, et al. (1998) Transfectant Influenza A Viruses with Long Deletions in the NS1 Protein Grow Efficiently in Vero Cells J Virol 72: 6437-6441.
  33. Wang X, Basler CF, Williams BR, Silverman RH, Palese P, et al. (2002) Functional replacement of the carboxy-terminal two-thirds of the influenza A virus NS1 protein with short heterologous dimerization domains. J Virol 76: 12951-12962.
  34. Tabynov K, Sansyzbay A, Kydyrbayev Z, Yespembetov B, Ryskeldinova S, et al. (2014) Influenza viral vectors expressing the Brucella OMP16 or L7/L12 proteins as vaccines against B. abortus infection. Virol J 11: 69.
  35. Romanova J, Krenn BM, Wolschek M, Ferko B, Romanovskaja-Romanko E, et al. (2009) Preclinical Evaluation of a Replication-Deficient Intranasal ΔNS1 H5N1 Influenza Vaccine. PLoS ONE 4: e5984.
  36. Wacheck V, Egorov A, Groiss F, Pfeiffer A, Fuereder T, et al. (2010) A novel type of influenza vaccine: safety and immunogenicity of replication-deficient influenza virus created by deletion of the interferon antagonist NS1. J Infect Dis 201: 354-62.

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