BackgroundWe generated novel, effective candidate vaccine against Brucella abortus based on recombinant influenza viruses expressing the Brucella ribosomal protein L7/L12 or outer membrane protein (Omp)-16 from the NS1 open reading frame. The main purpose of this work was to evaluate the safety, immunogenicity and protectiveness of vaccine candidate in laboratory animals.Methods and ResultsFour recombinant influenza A viral constructs of the subtypes Н5N1 or H1N1 expressing the Brucella proteins L7/L12 or Omp16 were obtained by a reverse genetics method: Flu-NS1-124-L7/L12-H5N1, Flu-NS1-124-Omp16-H5N1, Flu-NS1-124-L7/L12-H1N1 and Flu-NS1-124-Omp16-H1N1. Despite of substantial modification of NS1 gene, all constructs replicated well and were retain their Brucella inserts over five passages in embryonated chicken eggs (CE). Administration of the mono- or bivalent vaccine formulation via prime-boost intranasal (i.n.), conjunctival (c.) or subcutaneous (s.c.) immunization was safe in mice; no deaths, body weight loss or pathomorphological changes were observed over 56 days. Moreover, guinea pigs vaccinated i.n. with vaccine vectors did not shed the vaccine viruses through their upper respiratory tract after the prime and booster vaccination. These findings confirmed the replication-deficient phenotype of viral vectors. The highest antibody response to Brucella antigen was obtained with constructs expressing L7/L12 (ELISA, GMT 242.5-735.0); whereas the highest T-cell immune response- with construct expressing Omp16 (ELISPOT, 337 ± 52-651 ± 45 spots/4×105cells), which was comparable (P > 0.05) to the response induced by the commercial vaccine B. abortus 19. Interestingly, c. immunization appeared to be optimal for eliciting T-cell immune response. In guinea pigs, the highest protective efficacy after challenge with B. abortus 544 was achieved with Omp16 expressing constructs in both monovalent or bivalent vaccine formulations; protective efficacy was comparable to those induced by a commercial live B. abortus 19 vaccine.ConclusionThus, influenza vectors expressing Brucella protective antigens can be developed as novel influenza vectored vaccine against B. abortus infection.

This paper presents the results of a study of the immunogenicity and protectiveness of new candidate vector vaccine against Brucella abortus - a bivalent vaccine formulation consisting of a mixture of 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 candidate vaccine, adjuvants such as Montanide Gel01 or chitosan were included in its composition. Immunization of cattle (heifers aged 1-1.5 years, 5 animals per group) with the viral constructs vaccine formulation only, or its combination with adjuvants Montanide Gel01 or chitosan, was conducted 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. Vaccine candidates were evaluated in comparison with the positive (B. abortus S19) and negative (PBS) controls. The viral constructs vaccine formulations, particularly in combination with Montanide Gel01 adjuvant promoted formation of IgG antibodies (with a predominance of antibodies of isotype IgG2a) against Brucella L7/L12 and Omp16 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 S19 vaccine and superior to the B. abortus S19 vaccine in combination with Montanide Gel01 adjuvant. Based on these findings, we recommended the bivalent vaccine formulation containing the adjuvant Montanide Gel01 for practical use in cattle.

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bIn this study, we assessed in humans the immunogenicity and safety of one dose (7.5 or 15 g of hemagglutinin [HA]) of a whole-virion inactivated prepandemic influenza vaccine adjuvanted with aluminum hydroxide. The vaccine strain was made by reverse genetics from the highly pathogenic avian A/Chicken/Astana/6/05 (H5N1) clade 2.2 strain isolated from a dead bird in Kazakhstan. The humoral immune response was evaluated after a single vaccination by hemagglutination inhibition (HI) and microneutralization (MN) assays. The vaccine was safe and immunogenic, inducing seroconversion in 55% of the evaluated patients, with a geometric mean titer (GMT) of 17.1 and a geometric mean increase (GMI) of 3.42 after a dose of 7.5 g in the HI test against the vaccine strain. The rate of seroconversion increased up to 70% when the dose of 15 g was used. The percentages of individuals achieving anti-HA titers of >1:40 were 52.5% and 57.5% for the 7.5-and 15-g dose groups, respectively. Similar results were obtained when antibodies were analyzed in an MN test. Substantial cross-neutralization titers (seroconversion in 35% and 52.5% of subjects in the two dose groups, respectively) were detected against heterologous clade 1 strain NIBRG14 (H5N1). Thus, one dose of this whole-virion prepandemic vaccine adjuvanted with aluminum has the potential to be effective against H5N1 viruses of different clades.

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