A new candidate vaccine for human brucellosis based on influenza viral vectors: a preliminary investigation for the development of an immunization schedule in a guinea pig model

Abstract

Background: A new candidate vector vaccine against human brucellosis based on recombinant influenza viral vectors (rIVV) subtypes H5N1 expressing Brucella outer membrane protein (Omp) 16, L7/L12, Omp19 or Cu-Zn SOD proteins has been developed. This paper presents the results of the study of protection of the vaccine using on guinea pigs, including various options of administering, dose and frequency. Provided data of the novel vaccine candidate will contribute to its further movement into the preclinical stage study.

Methods: General states of guinea pigs was assessed based on behavior and dynamics of a guinea pig weight-gain test. The effectiveness of the new anti-brucellosis vector vaccine was determined by studying its protective effect after conjunctival, intranasal and sublingual administration in doses 10⁵ EID₅₀, 10⁶ EID₅₀ and 10⁷ EID₅₀ during prime and boost vaccinations of animals, followed by challenge with a virulent strain of B. melitensis 16 M infection. For sake of comparison, the commercial B. melitensis Rev.1 vaccine was used as a control. The protective properties of vaccines were assessed by quantitation of Brucella colonization in organs and tissues of infected animals and compared to the control groups.

Results: It was observed a gradual increase in body weight of guinea pigs after prime and booster immunization with the vaccine using conjunctival, intranasal and sublingual routes of administration, as well as after using various doses of vaccine. The most optimal way of using the vaccine has been established: double intranasal immunization of guinea pigs at a dose of 10⁶ EID₅₀, which provides 80% protection of guinea pigs from B. melitensis 16 M infection (P < 0.05), which is comparable to the results of the effectiveness of the commercial B. melitensis Rev.1 vaccine.

Conclusions: We developed effective human vaccine candidate against brucellosis and developed its immunization protocol in guinea pig model. We believe that because of these studies, the proposed vaccine has achieved the best level of protection, which in turn provides a basis for its further promotion.

Keywords: Guinea pigs; Human brucellosis; Immunization route; Influenza viral vectors; Protection; Vaccination dose; Vaccine candidate.

Fig. 1

Schematic representation of the recombinant influenza viral vector construction destined to generate a vaccine against brucellosis. a Full size NS1 protein virulence factor for antagonizing interferon system and b deletion part of NS1 replaced by Brucella immunodominant proteins NS1-Omp 16, NS1-Omp 19, NS1-L7/L12 and NS1-Cu–Zn SOD. The blue box and red box b represent RNA binding site of NS1 and part of NS1 protein at amino acid position 80 for insertion brucellosis segment, respectively. PB2, PB1 and PA: Influenza A virus RNA polymerase subunits; HA: Hemagglutinin; NP: Nucleoprotein; NA: Neuraminidase; M: Matrix; NS1: Non–structural protein 1; NEP: Nuclear export protein. The scheme is not drawn according to scale

Fig. 2

Dynamics of body weight change of guinea pigs on day 42 after prime-boost immunization. Animals were immunized with vaccine candidate against human brucellosis at different routes of immunization (a) and various doses (b). Statistical analysis was performed with two-way ANOVA followed by Dunnett’s multiple comparisons test showed that during 42-day body weight measurement between the PBS control and vaccinated groups were not significant. P < 0.05 values were considered significant

Fig. 3

Protective efficacy of the vaccines in guinea pigs when administered by different routes. Protective efficacy of vaccines as evaluated by the effectiveness of vaccination (a), index of infection (b) and isolation rate of Brucella (c) from the tissues of control and experimental groups of guinea pigs on day 30 after challenging with the virulent strain of B. melitensis 16 M. Animals were vaccinated with the vector vaccine by prime-boost conjunctival (c.), intranasal (i.n.), sublingual (s.l.) at interval of 21 days, and with B. melitensis Rev.1 by single subcutaneous (s.c.) vaccination. Guinea pigs in negative control group were injected with PBS. The challenge of animals was performed with B. melitensis 16 M at a dose of 20 CFU/animal using s.c. route. Bacteriological evaluation was assessed by the index of infection in animals (the arithmetic mean ± standard error was given for each group; the number of organs and lymph nodes from which Brucella was isolated for each animal) and by counting Brucella colonies in tissues, where data is expressed as log₁₀ CFU/g. Statistical difference between groups was indicated with asterisks and statistical analysis for (B) was performed using a one-way ANOVA followed by Dunnett's multiple comparisons test and (*, P < 0.01; **, P < 0.002) and for c two-way ANOVA, Tukey’s multiple comparisons test (*P = 0.04–0.01; **P = 0.009–0.001; ***P = 0.0004–0.0002, ****P < 0.0001)

Fig. 4

Protectiveness of vaccine samples at different doses in guinea pigs. a–c Colonization of B. melitensis in organs and tissues of prime-boost vaccinated guinea pigs and c index of Brucella infection upon challenge with B. melitensis 16 M. Guinea pigs were immunized twice i.n. 21 days apart with a new vaccine candidate at dose 10⁵ EID₅₀, 10⁶ EID₅₀ and 10⁷ EID₅₀ or a single delivery of commercial vaccine B. melitensis Rev.1. via s.c. immunization. Guinea pigs of positive control groups were injected with PBS. Animals challenged with virulent strain of B. melitensis 16 M at a dose of 20 CFU/animal using s.c. route. Bacteriological evaluation was assessed by counting Brucella colonies in tissues, where data is expressed as log₁₀ CFU/g and the index of infection of infected animals and compared to the control groups (the arithmetic mean ± standard error was given for each group; number of tissues from where Brucella was isolated for each animal). Statistical analysis for (A-C) was performed using a one-way ANOVA followed by Dunnett's multiple comparisons test and for (D) using two-way ANOVA, Tukey’s multiple comparisons test