RESEARCH Open Access

Influenza viral vectors expressing the Brucella OMP16 or L7/L12 proteins as vaccines against B. abortus infectio

Development of Human Vectored Brucellosis Vaccine Formulation: Assessment of Safety and Protectiveness of Influenza Viral Vectors Expressing Brucella Immunodominant Proteins in Mice and Guinea Pigs

In this paper, we first used recombinant influenza viral vector (rIVV) subtype H5N1 expressing from the open reading frame of NS1 80 and NS1 124 amino acids of Brucella outer membrane proteins (Omp) 16 and 19, ribosomal L7/L12, and Cu-Zn superoxide dismutase (SOD) proteins to develop a human brucellosis vaccine. We made 18 combinations of IVVs in mono-, bi-, and tetravalent vaccine formulations and tested them on mice to select the safest and most effective vaccine samples. Then, the most effective vaccine candidates were further tested on guinea pigs. Safety of the rIVV-based vaccine candidate was evaluated by a mouse weight-gain test. Mice and guinea pigs were challenged with the virulent strain B. melitensis 16M. The protective effect of the rIVV-based vaccine candidate was assessed by quantitation of Brucella colonization in tissues and organs of challenged animals. All vaccine formulations were safe in mice. Tested vaccine formulations, as well as the commercial B. melitensis Rev.1 vaccine, have been found to protect mice from B. melitensis 16M infection within the range of 1.6 to 2.97 log10 units (P<0.05). Tetravalent vaccine formulations from the position of NS1 80 amino acids (0.2±0.4), as well as the commercial B. melitensis Rev.1 vaccine (1.2±2.6), have been found to protect guinea pigs from B. melitensis 16M infection at a significant level (P<0.05). Thus, tetravalent vaccine formulation Flu-NS1-80-Omp16+Flu-NS1-80-L7/L12+Flu-NS1-80-Omp19+Flu-NS1-80-SOD was chosen as a potential vaccine candidate for further development of an effective human vaccine against brucellosis. These results show a promising future for the development of a safe human vaccine against brucellosis based on rIVVs

Improved influenza viral vector based Brucella abortus vaccine induces robust B and T-cell responses and protection against Brucella melitensis infection in pregnant sheep and goats.

We previously developed a potent candidate vaccine against bovine brucellosis caused by Brucella abortus using the influenza viral vector expressing Brucella Omp16 and L7/L12 proteins (Flu-BA). Our success in the Flu-BA vaccine trial in cattle and results of a pilot study in non-pregnant small ruminants prompted us in the current study to test its efficacy against B. melitensis infection in pregnant sheep and goats. In this study, we improved the Flu-BA vaccine formulation and immunization method to achieve maximum efficacy and safety. The Flu-BA vaccine formulation had two additional proteins Omp19 and SOD, and administered thrice with 20% Montanide Gel01 adjuvant, simultaneously by both subcutaneous and conjunctival routes at 21 days intervals in pregnant sheep and goats. At 42 days post-vaccination (DPV) we detected antigen-specific IgG antibodies predominantly of IgG2a isotype but also IgG1, and also detected a strong lymphocyte recall response with IFN-γ production. Importantly, our candidate vaccine prevented abortion in 66.7% and 77.8% of pregnant sheep and goats, respectively. Furthermore, complete protection (absence of live B. melitensis 16M) was observed in 55.6% and 66.7% of challenged sheep and goats, and 72.7% and 90.0% of their fetuses (lambs/yeanlings), respectively. The severity of B. melitensis 16M infection in vaccinated sheep and goats and their fetuses (index of infection and rates of Brucella colonization in tissues) was significantly lower than in control groups. None of the protection parameters after vaccination with Flu-BA vaccine were statistically inferior to protection seen with the commercial B. melitensis Rev.1 vaccine (protection against abortion and vaccination efficacy, alpha = 0.18-0.34, infection index, P = 0.37-0.77, Brucella colonization, P = 0.16 to P > 0.99). In conclusion, our improved Flu-BA vaccine formulation and delivery method were found safe and effective in protecting pregnant sheep and goats against adverse consequences of B. melitensis infection

Colonization and incidence of recovery of <i>B</i>. <i>melitensis</i> in tissues after challenge with <i>B</i>. <i>melitensis</i> 16M.

<p>Colonization and incidence of recovery of <i>B</i>. <i>melitensis</i> in tissues after challenge with <i>B</i>. <i>melitensis</i> 16M.</p

Index of infection for sheep and goats challenged with <i>B</i>. <i>melitensis</i> 16M at 113–120 days post-vaccination.

<p>Pregnant sheep and goats in the group I (Flu-BA_Omp19-SOD) were immunized twice concurrently via the subcutaneous and conjunctival routes of administration at an interval of 21 days with vaccines generated from the influenza viral vectors (IVV) subtypes H5N1 (prime vaccination) and H1N1 (booster vaccination). The vaccination of animals of group II (Flu-BA_Omp19-SOD_TV) was carried out in the same way as in group I, but only the vaccine generated from IVV subtypes H5N1 was used, which was administered three times at 21 days intervals. Sheep and goats in the negative control group (IV) were vaccinated with 20% Montanide Gel01 adjuvant in PBS three times at 21 days intervals. Animals in the positive control group (III) were immunized once subcutaneously in the axillary region (right side) with commercial vaccine <i>B</i>. <i>melitensis</i> Rev.1 according to the manufacturer's instructions. Challenge with the virulent strain <i>B</i>. <i>melitensis</i> 16M was performed via the subcutaneous route (10⁶ CFU/animal). The index of infection is the number of animals from which <i>Brucella</i> was isolated from the organs and lymph nodes. The data presented as mean ± standard error; * <i>P <0</i>.<i>0001</i> vs. appropriate control group IV. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparisons test. <i>P</i> values < 0.05 were considered significant.</p

Experimental design.

<p>Experimental design.</p

Improved influenza viral vector based <i>Brucella abortus</i> vaccine induces robust B and T-cell responses and protection against <i>Brucella melitensis</i> infection in pregnant sheep and goats - Fig 4

<p>Lymphocyte stimulation index (A, C) and levels of IFN-γ (B, D) in the supernatants of PBMCs of pregnant sheep and goats at 42 and 63 days post-vaccination (DPV). Pregnant sheep and goats in the group I (Flu-BA_Omp19-SOD) were immunized twice concurrently via the subcutaneous and conjunctival routes at an interval of 21 days with vaccines generated from the influenza viral vectors (IVV) subtypes H5N1 (prime vaccination) and H1N1 (booster vaccination). The vaccination of animals of group II (Flu-BA_Omp19-SOD_TV) was carried out in the same way as in group I, but only the vaccine generated from IVV subtypes H5N1 was used, which was administered three times at 21 days intervals. Sheep and goats in the negative control group (IV) were vaccinated with 20% Montanide Gel01 adjuvant in PBS three times at 21 days intervals. All data are presented as mean ± standard error; * <i>P = 0</i>.<i>02 to P<0</i>.<i>0001</i> vs. appropriate control group; † <i>P = 0</i>.<i>02–0</i>.<i>01</i> vs. Day 42 DPV. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparisons test. <i>P</i> values < 0.05 were considered significant. NI—not investigated.</p

Rates of abortion, parturition and infection in the sheep and goats after challenge with the virulent strain <i>B</i>. <i>melitensis</i> 16M.

<p>Rates of abortion, parturition and infection in the sheep and goats after challenge with the virulent strain <i>B</i>. <i>melitensis</i> 16M.</p