Influenza viral vector based Brucella abortus vaccine: a novel vaccine candidate for veterinary practice

KEYWORDS:

• Brucella abortus
• vaccine
• influenza viral vector
• safety
• immunogenicity
• T cell immune response
• protection
• cattle

Bovine brucellosis is caused by the gram-negative Brucella abortus bacterium and induces abortion and decreased fertility that lead to significant economic losses in agriculture, and is also responsible for chronic zoonotic infections in humans [1]. Vaccination of livestock against brucellosis is a cost-effective strategy that can protect public health in endemic areas [2], as well as the most important tool in the control and eradication of the disease among livestock [3]. At present, specific prophylaxis of bovine brucellosis in cattle is mainly carried out using the live attenuated B. abortus S19 and RB51 vaccines. These vaccines are highly effective [4,5] in cattle, but possess a number of serious shortcomings, not least including their ability to induce abortions, virulence in humans, and their pronounced agglutinogenic properties (except for RB51) that prevent subsequent differential diagnosis [6–8]. Furthermore, the RB51 vaccine is resistant to the antibiotic rifampin, which is used to treat the disease [9,10]. These drawbacks limit the global use of these vaccines, and have created the need to develop new, safe, effective vaccines.

In an attempt to create a safe and effective B. abortus vaccine, different research groups have developed attenuated mutant, subunit (recombinant proteins), DNA, and vector vaccines [11]. The primary aim of these vaccine candidates is to promote formation of an antigen-specific T-cell immune response and protection against B. abortus infection in mice, which in some cases is comparable to commercial vaccines. However, critically, none of these vaccine candidates have been tested in the natural host. Only a single safety and immunogenicity trial has been reported, for the double mutant strain B. abortus htrA cycL in cattle, though the animals were not challenged with a virulent B. abortus strain to confirm vaccine protectiveness [12]. Therefore, due to the lack of any information on their effectiveness in the natural animal host, these vaccine candidates are far from practical application.

At present, a single novel vector vaccine, Flu-BA, which is based on recombinant influenza viruses of the subtypes H5N1 and H1N1 expressing the Brucella immunodominant Omp16 and L7/L12 proteins, is the sole vaccine candidate with confirmed safety and efficacy in cattle. Influenza A possesses a segmented viral genome composed of eight negative-strand RNA fragments. The smallest fragment of the Influenza A genome, NS, encodes two proteins (nuclear export protein and viral nonstructural protein [NS1]) and provides a convenient target for genetic manipulation as it can tolerate insertion of long foreign sequences [13]. Thus, the Brucella sequences were inserted into the NS1 open reading frame. The strain А/Puerto Rico/8/34 (H1N1) was selected as the backbone to generate influenza A viral vectors expressing the Brucella L7/L12 or Omp16 proteins fused to the N-terminal 124 amino acids of NS1. Influenza A viruses were selected as the vector, as it has been demonstrated that they can infect cattle and induce serological reactions, and even clinical disease [14–16]. Based on a previous study [17] and to maximize the Brucella protein expression levels and T-cell immune response in vivo, a cross vaccination schedule employing both subtypes of influenza viral vector (IVV), with Н5N1 as a prime vaccination and H1N1 as a booster vaccination, was used to immunize cattle. This immunization strategy effectively overcame the immune background elicited toward the viral vector after prime vaccination. In addition, the commercial adjuvant Montanide Gel01 (Seppic, France) was included in the formulation to enhance its effectiveness; according to the manufacturer’s recommendations, this adjuvant is suitable for both mucosal and subcutaneous administration. The benefits of the adjuvant Montanide Gel01 in various vaccine compositions have been confirmed. In cattle, Montanide Gel01 was shown to significantly boost the efficacy of vaccine compared to control without adjuvant [18].

Previous studies showed that the vaccine formulated with Montanide Gel01 adjuvant is safe [19], induces humoral and T-cell immune responses, and provides a high level of protection against B. abortus 544 infection in cattle [18], including pregnant animals [20]. Notably, conjunctival or subcutaneous administration provide complete protection against B. abortus 544 infection (in 70–80% animals) and abortion (in 80–90% animals) in first-calf heifers. Based on these indicators, the vaccine is equivalent to the most effective commercial B. abortus S19 vaccine [20]. According to our latest unpublished data, the level of protection against B. abortus 544 infection in cattle (abortion protection, 100%; infection protection in pregnant heifers 88.8% and fetuses/calves 100%) provided by simultaneous subcutaneous and conjunctival administration of Flu-BA even exceeds that of the commercial B. abortus S19 vaccine. This improved method of vaccine administration is only recommended (in combination with other epidemic countermeasures) for recovery of farms with a high prevalence of brucellosis or high risk of disease occurrence; in other situations, the vaccine can simply be administered subcutaneously (as the conjunctival route has limitations like low efficacy and difficulty in controlling the vaccine dose). Most significantly, Flu-BA induces formation of a long-term protective immune response against B. abortus infection in vaccinated cattle (at least 12 months after booster vaccination) [21]. Moreover, the vaccine also provides good cross-protection against B. melitensis infection in cattle [22], which is important as B. melitensis can be transmitted and become pathogenic among cattle due to poor management on mixed livestock farms.

Our previous studies [17–22] demonstrate Flu-BA is the first candidate vaccine to comply with most of the criteria for an ‘ideal Brucella vaccine’ suggested by Schurig et al. [23] and Ko and Splitter [24]. Flu-BA is a live vaccine based on IVV that induces a strong Th-1 immune response in cattle [18]. Additionally, Brucella agglutinogen antibodies are not produced in vaccinated cattle, making it simple to identify vaccinated and infected animals. Moreover, as IVV expressing the truncated interferon antagonist NS1 protein has limited replicative capacity (and IVV subtype H5 was further attenuated by replacing its polybasic cleavage site with a trypsin-dependent sequence), the attenuated Flu-BA vaccine cannot cause disease in cattle (within 28 days after the prime or booster vaccination) [19] or humans [25,26]. Furthermore, vaccinated animals do not shed IVV into the environment; therefore, the virus cannot be transmitted to other animals or humans [25,26]. Indeed, we previously confirmed IVV expressing the Brucella proteins were not shed from vaccinated guinea pigs or cattle (observation period – 7 days after the prime or booster vaccination) [17,19]. In addition, Flu-BA is genetically stable, retaining its basic biological properties (including the markers of attenuation) and does not lose the Brucella protein inserts after at least five repeated passage in the chicken embryo (CE) culture system [17]. However, the stability of this vaccine cannot be established in the host, as it is not shed into the environment by vaccinated cattle [19]. Finally, Flu-BA will be easily manufactured on the large scale at facilities that use CEs. Based on the average IVV titer (8.5–9.5 log₁₀ EID₅₀/CE) and dose required for cattle (6.0 log₁₀ EID₅₀/animal), a single CE can produce between 300 and 3000 doses at the low cost of 0.01–0.001 euro/vaccine dose, which will be combined with the commercial adjuvant Montanide Gel01 (1 kg/10,000 vaccine doses; 0.012 euro/vaccine dose). Therefore, the basic low material cost of this vaccine (the estimated market value – 0.4 euro/two vaccine doses) is comparable to that of the commercially available B. abortus S19 and RB51 vaccines (the market price on the Kazakhstan market – 0.2 and 1.3 euro/vaccine dose, respectively). Consequently, the cost of this vaccine will be acceptable for the majority of Brucella-infected developing countries.

In fact, for the first time in 20 years since the introduction of the B. abortus RB51 vaccine, a real vaccine candidate exists, which has not only been comprehensively studied in the natural host animals, but is also close to entering field trials. During the development of this vaccine to evaluate its safety, immunogenicity, protectiveness, and method of application (immunization method and dose) and measure the duration of the immunoprotective response in animals and other vaccine properties, a total of 273 cattle were used, including 167 pregnant animals aged between 17 months and 2 years. Total vaccine development costs in 2012–2014 amounted to about 1.1 million US dollars. Currently, the preliminary organization and initiation of vaccine field trials in Kazakhstan is underway; the estimated date of completion is 2017. After confirming its safety and efficacy in field trials, commercial production and practical application of the Flu-BA vaccine is planned in Kazakhstan, which to this day remains endemic for brucellosis with a high prevalence of infection.

Declaration of interest

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Additional information

Funding

This work was carried out under the project 0402 ‘Development of Products for Preventing Bovine Brucellosis’ as part of the research program O.0600 ‘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.