A new candidate vaccine for human brucellosis based on influenza viral vectors: a preliminary investigation for the development of an immunization…

Posted: February 17, 2021 at 1:54 am

To our knowledge, this study is the first trial conducted in guinea pigs to evaluate the protective properties of a new candidate for vector vaccine against human brucellosis. This phase in vaccine trials is an important step in making an experimental vaccine a promising candidate for further human clinical trials to determine its effectiveness. In this study, double i.n. immunization with a vector vaccine based on influenza viral vectors expressing the immunodominant brucellosis proteins Omp16, L7/L12, Omp19 and CuZn SOD at a dose of 106 EID50 ensured protection against B. melitensis 16M infection comparable to the effect of commercial B. melitensis Rev.1 vaccine.

The choice of guinea pigs as model animals for evaluation of body gain changes and vaccine candidate protection was determined by their natural resistance to influenza infection in comparison with laboratory mice. In this case, the use of a more resistant model animal seemed to be a key condition in the study of protection, since, in the long run, the vaccine is designed for humans.

The previous success of using IVV in the development of the anti-brucellosis vaccine Flu-BA for cattle [12], which is now at the stage of commercialization in Kazakhstan, served as the basis for this study. The idea of developing an anti-brucellosis human vaccine is that the high efficacy of the vaccine is achieved in cattle which naturally resistant to influenza infection (i.e., as a non-replicable viral vector), and, in our opinion, should be even more pronounced in humans. This assumption is based on the fact that humans are a natural host for the influenza virus (Influenza A), including the influenza viral vectors we use.

It should be noted that we used an influenza viral vector (IVV) of the H5N1 subtype, because there is no immune background to this type of pathogen [22] in the human population and IVV of the H5N1 subtype has a greater potential as a vaccine vector.

We began the process of creating an effective anti-brucellosis vector vaccine for humans with the formation of requirements for the developed product, production technology and methods of its application and testing in healthcare practice. To this end, we have accumulated the existing experience in the development of vector vaccines for public health, have chosen the most generally accepted requirements for such vaccines and their manufacturing technologies, the method and frequency of their use and have developed criteria for assessing the effectiveness and safety of vaccine candidates.

An analysis of the compliance of the developed vaccine with the above requirements (which are more general in nature than specific) showed that the viral vectors we selected, as well as the method for preparing and using the vaccine, correspond to them. In particular, we use non-pathogenic influenza viral vectors, the general safety of which has been confirmed by studies on guinea pigs with various ways of administering and dose of immunization.

In order to obtain influenza viral vectors (IVV), we used A/Puerto Rico/8/34 (H1N1) with a length-modified NS1-80 gene encoding 80 amino acids in the N-terminal region of the protein as the initial strain. The surface genes of hemagglutinin (HA) and neuraminidase (NA) were taken from A/chicken/Astana/6/05 strains (H5N1, with the HA cleavage site preliminary removed). The safety or attenuation of IVV is ensured by the truncated NS1 protein (interferon antagonist), which results in their limited replicative capabilities (they make one cycle of reproduction in the cell and do not leave it) [14]. It is known that the degree of IVV attenuation is directly dependent on the length of the NS1 protein [23]. We have an IVV with NS1 length in 80 amino acid. NS1-124 was used to create a veterinary brucellosis vaccine as it for use in cattle a more aggressive IVV was required. As for humans, far preferable may be IVV with NS1-80. With IVV subtype H5N1 (a pathogenic variety of influenza virus), the attenuation was additionally achieved by removing the proteolytic cleavage site in the HA protein, that is, double attenuation was performed. During repeated re-inoculation in chick embryos, IVV retained all their basic biological properties, including signs of attenuation, and did not lose the brucellosis insertion segment [14], which indicates their genetic stability. In addition, the influenza viral vectors we use are RNA-containing viruses that are limited to cytoplasmic replication, thus eliminating the risk of integration and long-term persistence.

The next important phase of our research was devoted to the study of the general safety control of the vaccine candidate at the early stage with different ways of administration and dose of use in guinea pigs. The vaccine has been found to be safe for guinea pigs when administered c., i.n. and s.l. The experimental animals did not show death or signs of any disease; by the end of observation (on day 14 after the prime-boost vaccinations), the body weight gain in guinea pigs was observed both after prime and after boost immunization. At the same time, the increase in body weight of guinea pigs in the experimental groups was comparable to the control group of animals that were injected with PBS. As a result of this work, the vaccine was recognized as a safe drug and was used in the future to assess its protectiveness depending on the immunization schedule.

Further assessment of the effectiveness of the vaccine with different routes of administration to mucosal areas was determined using c., i.n. and s.l. methods of vaccine immunization in prime-boost mode. Since the influenza virus has a tropism for mucosal surfaces, it was assumed that the optimal way to administer a vaccine based on an influenza viral vector would be one of the tested mucosal routes. Since Brucella should be considered as a mucosal pathogen penetrating mucous surfaces, the gates of infection are the mucosal surfaces of the nose or mouth. Consequently, mucosal vaccination is capable to generate protective responses against pathogens at the site of the infection gate [24]. Our bacteriological study demonstrated that significant protection of guinea pigs after challenging with virulent strain of B. melitensis 16M infection was achieved through i.n. administration of the vaccine in comparison with other methods of application.

The next important step in our study was devoted to the choice of the vaccination dose and, at the same time, the frequency of vaccination, where the study of the protection and immunogenicity of the vaccine candidate was evaluated in animals by the ability to retain bacteria in organs and lymph nodes after animal infection with standard methods. Another distinctive feature of our studies was that the vaccine protection was assessed not only by the Brucella culture isolation from the tissues of vaccinated and unvaccinated animals, but also by such aspects as vaccination efficiency and infection index. It is believed that these indicators jointly provide a more complete and objective characterization of the vaccine protection. The new vaccine induced significant protection in response to B. melitensis 16M infection within a range of 6080% when administered i.n. in a double vaccination mode for all tested doses, and it was not inferior in efficiency to B. melitensis Rev.1, which is currently used in veterinary practice as the most immunogenic brucellosis vaccine. The level of protection of the B. melitensis Rev.1 vaccine obtained in our studies corresponds to the science literature data [25]. At the same time, it was found that the new vaccine candidate does not possess protection after primary vaccination, regardless of the dose. When choosing an immunizing dose of the vaccine, it is recommended to use a vaccination dose of 106 EID50, since the protection at the 106 EID50 dose (80% efficiency) was higher than 105 EID50 (60% efficiency) and similar to 107 EID50 (80% efficiency). The choice of an immunizing dose of 106 EID50 is determined by the reduction of possible adverse effect of vaccination and the cost of the production process of the vaccine. The vaccine is targeted at a specific risk grouplaboratory scientists working with the pathogen, veterinarians, slaughterhouse workers and people involved in animal care industry. The next step in the further vaccine development will be devoted to the preclinical studies where will be evaluated the safety, immunogenicity and protectiveness of a new human vaccine candidate against brucellosis.

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A new candidate vaccine for human brucellosis based on influenza viral vectors: a preliminary investigation for the development of an immunization...

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