Preclinical studies of safety, immunogenicity and protective activity of attenuated Bordetella pertussis bacteria on the Macaca mulatta model

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Introduction

Despite wide-scale vaccination against pertussis in many countries since the early 1950s, pertussiscausing bacteria have not been eliminated so far. Even with many underdiagnosed pertussis cases, there are annually reported more than 16 million cases of pertussis of different severity and around 200 thousand deaths worldwide [1]. Laboratory-confirmed pertussis cases among adolescents and adults have been relentlessly increasing over the past decade [2][3]; the number of subclinical and asymptomatic cases of Bordetella pertussis (BP) carriage is also climbing [2][4][5]. In the United States, where pertussis vaccination (PV) coverage in children is 95%, the pertussis incidence has been dramatically increasing since the early 2000s, nearly reaching the before-vaccination levels [6][7]. An increase in incidence has been reported by Italy and England [8][9]. In Russia, pertussis cases more than doubled from 2017 to 2018. The upward trend continued both in 2019 and at the beginning of 2020. [10]. In previous years, the increased incidence was mainly recorded in Moscow and St. Petersburg, which, most likely, can be explained by the high quality of diagnostics [11].

At present, pertussis prevention is built on vac­cines containing a corpuscular pertussis component (whole-cell pertussis vaccines (wPV)) or an acellular pertussis component (acellular pertussis vaccine (aPV)) combined with inactivated diphtheria and tetanus ana­toxins. Sometimes, wPV or aPV are used as monovac­cines. Although aPVs are believed to be less reactogenic, the direct studies on primates showed that these vac­cines do not provide antibacterial immunity and do not protect the animals from experimental pertussis infec­tion [12]. The comparative assessment of the incidence in the vaccinated and unvaccinated groups demonstra­ted low efficacy in adolescents and adults revaccinated with aPV [13][14].

The short duration of vaccine-acquired immuni­ty is another significant shortcoming of the currently available PVs. The evaluation of efficacy of different types of PVs shows that the duration of post-vacci­nation immunity does not exceed 5 years. The infec­tion-acquired immunity wanes after 10 to 15 years [15].

All contemporary PVs are administered to chil­dren over 2 months of age as a three-dose series. There­fore, the full vaccination series is completed when chil­dren reach the age of 6 months, which implies a high risk of infection during the first few months of their life when they are most vulnerable to pertussis.

An increasing number of pertussis cases, includ­ing older children and adults, culminated in understand­ing the importance of revaccination of adolescents and adults. Maternal vaccination leading to building of "family immunity" is placed on the agenda [3][4][16][17]. The only recommended vaccine is aPV [4], which, as mentioned above, does not provide children and adults with protection against infection acquisition and transmission. Thus, we have to admit that although there are solid grounds for revaccination of adolescents and adults as well as for building of family immunity, none of the currently available vaccines can be used for these purposes. As for wPVs, they are not recommend­ed by WHO for using in adults, and contemporary aPVs are most likely inefficient. The aPV demonstrated its efficacy and safety as an alternative to the wPV in vac­cination of infants. Such vaccination controls mortality and disease severity in infants who are most vulnerable to pertussis. However, similar to the wPV, it is admin­istered as a 3-4-dose series vaccination, which, if not completed, results in substantially decreased vaccine protective efficacy in children.

Based on preclinical studies, we demonstrated safety of the intranasal vaccination of laboratory ani­mals with attenuated BP 4MKS bacteria and the protec­tive effect produced by the mice vaccination providing protection in their intracerebral and intranasal infection with virulent BP bacteria [18]. The recent studies have demonstrated the viability of using the experimental model of nonhuman primates in studying of immuno- biological characteristics of the pertussis pathogen and

PV immunogenicity [19][20][21][22][23]. The studies have shown that the experimental infection of monkeys results in laboratory test presentation of pertussis infection, naso­pharyngeal hyperemia, long-term BP persistence and an increased titer of specific immunoglobulins in blood serum of animals. The studies on hamadryas baboons proved that infection can be transmitted from a human to a primate and among primates [23].

The purpose of this study is evaluation of safety, immunogenicity and protective activity of the new con­structed attenuated BP bacteria 4MKS by infecting im­munized Macaca mulatta monkeys (rhesus macaque; RM) intranasally with virulent bacteria of the pertussis pathogen.

Materials and methods

BPs were grown in solid casein-charcoal agar (CCA) media supplemented with 10% defibrinated sheep blood, at 36°C. Attenuated BPs 4MKS collected from nasopharyngeal washings were seeded in the CCA medium containing 200 μg/ml of streptomycin.

To identify the serotype composition of the cul­ture, we used diagnostic pertussis sera for agglutino­gens of BP 1, 2, 3 bacteria, adsorbed, for agglutination test, dry (Gamaleya Federal Research Center for Epi­demiology and Microbiology, Federal State Budgetary Institution) in accordance with the manufacturer’s re­commendations.

Five adult, clinically healthy RMs aged 3-4 years were used for immunization and experimental infec­tion. The re-immunization was performed in 6 months. Three non-immunized RMs of the same age were used as controls. The tests were conducted with animals from the Sukhumi monkey breeding farm (The Research In­stitute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia). The studies in­volving animals were conducted in compliance with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes, and in compliance with the applicable Rus­sian regulations.

Protective activity of the attenuated bacteria was assessed by comparing rates of elimination of virulent BP 475 bacteria from the nasopharynxes of the vacci­nated and non-immunized, control RMs, by comparing the development of immune responses, clinical symp­toms and laboratory presentation of pertussis.

Blood samples were taken from non-anesthetized RMs in squeeze cages.

To identify BP bacteria DNA, we used nasopha­ryngeal swabs washed with 500 μl of saline. After centrifugation, DNA was isolated through standard treatment with guanidine thiocyanate solution and sub­sequent sorption by using the magnetic sorbent (Promega). We used the real-time PCR test designed by us for identification of BP bacteria DNA [24][25].

RM blood sera were examined for presence of pertussis toxin and filamentous hemagglutinin-specific IgG and IgM as well as non-specific IgE after the sin­gle-dose and double-dose intranasal vaccination by us­ing Ridascreen test systems. Vector-Best test was used in identification of non-specific IgE.

The obtained results were analyzed by using Stu­dent’s t-test. The differences are significant atp ≤ 0.05.

Results

The RMs' overall condition and blood test results after the immunization and infection with virulent BPs 475

After the first and second intranasal immuniza­tion, RMs did not show any abnormalities in their be­havior, overall condition, blood count, white blood cell count and glucose levels, any changes in the activities of the aspartate and alanine aminotransferase, and any signs of inflammation or any other reactions in the nasopharynx (Table 1).

 

 

The experimental infection of the immunized mon­keys with virulent BP bacteria 12 months after the re-im­munization also did not demonstrate any deviations from the normal values (Table 1). At the same time, in the con­trol group, the infection of native monkeys with virulent bacteria resulted in significantly increased white blood cell counts and decreased glucose levels (Table 1), nasal mucus and nasopharyngeal inflammation developed on the 3^rd to 10^th day of the infection. No cough was ob­served in the control and vaccinated monkeys.

The measurement results for non-specific IgE in blood sera of the immunized and infected animals are given in Table 2.

 

Table 2 shows that out of all the immunized ani­mals, only monkey # 31883, and out of all the control animals, only monkey # 31927 has the IgE levels close to the negative control values in humans (20-30 IU/ml). In all the other monkeys, the IgE values range from 130 to 250 in # 31882 and from 390 to 720 in # 31901.

No steady increase in IgE levels was observed after the immunization and re-immunization. On the contrary, monkey # 31882 demonstrated a decreasing tendency in its IgE concentration immediately after the re-immunization; all the animals had significantly de­creased IgE levels after they were infected with virulent BP 475 bacteria.

The BP bacterial load in the monkeys' nasopharynx after the immunization and infection with virulent BP 475

The bacteriological technique is the "gold stan­dard" in pertussis diagnostics. The washings of the na­sopharyngeal swabs collected from the posterior wall of the monkeys’ nasopharynxes were seeded on the blood-containing CCA medium with or without strepto­mycin. The swabs were taken one hour after the immu­nization or the experimental infection and then in 3, 7, 10, 14 days, etc. The growth of bacteria in the CCA me­dium was measured on the 4^th-5^th day after the seeding. The grown colonies were typified by using agglutino­gen 1, 2 and 3 specific sera. The growth of BP bacteria on plates was recorded during the first 2 weeks and, in rare cases, 3-4 weeks after the inoculation. The pres­ence of foreign microflora posed an additional prob­lem, especially for the analysis of the bacteria grown in the antibiotic-free CCA medium. Taking this fact into account and considering the low effectiveness of the bacteriologic culture technique, we used our real-time PCR method to diagnose pertussis and to measure the number of bacteria in the nasopharynx of the animals.

The seeding and PCR analysis of the washings from the nasopharyngeal swabs from the control ani­mals did not reveal any growth of colonies and DNAs of the pertussis pathogen.

Titer of specific IgG in the blood serum of the vaccinated RMs after the intranasal infection with BP

All the tests described in the previous section were accompanied by the analysis of the changes in the levels of BP-specific IgG in the blood serum of the in­fected animals. After the 1^st experimental infection, the levels of specific IgG in the blood serum of the animals were increasing (Fig. 3, 4), starting from the 10^th to 14^th day, and reached its maximum by the 28^th day in the monkeys infected with virulent and attenuated BP 475 bacteria, and by the 35^th - 48^th day after the infection with BP 18323. After the repeated infection with BP 18323 bacteria, the IgG levels reached their maximum values by the 14^th day after the inoculation with bacteria of any strain.

 

Discussion

The preclinical studies of acute toxicity on in­fant rates and mice, leukocytosis -promoting and his­tamine-sensitizing activity of the pertussis toxin and weight toxicity of the suspension of attenuated BP bac­teria in classical tests on linear mice, activity of dermonecrotic endotoxin and hypoallergenicity in tests on rabbits and guinea pigs demonstrated the safety of intranasal administration of the new constructed live recombinant pertussis vaccine (LPV) [18].

The performed studies on adult nonhuman pri­mates showed that experimental intranasal infection of monkeys with virulent BP bacteria results in develop­ment of laboratory presentation of pertussis infection in RMs, hamadryas baboons, Javanese macaques and Japanese macaques [22]. Juvenile Anubis baboons de­veloped a whooping cough typical of pertussis [19]. The immunization of Anubis baboons with LPV based on attenuated BP BPZE1 bacteria demonstrated safety and immunogenicity of the vaccine [21]. The results pointed at viability of using the experimental model of nonhuman primates in the studies of immunobiological characteristics of the pertussis pathogen and immunogenicity of pertussis vaccines.

The obtained results showed absence of any changes in blood counts and site responses in RMs after the intranasal inoculation with attenuated BP bacteria, thus fully corresponding to the results of the tests on smaller laboratory animals. No inflammatory processes were detected in the nasopharynxes of the animals monitored after the experimental infection with virulent bacteria after the first and repeated vaccination. No increase in total IgE was found in the monkeys’ blood serum after the vaccination and re-vaccination. All the immunized animals demonstrated a decrease in IgE after being infected with virulent BP 475 bacteria. These findings are consistent with the results obtained by R. Li et al. [26] who showed that intranasal vaccination of mice with attenuated BP BPZE1 bacteria not only causes no allergic reactions, but also protects animals from experimental allergic inflammation and decreases IgE levels in blood sera.

It should be noted that the IgE Ridascreen test designed for human blood serum testing did not detect any IgE in the monkeys’ sera. The IgE levels in the monkeys’ blood were measured with the IgE Vector- Best test and the results are shown in Table 3. This test had never been used for evaluation of monkeys’ IgE levels; therefore, the obtained values cannot be used in quantitative measurements, though they can be used for qualitative assessment of changes in IgE levels after the animals’ immunization.

The growth of virulent BP bacteria in the nasopharynx and their persistence in a human body are critical characteristics of pertussis infection. Based on the available data, the immunization of monkeys with wPVs and attenuated bacteria, as opposed to immunization with aPVs, results in development of mucosal immunity preventing the growth of virulent BP bacteria when they enter the body of a human or a monkey. The charts shown in Fig. 1 and 2 demonstrate the highly similar dynamics of the growth/eradication of attenuated and virulent bacteria of different strains in the nasopharynx of RMs. Individual BP genomeequivalents are detected in nasopharyngeal isolates during 6 months by using the real-time PCR. We received similar results during examination of children of different age, recovering from pertussis, where in 15-20% cases, the pertussis pathogen was detected by the real-time PCR 6 months after the diagnosis [27].

The similar picture was observed after the repeated inoculation of monkeys with attenuated and virulent bacteria and after the experimental infection of the immunized animals with virulent bacteria. The curves of growth/eradication after the repeated inoculation of bacteria (Fig. 1 and 2) do not demonstrate any qualitative differences, though they fundamentally differ from the picture observed after the 1 ^st inoculation by absent accumulation and higher rates of bacteria eradication. For example, by the 28^th day after the repeated infection (immunization), bacteria were not detected or were detected only individual cases, while during the same period after the 1^st infection, the number of BP genome-equivalents could reach several dozen. It should be noted that the sensitivity of our test is 0.01-0.1 genome-equivalent in 5 μl of the solution and is based on using multi-copy sequence of IS481 as the amplification target [24][25].

During the entire development of the 1^st infection, the monkey infected with BP bacteria at a dose of 10⁷ CFU demonstrated significantly lower amounts of BP genome-equivalents in the animals’ nasopharynxes (Fig. 2). This circumstance was taken into account in our decision on the vaccination dose for ongoing clinical trials of LPV on healthy volunteers.

It should be noted that our previous studies showed that the phase composition of the population undergoes changes during the persistence of pertussis bacteria in the monkeys’ bodies. While during the first hours after the infection, most of the BP bacteria are in a virulent state characterized by the native structure of the bvgAS operon, during the infection development, the heterogeneity of the BP bacteria population increases due to the increasing levels of non-virulent mutants of the pertussis pathogen, which carry the IS481 insertion in the bvgAS BP operon. The process is especially pronounced after repeated inoculations when the number of insertion mutants of BP bacteria in the persisting bacteria population can reach 50% of the total number of the recorded bacteria [28]. This observation suggests the possible existence of the mechanism responsible for forming long-term persisting bacteria that secure the survival of the pathogen and its transmission to a new host.

Fig. 3 and 4 show that the changes in the titer of specific IgG in the blood sera of the vaccinated monkeys experimentally infected with virulent BP bacteria show resemblance with the respective curve obtained after the repeated infection of RMs and are significantly different from the changes in the IgG levels after the 1^st experimental infection with virulent and attenuated bacteria. All the animals re-immunized and infected after the immunization with virulent BP bacteria demonstrated a rapid increase in the levels of antibodies, which reached maximum values by the 10^th - 14^th day. These results are totally consistent with the results for IgG levels after the repeated infection with virulent BP 475 bacteria and correlate with the above results capturing changes in the accumulation of bacteria in the nasopharynxes of the infected animals. The rapidly increasing levels of specific antibodies after the repeated exposure to infection contribute to suppression of the bacterial growth and bacteria elimination from the nasopharynxes of the animals.

Thus, the experimental intranasal infection of RMs with attenuated and virulent BP bacteria results in developing of a protective reaction to repeated infection, including suppression of the bacterial growth, increased rates of bacteria elimination from nasopharynxes of the animals and development of a humoral immune response to infection. The development of immunity against repeated pertussis infection is accompanies by a pronounced booster effect regardless of the bacteria strain. The obtained results imply common mechanisms of development of post­vaccination immunity after intranasal immunization of the animals with LPV and post-infection pertussis immunity, both of them providing protection against re­infection with BP bacteria and against development of clinical symptoms of pertussis. The obtained data are consistent with the results obtained by C. Locht at al. [22], who demonstrated the presence of a pronounced protective effect from the experimental infection with virulent BP D420 bacteria through the example of Anubis baboons immunized with live attenuated BP bacteria. Inactivated bacteria of the pathogen (wPVs) have a less pronounced protective effect, while aPVs do not provide protection against the bacterial growth after the repeated infection [20][21].

The absence of monkey-specific enzyme-linked immunoassay (ELISA) tests makes quantitative measurement of pertussis antibodies in these animals highly challenging. While the Ridascreen human IgG enzyme immunoassay kit was suitable for measurement of IgG levels in RMs, hamadryas baboons, Javanese macaques and Japanese macaques, the test from the same manufacturer for measurement of human IgA levels was totally insensitive to simian IgA. We were able to measure IgM levels in different periods of the experimental pertussis infection of the monkeys by using Ridascreen ELISA test for human IgM, though low absolute values and widely scattered results make their discussion unproductive. Most likely, the observed picture can be explained by the insufficient efficiency of the commercial tests for monkeys’ sera. The obtained results emphasize the urgency of designing ELISA tests specific for immunoglobulins of nonhuman primates, so that they could be further used as an experimental model.

Conclusion

The single-dose intranasal inoculation and re­inoculation with attenuated BP bacteria did not cause any inflammatory processes in the RMs’ nasopharynxes and any changes in the blood counts after the experimental infection of nonhuman primates with virulent bacteria.

No increase in the levels of total IgE in the mon­keys’ blood serum was detected after single-dose and double-dose immunization.

Experimental infection of monkeys with attenuat­ed and virulent BP bacteria induces the development of protective reaction to repeated infection, including suppression of the bacterial growth, increased rates of bacteria elimination from animals’ nasopharynxes and development of a humoral immune response to infec­tion. The development of immunity against the repeat­ed pertussis infection is accompanied by a pronounced booster effect regardless of the bacteria strain.

The obtained results suggest common mecha­nisms of development of post-vaccination immunity af­ter intranasal vaccination of animals and post-infection pertussis immunity, which provide protection against repeated infection with BP bacteria and development of clinical symptoms of pertussis.

About the authors

Alisa Yu. Medkova

Federal Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya;
Central Сlinical Hospital with a Polyclinic, Office of the President of the Russian Federation

Email: fake@neicon.ru

senior researcher, Laboratory of genetics of bacteria

Head, Department of paediatric infectious diseases

Россия

Lyudmila N. Sinyashina

Federal Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya

Email: fake@neicon.ru
ORCID iD: 0000-0003-1708-5453
leading researcher, Laboratory of genetics of bacteria Россия

Astanda A. Amichba

Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia

Email: fake@neicon.ru
ORCID iD: 0000-0002-4986-1392
junior researcher, Laboratory of virology and immunology

Evgeniy G. Semin

Federal Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya

Email: fake@neicon.ru
ORCID iD: 0000-0001-6696-8362
researcher, Laboratory of genetics of bacteria Россия

Zinaida V. Shevtsova

Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia;
Abkhazian State University

Email: fake@neicon.ru
ORCID iD: 0000-0002-6471-1608

D. Sci. (Med.), chief researcher, Laboratory of virology and immunology

Prof., Department of experimental biology and medicine

Alisa Z. Matua

Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia;
Abkhazian State University

Email: fake@neicon.ru
ORCID iD: 0000-0003-3275-0941

PhD (Biol.), Deputy Director for science, Head, Laboratory of immunology and virology

Assoc. Prof., Department of experimental biology and medicine

Anush A. Djidaryan

Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia

Email: fake@neicon.ru
ORCID iD: 0000-0002-8792-5289
senior laboratory assistant, Laboratory of virology and immunology

Dzhenni T. Kubrava

Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia

Email: fake@neicon.ru
ORCID iD: 0000-0001-9104-4014
junior researcher, Laboratory of virology and immunology

Irina G. Kondzhariya

Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia;
Abkhazian State University

Email: fake@neicon.ru
ORCID iD: 0000-0003-3707-4874

PhD (Biol.), senior researcher, Laboratory of virology and immunology

Assoc. Prof., Department of experimental biology and medicine

Vladimir S. Barkaya

Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia

Email: fake@neicon.ru

Zurab Ya. Mikvabiya

Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia

Email: fake@neicon.ru
ORCID iD: 0000-0002-0729-6516
D. Sci. (Med.), Prof., director

Gennadiy I. Karataev

Federal Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya

Author for correspondence.
Email: karataevgi@rambler.ru
ORCID iD: 0000-0001-8771-6092
D. Sci. (Biol.), Head, Laboratory of genetics of bacteria Россия

References

Supplementary files