Preview

Journal of microbiology, epidemiology and immunobiology

Advanced search

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

https://doi.org/10.36233/0372-9311-2020-97-4-3

Abstract

Introduction. An increasing incidence of pertussis among different groups of population and shortcomings of the existing preventive solutions pinpoint urgency of development of new safe vaccines suitable for immunization of infants and for booster immunization of adolescents and adults.
The purpose of this study is evaluation of safety, immunogenicity and protective activity of the new constructed attenuated Bordetella pertussis bacteria 4MKS by infecting immunized Macaca mulatta monkeys intranasally with virulent bacteria of the pertussis pathogen.
Materials and methods. Five adult, clinically healthy Macaca mulatta monkeys aged 3–4 years were used for immunization and experimental infection. The re-immunization was performed in 6 months. Three non-immunized animals of the same age were used as controls.
Results. The intranasal single-dose inoculation and re-inoculation of attenuated B. pertussis bacteria did not cause any nasopharyngeal inflammation in the rhesus monkeys and any changes in the blood lab test values after the nonhuman primates had been infected with virulent bacteria. No elevation of total IgE was detected in blood serum of the Macaca mulatta monkeys after the single-dose and double-dose immunization. When the monkeys were intranasally immunized with attenuated and virulent B. pertussis bacteria, they developed a defensive reaction to re-infection, namely suppression of the bacterial growth, increased rates of elimination of bacteria from the animals’ nasopharynxes and development of a humoral immune response to the infection. The development of immunity against pertussis re-infection is accompanied by a pronounced booster effect.
Discussion. The obtained results suggest common mechanisms of development both of post-vaccination immunity after intranasal vaccination of animals and infection-acquired immunity against pertussis. Both of them provide protection against re-infection with B. pertussis bacteria and prevent development of clinical symptoms of pertussis.

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.

Prior to the procedures (immunization, experi­mental infection, collection of nasopharyngeal swabs), RMs were anesthetized with intramuscular injections of 0.03-0.04 ml of Zoletil (Virbac, France) at a con­centration of 100 mg/ml (after the premedication with xylazine hydrochloride, 20 mg/ml). The experimental infection and vaccination (107-1010 bacteria) were per­formed by inoculation of 0.5 ml of suspension contain­ing virulent or attenuated bacteria into each nostril o the animals in a supine position.

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).

 

Table 1. RMs' blood biochemistry after their intranasal immunization with attenuated BP bacteria and experimental infection with virulent BP 475 bacteria

Intranasal inoculation

Days

Alanine aminotransferase, U/ml

Aspartate aminotransferase, U/ml

Glucose, Mm/l

White blood cells, ×103

First immunization

Background

34,5 ± 4,4

36,5 ± 12,5

5,4 ± 1,1

10,1 ± 2,8

 

3

38,8 ± 10,2

36,2 ± 10,4

4,7 ± 0,3

8,2 ± 1,9

 

7

42,1 ± 6,1

33,1 ± 2,6

5,5 ± 0,5

9,8 ± 2,7

 

14

46,1 ± 6,7

41,1 ± 10,6

4,7 ± 0,7

8,8 ± 2,3

Re-immunization

Background

39,1 ± 4,6

43,5 ± 11,5

4,4 ± 0,6

9,1 ± 2,6

 

3

37,2 ± 3,9

38,2 ± 8,4

4,7 ± 0,4

7,2 ± 1,9

 

7

40,0 ± 3,1

34,1 ± 5,6

5,3 ± 0,5

8,0 ± 3,1

 

14

41,8 ± 4,0

39,1 ± 7,1

4,7 ± 0,4

8,3 ± 2,5

Infection of native monkeys with BP 475 bacteria

Background

42,2 ± 6,4

42,0 ± 6,5

6,2 ± 0,3

12,1 ± 1,4

3

44,2 ± 4,9

39,2 ± 7,4

5,7 ± 0,7

17,8 ± 2,9

 

7

40,4 ± 4,1

41,1 ± 5,5

4,4 ± 0,6*

19,3 ± 2,9*

 

14

39,8 ± 3,8

40,1 ± 5,1

4,7 ± 0,4*

19,3 ± 2,9*

Note. *p < 0.05 as compared to the background.

 

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 3rd to 10th 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. Values of IgE in blood sera of RMs vaccinated and re-vaccinated with attenuated BP bacteriа

Day after infection

Monkey identification number

31881

31882

31883

31901

31908

31926

31927

31843

31870

31888

First immunization

Background

320

250

66

680

678

 

 

 

 

 

7

290

190

73

-

-

 

 

 

 

 

14

285

220

56

845

693

 

 

 

 

 

24

427

350

48

690

578

 

 

 

 

 

64

469

191

53

662

750

 

 

 

 

 

180

668

117

63

609

555

 

 

 

 

 

Re-immunization in 6 months

Background

668

117

63

609

555

 

 

 

 

 

7

720

141

31

745

449

 

 

 

 

 

14

518

187

43

722

458

 

 

 

 

 

24

562

203

62

648

-

 

 

 

 

 

64

669

177

47

393

524

 

 

 

 

 

180

580

258

-

-

180

 

 

 

 

 

Infection with virulent BP bacteria

Background

219

22

45

58

55

656

48

559

376

257

7

73

13

15

45

32

732

37

635

281

201

14

48

13

17

10

14

651

25

759

351

224

28

43

7

9

23

9

515

52

569

390

356

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 4th-5th 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.

Fig. 1 and Fig. 2 demonstrate changes in the num­ber of bacteria genome-equivalents in a conventional milliliter of washings from the nasopharyngeal swabs (aspirate) of the monkeys one-time and two-time in­fected with virulent and attenuated BP bacteria. The repeated inoculation with attenuated BP bac­teria was performed 6 months after the 1st dose and the experimental infection with virulent BP bacteria was performed 12 months after the re-immunization. To clarify the relationship between the elimina­tion and bacterial strains as well as the infecting dose, adult RMs were infected with two doses (107 CFU and 109-1010 CFU) of virulent BP 18323 bacteria. Each of the doses was administered intranasally to 3 RMs. All the RMs were infected for a second time with one dose of 109-1010 bacteria. The repeated experimental infec­tion with virulent BP 475 or BP 18323 was performed 4-6 months after the 1 st inoculation. Fig. 1 and 2 show that the number of bacteria in the RMs’ nasopharynxes reaches its maximum 7-14 days after the experimen­tal infection with attenuated BP 4MKS bacteria (tmax= 7-14 days), 14-21 days after the infection with isogen­ic virulent BP 475 bacteria and 7-10 days after the in­tranasal inoculation with virulent BP 18323 bacteria.

Fig. 1. Changes in the number of BP genome-equivalents in the RMs' nasopharynx after the first and repeated intranasal vaccination with BP bacteria.

N — the number of BP genome-equivalents in 1 ml of the nasopharyngeal aspirate. 1 — the first vaccination with virulent BP bacteria; 2 — the repeated vaccination with virulent BP bacteria; 3 — the first vaccination with attenuated BP bacteria; 4 — the repeated vaccination with attenuated BP bacteria; 5 — the infection with virulent BP bacteria 12 months after the vaccination with attenuated BP bacteria.

Fig. 2. Changes in the number of BP 18323 genome-equivalents after the 1st and 2nd experimental intranasal infection of RMs. 1 — the 1st experimental intranasal infection at a dose of 107 CFU; 2 — the 1st experimental intranasal infection at a dose of 109 CFU; 3 — repeated experimental infection with BP 18323 at a dose of 109-1010 CFU.

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

Fig. 3. Changes in the IgG level in the RMs' blood serum after the 1st and repeated intranasal vaccination with BP bacteria. On the vertical axis — the relative value of IgG levels (%): ODC+/ODi, where ODC+ — optical density of the positive control, ODi — optical density in the well with the studied serum.

1 — the 1st vaccination with attenuated BP bacteria; 2 — the 1st vaccination with virulent BP bacteria; 3 — the infection with virulent BP bacteria 12 months after the vaccination with attenuated BP bacteria; 4 — the repeated vaccination with virulent BP bacteria; 5 — the repeated vaccination with attenuated BP bacteria.

Fig. 4. Changes in the IgG relative levels in the RMs' blood serum after the 1st and repeated intranasal infection of the animals with BP 18323 bacteria.

1 — the 1st experimental intranasal infection at a dose of 107 CFU; 2 — the 1st experimental intranasal infection at a dose of 109 CFU; 3 — the repeated experimental infection with BP 18323 at a dose of 109 CFU.

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 28th day after the repeated infection (immunization), bacteria were not detected or were detected only individual cases, while during the same period after the 1st 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].

To analyze the relationship between the changes in the bacterial growth and development of an immune response from the infecting dose and the bacteria strains, along with isogenic virulent and attenuated BP 475 bacteria and 4MKS bacteria, we used virulent BP18323 bacteria, which are used for evaluation of protective activity of wPVs. The bacteria were vaccinated to 3 RMs at a dose of 109-1010 CFU and to 3 RMs at a dose of 107 CFU. The number of bacteria and changes in growth/eradication after the experimental infection of RMs vaccinated with 109 bacteria did not show any statistically significant differences as compared to the results obtained from infection of animals with other BP strains. The tmax values we measure for BP 475, 18323 and 4MKS are similar and correlate with the values given in the work of J.M. Warfel et al. [19] for BP D420 bacteria; the latter values were obtained after infecting of Anubis infant baboons.

During the entire development of the 1st 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 1st 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 10th - 14th 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.

The similar dynamics was observed during 2 and 3-time experimental infections of different species of monkeys of the Old World monkeys with virulent BP bacteria, including infection at a dose of 1010-1011 CFU. All the cases demonstrated a pronounced boosted humoral immune response and rapid elimination of the pathogen after the repeated infection [22]. The presence ofthe booster effect produced by the repeated vaccination at low or even zero levels of IgG in some animals after the 1st vaccination as well as the rapid elimination of bacteria in all the animals after the repeated vaccination suggest that even one-time intranasal vaccination with attenuated bacteria can be sufficient and can provide protection against experimental infection.

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.

References

1. Pertussis vaccines: WHO position paper – September 2015. Wkly. Epidemiol. Rec. 2015; 90(35): 433-60.

2. Wood N., McIntyre P. Pertussis: review of epidemiology, diagnosis, management and prevention. Paediatr. Respir Rev. 2008; 9(3): 201-11. DOI: http://doi.org/10.1016/j.prrv.2008.05.010

3. Wiley K.E., Zuo Y., Macartney K.K., McIntyre P. Sources of pertussis infection in young infants: a review of key evidence informing targeting of the cocoon strategy. Vaccine. 2013; 31(4): 618-25. DOI: http://doi.org/10.1016/j.vaccine.2012.11.052

4. Pinto M.V., Merkel T.J. Pertussis disease and transmission and host responses: insights from the baboon model of pertussis. J. Infect. 2017; 74(Suppl. 1): S114-9. DOI: http://doi.org/10.1016/S0163-4453(17)30201-3

5. Medkova A.Yu., Alyapkina Yu.S., Sinyashina L.N., Amelina I.P., Alekseev Ya.I., Karataev G.I., et al. The prevalence of subclinical forms of pertussis and analysis of phase states of bacteria Bordetella pertussis. Detskie infektsii. 2010; 9(4): 19-22. (in Russian)

6. CDC. Pertussis epidemic – Washington, 2012. MMWR Morb. Mortal. Wkly Rep. 2012; 61(28): 517-22.

7. Rosewell A., Spokes P., Gilmour R.E. NSW Annual vaccine-preventable disease report, 2011. NSW Public Health Bull. 2012; 23(9-10): 171-8. DOI: http://doi.org/10.1071/NB12086

8. Gonfiantini M.V., Carloni E., Gesualdo F., Pandolfi E., Agricola E., Rizzuto E., et al. Epidemiology of pertussis in Italy: disease trends over the last century. Euro Surveill. 2014; 19(40): 20921. DOI: http://doi.org/10.2807/1560-7917.es2014.19.40.20921

9. Health Protection report. Confirmed pertussis in England and Wales: data to end – November 2012. Available at: http://webarchive.nationalarchives.gov.uk/20140505162355/http://www.hpa.org.uk/hpr/archives/2012/news5112.htm

10. The Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing. Infectious diseases in the Russian Federation in January–October 2018. Moscow; 2018. (in Russian)

11. Lobzin Yu.V., Kharit S.M. The problem of vaccination: a brief history, state-of-the-art, and ways of solution. Epidemiologiya i infektsionnye bolezni. Aktual'nye voprosy. 2014; (6): 30-7. (in Russian)

12. Warfel J.M., Zimmerman L.I., Merkel T.J. Acellular pertussis vaccines protect against disease but fail to prevent infection and transmission in a nonhuman primate model. Proc. Natl. Acad. Sci. USA. 2014; 111(2): 787-92. DOI: http://doi.org/10.1073/pnas.1314688110

13. Kilgore P.E., Salim A.M., Zervos M.J., Schmitt H.J. Pertussis: microbiology, disease, treatment, and prevention. Clin. Microbiol. Rev. 2016; 29(3): 449-86. DOI: http://doi.org/10.1128/CMR.00083-15

14. Chen Z., He Q. Immune persistence after pertussis vaccination. Hum. Vaccin. Immunother. 2017; 13(4): 744-56. DOI: http://doi.org/10.1080/21645515.2016.1259780

15. Guiso N., Njamkepo E., Vié le Sage F., Zepp F., Meyer C.U., Abitbol V., et al. Long-term humoral and cell-mediated immunity after acellular pertussis vaccination compares favourably with wholecell vaccines 6 years after booster vaccination in the second year of life. Vaccine. 2007; 25(8): 1390-7. DOI: http://doi.org/10.1016/j.vaccine.2006.10.048

16. Amirthalingam G., Andrews N., Campbell H., Ribeiro S., Kara E., Donegan K., et al. Effectiveness of maternal pertussis vaccination in England: an observational study. Lancet. 2014; 384(9953): 1521-8. DOI: http://doi.org/10.1016/S0140-6736(14)60686-3

17. Semin E.G., Sinyashina L.N., Medkova A.Yu., Karataev G.I. Construction of recombinant attenuated Bordetella pertussis bacteria PtxP3. Journal of microbiology, epidemiology and immunobiology. 2018; (4): 33-41. DOI: https://doi.org/10.36233/0372-9311-2018-4-33-4118 (In Russ.)

18. Sinyashina L.N., Semin E.G., Medkova A.Yu., Syundyukova R.A., Karataev G.I. Pre-clinical toxicity study and safety assessment of candidate live pertussis vaccine for intranasal administration. Epidemiologiya i vaktsinoprofilaktika. 2018; 17(6): 98-108. DOI: http://doi.org/10.31631/2073-3046-2018-17-98-108 (in Russian)

19. Warfel J.M., Beren J., Kelly V.K., Lee G., Merkel T.J. Nonhuman primate model of pertussis. Infect. Immun. 2012; 80(4): 1530-6. DOI: http://doi.org/10.1128/IAI.06310-11

20. Warfel J.M., Zimmerman L.I., Merkel T.J. Comparison of three whole-cell pertussis vaccines in the baboon model of pertussis. Clin. Vaccine Immunol. 2015; 23(1): 47-54. DOI: http://doi.org/10.1128/CVI.00449-15

21. Locht C., Papin J.F., Lecher S., Debrie A.S., Thalen M., Solovay K., et al. Live attenuated pertussis vaccine BPZE1 protects baboons against B. pertussis. J. Infect. Dis. 2017; 216(1): 117-24. DOI: http://doi.org/10.1093/infdis/jix254

22. Kubrava D.T., Medkova A.Yu., Sinyashina L.N., Shevtsova Z.V., Matua A.Z., Kondzhariya I.G., et al Experimental whooping cough of nonhuman primate. Vestnik Rossiyskoy akademii meditsinskikh nauk. 2013; 68(8): 28-33. (in Russian)

23. Medkova A.Yu., Karataev G.I., Shevtsova Z.V., Matua A.Z., Semin E.G., Amichba A.A., et al. Epizootic pertussis focus of hamadryad baboons. Zhurnal infektologii. 2015; 7(3): 103-11. DOI: http://doi.org/10.22625/2072-6732-2015-7-3-103-111 (in Russian)

24. Medkova A.Yu., Alyapkina Yu.S., Sinyashina L.N., Amelina I.P., Alekseev G.I., Bokovoy Ya.I., et al. Detection of avirulent insertional Bordetella pertussis bvg-mutants in patients with pertussis and with upper respiratory tract infection and in seemingly healthy people. Molekulyarnaya genetika, mikrobiologiya i virusologiya. 2010; (4): 27-31. (in Russian)

25. Bidet P., Liguori S., De Lauzanne A., Caro V., Lorrot M., Carol A., et al. Real-time PCR measurement of persistence of Bordetella pertussis DNA in nasopharyngeal secretions during antibiotic treatment of young children with pertussis. J. Clin. Microbiol. 2008; 46(11): 3636-8. DOI: http://doi.org/10.1128/JCM.01308-08

26. Li R., Cheng C., Chong S.Z., Lim A.R., Goh Y.F., Locht C., et al. Attenuated Bordetella pertussis BPZE1 protects against allergic airway inflammation and contact dermatitis in mouse models. Allergy. 2012; 67(10): 1250-8. DOI: http://doi.org/10.1111/j.1398-9995.2012.02884.x

27. Nesterova Yu.V., Medkova A.Yu., Babachenko I.V., Semin E.G., Kalisnikova E.L., Sinyashina L.N., et al. Clinical-diagnostic value of Bordetella pertussis genetic markers in contact persons in familial foci. Zhurnal infektologii. 2019; 11(1): 17-24. DOI: http://doi.org/10.22625/2072-6732-2019-11-1-17-24 (in Russia)

28. Karataev G.I., Sinyashina L.N., Medkova A.Yu., Semin E.G., Shevtsova Z.V., Matua A.Z., et al. Insertional inactivation of virulence operon in population of persistent Bordetella pertussis bacteria. Genetika. 2016; 52(4): 422-30. DOI: http://doi.org/10.7868/S0016675816030085 (in Russian)


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
Russian Federation

senior researcher, Laboratory of genetics of bacteria

Head, Department of paediatric infectious diseases



Lyudmila N. Sinyashina
Federal Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya
Russian Federation
leading researcher, Laboratory of genetics of bacteria


Astanda A. Amichba
Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia
Abkhazia
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
Russian Federation
researcher, Laboratory of genetics of bacteria


Zinaida V. Shevtsova
Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia; Abkhazian State University
Abkhazia

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
Abkhazia

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
Abkhazia
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
Abkhazia
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
Abkhazia

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
Abkhazia


Zurab Ya. Mikvabiya
Research Institute of Experimental Pathology and Therapy of the Academy of Sciences of Abkhazia
Abkhazia
D. Sci. (Med.), Prof., director


Gennadiy I. Karataev
Federal Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya
Russian Federation
D. Sci. (Biol.), Head, Laboratory of genetics of bacteria


Views: 141


Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.


ISSN 0372-9311 (Print)
ISSN 2686-7613 (Online)