Immunopotentiation of live brucellosis vaccine by adjuvants

Vaccine 28S (2010) F17–F22

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Immunopotentiation of live brucellosis vaccine by adjuvants Alexander A. Denisov a,∗ , Yulia S. Korobovtseva a , Olga M. Karpova a , Alla V. Tretyakova a , Larisa V. Mikhina a , Arkadyi V. Ivanov b , Konstantin M. Salmakov b , Roman V. Borovick a a State Federal Enterprise for Science “Research Centre for Toxicology and Hygienic Regulation of Biopreparations” at Federal Medico-Biological Agency (RCT&HRB), Bld.102A, Lenin Str., Serpukhov, Moscow Region 142253, Russia b Federal State Institution “Federal Centre of Toxicological and Radiation Safety of Animals–All-Russian Research Veterinary Institute” (ARVI), Nauchni Gorodok-2, Kazan, Tatarstan 420075, Russia

a r t i c l e

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Article history: Received 18 March 2010 Accepted 19 March 2010 Available online 1 April 2010 Keywords: Adjuvant Brucellosis Live vaccines Brucella abortus Immune response

a b s t r a c t In a series of studies in SPF and conventional guinea pigs, various adjuvants (larifan, polyoxidonium—PO, natrium thiosulphate—NT, TNF-␤ and Ribi adjuvant system—RAS) were evaluated for their ability to enhance immune responses to the live brucellosis vaccine, Brucella abortus strain 82-PS (penicillinsensitive). Combining adjuvants with S82-PS increased synthesis of antibodies against rough (R) and smooth (S) Brucella antigens. Dynamics and levels of antibodies differed dependent upon the adjuvant. Adjuvants enhanced cell-mediated responses to S82-PS, and phagocytosis by macrophages. Humoral and cellular immune responses stimulated by the adjuvants correlated with increased vaccine protection against experimental challenge. The highest protection was demonstrated by combining TNF-␤ or PO with S82-PS. Our data demonstrates the potential of adjuvants to improve immunogenic properties of live brucellosis vaccines. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction As vaccination remains the single most effective method for preventing infectious diseases, development of new and more efficacious vaccines is of importance in both veterinary and human medicine. The greatest challenge for development of vaccines against bacterial diseases is the development of vaccines against intracellular pathogens [1]. Among such pathogens—bacteria of Brucella genus, which cause chronic infection affecting life-support systems of many mammals including farm animals and humans [2,3]. Cellular immunity involving CD4+ , CD8+ , and ␥␦ T cells is crucial for protection against brucellosis and other infections caused by intracellular bacteria [4]. These different T cell types play distinct and complementary roles in protective immunity. For example, CD4+ T cells produce a range of cytokines that orchestrate the immune response and activate host cells, such as macrophages, to kill the pathogen. CD8+ T cells (cytotoxic T cells) are able to directly kill infected cells [4]. Activated ␥␦ T cells might contribute to protection by their cytolytic activity and their ability to produce inflammatory cytokines [5]. Concurrently, antibodies specific to Brucella outer-membrane proteins may enhance phagocytosis which may contribute to protection [6,7].

∗ Corresponding author. Tel.: +7 4967 39 97 38; fax: +7 4967 39 97 38. E-mail address: [email protected] (A.A. Denisov). 0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.03.054

Live attenuated vaccines which broadly stimulate immune responses, including CD8+ responses, are often the most effective vaccines against intracellular pathogens. These live vaccines are highly immunogenic and can induce high levels of protection with administration of a single dose. Replication of the vaccine strain in vivo and its persistence in an attenuated state within the host stimulates long-term and repeatable protective immunity. Brucella abortus strain 19, developed by U.S. researcher Buck, is an example of a live attenuation brucellosis vaccine [8]. Studies have established that the immune response is controlled by genes (Ir-genes) of the main histocompatibility complex. Therefore, the immunological ability to respond to an infection is not associated with characteristics of the antigen or host-specificity; but is controlled genetically in the host [9]. This may be why currently available live Brucella vaccines (B. abortus 19, RB51) which are highly effective in cattle are less efficacious in other hosts [10–13]. The ideal brucellosis vaccine, that provides protection against all species of brucella in all species of animals, has not been developed, one possible mechanism to improve efficacy is to use immunopotentiating compounds with currently available live vaccines. Adjuvants augment immune responses by direct or indirect immunomodulation, formation of antigen–adjuvant depots, chemoattraction of immune cells to the site of administration, and/or influencing delivery to antigen presenting cells [14,15]. Identification of novel adjuvants and/or adjuvant combinations that can enhance and modulate antigen-specific immune responses

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could be of great importance in the development of more efficacious vaccines [16]. Adjuvants have many different compositions and properties: proteins, lipids, chemical compounds, natural substances of plant or animal origin, immunomodulators, metal salts, etc. [17]. Recently, greater knowledge on the influence of cytokines on the immune system has suggested that cytokines may function as vaccine adjuvants [18]. Cytokine adjuvants may compensate for the deficiency of soluble immunologic regulatory factors particularly when the immune system has been impaired or suppressed [19–21]. However, improper application of cytokine adjuvants could unbalance the immune system by feedback mechanisms. In some instances, cytokine adjuvants have greater clinical effects when combined with other adjuvants. In this study, the effects of different adjuvants are compared on humoral or cellular immune responses to B. abortus strain 82-PS in guinea pigs. Additionally, the adjuvant influence on protection against experimental challenge with virulent Brucella strains was characterized.

cycle and given feed from “Ssniff” (Germany); their clinical parameters were followed. At 35 days after experimental challenge animals were euthanized, and samples of internal organs and lymph nodes obtained for bacteriological analysis. Each sample was plated on liver broth and erythrite agar and incubated at 37 ◦ C for 15 days. Colonization was evaluated by infection index (II) and index of spleen weight (ISW). II was calculated by the formula: II =

A × 100 B×C

where II—infection index in %; A—number of organs and lymph nodes from which Brucella cultures were isolated; B—number of guinea pigs in the experiment; C—number of organs and lymph nodes taken for plating from each guinea pig. ISW was calculated by the formula: ISW =

Spleen weight (mg) × 1000 Guinea pig weight (g)

2. Materials and methods 2.1. Bacterial strains Live brucellosis vaccine from B. abortus 82-PS strain was obtained as a lyophilized culture from the ARVI (Kazan, Tatarstan) [22–24]. After growth on erythrite agar at 37 ◦ C for 48 h, bacterial suspensions were diluted with buffered physiological saline to 109 CFU/ml for immunization of guinea pigs. For challenging animals, virulent B. abortus strain 54-M was grown for 3 days at 37 ◦ C on liver-peptone–glucose–glycerin agar (LPGGA), pH 7.2. Bacteria were washed off the agar with sterile 0.9% NaCl, and the bacterial concentration adjusted to 83 CFU/ml. Concentration of bacteria in the challenge inoculum was determined by standard plate counts. 2.2. Adjuvants The adjuvants polyoxidonium (co-polymer of N-oxide 1,4ethylene piperazine and [N-carboxyethyl]-1,4-ethylene piperazinium bromide) (Immapharma Co., Institute of Immunology, Russia) [25], natrium thiosulphate (DiaM Co., Russia) [26], larifan (a double-chain ribonucleic acid isolated from a bacteriophage of E. coli) (A. Kirchenstein Institute of Microbiology & Virology University of Latvia) [27], Ribi adjuvant system (RAS) (Sigma, USA) [28] and tumor necrosis factor-␤ (TNF-␤) (RCT&HRB, Russia) were used [29,30] in the study. 2.3. Animals, immunization and challenging Both SPF (n = 120) and conventional (n = 80) male guinea pigs (350–400 g) were obtained from the Animal Breeding Center of the Russian Academy of Sciences or the Charles River Company (Germany). After acclimation for 21 days, animals were s.c. (subcutaneously) immunized in the inguinal region with 1 × 109 CFU of B. abortus strain 82-PS (1 ml volume) simultaneously with adjuvants. Adjuvants were also s.c. administered in the inguinal region with vaccine or in the opposite leg using a previously determined dosages (larifan—0.5 mg total dose; polyoxidonium—0.04 mg total dose; natrium thiosulphate—150 mg total dose; RAS—0.5 ml total dose; and TNF-␤—1 × 105 U total dose). Animals were bled at 2, 4, 8, 12, and 24 weeks after vaccination. At 12 weeks after vaccination, animals received an experimental s.c. challenge containing 83 CFU of B. abortus strain 54-M (1 ml volume) in the opposite inguinal region where vaccination was administered. Animals were maintained for 6 months at 24-h day–night

2.4. ELISA Both rough and smooth Brucella lipopolysaccharide (LPS) antigens were diluted in 0.05 M carbonate–bicarbonate buffer (pH 9.6) and 100 ␮L was placed into separate wells of microtiter plates. After overnight incubation at 4 ◦ C and 4× washing with 0.01 M phosphate buffer (pH 7.4) containing Tween-20 (PB-T), 150 ␮L of 0.01 M phosphate buffer (pH 7.4) containing 1% (w/v) bovine serum albumin was added to each well to prevent non-specific adsorption. After incubation at room temperature (RT) for 1 h, plates were 3× washed with PB-T. For evaluation of antibody responses against Brucella antigens, 0.2 ml of initially 1:2500 (for detection rough antigen) or 1:5 (for detection of smooth antigen) diluted guinea pig’s sera were added to wells containing the rough and smooth antigens. Samples of sera were diluted 1:2 in PB-T across the microtiter plate. Each plate contained a reagent control (RC) well in which the diluting buffer was added instead of serum. After incubation with shaking for 1 h at 37 ◦ C, and 4× washing in PB-T, a peroxidase conjugated goat anti-guinea pig IgG secondary antibody (Sigma) was added at 1:2000 dilution. After washing with PB-T, the reaction was visualized using 0.01% 3,3 -5,5 -Tetramethylbenzidine Liquid Substrate System for ELISA (Sigma), for 30 min and terminated by addition of 50 ␮l of 1.0 M H2 SO4 to each well. Optical density (OD) was read at 450 nm on a microtiter plate reader (“BIO-TEK”, USA). Results were given in indices of reaction (IR) calculated by the formula: IR =

ODsample Av ODRC

where ODsample —optical density of test sample and ODRC —average value of optical density of reagent control.

Av

2.5. Lymphocytes proliferation Blood was collected into citrate buffer. Peripheral lymphocytes were obtained by centrifugation at 1000 rpm (×g) for 10 min. Cells were re-suspended at 1 × 107 cells/ml in medium 199 and 50 ␮L of the lymphocyte suspensions were added to wells of a microtiter plate. Wells also contained 100 ␮L of medium 199 alone (control), or 100 ␮L of medium 199 media containing Brucella antigen (killed S82 cells), 5 ␮g/ml phytohemagglutinine (PHA), or 5 ␮g/ml lipopolysaccharide (LPS) conducted in triplicate. Cell cultures were incubated for 4 days at 37 ◦ C in 5% CO2 . Spontaneous and induced proliferation was evaluated by counting cells. Index of proliferation (IP) was calculated by the

A.A. Denisov et al. / Vaccine 28S (2010) F17–F22

F19

formula: IP =

O−K K

where O—number of cells in wells with mitogen and K—number of cells in wells without mitogen. 2.6. Phagocytosis Peritoneal macrophages were obtained from flushing the abdominal cavity of recently euthanized guinea pigs with physiological saline containing 10 U/ml heparin. After washing in physiological saline, monolayers of peritoneal macrophages were obtained on glass cover slips by adding 106 cell/ml in medium 199 containing 10% of fetal calf serum and 2.0 mM l-glutamine and incubating at 37 ◦ C and 5% CO2 for 60 min. Heat-inactivated Staphylococcus aureus (5 × 107 CFU) were added to 1 ml of medium 199 containing cover slips with adherent macrophages at a bacteria/macrophage ratio of 50:1. After incubation at 37 ◦ C for 40 min, cover slips were washed, stained with azur-eosine, and examined under a microscope. Phagocytosis was assessed by: (1) phagocytosis activity (PA)—percentage of total number of macrophages demonstrating uptake of S. aureus, and (2) phagocytosis index (PI)—average amount of bacteria phagocytized per macrophage. 2.7. Statistic analysis Data were analyzed using the one-sided Student’s t-test. Differences were considered to be statistically significant when P < 0.05. 3. Results 3.1. Serological studies Analysis of reaction of agglutination (RA) data did not reveal positive responses to Brucella in any sample indicating the absence or low level titters against the smooth antigens of Brucella LPS. 3.2. Immune-enzyme assay Combination of B. abortus 82-PS with adjuvant treatments induced synthesis of specific IgG to rough LPS antigens in almost all guinea pigs (Fig. 1). Co-administration of natrium thiosulphate with strain 82-PS induced the highest antibody responses on the ELISA. At 2 and 4 weeks after vaccination, humoral responses

Fig. 2. Dynamics of S-antibodies formation in blood serum of guinea pigs after administration of B. abortus 82-PS strain and adjuvants.

of guinea pigs that received the adjuvant natrium thiosulphate had greater responses than animals inoculated with strain 82-PS alone. Administration of larifan, polyoxidonium, RAS or TNF-␤ with strain 82-PS had peak antibody titers at 2 weeks after vaccination. By 8 weeks after vaccination, serum antibodies as detected by the ELISA had decreased but low titers continued to be detected at 6 months after immunization. ELISA titers (IgG) when smooth LPS antigens were used were lower when compared to titers to rough LPS antigens of Brucella. Use of RAS or larifan caused all animals to develop titers to smooth LPS antigens sooner than other adjuvant treatments. However, animals in all groups had developed titers against smooth LPS antigens by 2 months after vaccination when antibody titers peaked (Fig. 2). Highest titers to smooth LPS antigens were noted with RAS (IR = 10.39 ± 2.43), larifan (IR = 9.82 ± .74) and TNF-␤ (IR = 9.29 ± 2.53) adjuvants. Low titers to smooth LPS antigens were detected in some animals in all treatments up to 6 months after vaccination. 3.3. T cell proliferation Stimulation of cell immunity in guinea pigs following administration of adjuvants simultaneously with vaccine was not clearly pronounced. Enhanced proliferation of lymphocytes following administering of RAS (stimulated by PHA and LPS), larifan or PO (stimulated by PHA), was noted 2 weeks, and with NT—7 weeks after vaccination. TNF-␤ administered simultaneously with vaccine stimulated both spontaneous and induced by mitogens response of guinea pig blood lymphocytes. Proliferate response induced by PHA achieved its maximum by 4th week, and by LPS—after 7 weeks. During 3 months spontaneous and induced proliferate responses in this group of animals remained a little higher compared to control. These data correlated with data on spleen mass coefficients in animals treated with vaccine and TNF-␤. After 3 and 6 months following administration of adjuvants and vaccine the level of cell immunity did not differ from the control. 3.4. Phagocytosis

Fig. 1. Dynamics of R-antibodies formation in blood serum of guinea pigs after administration of B. abortus 82-PS strain and adjuvants.

All adjuvants administered simultaneously with vaccine stimulated phagocytosis activity of peritoneal macrophages within short time after the vaccination, maximal activity was achieved by 4th week and decreased by 8th week. In this case, both general phagocytosis activity of macrophages (PA index) and individual activity of separate macrophage (PI index) were enhanced. The highest PA index within 2–4 weeks was noted when using vaccine and adjuvants RAS, TNF-␤ or NT; PA index for TNF-␤ was the most stable within the whole period of testing. By week 8, the number of

90.0 9

5

9

5

1

–

5

0 2 0 0 1 – 4 7 7 9 – – – – – – – – – – – 2 – – 1 – 4 7 7 9 9 6 8 4 7 10 6 2 3 1

–

% Total Non-ster Ster© Total

1 – 1.0

3 28.0 ± 16.3

2

9 6 6 4 5 10 4 2 – 1

– 1.44 ± 0.06 10

1

– 1.51 ± 0.12 10

28

Virulent strain

82 51 53 30 32 34 29 17 3 9 – – – – – – – – – – 0.44 0.54 0.48 0.28 1.12 0.23 0.10 0.21 0.18 0.15 ± ± ± ± ± ± ± ± ± ± 3.43 3.06 3.31 3.23 2.14 2.92 1.59 1.70 1.82 1.31 9 8 8 8 8 10 10 9 10 10

1. Physiological saline 2. Polyoxidonium 3. Natrium thiosulphate 4. RAS 5. Larifan 6. TNF–␤ 7. B. abortus 82-PS 8. B. abortus 82-PS + Larifan 9. B. abortus 82-PS + RAS 10. B. abortus 82-PS + polyoxidonium 11. B. abortus 82-PS + natrium thiosulphate 12. B. abortus 82-PS + TNF-␤

Vaccine strain

91.1 ± 2.0 63.8 ± 14.4 66.3 ± 13.0 75.0 ± 6.5 53.3 ± 16.5 42.5 ± 11.5 29.0 ± 10.9 18.9 ± 15.4 3.0 ± 1.5 9.0

– – 2 – 2 – 2 – 3 –

Reg Isolated Brucella cultures

II (b) with virulent culture (%)

Gen

Immune animals Infected animals Bacteriological study

Pathomorphological alterations were most frequently noticed in regional inguinal and submandibular lymph nodes, less—in tracheal and mesenteric lymph nodes and were almost absent in the spleen. The character of alterations in inguinal and mesenteric lymph nodes evidenced the reaction of immune system in the form of follicle hyperplasia at the expense of plasmatic cells, lymphocytes and macrophages. The most pronounced alterations were observed within 4 weeks after administration of vaccine in combination with adjuvants larifan (increase of plasmatic cells in inguinal and submandibular lymph nodes and decrease of lymphocytes in mesenteric and tracheal lymph nodes) or PO (hyperplasia in inguinal and submandibular lymph nodes). After 7 weeks, the alterations in inguinal and submandibular lymph nodes were weakly pronounced and almost absent in tracheal and mesenteric lymph nodes. 3.6. Bacteriological studies

Number of animals in group

ISW (a)

macrophages able to participate in phagocytosis had decreased in all groups. Comparison of the dynamics of phagocytosis activity indexes in the groups immunized with the listed adjuvants demonstrated that RAS provides the most efficient stimulation of phagocytosis activity of peritoneal macrophages. Adjuvants administered simultaneously with the vaccine strain also enhanced the ability of separate macrophage to ingest S. aureus cells. Among all the adjuvants RAS, NT or TNF-␤ provided the most noticeable increase of macrophage activity (PI). Therefore, according to the data on individual activity of separate macrophages and general phagocytosis activity, the highest activation of phagocytosis was observed in animals immunized with B. abortus 82-PS vaccine and simultaneous administration of adjuvants RAS, NT or TNF-␤. 3.5. Pathomorphological studies

Test preparations

Table 1 Results of comparative study of adjuvants’ potential for enhancing immunogenic properties of vaccine B. abortus 82-PS strain on guinea pigs (Mean ± S.E.).

50.0

A.A. Denisov et al. / Vaccine 28S (2010) F17–F22

0.0 25.0?? 0.0 0.0 12.5 0.0 40.0 77.7 70.0 90.0

F20

Bacteriological studies of guinea pigs vaccinated with B. abortus 82-PS strain simultaneously with adjuvant administration and challenged in 12 weeks after the vaccination with virulent B. abortus 54-M strain in dose 83 CFU (according to the data obtained by plating on triptose agar) were conducted. These studies demonstrated that all control animals dosed with physiological saline were infected with brucellosis (Table 1). A generalized form of infection was observed in these animals. From 10 guinea pigs 82 cultures of virulent strain were isolated. Index of infection (II) in this group of animals was high (91.1 ± 2.0%). Adjuvants without vaccine did not protect guinea pigs against brucellosis infection. In these groups of animals, high II values (from 42.5 ± 11.5 to 63.8 ± 14.4) were noted; the virulent strain was isolated from many organs. In most animals the generalized form of infection was observed. B. abortus 82-PS vaccine strain administered without adjuvants partially protected animals from brucellosis infection. Among 10 animals, 6 animals were infected: generalized forms of infection were observed in 4 guinea pigs and regional forms of infection—in 2 animals. Low ISW indexes were noticed in animals of this group, and II was considerably lower compared to control. Number of immune animals was 40%. All tested adjuvants administered simultaneously with vaccine enhanced its immunogenic properties: NT by 10%, RAS by 30%, larifan by 37.7%. TNF-␤ and PO adjuvants enhanced immunogenicity of vaccine by 50%. Of these 2 adjuvants TNF-␤ showed more prophylactic potential. Despite only one guinea pig was infected in both groups, animals inoculated with vaccine + PO had generalized form of infection, and 9 cultures of virulent strain were isolated from their internal organs (II = 9%). On the contrary, in one infected

A.A. Denisov et al. / Vaccine 28S (2010) F17–F22

guinea pig inoculated with vaccine and TNF-␤ had regional form of infection and only one culture of virulent strain was isolated from this animal (II = 1%). Therefore, the studies have established stimulating effect of the tested adjuvants on protective immunity of B. abortus 82-PS vaccine strain. The highest protective effect was achieved after simultaneously administration of vaccine with TNF-␤ or PO.

4. Discussion Our studies demonstrated that tested adjuvants had different stimulating effect after their simultaneous administration with live brucellosis vaccine from B. abortus 82-PS strain. Some of adjuvants were more efficacious in inducing of humoral responses by increasing the levels of specific IgG (TNF-␤, RAS, NT). In contrast to administration of vaccine without adjuvants, simultaneous administration of vaccine with these adjuvants induced high synthesis of not only specific IgG to Brucella R-antigen, but also to S-antigen. Dynamics of their synthesis differed depending on the adjuvant selected. These and other adjuvants (larifan, PO) demonstrated their efficiency in enhancing spontaneous and induced by mitogens cell-mediated responses to the vaccine, as well as in enhancing phagocytosis activity of macrophages. However, these effects were less evident. It is noteworthy, that activation of humoral, cellular and nonspecific immunity by the tested adjuvants directly correlated with their contribution in improvement of protective activity of B. abortus 82-PS vaccine against brucellosis infection. In our studies, the highest protective immunity was observed by TNF-␤ or PO. Simultaneous administration of these adjuvants with vaccine provided protection of 90% animals against challenging with virulent B. abortus 54-M strain in 16-fold minimal challenging dose. Our studies confirmed the already obtained data on the role of some cytokines in stimulation of humoral and cellular immunity and enhancing protective immunity against infections [20,21]. The studies also established a critical role of TNF-␤ in increasing the level of brucellosis IgG, T cell responses and general protection against brucellosis infection. A significant outcome of the conducted research was the determination of inducing different immune responses by adjuvants. It is generally recognized that induction of specific immunologic responses to brucellosis vaccines in animals is species-specific. Current knowledge suggests that there are phenotypic differences in bison and elk compared to cattle regarding their response to infection by virulent B. abortus strains or by vaccination with Brucella 19 or RB51 strains [31]. In bison cell immune response was noted while in elk vaccination failed to develop robust cellmediated responses but induced strong humoral response [31]. Strong cellular immune responses are the best indicators of protective immunity against brucellosis known at this time. The lack of cellular immune responses after vaccination may explain why elk are not protected by current Brucella vaccines. Therefore, to increase protection against brucellosis in bison it will be advisably to use with current brucellosis vaccines adjuvants primarily stimulating B-cell responses. In elk, on the contrary, application of adjuvants primarily stimulating T cell responses is more preferable. These studies demonstrated that, adjuvants may be successfully used for stimulation of humoral and cellular immune responses to live brucellosis vaccine. The selection of specific adjuvant depends on type of immunity to be induced and on host specific immune response. To provide effective protection the adjuvants primarily stimulating a weak link of immunity in the target animal should be used.

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Acknowledgements The work was conducted within the framework of the ISTC #2434 Project. The authors thank Prof. Fomin A.M., Plotnikova E.M., Safin G.M., Salmakova A.V., and Kruchkov R.A for assistance in conducting bacteriological studies. Conflict of interest: A. Denisov states that this article was prepared for publication in framework of the ISTC #2434 Project Grant. The PCT patent No. PCT/RU2007/000505 (from 19.09.2007) titled “Device for remote delivery of vaccines and veterinary preparations” was obtained. All other authors declare no conflict of interest.

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