Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544

Biologicals xxx (2015) 1e8

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Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544 Lata Jain a, Mayank Rawat a, *, Awadhesh Prajapati a, Ashok Kumar Tiwari a, Bablu Kumar b, V.K. Chaturvedi b, H.M. Saxena c, Sarvanan Ramakrishnan d, Jatin Kumar a, Priscilla Kerketta e a

Division of Biological Standardization, Indian Veterinary Research Institute, Izatnagar, UP 243122, India Division of Biological Products, Indian Veterinary Research Institute, Izatnagar, UP 243122, India c Department of Veterinary Microbiology, College of Veterinary and Animal Sciences, Guru Angad Dev Veterinary and Animal Science University, Ludhiana, Punjab, India d Immunology Section, Indian Veterinary Research Institute, Izatnagar, UP 243122, India e Department of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar, UP 243122, India b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 January 2015 Received in revised form 9 June 2015 Accepted 10 June 2015 Available online xxx

The prophylactic efficacies of plain and alum adsorbed lysate were evaluated by direct virulent challenge in mice model. A recently isolated brucellaphage ‘fLd’ was used for generation of lysates. Twenty four h incubated Brucella abortus S19 broth cultures standardized to contain approximately 108 CFU/ml were found suitable for generation of lysates. Three lysate batches produced through separate cycles did not show any significant variation with respect to protein and polysaccharide contents, endotoxin level and phage counts, indicating that compositionally stable lysate preparations can be generated through an optimized production process. Three polypeptides of ~16, 19 and 23 kDa could be identified as immunodominant antigens of the lysate which induced both humoral and cell-mediated immune responses in a dose dependent manner. Results of efficacy evaluation trial confirmed dose-dependent protective potencies of lysate preparation. The lysate with an antigenic dose of 0.52 mg protein and 60 mg CHO adsorbed on aluminium gel (0.1 percent aluminium concentration) exhibited the highest protective potency which was greater than that induced by standard S19 vaccine. Phage lysate methodology provides a very viable option through which an improved immunizing preparation with all desirable traits can be developed against brucellosis, and integrated with immunization programmes in a more efficient manner. © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Keywords: Brucellosis Phage lysate Humoral response Cell mediate immune response Protective efficacy

1. Introduction Brucellosis is an economically important, re-emerging zoonotic infectious disease of worldwide occurrence [1]. The only ways of controlling and eradicating brucellosis are by test and segregation of infected animals, and vaccination of all susceptible hosts. Despite the availability of a WHO recommended smooth live vaccine S19, a rough attenuated strain RB51 for cattle [2], and Rev1 for small ruminants, the search for improved preparations against brucellosis

* Corresponding author. E-mail addresses: [email protected], [email protected] (M. Rawat).

has never ended because none of these vaccines fulfils the requirements of an ideal immunizing agent [3]. Firstly, the residual virulence of live vaccines for humans [4], and the residual abortifacient potential of smooth vaccines for pregnant animals [5] make them less than ideal preparations. No safe and effective vaccine for human use is still available. In bovines, the success of these preparations in field is related to their strategic use in animals of a particular age group. Inability of these vaccines to induce an efficacious cross protection against different Brucella species affecting different animal species limits their applicability [6]. Additionally, when S19 or RB51 preparations are used in animals, the interference of vaccine induced antibody response with

http://dx.doi.org/10.1016/j.biologicals.2015.06.006 1045-1056/© 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Jain L, et al., Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544, Biologicals (2015), http://dx.doi.org/10.1016/j.biologicals.2015.06.006

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L. Jain et al. / Biologicals xxx (2015) 1e8

conventional serological tests [7e11] also creates problems during the surveillance programs. Secondly, production of these vaccines is complicated. A large amount of bacterial biomass is required for a standard dose of S19 or RB51. This poses problems for their availability in the field. The development of an efficacious vaccine for brucellosis has therefore been a challenge for scientists for many years now. To develop better candidates, antigens with different approaches have been tried since last 50 years. Efforts were made towards developing genetically modified and naturally attenuated strains in different adjuvant formulations [12e18], DNA Vaccines [19e23] and defined live mutants [24]. Though variable successes have been claimed, none found the results equivalent to the strain 19 vaccine. Some new Brucella vaccine strains were also proposed, one of which Brucella abortus 82 (Russia) was found to be promising with respect to its pronounced immunogenic and weak agglutinogenic properties [25]. Though the live vaccine from strain 82 was adopted in veterinary practice in Russian Federation, it cannot be regarded as an ideal immunizing preparation since it still possesses all the drawbacks of a live vaccine. In general, live vaccines may provide an appropriate immune response, yet they are generally less favoured than non-living vaccines because all of them also suffer from limitations concerned with safety, and practicability of production on a large scale. The use of lytic phages to inactivate bacteria and the application of the phage lysate as a vaccine antigen has not been properly investigated till now. We have explored this approach to improve upon some of the major drawbacks of currently available preparations against Brucellosis. Bacterial phage-lysis can be considered as a method of antigen extraction by lysing whole bacteria, which does not denature antigenic epitopes. Thus, phage lysates comprise a means of effectively killing bacteria without altering the antigenicity active antigens [26]. The phage lysate contains bacteriophage particles and bacterial antigenic components as a result of cell lysis caused by lytic bacteriophage. The former is primarily responsible for the first phase of therapeutic protection mediated by phage and the latter are associated the second phase of protective response mediated by antibodies or effector cells. A preparation containing complete range of structurally unaltered antigenic moieties of the bacterial cell can be expected to mimic a non-replicating live organism in the host and induce the desirable ‘cross protective’ response at lower doses compared to killed, subunit or live attenuated vaccines. Since the earliest days of phage research, it has been reported that bacteriophage lysates could be effective at eliciting protective immunity against a variety of bacterial pathogens [27] Many workers of the period reported that phage lysates were superior to preparations of whole bacteria for vaccination to prevent bacterial diseases [28,29]. Later, in 1928, Compton [30] compared efficacies of plain-formalinized and phage-lysed plague vaccine preparations, and confirmed that the phage-lysate was superior to the formalin inactivated preparations. Under the current scenario, this methodology appears to provide a better practical and technologically feasible option than all the other conventional and molecular approaches used for improving the Brucella vaccines. We therefore find it desirable to investigate the methodology that is expected to provide an almost ideal immunizing preparation which may be (i) safe and capable of inducing a cross protective response so that the same preparation may be used for prevention of brucellosis in other animal species (ii) capable of inducing an antibody response that declines after a desirable period so that vaccinated reactors are not detected during serological investigations (iii) can circumvent the problem of requirement of large amounts of “antigenic biomass” for the vaccine doses, so that a cheaper commercial preparation with better

keeping quality, transportability and ease of administration could be developed for the immunization programmes. In the present communication we report the methodology of generating compositionally stable phage lysates against S19 with evaluation of the efficacy of the preparation in mice against direct virulent challenge of B. abortus 544. 2. Materials and methods 2.1. Ethics statement All the experimental protocols carried out on laboratory animals were approved by the Animals Ethics Committee (AEC) of Indian Veterinary Research Institute (IVRI), Izatnagar-243122 (India). Animals were kept in AEC approved facilities and received water and food ad lib. For retro-orbital bleeding, mice were anaesthetized using chloroform. For euthanization, an overdose of carbon dioxide was used followed by cervical dislocation. 2.2. Bacterial strains and brucellaphage 2.2.1. Bacteria B. abortus S19, the most widely used vaccine strain for brucellosis, to which other vaccines are compared [31], was obtained from the Brucella Referral Laboratory, Division of Veterinary Public Health, IVRI, and validated for its desired characteristics by the recommended tests. It showed normal properties of CO2-independent biovar 1 strain of B abortus, and did not grow in presence of standard concentrations of benzyl penicillin, thionin blue and ierythritol. Similarly, B. abortus 99, B. abortus 544, Brucella melitensis 16M, Brucella suis 1330 procured were from the Referral Lab. The identity of each Brucella strain was confirmed by morphological, biochemical and serological examinations. The strains were maintained by periodic sub-culturing on Brucella agar (Difco) slopes throughout the period of the study. 2.2.2. Bacteriophage A phage (ɸ LD; Guru Angad Dev Veterinary and Animal Science University, Ludhiana, Punjab, India), which showed consistent lytic activity against B. abortus S19 was isolated and characterized [32e34]. 2.3. Generation and characterization of phage-lysates 2.3.1. Revival, propagation and master phage stock The stock suspension of the phage was sterilized by passing through 0.22 mm PVDF filter (Millipore) before confirming its lytic activity against S19 on Brucella medium soft agar overlay [35,36]. An overlay plate showing plaques visible to the naked eyes was selected for further purification of the phage through three repeated cycles of streaking a single plaque on soft Brucella agar overlay pre-inoculated with purity-checked, 48 h incubated broth culture of S19. Using SM buffer [35], (50 mM Tris-Cl [pH7.5], 0.1 M NaCl, 8 mM MgSO4.7H2O, 0.01% Gelatin) the purified phage was harvested from several plates showing clear lysis around streaked lines, and pooled. The harvest (about 25 ml) was clarified by centrifugation at 4
C and filter sterilized. The phage count of the pool was determined before storing it in small aliquots at 4
C, and under freeze-dried condition as master stock (BrPMSL-I). All future experiments for production of lysates were conducted using the master stock of the phage. The phage titers of the preserved master lot were monitored at regular intervals during the entire period of study. Lytic range of phage: The lytic range of the phage was determined against several gram-positive and gram-negative bacterial

Please cite this article in press as: Jain L, et al., Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544, Biologicals (2015), http://dx.doi.org/10.1016/j.biologicals.2015.06.006

L. Jain et al. / Biologicals xxx (2015) 1e8

species that included B. abortus strain 99, B. abortus 544, B. melitensis 16M, B. suis 1330, Pasteurella multocida (P52), Salmonella Gallinarum, S. Thyphimurium, S. Enteritidis and field isolates of Escherichia coli and Staphylococcus aureus, using spot-inoculation technique [36]. Multiplicity of Infection (MOI) for S19: The optimum phage: bacteria ratio required to achieve maximum lysis of the indicator S19 within shortest period incubation was determined as MOI [36]. 2.3.2. Generation of phage lysates Optimization of conditions and generation of phage-lysates: Conditions for the generation of stable lysates of S19 were optimised by selecting an appropriate medium and suitable methods for harvesting, preparation of bacterial suspensions, viable counts, and Brown's turbidity equivalents for lysis to assess the optimum MOI of phage to be added for lysis as well as duration and temperature of the incubation with phage to ascertain inactivation of B. abortus S19. Three batches of bacteriologically sterile lysates were then prepared using Liquid Cultures [37], and designated as LCPL-I, II and III. All preparations were stored both at 4
C, and under lyophilized condition.

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recommendations for inactivated vaccines before the commencement of immunization. Safety in mice: The test was conducted on both preparations by injecting 0.5 ml of test preparation through intramuscular, subcutaneous, intravenous or intra-peritoneal route into each of five mice kept in 4 separate groups. The animals were observed for untoward reactions or mortality till 7th day of inoculation. 2.4.2. Experimental population and immunization Female Swiss albino mice (4e5 weeks age, 18 ± 2 gms) obtained from Laboratory Animal Research Unit of IVRI. The animals were randomly distributed into eight experimental groups consisting of twelve mice each. The overall experimental schedule has been summarized in Table 2. Each mouse of the groups III to VIII was injected with PL-Plain or AD-Alum adjuvanted lysate in a dose volume of 50, 100, or 200 ml through subcutaneous route. As per the accepted method of OIE [31], each mice of the II group received Brucella S-19 vaccine (1  105 cfu/mice; subcutaneously) and kept as positive control. Mice of group I, kept as negative control, received Buffered Brucella Saline (BSS). 2.5. Evaluation of humoral immune-responses

2.3.3. Characterization of phage lysates Biochemical composition: Biochemical composition of each lysate preparation was determined by estimating total protein [38] and carbohydrate concentrations [39]. The endotoxin content of each was estimated by Limulus Amoebocyte Lysate Assay using EToxate Kit (Sigma) as per the manufacturer's protocol. The phage count was also determined by usual serial dilution method. Protein profiling of phage-lysate: Hyperimmune sera against phage-lysate were produced in rabbits (HIRS) and mice (HIMS) [16]. Acetone precipitated protein concentrates of phage-lysates [40], along with physico-chemically extracted cell-wall proteins of different Brucella species [41] were characterized and compared by SDS-PAGE and Western-blot analyses.

Serum IgM and IgG: Blood samples of 3 randomly selected mice from each group were collected on 0, 20th and 30th day of immunization (DPI) by and pooled to draw serum. All sera samples were stored at 20
C till used for determination of serum IgM and IgG levels against lysate antigens by single dilution method of ELISA [43]. Anti- Brucella abortus hyper immune mice serum (HIMS) and, pre-vaccination mice serum were used as positive and negative controls respectively. 1:6000 diluted mouse anti-IgM and IgG1 and IgG2a conjugates (Santa cruz, USA) were used as secondary antibodies. The readings were taken by an ELISA Reader (Anthos Lab Tech., HT2, Austria) at 492 nm. The Ig titers of individual serum sample were expressed as positive/negative (P/N) ratio. 2.6. Cytokine profile

2.4. Evaluation of immune responses and protective efficacy of lysates 2.4.1. Test preparations B. abortus S19 vaccine was procured from the Division of Biological Products, IVRI, Izatnagar (India). For comparative evaluation of protective efficacy and immune responses of the phage-lysate and the standard vaccine, a pool of equal volumes of the 3 lysate batches (PL-Plain) was used as the final test preparation. The antigenic composition of the preparation been presented in Table 1. Adjuvantation: Sterilized 1 percent aluminium hydroxide gel suspension in saline was mixed with the test lysate preparation in ratio of 1:10 (final aluminium concentration: 0.1 percent), and incubated at 37
C for 24 h. The amount of protein adsorbed was determined by calculating the residual protein in supernatant after centrifugation at 11000 g for 10 min. The preparation was designated as (PL-Ad). Sterility test: Sterility tests on both preparations were carried out as per the Indian Pharmacopoeia, Section 2.2.11 [42]

IL-10, IL-17A, TNF, IFN, IL-6, IL-4 and IL-2: Serum samples used for estimation of Ig titers were also subjected to determination of cytokine levels using the kit procured from BD Biosciences. Manufacturer's protocol was followed for the analysis using BD FACS Calibur instrument for Flow Cytometry. Cytokine levels were measured from standard curves calibrated from standard reagents using the Flow Cytometer's “BD Cell Quest” software and the samples values were evaluated in pg/ml. For data analysis, readings falling below the detectable threshold (0) were disregarded in the statistical analysis. Data was analysed using the using EXCEL statistical software. The test and control groups were analysed using ANOVA. 2.7. Evaluation of cell-mediated response (CMI) CD4þ and CD8þ T-cell population: Blood samples of 3 randomly selected mice from each group were collected on 14th and 28th day

Table 1 Composition of phage lysates and finished (Test) preparations. Preparation

Total protein (mg/ml)

Total carbohydrate (mg/ml)

Endotoxin (EU/ml)

Phage count (PFU/ml)

LCPL-I LCPL-II LCPL-III Test preparation (PL-Plain)a

5.3 5.5 4.8 5.2

590 640 570 600

<2 <2 <2 <2

1.5  107 1.8  107 1.3  107 107

a

Alum adsorbed Test preparation PL-Ad contained 0.1% Aluminium.

Please cite this article in press as: Jain L, et al., Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544, Biologicals (2015), http://dx.doi.org/10.1016/j.biologicals.2015.06.006

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L. Jain et al. / Biologicals xxx (2015) 1e8

Table 2 Schedule for immunization, immune response and challenge studies. Groups N ¼ 12

Immunization (single injection through S/C route)

I II

Unvaccinated controls (0.2 ml BBS) Standard B. abortus strain 19 vaccine (105 cfu) Phage Lysate (PL) Plain 50 ml PL Plain 100 ml PL Plain 200 ml Alum gel-lysate (PL-Ad) 50 ml PL-Ad 100 ml PL-Ad 200 ml

III IV V VI VII VIII

Bleeding For ELISA and cytokine assays N ¼ 3 (randomly selected from each group)

For T cell assays N ¼ 3 (randomly selected from each group)

Pre-vaccination; 10 and 20th DPI; Pre-challenge

14th and 28th DPI

of immunization (DPI) in sterile tubes containing sterile preservative-free heparin (50 units/ml). The CD4þ and CD8þ T-cell response was evaluated by Flow Cytometric Analysis using Mouse T lymphocyte Subset Antibody Cocktail (BD Pharmingen TM; Cat. No. 558391), following manufacturer's protocol. Splenocyte culture and Lymphocyte proliferation assay: On 21st and 28th DPI, 2 randomly selected mice from each group were sacrificed, and their spleens were removed under aseptic conditions. Single-cell suspensions were prepared from the spleens, and the red blood cells were lysed with (150 mM NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA, pH 7.3) solution. Splenocytes were cultured at 37
C in 5% CO2 in a 96-well flat-bottom plate at a concentration of 2  105st cells/well in RPMI 1640 medium supplemented with 2 mM L-glutamine and 10% heat-inactivated fetal calf serum (Sigma), in the presence of phage lysate (0.5 mg protein), ConA (10 mg/ml) or no additives (unstimulated control). At the end of incubation, 20 ml of MTT (5 mg/ml) [3-(4, 5-dimethylthiazol-2-yl-2, 5-diphenyl-tetrazolium bromide; Sigma] was added and reincubated for another 4 h. A 100 ml of culture supernatant was collected carefully from each well. The formazan crystals were dissolved by adding 100 ml of Dimethyl sulfoxide (Amresco, USA) and A570 optical density (OD) reading was taken on a microplate ELISA reader. Blastogenic response was expressed as the mean Stimulation Index (SI), calculated by dividing the mean OD of the stimulated cultures by the mean OD of unstimulated control cultures. 2.8. Determination of protective efficacy against challenge Challenge and collection of spleen samples: Thirty days post immunization, mice were challenged intra-peritoneally with 0.1 ml saline containing 2  105 cfu virulent B. abortus 544 following recommended procedures [31]. At day 15 post challenge all mice were sacrificed by cervical dislocation and spleens were removed aseptically in biosafety cabinet. The body weight of each mice and its spleen was determined. Spleens were stored at 20
C. The efficacy of the phage lysate formulations was determined by estimating total B. abortus S544 counts per spleen and, by estimation of Splenic Weight Index (SWI) [31]. Viable S544 counts of spleens: The total viable counts (TVC) of Brucella abortus S544 per spleen were first recorded as X and expressed as Mean Protective Response (Y), after the following transformation: Mean protective response (Y) ¼ log (X/log X) Mean and Standard Deviation, which are the responses of each group of 6 mice, were calculated. Statistical analysis of this experiment was performed accordingly by one-way analysis of variance

Sacrificed for splenocyte culture (MTT) assay N ¼ 2

Direct challenge N ¼ 6

21st and 28th DPI

On 30th DPI with 2  105 cfu of B. abortus strain 544

(ANOVA) following Duncan's test using an error of 0.05. The test preparation was considered to be protective, if the immunogenicity value obtained in mice was significantly lower than that obtained in the unvaccinated controls and did not differ significantly from that obtained in mice vaccinated with the reference vaccine. Splenic weight index (SWI): SWI was determined by the following formula [31]: SI ¼ Spleen weight (mg)/Body weight of mice (g)  100

2.9. Statistical analysis The data were analysed by one-way analysis of variance (ANOVA) following Duncan's test using an error of 0.05. 3. Results and discussion The first and the most important requirement for developing a phage-based ‘immunotherapeutic’ preparation is selection of consistently lytic phage/phages against the target organism. The anti- B. abortus S19 phage (ɸ LD) was isolated from Punjab (India), and was partially characterized in-terms of its morphological, structural and genetic properties [32e34]. A purified master lot of the phage was raised and maintained by appropriate methods for the present investigations. The phage was further characterized for its biological activities in-terms of its lytic range. The phage showed lytic activity against B. abortus isolates S99 and S544, and also lysed species B. suis 1330 and B. melitensis Rev1. No activity against any other gram-negative or gram-positive organism could be observed. Determination of the lytic range of phages is an important step that helps in selection of phage candidate/candidates for a particular purpose. Phages having across-species or across-genus lytic activity are preferred when they are intended to be used for therapeutic applications. From a vaccinologists' point of view, development of a new vaccine candidate requires experimental data to confirm that consistently stable batches of the finished product can be generated through repeated production cycles and, the batches prepared through a production process pass the mandatory requirements of being safe and immunogenic (clinically protective against challenge) in target animal species in which the composition is intended to be used [44]. B. abortus S19 cultured in Brucella broth (Difco) for 24 h, generated approximately 108 CFU/ml which were an optimal substrate for phage mediated lysis. Following addition of phage at 1MOI, about 90% reduction of visible turbidity with slightly less than 1 log reduction in TVC could be observed after 48 h incubation at 37
C. Lysates batches produced through 3 separate cycles did not

Please cite this article in press as: Jain L, et al., Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544, Biologicals (2015), http://dx.doi.org/10.1016/j.biologicals.2015.06.006

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scale production in countries where the disease exists. In addition, processes involving physico-chemical means of crude or purified antigen extraction alter the structural epitopes of the cell wall antigens [51] resulting in their poor, mono-specific immune response that requires high antigenic mass. Preparations incorporating such antigens are considered to be of limited value as they do not impart protection against challenge or infection [52e54] and, are therefore unsuitable vaccine candidates. A more feasible proposition may be a whole cell based adjuvant formulation in which the organisms, grown in a medium that permits full expression of all antigenic components are disrupted by such a ‘process’ that allows all cell wall antigens to be liberated in their structurally unaltered state. As compared to the other physico-chemical methods, bacterial phagelysis can be considered as a method of inactivation or antigen extraction that does not or minimally alter the configurations of antigenic epitopes of the organism. A phage-lysate preparation containing complete range of structurally unaltered antigenic moieties of the bacterial cell in a solubilized state is therefore expected to be a significantly better immunizing composition than any of the preparation containing purified, recombinant or synthetic protein antigen/antigens. To compare the immunological responses of S19 vaccine and phage lysate, immunoglobulin titers, cytokine profiles, T-cell responses and lymphocyte proliferation assay (LTT) or delayed hypersensitivity (DTH) of the vaccinated mice groups were evaluated by appropriate techniques. The data on immunoglobulin (Table 3) and cytokine profiles (Table 4), CD4þ and CD8þ T cell response (Table 5) and LTT (Table 6) indicate that the phage lysates induced both humoral and CMI responses in a dose dependent manner. The group that received antigenic dose of 0.52 mg proteins and 60 mg carbohydrates with 0.1% alum showed serum IgM and IgG titers and CMI responses comparable to that of standard S19 vaccinate group. In order to develop better vaccine candidates for brucellosis, an understanding of the protective immune-response to Brucella is required. Since the organism is considered to be a facultative intracellular pathogen, cell mediated immune mechanisms have been over emphasized since long [55] leading to a dogmatic preference for living vaccines. Experiments with passively transferred CD4þ, CD8þ or whole T cell populations from immunized mice to naïve mice however, have not conclusively proven a predominant role of CMI in affording “clinical protection” against the organism. On the contrary, most experiments with passively transferred antibodies from immunized mice to naïve mice have indicated that antibodies alone are capable of providing protection against Brucella infection, and the protection against the organism is largely due to anti-lipopolysaccharide (LPS) antibodies, with T-cell mediated responses having a subsidiary role [56e58]. Recent insights on molecular mechanisms of entry of Brucella in host cells also support the major inhibitory role of antibodies in initial establishment, subsequent spread and intracellular persistence of infection [3,59].

show any significant variation with respect to protein, polysaccharide and endotoxin contents, and phage counts, indicating that compositionally stable phage lysate preparations can be generated through an optimized production process (Table 1). Endotoxin levels of the 3 batches were found to be < 200 ng/50 ml which was well within the accepted safe limits for the lysate preparations intended to be used as vaccines, where the safety limit is 500,000 EU/ml [26]. Strains of B. abortus, B. suis and B. melitensis showed strong agglutination activity with hyperimmune rabbit serum (HIRS) raised against lysate. Brucella ovis reacted weakly in rapid slide agglutination tests. This indicates that S19 possesses cell wall antigens that are able to generate the antibodies that cross-react with different species of the Brucella genus. One conclusion that can be drawn in the light of this observation is that, development of a single immunizing composition with cross-protective capabilities against brucellosis of different animal species and human beings is possible with phage lysate preparations. A comparative analysis of SDS PAGE and immunoblot profiles of acetone precipitated phage lysate and physico-chemically extracted cell wall preparations of S19 was carried out to identify the major immuno-dominant cell wall proteins in the preparations. Phage lysates revealed presence of about 15 polypeptide bands with apparent molecular mass ranging between 10 kDa and 180 kDa in SDS PAGE. In comparison, cell wall preparations of indicator strain revealed ~21 detectable bands between 10 kDa and 180 kDa. Immuno-blots showed presence of 7 polypeptides of about 95 kDa, 85 kDa, 65 kDa, 50 kDa, 23 kDa, 19 kDa and 16 kDa in both the preparations. Out of these, 3 polypeptides having molecular weight of 16, 19 and 23 kDa reacted strongly with HIRS and were identified as immuno-dominant antigens of the test lysate. The role of various cell wall and cytoplasmic proteins in provoking humoral and cell mediated immune response against Brucella has been investigated in cattle and murine models [12e18]. The efficacies of individual native or rOMPs of Brucella in affording protection against experimental challenge in laboratory animals and cattle heifers have also been reported earlier by several workers [18,45e50] with contradictory and inconclusive findings. Based on the results of these findings, it may be concluded that many Brucella proteins to which infected or vaccinated animals develop appropriate humoral and cell-mediated responses may not play a crucial role in host-acquired protective immune mechanisms to brucellosis. It appears unlikely that any single antigenic component of B. abortus is solely responsible for inducing desirable protective response. All cell wall fractions including LPS, proteins and polysaccharide contribute towards development of ‘clinical protection’ in experimental or target host/hosts. Therefore development of an effective, single antigen based preparation is not possible in near future. In any event, vaccines based on purified extracts or recombinant antigens will not lend themselves to large

Table 3 Serum immunoglobulin response of mice immunized with various phage lysates. Groups/day post immunization

IgG (mean P/N ratio ± SD)* 10th

Unvaccinated control Brucella abortus S19 vaccine PL-Plain (50 ml) PL-Plain (100 ml) PL-Plain (200 ml) PL-Ad (50 ml) PL-Ad (100 ml) PL-Ad (200 ml)

1.264 2.830 2.326 2.748 2.758 2.799 2.912 3.111

IgM (mean P/N ratio ± SD)

20th ± ± ± ± ± ± ± ±

a

0.032 0.075d 0.037b 0.042c 0.049cd 0.025cd 0.033e 0.019f

1.406 4.897 2.465 3.959 4.486 3.714 4.796 4.742

30th ± ± ± ± ± ± ± ±

a

0.084 0.029f 0.219b 0.083c 0.040d 0.067e 0.070f 0.074f

1.341 5.757 3.178 4.992 5.223 4.488 5.460 3.648

± ± ± ± ± ± ± ±

a

0.060 0.052h 0.086b 0.062e 0.055f 0.047d 0.108g 0.213c

IgG1/IgG2a

10th

20th

30th

10th

20th

30th

1.176 2.126 1.857 2.291 2.165 2.277 1.755 1.885

1.179 2.585 2.311 2.434 2.595 2.233 3.615 2.689

1.261 3.140 1.389 1.604 2.198 2.070 3.395 2.019

0.994 1.065 1.109 1.167 1.064 1.070 0.943 1.001

1.000 1.176 1.236 1.061 1.037 1.175 1.218 1.107

1.106 1.254 1.136 1.200 1.351 1.291 1.346 1.353

*Different superscripts (a, b, c, d, e, f, g, h) differ significantly (p < 0.05).

Please cite this article in press as: Jain L, et al., Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544, Biologicals (2015), http://dx.doi.org/10.1016/j.biologicals.2015.06.006

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L. Jain et al. / Biologicals xxx (2015) 1e8

Table 4 Mean serum cytokine profile of mice groups vaccinated with various phage lysates. Group

Serum concentration of various cytokines in pg/ml (mean ± SD*) on day 10 PV IL-10

Control S19 PL-Plain 50 ml PL-Plain 100 ml PL-Plain 200 ml PL-Ad 50 ml PL-Ad 100 ml PL-Ad 200 ml Group

IL-17A

17.87 607.00 26.04 39.91 56.27 129.02 305.86 342.11

± ± ± ± ± ± ± ±

a

2.57 24.56f 1.90ab 3.21ab 4.12b 4.81c 14.84d 29.44e

1.55 42.34 16.56 19.62 23.51 27.64 37.61 24.48

± ± ± ± ± ± ± ±

TNF a

0.35 4.60e 1.27b 1.86bc 1.05cd 1.82d 4.32e 1.47cd

37.65 242.48 41.39 54.76 67.83 54.50 110.51 137.26

IFN ± ± ± ± ± ± ± ±

a

1.48 8.99f 1.51a 1.24b 1.97c 2.64b 6.05d 3.57e

11.061 120.87 46.17 65.28 87.85 88.78 120.06 77.76

IL-6 ± ± ± ± ± ± ± ±

a

2.39 4.80f 2.64b 2.76c 2.03de 4.64e 9.29f 1.84d

± ± ± ± ± ± ± ±

2.23 6.34g 2.18b 2.16b 1.83c 3.05d 4.84f 1.72e

± ± ± ± ± ± ± ±

1.36a 11.94h 1.53b 1.02c 3.53d 8.06e 9.16g 1.39f

± ± ± ± ± ± ± ±

1.93a 8.68h 1.88b 1.09c 4.39d 4.56f 1.77g 2.41e

3.91 70.29 27.26 41.18 43.50 45.48 62.79 47.30

IL-2 a

± ± ± ± ± ± ± ±

0.92 3.89e 1.73b 2.06c 1.63c 3.05c 4.14d 1.34c

± ± ± ± ± ± ± ±

0.53a 2.06e 2.77b 2.55c 1.45c 5.61c 5.65e 2.10d

± ± ± ± ± ± ± ±

0.74a 2.52g 1.54b 1.99c 1.53d 2.39d 4.50f 2.22e

3.98 42.11 12.82 18.91 24.65 28.36 36.49 32.12

± ± ± ± ± ± ± ±

1.07a 1.78h 0.78b 1.23c 0.98d 1.77e 1.68g 0.42f

± ± ± ± ± ± ± ±

0.91a 2.98g 0.52b 2.00c 0.16d 0.86de 1.00f 0.78e

± ± ± ± ± ± ± ±

0.75a 1.09f 0.57b 1.16c 0.13d 0.64d 0.71e 0.91d

Serum concentration of various cytokines in pg/ml (Mean ± SD*) on day 20 PV IL-10

IL-17A ± ± ± ± ± ± ± ±

1.37a 33.11g 2.79b 5.50bc 2.47c 25.88d 13.17f 10.77e

2.33 26.68 9.22 9.92 12.67 13.26 16.32 12.75

± ± ± ± ± ± ± ±

TNF 0.24a 2.77f 0.83b 0.51bc 0.72cd 0.73d 1.64e 0.53cd

39.69 287.56 62.41 90.27 95.51 100.38 252.25 180.10

IFN ± ± ± ± ± ± ± ±

1.23a 5.53g 1.90b 1.59c 2.80cd 3.76d 5.82f 2.91e

9.51 133.73 51.38 56.68 64.61 66.72 88.69 62.48

IL-6 ± ± ± ± ± ± ± ±

2.61a 6.61e 0.98b 2.78bc 1.07c 3.68c 8.00d 1.51c

Control S19 PL-Plain 50 ml PL-Plain 100 ml PL-Plain 200 ml PL-Ad 50 ml PL-Ad 100 ml PL-Ad 200 ml

21.31 815.46 58.70 79.36 105.385 160.39 535.43 406.28

Group

Serum concentration of various cytokines in pg/ml (Mean ± SD*) on day 30 PV IL-10

Control S19 PL-Plain 50 ml PL-Plain 100 ml PL-Plain 200 ml PL-Ad 50 ml PL-Ad 100 ml PL-Ad 200 ml

23.63 207.78 67.30 71.96 93.79 114.44 175.96 156.84

IL-4 a

IL-17A

20.29 723.11 44.47 65.61 76.72 153.38 446.28 263.10

± ± ± ± ± ± ± ±

2.33a 23.38f 2.00ab 3.15b 3.06b 19.46c 23.24e 7.33d

2.48 20.45 6.8 7.79 9.41 7.68 12.32 7.68

± ± ± ± ± ± ± ±

TNF 0.87a 3.35d 0.76b 0.52b 0.22bc 0.42b 1.54c 0.24b

38.69 331.95 93.37 109.06 147.97 179.21 309.20 202.16

20.82 347.60 94.26 113.34 129.88 158.37 221.90 207.14

IFN ± ± ± ± ± ± ± ±

1.60a 9.99h 2.49b 2.08c 3.94d 4.53e 4.70g 9.08f

11.74 105.19 24.2 30.29 40.26 51.37 77.65 37.63

IL-4

IL-6 ± ± ± ± ± ± ± ±

1.06a 3.13g 1.37b 2.04c 1.15d 1.73e 1.16f 1.48d

20.33 490.62 107.06 168.64 186.52 313.94 363.82 303.46

4.75 94.53 41.31 60.41 67.17 66.66 89.27 80.00

IL-2 4.72 59.25 16.15 23.90 37.21 40.04 45.44 41.26

IL-4 3.37 167.65 52.67 73.24 84.34 87.66 132.51 95.30

IL-2 3.16 27.08 9.22 13.96 16.41 17.86 21.85 16.82

*Different superscripts (a, b, c, d, e, f, g) in each column differ significantly (p < 0.05).

Table 5 T-cell response of mice immunized with various phage lysates. Group

14th DPI

28th DPI

% CD4þ Control S19 PL-Plain 50 ml PL-Plain 100 ml PL-Plain 200 ml PL-Ad 50 ml PL-Ad 100 ml PL-Ad 200 ml

17.60 25.92 17.96 18.87 23.46 21.70 23.05 19.17

± ± ± ± ± ± ± ±

% CD8þ 0.41 0.50 0.39 0.40 0.85 0.35 1.04 0.28

11.13 16.83 11.31 12.62 13.4 13.32 16.05 12.07

± ± ± ± ± ± ± ±

0.30 0.79 0.32 0.42 0.13 0.44 0.38 0.26

CD4þ/CD8þ ratio

%CD4þ

1.5813:1 1.540:1 1.5880:1 1.496:1 1.751:1 1.6291:1 1.4361:1 1.588:1

17.33 19.04 14.68 17.40 17.89 16.96 16.78 17.66

In present investigation, the bacteriophage lysate, an inactivated composition of complete and structurally unaltered antigenic repertoire of Brucella abortus, has shown to induce both humoral and CMI responses at a very low dose as compared to the live vaccine indicating that the immunizing agents generated through this methodology hold great potential to be developed into an improved vaccine. With the objectives to evaluate the protective efficacies of plain and alum-adsorbed phage lysate preparations in comparison with the standard S19 vaccine, and to determine their minimum protective dose in terms of protein and carbohydrate concentrations, challenge infection of various immunized mice groups with S544 was conducted. Protective efficacy of each preparation was evaluated in terms of Mean Protective Response (Y) ¼ log (X/log X) for its group; where X is TVC of S544 in each spleen. Protective values (Y) for each of 6 mice of all groups have been presented in Table 7. The protective efficacies of test preparations have been summarized in

± ± ± ± ± ± ± ±

%CD8þ 0.36 0.27 0.42 0.35 0.38 0.34 0.75 0.29

10.55 13.44 10.92 11.36 12.61 12.83 13.23 12.95

± ± ± ± ± ± ± ±

CD4þ/CD8þ ratio 0.44 0.58 0.17 0.24 0.25 0.43 0.25 0.44

1.547:1 1.416:1 1.344:1 1.531:1 1.418:1 1.321:1 1.268:1 1.363:1

Table 8. Protection was defined as a significant reduction in the TVC of S544 in spleen of various immunized group in terms of Y value. On day 15 post challenge, all the lysate vaccinated groups showed significant (P < 0.05) reduction in bacterial load or TVC in Table 6 Splenocyte Proliferation Response of mice immunized with various phage lysates. Group

Stimulation index (SI) ± SD* 21st DPI

Unvaccinated Control Brucella abortus S19 vaccine PL-Plain (50 ml) PL-Plain (100 ml) PL-Plain (200 ml) PL-Ad (50 ml) PL-Ad (100 ml) PL-Ad (200 ml)

1.107 1.649 1.311 1.462 1.535 1.420 1.560 1.545

± ± ± ± ± ± ± ±

30th DPI a

0.025 0.128f 0.022b 0.042cd 0.019de 0.033c 0.012e 0.013de

1.083 2.434 1.390 1.498 1.793 1.582 1.941 1.609

± ± ± ± ± ± ± ±

0.037a 0.047g 0.024b 0.024c 0.042e 0.032d 0.062f 0.026d

*

Different superscripts (a, b, c, d, e) differ significantly (p < 0.05).

Please cite this article in press as: Jain L, et al., Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544, Biologicals (2015), http://dx.doi.org/10.1016/j.biologicals.2015.06.006

L. Jain et al. / Biologicals xxx (2015) 1e8

7

Table 7 Determination of protective response (Y) in mice. Mean Y ± SE*

Transformation value (Y) ¼ log(Xa/log X) of each of the six mice in a group

Group

Control S19 PL-Plain (50 ml) PL-Plain (100 ml) PL-Plain (200 ml) PL-Ad (50 ml) PL-Ad (100 ml) PL-Ad (200 ml)

1

2

3

4

5

6

4.6751 2.4626 3.6306 2.9835 2.1486 2.7007 2.5038 2.8590

4.5961 0.8545 3.0905 2.8667 2.8007 1.8193 2.2820 2.4878

4.7643 2.0181 3.9391 3.4996 1.7888 2.0209 0.8545 2.7076

5.1103 2.5907 2.8815 3.2316 2.0584 3.2196 2.1118 2.3910

4.6460 2.7426 3.7546 3.3979 3.0259 2.3637 0.8545 2.2294

5.0485 2.5153 3.7546 3.6474 3.1532 2.6483 2.1939 2.6148

4.81 2.20 3.47 3.27 2.50 2.46 1.80 2.55

± ± ± ± ± ± ± ±

0.09d 0.29ab 0.17c 0.12c 0.23b 0.21b 0.30a 0.09b

*Different superscripts (a, b, c, d) differ significantly (p < 0.05). a Total Viable Count of Brucella abortus S544 for the mouse spleen.

Table 8 Evaluation of protective efficacy. Group

1 2 3 4 5 6 7 8

Treatment

Control Unvaccinated S 19 Vaccine 105 CFU/mice PL-Plain (50 ml) PL-Plain (100 ml) PL-Plain (200 ml) PL-Ad (50 ml) PL-Ad (100 ml) PL-Ad (200 ml)

Antigenic content administered

Protective response

Protein (mg)

CHO (mg)

By mean SWIa

e e 0.26 0.52 1.04 0.26 0.52 1.04

e e 30 mg 60 mg 120 mg 30 mg 60 mg 120 mg

1.668 0.495 0.942 0.716 0.620 0.650 0.517 0.566

mg mg mg mg mg mg

± ± ± ± ± ± ± ±

0.158e 0.129a 0.157d 0.107c 0.129abc 0.065bc 0.118ab 0.051abc

Mean Y 4.81 2.20 3.47 3.27 2.50 2.46 1.80 2.55

± ± ± ± ± ± ± ±

0.09d 0.29ab 0.17c 0.12c 0.23b 0.21b 0.30a 0.09b

Protective index ¼ (mean Y (Mean Y vaccinated)

control)

e

Nil 2.61 1.34 1.54 2.31 2.35 3.01 2.26

*Mean value bearing Different superscripts (a, b, c, d) differ significantly (p < 0.05). a Different superscript a, b differ significant (p < 0.05).

the spleen (Y value from 3.47 to 1.80) as compared to unvaccinated control group (Y value 4.81) confirming dose-dependent protective efficacy of the phage lysate. Interestingly, the Y value (1.80) for the vaccinate group VII receiving 100 ml lysate containing 0.52 mg protein and 60 mg carbohydrate with 0.1 percent aluminium gel, was found lower than the Y value (2.20) obtained for the standard S19 vaccine group, indicating that the preparation induced a protective response comparable to the response induced by the standard reference vaccine at a very low antigenic dose. Splenic Weight Index (SWI) values, an indicator of splenomegaly, further confirmed the protective efficacy evaluated by total bacterial load of spleens. Significant increase in SWI was found in the unvaccinated group as compared to the vaccinated groups. The mice group VIII receiving higher immunizing dose, showed reduced survivability score as compared to that of group VII. This observation is in agreement with the unexplained conclusions of de’Herelle [60] that higher doses of phage lysates of Haemorrhagic septicaemia (HS)-causing P. multocida induce weaker protective response that requires more time to be established. In light of the current knowledge of immunology, this observation can now be partially explained through phenomenon of immunological tolerance [61], in which the animals administered with a higher and lower quantity of antigen than what is optimum; develop tolerance against the antigen without inducing a response. The observation on the antigenic contents of lysate is also critical form the point of view the production process of live S19 vaccine. The minimum field dose of S19 (or RB51) vaccines is very high (not lower than 4.0  1010 CFU/dose). A large amount of living Brucella biomass is therefore needed for the production of a very low number of live vaccine doses. This makes production process complex and uneconomical. From the data of the investigation, it may be concluded that through phage lysis, a significantly higher number of doses can be produced from the same antigenic biomass of Brucella required for a single dose of live product.

On the basis of the present investigations, and by reviewing the current status and options for developing better immunizing preparations for brucellosis, it can be concluded that phage-lysate appears to be a methodology with great potential. Such preparations are expected not only to circumvent most limitations of live vaccines, but also have an additional advantage. Being inactivated preparations, their field use at the time of insemination or during late pregnancy will be possible without any danger of abortions. The live vaccine can only be used at calf-hood stage. Further immunogenicity trials in bovines are needed to develop safe dose formulation that is capable of imparting clinical protection during pregnancy period without inducing persistent anti- Brucella antibody titres.

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biologicals.2015.06.006.

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Please cite this article in press as: Jain L, et al., Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544, Biologicals (2015), http://dx.doi.org/10.1016/j.biologicals.2015.06.006