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Brucella phage lysate bacterin induces elevated TLRs and cytokines response in murine model.

Brucellosis is a major bacterial zoonoses worldwide (Manish et al, 2013) caused by Brucella species. B. abortus causes spontaneous abortion in cattle, lead to major economical losses in dairy and chronic infection in humans (Moriyon et al., 2004; Pappas et al., 2005). Vaccination plays a central role in bovine brucellosis control programs and has been successfully used worldwide for decades to control the disease (Olsen and Stoffregen, 2005). The development of an efficacious and safe vaccine for brucellosis has been a challenge for scientists for many years. Despite enormous advances and the development of B. abortus S19 (Graves, 1943) and RB51 (Schurig et al., 2002; Stern et al., 1971) vaccines, the search for improved vaccines has never ended. Although the available vaccines are effective in controlling brucellosis, they have numerous drawbacks like abortifacient potential for pregnant animals (Smith and Ficht, 1990), residual virulence ability for human (Spink et al., 1962), the complex manufacturing process etc. Apart from that, when S19 or RB51 preparations are used in animals, the interference of vaccine induced antibody response with conventional serological tests (Stevens et al., 1994; Stevens et al., 1995; Schurig et al., 2002) also creates problems during the surveillance programs. In past, inactivated Brucella vaccines have also been tried with limited success (Plommet et al., 1970). The major drawback of chemically killed vaccines include high production cost, poor protection, residual virulence, batch to batch variation, denaturation of conformational epitopes, unaccepted local reaction, serological problems (Moriyon et al., 2004).

Continuous efforts by various researchers have been made in past to develop alternate effective and safe vaccine candidate against brucellosis. Till date there is no licensed human vaccine against brucellosis. Several new generation vaccines like recombinant vaccine (Al-Mariri et al., 2001; Oliveira et al.,1996), DNA vaccines (Munoz et al, 2004), genetically modified vaccines, vectored vaccines (Ruecker and Guzman, 2012) have been tried with varying degree of success but none showed the results equivalent to the Brucella S19 vaccine (Schurig et al., 2002).

The use of lytic phages to inactivate bacteria and the application of the phage lysate as a vaccine antigen is an alternate approach to develop candidate vaccine against bacterial disease but it has been poorly explored. Recently the protective ability of phage lysate bacterins against brucellosis (Jain et al., 2015) and Salmonellosis (Verma et al., 2012) has been evaluated by various workers. The exact mechanism for protective ability of this candidate vaccine has been poorly understood. It is hypothesized that due to specific lysis of bacteria by bacteriophage presents large number of unaltered conformational epitopes which can stimulate both innate and adaptive immune response to elicit robust immune response in the body. Amongst various antigens LPS, lipoprotein, Outer membrane proteins, DNA are the major antigens supposed to be present in phage lysate preparation which are considered as important PAMPs to be recognized by innate immune system of defense.

In the recent years, there has much emphasis on improvement of vaccine utilizing better adjuvant targeting innate immune response to enhance both humoral and cell-mediated immunity. The profound capacity of TLRs mediated immunity can be exploited to augment the antigen specific immune response for many of the vaccine antigens. Emerging evidences indicate that engagement of multiple TLRs with their respective agonists can result in cross-talk between them. In an attempt to understand mechanism of immune response elicited by phage lysate in present study we are reporting the TLR mediated immune response of plain and alum adjuvanated phage lysate of B. abortus strain 19 in mice.

MATERIALS AND METHODS

Mice

Apparently healthy female Swiss albino mice (4 to 6 week age, about 20 grm) were obtained from the Laboratory Animal Research (LAR) section unit, IVRI, Izatnagar. The animals were kept under conventional housing condition and provided feed and water ad lib. All animal studies have been conducted as approved by the Ethics Committee of the Indian Veterinary Research Institute, Izatnagar.

Bacterial strain

Culture of B. abortus S19 maintained in Brucella Laboratory, Biological product division, Indian Veterinary Research Institute (IVRI), Izatnagar was used in the study. The purity and identity of culture were tested by morphological, cultural and biochemical examination. Brucellaphage

A phage showing consistent lytic activity against B. abortus S19 was isolated in the Department of Veterinary Microbiology, Veterinary College, Guru Angad Dev Veterinary and Animal Science University, Ludhiana, Punjab, India and used for the present studies.

Generation of phage lysate

Conditions for generation of stable phage lysate of Brucella S19 were optimized in our laboratory system as per Jain et al., 2015 with slight modification. For comparative evaluation of TLR response of phage lysate and the standard Brucella S19 vaccine, a pool of equal volume of 3 lysate batches was used as the final test preparation. Safety test was conducted by injecting 0.5 ml of test preparation through intramuscular, subcutaneous, intravenous or intraperitoneal rout into 5 mice kept in 4 separate groups. The animals were observed for any untoward reaction or mortality till 21st day of inoculation.

Characterization of phage lysate

Biochemical composition of each batch of lysate was determined by estimating total protein (Lowry, 1940) and carbohydrate (concentration Dubois et al., 1956) concentration. The phage count was determined by using serial dilution method.

Experimental population and immunization

4-5 week old healthy swiss albino female mice obtained from Laboratory Animal Research (LAR) unit IVRI Izatnagar were randomly distributed in to four experimental groups consisting of 6 animals each. Each mouse of group I and II were injected with plain phage lysate and alum adjuvanated phage lysate respectively in a dose volume of 0.1 ml through subcutaneous route. Mice of group III received Brucella S19 vaccine (1 X [10.sup.5] cfu/mice: subcutaneously) and kept as positive control. Mice of group IV kept as negative control, received buffered Brucella saline (BSS). Collection of sample

Two randomly selected mice from each group were sacrificed on 3, 7 and 14th day post immunization (DPI). Spleen tissue was collected aseptically, washed three times with PB S and stored in RNA Later (Sigma Aldrich, USA) at -20[degrees]C for RNA isolation.

RNA isolation and cDNA synthesis

Spleen tissues were processed for total RNA isolation using RibozolTM (Amresco, USA) as per the manufacturer's protocol and the complementary DNA (cDNA) was synthesized from total RNA using RevertaidTM First Strand cDNA Synthesis Kit (Thermo Scientific, USA), following manufacturer's instructions. 1 [micro]g of total RNA was treated with 1 [micro]l of DNaseI (Fermentas U SA) prior to reverse transcription to remove any DNA contamination from extracted RNA. The extracted RNA was assessed for quantity and purity using nanodrop Spectrophotometer (nanodrop Ltd, UK) and stored at -80[degrees]C until further use. The reverse transcription was performed using M-MuLVReverse Transcriptase (Thermo scientific, USA). Briefly, 1 [micro]g of total RNA was mixed with 1 [micro]l of random hexamer primers (Thermo scientific, USA) in a 0.2 ml PCR tube and incubated at 65[degrees]C for 5 min to remove any secondary structure within the RNA. Then the tubes were snap chilled on ice which was followed by addition of 4 [micro]l of 5x RT-buffer, 1.5 [micro]l of 10Mm dNTP mix, 1 [micro]l of reverse transcriptase enzyme, 1 [micro]l of RiboLock RNase inhibitor in a final 20 [micro]l volume of reaction. The contents were mixed properly and incubated at 420 C for 1h in a thermo cycler for reverse transcription to occur. Finally the reactions were terminated by heating at 90[degrees]C for 2 min to inactivate M-MuLVRT enzyme. The cDNA prepared was stored at -80[degrees]C for further use.

Real time quantification

The differential mRNA Expression levels of TLR2, TLR4, TLR9, IL-4, [INF-.sup.3] and IL-12 genes were analyzed by real time PCR using the Maxima SYBR Green (Thermo Scientific, USA) on ABI 7900 real time PCR system (Applied Biosystem, USA) using published gene specific primers (Table 1). (2)-Actin was used as the reference gene. Expression levels of the target genes were calculated relative to the expression of the 2-Actin gene and expressed as n-fold increase or decrease relative to the control. Real time RT-PCR was conducted in a final volume of 10 [micro]l containing 5 [micro]l SYBR green PCR master mix (Thermo scientific, USA), 1 [micro]l cDNA as the PCR template and 1 [micro]l (5 pmol) forward and reverse primers. Real time RT-PCR was performed using the following cycling parameters: 1 cycle at 95[degrees]C for 5 min followed by 40 cycles of 95[degrees]C for 15s and annealing at temperature as shown in table 1 for 30s, and a dissociation stage of 95[degrees]C for 15s, 60[degrees]C for 30s, 95[degrees]C for 15s and 60[degrees]C for 15s. The change in the gene expression was calculated as 2-""Ct and the value indicated in an n-fold difference relative to the calibrator.

Statistical analysis

The relative expression of target gene (TLR) were evaluated by analysis of variance (ANOVA) followed by Duncan's post hoc test in IBM SPSS statistics 20 software. Result are expressed as Mean [+ or -] SEM. The level of significance was set at P < 0.05.

RESULTS

Production and characterization of Lysate: Lysate batches produced through 3 separate cycles did not show any significant variation with respect to protein and polysaccharide contents. Carbohydrate and protein concentration of the pooled lysate were found to be 360 [micro]g and 62 [micro]g/ml respectively.

Safety result: Test preparation did not produce any untoward effect in mice till 21st DPI. The relative expression of TLRs gene

Expression level of TLR2 gene was significantly (P < 0.05) up-regulated in all the immunized group at all the time interval under study as compared to unimmunized control group (Fig. 1). Expression of TLR2 was highest in all the immunized group at 3rd DPI which gradually declined up to day 14th. However in alum adjuvanated Phage lysate group after attaining peak on 3rd DPI, declined on 7th DPI and increased again on 14th DPI. TLR2 expression was invariably higher in mice immunized with Brucella S19 vaccine at almost all day interval. The relative expression of TLR4 transcripts were significantly (P < 0.05) up regulated in all the immunized group at all the time intervals under study and peaked at 3rd DPI in all the groups and gradually declined thereafter up to 14th DPI as compared to unimmunized mice group (Fig. 2). But alum adjuvanated phage lysate group showed almost equivalent relative expression level of TLR4 as compared to Brucella S19 on 7th DPI and 14th DPI. Similar trends were observed in expression level of TLR9 gene (Fig. 3)

Cytokine expression profile

There was significant (P < 0.05) increase in IL-4 expression at 3rd, 7th and 14th DPI (Fig. 4) and peaked on 7th DPI in Brucella S19 and plain lysate group but on 14th DPI in alum adjuvanated group. The expression of IFN-3 (Fig. 5) peaked on 3rd DPI in all the immunized group and declined up to 14th day. In case of IL-12, significantly higher (P < 0.01) expression was observed at all the time interval studied after immunization (Fig. 6). IL-12 expression level peaked on 7th DPI at all immunized group and persisted up to 14th DPI. However in group immunized with standard Brucella S19 vaccine the level of expression of almost all genes were significantly higher than lysate group.

DISCUSSION

In this study, we have evaluated the expression of toll like receptor and cytokine transcripts in spleen of mice model against phage lysate bacterin of Brucella S19 to understand the immuno-protective mechanism of Phage lysate bacterin. Phage lysate which contains varied type of microbial components is expected to stimulate innate immune response which can guide towards better adaptive immune response in the body. The most common PAMPs of bacteria such as lipopolysaccharide (LPS), lipoproteins and unmethylated CpG are recognized by TLR4, TLR2 and TLR9 respectively (Kawai and Akira, 2007). TLR signaling induced production of several proinuammatory cytokines (Kawai and Akira, 2006). But no such systemic studies have been conducted in this regard. So in an effort to understand mechanism of protection in the present report, we are reporting TLRs and cytokines mediated immune responses of phage lysate bacterin of B. abortus S19 in mice model via evaluation of TLR2, TLR4, TLR9, IL-4 IL-12 and [INF-.sup.3]. Such kind of study will be helpful in understanding the mechanism of immune protection of phage lysate bacterin. As per previous reports, acute phase of brucellosis start from the 3rd day to the 2nd-3rd week which is marked by rapid increase the bacterial load in target organs, inflammation of the spleen and lymph node (Hort et al, 2003). Phage lysate bacterin contains several unaltered conformational epitopes which recognized by TLRs found on the surface and endosomal membrane of different types of immune cells (macrophage, DC, neutrophile etc.). Among all the TLRs, TLR4, TLR2 and TLR9 play important role in controlling Brucella infection (Oliveira et al., 2008). Up-regulation of TLRs and cytokines transcripts were observed in immunized mice groups in comparison to unimmunized group in present study.

In our study TLR9, IL-12 and INF-[alpha] expression were significantly up-regulated at every interval which indicate bacterial CpG motif of phage lysate bacterin act as TLR9 agonist and reaffirm the previous observations that activation of intracellularly located TLR9 with bacterial CpG motif lead to the production of IL12, which drive the Th1 immune response (Oliveira et al., 2011) and increases the immunogenicity of peptide, DNA, tumor cells or DC based vaccines (van Duin et al., 2006). TLR9 expression was higher in mice group immunized with Brucella S19 in compared to plain and alum adjuvanated phage lysate group at 3rd, 7th and 14th DPI which indicate that CMI response is relatively higher in Brucella S19 in comparison to plain and alum adjuvanated phage lysate bacterin. However the CMI response of phage lysate bacterin was sufficient enough to protect intracellular infection of B. abortus in mice as reported by Jain et al., 2015. The relative higher expression of TLR9 in live Brucella S19 vaccine group may be due to mimicking of natural way of releasing and sensing of bacterial CpG motif by TLR9 in effective manner and higher antigen load.

In our study, TLR2, TLR4 and IL-12 expression were also significantly up-regulated in all immunizing groups as compared to negative control at every interval which suggest that LPS as a TLR4 agonist and lipoprotein as TLR2 agonist of Phage lysate bacterin augment the immunoprotective mechanism of phage lysate bacterin. Our findings were in accordance with previous reports, TLR2 recognize B. abortus lipoprotein omp 19 and omp16 induce the expression of IL-12 and other pro inflammatory cytokines (Barrionuevo et al., 2008). TLR4 activation strongly enhances Th1 type cellular and humoral immune responses and typically induces robust IL-12 production (Ulrich et al., 1995 and Evans et al., 2003). Although TLR2 and TLR4 expression were found to be higher in mice immunized with Brucella S19 in compared to alum adjuvanated phage lysate at 3rd DPI however TLR2 expression was almost similar in both group at 14th DPI and TLR4 expression was almost similar in both group at 7th and 14th DPI, which suggest that alum adjuvanated phage lysate bacterin may be alternative approach to combat the brucella infection at very low antigenic biomass while live Brucella S19 vaccine require high antigenic biomass. Expression of IL4 transcript significantly up-regulate in all the immunized groups in comparison to unimmunized group. In Brucella S19 group, the maximum IL-4 expression was observed at 7th DPI which reduced at 14th DPI. IL-4 expression was significantly higher in alum adjuvanated phage lysate group than Brucella S19 group and plain phage lysate at 14th DPI which reaffirm the previous observations that alum adjuvant drive Th2 immune response preferentially and help in early protection.

CONCLUSION

On the basis of present observation, we found that alum adjuvanated phage lysate bacterin produce sufficient expression level of TLRs and cytokines which in turn suggest alum adjuvanated phage lysate bacterin in comparison to plain lysate produce sufficient immune-protection in form of Th1, Th2, cell mediated and humoral immune response.

ACKNOWLEDGMENTS

The authors are thankful to the director, Indian Veterinary Research Institute for providing the facilities to carry out the work.

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Ankita Jain [1], Mayank Rawat [3], Soumendu Chakaravarti [2], Abhishek [1], Vinod Chaturvedi [2], Losa Chesti [1], Ankush K. Niranjan [1], Rajat Varshney [1] and Bablu Kumar [2] *

[1] Division of Bacteriololy and Mycology, Indian Veterinary Research Institute, Bareilly--243 122, India.

[2] Division of Biological Products, Indian Veterinary Research Institute, Bareilly--243 122, India.

[3] Division of Biological Standardization, Indian Veterinary Research Institute, Bareilly--243122, India.

(Received: 30 January 2016; accepted: 20 February 2016)

* To whom all correspondence should be addressed.

E-mail: babbacteriol@gmail.com

Caption: Fig. 1. Relative expression profile of TLR2 in Brucella phage lysate (PL) bacterin, alum adjuvanated phage lysate and Brucella S19 immunized mice. The data are expressed as mean [+ or -] SDE.

Caption: Fig. 2. Relative expression profile of TLR4 in Brucella phage lysate (PL) bacterin, alum adjuvanated phage lysate and Brucella S19 immunized mice. The data are expressed as mean [+ or -] SDE.

Caption: Fig. 3. Relative expression profile of TLR9 in Brucella phage lysate (PL) bacterin, alum adjuvanated phage lysate and Brucella S19 immunized mice. The data are expressed as mean [+ or -] SDE

Caption: Fig. 4. Relative expression profile of IL4 in Brucella phage lysate (PL) bacterin, alum adjuvanated phage lysate and Brucella S19 immunized mice. The data are expressed as mean [+ or -] SDE

Caption: Fig. 5. Relative expression profile of [INF.sup.3] in Brucella phage lysate (PL) bacterin, alum adjuvanated phage lysate and Brucella S19 immunized mice. The data are expressed as mean [+ or -] SDE

Caption: Fig. 6. Relative expression profile of IL12 in Brucella phage lysate (PL) bacterin, alum adjuvanated phage lysate and Brucella S19 immunized mice. The data are expressed as mean [+ or -] SDE
Table 1.

Gene           Primer    Annealing      Primer sequence
                         ([degrees]C)

TLR2           Forward   54             5'-AAGAGGAAGCCCAAGAAAGC-3'
               Reverse                  5'-CGATGGAATCGATGATGTTG-3'
TLR4           Forward   54             5'-ACCTGGCTGGTTTACACGTC-3'
               Reverse                  5'-CTGCCAGAGACATTGCAGAA-3'
TLR9           Forward   54             5'-ACTGAGCACCCCTGCTTCTA-3'
               Reverse                  5'-AGATTAGTCAGCGGCAGGAA-3'
IL12           Forward   54             5'-TTGCTGGTGTCTCCACTCATG-3'
               Reverse                  5'-GTCACAGGTGAGGTTCACTGTTTC-3'
INF3           Forward   54             5'-GCTTTGCAGCTCTTCCTCATG-3'
               Reverse                  5'-CTTCCACATCTATGCCACTTGAG-3'
IL4            Forward   54             5'-GAGACTCTTTCGGGCTTTTCG-3'
               Reverse                  5'-CAGGAAGTCTTTCAGTGATGTGG-3'
[beta]-actin   Forward   60             5'-CAATAGTGATGACCTGGCCGT-3'
               Reverse   60             5'-AGAGGGAAATCGTGCGTGAC-3'

Gene           Primer    Reference

TLR2           Forward   Renshew et al., 2002
               Reverse   Renshew et al., 2002
TLR4           Forward   Renshew et al., 2002
               Reverse   Renshew et al., 2002
TLR9           Forward   Renshew et al., 2002
               Reverse   Renshew et al., 2002
IL12           Forward   Chan et al., 2012
               Reverse   Chan et al., 2012
INF3           Forward   Chan et al., 2012
               Reverse   Chan et al., 2012
IL4            Forward   Chan et al., 2012
               Reverse   Chan et al., 2012
[beta]-actin   Forward   Overbergh et al. 1999
               Reverse   Overbergh et al. 1999
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Author:Jain, Ankita; Rawat, Mayank; Chakaravarti, Soumendu; Abhishek; Chaturvedi, Vinod; Chesti, Losa; Nira
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Sep 1, 2016
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