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Antigenic potential of a recombinant polyvalent DNA vaccine against pathogenic leptospiral infection
Microbial Pathogenesis 124 (2018) 136–144
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Antigenic potential of a recombinant polyvalent DNA vaccine against pathogenic leptospiral infection
T
Bashiru Garbaa,b, Abdul Rani Bahamanb,∗, Zunita Zakariab, Siti Khairani Bejob, Abdul Rahim Mutalibc, Faruku Bandeb,d, Nasiru Suleimane a
Veterinary Public Health Lab, Department of Veterinary Public Health and Preventive Medicine, Faculty of Veterinary Medicine, Usmanu Danfodiyo University, Sokoto, Nigeria b Bacteriology Lab, Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia c Department of Veterinary Laboratory Diagnostics Services Unit, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia d Department of Veterinary Services, Ministry of Animal Health and Fisheries Development, Usman Faruk Secretariat Complex, 840245, Sokoto State, Nigeria e Department of Veterinary Physiology and Biochemistry, Faculty of Veterinary Medicine, Usmanu Danfodiyo University, Sokoto, Nigeria
A R T I C LE I N FO
A B S T R A C T
Keywords: Multi-epitope DNA vaccine Leptospira infection Immune response Antigenic epitopes Agglutinating and neutralizing antibodies
Leptospirosis is a serious epidemic disease caused by pathogenic Leptospira species. The disease is endemic in most tropical and sub-tropical regions of the world. Currently, there is no effective polyvalent vaccine for prevention against most of the circulating serovars. Moreover, development of an efficient leptospiral vaccine capable of stimulating cross-protective immune responses against a wide range of serovars remains a daunting challenge. This, in part, is associated with the extensive diversity and variation of leptospiral serovars from region to region. In this study, a multi-epitope DNA vaccine encoding highly immunogenic epitopes from LipL32 and LipL41 was designed using in-silico approach. The DNA encoding antigenic epitopes was constructed from conserved pathogenic Leptospira genes (LipL32 and LipL41). Immunization of golden Syrian hamsters with the multi-epitope chimeric DNA vaccine resulted in the production of both agglutinating and neutralizing antibodies as evidence by MAT and in-vitro growth inhibition tests respectively. The antibodies produced reacted against eight different serovars and significantly reduced renal colonization following in vivo challenge. The vaccine was also able to significantly reduce renal colonization which is a very important factor responsible for persistence of leptospires among susceptible and reservoir animal hosts. In conclusion, the leptospiral multi-epitope chimeric DNA vaccine can serve as a potentially effective and safe vaccine against infection with different pathogenic leptospiral serovars.
1. Introduction The importance of leptospirosis as a zoonotic disease is becoming evident with the increasing number of cases and mortality worldwide [2]. The epidemiology of the disease is gradually changing and the importance of indirect transmission following exposure to contaminated environment during recreational activities is also becoming relevant [15,29,37]. Other factors that increases the risk of human infection include water sport, climatic changes and the global economic hardships [16]. The disease is endemic in most tropical countries especially regions with high rainfall and large population of domestic and wild animal reservoirs especially rats, cattle and dogs [22]. Despite the health burden the disease exert on humans as well as its effect on livestock productivity, there is currently no universally
∗
approved vaccine that can protect against most of the circulating serovars [3,18]. Moreover, the currently available killed vaccines were developed based on surface exposed lipopolysaccharide coat which is characterized by variable antigenic differences between strains, thus limiting cross-protection [4]. Furthermore, the lack of complete knowledge of the mechanisms of protective immunity against leptospiral infection is also making the prospects of developing highly efficacious vaccine seemingly impossible. Epitope based DNA vaccines represent an alternative strategy for the development of effective leptospiral vaccine that can provide heterologous protection. The potential advantages include; increased safety, opportunity to rationally engineer epitopes for increased potency and ability to focus immune response on conserved epitopes [31]. DNA vaccine also provide prolonged antigen expression and
Corresponding author. E-mail addresses: [email protected] (B. Garba), [email protected] (A.R. Bahaman).
https://doi.org/10.1016/j.micpath.2018.08.028 Received 24 March 2018; Received in revised form 2 August 2018; Accepted 18 August 2018 Available online 21 August 2018 0882-4010/ © 2018 Published by Elsevier Ltd.
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optimized for mammalian codon usage. The DNA molecule designated LipDNA-01 was generated via chemical gene synthesis (GeneScript gene synthesis). The synthesized DNA was then cloned into pBudCE4.1 (Invitrogen™) expression vector which contains cMyc and V5 epitope tags for rapid detection. The final plasmid DNA was designated LipDNA-01pBudCE4.1.
amplification of the immune response, whilst simultaneously offering several advantages over other vaccine preparations such as ease of construction, low cost of mass production, high level temperature stability and the ability to stimulate both humoral and cell mediated immune response [26]. Essentially, the key element for the construction of an effective DNA vaccine is the immune-dominant gene. While traditional DNA vaccines are developed after cultivation of the pathogen and extraction and purification of the DNA samples. This approach seems to affect the quality and efficiency of the vaccine. In addition, there is lack of facilities and difficulties in culturing leptospires as well as the associated risk of infection. Leptospires are also characterized by a diverse serovars with varying genomic features. Hence, gene synthesis offers a highly effective alternative that allows detailed evaluation of gene function and analysis of protein-nucleic acid interactions [41]. Chemical gene synthesis also allows the synthesis of DNA sequence and assembly of these epitopes derived from multiple antigens into a relatively more specific gene. Such vaccines have been reported to induce powerful cross-reactive immune responses against multiple antigens [38]. In this experiment, a multi-epitope chimeric leptospiral DNA vaccine encoding 10 dominant B-cell epitopes from two highly conserved immunogenic protein genes was developed. The ability of the DNA vaccine to stimulate both agglutinating and neutralizing antibodies in immunized hamsters was evaluated as well as histopathological analysis. While Microscopic agglutination test is the WHO recommended test to identify agglutinating antibodies to leptospires, the in vitro growth inhibition test is the preferred test to evaluate the presence of neutralizing antibodies in the serum of vaccinated animals [7,42]; Tripathy et al., 1971.
2.3. Physicochemical properties The amino acid composition, molecular weight, instability index, aliphatic index and grand average of hydropathicity of the protein sequences were analyzed using ProtParam tool (http://web.expasy.org/ tools/protparam/protpar-ref.html) based on the accession number of each of the proteins. 2.4. Cloning and expression of recombinant chimeric DNA The chimeric gene insert was obtained after digestion from the pUC57 vector it was supplied with using NotI and XhoI restriction enzymes. This was then followed by ligation downstream of the V5 epitope tag of the pBudCE4.1 expression vector after gel purification of the insert fragment and designated LipDNA-pBudCE01. In addition to the restriction enzyme analysis, the cloned plasmid was also sequenced using ABI 3730xl automatic sequencer (GeneScript®). The lipid-based Lipofectamin® LTX transfection method was used to transfect 70–90% confluent CHO cells grown on a cover slip in a 6 well cell culture plate. Six well plates were seeded with 106 cells per well resuspended in F12-K1 medium containing 10% FBS and incubated for 48 h until 70–90% confluence was attained. A pBudCE4.1 plasmid containing multi-epitope gene and an empty control plasmid were used to transfect the confluent cells, according to the manufacturer's instructions (Invitrogen™ USA). The expression of the recombinant protein was analyzed by indirect immunofluorescence antibody test. The commercially available V5 antibody was used as the primary antibody while the secondary antibody used was Alexafluor 488 goat-anti-hamster IgG (Invitrogen™).
2. Materials and methods 2.1. Experimental animals Three to four weeks old Golden Syrian hamsters were purchased from the laboratory animal breeding unit of Monash University Malaysia, Bandar Sunway Selangor, Malaysia. The hamsters were kept in the Laboratory Animal Research facility of the Faculty of Veterinary Medicine, Universiti Putra Malaysia, for two weeks to acclimatize and then screened for leptospirosis using microscopic agglutination test (MAT) based on a panel of 18 reference strains (Australis, Autumnalis, Ballum, Bataviae, Canicola, Cellodoni, Cynopteri, Djasiman, Grippotyphosa, Hardjo prajitno, Hardjobovis 117123, Hebdomadis, Icterohemorrhagiae, Javanica, Lai, Malaysia Bejo Iso-9, Pomona and Pyrogenes) available in the Bacteriology Laboratory of the Faculty of Veterinary Medicine, Universiti Putra Malaysia. All animal procedure was approved by the Institutional Animal Care and Use Committee (IACUC) of the Universiti Putra Malaysia (UPM/IACUC/AUP-R0012/ 2016).
2.5. Immunization of hamsters with the leptospiral DNA vaccine The LipDNA01-pBudCE4.1 plasmid propagated in Top10 E. coli cells was extracted using Qiagen EndoFree® Plasmid Mega Kit (Qiagen®) for the immunizations. 4–5 weeks old golden Syrian hamsters were divided into two groups (12 hamsters per group). The hamsters were immunized twice in the quadriceps muscle at two weeks' interval. Each animal received 300 μl containing 150 μg of LipDNA01-pBudCE.4.1 in equal volume of incomplete Freund's adjuvant and then topped up to 300 μl with PBS. However, the booster immunization was administered without any adjuvant, i.e only 150 μg of the vaccine topped up to 300 μl with sterile PBS. Similarly, one control group (plasmid control) immunized with pBudCE4.1 plasmid alone without any insert was used in this study. At the end of the immunization, blood samples were collected via the retro-orbital sinus of the hamsters under general anaesthesia for further analysis.
2.2. In-silico prediction and selection of vaccine epitopes The glycoprotein's sequences of LipL32 and LipL41 were retrieved from the UniProt knowledgebase (UniprotKB) NCBI database using UniProtKB/Swiss-Prot-non-redundant protein sequences (nr), locus Q72SM7_LEPIC, accession Q72SM7 parameters. The antigenic epitopes of the glycoprotein sequence were predicted using BepiPred Prediction Software. Computational analysis of the consensus sequences of potential B cell epitopes was done using the IEDB Bepipred 1.0 prediction server which predicts the location of linear B cell epitopes using a combination of a hidden Markov model and a propensity scale method [21]. Five epitopes were selected and further subjected to VaxiJen 2.0 analysis which is a server for the prediction of protective antigens and subunit vaccines. These five selected epitopes were engineered and linked with Glycine-Serine spacer. Restriction enzyme sites were inserted in the chimeric gene encoding multiple epitopes and then
2.6. Microscopic agglutination test MAT analysis The microscopic agglutination test was performed using a panel of 17 reference Leptospira serovars (Australis, Autumnalis, Ballum, Bataviae, Pyrogenes, Icterohaemorrhagiae, Javanica, Pomona, Canicola, Cellodoni, Cynopteri, Djasiman, Grippotyphosa, Hardjo bovis, HardjoHardjoprajitno, Lai and Malaysia-Bejo) according to the microtechnique described by Ref. [43]. About 7–10 day-old culture of the reference serovars grown in EMJH medium supplemented with serum albumin were used as antigens. A two-fold dilution of all serum samples was prepared using phosphate buffer saline (PBS) in a 96-well microtiter plate starting from 1:20 dilution (1:20, 1:40, 1:80 and 1:160). 50 μl of 137
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Table 1 LipL32 Predicted epitopes. No
Position
Epitope sequence
Peptide length
Antigenicity score
2 4
62–77 148–177
YVKPGQAPDGLVDGNK IAKAAKAKPVQKLDDDDDGDDTYKEERHNK
16 30
0.9096 1.2556
comparative statistical analysis was conducted based on the scores for the overall experiments between control and immunized groups with the help of SPSS statistical package.
PBS was added into all wells of the 96 well plate followed by additional 40 μl PBS into the second well. 10 μl of serum sample was then added to the second well, mixed by pipetting (at least 5 times) and then 50 μl volume was transferred from the second well into the preceding well. The same was done to the remaining wells until the last well. Finally, 50 μl of antigen was added to each well, including the first well and incubated at 37 °C in a shaking incubator for an initial 5 min followed by 2 h' incubation without shaking. One positive and negative standard serum controls were used. Titers of ≥1:80 were considered positive. This endpoint titre was determined as the highest serum dilution showing agglutination of at least 50% of the leptospires.
2.10. Statistical analysis The mean for the quantitative variables was obtained with the help of SPSS statistical software. The hamster immunizations were analyzed by comparison of renal colonization based on recovery of leptospires with agglutinating and neutralizing antibody mean titers. The means of agglutinating and growth inhibition antibody titers was analyzed by Two-tailed t-Test using SPSS, 12.0 version program.
2.7. In-vitro leptospiral growth inhibition test IGIT 3. Results To detect neutralizing antibodies produced as a result of the DNA immunization, equal amount each of serum pools containing equal amounts of serum from all hamsters in LipDNA01, and pBudCE4.1 groups was prepared. The serum was inactivated at 56 °C for 30min in a water bath. In order to confirm the serum is not contaminated with saprophytic bacteria, 50 μl of each serum pool was cultured in Tryptic Soy Broth (TSB) at 37 °C for 48 h. The growth inhibition test was carried out using 200 μl of the twofold diluted (1:1) serum pools from each group. The 200 μl diluted serum was added into a 15 ml falcon tube containing 2.5 ml EMJH medium and 100 μl of 7–10 day old leptospiral culture containing approximately 108 leptospires. The mixture was incubated at 37 °C for 10 days and then examined for growth under darkfield microscope. As a control, 200 μl PBS was added to the 2.5 ml EMJH medium containing 100 μl leptospiral culture and incubated together. Dark-field microscopy was used to examine for the dpresence or absence of leptospiral growth. The result was analyzed based on the method of Tripathy et al. (1971) where tubes that had less than 10 leptospires per microscope field were scored as positive growth inhibition while negative growth inhibition was determined when the number of leptospires in the tube is similar to the number observed in the control tubes in which PBS (instead of test serum) was added to the media containing leptospires.
3.1. Prediction and selection of vaccine epitopes In-silico bioinformatics tools were employed in the construction of the multi-epitope chimeric DNA vaccine. Five antigenic epitopes [Tables 1 and 2] from LipL32 and LipL41 glycoproteins were predicted, engineered and assembled into a synthetic gene linked together with the help of GGGGS spacer between each epitope peptide as shown in Figs. 1 and 2. The full length multi-epitope chimeric gene construct contained a CpG motif at both the 5′ and 3′ end of the gene in order to enhance its immune stimulating ability (Fig. 3). Cloned expression plasmid (containing DNA fragment) was confirmed by restriction enzyme digestion and sequencing (Fig. 4). This recombinant plasmid LipDNA-01-pBudCE4.1 was then used as multi-epitope chimeric DNA vaccine to produce agglutinating and neutralizing antibodies against leptospira. 3.2. Physicochemical properties ProtParam tool was used to analyze the various physical and chemical parameters of the protein. The results indicate that LipL32 had 272 aa while LipL41 has 355 aa. Other parameters analyzed are a shown in Table 3 below.
2.8. Recovery of leptospires from kidneys by culture 3.3. Cloning and expression of recombinant chimeric DNA In order to assess kidney infection among the challenged hamsters by standard bacteriological method, kidney tissues were aseptically harvested after euthanizing the animal via exsanguination under general anaesthesia. The kidney was rinsed in sterile PBS, exsheathed and then crushed by passing it through the narrow opening of a 3 ml syringe into a 15 ml falcon tube containing 5 ml liquid EMJH medium with 5fluorouracil (200 μg/ml). The medium was then incubated for 10 days at 37 °C and then examined for growth of Leptospira under dark-field microscope.
The synthetic gene fragment after restriction enzyme digestion was successfully ligated at the multiple cloning site of the pBudCE expression plasmid downstream of the V5 epitope tag as shown in Figs. 5 and 6. The expression of the chimeric epitopes was achieved following transfection of Chinese hamster ovary cell line with LipDNA01pBudCE4.1. Indirect immunofluorescence antibody test was used to evaluate the level of expression. V5 antibody was used as the primary antibody while the secondary antibody used was Alexafluor 488 goatanti-hamster IgG (Invitrogen). Cytoplasmic expression of the chimeric DNA in CHO cells was evident by the green fluorescein color produced
2.9. Histopathological analysis
Table 2 LipL41 Predicted epitopes.
Kidney samples were aseptically collected from all hamsters at postmortem, rinsed with normal saline solution and then fixed in 10% (vol/vol) buffered formalin for 8 h and then transferred to 70% ethanol [17]. The fixed kidney tissues were sectioned at 5 μm with the aid of a microtome, stained with hematoxylin and eosin, and examined by light microscopy. The renal lesions for hamsters indicative of infection, were graded with 0 as normal, 1 as mild, 2 as moderate, and 3 as severe. A 138
No
Position
Epitope sequence
Peptide length
Antigenicity score
1 2 4
29–38 54–60 163–180
PVFPKDKEGR VEAPEKS ATGKDVNTGNEPVSKPTG
10 7 18
0.8006 0.9825 0.9697
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Fig. 1. IEDB BepiPred antigenic epitope prediction showing potential B cell epitopes for gene LipL32.
by the cells as shown in Fig. 7. The fluorescence emission of FITC (Fluorescein isothiocyanate) conjugates produced green fluorescence while DAPI which is a nucleic acid stain that binds to A-T rich regions of DNA stains the nucleus blue.
The arithmetic mean titer and standard error recorded for the plasmid control was 0.053 and 0.031 respectively. LipDNA01 vaccine group produced neutralizing antibodies against 8/19 serovars tested. The difference observed at 95% confidence interval between vaccine group and control was statistically significant at (p < 0.05).
3.4. Microscopic agglutination test MAT 3.6. Recovery of leptospires from kidneys by culture Prior to commencement of vaccination, all hamsters (day 0), were negative for agglutinating MAT titre. Agglutinating antibody titres was measured using the gold standard microscopic agglutination test based on their reactivity against 19 different pathogenic serovars. The antibody response was measured by calculating the arithmetic mean titre for both immunized and control groups. The obtained results indicate no detectable antibody response in the plasmid control. However, a significant raise in agglutinating antibody titre was observed in the vaccine groups (p < 0.05) which increased after booster immunization (Fig. 8). Traditionally, a positive reaction at 1:50 dilution is considered positive MAT, but in this study, only titre above the 1:80 dilutions was considered. The vaccines showed varying reactivity against antigens from serovars Bataviae, Copenhageni, Djasiman, Grippotyphosa, Icterohemorrhagiae, Javanica, Lai, and Hardjo bovis.
There was a significant decrease (p < 0.05) in the amount of leptospires recovered from the kidney of the vaccinated hamsters 28 days after challenge with sub-lethal dose of L. interrogans Copenhageni Fiocruz compared with the pBudCE4.1 control group. Although a large number of the cultures were heavily contaminated even after filtration, the decrease in leptospires observed was significantly lower in hamsters vaccinated with LipDNA01-pBudCE4.1. On the other hand, none of the unvaccinated control group, died after sub-lethal challenge with Copenhageni Fiocruz, probably because of the low dose or loss of virulence of the challenge strain due to repeated subculturing. However, a significant amount of leptospires was recovered (p < 0.05). Statistical analysis indicating the level of significance based on the mean between different vaccine groups is presented in Table 5.
3.5. Invitro growth inhibition test 3.7. Histopathological analysis The result for the invitro leptospira growth inhibition test is presented in Table 4 below. The table represents reactivity to each of the vaccine against all the serovars tested.
No obvious gross pathological lesion was observed in any of the kidneys examined at postmortem. Meanwhile, after H & E staining
Fig. 2. IEDB BepiPred antigenic epitope prediction showing potential B cell epitopes for gene LipL41. 139
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Fig. 3. Schematic representation of assembled multi-epitope chimeric gene indicating linked epitopes, oligonucleotide sequence and restriction enzymes.
4. Discussion Despite the increased level of awareness on the public health importance of leptospirosis, the incidence of the disease is increasing annually from an initial estimate of 500,000 cases in 1999 to more than double with about 60,000 fatalities in 2015 [6,35]. Leptospirosis constitutes an important public health problem in developing countries particularly those that are impoverished. The disease is responsible for economic losses in animal production as well as exerts a burden on human health. Available vaccines can only provide limited protection, which is short lived. The most commonly used leptospiral vaccines are based on inactivated whole-cell bacterins or leptospiral cell membrane components. These preparations although effective, are associated with several side-effects like pain, irritation and discomfort in addition to limited protection. In addition, the vast majority of the vaccines are for animal use, albeit for few countries that have licensed bacterins for human use [9,25]. As a result, the last few decades have seen research efforts shifting focus to the classical identification of antigens for the development of recombinant vaccines against leptospira. While significant progress have been recorded, the development of a broad-range leptospiral vaccine still remains elusive [8,39]. In this regard, reverse vaccinology which explores the entire genome of the pathogen and uses in-silico bioinformatics to produce highly specific recombinant antigens is currently being explored with a promising result. DNA vaccines containing multi-epitope encoded gene have been reported to confer protection against many infectious diseases [11,30,33]. DNA vaccine stimulates antibody production in a similar way as foreign antigens are processed and presented during natural infection. Similarly, multi-epitope DNA vaccines was reported to induce more potent immunoreactions than whole protein vaccines [41]. Such vaccines are also able to induce powerful cross-reactive immunological response due to the fact they are derived from multiple antigens packaged into a relatively small chimeric molecule. Moreover, immunity induced by multi-epitope DNA vaccine includes both the humoral and cellular immune component. Hence they are considered suitable for protection against a wide range of serovars especially in endemic regions. The construction of such highly complex synthetic vaccine may potentially have higher efficacy than assembly of naturally occurring sequences [40]. Chemically synthesized genes could produce a significant impact on the immuno-reactivity against diverse leptospiral outer membrane proteins [27,36]. In-silico bioinformatics approach can
Fig. 4. pBudCE4.1 plasmid carrying insert. Lane, 1 and 6. 1.5 kb DNA marker; lane 2–3, double digested plasmid, lane 4–5 linearized pBudCE4.1. Table 3 Physicochemical properties of amino acid sequences based on the ProtParam.
Molecular weight Theoretical pI Instability Index Aliphatic Index Grand average of hydropathicity
LipL32
LipL41
29612.9 6.34 32.53 85.74 −0.257
38939.7 6.01 26.19 87.15 −0.244
varying degrees of histopathological changes was observed (Fig. 9) The most common pathology was interstitial nephritis that consisted of inflammatory infiltrates like lymphocytes, plasma cells and macrophages. Based on the lesion scoring method, the intensity of the lesions for the vaccinated groups was less severe compared to the challenged control group. The lesions were predominantly located in the kidney cortex and medullary junction. The mean kidney score was mild and generally lower in the LipDNA01 immunized group and in comparison with the control groups, the difference was statistically significant at (p < 0.0109).
3.8. Morphological diagnosis Necro-hemorrhagic interstitial nephritis.
Fig. 5. Insilico clonning of LipDNA01 gene fragment (green) downstream of V5 epitope tag. The gene was inserted with the help of NotI and XhoI restriction enzymes and placed under the control of EF1α promoter. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 140
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Fig. 6. The pBudCE plasmid map showing cloned insert (green) downstream of the V5 epitope. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Fig. 7. Indirect Immunoflourecence test showing the expression of plasmid DNA constructs in CHO-K1 cells. LipDNA01pBudCE.4.1 expressed proteins in the cytoplasm is indicated by the diffused green fluorescence emission of FITC (Left) while the untransfected control shows only the DAPI stained nucleus (Right). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Table 4 Overall invitro growth inhibition test result.
Bataviae Canicola Copenhageni Djasiman Grippotyphosa Hardjobovis Icterohemorrhagiae Lai
LipDNA01
pBudCE4.1
Positive Positive Positive Positive Positive Positive Positive Positive
Negative Negative Positive Negative Negative Negative Negative Negative
Table 5 Recovery of leptospires from kidney samples of immunized.
Fig. 8. Agglutinating antibody titre after hamster immunization with plasmid DNA vaccine and controls. (2 = LipDNA01; 7 = pBudCE4.1).
Vaccine group
Number of samples
Contamination
Mean
Standard deviation
Standard error
LipDNA01 pBudCE4.1
12 12
6 6
0.2 0.6
0.422 0.516
0.075 0.092
LipL41 immunodominant glycoproteins of pathogenic leptospira were selected after prediction and evaluation by Bepipred and VaxiJen software. Immune response during leptospirosis is antibody based; hence humoral mediated immunity is of utmost importance [44,45]. In silico analysis of the physicochemical properties of the proteins showed that these selected epitopes are highly immunogenic and stable. While VaxiJen allocate a score of 0.4 as antigenic, the amino acid sequences
be employed to engineer synthetic proteins to induce antibody production that can match the highly complex antigenic variations of various pathogenic serovars to improve vaccine efficiency. In this study, a multi-epitope chimeric DNA vaccine was constructed against pathogenic leptospira species based on sequences in the NCBI data base. Five dominant immunogenic epitopes capable of inducing Bcell immunoreaction corresponding to segments in the LipL32 and 141
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Fig. 9. Photomicrographs of kidney sections. Control group (A) cortical section showing congested glomeruli (G), necrotic renal tubules containing bacterial cast (leptospira colonies; T), and presence of interstitial inflammatory response typified by infiltration of lymphocytes and few neutrophils, H&E x 400 and (B) cortical area showing an extensive area of interstitial hemorrhage (yellow arrow) with surrounding interstitial inflammatory response (black arrow), H&E x 400: LipDNA01 vaccine group (C) medullary section showing moderate tubular degeneration and necrosis (black arrow) of renal tubules and ducts accompanied by multifocal interstitial inflammatory response (yellow arrow) and few pinkish homogeneous cast in the tubular lumen, H&E x 100 (D) medullary section showing severe tubular degeneration and necrosis of renal tubules and ducts (black arrow) resulting in epithelial cast in the tubular lumen, multifocal areas of interstitial hemorrhage (yellow arrow) and marked interstitial inflammatory response (blue arrow), Normal renal parenchyma showing no pathological lesion H&E x 100. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
where found to persist for almost 5 months after vaccination [42]. This persistence is presumed to be as a result of longer opportunity for antibodies to react with the antigens in the invitro growth inhibition test as compared to MAT. Moreover, the steady increase in antibody titre may suggest continued expression of the DNA vaccine. This may also result in production of heightened immune response as previously reported where both LipL32 and LipL41 were expressed by invitro expression systems and they protect against lethal leptospira challenge [5]; [23]. These OM proteins are readily expressed in-vivo and are highly immunogenic. They are also conserved among pathogenic leptospira. Both MAT and GIT have been used to evaluate the immune response in buffalo calves vaccinated with heat-killed pentavalent bacterin vaccines [7]. In some studies, GIT cross-reacting antibodies have been demonstrated in buffaloes vaccinated with three experimental vaccines [7]. This is similar to the result of our study where the vaccines reacted to multiple serovars from different pathogenic species. This may indicate the potentials of the vaccine to provide broad range of protection against the commonly circulating serovars of pathogenic leptospira species. The in-vitro leptospira growth inhibition test is said to be a good method for assessing the potency of leptospira vaccines and may also be used in the analysis of vaccine efficacy [7]. Evaluation of protection from renal colonization as well as development of histopathological lesions on kidney of vaccinated and challenged hamsters show some mild to moderate pathologies among the vaccinated group while the control group show severe lesions. Majority of the lesions observed were located either in the cortical, corticomedullary or medullary region. They range from renal tubulointerstitial lesions, such as interstitial nephritis and cellular infiltration. Glomerular lesions with minimal intensity were also observed among the control group. While the vaccine group showed minimal to no obvious pathology in the kidney tissues, samples analyzed from the plasmid control group showed signs of severe damage with cellular infiltration and severe interstitial hemorrhages. This finding is supported by previous evidence that hamsters vaccinated with a combination of OmpL1 and LipL41 developed protective immunity and
considered in this study has antigenicity scores between 0.8 and 1.2, indicating they are highly antigenic. Moreover, the Aliphatic index is generally regarded as a positive factor for the increase of thermostability of proteins which is necessary for prolonged activity [46]. Bioinformatics has given room for selecting potential epitopes without the risk involved in propagating the pathogen of interest. This technique represent a huge advantage over conventional methods of vaccine production in addition to faster output and lower cost [34]. The result after immunization of golden Syrian hamsters revealed a raising antibody titre that increases after the booster immunization. In recent years, the use of epitopes in vaccines has become a valid alternative for improving the efficacy of vaccines [31,32]. Multi-epitope peptide DNA vaccines are effective against some viruses and they have recently being shown to have potential efficacy against some bacterial diseases including leptospires [10,11]. Similarly, in response to antigen stimulation, the chimeric DNA vaccine elicited both agglutinating and neutralization antibodies with the levels of the antibody being higher in the vaccine group compared to the plasmid control group. The fact that the DNA was expressed efficiently invitro is indicative of the fact that the DNA is correspondingly expressed after immunization as observed from the protection rendered. A significant correlation has also been previously reported between intra-cytoplasmic expression and elevation of serum level of proteins (Nozomi et al., 2004). Furthermore, although MAT is the most preferred test for leptospira infection, its sensitivity is relatively low, hence the recommendation that only a four-fold raise in antibody titres should be considered. Interestingly, the present result showed that the candidate vaccine is able to cause reaction against eight out of a panel of nineteen serovars tested by MAT. Similarly, it also prevented the growth of eight serovars according to the invitro growth inhibition test. This is in agreement with earlier reports where growth inhibition antibodies were used to measure cross-neutralizing antibodies in hamsters (Rosana et al., 2010). The growth inhibition antibodies were found to increase consistently and persisted longer that agglutinating antibodies by MAT. This observation is also similar to earlier reports where neutralizing antibodies 142
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resistance to renal colonization [19]. Immunization with a DNA vaccine encoding the conserved amino-terminal regions of LigA and LigB have also been reported to provide partial protection and sterilizing immunity [13,14]. In conclusion, a multi-epitope chimeric DNA vaccine against pathogenic leptospira was constructed, and the vaccine was able to elicit strong humoral immune responses as demonstrated by the high level agglutinating as well as neutralizing antibodies. Although, the vaccine was not able to provide full protection, but it was able to significantly reduced renal colonization. This is even more important when we consider that the protection rendered was against challenge with 106 leptospires which can hardly occur under natural situations. However, incorporating T-cell epitopes may likely improve the potency of the vaccine as Th-1 type immune response has previously been demonstrated in cattle vaccinated with killed L. hardjo vaccine [47]. Overall, the present novel immunogenic multi-epitope DNA vaccine developed by chemical gene synthesis and delivered as a plasmid DNA vaccine may serve as a new candidate target for leptospiral vaccine development.
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Conflicts of interest The authors wish to declare that they had no conflict of interest. Funding The work was supported by Universiti Putra Malaysia grant Geran Putra IPB (Sub-Projek)/IPM/IPS VOT 9472400 under Assoc. Prof. Siti Khairani-Bejo. Additional funds were also provided by Assoc. Prof. Zunita Zakaria, Faculty of Veterinary Medicine, Universiti Putra Malaysia. Acknowledgment The authors wish to acknowledge the contribution and assistance of Mr. Azri Muhammad Roslan, Mrs. Krishnammah Kuppusamy and Rabiatuladawiyah Rosli of the Bacteriology Lab, Faculty of Veterinary Medicine, Universiti Putra Malaysia. Appendix A. Supplementary data Supplementary data related to this article can be found at https:// doi.org/10.1016/j.micpath.2018.08.028. References [2] A.T. Amilasan, M. Ujiie, M. Suzuki, E. Salva, M.C.P. Belo, N. Koizumi, K. Yoshimatsu, W.-P. Schmidt, S. Marte, E.M. Dimaano, J.B. Villarama, K. Ariyoshi, Outbreak of leptospirosis after flood, the Philippines, 2009, Emerg. Infect. Dis. 18 (2012) 91–94 https://doi.org/10.3201/eid1801.101892. [3] G. Bashiru, A. Bahaman, Advances & challenges in leptospiral vaccine development, Indian J. Med. Res. 147 (15) (2018), https://doi.org/10.4103/ijmr.IJMR_1022_ 16Pubmed. [4] A.R. Bharti, J.E. Nally, J.N. Ricaldi, M.A. Matthias, M.M. Diaz, M.A. Lovett, P.N. Levett, R.H. Gilman, M.R. Willig, E. Gotuzzo, J.M. Vinetz, Leptospirosis: a zoonotic disease of global importance, Lancet Infect. Dis. 3 (2003) 757–771 https:// doi.org/10.1016/S1473-3099(03)00830-2. [5] M.-Y. Chang, Y.-C. Cheng, S.-H. Hsu, T.-L. Ma, L.-F. Chou, H.-H. Hsu, Y.-C. Tian, Y.C. Chen, Y.-J. Sun, C.-C. Hung, R.-L. Pan, C.-W. Yang, Leptospiral outer membrane protein LipL32 induces inflammation and kidney injury in zebrafish larvae, Sci. Rep. 6 (2016) 27838 https://doi.org/10.1038/srep27838. [6] F. Costa, J.E. Hagan, J. Calcagno, M. Kane, P. Torgerson, M.S. Martinez-Silveira, C. Stein, B. Abela-Ridder, A.I. Ko, Global morbidity and mortality of leptospirosis: a systematic review, PLoS Neglected Trop. Dis. 9 (2015) e0003898https://doi.org/ 10.1371/journal.pntd.0003898. [7] G. de Nardi Júnior, M.E. Genovez, M.G. Ribeiro, V. Castro, A.M. Jorge, An in vitro growth inhibition test for measuring the potency of Leptospira spp. Sejroe group vaccine in buffaloes, Biologicals 38 (2010) 474–478 https://doi.org/10.1016/j. biologicals.2010.02.014. [8] O. Dellagostin, A. Grassmann, Recombinant vaccines against leptospirosis, Vaccines 7 (2011) 1215–1224 https://doi.org/10.4161/hv.7.11.17944.
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Antigenic potential of a recombinant polyvalent DNA vaccine against pathogenic leptospiral infection
T
Bashiru Garbaa,b, Abdul Rani Bahamanb,∗, Zunita Zakariab, Siti Khairani Bejob, Abdul Rahim Mutalibc, Faruku Bandeb,d, Nasiru Suleimane a
Veterinary Public Health Lab, Department of Veterinary Public Health and Preventive Medicine, Faculty of Veterinary Medicine, Usmanu Danfodiyo University, Sokoto, Nigeria b Bacteriology Lab, Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia c Department of Veterinary Laboratory Diagnostics Services Unit, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia d Department of Veterinary Services, Ministry of Animal Health and Fisheries Development, Usman Faruk Secretariat Complex, 840245, Sokoto State, Nigeria e Department of Veterinary Physiology and Biochemistry, Faculty of Veterinary Medicine, Usmanu Danfodiyo University, Sokoto, Nigeria
A R T I C LE I N FO
A B S T R A C T
Keywords: Multi-epitope DNA vaccine Leptospira infection Immune response Antigenic epitopes Agglutinating and neutralizing antibodies
Leptospirosis is a serious epidemic disease caused by pathogenic Leptospira species. The disease is endemic in most tropical and sub-tropical regions of the world. Currently, there is no effective polyvalent vaccine for prevention against most of the circulating serovars. Moreover, development of an efficient leptospiral vaccine capable of stimulating cross-protective immune responses against a wide range of serovars remains a daunting challenge. This, in part, is associated with the extensive diversity and variation of leptospiral serovars from region to region. In this study, a multi-epitope DNA vaccine encoding highly immunogenic epitopes from LipL32 and LipL41 was designed using in-silico approach. The DNA encoding antigenic epitopes was constructed from conserved pathogenic Leptospira genes (LipL32 and LipL41). Immunization of golden Syrian hamsters with the multi-epitope chimeric DNA vaccine resulted in the production of both agglutinating and neutralizing antibodies as evidence by MAT and in-vitro growth inhibition tests respectively. The antibodies produced reacted against eight different serovars and significantly reduced renal colonization following in vivo challenge. The vaccine was also able to significantly reduce renal colonization which is a very important factor responsible for persistence of leptospires among susceptible and reservoir animal hosts. In conclusion, the leptospiral multi-epitope chimeric DNA vaccine can serve as a potentially effective and safe vaccine against infection with different pathogenic leptospiral serovars.
1. Introduction The importance of leptospirosis as a zoonotic disease is becoming evident with the increasing number of cases and mortality worldwide [2]. The epidemiology of the disease is gradually changing and the importance of indirect transmission following exposure to contaminated environment during recreational activities is also becoming relevant [15,29,37]. Other factors that increases the risk of human infection include water sport, climatic changes and the global economic hardships [16]. The disease is endemic in most tropical countries especially regions with high rainfall and large population of domestic and wild animal reservoirs especially rats, cattle and dogs [22]. Despite the health burden the disease exert on humans as well as its effect on livestock productivity, there is currently no universally
∗
approved vaccine that can protect against most of the circulating serovars [3,18]. Moreover, the currently available killed vaccines were developed based on surface exposed lipopolysaccharide coat which is characterized by variable antigenic differences between strains, thus limiting cross-protection [4]. Furthermore, the lack of complete knowledge of the mechanisms of protective immunity against leptospiral infection is also making the prospects of developing highly efficacious vaccine seemingly impossible. Epitope based DNA vaccines represent an alternative strategy for the development of effective leptospiral vaccine that can provide heterologous protection. The potential advantages include; increased safety, opportunity to rationally engineer epitopes for increased potency and ability to focus immune response on conserved epitopes [31]. DNA vaccine also provide prolonged antigen expression and
Corresponding author. E-mail addresses: [email protected] (B. Garba), [email protected] (A.R. Bahaman).
https://doi.org/10.1016/j.micpath.2018.08.028 Received 24 March 2018; Received in revised form 2 August 2018; Accepted 18 August 2018 Available online 21 August 2018 0882-4010/ © 2018 Published by Elsevier Ltd.
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optimized for mammalian codon usage. The DNA molecule designated LipDNA-01 was generated via chemical gene synthesis (GeneScript gene synthesis). The synthesized DNA was then cloned into pBudCE4.1 (Invitrogen™) expression vector which contains cMyc and V5 epitope tags for rapid detection. The final plasmid DNA was designated LipDNA-01pBudCE4.1.
amplification of the immune response, whilst simultaneously offering several advantages over other vaccine preparations such as ease of construction, low cost of mass production, high level temperature stability and the ability to stimulate both humoral and cell mediated immune response [26]. Essentially, the key element for the construction of an effective DNA vaccine is the immune-dominant gene. While traditional DNA vaccines are developed after cultivation of the pathogen and extraction and purification of the DNA samples. This approach seems to affect the quality and efficiency of the vaccine. In addition, there is lack of facilities and difficulties in culturing leptospires as well as the associated risk of infection. Leptospires are also characterized by a diverse serovars with varying genomic features. Hence, gene synthesis offers a highly effective alternative that allows detailed evaluation of gene function and analysis of protein-nucleic acid interactions [41]. Chemical gene synthesis also allows the synthesis of DNA sequence and assembly of these epitopes derived from multiple antigens into a relatively more specific gene. Such vaccines have been reported to induce powerful cross-reactive immune responses against multiple antigens [38]. In this experiment, a multi-epitope chimeric leptospiral DNA vaccine encoding 10 dominant B-cell epitopes from two highly conserved immunogenic protein genes was developed. The ability of the DNA vaccine to stimulate both agglutinating and neutralizing antibodies in immunized hamsters was evaluated as well as histopathological analysis. While Microscopic agglutination test is the WHO recommended test to identify agglutinating antibodies to leptospires, the in vitro growth inhibition test is the preferred test to evaluate the presence of neutralizing antibodies in the serum of vaccinated animals [7,42]; Tripathy et al., 1971.
2.3. Physicochemical properties The amino acid composition, molecular weight, instability index, aliphatic index and grand average of hydropathicity of the protein sequences were analyzed using ProtParam tool (http://web.expasy.org/ tools/protparam/protpar-ref.html) based on the accession number of each of the proteins. 2.4. Cloning and expression of recombinant chimeric DNA The chimeric gene insert was obtained after digestion from the pUC57 vector it was supplied with using NotI and XhoI restriction enzymes. This was then followed by ligation downstream of the V5 epitope tag of the pBudCE4.1 expression vector after gel purification of the insert fragment and designated LipDNA-pBudCE01. In addition to the restriction enzyme analysis, the cloned plasmid was also sequenced using ABI 3730xl automatic sequencer (GeneScript®). The lipid-based Lipofectamin® LTX transfection method was used to transfect 70–90% confluent CHO cells grown on a cover slip in a 6 well cell culture plate. Six well plates were seeded with 106 cells per well resuspended in F12-K1 medium containing 10% FBS and incubated for 48 h until 70–90% confluence was attained. A pBudCE4.1 plasmid containing multi-epitope gene and an empty control plasmid were used to transfect the confluent cells, according to the manufacturer's instructions (Invitrogen™ USA). The expression of the recombinant protein was analyzed by indirect immunofluorescence antibody test. The commercially available V5 antibody was used as the primary antibody while the secondary antibody used was Alexafluor 488 goat-anti-hamster IgG (Invitrogen™).
2. Materials and methods 2.1. Experimental animals Three to four weeks old Golden Syrian hamsters were purchased from the laboratory animal breeding unit of Monash University Malaysia, Bandar Sunway Selangor, Malaysia. The hamsters were kept in the Laboratory Animal Research facility of the Faculty of Veterinary Medicine, Universiti Putra Malaysia, for two weeks to acclimatize and then screened for leptospirosis using microscopic agglutination test (MAT) based on a panel of 18 reference strains (Australis, Autumnalis, Ballum, Bataviae, Canicola, Cellodoni, Cynopteri, Djasiman, Grippotyphosa, Hardjo prajitno, Hardjobovis 117123, Hebdomadis, Icterohemorrhagiae, Javanica, Lai, Malaysia Bejo Iso-9, Pomona and Pyrogenes) available in the Bacteriology Laboratory of the Faculty of Veterinary Medicine, Universiti Putra Malaysia. All animal procedure was approved by the Institutional Animal Care and Use Committee (IACUC) of the Universiti Putra Malaysia (UPM/IACUC/AUP-R0012/ 2016).
2.5. Immunization of hamsters with the leptospiral DNA vaccine The LipDNA01-pBudCE4.1 plasmid propagated in Top10 E. coli cells was extracted using Qiagen EndoFree® Plasmid Mega Kit (Qiagen®) for the immunizations. 4–5 weeks old golden Syrian hamsters were divided into two groups (12 hamsters per group). The hamsters were immunized twice in the quadriceps muscle at two weeks' interval. Each animal received 300 μl containing 150 μg of LipDNA01-pBudCE.4.1 in equal volume of incomplete Freund's adjuvant and then topped up to 300 μl with PBS. However, the booster immunization was administered without any adjuvant, i.e only 150 μg of the vaccine topped up to 300 μl with sterile PBS. Similarly, one control group (plasmid control) immunized with pBudCE4.1 plasmid alone without any insert was used in this study. At the end of the immunization, blood samples were collected via the retro-orbital sinus of the hamsters under general anaesthesia for further analysis.
2.2. In-silico prediction and selection of vaccine epitopes The glycoprotein's sequences of LipL32 and LipL41 were retrieved from the UniProt knowledgebase (UniprotKB) NCBI database using UniProtKB/Swiss-Prot-non-redundant protein sequences (nr), locus Q72SM7_LEPIC, accession Q72SM7 parameters. The antigenic epitopes of the glycoprotein sequence were predicted using BepiPred Prediction Software. Computational analysis of the consensus sequences of potential B cell epitopes was done using the IEDB Bepipred 1.0 prediction server which predicts the location of linear B cell epitopes using a combination of a hidden Markov model and a propensity scale method [21]. Five epitopes were selected and further subjected to VaxiJen 2.0 analysis which is a server for the prediction of protective antigens and subunit vaccines. These five selected epitopes were engineered and linked with Glycine-Serine spacer. Restriction enzyme sites were inserted in the chimeric gene encoding multiple epitopes and then
2.6. Microscopic agglutination test MAT analysis The microscopic agglutination test was performed using a panel of 17 reference Leptospira serovars (Australis, Autumnalis, Ballum, Bataviae, Pyrogenes, Icterohaemorrhagiae, Javanica, Pomona, Canicola, Cellodoni, Cynopteri, Djasiman, Grippotyphosa, Hardjo bovis, HardjoHardjoprajitno, Lai and Malaysia-Bejo) according to the microtechnique described by Ref. [43]. About 7–10 day-old culture of the reference serovars grown in EMJH medium supplemented with serum albumin were used as antigens. A two-fold dilution of all serum samples was prepared using phosphate buffer saline (PBS) in a 96-well microtiter plate starting from 1:20 dilution (1:20, 1:40, 1:80 and 1:160). 50 μl of 137
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Table 1 LipL32 Predicted epitopes. No
Position
Epitope sequence
Peptide length
Antigenicity score
2 4
62–77 148–177
YVKPGQAPDGLVDGNK IAKAAKAKPVQKLDDDDDGDDTYKEERHNK
16 30
0.9096 1.2556
comparative statistical analysis was conducted based on the scores for the overall experiments between control and immunized groups with the help of SPSS statistical package.
PBS was added into all wells of the 96 well plate followed by additional 40 μl PBS into the second well. 10 μl of serum sample was then added to the second well, mixed by pipetting (at least 5 times) and then 50 μl volume was transferred from the second well into the preceding well. The same was done to the remaining wells until the last well. Finally, 50 μl of antigen was added to each well, including the first well and incubated at 37 °C in a shaking incubator for an initial 5 min followed by 2 h' incubation without shaking. One positive and negative standard serum controls were used. Titers of ≥1:80 were considered positive. This endpoint titre was determined as the highest serum dilution showing agglutination of at least 50% of the leptospires.
2.10. Statistical analysis The mean for the quantitative variables was obtained with the help of SPSS statistical software. The hamster immunizations were analyzed by comparison of renal colonization based on recovery of leptospires with agglutinating and neutralizing antibody mean titers. The means of agglutinating and growth inhibition antibody titers was analyzed by Two-tailed t-Test using SPSS, 12.0 version program.
2.7. In-vitro leptospiral growth inhibition test IGIT 3. Results To detect neutralizing antibodies produced as a result of the DNA immunization, equal amount each of serum pools containing equal amounts of serum from all hamsters in LipDNA01, and pBudCE4.1 groups was prepared. The serum was inactivated at 56 °C for 30min in a water bath. In order to confirm the serum is not contaminated with saprophytic bacteria, 50 μl of each serum pool was cultured in Tryptic Soy Broth (TSB) at 37 °C for 48 h. The growth inhibition test was carried out using 200 μl of the twofold diluted (1:1) serum pools from each group. The 200 μl diluted serum was added into a 15 ml falcon tube containing 2.5 ml EMJH medium and 100 μl of 7–10 day old leptospiral culture containing approximately 108 leptospires. The mixture was incubated at 37 °C for 10 days and then examined for growth under darkfield microscope. As a control, 200 μl PBS was added to the 2.5 ml EMJH medium containing 100 μl leptospiral culture and incubated together. Dark-field microscopy was used to examine for the dpresence or absence of leptospiral growth. The result was analyzed based on the method of Tripathy et al. (1971) where tubes that had less than 10 leptospires per microscope field were scored as positive growth inhibition while negative growth inhibition was determined when the number of leptospires in the tube is similar to the number observed in the control tubes in which PBS (instead of test serum) was added to the media containing leptospires.
3.1. Prediction and selection of vaccine epitopes In-silico bioinformatics tools were employed in the construction of the multi-epitope chimeric DNA vaccine. Five antigenic epitopes [Tables 1 and 2] from LipL32 and LipL41 glycoproteins were predicted, engineered and assembled into a synthetic gene linked together with the help of GGGGS spacer between each epitope peptide as shown in Figs. 1 and 2. The full length multi-epitope chimeric gene construct contained a CpG motif at both the 5′ and 3′ end of the gene in order to enhance its immune stimulating ability (Fig. 3). Cloned expression plasmid (containing DNA fragment) was confirmed by restriction enzyme digestion and sequencing (Fig. 4). This recombinant plasmid LipDNA-01-pBudCE4.1 was then used as multi-epitope chimeric DNA vaccine to produce agglutinating and neutralizing antibodies against leptospira. 3.2. Physicochemical properties ProtParam tool was used to analyze the various physical and chemical parameters of the protein. The results indicate that LipL32 had 272 aa while LipL41 has 355 aa. Other parameters analyzed are a shown in Table 3 below.
2.8. Recovery of leptospires from kidneys by culture 3.3. Cloning and expression of recombinant chimeric DNA In order to assess kidney infection among the challenged hamsters by standard bacteriological method, kidney tissues were aseptically harvested after euthanizing the animal via exsanguination under general anaesthesia. The kidney was rinsed in sterile PBS, exsheathed and then crushed by passing it through the narrow opening of a 3 ml syringe into a 15 ml falcon tube containing 5 ml liquid EMJH medium with 5fluorouracil (200 μg/ml). The medium was then incubated for 10 days at 37 °C and then examined for growth of Leptospira under dark-field microscope.
The synthetic gene fragment after restriction enzyme digestion was successfully ligated at the multiple cloning site of the pBudCE expression plasmid downstream of the V5 epitope tag as shown in Figs. 5 and 6. The expression of the chimeric epitopes was achieved following transfection of Chinese hamster ovary cell line with LipDNA01pBudCE4.1. Indirect immunofluorescence antibody test was used to evaluate the level of expression. V5 antibody was used as the primary antibody while the secondary antibody used was Alexafluor 488 goatanti-hamster IgG (Invitrogen). Cytoplasmic expression of the chimeric DNA in CHO cells was evident by the green fluorescein color produced
2.9. Histopathological analysis
Table 2 LipL41 Predicted epitopes.
Kidney samples were aseptically collected from all hamsters at postmortem, rinsed with normal saline solution and then fixed in 10% (vol/vol) buffered formalin for 8 h and then transferred to 70% ethanol [17]. The fixed kidney tissues were sectioned at 5 μm with the aid of a microtome, stained with hematoxylin and eosin, and examined by light microscopy. The renal lesions for hamsters indicative of infection, were graded with 0 as normal, 1 as mild, 2 as moderate, and 3 as severe. A 138
No
Position
Epitope sequence
Peptide length
Antigenicity score
1 2 4
29–38 54–60 163–180
PVFPKDKEGR VEAPEKS ATGKDVNTGNEPVSKPTG
10 7 18
0.8006 0.9825 0.9697
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Fig. 1. IEDB BepiPred antigenic epitope prediction showing potential B cell epitopes for gene LipL32.
by the cells as shown in Fig. 7. The fluorescence emission of FITC (Fluorescein isothiocyanate) conjugates produced green fluorescence while DAPI which is a nucleic acid stain that binds to A-T rich regions of DNA stains the nucleus blue.
The arithmetic mean titer and standard error recorded for the plasmid control was 0.053 and 0.031 respectively. LipDNA01 vaccine group produced neutralizing antibodies against 8/19 serovars tested. The difference observed at 95% confidence interval between vaccine group and control was statistically significant at (p < 0.05).
3.4. Microscopic agglutination test MAT 3.6. Recovery of leptospires from kidneys by culture Prior to commencement of vaccination, all hamsters (day 0), were negative for agglutinating MAT titre. Agglutinating antibody titres was measured using the gold standard microscopic agglutination test based on their reactivity against 19 different pathogenic serovars. The antibody response was measured by calculating the arithmetic mean titre for both immunized and control groups. The obtained results indicate no detectable antibody response in the plasmid control. However, a significant raise in agglutinating antibody titre was observed in the vaccine groups (p < 0.05) which increased after booster immunization (Fig. 8). Traditionally, a positive reaction at 1:50 dilution is considered positive MAT, but in this study, only titre above the 1:80 dilutions was considered. The vaccines showed varying reactivity against antigens from serovars Bataviae, Copenhageni, Djasiman, Grippotyphosa, Icterohemorrhagiae, Javanica, Lai, and Hardjo bovis.
There was a significant decrease (p < 0.05) in the amount of leptospires recovered from the kidney of the vaccinated hamsters 28 days after challenge with sub-lethal dose of L. interrogans Copenhageni Fiocruz compared with the pBudCE4.1 control group. Although a large number of the cultures were heavily contaminated even after filtration, the decrease in leptospires observed was significantly lower in hamsters vaccinated with LipDNA01-pBudCE4.1. On the other hand, none of the unvaccinated control group, died after sub-lethal challenge with Copenhageni Fiocruz, probably because of the low dose or loss of virulence of the challenge strain due to repeated subculturing. However, a significant amount of leptospires was recovered (p < 0.05). Statistical analysis indicating the level of significance based on the mean between different vaccine groups is presented in Table 5.
3.5. Invitro growth inhibition test 3.7. Histopathological analysis The result for the invitro leptospira growth inhibition test is presented in Table 4 below. The table represents reactivity to each of the vaccine against all the serovars tested.
No obvious gross pathological lesion was observed in any of the kidneys examined at postmortem. Meanwhile, after H & E staining
Fig. 2. IEDB BepiPred antigenic epitope prediction showing potential B cell epitopes for gene LipL41. 139
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Fig. 3. Schematic representation of assembled multi-epitope chimeric gene indicating linked epitopes, oligonucleotide sequence and restriction enzymes.
4. Discussion Despite the increased level of awareness on the public health importance of leptospirosis, the incidence of the disease is increasing annually from an initial estimate of 500,000 cases in 1999 to more than double with about 60,000 fatalities in 2015 [6,35]. Leptospirosis constitutes an important public health problem in developing countries particularly those that are impoverished. The disease is responsible for economic losses in animal production as well as exerts a burden on human health. Available vaccines can only provide limited protection, which is short lived. The most commonly used leptospiral vaccines are based on inactivated whole-cell bacterins or leptospiral cell membrane components. These preparations although effective, are associated with several side-effects like pain, irritation and discomfort in addition to limited protection. In addition, the vast majority of the vaccines are for animal use, albeit for few countries that have licensed bacterins for human use [9,25]. As a result, the last few decades have seen research efforts shifting focus to the classical identification of antigens for the development of recombinant vaccines against leptospira. While significant progress have been recorded, the development of a broad-range leptospiral vaccine still remains elusive [8,39]. In this regard, reverse vaccinology which explores the entire genome of the pathogen and uses in-silico bioinformatics to produce highly specific recombinant antigens is currently being explored with a promising result. DNA vaccines containing multi-epitope encoded gene have been reported to confer protection against many infectious diseases [11,30,33]. DNA vaccine stimulates antibody production in a similar way as foreign antigens are processed and presented during natural infection. Similarly, multi-epitope DNA vaccines was reported to induce more potent immunoreactions than whole protein vaccines [41]. Such vaccines are also able to induce powerful cross-reactive immunological response due to the fact they are derived from multiple antigens packaged into a relatively small chimeric molecule. Moreover, immunity induced by multi-epitope DNA vaccine includes both the humoral and cellular immune component. Hence they are considered suitable for protection against a wide range of serovars especially in endemic regions. The construction of such highly complex synthetic vaccine may potentially have higher efficacy than assembly of naturally occurring sequences [40]. Chemically synthesized genes could produce a significant impact on the immuno-reactivity against diverse leptospiral outer membrane proteins [27,36]. In-silico bioinformatics approach can
Fig. 4. pBudCE4.1 plasmid carrying insert. Lane, 1 and 6. 1.5 kb DNA marker; lane 2–3, double digested plasmid, lane 4–5 linearized pBudCE4.1. Table 3 Physicochemical properties of amino acid sequences based on the ProtParam.
Molecular weight Theoretical pI Instability Index Aliphatic Index Grand average of hydropathicity
LipL32
LipL41
29612.9 6.34 32.53 85.74 −0.257
38939.7 6.01 26.19 87.15 −0.244
varying degrees of histopathological changes was observed (Fig. 9) The most common pathology was interstitial nephritis that consisted of inflammatory infiltrates like lymphocytes, plasma cells and macrophages. Based on the lesion scoring method, the intensity of the lesions for the vaccinated groups was less severe compared to the challenged control group. The lesions were predominantly located in the kidney cortex and medullary junction. The mean kidney score was mild and generally lower in the LipDNA01 immunized group and in comparison with the control groups, the difference was statistically significant at (p < 0.0109).
3.8. Morphological diagnosis Necro-hemorrhagic interstitial nephritis.
Fig. 5. Insilico clonning of LipDNA01 gene fragment (green) downstream of V5 epitope tag. The gene was inserted with the help of NotI and XhoI restriction enzymes and placed under the control of EF1α promoter. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 140
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Fig. 6. The pBudCE plasmid map showing cloned insert (green) downstream of the V5 epitope. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Fig. 7. Indirect Immunoflourecence test showing the expression of plasmid DNA constructs in CHO-K1 cells. LipDNA01pBudCE.4.1 expressed proteins in the cytoplasm is indicated by the diffused green fluorescence emission of FITC (Left) while the untransfected control shows only the DAPI stained nucleus (Right). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Table 4 Overall invitro growth inhibition test result.
Bataviae Canicola Copenhageni Djasiman Grippotyphosa Hardjobovis Icterohemorrhagiae Lai
LipDNA01
pBudCE4.1
Positive Positive Positive Positive Positive Positive Positive Positive
Negative Negative Positive Negative Negative Negative Negative Negative
Table 5 Recovery of leptospires from kidney samples of immunized.
Fig. 8. Agglutinating antibody titre after hamster immunization with plasmid DNA vaccine and controls. (2 = LipDNA01; 7 = pBudCE4.1).
Vaccine group
Number of samples
Contamination
Mean
Standard deviation
Standard error
LipDNA01 pBudCE4.1
12 12
6 6
0.2 0.6
0.422 0.516
0.075 0.092
LipL41 immunodominant glycoproteins of pathogenic leptospira were selected after prediction and evaluation by Bepipred and VaxiJen software. Immune response during leptospirosis is antibody based; hence humoral mediated immunity is of utmost importance [44,45]. In silico analysis of the physicochemical properties of the proteins showed that these selected epitopes are highly immunogenic and stable. While VaxiJen allocate a score of 0.4 as antigenic, the amino acid sequences
be employed to engineer synthetic proteins to induce antibody production that can match the highly complex antigenic variations of various pathogenic serovars to improve vaccine efficiency. In this study, a multi-epitope chimeric DNA vaccine was constructed against pathogenic leptospira species based on sequences in the NCBI data base. Five dominant immunogenic epitopes capable of inducing Bcell immunoreaction corresponding to segments in the LipL32 and 141
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Fig. 9. Photomicrographs of kidney sections. Control group (A) cortical section showing congested glomeruli (G), necrotic renal tubules containing bacterial cast (leptospira colonies; T), and presence of interstitial inflammatory response typified by infiltration of lymphocytes and few neutrophils, H&E x 400 and (B) cortical area showing an extensive area of interstitial hemorrhage (yellow arrow) with surrounding interstitial inflammatory response (black arrow), H&E x 400: LipDNA01 vaccine group (C) medullary section showing moderate tubular degeneration and necrosis (black arrow) of renal tubules and ducts accompanied by multifocal interstitial inflammatory response (yellow arrow) and few pinkish homogeneous cast in the tubular lumen, H&E x 100 (D) medullary section showing severe tubular degeneration and necrosis of renal tubules and ducts (black arrow) resulting in epithelial cast in the tubular lumen, multifocal areas of interstitial hemorrhage (yellow arrow) and marked interstitial inflammatory response (blue arrow), Normal renal parenchyma showing no pathological lesion H&E x 100. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
where found to persist for almost 5 months after vaccination [42]. This persistence is presumed to be as a result of longer opportunity for antibodies to react with the antigens in the invitro growth inhibition test as compared to MAT. Moreover, the steady increase in antibody titre may suggest continued expression of the DNA vaccine. This may also result in production of heightened immune response as previously reported where both LipL32 and LipL41 were expressed by invitro expression systems and they protect against lethal leptospira challenge [5]; [23]. These OM proteins are readily expressed in-vivo and are highly immunogenic. They are also conserved among pathogenic leptospira. Both MAT and GIT have been used to evaluate the immune response in buffalo calves vaccinated with heat-killed pentavalent bacterin vaccines [7]. In some studies, GIT cross-reacting antibodies have been demonstrated in buffaloes vaccinated with three experimental vaccines [7]. This is similar to the result of our study where the vaccines reacted to multiple serovars from different pathogenic species. This may indicate the potentials of the vaccine to provide broad range of protection against the commonly circulating serovars of pathogenic leptospira species. The in-vitro leptospira growth inhibition test is said to be a good method for assessing the potency of leptospira vaccines and may also be used in the analysis of vaccine efficacy [7]. Evaluation of protection from renal colonization as well as development of histopathological lesions on kidney of vaccinated and challenged hamsters show some mild to moderate pathologies among the vaccinated group while the control group show severe lesions. Majority of the lesions observed were located either in the cortical, corticomedullary or medullary region. They range from renal tubulointerstitial lesions, such as interstitial nephritis and cellular infiltration. Glomerular lesions with minimal intensity were also observed among the control group. While the vaccine group showed minimal to no obvious pathology in the kidney tissues, samples analyzed from the plasmid control group showed signs of severe damage with cellular infiltration and severe interstitial hemorrhages. This finding is supported by previous evidence that hamsters vaccinated with a combination of OmpL1 and LipL41 developed protective immunity and
considered in this study has antigenicity scores between 0.8 and 1.2, indicating they are highly antigenic. Moreover, the Aliphatic index is generally regarded as a positive factor for the increase of thermostability of proteins which is necessary for prolonged activity [46]. Bioinformatics has given room for selecting potential epitopes without the risk involved in propagating the pathogen of interest. This technique represent a huge advantage over conventional methods of vaccine production in addition to faster output and lower cost [34]. The result after immunization of golden Syrian hamsters revealed a raising antibody titre that increases after the booster immunization. In recent years, the use of epitopes in vaccines has become a valid alternative for improving the efficacy of vaccines [31,32]. Multi-epitope peptide DNA vaccines are effective against some viruses and they have recently being shown to have potential efficacy against some bacterial diseases including leptospires [10,11]. Similarly, in response to antigen stimulation, the chimeric DNA vaccine elicited both agglutinating and neutralization antibodies with the levels of the antibody being higher in the vaccine group compared to the plasmid control group. The fact that the DNA was expressed efficiently invitro is indicative of the fact that the DNA is correspondingly expressed after immunization as observed from the protection rendered. A significant correlation has also been previously reported between intra-cytoplasmic expression and elevation of serum level of proteins (Nozomi et al., 2004). Furthermore, although MAT is the most preferred test for leptospira infection, its sensitivity is relatively low, hence the recommendation that only a four-fold raise in antibody titres should be considered. Interestingly, the present result showed that the candidate vaccine is able to cause reaction against eight out of a panel of nineteen serovars tested by MAT. Similarly, it also prevented the growth of eight serovars according to the invitro growth inhibition test. This is in agreement with earlier reports where growth inhibition antibodies were used to measure cross-neutralizing antibodies in hamsters (Rosana et al., 2010). The growth inhibition antibodies were found to increase consistently and persisted longer that agglutinating antibodies by MAT. This observation is also similar to earlier reports where neutralizing antibodies 142
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resistance to renal colonization [19]. Immunization with a DNA vaccine encoding the conserved amino-terminal regions of LigA and LigB have also been reported to provide partial protection and sterilizing immunity [13,14]. In conclusion, a multi-epitope chimeric DNA vaccine against pathogenic leptospira was constructed, and the vaccine was able to elicit strong humoral immune responses as demonstrated by the high level agglutinating as well as neutralizing antibodies. Although, the vaccine was not able to provide full protection, but it was able to significantly reduced renal colonization. This is even more important when we consider that the protection rendered was against challenge with 106 leptospires which can hardly occur under natural situations. However, incorporating T-cell epitopes may likely improve the potency of the vaccine as Th-1 type immune response has previously been demonstrated in cattle vaccinated with killed L. hardjo vaccine [47]. Overall, the present novel immunogenic multi-epitope DNA vaccine developed by chemical gene synthesis and delivered as a plasmid DNA vaccine may serve as a new candidate target for leptospiral vaccine development.
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