Introduction
Leptospirosis is a standout among the most well-known zoonotic infections, resulting in very high morbidity and mortality in humans and also affecting global livestock production.[ 1 , 2 ] In India, the annual morbidity rate of leptospirosis has been reported to be 19.6 cases per 100,000 population.[ 3 ]
Outer membrane proteins (OMPs) constitute about half of the mass of the bacterial outer membrane (OM) and play a vital role in virulence mechanisms of pathogens as well as putative vaccinogen and diagnostic candidates. It is observed that different leptospiral extracts or protein fraction of Leptospira interrogans serovars can provide cross-protection against heterologous challenge. There have been several efforts for developing recombinant leptospiral vaccines with OMPs of the leptospires. Despite the identification of leptospiral antigens such as OmpL1, LipL41, LipL36, LipL32, and LipL21, only a few attempts have been made to utilize these leptospiral antigens in a recombinant vaccine. Currently, the leptospiral vaccine preparations using bacterin used for immunization of humans and animals suffer from several limitations, including severe side effects, including both systemic and local reactions. This is due to the presence of serovar-specific Lipopolysaccharide (LPS) in its preparations as a major antigenic component. In recent years, several studies have evaluated different formulations of recombinant vaccine candidates with the intention of improving leptospiral vaccines.[ 4 , 5 ] Targeting the surface proteins as a potential vaccine candidate for the development of new and effective vaccines in the prevention of leptospirosis holds promise, and protein with conserved immunogenic domain can confer cross-protection against heterologous serovars of L. interrogans .
The computational approaches for vaccine development can provide the complete information of protein structure, model, and also the immunogenic epitopes, emerging as a very useful tool for predicting and studying novel and potential vaccine candidates. In our previous study, LipL21 was evaluated for its diagnostic[ 6 ] and protective efficacy,[ 7 ] which also showed that it is a conserved protein and has a prominent role in diagnosis. This study involved the screening of major OMPs (OmpL1, LipL41, LipL32, and LipL21) of L. interrogans using bioinformatic tools for defining a better immunogenic and conserved OMP.
Materials and Methods
Using in silico techniques, the B-cell and T-cell epitopes were analyzed for providing both humoral as well as protective immunity, which could be universally used as a vaccine for the prevention of leptospirosis.
LipL21, LipL32, LipL41, and OmpL1 sequences were retrieved from the Swiss-Prot, a public domain protein database. The antigenicity of the four OMPs was determined using the VaxiJen v2.0 server. The proteins with an antigenic score of >0.4 were further analyzed. VaxiJen is the first server for alignment-independent prediction of potential antigens. The cutoff of the VaxiJen server is predefined for identifying a protein as an antigen or nonantigen. The value of 0.4 is based on the tool’s internal models and validation, which provides a balance between sensitivity and specificity in identifying the potential antigens.
A total of 23 sequences were retrieved for LipL21 from UniProtKB and subjected to the Clustal Omega server to check the similarity index of all the protein sequences of LipL21 ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ). To study a protein for its vaccine or diagnostic potential, its location in the cell is very significant. The protein sequence with the highest VaxiJen score was subjected to the CELLO V.2.5 server[ 8 ] to confirm the location of the protein, as location is significant in immunogenicity. Motifs in the LipL21 sequence were scanned using the SOSUI server[ 9 ] for hydropathicity and presence of transmembrane protein.
A total of 38 sequences of lipoproteins, such as LipL32, LipL41, OmpL1, and 34 different pathogenic sequences of LipL21 from L. interrogans serovar autumnalis, were retrieved from the UniProt database. The L. interrogans serovar autumnalis was taken in this study, as autumnalis is one of the most dominant serovars in India. The sequences retrieved were subjected to multiple sequence alignment (Clustal W). The purpose of the molecular phylogenetic tree was to estimate the relationships among the species represented by the sequences and to understand the relationships among the sequences themselves without any relation to the host species, using MEGA (Pennsylvania State University, USA).
For physicochemical characterization, isoelectric point (pI), molecular weight, total number of positive and negative residues, extinction coefficient,[ 10 ] instability index (II),[ 11 ] aliphatic index,[ 12 ] and grand average hydropathicity[ 13 ] were computed using the ExPASy ProtParam server.[ 14 ]
The analysis of the secondary and tertiary structure of protein sequences was based only on knowledge of their primary structure. The secondary structure feature of the protein sequences from L. interrogans was identified by GOR IV. The modeling of three-dimensional (3D) structure of the sequences was executed by Swiss-Modeler ( http://swissmodel.expasy.org/ ) program.[ 15 , 16 ]
The Kolaskar and Tongaonkar method was applied to define the linear B-cell epitopes (tool.iedb.org/). The accuracy of this method for the prediction of B-cell epitope is about 75%.[ 17 ] ElliPro from Immune Epitope Database (IEDB) analysis was used for the prediction of the conformational B-cell epitopes of LipL21 ( http://tools.iedb.org/ellipro/result/predict/ ).[ 18 ] The immunogenic B-cell epitope of LipL21 was selected for the prediction of T-cell epitopes using MHCPred analysis as shown in previous studies ( http://www.jenner.ac.uk/MHCPred ).[ 6 ]
Results
The in silico study with VaxiJen 2.0 of LipL21, LipL32, LipL41, and OmpL1 was done to identify and select a probable antigen, which is a critical step in vaccine design. The four OMPs were analyzed by VaxiJen 2.0, which revealed that LipL21 has the highest VaxiJen score as compared to OmpL1, LipL41, and LipL32 [ Table 1 ]. LipL21 was thus selected for further study on the basis of VaxiJen score of the selected OMPs.
Table 1
Antigenicity of the different outer membrane proteins using VaxiJen 2.0
The physiochemical and secondary structure analysis of LipL21 of Leptospira interrogans is further highlighted
The Clustal Omega analysis of LipL21 showed that it is conserved in all the pathogenic serovars. A total of 23 sequences from different pathogenic serovars of L. interrogans showed 100% similarity [ Figure 1 ]. This shows that LipL21 can be widely used in vaccine development as well as in diagnosis.
The phylogenetic tree was constructed using 38 protein sequences, comprising 37 sequences from pathogenic serovars of L. interrogans and one outlier group. The phylogenetic analysis revealed that all the proteins clustered in region [ Figure 1 ].
The ProtParam results exhibited the physicochemical parameters of the protein of LipL21. It was predicted to have 186 amino acids with the molecular weight of 18890.19 Daltons and the theoretical pI of 7.4 [ Table 1 ]. This states that LipL21 is basic in nature. The pI point of a protein is useful in developing a standard buffer system when the proteins are purified. The II was computed to be 32.68, which implies that the sequence of LipL21 is stable. The sequence has about 21 negatively charged residues (aspartic acid + glutamic acid) and 22 positively charged residues (arginine + lysine). The extinction coefficient for LipL21 was 28210 -1 Mcm 1 . The extinction coefficient is useful in determining protein–protein and protein–ligand interactions. The aliphatic index was calculated as 59.94, determining the thermal stability of globular proteins. The aliphatic index decides the stability of LipL21 over a wide temperature range. The grand average of hydropathicity (GRAVY) was calculated to be −0.425, indicating that the protein has better interaction with water. The negative value of the protein defines its hydrophilic nature, where most of the residues are localized on surface enhancing the interaction of LipL21 with other protein. The subcellular localization of protein using CELLO predicted that LipL21 is a cytoplasmic protein. SOSUI was used to determine the nature of the protein, i.e., soluble or transmembrane. SOSUI results predicted that it is a soluble protein with a hydrophobicity of − 0.458602. The secondary structural analysis of the protein by GOR IV showed that the random coil was found to be more frequent (47.31%), followed by the extended strand, to be the least frequent (34.41%), and the alpha helix was 18.28%.
The 3D model for LipL21 was generated using Swiss modeler program and validated using Ramachandran plot in the Procheck server. The analysis revealed that more than 90% of residues lie in the favoured region. The Q-MEAN Z of the model was −3.82, which defines the “degree of nativeness” of the structure. QMEAN Z around zero indicates a good agreement between the predicted model and experimental structure.
B-cell epitope prediction was done using the IEDB server. A total of six peptide sequences were obtained after analysis. The Kolaskar and Tongaonkar’s[ 17 ] method predicts the antigenic peptides, which involves the analysis of the physicochemical properties of the amino acids and their availability in the antigenic epitopes. LipL21 protein sequence, comprised 186 amino acids, has five antigenic peptide domains lying in the range of 8–22 amino acid length [ Table 2 ]. The conformational B-cell epitope in 3D structure was defined using the ElliPro server. The conformational B-cell epitopes with a protrusion index of 0.5 were selected. The PI score determines the percentage of protein atoms that are responsible for antibody binding.[ 19 ] The highest probability of conformational epitopes was 75.5% (0.755). The amino acid residues deduced by ElliPro are shown in Table 2 . T-cell epitopes on LipL21 were determined using MHCPred for binding affinity prediction. Peptides with the best predicted binding affinities for each allele were presented (data already shown in a previous study).[ 6 ]
Table 2
Various predictions and validation parameters of LipL21 of Leptospira interrogans
Prediction of hydrophobicity and presence of transmembrane domain, 3D structure validation, tertiary structure prediction, and the epitope prediction. RMSD=Root mean square deviation
| Prediction of hydrophobicity and presence of transmembrane domain of LipL21 | ||||
| Accession number | Average hydrophobicity | Protein type | Transmembrane region | Length |
| LipL21 | −0.458602 | Soluble protein | - | - |
| Validation of parameters computed for the modeled 3D structure of LipL21 | ||||
| Target | Ramapage percentage of residue favored region | RMSD (Å) | Quality of the model | |
| LipL21 | 90.6 | 1.83 | Fairly good | |
| Tertiary structure prediction of LipL21 sequence | ||||
| Accession number | Modeled residue range | Template | Sequence identity | QMEAN value |
| LipL21 | 32–129 | 5ok8.3.A | 14.16 | −3.82 |
| Predicted linear epitope (s) | ||||
| Number | Start | End | Peptide | Number of residues |
| 1 | 5 | 20 | LIALSLATMIFAACSS | 16 |
| 2 | 73 | 79 | GKAVAKK | 7 |
| 3 | 97 | 104 | ASDVVKKM | 8 |
| 4 | 121 | 142 | TASVIVSQSQGVVKGVGVYECK | 22 |
| 5 | 160 | 168 | ECQCVIYAK | 9 |
| Predicted discontinuous (confirmational) epitope (s) | ||||
| Number | Residues | Number of residues | Score | |
| 1 | A: T53, A: P54, A: R55, A: D56, A: T57, A: N58, A: P59, A: K60, A: D61 | 9 | 0.755 | |
| 2 | A: S68, A: S69, A: R70, A: A71, A: S72, A: G73, A: K74, A: A75, A: V76, A: A77, A: K78, A: K79, A: S80, A: Q81, A: M84 | 15 | 0.722 | |
| 3 | A: E46, A: D50, A: G51, A: K52 | 4 | 0.679 | |
| 4 | A: T108, A: V109, A: E110, A: S111, A: A112, A: S113, A: G114, A: V115, A: S116, A: D117, A: G118, A: E119, A: A120, A: T121, A: A122, A: S123, A: V124, A: I125, A: S127, A: Q128, A: S129 | 21 | 0.677 | |
| 5 | A: G32, A: D33, A: G34, A: G35, A: W36, A: T37 | 6 | 0.502 |
LipL21 is the second most abundant protein in L. interrogans serovars’ OMPs.[ 20 ] In this study, LipL21 was found to be highly antigenic with a score >0.4. LipL21 was selected for determining the primary, secondary, and tertiary structure using a bioinformatics tool, and also the B-cell epitope and T-cell epitope of the protein were defined. The ClustalW analysis of LipL21 revealed that it is conserved in all the pathogenic serovars. LipL21 is known to be conserved across the pathogenic serovars and is absent in nonpathogenic serovars.[ 6 ] The GRAVY was calculated to be −0.425 [ Table 2 ]. This low range of value indicates that the protein has better interaction with water. The negative value of the protein defines its hydrophilic nature, which states that the most of the residues are localized on surface. This also gives the insight that the probability of interaction of LipL21 with other proteins is more, which is in accordance with the data published by Raja and Natarajaseenivasan[ 21 ] that LipL21 is an OMP.
Discussion
A vaccine candidate is more effective when it comes in combination with B-cell and T-cell capable of producing both humoral and cell-mediated immunity.[ 18 ] B-cell epitope in an antigen is the most crucial step in the determination of peptide-based vaccines, the development of immunodiagnostic assay, and also in the production of antibodies. LipL21 has both B-cell and T-cell epitopes and thus can be targeted as a vaccine candidate for providing protection against Leptospira . The phylogenetic tree analysis also revealed that the protein sequences are clustered in the region, and also the sequences are conserved, making LipL21 a better candidate for vaccine development.
The currently available laboratory diagnostic tests are based on serological methods and are less sensitive and specific. The microscopic agglutination test is a less sensitive test with acute samples and is time-consuming.[ 22 ] There is reduced sensitivity during the acute stage of the disease and is time-consuming. The conventional vaccines, which are being used, do not provide heterologous protection and also have limited memory and thus cannot be used for long-term protection. Hence, the identification of a suitable vaccine candidate which could provide heterologous protection will be helpful in managing leptospirosis. The OMPs have been reported to be immunogenic and have a role in pathogenesis, as these are reported for cellular structure, attachment to various substrates, and in providing humoral and cellular immunity in humans and animals. The conserved nature of LipL21 in pathogenic leptospires gives insight that the recombinant protein will be effective in vaccine development regardless of serovar diversity. LipL21 protein with immunogenic epitopes having a higher VaxiJen score may be an ideal candidate to be utilized as a potential antigen. This suggests that LipL21 may be an ideal vaccine candidate for the protective immunity against leptospirosis.
Conclusion
There is a great need to develop a new vaccine candidate that can provide protection against heterologous serovars. In this study, the tools used for deducing the chemical and physical properties of the protein are reliable. B-cell epitope and T-cell epitopes of LipL21 analysis revealed that it is immunogenic in nature and can be targeted as a potential vaccine candidate.
Conflicts of interest
There are no conflicts of interest.
Funding Statement
Nil.
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