The 3D structure was predicted, refined, and validated using bioinformatics tools. Protein-protein docking of the chimeric vaccine peptide with the TLR4 protein predicted efficient binding. Overall, the constructed recombinant putative peptide demonstrated antigenicity superior to current vaccine candidates. Human onchocerciasis river blindness , caused by a parasitic nematode Onchocerca volvulus , is transmitted by the repeated bites of infected Simulium black flies.
It remains one of the most debilitating yet neglected tropical diseases NTDs 1. Recent estimates indicate that approximately An additional million at-risk people are reported to be in need of preventive chemotherapy 3. Although O. Some severely affected people also live in Venezuela and Brazil 2. The public health concern and socio-economic burden of onchocerciasis has led to the creation of various control programmes 7 , and large-scale control efforts have been ongoing for over 40 years 8.
Onchocerciasis control programmes have been successful in the Americas; the number of endemic foci decreased from 13 in six countries to two in two countries during the 4-year period from — The success of onchocerciasis control programmes in Africa however, has been limited to a few isolated communities 9 , 10 , The disease distribution worldwide, as well as the status of preventive chemotherapy in endemic countries was recently published by the World Health Organization WHO In addition, disease modelling studies have suggested that depending on compliance, therapeutic coverage, and levels of parasite transmission, it may not be possible to achieve onchocerciasis elimination even after 50 years of annual IVM treatments It has therefore been suggested that implementation of MDA programs using IVM alone will not be sufficient to achieve onchocerciasis elimination 16 especially in Africa where the disease burden is highest There is therefore a need for new tools to combat this disease 18 , 19 , Several observations support the feasibility of a vaccine approach to combat onchocerciasis.
Firstly, studies of human populations have provided evidence that naturally acquired immunity against O. Similar observations have been reported in cattle that are infected by a related parasite species, Onchocerca ochengi Specific antibody responses against the infective L3 larval stage develop over years of exposure in endemic regions 23 ; this likely explains the effective anti-L3 immunity acquired with age 24 , consistent with the concept of concomitant immunity In addition, immunisation of cattle and mice with irradiated L3 larvae protects against Onchocerca infection 22 , Finally, immuno-epidemiological evidence has led to the conclusion that the concurrent and predominant transmission of O.
It was of course not the first call for vaccine development initiatives. The selection of vaccine candidates reported in previous studies included either antigens immunogenic in putatively immune individuals or molecules supposedly important for parasite development 2. The most recent vaccine development initiatives led to the selection of two candidate vaccine antigens, Ov-RAL-2 and Ov, for further clinical development 2.
It has however been reported in the majority of cases that single recombinant antigens used for immunoprophylaxis induced insufficient immunity and thus elicited limited protective effects 27 , The L3 larval stage, together with the moulting larva mL3 stage, is where O. Thus, an antigen promoting anti-L3 larval immunity would block infection 29 and result in a preventative vaccine.
On the other hand, a vaccine targeted at the mf stage, the parasite stage which ensures transmission to the insect vector and which is associated with most of the pathology, would be both therapeutic and help to prevent transmission but would not eliminate the infection Most previous studies have focused on the use of either irradiated parasites 31 , 32 , 33 or single recombinant antigens 34 , 35 ; however, the immune responses generated have been inadequate to warrant their use in the development of an effective protective tool Alternatively, we propose the development of a multi-epitope vaccine candidate which could lead to the generation of a more potent protective immune response Multi-epitope vaccine candidates have already been designed for several diseases, including leishmaniasis, cholera, dengue 36 , 37 , 38 , and cancers 39 , 40 , and their efficacies have been further reported in Helicobacter, Schistosoma and cancer 27 , 41 , 42 , This work is an attempt to respond to the need for novel tools to help achieve the onchocerciasis elimination goal.
According to studies in human and animal models, immunity to O. IL-5 and IL-4 are deemed necessary for protection against irradiated L3 larva as elimination of either of these cytokines using either monoclonal antibodies or knockout mice significantly reduced the protective effects of vaccination against larval O. Immunity to microfilariae in a mouse model has however been demonstrated to be independent of IL-4 TLR4 involvement has also been reported in immune protective responses These antigens are recognized by sera from the putatively immune individuals who have never developed a patent infection with microfilaridermia To address the cross-protection potential of the designed vaccine candidate, the constituent proteins and predicted epitopes were evaluated for amino acid conservation with homologous proteins in other related parasites selected based on their importance in livestock or humans.
These include Onchocerca ochengi and Onchocerca flexuosa responsible for onchocerciasis in livestock leading to huge financial losses 50 , Loa loa which is a human filarial nematode that poses a significant challenge to onchocerciasis control programs 51 , and lastly Brugia malayi and Wuchereria bancrofti which are responsible for lymphatic filariasis, another debilitating filaria disease The amino acid sequences for six proteins were retrieved from the WormBase database and used to design a potential multi-epitope vaccine against onchocerciasis.
No signal peptides were predicted for the other proteins. The functional sequences for the proteins were then subjected to linear B-cell and T-cell epitope prediction. Immune adjuvants are a key requirement in vaccine formulation and play a critical role in enhancing the efficacy of vaccines Linear B-cell epitopes of varying residue lengths were predicted using different servers, but recurrent epitopes simultaneously predicted with BepiPred 2.
Some of the predicted linear B-cell epitopes were also predicted to be T-cell epitopes and used in generating the chimera. A total of eight high-binding HTL epitopes were selected for the final vaccine peptide. A BLAST search of the selected proteins against the UniProt database revealed a high degree of conservation for all the proteins in related nematodes ranging from Two of the proteins were found to be absent in at most one of the selected nematodes Onchocerca ochengi , Onchocerca flexuosa, Loa loa, Brugia malayi, Wuchereria bancrofti.
Multiple sequence alignment of the predicted B-cell and T-cell epitopes also revealed a large degree of conservation across the selected homologous proteins. The total number of predicted epitopes used in designing the chimera were 14 linear B-cell epitopes, 14 CTL epitopes, and eight HTL epitopes. P9WHE3, was chosen as an adjuvant and added to the amino terminus of the vaccine peptide using an EAAAK linker in order to potentiate antigen-specific immune responses. In addition, a 6xHis tag was added at the C-terminal to aid in protein purification and identification.
The final vaccine peptide generated consisted of amino acid residues derived from 22 merged peptide sequences Fig. Schematic presentation of the final multi-epitope vaccine peptide. The amino acid long peptide sequence containing an adjuvant green at the amino terminal end linked with the multi-epitope sequence through an EAAAK linker cyan. A 6x-His tag is added at the Carboxy terminus for purification and identification purposes. On the other hand, a total of potential epitopes having both negative and positive prediction scores were predicted for the main vaccine sequence by the IFNepitope server which predicts epitopes using the MERCI software.
C-ImmSim simulation of the cytokine levels induced by three injections given 4 weeks apart. The main plot shows cytokine levels after the injections. The insert plot shows IL-2 level with the Simpson index, D indicated by the dotted line. D is a measure of diversity. Increase in D over time indicates emergence of different epitope-specific dominant clones of T-cells.
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The smaller the D value, the lower the diversity. The antigenicity of the final sequence including the adjuvant sequence was predicted by the VaxiJen 2. The main vaccine sequence without adjuvant gave scores of 0. The results indicate that the generated sequences with and without adjuvant are both antigenic in nature. The vaccine sequence with and without the adjuvant were both predicted to be non-allergenic on both the AllerTOP v.
The molecular weight MW of the final protein was predicted to be The protein is predicted to be slightly acidic in nature based on the pI. The protein was predicted to be soluble upon expression with a solubility score of 0. An instability index II of The estimated aliphatic index was predicted to be The negative value indicates the protein is hydrophilic in nature and can interact with water molecules Graphical representation of secondary structure features of the final subunit vaccine sequence.
A The protein is predicted to comprise alpha-helices All the 10 chosen templates showed good alignment as per their Z-score values ranging from 1. The model with the highest C -score from the homology modelling was selected for further refinement Fig. This model had an estimated TM-score of 0. The TM-score has been proposed as a scale for measuring the structural similarity between two structures These cut-off values are independent of protein length.
Protein modelling, refinement and validation. Validation of the refined model with C Ramachandran plot analysis showing Based on model quality scores for all refined models, model 2 was found to be best based on various parameters including GDT-HA 0. The clash score was This model was chosen as the final vaccine model for further analysis Fig. The Ramachandran plot analysis of the modelled protein revealed that This is consistent with the Additionally, 4.
The chosen model after refinement had an overall quality factor of The protein fell outside the score range commonly found for native proteins of comparable size. A total of residues were predicted to be located in sixteen discontinuous B-cell epitopes with scores ranging from 0. The conformation epitopes ranged in size from five to 40 residues. The CASTp server was used to determine the protein binding and hydrophobic interaction sites on the protein surface. A binding pocket was identified between residue one and that could act as a potential binding site for TLR4.
The molecular surface area of the pocket was The immune response of TLR-4 against vaccine construct was estimated by analysing the overall conformational stability of vaccine protein-TLR4 docked complex with respect to that of adjuvant-TLR4 docked complex after performing data-driven docking of the complexes.
Based on input data used to drive docking, further examination of interface residues common to both complexes were performed as a validation of protocols used for their selection. A41 and A43 of the adjuvant sequences free adjuvant and adjuvant section of chimeric protein were involved in interactions at both interfaces, with three distinct residues D50, Y72 and F75 on the TLR4 B chain. These data not only validate selected docked complexes but may also explain the positive conformational shift on the adjuvant structure A41 and A43 both interact with D50 of TLR4, in the chimeric protein-TLR4 complex while in the adjuvant-TLR4 complex, A41 and A43 respectively interact with F75 and Y72 resulting from binding of the chimeric protein Fig.
Molecular docking of subunit vaccine with immune receptor TLR4. A Docked complexes for adjuvant-TLR4 complex with adjuvant colored light pink, chain B of TLR4 colored light blue and the interface colored hot pink and blue, and B chimeric protein-TLR4 complex with protein colored light pink, B chain of TLR4 colored light blue and the interface colored in blue and hot pink.
C Interface active residues for adjuvant-TLR4 complex with adjuvant active residues colored hotpink and TLR4 active residues colored in blue, and D chimeric protein-adjuvant complex with protein active residues colored hotpink and TLR4 active residues colored blue. In order to optimize codon usage of the vaccine construct in E. The length of the optimized codon sequence was 1, nucleotides.
The His-tag is located at the Carboxy-terminal end. C-ImmSim server immune simulation yielded results consistent with actual immune responses as evidenced by a general marked increase in the generation of secondary responses.
New approaches and omics tools for mining of vaccine candidates against vector-borne diseases
The primary response was characterized by high levels of IgM. This profile indicates development of immune memory and consequently increased clearance of the antigen upon subsequent exposures Fig. A similarly high response was seen in the T H helper and T C cytotoxic cell populations with corresponding memory development Fig. C-ImmSim presentation of an in silico immune simulation with the chimeric peptide. A Immunoglobulin production in response to antigen injections black vertical lines ; specific subclasses are indicated as coloured peaks.
B The evolution of B-cell populations after the three injections. C The evolution of T-helper, and D T-cytotoxic cell populations per state after the injections. The resting state represents cells not presented with the antigen while the anergic state represents tolerance of the T-cells to the antigen due to repeated exposures. Though chemotherapeutic measures do exist for many of these diseases, they are not sustainable countermeasures on their own due to rates of reinfection, risk of drug resistance, and inconsistent maintenance of drug treatment programs. Lustigman et al.
Many vaccines against onchocerciasis, including subunit and whole-organism vaccines, have been tested in animal models 2 , 28 , 60 , 61 , 62 ; however, none have been translated for use in humans. The focus has recently shifted towards the development of subunit vaccines as they are associated with better safety profiles and are logistically more feasible Epitope-based vaccines represent a novel approach for generating a specific immune response and avoiding responses against other unfavourable epitopes like epitopes that may drive immunopathogenic or immune modulating responses in the complete antigen Potential advantages of epitope-based vaccines also include increased safety, the opportunity to rationally engineer the epitopes for increased potency and breadth, and the ability to focus immune responses on conserved epitopes This work therefore focussed on the in silico design and development of a potential multi-epitope vaccine peptide for onchocerciasis using six proteins expressed in one of two parasite stages the microfilarial and infective larval stages.
These stages are preferentially targeted in vaccine development approaches, as any vaccine generated may have both prophylactic and therapeutic potentials. The potential for cross-protection also exists as the selected proteins and predicted epitopes used in generating the chimeric peptide exhibited considerable conservation across the related nematodes analysed. Cross-protection following vaccination with either purified antigens or whole attenuated parasites has been reported in the literature 26 , 62 , The proteins that we selected had exhibited potential to be vaccine candidates in immunomic studies It has been reported that immunity to onchocerciasis is dependent on both B- and T-cells 33 , The role for antibody-dependent cell-mediated cytotoxicity 46 , 66 , 67 and the TLR4 pathway in immunity to onchocerciasis has also been documented The potential role of TLR in mediating interactions between helminth parasites and the host immune system has been described Many filarial parasites including O.
It has been reported that signalling through TLR in filarial nematodes is associated with the major surface protein of Wolbachia wsp In the same study, Brattig et al. Mice deficient in either of these TLRs failed to elicit the same response. Studies have reported reduced expression of TLR1, 2, 4, and 9 on B-cells, both at the protein and mRNA levels, in subjects infected with lymphatic filariasis compared to uninfected controls. It was thus concluded that down-regulation of TLR1, 2, 4, and 9 appears to be an important mechanism of immune evasion in filarial infections It has been demonstrated in a murine model of T.
We first predicted B-cell and T-cell epitopes from the selected proteins and fused them using appropriate linkers in order to generate a multi-epitope peptide. Vaccine design is improved through the use of specialized spacer sequences Previously reported AAY and GPGPG linkers 36 , 37 were incorporated between the predicted epitopes to produce sequences with minimized junctional immunogenicity, therefore allowing the rational design construction of a potent multi-epitope vaccine The EAAAK linker, previously reported for bifunctional proteins 72 , was also added between the adjuvant sequence and the fused epitopes in order to reach a high level of expression and improved bioactivity of the fusion protein.
The lack of allergenic properties of the designed protein chimera further strengthens its potential as a vaccine candidate. To further assess the potential of the vaccine peptide developed through our work, its antigenicity was compared with that of Ov-RAL-2 and Ov The latter are reported to be lead vaccine candidates for onchocerciasis Vaccines carrying multiple epitopes are often poorly immunogenic and require coupling to adjuvants 27 ; however, the designed protein showed similar antigenicity scores with and without the addition of an adjuvant sequence.
The absence of the TLR4 agonist sequence even led to slightly higher predicted antigenicity. These results suggest that it may be worthwhile to express the chimeric peptide without the adjuvant used here and assess it in various tests with other adjuvants. Different adjuvants used in immunoprophylactic studies in onchocerciasis have been reported to influence the nature of the elicited immune response 74 , Ov-RAL-2 was predicted to be unstable.
The molecular weight of our vaccine candidate is The solubility of the overexpressed recombinant protein in the E. The theoretical pI is predicted to be 5. Also, the predicted instability index indicates that the protein will be stable upon expression, thus further strengthening its potential for use. The aliphatic index showed that the protein contains aliphatic side chains, indicating potential hydrophobicity.
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All these parameters indicate the recombinant protein is thermally stable and therefore suited for use in endemic areas, most of which are found in sub-Saharan Africa. The knowledge of secondary and tertiary structures of the target protein is essential in vaccine design These two structural forms, when tested in synthetic peptides, have the ability to fold into their native structure and thus be recognized by antibodies naturally induced in response to infection The 3D structure of the vaccine candidate improved markedly after the refinement and showed desirable properties based on Ramachandran plot predictions.
The Ramachandran plot shows that most of the residues are found in the favoured and allowed regions Previous studies have reported the involvement of TLR4 in immune protection against O. To assess potential immune interaction between TLR4 and the chimeric vaccine peptide, a data-driven protein-protein docking analysis was performed since a TLR4 agonist was used as an adjuvant in the designed chimera.
Though, molecular dynamic simulation is more appropriate to predict protein-protein binding efficiency, this option was not chosen because the interface between the TRL4 and the adjuvant was already reported Our findings support the idea that this interface is not perturbed upon fusion of the adjuvant to our polypeptide.
Moreover, the binding energies obtained from the adjuvant alone and the adjuvant-chimera interactions with TLR4 indicated that our designed chimera could indeed have a higher stimulation effect. In vitro , purified TLR4 and the recombinant protein could be investigated for such direct interaction. In vivo , this chimeric protein is therefore expected to interact with the TLR4 on professional antigen presenting cells, eliciting a potentially protective immune response. Immune simulation showed results consistent with typical immune responses. Following repeated exposure to the antigen, there was a general increase in the generated immune responses.
In onchocerciasis, IgG 1 , IgG 3 and IgE responses to parasite antigens are implicated in disease protection 24 , 46 , The development of memory B-cells and T- cells was evident, with memory in B-cells lasting several months. Helper T cells were particularly stimulated. This indicates high levels of T H cells and consequently efficient Ig production, supporting a humoral response. The Simpson index, D for investigation of clonal specificity suggests a possible diverse immune response.
This is plausible considering the generated chimeric peptide is composed of several B and T epitopes. One of the first steps in validating a candidate vaccine is to screen for immunoreactivity through serological analysis This requires the expression of the recombinant protein in a suitable host. Escherichia coli expression systems are the preferred choice for the production of recombinant proteins 86 , In order to achieve high-level expression of our recombinant vaccine protein in E.
Both the codon adaptability index 0. The next step projected at the moment, is to express this peptide in a bacterial system and perform the various immunological assays needed to validate the results obtained here through immuno-informatics analyses. The elimination of onchocerciasis will not be achieved without novel control methods These involve diagnostic and chemotherapeutic tools as well as a vaccine if possible 2.
In this study, immuno-informatics tools were employed to design a potential vaccine peptide coding for multiple B-cell and T-cell HTL and CTL epitopes. Given that the proteins containing these epitopes are expressed in the microfilariae and infective stages of the parasite, the vaccine peptide could potentially provide both prophylactic and therapeutic benefits.
This chimeric vaccine peptide could potentially be used as complementary tool to achieve onchocerciasis elimination. As a result of the cross-protection expected from the engineered vaccine candidate, other NTD control programs focusing on related filarial diseases such as lymphatic filariasis and loiasis, as well as control programs for onchocerciasis in livestock, could benefit from this initiative.
These proteins, which are highly expressed in the microfilariae or L3 larval stages of the parasite, were selected based on previous reports of reactivity with serum from putative immune individuals 2. The proteins were then subjected to signal peptide analyses using SignalP 4. B-cell epitopes are antigenic determinants recognized by the immune system and represent the specific piece of the antigen to which B lymphocytes bind. These play a vital role in vaccine design.
Linear B-cell epitopes were predicted predominantly using the BepiPred This method was superior to other available tools for sequence-based epitope prediction with regards to both epitope data derived from solved 3D structures and a large collection of linear epitopes downloaded from the IEDB database In addition, the proteins were subjected to linear epitope prediction using three other selected servers. Initially, epitopes for mers were predicted using ABCpred, with a default threshold of 0. The use of multiple tools in epitope prediction has been reported to improve the rate of true positives MHC class I binding and proteasomal cleavage are performed using artificial neural networks.
TAP transport efficiency is predicted using a weight matrix The threshold value for epitope identification was set at 0. The prediction of MHC II epitopes was based on receptor affinity, which can be inferred from the IC 50 values and percentile ranks assigned to each predicted epitope. The percentile rank may therefore be inversely related to the affinity of the epitope and directly related to the IC To assess the possibility of cross-protection conferred against other related nematode infections by the chosen proteins and predicted epitopes, a BLAST search was conducted on the UniProt database and the percentage identities for homologous relatives of the selected protein were obtained.
The percentage conservation of epitopes was calculated for the different predicted epitopes following multiple sequence alignment of homologous proteins in related nematodes. The nematodes selected for comparison included Onchocerca ochengi, Onchocerca flexuosa, Loa loa, Brugia malayi , and Wuchereria bancrofti. Either by signing into your account or linking your membership details before your order is placed.
Your points will be added to your account once your order is shipped. Click on the cover image above to read some pages of this book! This text provides a practical guide providing step-by-step protocol to design and develop vaccines.
Proof of principle for epitope-focused vaccine design
Chapters detail protocols for developing novel vaccines against infectious bacteria, viruses, fungi, and parasites for humans and animals. Volume 1: Vaccines for Human Diseases has an introductory section on how vaccines impacted diseases, the immunological mechanism of vaccines, future challenges for vaccinologists, and current trends in vaccinology. The design of human vaccines for viral, bacterial, fungal, parasitic and prion diseases as well as vaccines for drug abuse, allergy, and tumor vaccines are also described in this volume.
As a volume in the highly successful Methods in Molecular Biology series, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and tips on troubleshooting and avoiding known pitfalls. Help Centre.
My Wishlist Sign In Join. Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts see more benefits. Buy eBook. Buy Hardcover. Buy Softcover. FAQ Policy. About this book This text provides a practical guide providing step-by-step protocol to design and develop vaccines.
Authoritative and practical, Vaccine Design: Methods and Protocols, Volume 1: Vaccines for Human Diseases aims to ensure successful results in the further study of this vital field.