Immune targeting of filarial glutaredoxin through a multi-epitope peptide-based vaccine: A reverse vaccinology approach.

Despite the efforts of global programme to eliminate lymphatic filariasis (GPELF), the threat of lymphatic filariasis (LF) still looms over humanity in terms of long-term disabilities, and morbidities across the globe. In light of this situation, investigators have chosen to focus on the development of immunotherapeutics targeting the physiologically important filarial-specific proteins. Glutaredoxin (16.43 kDa) plays a pivotal role in filarial redox biology, serving as a vital contributor. In the context of the intra-host survival of filarial parasites, this antioxidant helps in mitigating the oxidative stress imposed by the host immune system. Given its significant contribution, the development of a vaccine targeting glutaredoxin holds promise as a new avenue for achieving a filaria-free world. Herein, multi-epitope-based vaccine was designed using advanced immunoinformatics approach. Initially, 4B-cell epitopes and 6 T-cell epitopes (4 MHC I and 2 MHC II) were identified from the 146 amino acid long sequence of glutaredoxin of the human filarid, Wuchereria bancrofti. Subsequent clustering of these epitopes with linker peptides finalized the vaccine structure. To boost TLR-mediated innate immunity, TLR-specific adjuvants were incorporated into the designed vaccine. After that, experimental analyses confirm the designed vaccine, Vac4 as anefficient ligand of human TLR5 to elicit protective innate immunity against filarial glutaredoxin. Immune simulation further demonstrated abundant levels of IgG and IgM as crucial contributors in triggering vaccine-induced adaptive responses in the recipients. Hence, to facilitate the validation of immunogenicity of the designed vaccine, Vac4 was cloned in silico in pET28a(+) expression vector for recombinant production. Taken together, our findings suggest that vaccine-mediated targeting of filarial glutaredoxin could be a future option for intervening LF on a global scale.

Programmed cell death pathways as targets for developing antifilarial drugs: Lessons from the recent findings.

More than half a century has passed since the introduction of the National Filariasis Control Program; however, as of 2023, lymphatic filariasis (LF) still prevails globally, particularly in the tropical and subtropical regions, posing a substantial challenge to the objective of worldwide elimination. LF is affecting human beings and its economically important livestock leading to a crucial contributor to morbidities and disabilities. The current scenario has been blowing up alarms of attention to develop potent therapeutics and strategies having efficiency against the adult stage of filarial nematodes. In this context, the exploration of a suitable drug target that ensures lethality to macro and microfilariae is now our first goal to achieve. Apoptosis has been the potential target across all three stages of filarial nematodes viz. oocytes, microfilariae (mf) and adults resulting in filarial death after receiving the signal from the reactive oxygen species (ROS) and executed through intrinsic and extrinsic pathways. Hence, it is considered a leading target for developing antifilarial drugs. Herein, we have shown the efficacy of several natural and synthetic compounds/nanoformulations in triggering the apoptotic death of filarial parasites with little or no toxicity to the host body system.

Comparative Binding Ability of Human Monoclonal Antibodies against Omicron Variants of SARS-CoV-2: An In Silico Investigation.

Mutation(s) in the spike protein is the major characteristic trait of newly emerged SARS-CoV-2 variants such as Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Delta-plus. Omicron (B.1.1.529) is the latest addition and it has been characterized by high transmissibility and the ability to escape host immunity. Recently developed vaccines and repurposed drugs exert limited action on Omicron strains and hence new therapeutics are immediately needed. Herein, we have explored the efficiency of twelve therapeutic monoclonal antibodies (mAbs) targeting the RBD region of the spike glycoprotein against all the Omicron variants bearing a mutation in spike protein through molecular docking and molecular dynamics simulation. Our in silico evidence reveals that adintivimab, beludivimab, and regadanivimab are the most potent mAbs to form strong biophysical interactions and neutralize most of the Omicron variants. Considering the efficacy of mAbs, we incorporated CDRH3 of beludavimab within the framework of adintrevimab, which displayed a more intense binding affinity towards all of the Omicron variants viz. BA.1, BA.2, BA.2.12.1, BA.4, and BA.5. Furthermore, the cDNA of chimeric mAb was cloned in silico within pET30ax for recombinant production. In conclusion, the present study represents the candidature of human mAbs (beludavimab and adintrevimab) and the therapeutic potential of designed chimeric mAb for treating Omicron-infected patients.

Reverse vaccinology assisted design of a novel multi-epitope vaccine to target Wuchereria bancrofti cystatin: An immunoinformatics approach.

Proteases are the critical mediators of immunomodulation exerted by the filarial parasites to bypass and divert host immunity. Cystatin is a small (∼15 kDa) immunomodulatory filarial protein and known to contribute in the immunomodulation strategy by inducing anti-inflammatory response through alternative activation of macrophages. Recently, Wuchereria bancrofti cystatin has been discovered as a ligand of human toll-like receptor 4 which is key behind the cystatin-induced anti-inflammatory response in major human antigen-presenting cells. Considering the pivotal role of cystatin in the immunobiology of filariasis, cystatin could be an efficacious target for developing vaccine. Herein, we present the design and in-silico analyses of a multi-epitope-based peptide vaccine to target W. bancrofti cystatin through immune-informatics approaches. The 262 amino acid long antigen construct comprises 9 MHC-I epitopes and MHC-II epitopes linked together by GPGPG peptide alongside an adjuvant (50S ribosomal protein L7/L12) at N terminus and 6 His tags at C terminus. Molecular docking study reveals that the peptide could trigger TLR4-MD2 to induce protective innate immune responses while the induced adaptive responses were found to be mediated by IgG, IgM and Th1 mediated responses. Notably, the designed vaccine exhibits high stability and no allergenicity in-silico. Furthermore, the muti epitope-vaccine was also predicted for its RNA structure and cloned in pET30ax for further experimental validation. Taken together, this study presents a novel multi-epitope peptide vaccine for triggering efficient innate and adaptive immune responses against W. bancrofti to intervene LF through immunotherapy.

Andrographolide induces anti-SARS-CoV-2 response through host-directed mechanism: an in silico study.

Aim: Considering the present alarming situation of COVID-19 pandemic, we concentrated on evaluating the efficacy of a novel natural antiviral drug-candidate andrographolide against SARS-CoV-2 through an in silico model of study. Materials & methods: Interaction of andrographolide against the major host molecules that are responsible for SARS-CoV-2 pathogenesis were determined using bio-computational tools, in other words, molecular docking, molecular dynamics simulation and pharmacodynamics-pharmacokinetics analysis. Result: Computational findings represent that andrographolide efficiently interacts with the major human-host-associated putative drug-targets of viral-entry points like furin (-10.54 kcal/mol), TMPRSS-2 (-9.50 kcal/mol), ACE2 (-8.99 kcal/mol) and Cathepsin L (-8.98 kcal/mol). Moreover, it also blocks the inflammatory regulators including TLR4-MD2 and IL-6, which promote virus-induced inflammation leading to cytokine storm in the host body. Conclusion: This work elucidates that, the candidature of andrographolide can be utilized as a potent natural agent for the therapeutic intervention of SARS-CoV-2 through host-directed treatment.

Designing efficient multi-epitope peptide-based vaccine by targeting the antioxidant thioredoxin of bancroftian filarial parasite.

Thioredoxin is a low molecular weight redox-active protein of filarial parasite that plays a crucial role in downregulating the host immune response to prolong the survival of the parasite within the host body. It has the ability to cope up with the oxidative challenges posed by the host. Hence, the antioxidant protein of the filarial parasite has been suggested to be a useful target for immunotherapeutic intervention of human filariasis. In this study, we have designed a multi-epitope peptide-based vaccine using thioredoxin of Wuchereria bancrofti. Different MHC-I and MHC-II epitopes were predicted using various web servers to construct the vaccine model as MHC-I and MHC-II epitopes are crucial for the development of both humoral and cellular immune responses. Moreover, TLRs specific adjuvants were also incorporated into the vaccine candidates as TLRs are the key immunomodulator to execute innate immunity. Protein-protein molecular docking and simulation analysis between the vaccine and human TLR was performed. TLR5 is the most potent receptor to convey the vaccine-mediated inductive signal for eliciting an innate immune response. A satisfactory immunogenic report from an in-silico immune simulation experiment directed us to propose our vaccine model for experimental and clinical validation. The reverse translated vaccine sequence was also cloned in pET28a(+) to apply the concept in a wet lab experiment in near future. Taken together, this in-silico study on the design of a vaccine construct to target W. bancrofti thioredoxin is predicted to be a future hope in saving human-being from the threat of filariasis.

In-silico evidences on filarial cystatin as a putative ligand of human TLR4.

Cystatin is a small molecular weight immunomodulatory protein of filarial parasite that plays a pivotal role in downregulating the host immune response to prolong the survival of the parasite inside the host body. Hitherto, this protein is familiar as an inhibitor of human proteases. However, growing evidences on the role of cystatin in regulating inflammatory homeostasis prompted us to investigate the molecular reasons behind the explicit anti-inflammatory trait of this protein. We have explored molecular docking and molecular dynamics simulation approaches to explore the interaction of cystatin of Wuchereria bancrofti (causative parasite of human filariasis) with human Toll-like receptors (TLRs). TLRs are the most crucial component of frontline host defence against pathogenic infections including filarial infection. Our in-silico data clearly revealed that cystatin strongly interacts with the extracellular domain of TLR4 (binding energy=-93.5 ± 10 kJ/mol) and this biophysical interaction is mediated by hydrogen bonding and hydrophobic interaction. Molecular dynamics simulation analysis revealed excellent stability of the cystatin-TLR4 complex. Taken together, our data indicated that cystatin appears to be a ligand of TLR4 and we hypothesize that cystatin-TLR4 interaction most likely to play a key role in activating the alternative activation pathways to establish an anti-inflammatory milieu. Thus, the study provokes the development of chemotherapeutics and/or vaccines for targeting the cystatin-TLR4 interaction to disrupt the pathological attributes of human lymphatic filariasis. Our findings are expected to provide a novel dimension to the existing knowledge on filarial immunopathogenesis and it will encourage the scientific communities for experimental validation of the present investigation. Communicated by Ramaswamy H. Sarma.

Therapeutic Efficacy of Anti-Bestrophin Antibodies against Experimental Filariasis: Immunological, Immune-Informatics and Immune Simulation Investigations.

Lymphatic filariasis (LF) is a debilitating parasitic disease caused by filarial parasites and it is prevalent across the underprivileged population throughout the globe. The inadequate efficacy of the existing treatment options has provoked the conception of alternative strategies, among which immunotherapy is steadily emerging as a promising option. Herein, we demonstrate the efficacy of an antibody-based immunotherapeutic approach in an experimental model of filariasis, i.e., Wistar rat infected with Setaria cervi (a model filarial parasite). The polyclonal antibodies were raised against filarial surface antigen bestrophin protein (FSAg) in mice using the purified Wuchereria bancrofti FSAg. The adoptive transfer of anti-FSAg antibody-containing serum resulted in the significant reduction of parasite burden in filaria-infected rats. Intriguingly, anti-FSAg sera-treated animals also displayed a reduction in the level of proinflammatory cytokines as compared to the infected but untreated group. Furthermore, our in silico immunoinformatics data revealed eight B-cell epitopes and several T-cell epitopes in FSAg and these epitopes were linked to form a refined antigen in silico. The immune simulation suggested IgM and IgG1 as the predominant immunoglobulins induced in response to FSAg. Taken together, our experimental and simulation data collectively indicated a therapeutic potential of anti-FSAg sera against LF.

Exploring the Differential Expression and Prognostic Significance of the COL11A1 Gene in Human Colorectal Carcinoma: An Integrated Bioinformatics Approach.

Colorectal cancer is one of the most common cancers of humans and the second highest in cancer-related death. Genes used as prognostic biomarkers play an imperative role in cancer detection and may direct the development of appropriate therapeutic strategies. Collagen type XI alpha 1 (COL11A1) is a minor fibrillary collagen that has an essential role in the regulation of cell division, differentiation, proliferation, migration, growth, and apoptosis of intestinal and colon cells. The present study seeks to evaluate the significance of the COL11A1 gene in the progression of colorectal cancer in humans across the various parameters using advanced bioinformatics approaches. The application of various databases and servers like ONCOMINE, UALCAN, and GEPIA were accessed for analyzing the differential expression of the COLL11A1 gene and its relative influence over the survival of the transformed subjects. In addition, oncogenomics of COL11A1 gene, mutations associated with this gene and interacting partners of the gene in the context of oncogenesis were studied using COSMIC, cBioPortal, GeneMANIA, and NetworkAnalyst. Our experimental data indicate that the COL11A1 gene is overexpressed in the transformed tissues across the various clinicopathological parameters reduces the probability of survival in both overall and disease-specific survival cases. Mutational studies imply that it can induce perturbations in various signaling pathways viz. RTK-RAS-PI3K, Wnt, TGF-ß, and TP53 pathways influencing cancer development. Also, a positive association and correlation amongst the THBS2, COL10A1, COL5A2, and COL1A2 genes were observed, which most likely to contribute to the upregulation of carcinogenesis. Conclusively, this comprehensive study indicates the COL11A1 gene to be a significant contributor in the etiology of colorectal cancer, henceforth this gene can be considered as a prognostic biomarker for the conception of diagnostic and therapeutic strategies against colorectal cancer in the near future.

In silico analyses on the comparative sensing of SARS-CoV-2 mRNA by the intracellular TLRs of humans.

The coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has already resulted in a huge setback to mankind in terms of millions of deaths, while the unavailability of an appropriate therapeutic strategy has made the scenario much more severe. Toll-like receptors (TLRs) are crucial mediators and regulators of host immunity and the role of human cell surface TLRs in SARS-CoV-2 induced inflammatory pathogenesis has been demonstrated recently. However, the functional significance of the human intracellular TLRs including TLR3, 7, 8, and 9 is yet unclear. Hitherto, the involvement of these intracellular TLRs in inducing pro-inflammatory responses in COVID-19 has been reported but the identity of the interacting viral RNA molecule(s) and the corresponding TLRs have not been explored. This study hopes to rationalize the comparative binding of the major SARS-CoV-2 mRNAs to the intracellular TLRs, considering the solvent-based force-fields operational in the cytosolic aqueous microenvironment that predominantly drives these interactions. Our in silico study on the binding of all mRNAs with the intracellular TLRs depicts that the mRNA of NSP10, S2, and E proteins of SARS-CoV-2 are possible virus-associated molecular patterns that bind to TLR3, TLR9, and TLR7, respectively, and trigger downstream cascade reactions. Intriguingly, binding of the viral mRNAs resulted in variable degrees of conformational changes in the ligand-binding domain of the TLRs ratifying the activation of the downstream inflammatory signaling cascade. Taken together, the current study is the maiden report to describe the role of TLR3, 7, and 9 in COVID-19 immunobiology and these could serve as useful targets for the conception of a therapeutic strategy against the pandemic.

Designing of a novel multi-epitope peptide based vaccine against Brugia malayi: An in silico approach.

In spite of the tremendous efforts of the World Health Organization, scientific and medical community to eradicate lymphatic filariasis (LF) within 2020, the disease is still taking a huge toll on mankind throughout the globe. The current therapeutic strategies and solution measures against this alarming condition are suffering from a number of limitations such as inadequate effectiveness of the drugs against the adult-stage parasites, low bioavailability, and emergence of resistance. Considering this situation, development of the new therapeutics are urgently needed to combat human LF, especially targeting the adult filarial nematodes. Brugia malayi, the causative parasite for the human brugian filariasis majorly found in the countries of the South-Asia. In this study, we have designed a vaccine candidate using B-cell and T-cell epitopes derived from the aspartic protease of B. malayi (BmASP-1) and found to display significant humoral and cell mediated immune responses using in-silico approaches. Protein-protein docking between the human Toll-like receptor 4 (TLR4) and the vaccine candidate helped us to predict the way of inductive signaling that leads to immune-response. Molecular dynamics (MD) simulation studies further confirmed the proper docking between the TLR4 and vaccine candidate. Moreover, in-silico cloning of the vaccine element within the expression vector was found useful to optimize the restriction sites as well as to determine the primer location. Taken together, the in-silico vaccine candidate depicted in this study promises could be a useful therapeutic option for treating LF and experimental validation of this study is expected to strengthen the candidature of the said vaccine in the future.

In Silico Analyses on the Comparative Potential of Therapeutic Human Monoclonal Antibodies Against Newly Emerged SARS-CoV-2 Variants Bearing Mutant Spike Protein.

Since the start of the pandemic, SARS-CoV-2 has already infected more than 250 million people globally, with more than five million fatal cases and huge socio-economic losses. In addition to corticosteroids, and antiviral drugs like remdesivir, various immunotherapies including monoclonal antibodies (mAbs) to S protein of SARS-CoV-2 have been investigated to treat COVID-19 patients. These mAbs were initially developed against the wild-type SARS-CoV-2; however, emergence of variant forms of SARS-CoV-2 having mutations in the spike protein in several countries including India raised serious questions on the potential use of these mAbs against SARS-CoV-2 variants. In this study, using an in silico approach, we have examined the binding abilities of eight mAbs against several SARS-CoV-2 variants of Alpha (B.1.1.7) and Delta (B.1.617.2) lineages. The structure of the Fab region of each mAb was designed in silico and subjected to molecular docking against each mutant protein. mAbs were subjected to two levels of selection based on their binding energy, stability, and conformational flexibility. Our data reveal that tixagevimab, regdanvimab, and cilgavimab can efficiently neutralize most of the SARS-CoV-2 Alpha strains while tixagevimab, bamlanivimab, and sotrovimab can form a stable complex with the Delta variants. Based on these data, we have designed, by in silico, a chimeric antibody by conjugating the CDRH3 of regdanivimab with a sotrovimab framework to combat the variants that could potentially escape from the mAb-mediated neutralization. Our finding suggests that though currently available mAbs could be used to treat COVID-19 caused by the variants of SARS-CoV-2, better results could be expected with the chimeric antibodies.

Toll-Like Receptors (TLRs) as Therapeutic Targets for Treating SARS-CoV-2: An Immunobiological Perspective.

INTRODUCTION: Coronavirus disease-19 (COVID-19) caused by SARS-CoV-2 is presently the biggest threat to mankind throughout the globe. Increasing reports on deaths, cases of new infection, and socioeconomic losses are continuously coming from all parts of the world. Developing an efficacious drug and/or vaccine is currently the major goal to the scientific communities. In this context, toll-like receptors (TLRs) could be the useful targets in adopting effective therapeutic approaches. METHODS: This chapter has been written by incorporating the findings on TLR-based therapies against SARS-CoV-2 demonstrated in the recently published research papers/reviews. RESULTS: TLRs are the essential components of host immunity and play critical roles in deciding the fate of SARS-CoV-2 by influencing the immunoregulatory circuits governing human immune response to this pathogen. Hitherto, a number of multi-subunit peptide-based vaccines and pharmacological agents developed against SARS-CoV-2 have been found to manipulate TLR function. Therefore, circumventing overt immunopathology of COVID-19 applying TLR-antagonists can effectively reduce the morality caused from "cytokine storm"-induced multiorgan failure. Similarly, pre-administration of TLR- agonists may be used as a prophylaxis to sensitize the immune system of the individuals having risk of infection. A lot of collaborative efforts are required for bench-to-bench transformation of these knowledges. CONCLUSION: This chapter enlightens the potentials and promises of TLR-guided therapeutic strategies against COVID-19 by reviewing the major findings and achievements depicted in the literatures published till date.