Vaccination and immunization strategies to design Aedes aegypti salivary protein based subunit vaccine tackling Flavivirus infection
Introduction
Flaviviruses are icosahedral, enveloped viruses having single-stranded positive-sense RNA as a genetic material with the genome size of 9.2–11 kb [1]. Flavivirus genera consisting of approximately 70 virus species, out of them, nearly 50 species are transmitted by arthropods [2]. The most severe disease-causing Flavivirus species include Dengue virus, Zika virus, and yellow fever virus. All these viral species have their specific endemic zone where their respective disease severity and the mortality rate are very high. The common symptom of all these viral diseases includes fever, chill, headache, nausea, vomiting while their severe form leads to the death. A single common link that exists between these Flavivirus species is their vector, to be specific, Aedes aegypti. It is responsible to spread the aforementioned viruses in its hitherto infected form leads to worldwide disease epidemiology [3]. Ae. aegypti originated in Africa and is presently distributed in urban regions throughout the tropical areas of Australia, South Pacific, Africa, America, Asia and the Middle East [4]. A literature survey has shown that the sandflies salivary proteins have anticoagulant, anti-platelet, anti-inflammatory, vasodilator and immunomodulatory properties [5]. Lutzomyia longipalpis salivary proteins are able to trigger IgG antibody in the person living in the endemic areas of visceral leishmaniasis [6,7]. Sandflies salivary proteins were also shown to influence the human antigen presenting cells and they were reported to inhibit the production of IL-10 and TNF-α while inducing the production of IL-8, IL-12p40 and IL-6 by the LPS activated antigen presenting cells [5]. Maxadilan (6.5 kDa) also belongs to the sandflies salivary protein having vasodilator and immunomodulatory properties. It was shown to intensify the skin lesion size in L. major infected mice as same as salivary gland sonicate [8]. Interestingly, mice immunized against maxadilan were protected against L. major infection [9], showing for the first time the opportunity of using a salivary protein as a vaccine directed against vector-borne pathogens. However, another study revealed that the use of Culex D7 salivary proteins actually increases the viral pathogenesis leads to the enhanced mice mortality, thereby emphasizing on the complexity of using salivary gland proteins in a vaccine strategy [10]. The salivary gland of Ae. aegypti also consists of immune factors along with vasodilatory, immune modulatory and anti-coagulant molecules that play a crucial role in blood meal collection, contributing towards the transmission of the aforementioned viruses [11]. It was also reported that after a mosquito bite, the human body develops anti-salivary antibody indicating the positive impact of a mosquito bite on human host immunity [12,13]. The immunogenic nature of Ae. aegypti salivary proteins may have the ability to alter the vertebrate immune response against Flavivirus virus infection, thereby modifying the infection kinetics in this manner [14]. The salivary protein namely putative 34-kDa protein (34kD: Q1HRW0) of Aedes aegypti has been reported to suppress type I IFN and anti-microbial peptide molecule during dengue virus infection [15,16]. To suppress the type I IFN gene expression, the 34 kDa protein represses the IRF-3 and IRF-7 transcription [16]. Salivary gland pair has also been reported to reduce CD4+ T cell activation and proliferation by suppressing IL-2 and IFN-γ during yellow fever virus infection. The secretion of pro-inflammatory cytokines like TNF-α, GM-CSF was also suppressed. Also, there was a reduced activation of macrophages and NK cells due to low IFN-γ titer [17]. Some of the most studied salivary proteins are aegyptin, anti-thrombin, apyrase, serine protease and venom allergen. Aegyptin (accession No: AAEL010235) is a collagen binding protein which involved in preventing platelet aggregation. It functions at both inoculation sites as well as in circulation. The concentration of cytokines and interleukins like GM-CSF, IFN-γ, IL-5, and IL-6 was increased by the functional activity of aegyptin [15]. Apyrase (Accession No: AAEL005672) is an inhibitor of ADP-dependent platelet aggregation, as well as neutrophil activation, leads to failure of platelet aggregation and mast cell degranulation [18]. Whereas other proteins namely serine protease (Accession No: Q1HRW0_AEDAE), venom allergen (Accession No: Q8T9U5_AEDAE) and anti-thrombin (Accession No: Q1HRTV7_AEDAE) suppress the IFN type-1, IRF-7, AMP responses of the host body. IRF-3 is suppressed by only serine proteases [15]. The aforementioned reports encourage us to use the salivary proteins of Ae. aegypti to design the subunit vaccine which may have the ability to enhance the host immunogenic response.
Till date, there is not even a single drug option to treat dengue infection and recently in 2015, the first dengue vaccine Dengvaxia was developed by Sanofi Pasteur and licensed in Mexico for use in an individual aged between 9 and 45 years. But nowadays it is in controversies because it has been proved to be effective only in patients with previous exposure to the virus. An infection after vaccination even results in severe diseased condition and the rest new vaccines are under the clinical trials. The yellow fever vaccine namely 17D was developed in the 1930s. It used live attenuated yellow fever virus and shown about 95% efficacy among the infected population. It had shown very rare side effects but those effects were very catastrophic, which includes post vaccine encephalitis and vaccine-associated viscerotropic disease leads to liver damage following systemic infection. Moreover, the use of this vaccine is prohibited in pregnant women, children below the age of 6 months, HIV infected and immuno-compromised people and also in a person allergic to eggs [19]. Zika virus vaccines are under clinical trials and none of them are licensed for the human use. In 2015, National Institute of Allergy and Infectious Diseases sponsored a vaccine against West Nile fever for clinical trials, no licensed vaccine is available till date. JEV has gain scientific attention resulted in the launch of four licensed vaccines which are live attenuated, live recombinant, inactivated Vero cell-derived, inactivated mouse brain-derived vaccines in nature. Although these vaccines are available, they all need booster doses and also have some adverse effect on some people.
Aforementioned drawbacks associated with the available vaccine needs a serious scientific concern to find a new vaccine candidate tackling these viral assaults. A step ahead, in the path of vaccine design, we developed a subunit vaccine based on the Ae. aegypti salivary protein with its capability to protect against aforementioned Flavivirus infection. As aforementioned, salivary proteins has shown to enhance the disease severity, therefore the subunit vaccine against salivary protein will generate the memory cell in the host body and on subsequent infection secondary antibody will neutralize the salivary proteins and will decreases the chances of pathogenic infection. Therefore, subunit vaccine was designed by the addition of B-cell epitope, CTL epitope, HTL epitope, Adjuvant, and linkers. All these constituents were added in a sequential manner to get an immunogenic, stable and non-allergic vaccine candidate. Further steps include 3D modeling, refinement, and validation followed by molecular docking and dynamics with the immune receptor (TLR-3). At last, in silico cloning was performed to obtain the clone of designed subunit vaccine into pET28a(+) vector for the microbial expression purposes. Overall this study applied an enthusiastic approach to tackle the problem of Flavivirus infection throughout the world.
Section snippets
Sequence procurement for vaccine design
We took five Ae. aegypti salivary protein from a literature survey and their respective FASTA sequences was obtained from National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/protein/?term=). Retrieved protein sequences were used for the vaccine development by employing sequential immunoinformatics approaches.
MHC-II specific helper T-lymphocyte epitope prediction and allelic population coverage analysis
Before vaccine designing, it was necessary to evaluate the shorted proteins for the presence of HTL epitopes. This objective was achieved by screening the
Salivary protein shorting and retrieval
Blood feeding is the essential step for the mosquito's survival as vertebrate host blood is important from the nutrition, survival and egg development perspective. The presence of numerous proteins in mosquito saliva helps in the successful blood feeding by the inhibition of hemostasis and the development of anti-inflammatory immune response. Eventually, the salivary proteins enhance the viral pathogen transmission via immunomodulatory and vasodilatory factors. Aedes aegypti saliva not only
Conclusion
Flavivirus species mainly Dengue virus, Zika virus, and, yellow fever virus causes mild to severe infection and sometimes may be fatal that affects the worldwide population. This study made an effort to design an immunogenic, non-allergenic, thermostable subunit vaccine which has the capacity to induce both humoral as well as cell-mediated immunity. We selected Ae. aegypti salivary protein sequences to design a subunit vaccine because of their role in mediating transmission of the
Author contributions
Protocol designed by RKP, VKP.
Methodology performed by RKP, VKP, SD.
Molecular Dynamics Simulations of the docked complex was performed by MJ.
Manuscript was written by RKP, SD, VKP and MJ.
Manuscript was improved by VKP and RS.
All authors have read and approved the manuscript.
Acknowledgment
RKP is thankful to Department of Science and Technology for providing INSPIRE fellowship. VKP is thankful to the Central University of Rajasthan for providing the computational facility. The project was partially supported by RS's JC Bose fellowship (SB/S2/JCB-071/2015). MJ and RS would also like to thank NCBS (TIFR) for infrastructural facilities.
Competing financial interests
The authors have declared no competing interest.
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