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Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line  [PDF]
James M. Wagner, J. David Pajerowski, Christopher L. Daniels, Patrick M. McHugh, Jessica A. Flynn, John W. Balliet, Danilo R. Casimiro, Shyamsundar Subramanian
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0094401
Abstract: Chikungunya virus-like particles (VLPs) have potential to be used as a prophylactic vaccine based on testing in multiple animal models and are currently being evaluated for human use in a Phase I clinical trial. The current method for producing these enveloped alphavirus VLPs by transient gene expression in mammalian cells presents challenges for scalable and robust industrial manufacturing, so the insect cell baculovirus expression vector system was evaluated as an alternative expression technology. Subsequent to recombinant baculovirus infection of Sf21 cells in standard culture media (pH 6.2–6.4), properly processed Chikungunya structural proteins were detected and assembled capsids were observed. However, an increase in culture pH to 6.6–6.8 was necessary to produce detectable concentrations of assembled VLPs. Since this elevated production pH exceeds the optimum for growth medium stability and Sf21 culture, medium modifications were made and a novel insect cell variant (SfBasic) was derived by exposure of Sf21 to elevated culture pH for a prolonged period of time. The high-pH adapted SfBasic insect cell line described herein is capable of maintaining normal cell growth into the typical mammalian cell culture pH range of 7.0–7.2 and produces 11-fold higher Chikungunya VLP yields relative to the parental Sf21 cell line. After scale-up into stirred tank bioreactors, SfBasic derived VLPs were chromatographically purified and shown to be similar in size and structure to a VLP standard derived from transient gene expression in HEK293 cells. Total serum anti-Chikungunya IgG and neutralizing titers from guinea pigs vaccinated with SfBasic derived VLPs or HEK293 derived VLPs were not significantly different with respect to production method, suggesting that this adapted insect cell line and production process could be useful for manufacturing Chikungunya VLPs for use as a vaccine. The adaptation of Sf21 to produce high levels of recombinant protein and VLPs in an elevated pH range may also have applications for other pH-sensitive protein or VLP targets.
Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells
Stefan W Metz, Corinne Geertsema, Byron E Martina, Paulina Andrade, Jacco G Heldens, Monique M van Oers, Rob W Goldbach, Just M Vlak, Gorben P Pijlman
Virology Journal , 2011, DOI: 10.1186/1743-422x-8-353
Abstract: Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits.Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections.Chikungunya virus (CHIKV) is an arthropod-borne (arbo)virus that causes epidemics in Africa, India and South-East Asia [1]. Recent outbreaks in Italy in 2007 [2] and autochthonous transmission events in France in 2010 [3] exemplify the threat of continued spread of CHIKV in the Western world, which correlates with the concurrent expanding distribution of its insect vector. CHIKV is maintained in a sylvatic transmission cycle of mosquitoes, rodents and primates, with Aedes aegyti as the primary vector. However, the responsible vector causing the severe CHIKV epidemic on the Reunion Islands in 2005/2006 was Ae. albopictus [4]. This vector switch made the virus endemic in more temperate regions and resulted in the first European cases (Italy, 2007) of transmission by local
Novel Chikungunya Vaccine Candidate with an IRES-Based Attenuation and Host Range Alteration Mechanism  [PDF]
Kenneth Plante,Eryu Wang,Charalambos D. Partidos,James Weger,Rodion Gorchakov,Konstantin Tsetsarkin,Erin M. Borland,Ann M. Powers,Robert Seymour,Dan T. Stinchcomb,Jorge E. Osorio,Ilya Frolov,Scott C. Weaver
PLOS Pathogens , 2011, DOI: 10.1371/journal.ppat.1002142
Abstract: Chikungunya virus (CHIKV) is a reemerging mosquito-borne pathogen that has recently caused devastating urban epidemics of severe and sometimes chronic arthralgia. As with most other mosquito-borne viral diseases, control relies on reducing mosquito populations and their contact with people, which has been ineffective in most locations. Therefore, vaccines remain the best strategy to prevent most vector-borne diseases. Ideally, vaccines for diseases of resource-limited countries should combine low cost and single dose efficacy, yet induce rapid and long-lived immunity with negligible risk of serious adverse reactions. To develop such a vaccine to protect against chikungunya fever, we employed a rational attenuation mechanism that also prevents the infection of mosquito vectors. The internal ribosome entry site (IRES) from encephalomyocarditis virus replaced the subgenomic promoter in a cDNA CHIKV clone, thus altering the levels and host-specific mechanism of structural protein gene expression. Testing in both normal outbred and interferon response-defective mice indicated that the new vaccine candidate is highly attenuated, immunogenic and efficacious after a single dose. Furthermore, it is incapable of replicating in mosquito cells or infecting mosquitoes in vivo. This IRES-based attenuation platform technology may be useful for the predictable attenuation of any alphavirus.
Jimit S Patel,Charoo S Garg,Kiran M Patel,Rinku K Patel
Pharmacie Globale : International Journal of Comprehensive Pharmacy , 2011,
Abstract: Chikungunya fever is a viral disease transmitted to humans by the bite of infected mosquitoes. The virus is a member of the genus Alphavirus, in the family Togaviridae. Chikungunya fever is a viral disease transmitted to humans by the bite of infected mosquitoes. Chikungunya virus is a member of the genus Alphavirus, in the family Togaviridae. The fever is diagnosed based on symptoms, physical findings (e.g., joint swelling), laboratory testing, and the possibility of exposure to infected mosquitoes. There is no specific treatment for chikungunya fever; care is based on symptoms. Chikungunya infection is not usually fatal. Steps to prevent infection with chikungunya virus include use of insect repellent, protective clothing, and staying in areas with screens. Chikungunya virus was first isolated from the blood of a febrile patient in Tanzania in 1953, and has since been cited as the cause of numerous human epidemics in many areas of Africa and Asia and most recently in limited areas of Europe.
A Rodent Model of Chikungunya Virus Infection in RAG1 -/- Mice, with Features of Persistence, for Vaccine Safety Evaluation  [PDF]
Robert L. Seymour?,A. Paige Adams?,Grace Leal?,Maria D. H. Alcorn?,Scott C. Weaver
PLOS Neglected Tropical Diseases , 2015, DOI: 10.1371/journal.pntd.0003800
Abstract: Chikungunya virus (CHIKV) is a positive sense, single stranded RNA virus in the genus Alphavirus, and the etiologic agent of epidemics of severe arthralgia in Africa, Asia, Europe and, most recently, the Americas. CHIKV causes chikungunya fever (CHIK), a syndrome characterized by rash, fever, and debilitating, often chronic arthritis. In recent outbreaks, CHIKV has been recognized to manifest more neurologic signs of illness in the elderly and those with co-morbidities. The syndrome caused by CHIKV is often self-limited; however, many patients develop persistent arthralgia that can last for months or years. These characteristics make CHIKV not only important from a human health standpoint, but also from an economic standpoint. Despite its importance as a reemerging disease, there is no licensed vaccine or specific treatment to prevent CHIK. Many studies have begun to elucidate the pathogenesis of CHIKF and the mechanism of persistent arthralgia, including the role of the adaptive immune response, which is still poorly understood. In addition, the lack of an animal model for chronic infection has limited studies of CHIKV pathogenesis as well as the ability to assess the safety of vaccine candidates currently under development. To address this deficiency, we used recombination activating gene 1 (RAG1-/-) knockout mice, which are deficient in both T and B lymphocytes, to develop a chronic CHIKV infection model. Here, we describe this model as well as its use in evaluating the safety of a live-attenuated vaccine candidate.
A Novel MVA Vectored Chikungunya Virus Vaccine Elicits Protective Immunity in Mice  [PDF]
James Weger-Lucarelli ,Haiyan Chu,Matthew T. Aliota,Charalambos D. Partidos,Jorge E. Osorio
PLOS Neglected Tropical Diseases , 2014, DOI: 10.1371/journal.pntd.0002970
Abstract: Background Chikungunya virus (CHIKV) is a re-emerging arbovirus associated with febrile illness often accompanied by rash and arthralgia that may persist for several years. Outbreaks are associated with high morbidity and create a public health challenge for countries affected. Recent outbreaks have occurred in both Europe and the Americas, suggesting CHIKV may continue to spread. Despite the sustained threat of the virus, there is no approved vaccine or antiviral therapy against CHIKV. Therefore, it is critical to develop a vaccine that is both well tolerated and highly protective. Methodology/Principal Findings In this study, we describe the construction and characterization of a modified Vaccinia virus Ankara (MVA) virus expressing CHIKV E3 and E2 proteins (MVA-CHIK) that protected several mouse models from challenge with CHIKV. In particular, BALB/c mice were completely protected against viremia upon challenge with CHIKV after two doses of MVA-CHIK. Additionally, A129 mice (deficient in IFNα/β) were protected from viremia, footpad swelling, and mortality. While high anti-virus antibodies were elicited, low or undetectable levels of neutralizing antibodies were produced in both mouse models. However, passive transfer of MVA-CHIK immune serum to na?ve mice did not protect against mortality, suggesting that antibodies may not be the main effectors of protection afforded by MVA-CHIK. Furthermore, depletion of CD4+, but not CD8+ T-cells from vaccinated mice resulted in 100% mortality, implicating the indispensable role of CD4+ T-cells in the protection afforded by MVA-CHIK. Conclusions/Significance The results presented herein demonstrate the potential of MVA to effectively express CHIKV E3-E2 proteins and generate protective immune responses. Our findings challenge the assumption that only neutralizing antibodies are effective in providing protection against CHIKV, and provides a framework for the development of novel, more effective vaccine strategies to combat CHIKV.
A Trivalent Virus-Like Particle Vaccine Elicits Protective Immune Responses against Seasonal Influenza Strains in Mice and Ferrets  [PDF]
Ted M. Ross, Kutub Mahmood, Corey J. Crevar, Kirsten Schneider-Ohrum, Penny M. Heaton, Rick A. Bright
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0006032
Abstract: There is need for improved human influenza vaccines, particularly for older adults who are at greatest risk for severe disease, as well as to address the continuous antigenic drift within circulating human subtypes of influenza virus. We have engineered an influenza virus-like particle (VLP) as a new generation vaccine candidate purified from the supernatants of Sf9 insect cells following infection by recombinant baculoviruses to express three influenza virus proteins, hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1). In this study, a seasonal trivalent VLP vaccine (TVV) formulation, composed of influenza A H1N1 and H3N2 and influenza B VLPs, was evaluated in mice and ferrets for the ability to elicit antigen-specific immune responses. Animals vaccinated with the TVV formulation had hemagglutination-inhibition (HAI) antibody titers against all three homologous influenza virus strains, as well as HAI antibodies against a panel of heterologous influenza viruses. HAI titers elicited by the TVV were statistically similar to HAI titers elicited in animals vaccinated with the corresponding monovalent VLP. Mice vaccinated with the TVV had higher level of influenza specific CD8+ T cell responses than a commercial trivalent inactivated vaccine (TIV). Ferrets vaccinated with the highest dose of the VLP vaccine and then challenged with the homologous H3N2 virus had the lowest titers of replicating virus in nasal washes and showed no signs of disease. Overall, a trivalent VLP vaccine elicits a broad array of immunity and can protect against influenza virus challenge.
Vimal Kumar Birendra,Singh Vishwabhan,Joon Uttam,Suvagiya Vishal
International Research Journal of Pharmacy , 2012,
Abstract: Mosquitoes transmit numerous arboviruses including dengue and chikungunya virus (CHIKV). Chikungunya is a re-emerging arthropod-borne viral disease caused by Chikungunya virus (CHIKV) belonging to the Togaviridae family of genus Alphavirus. It is a virus with a single stranded, positive sense RNA, as its genome. It is maintained in a sylvatic and urban cycle involving humans and the mosquito species Aedes aegypti and Aedes albopictus. It has a major health impact on humans as it causes fever, rashes, arthralgia and myalgia. Polyarthralgia is the most important feature of CHIKV infection which primarily affects the small joints of the wrists and fingers along with the large joints like shoulders and knees. Currently, there are no vaccines or treatment regimens available for CHIKV infection. The molecular mechanism underlying the chronic polyarthralgia observed in patients is not well understood. The abundance of bacteria from the Enterobacteriaceae family increased with CHIKV infection whereas the abundance of known insect endosymbionts like Wolbachia and Blattabacterium decreased. In this review we have summarized the CHIKV organization, replication, epidemiology, clinical manifestations and pathogenesis with emphasis on the arthralgia.
Deliberate Attenuation of Chikungunya Virus by Adaptation to Heparan Sulfate-Dependent Infectivity: A Model for Rational Arboviral Vaccine Design  [PDF]
Christina L. Gardner,Jozef Hritz,Chengqun Sun,Dana L. Vanlandingham,Timothy Y. Song,Elodie Ghedin,Stephen Higgs,William B. Klimstra,Kate D. Ryman
PLOS Neglected Tropical Diseases , 2014, DOI: 10.1371/journal.pntd.0002719
Abstract: Mosquito-borne chikungunya virus (CHIKV) is a positive-sense, single-stranded RNA virus from the genus Alphavirus, family Togaviridae, which causes fever, rash and severe persistent polyarthralgia in humans. Since there are currently no FDA licensed vaccines or antiviral therapies for CHIKV, the development of vaccine candidates is of critical importance. Historically, live-attenuated vaccines (LAVs) for protection against arthropod-borne viruses have been created by blind cell culture passage leading to attenuation of disease, while maintaining immunogenicity. Attenuation may occur via multiple mechanisms. However, all examined arbovirus LAVs have in common the acquisition of positively charged amino acid substitutions in cell-surface attachment proteins that render virus infection partially dependent upon heparan sulfate (HS), a ubiquitously expressed sulfated polysaccharide, and appear to attenuate by retarding dissemination of virus particles in vivo. We previously reported that, like other wild-type Old World alphaviruses, CHIKV strain, La Réunion, (CHIKV-LR), does not depend upon HS for infectivity. To deliberately identify CHIKV attachment protein mutations that could be combined with other attenuating processes in a LAV candidate, we passaged CHIKV-LR on evolutionarily divergent cell-types. A panel of single amino acid substitutions was identified in the E2 glycoprotein of passaged virus populations that were predicted to increase electrostatic potential. Each of these substitutions was made in the CHIKV-LR cDNA clone and comparisons of the mutant viruses revealed surface exposure of the mutated residue on the spike and sensitivity to competition with the HS analog, heparin, to be primary correlates of attenuation in vivo. Furthermore, we have identified a mutation at E2 position 79 as a promising candidate for inclusion in a CHIKV LAV.
A Review Of Chikungunya  [cached]
Bincy Thomas,A.R.Tekade,Pande V.V
Pharmaceutical Reviews , 2007,
Abstract: Vaccines and new drugs against chikungunya are urgently needed. Chikungunya is generally not fatal.However, in 2005-2007, many deaths have been associated with chikungunya worldwide and still it is continuing. Currently there is no vaccine and fully satisfied drugs to treat all symptoms of chikungunya.Treatment are just symptomatic and in many cases improper use of NSAIDS leads to more complications. Every year 1000s are getting infected and many are dying. So it is high time to turn health organisation’s concentration to find a solution for this health issue.IntroductionThe word chikungunya meaning "that which bends up", which is derived from its arthritic symptoms. It is the hottest topic of discussion among public today, so is necessary to know the causes, symptoms, treatment and preventive measures of this viral fever. Usually chikungunya virus or CHIKV is spread by mosquito bites from Aedes aegypti mosquitoes, Aedes albopictus (Tiger mosquito), Aedes luteocephalus, or A. taylori.. There is no reported case of human-human transmission of CHIKV. The outbreak of infection and studies about it reveals that people living near to forest and water stores are more prone to this viral desease, since these areas provide better environment for mosquito breeding.High fever and arthritic pain, especially severe pain on extremes is characteristic of chikungunya fever. In allopathic, treatment is based on the symptoms and no preventive drugs are available. Siddha system claims some medicines for both treatment and prevention, but is not scientifically proven. By taking proper precaution against mosquito bites by using insecticides, repellents and other measures, transmission of CHIKV can be prevented to greater extent.
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