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1.
Methods Mol Biol ; 2829: 247-255, 2024.
Article in English | MEDLINE | ID: mdl-38951340

ABSTRACT

The Baculovirus Expression Vector System (BEVS) is used with cultured insect cells to produce a wide variety of heterologous proteins, which can be secreted into the culture medium during the transient infection process (Smith et al. Mol Cell Biol 12:2156-2165, 1983). When the infection process is complete, centrifugation is often used to separate the desired protein from the spent insect cells. The desired product in the harvested supernatant is contaminated with baculovirus, amino acids, lipids, detergents, oils, lysed cells from the infection process, genomic DNA from the insect cells, and proteases due to the lytic nature of the baculovirus infection process and many other contaminants (Ikonomou et al. Appl Microbiol Biotechnol 62:1-20, 2003). All these contaminants that are present in the centrifuged supernatant with the desired secreted protein make the initial chromatographic capture step critical for effective purification of the desired protein. A purification scheme will be outlined for a slightly acidic secreted protein using cation exchange chromatography (Lundanes et al. Chromatography: basic principles, sample preparations and related methods, 1st edn. Wiley, 2013).


Subject(s)
Baculoviridae , Animals , Baculoviridae/genetics , Baculoviridae/metabolism , Chromatography, Ion Exchange/methods , Recombinant Proteins/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Insecta/cytology , Sf9 Cells , Genetic Vectors/genetics , Cell Line , Spodoptera
2.
Hum Vaccin Immunother ; 18(4): 2062971, 2022 11 30.
Article in English | MEDLINE | ID: mdl-35801956

ABSTRACT

While there are several SARS-CoV-2 vaccines currently available, additional options must be provided that are safe, effective, and affordable for the entire global population. We have developed a novel immune activating platform technology that will fill this need. This recombinant platform protein is produced in insect cells using baculoviral expression technology similar to what is currently used for several other approved vaccines as well as employed by myriad GMP facilities globally. Thus, infrastructure exists for rapid scale up following initial optimizations. Here we report initial results for a SARS-CoV-2 vaccine (OMN008) based on our platform technology. Unadjuvanted OMN008 vaccination resulted in robust antigenicity and neutralization. Additionally, OMN008 vaccination induced a specific CD8 T-cell response. All of these results taken together indicate OMN008 may be an excellent candidate to fill gaps left by the currently available vaccines. Further testing is necessary to fully optimize production; however, overall cost of production should remain low given the simple formulation of this recombinant platform.


Subject(s)
COVID-19 Vaccines , COVID-19 , Humans , COVID-19/prevention & control , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Subunit , Vaccine Development , Recombinant Proteins
3.
Methods Mol Biol ; 1403: 269-84, 2016.
Article in English | MEDLINE | ID: mdl-27076136

ABSTRACT

Given periodic outbreaks of fatal human infections caused by coronaviruses, development of an optimal coronavirus vaccine platform capable of rapid production is an ongoing priority. This chapter describes the use of an insect cell expression system for rapid production of a recombinant vaccine against severe acute respiratory syndrome coronavirus (SARS). Detailed methods are presented for expression, purification, and release testing of SARS recombinant spike protein antigen, followed by adjuvant formulation and animal testing. The methods herein described for rapid development of a highly protective SARS vaccine are equally suited to rapid development of vaccines against other fatal human coronavirus infections, e.g., the MERS coronavirus.


Subject(s)
Inulin/immunology , Severe acute respiratory syndrome-related coronavirus/immunology , Viral Vaccines/immunology , Adjuvants, Immunologic , Animals , Humans , Severe Acute Respiratory Syndrome/immunology , Spike Glycoprotein, Coronavirus/immunology
4.
Vaccine ; 27(2): 213-22, 2009 Jan 07.
Article in English | MEDLINE | ID: mdl-18996430

ABSTRACT

In this study, a recombinant truncated West Nile virus envelope protein antigen (rWNV-E) was produced in serum-free cultures of the expresSF+ insect cell line via baculovirus infection. This production system was selected based on its use in the production of candidate human and animal vaccine antigens. A defined fermentation and purification process for the rWNV-E antigen was established to control for purity and immunogenicity of each protein batch. The material formulated with aluminum hydroxide was stable for greater than 8months at 4 degrees C. The recombinant vaccine candidate was evaluated for immunogenicity and protective efficacy in several animal models. In mouse and hamster WNV challenge models, the vaccine candidate induced viral protection that correlated with anti-rWNV-E immunogenicity and WNV neutralizing antibody titers. The rWNV-E vaccine candidate was used to boost horses previously immunized with the Fort Dodge inactivated WNV vaccine and also to induce WNV neutralizing titers in naïve foals that were at least 14weeks of age. Furthermore, the vaccine candidate was found safe when high doses were injected into rats, with no detectable treatment-related clinical adverse effects. These observations demonstrate that baculovirus-produced rWNV-E can be formulated with aluminum hydroxide to produce a stable and safe vaccine which induces humoral immunity that can protect against WNV infection.


Subject(s)
Recombinant Proteins/metabolism , Spodoptera/metabolism , Viral Envelope Proteins/metabolism , West Nile Fever/prevention & control , West Nile Virus Vaccines/metabolism , West Nile virus/immunology , Animals , Antibodies, Viral/blood , Baculoviridae/genetics , Baculoviridae/metabolism , Cells, Cultured , Cricetinae , Disease Models, Animal , Horse Diseases/immunology , Horse Diseases/prevention & control , Horse Diseases/virology , Horses , Humans , Mice , Rats , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Spodoptera/virology , Viral Envelope Proteins/genetics , Viral Envelope Proteins/immunology , West Nile Fever/immunology , West Nile Virus Vaccines/administration & dosage , West Nile Virus Vaccines/immunology , West Nile virus/genetics
5.
Vaccine ; 24(12): 2176-85, 2006 Mar 15.
Article in English | MEDLINE | ID: mdl-16310896

ABSTRACT

Numerous human infections with avian influenza viruses in Asia in recent years have raised the concern that the next influenza pandemic is imminent. The most effective way to combat influenza is through the vaccination of the public. However, a minimum of 3-6 months is needed to develop an influenza vaccine using the traditional egg-based vaccine approach. The influenza hemagglutinin protein (HA), the active ingredient in the current vaccine, can be expressed in insect cells using the baculovirus expression vector system and purified rapidly. An influenza vaccine based on such a recombinant antigen allows a more timely response to a potential influenza pandemic. Here, we report an innovative monitoring assay for recombinant HA (rHA) expression and a rapid purification process. Various biochemical analyses indicate that the purified rHA is properly folded and biologically active.


Subject(s)
Hemagglutinin Glycoproteins, Influenza Virus/isolation & purification , Influenza A virus/chemistry , Vaccines, Synthetic/isolation & purification , Animals , Baculoviridae/genetics , Baculoviridae/metabolism , Gene Expression , Genetic Vectors , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Influenza Vaccines/immunology , Influenza Vaccines/therapeutic use , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
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