660 Production of Therapeutic Proteins in Transgenic Plants and Viral Vectors

Transgenic plants and plant viruses have potential advantages over other production systems for therapeutic proteins. 1) Plants are not susceptible to human and animal pathogens, such as viruses that can contaminate mammalian and avian cell lines used for production of many vaccines. Recent experiences of “mad cow” disease and theories of the possible origin of HIV from monkey cell lines have highlighted the need for increased product safety. 2) There are established protocols for preparing naturally occurring pharmaceuticals from plants. 3) Unlike bacteria, plants recognize the same glycosylation signals as other eukaryotic expression systems such as mammalian, insect, or yeast cell cultures and can thus produce glycosylated proteins. Although there are differences between plants and other eukaryotes in the types of sugar residues added to glycosylated proteins, it has been demonstrated several times that plant-produced proteins have similar stability and bioequivalence of function and that antigenicity is similar. 4) Plants can produce high yields; a single transgenic plant could yield as much human glucocerebrosidase as 500 placentae. We expressed an epitope from HIV-1 on the surface of bean yellow mosaic potyvirus (BYMV) coat protein (CP); protein produced in transgenic plants is recognized by a human monoclonal antibody that neutralizes most HIV-1 isolates. Epitope-modified BYMV-CP can be recovered from transgenic plants by incorporation into BYMV virions following infection of the transgenic plants. Modified virions display the HIV-1 epitope in a semi-regular array that should stimulate the immune system to a greater degree than free subunits. HIV epitope-bearing BYMV has been used to immunize mice to assess the immune response.

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EXPRESSION OF THE SURFACE ANTIGEN BRUCELLA ABORTUS OMR16 IN NICOTIANA BENTHAMIANA PLANT

REPORTS ◽

10.32014/2020.2518-1483.122 ◽

2020 ◽

Vol 5 (333) ◽

pp. 81-85

Author(s):

G.D. Ilgekbaeva, ◽

◽

E.Sh. Makhashov ◽

G. Tulepova ◽

D. Yessimkhankyzy ◽

...

Keyword(s):

Membrane Proteins ◽

Transgenic Plants ◽

Brucella Abortus ◽

Nicotiana Benthamiana ◽

Viral Vectors ◽

Viral Vector ◽

Molecular Genetic ◽

Genetic Research ◽

Plant Viruses ◽

Incidence Rates

Brucellosis is one of the most contagious and infectious diseases with high incidence rates of cattle and humans in Kazakhstan. Using modern biotechnology techniques to develop vaccines that are reliable and affordable for farmers is an alternative solution to the problem. Plant viruses are often used as a vector for obtaining the expression of antigens of the pathogen. The grape virus A (BAB) is widely used among plant viruses. Brucella membrane proteins are the main objects of this research for futher development of vaccines or diagnostic texts against brucellosis, Membrane proteins (OMPs) are cell specific surface antigens that are immunogenic. OMPs are ideal candidates for the production of recombinant brucellosis vaccines. The object of the study was the outer membrane protein (Omp16), which plays an important role in the suppression of TNF-α production in macrophages. In this study, molecular cloning and analysis of the expression of the Omp16 gene, which was used to express the recombinant protein in plants, was carried out. We selected brucella from the vaccine strain of Brucella abortus 19, and the plant Nicotiana benthamiana, as the subjects for our research, since they widely used for the production of recombinant proteins, and they both appropriate for molecular genetic research. A viral vector was constructed to express the brucellosis antigen Omp16 in Nicotiana benthamiana plants. Nineteen explants were used for the regeneration of transgenic plants. As a result of this studies, the introduced gene of Omp16 was under the subgenomic promoter control of the ORF4 and was successfully expressed while maintaining the efficiency of expression in transgenic plants. The efficiency of viral vectors was evaluated at the level of transcription during expression of the protein Omp16 with viral proteins. The entire leaf blade was infiltrated; the density of Agrobacteria was 0.7. We were able to obtained transgenic plants Nicotiana benthamiana carrying the gene of capsid protein BAB, and the expression of the membrane antigen Omp16 in the viral vector was achieved by replacing the ORF4 with the Omp16 gene. The development of transgenic plants was carried out using agrobacterial transformation.

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Molecular and genetic characterization of natural variants of HIV-1 Nef gene from North India and its functional implication in down-regulation of MHC-I and CD-4

Current HIV Research ◽

10.2174/1570162x18666200925160755 ◽

2020 ◽

Vol 18 ◽

Author(s):

J. Singh ◽

L. Ronsard ◽

M. Pandey ◽

R. Kapoor ◽

V.G. Ramachandran ◽

...

Keyword(s):

Biological Activities ◽

Sequence Similarity ◽

Eukaryotic Expression ◽

North India ◽

Cell Surface Expression ◽

Functional Domains ◽

Wild Type ◽

Down Regulation ◽

Subtype B ◽

Hiv 1

Background: HIV-1 Nef is an important accessory protein with multiple effector functions. Genetic studies of HIV-1 Nef gene shows extensive genetic diversity and the functional studies have been carried out mostly with Nef derived from regions dominated by subtype B (North America & Europe). Objective: This study was carried out to characterize genetic variations of the Nef gene from HIV-1 infected individuals from North-India and to find out their functional implications. Methods: The unique representative variants were sub-cloned in eukaryotic expression vector and further characterized with respect to their ability to down regulate cell surface expression of CD4 and MHC-1molecules. Results: The phylogenetic analysis of Nef variants revealed sequence similarity with either consensus subtype B or B/C recombinants. Boot scan analysis of some of our variants showed homology to B/C recombinant and some to wild type Nef B. Extensive variations were observed in most of the variants. The dN/dS ratio revealed 80% purifying selection and 20% diversifying selection implying the importance of mutations in Nef variants. Intracellular stability of Nef variants differed greatly when compared with wild type Nef B and C. There were some variants that possessed mutations in the functional domains of Nef and responsible for its differential CD4 and MHC-1 down regulation activity. Conclusion: We observed enhanced biological activities in some of the variants, perhaps arising out of amino acid substitutions in their functional domains. The CD4 and MHC-1 down-regulation activity of Nef is likely to confer immense survival advantage allowing the most rare genotype in a population to become the most abundant after a single selection event.

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HIV-1 Vaccine Strategies Utilizing Viral Vectors Including Antigen- Displayed Inoviral Vectors

Current HIV Research ◽

10.2174/1570162x12666140209135651 ◽

2014 ◽

Vol 11 (8) ◽

pp. 610-622 ◽

Cited By ~ 2

Author(s):

Kyriakos Hassapis ◽

Leondios Kostrikis

Keyword(s):

Viral Vectors ◽

Hiv 1

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Generation and characterization of a human monoclonal antibody that neutralizes diverse HIV-1 isolates in vitro

AIDS ◽

10.1097/00002030-199201000-00002 ◽

1992 ◽

Vol 6 (1) ◽

pp. 17-24 ◽

Cited By ~ 9

Author(s):

Douglas F. Lake ◽

Takashi Kawamura ◽

Takami Tomiyama ◽

W. Edward Robinson ◽

Yoh-ichi Matsumoto ◽

...

Keyword(s):

Monoclonal Antibody ◽

Human Monoclonal Antibody ◽

Hiv 1

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Human Immunodeficiency Virus Type 1 Replication in the Absence of Integrase-Mediated DNA Recombination: Definition of Permissive and Nonpermissive T-Cell Lines

Journal of Virology ◽

10.1128/jvi.75.17.7944-7955.2001 ◽

2001 ◽

Vol 75 (17) ◽

pp. 7944-7955 ◽

Cited By ~ 78

Author(s):

Noriko Nakajima ◽

Richard Lu ◽

Alan Engelman

Keyword(s):

Human Immunodeficiency Virus ◽

Cell Lines ◽

Dna Recombination ◽

Cell Types ◽

Class Ii ◽

Class I ◽

Immunodeficiency Virus ◽

T Cell Lines ◽

Hiv 1

ABSTRACT Functional retroviral integrase protein is thought to be essential for productive viral replication. Yet, previous studies differed on the extent to which integrase mutant viruses expressed human immunodeficiency virus type 1 (HIV-1) genes from unintegrated DNA. Although one reason for this difference was that class II integrase mutations pleiotropically affected the viral life cycle, another reason apparently depended on the identity of the infected cell. Here, we analyzed integrase mutant viral infectivities in a variety of cell types. Single-round infectivity of class I integration-specific mutant HIV-1 ranged from <0.03 to 0.3% of that of the wild type (WT) across four different T-cell lines. Based on this approximately 10-fold influence of cell type on mutant gene expression, we examined class I and class II mutant replication kinetics in seven different cell lines and two primary cell types. Unexpectedly, some cell lines supported productive class I mutant viral replication under conditions that restricted class II mutant growth. Cells were defined as permissive, semipermissive, or nonpermissive based on their ability to support the continual passage of class I integration-defective HIV-1. Mutant infectivity in semipermissive and permissive cells as quantified by 50% tissue culture infectious doses, however, was only 0.0006 to 0.005% of that of WT. Since the frequencies of mutant DNA recombination in these lines ranged from 0.023 to <0.093% of the WT, we conclude that productive replication in the absence of integrase function most likely required the illegitimate integration of HIV-1 into host chromosomes by cellular DNA recombination enzymes.

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