New Molecular Approaches for Advanced Safety and Purity Testing of Vaccines

Principal Investigator: Vladimir Chizhikov, PhD
Office / Division / Lab: OVRR / DVP / LMD

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Overview

Public Health Issue: A large class of biological products including vaccines, gene, and cell therapeutics are manufactured using living cells. The cell substrates can potentially be contaminated with a number of adventitious agents of viral and bacterial nature. Purity and safety testing by using conventional methods usually requires a long time, in some cases exceeding the shelf life of the products. This limitation of the currently used methods raises the need to continuously work on application of new cutting-edge technologies to improve product safety, particularly with respect to detection of potential contamination with agents that could cause adverse events. Therefore assessment of utility of new cutting edge methods for evaluation of safety of biological products is urgently needed.

Regulatory Contribution: Alternative approaches for assessment of purity and safety of cell substrates used in manufacture of vaccines as well as cell-derived biological products, should be extensively evaluated to minimize the risk of transmitting infectious agents through accidental or intentional contamination of the products during manufacture.

Research Approach: The goal of this program is to substantially increase the sensitivity, specificity, and throughput of safety analyses of biological samples by using novel molecular technologies. In recent years, there has been remarkable progress in the development of new techniques for isolation and amplification of nucleic acids, total-genome sequencing, microarray-based gene expression profiling, global SNP’s analysis, etc. The current program is focused on evaluation of the feasibility of emerging molecular techniques for development of advanced methods for elevation of safety and purity of biological products including live and inactivated vaccines. Nucleic acid amplification methods combined with oligonucleotide microarrays and other sequence-based identification assays represent a powerful tool for detection and genetic analysis of small amounts of contaminating materials in biological products. Use of improved methods for sample preparation based on biological enrichment or a sequence- or immuno-specific concentration and magnetic particle-based extraction of target material from complex biological mixtures ensures the high sensitivity, reliability, and reproducibility of detection of extraneous agents.

Mission Relevance & Outcomes: Our work will identify new approaches tosignificantly simplify and expedite analysis of biological products, while maintaining equivalent or increased sensitivity compared to conventional test methods. Implementation of the novel methods developed by our laboratory will provide improved measures of safety and purity.

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Publications

J Virol Methods 2008 Dec;154(1-2):27-40
Microarray-based assay for the detection of genetic variations of structural genes of West Nile virus.
Grinev A, Daniel S, Laassri M, Chumakov K, Chizhikov V, Rios M

Appl Environ Microbiol 2008 Sep;74(17):5383-91
Biological Enrichment of Mycoplasma Agents using Co-Cultivation with Permissive Cell Cultures.
Volokhov DV, Kong H, George J, Anderson C, Chizhikov VE

Mol Phylogenet Evol 2007 Aug;44(2):699-710
Genetic analysis of housekeeping genes of members of the genus Acholeplasma: Phylogeny and complementary molecular markers to the 16S rRNA gene.
Volokhov DV, Neverov AA, George J, Kong H, Liu SX, Anderson C, Davidson MK, Chizhikov V

J Med Virol 2006 Oct;78(10):1325-40
Microarray assay for detection and discrimination of Orthopoxvirus species.
Ryabinin VA, Shundrin LA, Kostina EB, Laassri M, Chizhikov V, Shchelkunov SN, Chumakov K, Sinyakov AN

J Clin Microbiol 2006 Oct;44(10):3752-9
Genotyping of measles virus in clinical specimens on the basis of oligonucleotide microarray hybridization patterns.
Neverov AA, Riddell MA, Moss WJ, Volokhov DV, Rota PA, Lowe LE, Chibo D, Smit SB, Griffin DE, Chumakov KM, Chizhikov VE

J Virol Methods 2006 Sep;136(1-2):8-16
New mosaic subgenotype of varicella-zoster virus in the USA: VZV detection and genotyping by oligonucleotide-microarray.
Sergeev N, Rubtcova E, Chizikov V, Schmid DS, Loparev VN

Appl Microbiol Biotechnol 2006 Aug;71(5):680-98
Sequencing of the intergenic 16S-23S rRNA spacer (ITS) region of Mollicutes species and their identification using microarray-based assay and DNA sequencing.
Volokhov DV, George J, Liu SX, Ikonomi P, Anderson C, Chizhikov V

J Microbiol Methods 2006 Jun;65(3):488-502
Microarray analysis of Bacillus cereus group virulence factors.
Sergeev N, Distler M, Vargas M, Chizhikov V, Herold KE, Rasooly A

Appl Environ Microbiol 2006 Apr;72(4):2439-48
Discovery of Natural Atypical Nonhemolytic Listeria seeligeri Isolates.
Volokhov D, George J, Anderson C, Duvall RE, Hitchins AD

J Med Microbiol 2005 Dec;54(Pt 12):1127-31
Microarray-based pncA genotyping of pyrazinamide-resistant strains of Mycobacterium tuberculosis.
Denkin S, Volokhov D, Chizhikov V, Zhang Y

J Mol Diagn 2005 Oct;7(4):486-94
A multiplex polymerase chain reaction microarray assay to detect bioterror pathogens in blood.
Tomioka K, Peredelchuk M, Zhu X, Arena R, Volokhov D, Selvapandiyan A, Stabler K, Mellquist-Riemenschneider J, Chizhikov V, Kaplan G, Nakhasi H, Duncan R

J Microbiol Methods 2005 Oct 19; [Epub ahead of print]
Microarray analysis of Bacillus cereus group virulence factors.
Sergeev N, Distler M, Vargas M, Chizhikov V, Herold KE, Rasooly A

Mol Cell Probes 2004 Dec;18(6):359-67
Genotyping of Clostridium perfringens toxins using multiple oligonucleotide microarray hybridization.
Al-Khaldi SF, Myers KM, Rasooly A, Chizhikov V

J Clin Microbiol 2004 Dec;42(12):5793-801
Mapping of genomic segments of influenza B virus strains by an oligonucleotide microarray method.
Ivshina AV, Vodeiko GM, Kuznetsov VA, Volokhov D, Taffs R, Chizhikov VI, Levandowski RA, Chumakov KM

Biosens Bioelectron 2004 Nov 15;20(4):684-98
Multipathogen oligonucleotide microarray for environmental and biodefense applications.
Sergeev N, Distler M, Courtney S, Al-Khaldi SF, Volokhov D, Chizhikov V, Rasooly A

Diagn Microbiol Infect Dis 2004 Jul;49(3):163-71
Identification of Bacillus anthracis by multiprobe microarray hybridization.
Volokhov D, Pomerantsev A, Kivovich V, Rasooly A, Chizhikov V

Appl Environ Microbiol 2004 Jul;70(7):4256-66
Natural Atypical Listeria innocua Strains with Listeria monocytogenes Pathogenicity Island 1 Genes.
Johnson J, Jinneman K, Stelma G, Smith BG, Lye D, Messer J, Ulaszek J, Evsen L, Gendel S, Bennett RW, Swaminathan B, Pruckler J, Steigerwalt A, Kathariou S, Yildirim S, Volokhov D, Rasooly A, Chizhikov V, Wiedmann M, Fortes E, Duvall RE, Hitchins AD

Diagn Microbiol Infect Dis 2004 Jun;49(2):89-97
Accurate mapping of mutations of pyrazinamide-resistant Mycobacterium tuberculosis strains with a scanning-frame oligonucleotide microarray.
Wade MM, Volokhov D, Peredelchuk M, Chizhikov V, Zhang Y

J Clin Microbiol 2004 May;42(5):2134-43
Simultaneous analysis of multiple staphylococcal enterotoxin genes by an oligonucleotide microarray assay.
Sergeev N, Volokhov D, Chizhikov V, Rasooly A

J Clin Microbiol 2004 Apr;42(4):1723-1726
Genotyping of Enteric Adenoviruses by Using Single-Stranded Conformation Polymorphism Analysis and Heteroduplex Mobility Assay.
Soares CC, Volotao EM, Albuquerque MC, Nozawa CM, Linhares RE, Volokhov D, Chizhikov V, Lu X, Erdman D, Santos N