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  1. Biologics Research Projects

New Ways to Improve Testing of Vaccines for Purity and Safety

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

General Overview

The major focus of our research program is to develop and evaluate novel tools to aid in the development of safe and effective vaccines against existing and emerging viral diseases.

The vaccine manufacturing process includes a vast array of in-process and release tests to ensure consistency in product quality and performance. Some of the most crucial of these tests are those that measure vaccine potency. Many such tests requiring use of animals are highly variable and cause significant pain and distress. One of the aims of our research program is therefore to reduce or eliminate the use of animals in potency testing.

Other tests performed during vaccine manufacture are designed to detect possible contaminants, including viruses and bacteria that can be inadvertently introduced into the process stream. These unwanted pathogens are referred to as adventitious agents. A major potential source of adventitious agents is the cell substrates used to produce the vaccines. Cell substrates are the living cells of mammals, birds, or insects that serve as tiny biological factories used to make viral products. There are already many types of tests developed that can detect adventitious agents; however, some agents may escape detection by these tests due to limitations in test sensitivity and specificity. To address this issue we are also engaged in efforts to develop new, more sensitive and specific detection methods involving use of novel concentration and amplification techniques and high throughput genetic sequencing.

Scientific Overview

The current research program is focused on the development and evaluation of novel technologies to expedite, simplify and improve vaccine quality control testing. Our specific focus is on minimizing the use of animals in potency testing and on developing more sensitive and specific tests for detection of adventitious agents that can potentially be introduced during vaccine manufacture.

Our current effort on minimizing the use of animals in product potency itesting is focused on rabies vaccines. Currently, rabies vaccine potency is assessed by evaluating the ability of the vaccine to protect mice against a lethal dose of live, virulent rabies virus. We are working on development and evaluation of an alternative potency method that is based on assessment of antibody levels generated in mice following immunization with the non-virulent product, combined with a non-animal based method to quantify the amount of rabies virus antigen in the vaccine. This alternative potency assessment approach is more humane (as it does not result in causing pain or distress in animals), uses far fewer animals than current testing and is able to yield less variable and more consistent results.

The evaluation and potential implementation of this approach will require developing and qualifying different reference materials that are necessary to demonstrate the validity of each assay and reliability and accuracy of the rabies vaccine potency quantification. The preparation of some reference materials for in vitro antigen quantification and in vivo potency assay will require using novel techniques such as DNA and RNA immunization, which also will allow us to develop expertise in assessing the safety and potency of DNA and RNA vaccines.

In contrast to conventional viral vaccines, which contain a complex array of viral and host-cell proteins and process-derived impurities, DNA and RNA vaccines are highly purified and contain only the genetic material encoding the proteins that will elicit the desired immune response.

With regard to adventitious agent testing, we are developing and validating methods that filter concentrates of these agents from in-process test samples, followed by application of deep sequencing techniques to detect and identify contaminants. In order to assess the detection limits and applicability of these new technologies we will use biological samples spiked with different amounts of various adventitious agents. We will compare the results of these tests with results from parallel testing of these samples using techniques currently available in our laboratory.


Appl Environ Microbiol 2017 Apr 17;83(9):e00211-17
Prevalence, genotype richness, and coinfection patterns of hemotropic mycoplasmas in raccoons (Procyon lotor) in environmentally protected and urbanized barrier islands.
Volokhov DV, Hwang J, Chizhikov VE, Danaceau H, Gottdenker NL

Vaccine 2017 Feb 7;35(6):966-71
Replacement of in vivo human rabies vaccine potency testing by in vitro glycoprotein quantification using ELISA--results of an international collaborative study.
Morgeaux S, Poirier B, Ragan CI, Wilkinson D, Arabin U, Guinet-Morlot F, Levis R, Meyer H, Riou P, Shaid S, Volokhov D, Tordo N, Chapsal JM

J Virol Methods 2017 Jan;239:17-25
Development of a microarray-based assay for rapid detection and monitoring of genetic variants of West Nile virus circulating in the United States.
Grinev A, Chancey C, Volkova E, Chizhikov v, Rios M

Euro Surveill 2016 Apr 14;21(15):30193
Environmental surveillance of viruses by tangential flow filtration and metagenomic reconstruction.
Furtak V, Roivainen M, Mirochnichenko O, Zagorodnyaya T, Laassri M, Zaidic SZ, Rehman L, Alam MM, Chizhikov V, Chumakov K

Biologicals 2015 Jan;43(1):23-30
Use of tangential flow filtration for improving detection of viral adventitious agents in cell substrates.
Furtak VA, Dabrazhynetskaya A, Volokhov DV, Chizhikov V

Genome Announc 2014 Mar 6;2(2):e00127-14
Genome Sequence of Mycoplasma hyorhinis Strain DBS 1050.
Dabrazhynetskaya A, Soika V, Volokhov D, Simonyan V, Chizhikov V

J Appl Microbiol 2014 Jan;116(1):100-8
Preparation of reference stocks suitable for evaluation of alternative NAT-based mycoplasma detection methods.
Dabrazhynetskaya A, Furtak V, Volokhov D, Beck B, Chizhikov V

Biologicals 2013 Nov;41(6):377-83
Collaborative study report: evaluation of the ATCC experimental mycoplasma reference strains panel prepared for comparison of NAT-based and conventional mycoplasma detection methods.
Dabrazhynetskaya A, Volokhov DV, Lin TL, Beck B, Gupta RK, Chizhikov V

Vet Microbiol 2013 Apr 12;163(1-2):149-56
Characterization of Pasteurella multocida strains isolated from geese.
Varga Z, Volokhov DV, Stipkovits L, Thuma A, Sellyei B, Magyar T

Appl Spectrosc 2012 Dec;66(12):1480-6
Enhanced mid-infrared chemical imaging (IRCI) detection of DNA microarrays.
Schoen B, Mossoba MM, Chizhikov V, Rashid A, Martinez-Diaz K, Al-Khaldi SF

Int J Syst Evol Microbiol 2012 Sep;62(Pt 9):2068-76
Lactobacillus brantae sp. nov., isolated from feces of Canada geese (Branta canadensis).
Volokhov DV, Amselle M, Beck BJ, Popham DL, Whittaker P, Wang H, Kerrigan E, Chizhikov VE

J Virol Methods 2012 Aug;183(2):219-23
Application of a full-genome microarray-based assay for the study of genetic variability of West Nile virus.
Grinev A, Lu Z, Chizhikov V, Rios M

J Clin Microbiol 2012 Mar;50(3):1113-7
Eosinophilic Fasciitis Associated with Mycoplasma arginini Infection.
Silló P, Pintér D, Ostorházi E, Mazán M, Wikonkál N, Pónyai K, Volokhov DV, Chizhikov VE, Szathmary S, Stipkovits L, Kárpáti S

Mol Phylogenet Evol 2012 Jan;62(1):515-28
RNA polymerase beta subunit (rpoB) gene and the 16S-23S rRNA intergenic transcribed spacer region (ITS) as complementary molecular markers in addition to the 16S rRNA gene for phylogenetic analysis and identification of the species of the family Mycoplasmataceae.
Volokhov DV, Simonyan V, Davidson MK, Chizhikov VE

J Appl Microbiol 2011 Oct;111(4):904-14
Preparation of reference strains for validation and comparison of mycoplasma testing methods.
Dabrazhynetskaya A, Volokhov DV, Ikonomi P, Brewer A, Chang A, Chizhikov V

J Microbiol Methods 2011 Sep;86(3):383-6
Identification of Mycoplasmas Using a Fluorophore-Free Microarray and Infrared Chemical Imaging (IRCI).
Mossoba MM, Chizhikov V, Volokhov DV, Martinez-Diaz Milians K, Schoen B, Al-Khaldi SF

Am Pharm Rev 2011 May-Jun;14(4):37370
Historical Overview of Mycoplasma Testing for Production of Biologics
Chandler DKF, Volokhov DV, Chizhikov VE

Vet Microbiol 2011 Apr 21;149(1-2):262-8
Novel hemotrophic mycoplasma identified in naturally infected California sea lions (Zalophus californianus).
Volokhov DV, Norris T, Rios C, Davidson MK, Messick JB, Gulland FM, Chizhikov VE

Mol Cell Probes 2011 Apr-Jun;25(2-3):69-77
Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques.
Volokhov DV, Graham LJ, Brorson KA, Chizhikov VE

J Appl Microbiol 2011 Jan;110(1):54-60
Detection of mycoplasma contamination in cell substrates using reverse transcription-PCR assays.
Peredeltchouk M, Wilson David SA, Bhattacharya B, Volokhov DV, Chizhikov V

Methods Mol Biol 2011;671:55-94
Oligonucleotide microarrays for identification of microbial pathogens and detection of their virulence-associated or drug-resistance determinants.
Volokhov DV, Kong H, Herold K, Chizhikov VE, Rasooly A

Appl Environ Microbiol 2010 May;76(9):2718-28
Evaluation of Mycoplasma inactivation during production of biologics: egg-based viral vaccines as a model.
David SA, Volokhov DV, Zhiping Y, Chizhikov V

Methods Mol Biol 2009;465:395-417
Molecular detection of drug-resistant Mycobacterium tuberculosis with a scanning-frame oligonucleotide microarray.
Volokhov DV, Chizhikov VE, Denkin S, Zhang Y

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

Appl Microbiol Biotechnol 2007 Nov;77(1):223-32
Application of cell culture enrichment for improving the sensitivity of mycoplasma detection methods based on nucleic acid amplification technology (NAT).
Kong H, Volokhov DV, George J, Ikonomi P, Chandler D, Anderson C, Chizhikov V

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 Clin Microbiol 2007 Aug;45(8):2641-8
Development and Validation of DNA Microarray for Genotyping Group A Rotavirus VP4 (P[4], P[6], P[8], P[9] and P[14]) and VP7 (G1-G6, G8-G10 and G12) Genes.
Honma S, Chizhikov V, Santos N, Tatsumi M, Timenetsky MD, Linhares AC, Mascarenhas JD, Ushijima H, Armah GE, Gentsch JR, Hoshino Y

Biochem Biophys Res Commun 2007 May 18;356(4):1017-23
Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples.
Hsia CC, Chizhikov VE, Yang AX, Selvapandiyan A, Hewlett I, Duncan R, Puri RK, Nakhasi HL, Kaplan GG

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, Chizhikov 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