Vaccines, Blood & Biologics

Vaccine Safety: Virus Detection and Latency

Principal Investigator: Philip R. Krause, MD
Office / Division / Lab: OVRR / DVP / LDV

General Overview

Our laboratory addresses two questions directly related to the safety of vaccines that are grown in cultured cells: 1) How can we prove that vaccines are free of infectious contaminants? and 2) How does viral latency affect vaccine safety? (Latency describes the phase when viruses remain within cells in an inactive state and there are no clinical symptoms in the individual harboring the viruses.)

Many vaccines are manufactured using human or animal cells that act as tiny factories to produce viruses or proteins that are components of the vaccine. In some cases, such cells may harbor or grow viruses other than the one that will be used to make a vaccine, and these unwanted viruses might be very difficult to detect. Our laboratory is developing and improving methods to detect potential contaminating viruses, While most tests for potential contaminating viruses are "specific" and will only detect certain contaminants, we are taking a general approach that aims to identify and detect even viruses that are not normally detected by more traditional tests. We are studying these powerful new techniques to determine how they may best be used in vaccine testing, and whether they might be able to complement or replace some of the currently used assays.

It is important to be able to evaluate the effect of vaccines on both the latent viruses contaminating cultured cells as well as on the latent viruses in humans that are the target of vaccines. Such evaluations help us to predict the effectiveness of these vaccines in preventing viral disease. Therefore, we are developing techniques to detect latent viruses and are studying the process of viral latency.

This work includes developing more effective ways to evaluate the risk posed by latent viruses in cell substrates. In addition to helping us understand how to detect latent viruses, our studies of viral latency also help to inform our regulatory decision-making about vaccines against herpesviruses, a class of viruses that can cause latent infections. The herpesviruses include HSV-1 (which causes cold sores and nervous system infections), HSV-2 (which causes genital herpes and nervous system infections), varicella-zoster virus (causes chickenpox and shingles), cytomegalovirus (major cause of congenital disease and severe infections in people with weakened immune systems), Epstein-Barr Virus (which causes infectious mononucleosis and some cancers), human herpesvirus 6 and 7 (which cause roseola, a childhood disease, and infections in individuals with weak immune systems), and human herpesvirus 8 (which causes some cancers).

In sum, our work directly supports the work of FDA regulators who make decisions about how manufacturers should make viral vaccines safe by preventing and detecting contamination and by supporting decision-making related to herpesvirus vaccines.

Scientific Overview

Our approach to developing new molecular techniques to ensure the absence of contaminants in vaccines and reagents used to produce them is to investigate schemes that are theoretically capable of identifying any viral sequence in a sample, whether from a known or an unknown virus. The goal is to use a system that can detect and identify any DNA or RNA by first enriching the samples for DNA or RNA that is likely from contaminating viruses. This will improve the chance of detecting any contaminating virus and reduce the chance of losing the virus signature in the normal cell DNA or RNA that is expected to be present. To that end, we have developed schemes that 1) distinguish viral from cellular sequences and 2) amplify and identify any sequences present.

After enriching samples for contaminating virus particles that contain DNA or RNA we use a generic PCR that amplifies all nucleic acid sequences that are of virus-genome size. The resulting amplicons are sequenced using high-throughput techniques and subjected to analysis using sophisticated computer programs that permit comparison with known viral sequences. This technique enables us to identify new viral sequences, including those that are only remotely related to previously described viruses. We are developing approaches to support the use of this type of powerful new assay in routine product testing.

In case viral sequences (other than vaccine virus) are identified using these techniques or otherwise suspected to be present in a vaccine or cell substrate, we develop highly specific and sensitive assays to determine 1) whether the finding can be confirmed and 2) whether the sequences are from free (non-infectious) nucleic acid, from encapsidated (potentially infectious) particles, or from particles that can be demonstrated to be infectious. For example, we recently studied the significance of the presence of porcine circovirus (PCV) DNA and virus in human rotavirus vaccines.

Our laboratory was the first to describe latently produced viral microRNAs (miRNAs, very small RNA pieces that play a role in regulation of gene expression) encoded by both HSV-1 and HSV-2. These miRNAs appear to play an important role in helping the virus establish latency and maintain the ability to reactivate at later times. We also have identified important viral sequences that allow HSV to establish its latent state in certain types of cells and to preferentially reactivate in certain body sites. Our laboratory uses molecular and biochemical techniques (including mutant and chimeric viruses) to further study these latently expressed viral miRNAs and other factors that interact with the viral sequences that control viral latency establishment and viral reactivation. The insights gained from these experiments are helping us to identify additional viral mechanisms that are important for evaluation of cell substrates and of herpesvirus vaccines.


Sci Transl Med 2014 Dec 3;6(265):265ra169
Inhibition of LSD1 reduces herpesvirus infection, shedding, and recurrence by promoting epigenetic suppression of viral genomes.
Hill JM, Quenelle DC, Cardin RD, Vogel JL, Clement C, Bravo FJ, Foster TP, Bosch-Marce M, Raja P, Lee JS, Bernstein DI, Krause PR, Knipe DM, Kristie TM

PDA J Pharm Sci Technol 2014 Nov-Dec;68(6):552-5
The potential role of advanced technologies for virus detection in development and regulation of vaccines.
McClenahan SD, Krause PR

Biologicals 2014 Jan;42(1):34-41
Optimization of virus detection in cells using massively parallel sequencing.
McClenahan SD, Uhlenhaut C, Krause PR

Vaccine 2013 Dec 17;32(1):4-10
Priorities for CMV vaccine development.
Krause PR, Bialek SR, Boppana SB, Griffiths PD, Laughlin CA, Ljungman P, Mocarski ES, Pass RF, Read JS, Schleiss MR, Plotkin SA

PLoS One 2013;8(8):e68777
Discovery of a bovine enterovirus in alpaca.
McClenahan SD, Scherba G, Borst L, Fredrickson RL, Krause PR, Uhlenhaut C

J Virol 2013 May;87(10):5820-30
Herpes Simplex Virus 2 expresses a novel form of ICP34.5, a major viral neurovirulence factor, through regulated alternative splicing.
Tang S, Guo N, Patel A, Krause PR

Vaccine 2013 Apr 18;31 Suppl 2:B197-203
Desirability and feasibility of a vaccine against cytomegalovirus.
Griffiths P, Plotkin S, Mocarski E, Pass R, Schleiss M, Krause P, Bialek S

Transpl Infect Dis 2012 Feb;14(1):79-85
Use of a novel virus detection assay to identify coronavirus HKU1 in the lungs of a hematopoietic stem cell transplant recipient with fatal pneumonia.
Uhlenhaut C, Cohen JI, Pavletic S, Illei G, Gea-Banacloche JC, Abu-Asab M, Krogmann T, Gubareva L, McClenahan S, Krause PR

PDA J Pharm Sci Technol 2011 Nov 1;65(6):681-4
Use of DOP-PCR in Non-Specific Virus Detection.
Uhlenhaut C, McClenahan S, Krause PR

PDA J Pharm Sci Technol 2011 Nov 1;65(6):557-62
Regulatory approaches for control of viral contamination of vaccines.
McClenahan S, Uhlenhaut C, Krause PR

Vaccine 2011 Jun 24;29(29-30):4745-53
Molecular and infectivity studies of porcine circovirus in vaccines.
McClenahan SD, Krause PR, Uhlenhaut C

J Virol 2011 May;85(9):4501-9
Herpes Simplex Virus-2 miR-H6 is a Novel LAT-associated MicroRNA, but Reduction of its Expression Does not Influence Viral Latency Establishment or Recurrence Phenotype.
Tang S, Bertke AS, Patel A, Margolis TP, Krause PR

Pediatrics 2011 May;127 Suppl 1:S78-86
Immunization-safety monitoring systems for the 2009 H1N1 monovalent influenza vaccination program.
Salmon DA, Akhtar A, Mergler MJ, Vannice KS, Izurieta H, Ball R, Lee GM, Vellozzi C, Garman P, Cunningham F, Gellin B, Koh H, Lurie N

J Virol 2011 Mar;85(6):3030-2
Spread of herpes simplex virus to the spinal cord is independent of spread to dorsal root ganglia.
Ohashi M, Bertke AS, Patel A, Krause PR

J Virol 2010 Jan;84(2):1189-92
Identification of viral microRNAs expressed in human sacral ganglia latently infected with herpes simplex virus 2.
Umbach JL, Wang K, Tang S, Krause PR, Mont EK, Cohen JI, Cullen BR

Biologicals 2009 Nov;37(6 Sp. Iss.):421-3
Panel discussion.
Smith Moderator D, Duchene M, Egan W, Jivapaisarnpong T, Knezevic I, Pierard I, Schofield T, Shin J, Southern J, Krause PR, Rapporteur

Biologicals 2009 Nov;37(6):355
Stability evaluation of vaccines.
Schofield T, Krause PR

Biologicals 2009 Nov;37(6):369-78
Goals of stability evaluation throughout the vaccine life cycle.
Krause PR

Biologicals 2009 Nov;37(6):412-5
A vaccine measured with a highly variable assay: rabies.
Jivapaisarnpong T, Schofield T, Krause PR

Biologicals 2009 Nov;37(6):410-1
An annual vaccine: seasonal influenza.
Jivapaisarnpong T, Krause PR

J Virol 2009 Oct;83(19):10007-15
Latency-associated transcript (LAT) exon 1 controls herpes simplex virus species-specific phenotypes: reactivation in the guinea pig genital model and neuron subtype-specific latent expression of LAT.
Bertke AS, Patel A, Imai Y, Apakupakul K, Margolis TP, Krause PR

J Virol 2009 Aug;83(16):7873-82
Investigation of the Mechanism by Which Herpes Simplex Virus Type 1 LAT Sequences Modulate Preferential Establishment of Latent Infection in Mouse Trigeminal Ganglia.
Imai Y, Apakupakul K, Krause PR, Halford WP, Margolis TP

J Clin Virol 2009 Apr;44(4):337-9
Use of a universal virus detection assay to identify human metapneumovirus in a hematopoietic stem cell transplant recipient with pneumonia of unknown origin.
Uhlenhaut C, Cohen JI, Fedorko D, Nanda S, Krause PR

J Virol 2009 Feb;83(3):1433-42
Novel less-abundant viral microRNAs encoded by herpes simplex virus 2 latency-associated transcript and their roles in regulating ICP34.5 and ICP0 mRNAs.
Tang S, Patel A, Krause PR

J Virol Methods 2008 Sep;152(1-2):18-24
Universal virus detection by degenerate-oligonucleotide primed polymerase chain reaction of purified viral nucleic acids.
Nanda S, Jayan G, Voulgaropoulou F, Sierra-Honigmann AM, Uhlenhaut C, McWatters BJ, Patel A, Krause PR

Proc Natl Acad Sci U S A 2008 Aug 5;105(31):10931-6
An acutely and latently expressed herpes simplex virus 2 viral microRNA inhibits expression of ICP34.5, a viral neurovirulence factor.
Tang S, Bertke AS, Patel A, Wang K, Cohen JI, Krause PR

MMWR Recomm Rep 2008 Jun 6;57(RR-5):1-30
Prevention of herpes zoster: recommendations of the Advisory Committee on Immunization Practices (ACIP).
Harpaz R, Ortega-Sanchez IR, Seward JF; Advisory Committee on Immunization Practices (ACIP) Centers for Disease Control and Prevention (CDC)

Clin Chim Acta 2008 Jan;387(1-2):145-9
Pseudohyperphosphatemia associated with high-dose liposomal amphotericin B therapy.
Lane JW, Rehak NN, Hortin GL, Zaoutis T, Krause PR, Walsh TJ

J Virol 2007 Jun;81(12):6605-1
Herpes Simplex Virus Latency-Associated Transcript (LAT) Sequence Downstream of the Promoter Influences Type-Specific Reactivation and Viral Neurotropism.
Bertke AS, Patel A, Krause PR

J Virol 2007 Feb;81(4):1872-8
HSV-2 Establishes Latent Infection In a Different Population of Ganglionic Neurons than HSV-1: Role of LAT.
Margolis TP, Imai Y, Yang L, Vallas V, Krause PR

Clin Infect Dis 2005 Sep 1;41(5):676-80
Development of herpes simplex virus disease in patients who are receiving cidofovir.
Wyles DL, Patel A, Madinger N, Bessesen M, Krause PR, Weinberg A

Virus Genes 2004 Jan;28(1):71-83
Simian cytomegalovirus encodes five rapidly evolving chemokine receptor homologues.
Sahagun-Ruiz A, Sierra-Honigmann AM, Krause P, Murphy PM



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