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  1. Biologics Research Projects

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 unexpected and undesired infectious agents? 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 that could potentially be present in 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 that can cause life-long disease.

Scientific Overview

We are using genomic approaches, including next generation sequencing, to develop new ways to address important regulatory questions that affect vaccines. This includes using these techniques to detect expected viruses that theoretically could be found in vaccines or other biological products. The basic approach involves 1) identifying samples appropriate for testing, 2) identifying useful ways to purify nucleic acids (DNA and RNA) from those samples, 3) obtaining unbiased sequence on those nucleic acids, and 4) determining whether those sequences could represent an unexpected virus. One area of recent interest has been to determine whether these types of genomic approaches could be used to supplement currently used cell culture-based assays, where a genomic readout might be more sensitive and reproducible than the more commonly used visual examination for cytopathic effect or other changes.

If 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.

We also use these techniques to develop improved understanding of antiviral immune responses. This includes study of antibodies that neutralize viruses, as well as understanding how HSV evades immune responses to reactivate from latency to cause disease.

Our studies of HSV latency also focus on virus genes that affect establishment, maintenance, and reactivation from latency. This includes factors that influence production of a viral gene called ICP34.5. The ICP34.5 gene plays a critical role in neurovirulence (the ability to replicate in neurons) and helps determine whether the virus can establish or reactivate from latency in different cells. 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 that influence expression of ICP34.5. 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.


N Engl J Med 2020 Apr 2;382(14):1366-9
Creating a framework for conducting randomized clinical trials during disease outbreaks.
Dean NE, Gsell PS, Brookmeyer R, Crawford FW, Donnelly CA, Ellenberg SS, Fleming TR, Halloran ME, Horby P, Jaki T, Krause PR, Longini IM, Mulangu S, Muyembe-Tamfum JJ, Nason MC, Smith PG, Wang R, Henao-Restrepo AM, De Gruttola V

J Infect Dis 2020 Mar 5;221(Suppl. 1):S103-8
Scientific and regulatory considerations for efficacy studies of cytomegalovirus vaccines.
Krause PR, Roberts J

PLoS Pathog 2019 Jun 17;15(6):e1007884
Hidden regulation of herpes simplex virus 1 pre-mRNA splicing and polyadenylation by virally encoded immediate early gene ICP27.
Tang S, Patel A, Krause PR

J Virol 2019 May 15;93(11):e00227-19
HSV-2 in autonomic ganglia: evidence for spontaneous reactivation.
Pieknik JR, Bertke AS, Krause PR

Vaccine 2019 Feb 8;37(7):1001-5
Towards dynamic monitoring of cell cultures using high throughput sequencing.
McClenahan SD, Krause PR

Vaccine 2019 Feb 4;37(6):863-8
Demonstrating vaccine effectiveness during a waning epidemic: A WHO/NIH meeting report on approaches to development and licensure of Zika vaccine candidates.
Vannice KS, Cassetti MC, Eisinger RW, Hombach J, Knezevic I, Marston HD, Wilder-Smith A, Cavaleri M, Krause PR

Biologicals 2018 Sep;55:1-16
Report of the international conference on next generation sequencing for adventitious virus detection in biologicals.
Khan AS, Benetti L, Blumel J, Deforce D, Egan WM, Knezevic I, Krause PR, Mallet L, Mayer D, Minor PD, Neels P, Wang G

J Virol 2018 Jun 29;92(14):e00642-18
Herpes simplex virus 2 Latency-Associated Transcript (LAT) region mutations do not identify a role for LAT-associated micro RNAs in viral reactivation in the guinea pig genital model.
Kawamura Y, Bosch-Marce M, Tang S, Patel A, Krause PR

Science 2018 Jun 22;360(6395):1308
Evaluating human trials: FDA's role.
Krause PR, Gruber MF

Viruses 2018 May 8;10(5):246
A VP26-mNeonGreen capsid fusion HSV-2 mutant reactivates from viral latency in the guinea pig genital model with normal kinetics.
Pieknik JR, Bertke AS, Tang S, Krause PR

J Infect Dis 2017 Dec 16;216(Suppl. 10):S964-70
Clinical development strategies and considerations for Zika vaccine licensure.
Gruber MF, Farizo KM, Pratt RD, Fink DL, Finn TM, Krause PR, Borio LL, Marks PW

J Clin Invest 2017 Jun 30;127(7):2626-30
Glutamine supplementation suppresses herpes simplex virus reactivation.
Wang K, Hoshino Y, Dowdell K, Bosch-Marce M, Myers TG, Sarmiento M, Pesnicak L, Krause PR, Cohen JI

Expert Rev Vaccines 2017 Jun;16(6):525-7
Regulating vaccines at the FDA: development and licensure of Zika vaccines.
Gruber MF, Krause PR

Proc Natl Acad Sci U S A 2016 Oct 25;113(43):12256-61
Herpes simplex virus ICP27 regulates alternative pre-mRNA polyadenylation and splicing in a sequence-dependent manner.
Tang S, Patel A, Krause PR

Lancet 2015 Aug 29;386(9996):831-3
Interim results from a phase 3 Ebola vaccine study in Guinea.
Krause PR

EBioMedicine 2015 Jun 30;2(8):857-65
Reverse engineering of vaccine antigens using high throughput sequencing-enhanced mRNA display.
Guo N, Duan H, Kachko A, Krause BW, Major ME, Krause PR

Lancet Infect Dis 2015 Jun;15(6):627-9
Approaches to demonstration of Ebola virus vaccine efficacy.
Krause PR, Cavaleri M, Coleman G, Gruber MF

J Virol 2015 May;89(9):4837-48
Characterization of HSV-2 primary miRNA transcript regulation.
Tang S, Bosch-Marce M, Patel A, Margolis TP, Krause PR

Sci Transl Med 2015 May 6;7(286):286ps11
Immunology of protection from Ebola virus infection.
Krause PR, Bryant PR, Clark T, Dempsey W, Henchal E, Michael NL, Regules JA, Gruber MF

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