Human Vaccines & Immunotherapeutics

ISSN: 2164-5515 (Print) 2164-554X (Online) Journal homepage: https://www.tandfonline.com/loi/khvi20

Emerging infectious disease laboratory and

diagnostic preparedness to accelerate vaccine
development

Christine C. Roberts

To cite this article: Christine C. Roberts (2019): Emerging infectious disease laboratory
and diagnostic preparedness to accelerate vaccine development, Human Vaccines &
Immunotherapeutics, DOI: 10.1080/21645515.2019.1634992

To link to this article: https://doi.org/10.1080/21645515.2019.1634992

Accepted author version posted online: 03

Jul 2019.

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Publisher: Taylor & Francis

Journal: Human Vaccines & Immunotherapeutics

DOI: 10.1080/21645515.2019.1634992
Emerging infectious disease laboratory and diagnostic preparedness to accelerate vaccine development.

Christine C. Roberts, Ph.D.

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GeneOne Life Science, Inc.

croberts@geneonels-us.com; Tel.: +1-484-965-9144

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Abstract

Rapid vaccine development in response to an outbreak of a new emerging infectious disease (EID)
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is a goal targeted by public health agencies worldwide. This goal becomes more complicated when there

are no standardized sets of viral and immunological assays, no accepted and well-characterized samples,
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standards or reagents, and no approved diagnostic tests for the EID pathogen. The diagnosis of

infections is of critical importance to public health, but also in vaccine development in order to track
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incident infections during clinical trials, to differentiate natural infection responses from those that are

vaccine-related and, if called for by study design, to exclude subjects with prior exposure from vaccine
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efficacy trials. Here we review emerging infectious disease biological standards development, vaccine
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clinical assay development and trial execution with the recent experiences of MERS-CoV and Zika virus

as examples. There is great need to establish, in advance, the standardized reagents, sample panels,
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controls and assays to support the rapid advancement of vaccine development efforts in response to EID

outbreaks.

Keywords: Emerging Infectious Disease; Vaccines; Immune Response; Virus Detection, Zika virus,

Flaviviruses, Clinical trials

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1. Introduction

The World Health Organization (WHO) has published a list of priority pathogens1 as those diseases

with high morbidity and mortality or with strong epidemic potential. One of the clear gaps affecting

both public health efforts and vaccine development programs for any of these pathogens is a lack of

standardized reagents and methods to test for evidence of current or prior infection.

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The need for fast and accurate diagnostic tests of infection in an outbreak situation is obvious:

identify the source or epicenter so that appropriate healthcare measures can be quickly instituted.

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Expanding that concept to the public health scale and attaining accurate infectious disease diagnoses

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allows for better understanding of the course and severity of an outbreak and aids decision-making for

population-level countermeasure implementation.

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The clinical assays with which the immune response and pathogen presence are measured in

vaccine trials become part of the basis for licensure for all vaccine products.2 Because vaccines are
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tested in healthy populations through all phases of clinical development for immune response and/or

pathogen presence, the methods selected to measure vaccine responses and endpoints are critical.
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While the identification of an immune correlate of protection for each new vaccine is highly desirable, it

is not always attainable.3

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Here we will use our recent experiences with the Middle East Respiratory Syndrome coronavirus
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(MERS-CoV) and Zika virus (ZIKV)4-9 outbreaks and ensuing public health countermeasures for

containment and vaccine development as examples of challenges faced during emerging infectious
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disease emergencies.

2. Biological Reference Materials and International Standards

International reference materials and standards allow for a common set of reagents for a given

pathogen to be available for the evaluation of the quality and consistency of clinical assays and to

enable comparisons of assay data between studies. Such reference materials are established with

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rigorous evaluation and collaborations between multiple international laboratories and are typically

assigned an international unit of measure at the completion of this process.10 The WHO also provides

guidance for the development of individual secondary standards and their calibration to accepted

international standards.11 This allows individual laboratories to maintain their own standards material

that is traceable to the accepted international standard and not deplete the limited supply of the

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standard prematurely.

Efforts are currently underway to alleviate the international standards issue for MERS-CoV and

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ZIKV. Antibody and nucleic acid standards are in development for MERS-CoV, though additional source

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material is being sought.10 In 2016, the WHO initiated a collaborative study effort for the development

of ZIKV nucleic acid standards12 and, in 2017, made available a plasma sample panel through the US
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Food and Drug Administration (FDA) for evaluation of ZIKV immunoassays.13 The acquisition,

characterization and standardization of relevant pathogen strains such that the strains reflect what is
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currently in circulation and not just a prototype strain is also of concern. For EIDs, there can be an

added complication for sharing such material if the disease is caused by a select agent requiring
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enhanced biosafety measures.

Many of the priority pathogens identified by WHO cause outbreaks that are difficult to predict and
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may be sporadic in nature.1 This complicates efforts to establish processes ahead of any outbreak to
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collect valuable acute and convalescent samples from individuals naturally infected by the respective

viruses. There was, and still is, a lack of well-characterized human specimens from naturally infected
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MERS-CoV and ZIKV subjects that vaccine development groups could use to for assay development and,

eventually, to establish a recognized set of standards that can be maintained over time.10

A potentially valuable source of relevant clinical samples is from ongoing epidemiological studies

and clinical trials. Study designers should take care to incorporate proper language into the informed

consents of participants to allow for the request of additional blood draws or sample collections and for

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the future use of their clinical samples for the purposes of standards and assay development.14-16 Such

studies should also ensure that well-established chain-of-custody, sample handling, and sample storage

procedures are in place to maintain the quality of these valuable specimens.17,18 A significant drawback

of this approach is that often the samples may not have adequate characterization, may be of limited

volume, and are proprietary to the study sponsors. It should also be taken into account that local laws

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or culture may be prohibitive to allowing specimens to be stored for future testing.16,19-23 A concerted

effort and willingness among researchers and companies to share available clinical specimens would be

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a valuable step forward in EID preparedness.24

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Sample collection & handling are critical to the quality of the specimen and its ability to be used in

assays, standards or controls.25 Even in vaccine studies conducted in developed nations, challenges to
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appropriate sample collection and handling can occur. We experienced an issue with the timing of

specimen intake and handling at a biorepository used for our ZIKV trials which required immediate
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corrective action to de-risk samples collected at the remaining study time points and prior to initiation

of a second study. Ensuring proper sample storage and shipping conditions, processing and aliquotting
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(with attention to contamination control), training of site and clinical research organization lab

personnel, a clear chain of custody from collection to final application, intermediate quality control
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checks and good data management is critical for collection of high quality samples that can be used in
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the future.17,26,27

3. Vaccine Clinical Assays, Reference Reagents, International controls

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In the case of EIDs, it can be difficult to predict what assays will be most useful or informative or

will perhaps even provide an immune correlate of protection for a vaccine in early development. Little

may be known about the basic virology or immunology of a new pathogen, though the need for

developing vaccines and therapeutics is urgent.9 Some typical methods used as vaccine clinical assays

are antibody-binding ELISAs, virus neutralization or bactericidal immunoassays, IFNγ-ELISpot or related

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cellular immune methodology using the target antigen or antigen-derived peptide pools, and detection

of the pathogen through molecular or culture assays. Vaccine clinical assays measuring humoral and

cellular immune responses developed early in a program will likely evolve as clinical development

progresses or as the scientific knowledge base of the pathogen and relevant immunology broadens. As

improvements in technology occur over time, early vaccine assays are often re-designed and bridged to

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assays with higher throughput, multiplexed detection, reduction of sample volumes, and automation to

support testing of large numbers of specimens for late stage clinical trials.28-35 Often, a variety of tests

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are evaluated early in the program and, based on the usefulness of the data, a down-selection occurs so

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that the most relevant few remain to support large Phase 3 clinical trials and licensure of the vaccine.
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Accepted “gold standards” of immunoassays for use at the onset of an EID vaccine program are

rare. Critical reagents, standards and controls must be monitored for consistent sourcing, batch-to-
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batch variability and overall quality over time. The implementation of partnerships between

governmental agencies, academic researchers and industry researchers to help secure these items for
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the identified priority pathogens before a need should arise is of great importance.

Reagents for newly emergent infectious diseases like MERS-CoV and ZIKV were not readily available
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from commercial vendors at the outset of vaccine development programs. As such, individual vaccine
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projects, including our own, had to rely on internally developed clinical assays to understand vaccine

related immune responses and to detect prior or current infections.4,5 Lack of standardization, however,
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can confound the interpretation of results from studies using different “home brew” assays across

multiple laboratories such that study results cannot be directly compared in the absence of an accepted

international standard or a proficiency panel of samples.2,10

The vast experience of our DNA vaccine consortium allowed us to develop MERS-CoV- and ZIKV-

specific tests such as ELISA, virus neutralization and ELISpot early in the development and pre-clinical

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testing of the respective plasmid DNA vaccine constructs.4-6,8 These assays were evaluated for

consistent performance throughout pre-clinical studies and we were able to adapt their use to support

our Phase 1 and Phase 1b/2a studies of GLS-5300 MERS-CoV vaccine and two Phase 1 GLS-5700 ZIKV

vaccine clinical trials.7 As these vaccines move further into clinical development, work will need to

continue to transition from our pre-clinical and early stage standards to well-sourced and characterized

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human control reagents. The concern always remains, however, that the inability to use formally

characterized and internationally accepted reagents and controls in early versions of vaccine clinical

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assays can result in maintenance challenges or regulatory hurdles later in the vaccine assays’ life cycle.37

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4. Diagnostics for Emerging Infectious Diseases

Diagnostic assays are often the same style tests as those used in vaccine development, like
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antibody binding or molecular detection. However, their intended purpose is to accurately identify the

infecting pathogen to enable healthcare professionals to initiate appropriate treatments and prevent
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further transmission of disease. Laboratory confirmation of a diagnosis for patient treatment must have

sufficient clinical sensitivity and specificity to be useful, the criteria for which may be different than
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analytical sensitivity and specificity criteria specified by assays for use in vaccine trials.38-45

Very few tests have gained Emergency Use Authorization (EUA) from the FDA. There are 2 MERS-
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CoV diagnostic tests, both molecular-based viral RNA detection, which received EUA in 2013 in response
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to the recognition of the significant potential for a future public health emergency.46 For ZIKV, currently

5 serological kits and 14 viral diagnostic kits have been granted EUA status.47 For ZIKV in particular, the
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response to the need for diagnostics was quite rapid with all EUA approvals rolling out over

approximately 19 months from February 2016 through September 2017,46 shortly after the declaration

of a public health emergency. Outside the US, the WHO’s Emergency Use Assessment and Listing

procedures (EUALs), recognizes the need and can grant authorization for use of diagnostic kits in

emergency situations.48 Although no MERS-CoV or ZIKV diagnostic kit, serological or viral, has been fully

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approved by the FDA to date, the FDA has worked collaboratively with developers to accelerate the

approval process when outbreak conditions warrant.49 A number of published studies have evaluated

EUA tests independently for relevant sensitivity and specificity performance or in comparative

studies32,39,50-56 to aid in the selection of appropriate tests for the needs of epidemiological surveillance

or public health diagnostics. Details outlining the relevant assay performance characteristics of each of

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the EUA-approved MERS-CoV and ZIKV assays can also be found on the FDA Medical Countermeasures

webpage.46,57,58

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In the instance of MERS, EUA diagnostics approvals were limited to use with select respiratory tract

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specimens from individuals with signs and symptoms of infection with MERS-CoV or with

epidemiological risk factors (i.e., contact with probable or confirmed MERS-CoV patient or having a
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history of travel to locations where MERS-CoV cases occur) for the detection of MERS-CoV.46

For ZIKV, a number of complications in diagnostic tests such as cross-reactivity of immunological

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assays and short window of viremia in various bodily fluids required establishment of an algorithm to

confirm ZIKV infection. This confirmatory algorithm guidance for healthcare providers was based on not
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only clinical symptoms, risk factors and diagnostic test results, 59,60 but also specifically for the use and

interpretation of EUA diagnostic ZIKV IgM tests to indicate recent exposure with or without
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accompanying molecular ZIKV test results.59 The types of specimens and timing of collection of
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specimens that would provide the most reliable results was also a consideration in the ZIKV diagnosis

algorithm.
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5. Future Preparedness for EIDs

The Coalition for Epidemic Preparedness Innovations (CEPI) and others are working to ensure that

rapid response mechanisms are in place to address emerging infectious diseases.24,61,62 The CEPI

coalition was launched in 2017 as an innovative concept to establish global partnerships between public,

private, philanthropic and civic organizations with the goal of accelerating the development of vaccines

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against emerging infectious diseases and strengthening vaccine access capabilities before an outbreak

situation is encountered. Funding to support development teams building the infrastructure necessary

for rapid vaccine design, manufacture and clinical assessment is being provided to help ensure that we

are prepared for EID outbreaks that may occur.62

EID public health and countermeasure programs have unique challenges for diagnostic and vaccine

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clinical assay development purposes.2,39,41,63-71 There may be incomplete understanding of the biology or

epidemiology of a new pathogen, which can delay or confound the selection of a relevant vaccine target

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and the subsequent assay development to be used to evaluate the candidates. The field may suffer from

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a lack of available reagent sources or with inconsistency in quantity and quality of those available,

especially early in the discovery and development process. The difficulty in obtaining or developing
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relevant human sample panels, reference materials and/or international standards for the evaluation of

test methods add to the challenges to support assay performance from early vaccine development
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through licensure.10

Although the speed at which the EID diagnostic or vaccine development field needs to move will be
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dependent upon the urgency of the pathogen outbreak and its impact on human life, scientific and

quality principles must still apply when developing vaccine or diagnostic assays. Biological assay
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standardization is critical.2,10,17,71,72 At a minimum, the development of relevant biological assays with

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adequate sensitivity and specificity for the application should use biostatistics to establish and verify

assay performance and to maintain the ability to produce stable and reproducible results over time to
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support diagnostic or vaccine program needs. The criteria for acceptability of any given test system will

be dependent upon the nature of the pathogen, our understanding of the immunology to fight the

pathogen (both of which may be poorly understood in an EID situation) and the assay platform. If assay

performance consistency and quality is not demonstrated, the validity of clinical study results may be

questioned. In the execution of vaccine clinical trials, assay methodology must be accurate, specific and

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robust with high quality procedures in place for sample collection, processing and storage to ensure

success.25,26,36,37 The translatability of assay methodology is also important if the acceleration of vaccine

development is dependent upon the use of animal challenge studies to establish efficacy or to help

define a correlate of protection for an EID pathogen.

Efforts to prepare reagents, collect well-characterized samples, develop research materials and

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international standards in advance for EIDs for whom alerts have already been raised, such as the WHO

priority pathogens, through collaborative partnerships with governmental and philanthropic agencies

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along with academic and industry based researchers will make us better suited to respond with speed to

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an outbreak.24,61,69,73 Assay standardization for these or any other newly emergent infectious disease will

be challenging and take time to develop, collect and characterize quality reagents, to achieve sufficient
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sources samples for the establishment of serological or molecular standards.10 The commitment of

researchers and companies invested in the research, diagnosis, treatment and prevention of EIDs to
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participate and contribute to organized efforts to create and validate internationally recognized

standardized reagents, assays and controls for priority pathogens in advance of an emergency is
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imperative and the time to start building this framework is now.

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Acknowledgments: The author would like to acknowledge Inovio Pharmaceuticals with whom we
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co-develop the GLS-5300 MERS-CoV and GLS-5700 Zika DNA vaccines, the David Weiner Laboratory at

the Wistar Institute, Philadelphia, PA, the Gary Kobinger Laboratory at Laval University, Quebec, Canada
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and the GeneOne Life Science Teams: Clinical Research in Blue Bell, PA and Basic Research &

Development in Seoul, Korea.

Conflicts of Interest: Author is an employee of GeneOne Life Science, Inc.

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