Personal View
Targeting the SARS-CoV-2 reservoir in long COVID
Amy D Proal, Soo Aleman, Morgane Bomsel, Petter Brodin, Marcus Buggert, Sara Cherry, Daniel S Chertow, Helen E Davies, Christopher L Dupont,
Steven G Deeks, E Wes Ely, Alessio Fasano, Marcelo Freire, Linda N Geng, Diane E Griffin, Timothy J Henrich, Stephen M Hewitt, Akiko Iwasaki,
Harlan M Krumholz, Michela Locci, Vincent C Marconi, Saurabh Mehandru, Michaela Muller-Trutwin, Mark M Painter, Etheresia Pretorius,
David A Price, David Putrino, Yu Qian, Nadia R Roan, Dominique Salmon, Gene S Tan, Michael B VanElzakker, E John Wherry,
Johan Van Weyenbergh, Lael M Yonker, Michael J Peluso
There are no approved treatments for post-COVID-19 condition (also known as long COVID), a debilitating disease
state following SARS-CoV-2 infection that is estimated to affect tens of millions of people. A growing body of evidence
shows that SARS-CoV-2 can persist for months or years following COVID-19 in a subset of individuals, with this
reservoir potentially driving long-COVID symptoms or sequelae. There is, therefore, an urgent need for clinical trials
targeting persistent SARS-CoV-2, and several trials of antivirals or monoclonal antibodies for long COVID are
underway. However, because mechanisms of SARS-CoV-2 persistence are not yet fully understood, such studies
require important considerations related to the mechanism of action of candidate therapeutics, participant selection,
duration of treatment, standardisation of reservoir-associated biomarkers and measurables, optimal outcome
assessments, and potential combination approaches. In addition, patient subgroups might respond to some
interventions or combinations of interventions, making post-hoc analyses crucial. Here, we outline these and other
key considerations, with the goal of informing the design, implementation, and interpretation of trials in this rapidly
growing field. Our recommendations are informed by knowledge gained from trials targeting the HIV reservoir,
hepatitis C, and other RNA viruses, as well as precision oncology, which share many of the same hurdles facing longCOVID trials.
Introduction
The COVID-19 pandemic has made it increasingly clear
that infectious pathogens can drive not just acute illness,
but also debilitating chronic disease. The US Centers for
Disease Control and Prevention estimates that approxi­
mately 6% of Americans infected with SARS-CoV-2
develop chronic symptoms or sequelae, referred to as
post-COVID-19 condition (also known as long COVID).1–3
Similar incidence has been noted in settings across the
globe.4 Although multiple biological factors are being
evaluated as contributors to long COVID,3,5 a growing
body of research centres around the persistence of
SARS-CoV-2 as a driver of disease in at least some
individuals.6–13 This SARS-CoV-2 reservoir might drive
inflammation, hinder virus-directed immune responses,
or disturb the function of infected cells, contributing to
long COVID and other complications.
If persistence of SARS-CoV-2 causes chronic disease,
then the virus is an obvious target for therapeutic studies.
Early trials of antivirals and monoclonal antibodies in
long COVID are underway. However, because of
uncertainty regarding the mechanisms and measures of
SARS-CoV-2 persistence, several con­siderations should
guide the design and interpretation of these and future
trials. Here, we draw from RNA virus biology, efforts to
target the HIV reservoir, and clinical oncology to outline
major considerations for the design and implementation
of trials targeting SARS-CoV-2 per­
sistence in long
COVID.
The SARS-CoV-2 reservoir
Although the initial expectation in the infectious disease
community was that SARS-CoV-2 infection would be
transient in immunocompetent individuals, multiple
studies now support the existence of a SARS-CoV-2
reservoir in at least a subset of people with long COVID
(appendix p 1). These studies have identified singlestranded or double-stranded SARS-CoV-2 RNA or
proteins well beyond the acute phase of infection in
tissues such as the gut7,8,14–18 or in host cells such as
platelets or megakaryocytes.19 Immune responses
suggestive of ongoing stimulation by viral antigens have
also been documented in individuals with long
COVID.20–22 Multiple teams have identified SARS-CoV-2
proteins in plasma up to 14 months after initial
infection,10,11,23–27 which are believed to be translated from
viral RNA in tissue reservoir sites and leak into the
circulation. Although SARS-CoV-2 persistence has been
documented even in people without long COVID,
evidence linking SARS-CoV-2 persistence to long COVID
is growing. In a large study from the National Institutes
of Health RECOVER programme, participants who
reported long-COVID symptoms affecting heart and
lung, brain, and musculoskeletal systems were
approximately twice as likely to have detection of
SARS-CoV-2 proteins circulating in their blood during
the post-acute phase.11 The totality of the evidence
suggests that some people with long COVID harbour a
tissue-based reservoir of SARS-CoV-2, with variable
detection in the circulation using present technologies,
which might drive inflammation or immune
dysregulation, and ultimately result in long COVID.3
Establishment and maintenance of the reservoir
The establishment of the reservoir begins during acute
SARS-CoV-2 infection. Both host and viral dynamics
likely play a role. A recent analysis of post-acute antigen
persistence in plasma showed that those with more
www.thelancet.com/infection Published online February 10, 2025 https://doi.org/10.1016/S1473-3099(24)00769-2
Lancet Infect Dis 2025
Published Online
February 10, 2025
https://doi.org/10.1016/
S1473-3099(24)00769-2
PolyBio Research Foundation,
Medford, MA, USA
(A D Proal PhD,
M B VanElzakker PhD);
Department of Infectious
Diseases and Unit of PostCOVID Huddinge, Karolinska
University Hospital, Stockholm,
Sweden (S Aleman MD);
Department of Medicine
Huddinge (S Aleman) and
Department of Women’s and
Children’s Health
(Prof P Brodin MD), Karolinska
Institutet, Stockholm, Sweden;
HIV entry and Laboratory of
Mucosal Immunity, Institut
Cochin, Paris, France
(Prof M Bomsel PhD); Université
Paris Cité, CNRS, INSERM,
Institut Cochin, Paris, France
(Prof M Bomsel); Department of
Immunology and Inflammation
(Prof P Brodin) and Medical
Research Council Laboratory of
Medical Sciences (Prof P Brodin),
Imperial College London,
London, UK; Center for
Infectious Medicine,
Department of Medicine
Huddinge, Karolinska
Institutet, Huddinge, Sweden
(M Buggert PhD); Department
of Pathology and Laboratory
Medicine (Prof S Cherry PhD),
Department of Microbiology
(M Locci PhD), Department of
Systems Pharmacology and
Translational Therapeutics
(Prof E J Wherry), and Institute
for Immunology and Immune
Health (M Locci PhD,
M M Painter PhD,
Prof E J Wherry PhD), Perelman
School of Medicine, University
of Pennsylvania, Philadelphia,
PA, USA; Emerging Pathogens
Section, Critical Care Medicine
Department, Clinical Center,
National Institutes of Health,
Bethesda, MD, USA
(D S Chertow MD); Laboratory of
Virology, National Institute of
Allergy and Infectious Diseases,
National Institutes of Health,
Hamilton, MT, USA
1
Personal View
SARS-CoV-2
Infected cell
Cytoplasm
Coreceptors
SARS-CoV-2 single-stranded RNA
remains, without necessarily being
translated into viral proteins or new
virions
SARS-CoV-2 RNA might be capable of
driving translation of viral proteins
without necessarily producing new
virions
SARS-CoV-2 RNA drives production of
new virions periodically, and perhaps
at low levels, which can potentially
infect new cells for long-term
reservoir maintenance
Figure 1: Potential forms of SARS-CoV-2 reservoir persistence
Each form of persistence likely requires targeting by different therapeutics, or combinations of therapeutics, for the
most effective treatment.
(D S Chertow); Department of
Respiratory Medicine,
University Hospital Llandough,
Cardiff, UK (H E Davies MD);
University School of Medicine,
University Hospital of Wales,
Cardiff, UK (H E Davies);
Division of Genomic Medicine,
Environment & Sustainability
(Prof C L Dupont PhD),
Department of Informatics
(Y Qian PhD) and Department
of Infectious Diseases
(G S Tan PhD, M Freire PhD),
J Craig Venter Institute,
University of California San
Diego, La Jolla, CA, USA;
Division of HIV, Infectious
Diseases, and Global Medicine
(Prof S G Deeks MD,
M J Peluso MD) and Division of
Experimental Medicine
(Prof T J Henrich MD), University
of California, San Francisco, CA,
USA; The Critical Illness, Brain
Dysfunction, Survivorship
Center at Vanderbilt University
Medical Center, Nashville, TN,
USA (Prof E W Ely MD); Veteran’s
Affairs Tennessee Valley
2
severe initial infection were more likely to have
subsequent antigen detection.10 Others have shown that
the duration of viral shedding or maximum viral load
during the acute phase correlates with subsequent long
COVID.28,29 These observations suggest that individuals
with a higher early viral burden might have a greater viral
inoculum that persists at primary infection sites such as
the lungs or gut, or that seeds distant tissue sites.
One autopsy study identified single-stranded and
subgenomic SARS-CoV-2 RNA or protein in dozens of
tissues including brain, nerve, and ocular tissue up to
230 days after COVID-19 onset.30 The seeding of distant
reservoir sites is reminiscent of post-Ebola syndrome,
where studies have identified viral reservoirs in immuneprivileged sites such as testes, eyes, and brain long after
acute infection.31
A related possibility is that host immune status or
failure of the immune system to adequately clear the
virus drives the establishment of a reservoir. For example,
some studies have connected long COVID to decreased
neutralising antibody function.32 Immune dysfunction
facilitating the seeding of a reservoir could partly explain
why some patients with long COVID report mild acute
infections but develop symptoms gradually over time.
Such hypotheses are compatible with studies showing
that efforts to reduce viral burden or enhance the
immune response (eg, antiviral treatment33–37 and
vaccination38,39) appear in some cases to protect against
the development of long COVID.
Once a reservoir is established, there are several
mechanisms by which it could be maintained (figure 1).
SARS-CoV-2 RNA could persist without producing new
extracellular virions, potentially via mutation or immune
escape, as observed with other single-stranded RNA
viruses.40 RNA could persist and be translated, provoking
an immune response even if it is not fully replicating.
The identification of viral proteins in plasma during the
post-acute phase10,11,23 suggests that translation is
occurring, as does higher expression of markers of
activation and exhaustion on SARS-CoV-2-specific CD8+
T cells among some individuals with long COVID in
comparison to those who fully recovered.41 Another
possibility is that the virus persists and replicates, at least
periodically or at low amounts, and is capable of infecting
new target cells. This scenario is supported by studies
that have identified double-stranded, antisense, or
subgenomic RNA (transcripts synthesised as products of
replication) in tissues, immune cells, or whole blood
from people with long COVID.7–9,19 However, these
transcripts could represent active or previous replication
of the virus, as it is possible the viral RNA persists for
some time in protected intracellular microenvironments.
Periodic viral replication is one potential explanation for
the fluctuating symptoms reported by many patients
with long COVID. Well designed studies targeting each
mechanism, together with careful monitoring of the
effects on reservoir biomarkers, will ultimately be needed
to reveal the primary drivers of reservoir persistence
(table 1).
Therapeutic approaches to target the reservoir
Here, we briefly review therapeutic strategies that could
target the reservoir in clinical trials meant to address
persistent SARS-CoV-2 as a driver of long COVID
(table 2).
Targeting the virus
Antivirals are straightforward candidates for reservoirtargeting trials. Antivirals do not directly destroy viruses
but rather disrupt specific components of their replication
cycle, preventing production of additional virions. They
are typically small molecules that target enzymes such as
RNA-dependent RNA polymerase or viral protease.
Antivirals such as nirmatrelvir–ritonavir, ensitrelvir, and
remdesivir are being tested in several long-COVID trials
(NCT05595369,
NCT05668091,43
NCT05823896,
NCT06161688, NCT05911906, and NCT06511063).
Although one trial (NCT05576662) showed no
symptomatic benefit of a 15-day treatment course of
nirmatrelvir–ritonavir compared with ritonavir placebo
among 155 people with long COVID,44 others are pursuing
www.thelancet.com/infection Published online February 10, 2025 https://doi.org/10.1016/S1473-3099(24)00769-2
Personal View
Mechanisms
Advantages
Considerations
Direct acting antivirals
Disrupt viral replication
Several approved; oral options
available; antiviral agents with different
synergistic mechanisms of action might
more successfully target reservoirs
Tissue penetration to sites of persistence requires
further study; extended courses might be needed if
reservoir persists within long-lived host cell types;
drug–drug interactions can complicate administration
Antisense oligonucleotides
and other CRISPR-based
approaches
Inactivate or destroy viral
ssRNA
Could potentially inactivate viral RNA
whether or not translating or
replicating, and even if it has escaped
immune clearance
Oligonucleotides and protein-based therapeutics often
need to be specifically directed to the tissue where RNA
persists and delivery should consider cellular
internalisation strategies
Bind extracellular proteins Can be updated for different variant
and virions
targets; maintain longer-term
therapeutic levels
Unlikely to work if virus is not extracellular or protein
not expressed on surface of infected cell; might or
might not have access to target tissue; might or might
not have retained effector function to facilitate
clearance of infected cells; evolving variants might
become resistant
Direct acting
Acquired and innate immunotherapies
Monoclonal antibodies
Cytokine-based therapies
Activate T and natural
(eg, interleukin superagonists) killer cell immunity or
innate defenses
Could still work even if persistent virus
has escaped immune-mediated
clearance
Stimulating the immune response might cause
patients to temporarily feel worse before feeling better
PD-1 and PD-L1 checkpoint
blockade therapies
Restore function of
exhausted T cells
Supports T cells to target intracellular
reservoir
Further investigation needed to elucidate which
patients with long COVID show T cell exhaustion;
potentially complex risk profile
Therapeutic vaccines
(including next generation
vaccines)
Increase immune response Can be updated for different variants
and antibody response
towards persistent
antigen
Development of next-generation vaccines that include
viral epitope targets beyond spike, and that elicit
antibody-dependent cell-mediated responses or T-cell
mediated responses might better direct clearance of
persistently infected cells
Table 1: Potential reservoir trial therapeutics and mechanisms of action
the approach in larger studies that include more targeted
phenotypes, along with longer treatment duration.
Monoclonal antibodies (mAbs) are also promising
therapeutic candidates, as these agents can have
two activities: directly neutralising replicating virus,
thereby serving as an antiviral to halt the replication cycle,
and promoting clearance of viral protein. If effector
function is maintained, they may also promote clearance
of infected cells expressing viral proteins at the cell surface
via antibody-dependent cellular cytotoxicity. Although no
agents of this class are currently approved or authorised
for treatment of COVID-19, several are available for
prophylaxis, and new agents are under development.
There are also approaches under development to
directly inactivate and degrade viral RNA, including
antisense oligonucleotides and CRISPR-based RNA
targeting.45–48 These therapies could target viral RNA even
if no replication is occurring. However, research is
needed to determine feasibility. In particular, under­
standing viral reservoir locations (eg, the gut) is
important since such treatments might need to be
directed to the tissue where RNA persists.
Enhancing the host immune system
The presence of a SARS-CoV-2 reservoir in long COVID
suggests that the virus might escape immune-mediated
clearance, perhaps due to dysfunctional immune
responses, or reduced activity of local cytotoxic T-cell and
natural killer (NK) cell responses in tissues, particularly
within immune-privileged sites. Murine norovirus—
another RNA virus—can persist in an immune-privileged
enteric niche via induction of a CD8+ T-cell differentiation
state that fails to detect and clear viral reservoirs.49
Cellular immune dysfunction and general immune
dysregulation have been observed in long COVID,41,50,51
and in some cases connected to SARS-CoV-2
persistence.25,52 Some,41,49 but not all,25 studies to date have
observed subtle changes in T-cell responses that warrant
further investigation to determine if SARS-CoV-2-specific
T cells are exhausted or otherwise dysfunctional in
patients with long COVID.
If T-cell exhaustion or NK cell dysfunction, or both, are
connected to SARS-CoV-2 persistence, enhancing these
innate and adaptive immune responses might facilitate
better control of persistent infection. Relevant therapies
include cytokines such as interleukin superagonists,
which are being studied in HIV to restore and boost T-cell
and NK cell responses53 or cytokines with antiviral
potential (eg, interferon and IL-15). PD-1 and PD-L1
checkpoint blockade therapies could be considered.
Whether therapeutic COVID-19 vaccination is beneficial
in inducing immune responses that clear the viral
reservoir could also be investigated. Thus far, although
some people with long COVID have reported symptom
benefit from vaccination,38,39 others have reported
worsening of symptoms.54 It should be noted that the
current SARS-CoV-2 vaccines such as mRNA and viralvectored vaccines generate neutralising antibody
www.thelancet.com/infection Published online February 10, 2025 https://doi.org/10.1016/S1473-3099(24)00769-2
Geriatric Research Education
Clinical Center, Nashville, TN,
USA (Prof E W Ely); Department
of Pediatrics (Prof A Fasano MD,
L M Yonker MD), Mucosal
Immunology and Biology
Research Center (Prof A Fasano,
L M Yonker), and Division of
Neurotherapeutics
(M B VanElzakker),
Massachusetts General
Hospital, Boston, MA, USA;
Harvard Medical School,
Boston, MA, USA (Prof A Fasano,
L M Yonker); J Craig Venter
Institute, Department of
Medicine, Stanford University
School of Medicine, Stanford,
CA, USA (L N Geng MD);
W Harry Feinstone Department
of Molecular Microbiology and
Immunology, Johns Hopkins
Bloomberg School of Public
Health, Baltimore, MD, USA
(Prof D E Griffin PhD);
Laboratory of Pathology,
Center for Cancer Research,
National Cancer Institute,
National Institutes of Health,
Bethesda, MD, USA
(S M Hewitt MD); Department
of Immunobiology
(Prof A Iwasaki PhD), and Center
for Infection and Immunity
(Prof A Iwasaki,
Prof H Krumholz MD), Yale
University School of Medicine,
New Haven, CT, USA; Howard
Hughes Medical Institute,
Chevy Chase, MD, USA
(Prof A Iwasaki); Center for
Outcomes Research and
Evaluation, Yale New Haven
Hospital, New Haven, CT, USA
(Prof H Krumholz MD); Section
of Cardiovascular Medicine,
Department of Internal
Medicine, Yale School of
Medicine, New Haven, CT, USA
(Prof H Krumholz); Department
of Health Policy and
Management, Yale School of
Public Health, New Haven, CT,
USA (Prof H Krumholz); Emory
University School of Medicine
and Rollins School of Public
Health, Atlanta, GA, USA
(Prof V C Marconi MD); Atlanta
Veterans Affairs Medical Center,
Decatur, GA, USA
(Prof V C Marconi); Precision
Immunology Institute
(Prof S Mehandru MD) and
Department of Rehabilitation
and Human Performance
(Prof D Putrino PhD), Icahn
School of Medicine at Mount
Sinai, New York, NY, USA;
Henry D Janowitz Division of
Gastroenterology, Department
of Medicine, Icahn School of
3
Personal View
NCT number
Therapeutic
Location
Recruitment target
Status
PREVAIL-LC
NCT06161688
Ensitrelvir fumaric acid (S-217622)
vs placebo
University of California, San Francisco,
CA, USA
40 participants
Active
RECOVER-VITAL
NCT05965726
Nirmatrelvir–ritonavir for 25 days or
15 days vs placebo ritonavir
Duke University, Durham. NC, USA
900 participants
Active
imPROving Quality of LIFe in the long COVID
patient
NCT05823896
Nirmatrelvir–ritonavir for 15 days
vs placebo ritonavir
Karolinska Institutet, Solna, Sweden
400 participants
Active
PaxLC
NCT05668091
Nirmatrelvir–ritonavir for 15 days
vs placebo ritonavir
Yale University, New Haven, CT, USA
100 participants
Completed
STOP-PASC
NCT05576662
Nirmatrelvir–ritonavir for 15 days
vs placebo ritonavir
Stanford University, Palo Alto, CA,
USA
168 participants
Completed
outSMART-LC
NCT05877508
AER002 administered once
intravenously vs placebo
University of California, San Francisco,
CA, USA
30 participants
Active
AT1001 for the treatment of long COVID
NCT05747534
Larazotide (AT1001) vs placebo in
children and young adults
Massachusetts General Hospital,
Boston, MA, USA
48 participants
Active
Antiviral clinical trial for long COVID-19
NCT06511063
Truvada or selzentry vs placebo
Icahn School of Medicine at Mount
Sinai, New York, NY, USA
90 participants
Active
ERASE-LC
NCT05911906
Remdesivir
University of Plymouth, Devon, UK
72 participants
Active
ESSOR
NCT05999435
LAU-7b vs placebo
Multiple hospitals, Canada
272 participants
Active
Study to evaluate the efficacy and safety of
ampligen in patients with post-COVID
conditions
NCT05592418
Rintatolimod vs placebo
Hunter-Hopkins Center, Charlotte,
NC, USA
80 participants
Completed
RSLV-132 in participants with long COVID
NCT04944121
RSLV-132 vs placebo
Resolve Therapeutics, Alabama and
Florida, USA
112 participants
Completed
ACTIV-2
NCT04518410
Amubarvimab and romlusevimab vs
placebo for acute COVID-19
Sites in the USA, Argentina, Brazil,
Mexico, Philippines, and South Africa
847 participants
Completed
SOLIDARITY
NCT04978259
Remdesivir during acute COVID-19
hospital stay up to 10 days in addition
to standard care
Clinical Urology and Epidemiology
Working Group, Helsinki, Finland
202 participants
Completed
PANORAMIC
ISRCTN30448031,
NIHR135366
Molnupiravir vs placebo during acute
COVID-19
University of Oxford, Oxfordshire, UK
783 participants
Completed
SCORPIO-SR*
jRCT2031210350
Ensitrelvir during acute COVID-19 vs
placebo
92 institutions in Japan, Viet Nam,
and South Korea
1821 participants,
2694 target
Completed
COVID-OUT*
NCT04510194
Metformin vs fluvoxamine
vs ivermectin vs metformin plus
fluvoxamine vs metformin plus
ivermectin vs placebo
University of Minnesota, Minneapolis, 1323 participants
MN, USA
Completed
Treatment of established long COVID
Prevention of long COVID
*The trial was not designed to target the reservoir, but secondary analysis found that metformin had an antiviral effect.
Table 2: Clinical trials designed to target or prevent the SARS-CoV-2 reservoir
Medicine at Mount Sinai,
New York, NY, USA
(Prof S Mehandru); Institut
Pasteur, Université Paris-Cité,
HIV, Inflammation and
Persistence Unit, Paris, France
(Prof M Muller-Trutwin PhD);
Department of Physiological
Sciences, Faculty of Science,
Stellenbosch University,
Stellenbosch, South Africa
(Prof E Pretorius PhD);
Department of Biochemistry
and Systems Biology, Institute
of Systems, Molecular and
Integrative Biology, Faculty of
Health and Life Sciences,
University of Liverpool,
Liverpool, UK (Prof E Pretorius);
Division of Infection and
Immunity (Prof D A Price MD)
4
responses and T-cell immunity only to the spike protein.
The development of next-generation vaccines that include
expanded viral protein targets or elicit antibodydependent cell-mediated responses might better direct
clearance of persistently infected cells. This effort mirrors
those to develop therapeutic vaccines in other chronic
viral infections such as HIV.
Key considerations for clinical trials targeting
the reservoir
The optimal design of reservoir-targeting trials requires
consideration of multiple factors (figure 2).
Measures of persistence
Because tissue-based studies have provided compelling
evidence of a SARS-CoV-2 reservoir, use of tissue biopsy is
an optimal approach for assessing the effect of trials
targeting the reservoir. Some tissues (eg, gut and lymph
nodes) can be safely sampled in living patients (figure 3).
After processing and preservation of the tissue,
investigators can assess the presence of RNA using
hybridisation, sequencing, or amplification approaches
(table 3). Detection of double-stranded RNA55 can be used
to infer the presence of ongoing or previous replicating
virus. Ideally, these assessments would occur before and
after intervention.
However, tissue collection is invasive, expensive, and
technically challenging. A small tissue specimen might
miss neighbouring cells harbouring SARS-CoV-2. The
reservoir might also persist in tissues such as the brain
or myocardium, which are likely inaccessible given the
risk of sampling such organs.
Hence, the field would benefit tremendously from a
persistence biomarker akin to the plasma HIV RNA level
www.thelancet.com/infection Published online February 10, 2025 https://doi.org/10.1016/S1473-3099(24)00769-2
Personal View
in HIV—an objective measurement that uses an easily
accessible body fluid (eg, blood), is sensitive and specific,
has a rapid turnaround time, is scalable, and for which a
change (eg, reduction) is clearly associated with a clinical
outcome.56 The development and validation of multiple
assays for SARS-CoV-2 persistence is among the most
urgent efforts in this field. But although multiple studies
have identified SARS-CoV-2 RNA or proteins in blood
obtained from patients with long COVID,9,10,25 detection
of these antigens is not nearly as reliable as gold standard
biomarkers, such as plasma HIV RNA, for several
reasons.
The degree to which antigens from tissue sites reach
the circulation is unknown, and participants harbouring
tissue reservoirs could fail to have antigen detected in
blood. Using current assays, detection in blood varies
within an individual over time.10,23 The cause of
such fluctuations, although unknown, might reflect
differences in translational activity in tissue, leading to
periods when proteins are released into the circulation at
different rates. It is also possible that only tiny amounts
of protein leak into circulation from reservoir sites; these
might be below the detection limit of many assays and
consequently missed. For this reason, ultrasensitive
assays (eg, Simoa) are used in some studies. However,
even these assays might require additional iterations to
accurately identify protein below the detection limit.10,11,25
SARS-CoV-2 RNA and protein in the blood of individuals
with long COVID might also persist inside host cells
including monocytes, megakaryocytes, or platelets.19
High-throughput assays capable of measuring cellassociated viral RNA, such as digital transcriptomics9 in
whole blood, might be needed to account for this
possibility.
Given these gaps, it is imperative to accelerate efforts to
further develop and optimise assays for SARS-CoV-2
RNA and protein in accessible matrices, such as whole
blood, plasma, stool, and saliva. These efforts should
include defining assay sensitivity, specificity, positive and
negative predictive value, reproducibility, determinants
of within-person and between-person variation, and the
likelihood of antigen detection in body fluids if the
reservoir is localised to tissue. The detection limitations
of the assay used should also be considered. For example,
blood samples from people with long COVID might have
high amounts of endogenous anti-SARS-CoV-2 IgG that
could form immune complexes, impairing antigen or
specific antibody detection with some assays. Protocols
that remove these endogenous IgGs or dissociate
immune complexes might be required before measuring
antigens via immunoassay. Ideally, assays would
distinguish inert from replicating virus, would quantify
viral transcripts, and would document if mutations occur
over time. Development of new assays, such as CRISPRbased approaches for viral RNA detection,57 or changes in
the immune system20 should also be prioritised. These
are key considerations because reliable persistence
Eligibility determination
Recruitment based on, or post hoc
analyses stratifying by, viral
persistence is recommended
Safety
Adverse effects of some drugs may
differ between acute and long
COVID, and some agents have
considerable drug–drug
interactions
Blinding and the placebo effect
Due to potential placebo effect,
inclusion of a control group is
essential for interpretation of
most studies
Duration and timing of
treatment
Longer treatment courses (months
rather than weeks) might be
needed to eradicate a reservoir
Tissue penetration of the drug
Reservoir-targeting therapeutics
must achieve adequate levels in
locations of persistence
On-study reinfections
Investigators should have a plan to
handle on-study reinfections,
including emphasising the
importance of testing
Measures of persistence
Tissue biopsy is ideal, but more
accessible biospecimens (eg,
blood, etc) should be collected for
certain persistence assays
Combination trials
Combination trials of drugs (eg,
antivirals and mAbs) might be
required to most effectively target
reservoirs
Evolving SARS-CoV-2 variants
Broadly neutralising SARS-CoV-2
antibodies might be needed to
target mixed reservoirs or those
established after infection with
modern variants
Prevention of long COVID
Interventions of acute COVID-19
treatment should include
post-acute viral persistence as a
prespecified outcome
Figure 2: Main considerations for clinical trials targeting the reservoir
mAbs=monoclonal antibodies.
measurables in blood and saliva would not only increase
our ability to interpret trials, but also allow trials to be
more inclusive, potentially enabling sample collection at
home for bedbound patients with severe disease.
Eligibility determination
Because SARS-CoV-2 can drive many forms of
physiological dysfunction, a portion of patients meeting
symptom-based long-COVID diagnostic criteria could
potentially be affected by underlying issues other than
SARS-CoV-2 persistence. Thus, to successfully trial
therapeutics targeting the SARS-CoV-2 reservoir in long
COVID, teams should recruit or analyse those patients
who have persistent virus. The recruitment of such
patients is not straightforward for the reasons outlined
above.
In general, there are two approaches to testing drugs
targeting the SARS-CoV-2 reservoir. Ideally, investigators
would recruit only participants confirmed to harbour a
SARS-CoV-2 reservoir and assess changes in reservoir
measures before and after the intervention. For example,
one trial of larazotide, a synthetic peptide regulator of gut
permeability, for long COVID requires confirmation of
spike antigenemia for eligibility (NCT05747534). Due to
variability in plasma protein detection (10–60%), and
questions about assay sensitivity, anywhere from
www.thelancet.com/infection Published online February 10, 2025 https://doi.org/10.1016/S1473-3099(24)00769-2
and Systems Immunity
Research Institute
(Prof D A Price), Cardiff
University School of Medicine,
University Hospital of Wales,
Cardiff, UK; Gladstone
Institutes (Prof N R Roan PhD)
and Department of Urology
(Prof N R Roan), University of
California, San Francisco, CA,
USA; Department of Infectious
Diseases, Institut Fournier,
Paris, France, (D Salmon MD);
Direction of International
Relations Assistance Publique
Hôpitaux de Paris, Paris, France
(D Salmon); Laboratory of
Clinical and Epidemiological
Virology, Rega Institute for
Medical Research, Department
of Microbiology, Immunology
and Transplantation, KU
Leuven, Leuven, Belgium
(J Van Weyenbergh PhD)
Correspondence to:
Dr Amy D Proal, PolyBio Research
Foundation, Medford,
MA 02155, USA
aproal@polybio.org
or
5
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Assist Prof Michael J Peluso,
Division of HIV, Infectious
Diseases, and Global Medicine,
University of California,
San Francisco, CA 94110, USA
michael.peluso@ucsf.edu
See Online for appendix
Biopsy
SARS-CoV-2 RNA and protein in the
numbered tissue reservoir sites can be
measured in samples collected via
biopsy.
Biopsy sites
1. Olfactory epithelium, tonsils, adenoids,
and salivary gland
2. Lungs
3. Lymph nodes
4. Gut
5. Endometrium
6. Muscle
7. Bone marrow
1
After appropriate tissue
processing and preservation, the
presence of RNA can be accessed
via hybridisation, sequencing, or
amplification approaches.
Assessment of viral replication,
for example via methods to
detect double-stranded RNA, can
be used to infer if active virus is
present.
6
3
2
Blood and other fluids
SARS-CoV-2 proteins in blood or other body fluids can be measured
via ultrasensitive single-molecule arrays. Protein measured via
these assays is likely translated by virus in tissue reservoir sites, and
leaks into the circulation via exosome transport where it can be
measured. However, even these assays may require further iteration
to improve their detection abilities.
7
5
4
Host cells
SARS-CoV-2 RNA and protein inside host cells, including cells of the immune
system (eg, monocytes, platelets, or megakaryocytes), can be measured via
methods such as digital transcriptomics or RNAScope probes. Analysis could
require collection of whole blood in which host cells are not depleted via
centrifugation as with plasma.
Figure 3: SARS-CoV-2 clinical trials persistence metrics
80 to 480 people might need to be screened to recruit the
sample size of 48 participants with spike antigenemia.
This effort might be warranted because the afore­
mentioned trial of nirmatrelvir–ritonavir44—which did
not reach its primary endpoint—did not have available
any validated or fit-for-purpose measure to inform
recruitment.43
An alternative, and more common, approach is to
recruit based on meeting an established case definition
for long COVID, and to store biospecimens for post-hoc
analyses focused on the reservoir. Orthogonal assays—
for example, using a combination of protein-based and
RNA-based measures in blood—can be used to assess
evidence of virus persistence at baseline, even if viral
persistence is not a key endpoint of the study. In such
cases, post-hoc analyses stratifying by the presence or
absence of viral persistence can be prespecified to
improve the validity of clinically significant findings.
This approach also allows for reanalysis of outcomes
should new persistence assays become available.
Depending on the proportion of people with long
COVID confirmed to have a viral reservoir, one might
not observe a benefit in the treatment group and might
only see an effect in the subgroup with persistence.
Investigators might consider calculating the sample
size needed to detect an effect assuming different
proportions (eg, 10%, 25%, and 50%) of participants
with SARS-CoV-2 persistence in the study cohort. This
calculation would inform the design and contextualise
the results.
6
Stratification by biological sex could also reveal subgroup
benefit. For example, a phase 2 study of RSLV-132, a
human RNase fused to IgG1 Fc that can degrade
extracellular RNA, was recently completed.47 Although the
primary endpoint of fatigue improvement at day 71 was
not met, earlier timepoints revealed statistically significant
improvement in fatigue. Importantly, women showed
greater responses to RSLV-132 than men, potentially
because TLR7, which detects viral RNA, is expressed at
higher concentrations in women.58
Combination trials
To date, most long-COVID trials have tested single
agents. If SARS-CoV-2 persistence is driven by active
replication, synergistic use of agents with different
mechanisms of action, including targeting viral proteins
at different phases of the viral life cycle (eg, viral proteases,
RNA-dependent RNA polymerase, or other proteins),
might be necessary. This possibility has been suggested
in acute COVID-19; although monotherapy is the current
standard, combining pyrimidine biosynthesis inhibitors
with antiviral nucleoside analogues synergistically
inhibits SARS-CoV-2 infection.59 Alter­natively, combining
direct-acting antivirals with mAbs could simultaneously
halt intracellular replication, enhance clearance of
infected cells, and neutralise extracellular virus and
protein. This approach needs to be tested directly.
Another consideration relates to the fact that the
relationships—that is, the connections and directionality—
between virus persistence and multiple other mechanisms
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Purpose of assay
Considerations
Quantitative PCR (real time or
droplet digital)
Quantitate the number of specific viral RNA target
sequences in blood, cell-free fluids, or bulk tissue
lysates
Nucleic acid sequence mutations might affect primer binding and
fragmentation of genetic material might lead to assay failure;
presence of RNA does not equate with active replication
Target-specific probe-based
assays
Examine specific viral RNA targets using probebased methods with or without concomitant
analysis of human gene transcription
Nucleic acid sequence mutations might affect primer binding and
fragmentation of genetic material might lead to assay failure;
presence of RNA does not equate with active replication; can also
multiplex assays to interrogate hundreds of viral and host targets in a
single reaction and set range of input RNA concentrations that can be
used at one time
In situ hybridisation
Examine the presence of the target in the native
tissue with high sensitivity
Contingent on tissue integrity and preservation; can target multiple
RNA regions simultaneously within a specific gene or subgenomic
region and provides spatial information, but background fluorescence
or non-specific binding or trapping of probe might complicate
interpretation
Digital spatial profiling
Examine specific viral RNA and human transcript
targets using either probe-based or less biased
RNA sequencing methods to provide spatial
information
Contingent on tissue integrity and preservation, can target multiple
RNA regions simultaneously within a specific gene or subgenomic
region and provides spatial information, but background fluorescence
or non-specific binding or trapping of probe might complicate
interpretation; some assays allow single-cell transcript resolution; low
throughput and data analysis is challenging
CRISPR-Cas target recognition
Direct detection of viral RNA that can allow
quantitative assessment of the extent of SARSCoV-2 persistence
Might need to be directed to the tissue where RNA persists
Metagenomic next-generation
sequencing
Unbiased method of identifying pathogen nucleic
acid fragments in tissue, cell-free fluids, or blood
Can be highly sensitive; RNA integrity might affect sensitivity, and
might pick up other pathogens present in sample (could also be prone
to environmental contamination)
Double-stranded RNA in situ
hybridisation
Suggests that some viral cycling is or has been
present
Target detection is not direct proof of replication
Viral outgrowth assays
Provide direct proof that virus is capable of
replication
Labour intensive; might be insensitive depending on location of
sample, and might not work on tissue samples; does not prove that
virus is replicating in vivo
Viral sequencing
Examine evolution and mutation
Detection of new viral mutations suggestive of ongoing replication
ELISA, single-molecule array
Detect the presence of viral proteins, which are
likely to be more stable than genetic material
Sensitivity uncertain; non-specific binding might lead to false-positive
signal, and interference by endogenous IgG or immune complexes
Immunohistochemistry
Detect the presence of viral proteins in relation to
tissue structure
Contingent on tissue integrity, background fluorescence, and nonspecific binding or trapping of probe
Nucleic acid
Viral replication
Viral protein
Indirect methods
Binding or neutralising antibody Detect and quantify the humoral immune
assays
response to virus
Suggests viral persistence in the absence of direct measurement;
might be useful to track responses over time, but might be altered by
re-exposure, re-infection, or vaccination
Plasmablast responses
Distinguish ongoing immune responses from
previous and historical responses
Suggests viral persistence in the absence of direct measurement;
might be useful to track responses over time, but might be altered by
re-exposure, re-infection, or vaccination
Cellular immune responses
Assess activation state of immune cells as a
biosensor of ongoing viral activity in tissues
Suggests viral persistence in the absence of direct measurement;
might be useful to track responses over time, but might be altered by
re-exposure, re-infection, or vaccination
Identify tissue location of viral persistence or
immune response to infection
Technologies in development but not yet available in clinical practice;
does not prove that virus is replicating in vivo
In vivo methods
Imaging with novel PET
radiotracers
Table 3: Potential methods for identification and characterisation of the SARS-CoV-2 reservoir
proposed to be important in long COVID have yet to be
confirmed. Perhaps a dysfunctional immune system,
driven by the reactivation of Epstein–Barr virus,60
fibrin formation,61 or microbiome perturbations, allows
SARS-CoV-2 to persist while simultaneously causing long
COVID. In such a case, addressing the primary insult, or
addressing multiple mechanisms simul­
taneously (eg,
a SARS-CoV-2 antiviral and a herpesvirus antiviral) might
be better than targeting the reservoir alone.
Combination trials to simultaneously address virus
persistence and dysregulated immunity might also be
important. There are multiple considerations for such
studies. The SARS-CoV-2 reservoir might persist in
immune-protected tissue microenvironments that are
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difficult to penetrate with single agents, or might require
recalibration of the immune system to address residual
virus. To address this issue in HIV cure research,
combination approaches have simultaneously targeted
the virus (with agents to reverse latency) and the immune
system (to improve innate and adaptive immune
responses) to reduce the reservoir or induce long-term
immunological control.62 Although the parallels are
imperfect, the scope of the challenge in addressing these
two reservoirs might be similar.
New takes on traditional randomised controlled trial
methodology, such as platform trials might also be useful.
Such approaches were taken for acute COVID-19 and are
being pursued for trials targeting Mycobacterium
tuberculosis.63 Properly conducted platform trials not only
allow testing of parallel combinations or sequences of
therapies, but as new information emerges these
experimental groups can be altered to incorporate the
success or failure of a particular intervention.64 This
approach is particularly useful in addressing conditions in
which the natural history is uncertain, in which effect sizes
and variance are not known, and for which new potential
therapies are expected to rapidly emerge; they also improve
efficiency by comparing multiple experimental treatments
against a shared control group.65–67
Duration and timing of treatment
The duration of treatment needed to clear the
SARS-CoV-2 reservoir is unknown. To date, the treatment
duration in reservoir-targeting trials has been limited to
the approved or authorised durations for acute COVID-19
(eg, short-course treatment); longer durations of recently
approved and authorised or novel agents would probably
require toxicology data in animals and humans. Most
antivirals have a short half-life, requiring dosing once or
twice per day, and generally do not maintain therapeutic
concentrations for more than a few days after
discontinuation. For antivirals that suppress replication,
subsequent immunological reactivity or spontaneous
turnover of infected cells would be necessary to clear the
reservoir. Although some cell types (eg, intestinal
epithelium) have higher turnover, other potential
target cells (eg, tissue macrophages and neurons) have
much longer lifespans and limited turnover. Agents
promoting immunological clearance of residual virus
(eg, mAbs) might have use with short courses. However,
mAbs can be modified to maintain therapeutic
concentrations for weeks or months, providing a longer
duration of therapeutic coverage with less frequent
administration. For example, the half-lives of mAbs can
be extended by LS mutation in their Fc portion, as
established in HIV.68
Ultimately, months of therapy or more might be
required, and early cessation of treatment might result in
symptom rebound after initial therapeutic benefit.69 For
example, previous studies suggest that 2 weeks of
nirmatrelvir–ritonavir treatment is insufficient, but
8
studies of longer-course therapy will be needed to
definitively address this issue. One trial (NCT05595369)
is testing 25 days of nirmatrelvir–ritonavir, but even this
might be insufficient. Treatment of hepatitis C, which
requires 8–12 weeks of antiviral treatment to achieve
eradication, is a good example of a case in which the
duration of treatment matters.70 The initial trials tested
24 weeks.71 In cats with feline infectious peritonitis—also
caused by a coronavirus—benefit has been shown with
12 weeks of treatment.72
It is also possible that eradication will not be achievable.
If some cells in which the SARS-CoV-2 reservoir persists
are long-lived, then maintaining durable suppression,
akin to antiretroviral therapy for HIV infection, might be
a more appropriate approach, even as the mechanism of
persistence is different.
Tissue penetration of the drug
Because the SARS-CoV-2 reservoir is probably tissuebased, any treatment targeting the reservoir needs to
reach and achieve adequate concentrations in the tissues
and cell types where the virus resides. Tissue penetration
of therapeutics has been an issue in HIV, with drug
penetrance differing among tissue reservoir sites.73
Although lung tissue concentrations and plasma
concentrations of many SARS-CoV-2 therapeutics have
been assessed, less is known about drug concentrations
in other potentially important anatomic reservoir spaces
such as the gut and lymph node. More research on this
front is needed, and trials might need to test different
drug doses to achieve optimal tissue penetration in some
body sites.
SARS-CoV-2 initially infects the respiratory epithelium
and studies have detected viral antigens in the gut,7,8,14
indicating that some component of the reservoir could be
localised in mucosal tissues. Therapeutics such as IgA
mAbs could consequently be directed to the mucosa or
designed to be delivered directly to mucosal sites—
eg, via a nasal spray.
Beyond mucosal sites, drug penetration into the CNS
might be essential, as several features of long COVID
(eg, prevalent neurocognitive symptoms74 and bio­
marker evidence of neurological inflammation75–77)
suggest that it could be a CNS-driven process in some
individuals. The CNS parenchyma is separated from
the periphery by the blood–brain barrier, which might
pose a challenge if CNS reservoirs exist because many
drugs cannot efficiently cross the intact blood–brain
barrier.
Evolving SARS-CoV-2 variants
Case reports of individuals with long COVID returning
to health following infusion of mAbs have increased
interest in this therapeutic modality.78 However, given
that individuals often do not know what variants they
harbour, effectively matching mAbs to SARS-CoV-2
variants present in a reservoir remains difficult.
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Monoclonals targeting the ancestral, delta, and early
omicron variants were previously approved. Trials can be
designed to target their use to specific patient populations.
For example, a US-based trial using a mAb therapy that
has activity against all variants that circulated in the USA
through Fall 2022 specifically recruited participants with
long COVID attributed to infection before that time
(NCT05877508). There is an urgent need to test other
mAbs, including both those targeting earlier variants and
those recently authorised for prevention, in long COVID.
It is unclear if long-COVID cases stemming from more
recent infections with variants that are not efficiently
neutralised by previous mAb therapy would benefit from
these treatments. A potentially complicating question is
whether so-called mixed reservoirs can exist—that is,
whether each subsequent infection also contributes to
the reservoir. In such a case, only broad-spectrum
therapeutics might be expected to work, and each
subsequent infection could increase the complexity of
treatment and diminish the likelihood of mAb treatment
success. Efforts are underway to develop broadly
neutralising SARS-CoV-2 mAbs targeting conserved
epitopes, which could hold promise in targeting potential
mixed reservoirs and prospective long-COVID cases.
Another concern is that reservoir persistence could be
driven by viral mutation and escape from immune
surveillance. Studies of chronic SARS-CoV-2 infection
in immunocompromised hosts provide proof-ofprinciple.79,80 This phenomenon requires further study in
immunocompetent individuals. Indeed, several RNA
viruses have been shown to persist via mutation, for
example via selection of less lytic viral variants to persist.40
If such evolution occurs, agents with activity only against
the initial infecting variant would likely fail. In addition,
some therapeutics could put selection pressure on the
virus.81 Determining if those with long COVID exhibit
viral evolution over the course of reservoir-targeting trials
is therefore likely to be informative. This potential
evolution could be assessed by viral genomic sequencing
before and after intervention, to provide insight into
compartmentalised virus populations, resistance to
treatment, and viral adaptation in different tissues.3,82
Blinding and the placebo effect
Clinical trials can be fraught with confounding,
necessitating the incorporation of rigorous design
features, including blinding and the use of a placebo.
Blinding can be challenging because some SARS-CoV-2
antivirals have clear and distinct side-effects, which
might inadvertently unblind the study (eg, dysgeusia
with nirmatrelvir–ritonavir). Recent clinical trials of long
COVID also revealed a strong placebo effect.44,47,83 Given
the challenges in defining the condition, its heterogeneity,
and the absence of persistence measurables, inclusion of
a placebo is currently essential for most trials. However,
there might be specific situations in which a single-arm
study without a placebo group is appropriate—for
example, in studies with pronounced and distinct
anticipated side-effects (eg, some immunotherapies) it
might not be possible to blind, as discussed earlier. In
such instances, it might be justifiable to conduct a singlearm study and set a threshold for a particular effect size,
but in general this approach should be kept to small
signal-finding studies or studies seeking to identify a
large effect.
Safety
In general, many drugs targeting the SARS-CoV-2
reservoir—either via activity against the virus or the
immune system—are likely to be safe and well tolerated.
Safety, however, must remain a key consideration,
especially in individuals with long COVID, where use of
these drugs has been limited to date. The adverse effects
of some drugs might differ between acute and long
COVID, and it is important to understand the effect of
these agents on pre-existing long-COVID symptoms and
other side-effects. This assessment is particularly
challenging concerning symptoms such as postexertional malaise,42 which can be exacerbated by study
participation.
Some agents (eg, protease inhibitors) also have
considerable drug–drug interactions, which is becoming
increasingly challenging, because many patients with
long COVID are on multiple medications to manage
symptoms. In some cases, discontinuing a medication in
anticipation of a clinical trial could exacerbate symptoms,
increasing the risk to the participant and making it
difficult to interpret the results of the study. Active
placebo groups have been used to address this in some
(NCT05576662 and NCT05595369) but not all
(NCT06161688) studies.
Immunotherapies targeting the reservoir might also
have challenges. For example, agents such as interleukin
superagonists and checkpoint inhibitors have well
known side-effect profiles, such as skin reactions,
inflammation, and other immune-related adverse events.
Careful monitoring for immune-related adverse events,
including autoimmunity, should be incorporated into the
study design. Because some immunotherapy drugs
trigger strong immune responses, these agents might
also cause a participant to feel worse before feeling better.
We can draw from experience gained in cancer and HIV
trials to help to manage these issues. For example, initial
data from studies of low-dose checkpoint inhibition in
HIV infection have shown that such treatment is
tolerable, and adverse effects manageable, in that
condition.84
Additionally, it is possible that the dose or duration of
immunomodulators in long-COVID trials might not
need to be as high or as long as those used in cancer
treatment, which could ameliorate the risks for adverse
events. Strategies to determine the minimum effective
dose that can stimulate an immune response should be
explored, with potentially lower thresholds than for other
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diseases. For example, combination trials with antivirals
and mAbs as a backbone therapy with lower doses of
immunomodulators as shorter cycles could be explored,
which could lower the risks for adverse events.
On-study reinfections
A major challenge for conducting SARS-CoV-2 reservoir
trials during an ongoing airborne pandemic is that many
participants might be re-exposed or reinfected during the
study, especially as public health restrictions are no
longer in place and the virus continues to spread.
Although reinfection poses challenges for all studies
focused on long-COVID symptoms, it is particularly
problematic for those testing the effect of a treatment on
SARS-CoV-2 reservoir measures, as reinfection can
potentially alter this outcome.
These issues are largely unavoidable. Investigators
designing studies might consider emphasising the
importance of testing, offering tests to symptomatic
participants, periodic testing even if participants are
asymptomatic, and including at least one endpoint early
in the study to ensure that at least some data and
specimens are collected before a reinfection occurs. For
example, a study with a primary endpoint at 90 days
following intervention might include an interim
assessment at 30 days, to increase the likelihood that at
least some assessments can occur before a reinfection.
Investigators should also include in the analysis plan an
approach for handling data (eg, censoring) or replacing
participants who are reinfected before the primary
endpoint. Although this issue will be less problematic in
large, randomised studies, it will continue to pose a
challenge for phase 2 or phase 3 studies with smaller
sample sizes.
Other clinical endpoints
Questions abound regarding assessment of clinical
outcomes in long-COVID trials targeting the SARSCoV-2 reservoir. Many have used a combination of
patient-reported and objective clinical endpoints. A
common toolbox of measurables would be a great
resource for the field. Wherever possible, trials should
strive to use common instruments to allow direct
comparisons between studies. Discussion of these
endpoints is beyond the scope of this Personal View but
should be a focus of future work.
Once endpoints are determined, there remain
challenges concerning their interpretation. This set of
concerns is especially true in smaller studies, which,
although crucial in parsing out the biological effects of
interventions targeting the SARS-CoV-2 reservoir, might
not be powered around clinical endpoints. To address
this issue, we recommend considering an approach
taken in many phase 2 cancer studies—the so-called
effect of interest. In such cases, the investigators set a
proportion at which the effect is thought to be negligible
(eg, 10% of participants experience a benefit, which
10
might be consistent with rates of expected improvement
in an untreated population) and then determine whether
there is an effect of interest in the intervention group—
whether the response rate in that group is statistically
different from the prespecified negligible effect.
It is also possible that agents tested in these trials
might have pleiotropic effects. For example, a monoclonal
antibody might enhance clearance of viral reservoirs, but
also modulate immune or inflammatory responses. If a
clinical benefit is observed, further work to identify the
mechanism driving the benefit will be crucial and will
help in the design of future trials to optimise treatment
approaches.
Other considerations
Prevention of long COVID
Thus far, most clinical trials of long COVID have focused
on treatment of established disease, and few studies have
connected events during acute infection to long-term
sequelae. The prevention agenda is equally important
and should be emphasised in the coming years, especially
as SARS-CoV-2 continues to spread and cause
reinfections. To that end, acute-phase interventions
should be studied for their effect on SARS-CoV-2
clearance and persistence. It remains possible, for
example, that minimising the viral burden in the days or
weeks following initial infection could alter the
establishment of the SARS-CoV-2 reservoir and mitigate
downstream consequences.28,29,33,85
Data on post-acute complications have been reported
from trials of some antivirals,86 either as a secondary
outcome80 or as post-hoc analyses comparing those who
received interventions against those who did not. In
several studies, treatment during acute infection reduced
long-COVID risk.34–36 Consequently, we advocate for a
research agenda focused on long-COVID prevention:
interventions during the acute phase of COVID-19 with a
focus on post-acute viral persistence as a primary
outcome to determine whether they can prevent the
formation of a SARS-CoV-2 reservoir. These approaches
could include trials testing longer durations of acute
treatment courses. Although studies would need to be
large, based on what we know about the incidence of
long COVID, they can and should be conducted.
Identifying a reduction in long-term virus persistence
would provide strong rationale for early treatment, even
without a clear reduction of symptom duration in the
acute phase.
Industry considerations
A challenge for long-COVID reservoir trials is that
pharmaceutical partners, who develop and have access to
drug products, have, to date, been unwilling or unable to
take on the financial cost of these studies. Some
pharmaceutical companies perceive risk in the space—
not only costs of engagement, but also risks of
jeopardising planned development pipelines in the event
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of studies that do not achieve the primary endpoint or in
which a serious adverse event occurs, especially when a
drug has been developed for another indication.
Although many small biotech companies are less riskaverse, they frequently do not have the resources for
drug development. Government and private funding
mechanisms focused on de-risking the long-COVID
reservoir trial space should consequently be a major
priority.
A similar situation existed for HIV in the late 1980s
and early 1990s. Efforts to de-risk the drug development
space via a combination of government and private
funding made multiple industry stakeholders more
willing to engage. There are now over 25 approved drugs
for HIV infection. The development of these treatments
rapidly transformed HIV from a life-threatening illness
to a manageable chronic condition over the course of a
few years.
Conclusion
Long COVID is one of many conditions in which patients
develop chronic symptoms following an acute infection.5
Other infection-associated chronic conditions in which
persistence of an initiating pathogen might be relevant
include chronic or post-treatment Lyme disease,88
myalgic encephalomyelitis/chronic fatigue syndrome,89
post-Ebola syndrome,31 and post-dengue fatigue
syndrome. Knowledge gained on mechanisms of
SARS-CoV-2 persistence and the lessons learned from
designing and implementing long-COVID reservoir
trials can provide proof-of-concept for these related
conditions and provide strong rationale for what
pathways might be pursued. Ultimately, efforts to target
the SARS-CoV-2 reservoir—a concept that did not exist at
the start of the pandemic but is now established as a
major area of investigation—have the potential to
transform our understanding of not just long COVID,
but also pre-2019 and future infection-associated chronic
conditions.
Contributors
ADP and MJP wrote the initial draft of the manuscript and helped
conceive of the figures and tables. MBV developed drafts of the figures,
which were edited substantially by SC and NRR. All authors made
substantial contributions to writing and editing the manuscript, and
approved the final version for publication.
Declaration of interests
ADP reports a leadership role at PolyBio Research Foundation.
SA reports research support from Gilead Sciences outside the submitted
work and reimbursement for participation on Gilead’s advisory board.
MB reports consulting fees or payment honoraria for lectures from
Bristol Myers Squibb, Mabtech, Pfizer, Gilead, and MSD outside of the
submitted work, and participation on an advisory board for Mabtech.
SGD reports research support from Aerium and Eli Lilly outside the
submitted work and consulting for BioVie, Enanta Pharmaceuticals, and
Pfizer. LNG reports research support from Pfizer outside the submitted
work. AF serves as Chief Science and Medical Officer at Mead Johnson
Nutrition. DEG reports research support from Gilead Sciences outside
the submitted work and the leadership role of vice president at the
National Academy of Sciences. AI reports being a member of the board
of directors for Roche Holding and Genentech, being member of the
Council for American Association of Immunologists, and being
co-founder and stockholder of RIGImmune, Xanadu Bio, and PanV.
In the past three years, HMK has received options for Element Science
and Identifeye and payments from F-Prime for advisory roles. He is a
cofounder of and holds equity in Hugo Health, Refactor Health, and
ENSIGHT-AI. He is associated with research contracts through Yale
University from Janssen, Kenvue, Novartis, and Pfizer. VCM reports
research support from Gilead Sciences, ViiV, CDC, NIH, Department of
Veterans Affairs, and Merck outside the submitted work, participation
on data safety monitoring or advisory boards for the IL-1b inhibitor
study, Outsmart, CLEAR HIV, ECLIPSE, and VB201, and role of study
section chair for NIH. YQ reports consulting for Moderna. DS reports
honoraria for conferences on long COVID for Pfizer, Gilead, and
Shionogi. EJW reports being an advisor for Arsenal Biosciences,
Coherus, Danger Bio, IpiNovyx, New Limit, Marengo, Pluto
Immunotherapeutics, Related Sciences, Santa Ana Bio, and Synthekine;
and being a founder of and holding stock in Coherus, Danger Bio, and
Arsenal Biosciences. MBV reports a leadership role at PolyBio Research
Foundation. JVW reports a speaker fee from Pfizer. MJP reports
consulting for Gilead Sciences, BioVie, and AstraZeneca; and receipt of
study drug from Aerium Therapeutics and Shionogi. All other authors
declare no competing interests.
Acknowledgments
We thank all members of the PolyBio Research Foundation-supported
LongCovid Research Consortium for helpful discussions and critical
analysis of the manuscript. PolyBio Research Foundation thanks its
generous donors including Kanro and the Silver Givings Foundation for
their support of the work. We thank the Steve & Alexandra Cohen
Foundation for its support. We thank John CW Carroll for help with
figure illustration and design. MJP is supported by NIH/NIAID
K23AI157875 and NIH/NINDSR01NS136197. This work was in part
supported by the intramural programmes of the National Institutes of
Health Clinical Center, National Cancer Institute, and National Institute
of Allergy and Infectious Diseases. This work was in part supported by
the Emory Center for AIDS Research (P30AI050409). We would like to
acknowledge our coauthor Diane E Griffin, who passed away in late
2024. Her expertise was crucial in guiding the development of the new
field of SARS-CoV-2 persistence. She is greatly missed as a colleague,
mentor, and friend.
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