A composite CoR: Antibody dynamics, serology in practice and
challenges, and expert
recommendations
The antibody component of a composite CoR should be developed under
defined conditions. To provide insight into these conditions, an
understanding of antibody dynamics is required.
SARS-CoV-2 antibody dynamics
Natural infection with SARS-CoV-2 elicits a diversity of antibodies
including those targeting S and nucleocapsid (N) antigens (59,92) and
the development of anti-RBD IgG antibodies is associated with improved
patient survival (93). A detailed systematic review of 66 studies
investigated antibody responses (94). Collectively, the evidence
supports the induction of IgM production in the acute phase of natural
infection (peak prevalence: 20 days) followed by IgA (peak prevalence:
23 days), IgG (peak prevalence: 25 days), and nAbs (peak prevalence: 31
days) after symptom onset (94).
Serum IgG has the longest half-life compared with the relatively
transient IgA or IgM (95). A longitudinal analysis of 4558 individuals,
measuring total anti-N antibodies, revealed that, whilst total
antibodies begin to decline after 90–100 days, they may persist for
over 500 days after natural infection (96). Specifically measuring nAb
via plaque reduction neutralization test (PRNT) shows that infection
yields a robust nAb response in most individuals (67). Some studies
report that anti-S antibodies show greater persistence than anti-N
antibodies (97,98).
Dramatic inductions of anti-S or anti-RBD IgG antibodies is indicative
of vaccination (59,99,100). Primary vaccination by some vaccines, (but
not all (101)), or boosters generates high nAb titers (100,102,103) or
neutralizing responses (99). Notably, nAbs wane over time (21) with a
half-life of 108 days (81) – although the level of decay may be assay
or variant dependent (102) – and multiple clinical factors affect the
duration of neutralization responses after primary vaccination (66).
Anti-SARS-CoV-2 antibody
testing
Commercial high-throughput immunoassays
Numerous immunoassays for the detection of antibodies against SARS-CoV-2
are available, differing in the immunoglobulin class detected, target
viral antigen, format, and output (qualitative, [semi]-quantitative)
(reviewed in detail (104,105)).
Head-to-head comparisons from the pre-Omicron era reveal variable levels
of performance between the assays (106-110), caused by numerous
technical factors including assay methodology, format and antibodies
used, timing of testing, and the targeted viral antigen. Comparison
studies show that sensitivity for detecting prior infection by different
serologic assays changes over time (111). Commercial assays developed
early during the pandemic are based on ancestral/wild-type antigens.
Subsequently, there is potential for differential performance in the
Omicron-era: in particular, S- and RBD-specific immunoassays have shown
significantly reduced performance (112-114), and decreased comparability
of quantitative results (115).
Most common commercial immunoassays detect both binding and nAbs without
differentiating between them, however certain assays measuring IgG or
total antibodies correlate well with neutralizing capacity
(14,78,116-122), acting as surrogates of neutralization. Cell-based
virus neutralization tests can be used to measure neutralizing
capability, but these are typically not readily available in clinical
laboratories due to inherent test performance challenges associated with
their methodology, time and cost (123).
Expert recommendations
Mature immune responses are dominated by IgG. Serologic assays that
measure IgG or total antibodies (if skewed towards IgG) that correlate
with neutralizing activity and focus on anti-RBD should be used for the
serologic component of a composite CoR; anti-N antibodies are unlikely
to be neutralizing as the N protein is located within the viral envelope
(59).
Assays should be adapted for accurate measurement of the modified
antigen, if applicable. However, frequent adaptation of assays is
unlikely if several variants are circulating in parallel and due to
regulatory requirements for assays. Therefore, studies are needed to
determine assay applicability in the present conditions, especially
since RBD mutations frequently occur and recombinant versions of RBD or
S are commonly used in immunoassays (105). Accordingly, the upper and
lower thresholds of any CoR may need modification.
External ring trials show poor comparability of assays from different
manufacturers (124,125) and there are significant challenges with the
current binding antibody units (BAU) standardization, due to multiple
factors, including different assay methods, antibody class(es) detected
and target antigen used. Of note, BAU reference materials were derived
from UK convalescent individuals infected in 2020 (126) (pre-Omicron),
and there are vastly different BAU standardized values (Kroidl et al
2023, submitted). Antibody measurements should be harmonized across
assays from different manufacturers, irrespective of the different
epitopes utilized, to reduce variability. To support this, there is an
urgent need for external quality assessment, production of robust
traceable certified reference materials, standards for different
variants, and improved documentation of the methods on laboratory
reports. Age-specific normalization of reference intervals in defined
groups, by means of z-log transformation and documentation in antibody
passes, may further improve the comparability of assays. Stakeholders
should agree on minimum performance-based criteria to develop the gold
standard for CoR, allowing validation of secondary assays.
Finally, systemic cellular assays could provide a comprehensive profile
of the immune response, especially in immunocompromised and susceptible
individuals who are not able to mount a robust antibody response.
Currently, they lack scientific evidence and their use in clinical
practice still remains uncertain.
Sample matrices
Systemic anti-SARS-CoV-2 antibody testing can be performed on blood,
plasma/serum, or dried blood spots (DBS) (105,127,128); Wieser et al
2023, submitted). An advantage of whole blood or DBS collection is the
ease in obtaining the sample. Whilst many methodologies focus on
systemic testing, infection with SARS-CoV-2 or vaccination against
COVID-19 induces mucosal antibodies (129,130), thus secretions such as
saliva offer another possibility. Antibody dynamics will differ
depending on the material in question (131), and sample types are
subject to specific idiosyncrasies, such as additional pre-processing,
that need to be accounted for (132). Currently secretion-based testing
is less suitable for a composite CoR as performance is variable (133).
Expert recommendations
A composite CoR will likely be sample matrix-specific. Our preference is
for plasma/serum, as this sample matrix has the largest evidence base,
shows the least variability, experiences less interference than whole
blood, and is consistent with CoRs established for other infectious
diseases. DBS would be also possible, but variability is high, and few
laboratories have an established workflow.
Serologic testing
formats
Formats include high-throughput automated enzyme immunoassay/
electrochemiluminescence immunoassay/enzyme-linked immunosorbent assay
(certified and used in central laboratories and hospitals),
point-of-care (POC) testing (used in emergencies and outpatients
setting), and direct-to-consumer testing (at-home use with online
services). POC testing is gaining in popularity, but methodological
variation is higher (134) and any method that relies upon sampling from
untrained individuals is less reliable for (semi)quantitative
measurements (135).
Expert recommendations
We recommend automated assays that are approved by location-specific
regulatory agencies and performed in certified and centralized
laboratories. Home sampling/DBS would contribute to a reduction in
clinician workload, particularly in high-density residential facilities,
but methods are not yet sufficiently robust. At this time, there is no
clear benefit in POC testing as urgent results are not critical.
Frequency of sampling and optimal time point
Considering antibody dynamics, several important questions arise: what
is the optimal time point for measurement; would the timing differ
depending on the vaccine schedule, and/or the presence of previous
infection of a specified severity; should antibody levels be measured
once or serially? Whilst single values can be plotted into modelled
curves showing decrease rates over time, serial measurements could
further refine the composite CoR. Only individuals with symptomatic
disease or vaccination are known to stabilise the curve — infections
that are sufficiently mild to lack detection will impact the composite
CoR model.
Expert recommendations
As most individuals have experienced infection or vaccination, and
titers are generally high and more stable than with single exposures,
sampling should be performed annually or less. Serologic evaluation
should be conducted more frequently in the elderly or immunocompromised
than the general population (time interval to be defined), depending on
any underlying disease and/or treatment.