Critical Reviews in Clinical Laboratory Sciences

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The COVID-19 pandemic: viral variants and vaccine

efficacy

Marco Ciotti, Massimo Ciccozzi, Massimo Pieri & Sergio Bernardini

To cite this article: Marco Ciotti, Massimo Ciccozzi, Massimo Pieri & Sergio Bernardini (2022) The
COVID-19 pandemic: viral variants and vaccine efficacy, Critical Reviews in Clinical Laboratory
Sciences, 59:1, 66-75, DOI: 10.1080/10408363.2021.1979462

To link to this article: https://doi.org/10.1080/10408363.2021.1979462

Published online: 01 Oct 2021.

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CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES
2022, VOL. 59, NO. 1, 66–75
https://doi.org/10.1080/10408363.2021.1979462

INVITED REVIEW

The COVID-19 pandemic: viral variants and vaccine efficacy

Marco Ciottia , Massimo Ciccozzib , Massimo Pieric,d and Sergio Bernardinic,e
a
Virology Unit, Polyclinic Tor Vergata Foundation, Rome, Italy; bUnit of Medical Statistics and Molecular Epidemiology, Campus
Bio-Medico of Rome, Rome, Italy; cDepartment of Experimental Medicine, University of Tor Vergata, Rome, Italy; dDepartment of
Laboratory Medicine, Polyclinic Tor Vergata Foundation, Viale Oxford, Rome, Italy; eEmerging Technologies Division (ETD) of the
International Federation Clinical Chemistry and Laboratory Medicine (IFCC), Milan, Italy

ABSTRACT ARTICLE HISTORY

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has prompted the Received 2 July 2021
scientific community and the pharmaceutical companies to put maximum efforts into developing Revised 15 August 2021
vaccines to contain the spread of this disease. Presently, many vaccines have been developed Accepted 8 September 2021
and authorized for use in human beings in different countries. In particular, in Europe to date,
KEYWORDS
the Pfizer-BioNTech, Moderna, AstraZeneca and Janssen COVID-19 vaccines have been author- SARS-CoV-2; vaccine anti-
ized. All of them are based on a version of the spike (S) glycoprotein characterized at the begin- SARS-CoV-2; SARS-CoV-2
ning of the pandemic. However, they differ by their level of efficacy against COVID-19. SARS- variants; COVID-19
COV-2, like other RNA viruses, mutates continually. Genome sequencing analysis shows a nucleo-
tide substitution rate of about 1
103 substitutions per year that leads to the emergence of
variants through point mutations, insertions, deletions and recombination. There is concern
about the ability of the current vaccines to protect against emerging viral variants. Mutations in
the S-glycoprotein may affect transmission dynamics and the risk of immune escape. In this
review, we address the different technological platforms in use for developing COVID-19 vac-
cines, the impact of emerging viral variants on virus transmission, hospitalization, and response
to current vaccines, as well as rare but important adverse reactions to them. Finally, different
methods for measuring antibody response to the vaccines, including the importance of using
the WHO International Standard to calibrate immunoassays accurately to an arbitrary unit, to
reduce interlaboratory variation and to create a common language for reporting results,
are reported.

Abbreviations: ACE2: angiotensin converting enzyme 2; COVID-19: coronavirus disease 19;

CoVLP: coronavirus-like-particles; EMA: European Agency of Medicine; FDA: Food and Drug
Administration; N: nucleocapsid; PF4: platelet factor 4; PRNT: plaque reduction neutralization test;
rAd: recombinant adenovirus; RBD: receptor binding domain; SARS-CoV-2: severe acute respira-
tory syndrome coronavirus 2; S: spike; VOC: variant of concern; VOI: variant of interest; WHO:
World Health Organization

Introduction in the pre-clinical stage [https://www.who.int/publica-

Since its beginning, the coronavirus disease 19 (COVID-
19) pandemic has caused millions of deaths worldwide
and an unprecedented crack in national health systems
and the global economy. Therefore, it has been impera-
tive for the scientific community, governments and the
pharmaceutical industry to develop a vaccine able to
contain the spread of severe acute respiratory syn-
drome coronavirus 2 (SARS-CoV-2) responsible for the
pandemic. In less than one year, several vaccines
have been approved and others are under
evaluation or in development. At the time of writing,
91 vaccines are in clinical development and 184 are
tions/m/item/draft-landscape-of-COVID-19-candidate-
vaccines]. The European Agency of Medicine (EMA) has
authorized four vaccines for use within the European
Union: Cominarty (BioNTech Manufacturing GmbH,
Mainz, Germany), COVID-19 Vaccine Moderna (Moderna
Biotech Spain, S.L.; Moderna Inc, Cambridge, MA, USA),
Vaxzevria (previously named COVID-19 Vaccine
AstraZeneca, AstraZeneca AB, Cambridge, UK), and
COVID-19 Vaccine Jansenn (Janssen-Cilag International
NV, Titusville, NJ, USA). Three are under rolling review,
CVnCoV (CureVac AG, Tubingen, Germany), NVX-
CoV2373 (Novavax CZ AS, Gaithersburg, MD, USA),

CONTACT Sergio Bernardini bernardini@med.uniroma2.it Department of Experimental Medicine, University of Tor Vergata, Via Montpellier 1, Rome
00133, Italy
ß 2021 Informa UK Limited, trading as Taylor & Francis Group
 CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 67

Sputnik V (Gam-COVID-Vac, Gamaleya National Center
of Epidemiology and Microbiology, Moscow, Russia),
and 52 clinical trials on COVID-19 and vaccines are
ongoing [https://www.clinicaltrialsregister.eu/ctr-search
/search?query=covid-19+AND+vaccine].
Different technological approaches have been used
for developing vaccines against SARS-CoV-2, an RNA
virus. RNA viruses are characterized by genetic instabil-
ity that make it challenging to develop an effective vac-
cine, although the replication machinery of coronavirus
is characterized by a higher fidelity rate than other RNA
viruses. To date, no effective vaccines are available for
hepatitis C virus or human immunodeficiency virus-1.
The present COVID-19 vaccines are based on the
expression of SARS-CoV-2 S-glycoprotein, which the
virus uses to bind to the angiotensin converting
enzyme 2 (ACE2) receptor and infect the host cells.
Based on the available data, the humoral immune
response against the S-protein, and in particular against
the receptor binding domain (RDB) of the virus, appears
to be the most protective. In patients who have recov-
ered from COVID-19, neutralizing antibodies are usually
present at high titer, and convalescent plasma has been
administered to critically ill patients with positive
effects on their clinical status [1]. However, the duration
of protective immunity and the role played by mucosal
immunity in fighting SARS-CoV-2 infection are
unknown. Indeed, secretory IgAs, together with IgM
and IgG antibodies, are rapidly formed during SARS-
CoV-2 infection [2,3]. Thus, the development of an
effective vaccine able to induce durable immunity is
key to containing the pandemic. Another important
concern is the protection against the variants offered
by the current vaccines.
In this review, we describe the different technological
platforms the vaccines are based on and the current evi-
dence of SARS-CoV-2 variants on infection, transmission
and hospitalization. Furthermore, the major serological
assays used for measuring antibody production after
vaccination, as well as the importance of their standard-
ization based on the World Health Organization (WHO)
International Standard, are described.
Technological platforms
Different technological approaches have been used for
the development of the current 91 vaccines in clinical
trials protein subunit, viral vector (replicating and non-
replicating), DNA, inactivated viruses, RNA, virus like
particle, viral vector replicating þ antigen presenting
cell (APC), live attenuated virus and viral vector non-
replicating þ APC. Sixteen of these candidate vaccines
are in phase 3 trials [https://www.who.int/publications/
m/item/draft-landscape-of-COVID-19-candidate-vac-
cines]. In the following sections, we describe the
technological platforms used for vaccines already
approved or under evaluation for approval (Table 1).
Vaccines based on protein subunit
Novavax vaccine (Novavax, Gaithersburg, MD, USA)
The NVX-CoV2373 vaccine by Novavax is based on
the full-length prefusion S-protein made using recom-
binant nanoparticles technology and saponin-based

Table 1. Platforms used for the development of vaccine candidates.

Platform Company Phase 3 Efficacy (%) Dose Storage ( C)
Protein subunit Novavax NCT04636697 60–89 2 (21-day interval) 2–8
GSK-Medicago NCT04636697 Unknown 2 (21-day interval) 2–8
Anhui Zhifeilongkoma ChiCTR2000040153 ? ? ?
Biopharmaceutical/
Chinese Academy
of Sciences
Viral vector AstraZeneca NCT04324606, 62–90 2 (28-day interval) 2–8
NCT04400838, and
NCT04444674
Gameleya NCT04530396 91.6 2 (21-day interval) –18,
Johnson & Johnson NCT04505722 66–85.4 1 2–8
CanSinoBio NCT04526990 ? 1 2–8
DNA Anges NCT04655625 ? 2 (14- and 28- Room temperature
day interval)
Inovio NCT04642638 ? 2 (28- day interval) Room temperature
Inactivated Sinopharm NCT04510207 79 2 (21-day interval) 2–8
viruses 73 (WIV04)
78 (HB02)
Chinese Academy NCT04659239 ? 2 (14-day interval) ?
Medical Science
Sinovac NCT04582344 50 2 (14-day interval) 2–8
Bharat Biotech NCT04641481 2 (28 days apart) 2–8
mRNA Pfizer/BioNTech NCT04368728 95 2 (21 days apart) 70
Moderna NCT04470427 94 2 (28 days apart) 20
CureVac NCT04652102 47 2 (28 days apart) 2–8
68 M. CIOTTI ET AL.
Matrix-MTM adjuvant that enhances antigen presenta-
tion in local lymph nodes, boosting the immune
response, and induces the response of neutralizing anti-
bodies. NVX-CoV2373 contains the purified protein anti-
gen, which is produced in insect cells. The vaccine
showed an efficacy of 60–83% in the phase 3 clinical
trial. It is stable at 2–8  C, and is available in a ready-to-
use liquid formulation [https://ir.novavax.com/news-
releases/news-release-details/novavax-covid-19-vaccine-
demonstrates-893-efficacy-uk-phase-3]. The vaccine is
administered in 2 doses 21 days apart.
GSK-Medicago vaccine (Quebec City, Canada)
Medicago’s plant-derived vaccine candidate uses cor-
onavirus-like-particles (CoVLP) technology. The vaccine
contains a recombinant S-protein expressed as a virus-
like particle administered with GSK’s pandemic adju-
vant. The vaccine is administered in 2 doses
21 days apart.
Vaccine of Anhui Zhifeilongkoma Biopharmaceutical
Co., Ltd. and Institute of Microbiology, Chinese
Academy of Sciences (Beijing, China)
This group is developing a vaccine based on the recom-
binant RBD. No data is currently available regarding
vaccine efficacy, dose and storage conditions.
Vaccine based on viral vector
AstraZeneca vaccine (Cambridge, UK)
The ChAdOx1 nCoV-19 vaccine, AZD1222 uses a replica-
tion deficient chimpanzee adenoviral vector ChAdOx1
that contains the S-gene of SARS-CoV-2 encoding for
the surface S-glycoprotein. Interim analysis from four
studies performed in United Kingdom, Brazil and South
Africa showed an acceptable safety profile and a vac-
cine efficacy ranging from 62.1% (participants vacci-
nated with two standard doses) to 90% (participants
who received a low dose followed by a standard dose).
Overall, the efficacy in both groups was 70.4% [4].
Sputnik V vaccine (Gamaleya National Center of
Epidemiology and Microbiology, Moscow, Russia)
The Sputnik V vaccine (Gam-COVID-Vac) is a recombin-
ant adenovirus (rAd)-based vaccine that uses replication
deficient rAd type 26 and rAd type 5 containing the
genetic material encoding for the full-length SARS-CoV-
2 S-glycoprotein [5]. The use of the adenoviral vector
may stimulate an immune response against parts of the
vector and lessen the response induced by the antigen.
This event has been largely attenuated with the strat-
egy of prime-boost heterologous vaccination with two
different vectors. rAd26 is administered first followed
by injection of rAd5 after 21 days. Gam-COVID-Vac
showed an efficacy of 91.6% against COVID-19 based
on the results of the phase 3 trial [5].
Johnson & Johnson/Janssen vaccine (Johnson &
Johnson/Janssen, Titusville, NJ, USA)
Janssen used the proprietary AdVacV R viral vector tech-
nology to develop its vaccine against COVID-19. The
same technology was used for developing the vaccine
against Ebola virus disease (Ad26 ZEBOV and MVN-BN-
Filo). The Ad26.COV2.S vaccine is based on a recombin-
ant replication incompetent Adv type 26 that carries
genetic material encoding for the whole S-protein of
SARS-CoV-2 in a prefusion-stabilized conformation. The
vaccine is 66% effective in preventing both moderate
and severe COVID-19 28 days after vaccine administra-
tion, including in individuals infected with the viral vari-
ant 20H/501Y.V2 (64% efficacy). It was higher against
severe-critical COVID-19 (85.4%) for onset at  28 days
[6]. The vaccine is administered as a single injection,
and it is stored at refrigerator temperature.
CanSinoBio vaccine (CanSinoBio inc., Tianjin, China)
CanSinoBio is developing a vaccine against COVID-19
using a recombinant Adv type 5 vector containing gen-
etic material encoding for the SARS-CoV-2 S-protein.
The vaccine is given as a single shot and is expected to
induce an immune response against the S-RBD of SARS-
CoV-2 [https://clinicaltrials.gov/ct2/show/NCT04526990].
Vaccine based on DNA
Anges vaccine (Anges Company, Osaka, Japan)
This company is developing a vaccine against COVID-19
using plasmid DNA technology expressing the S-protein
of SARS-CoV-2. The plasmid can be produced in E. coli
in large amounts. Five-hundred participants have been
enrolled and divided in two groups, A and B, with part
of each group being a control group. The vaccine is
administered in two doses at a 14-day interval for
group A and at a 28-day interval for group B. Antibody
response against the S-protein will be measured
[https://clinicaltrials.gov/ct2/show/NCT04655625].
Inovio vaccine (Inovio, CA, USA)
Inovio has developed a plasmid-based DNA vaccine,
INO-4800, expressing the S-protein of SARS-CoV-2. The
vaccine is administered to subjects at high risk of
exposure to SARS-CoV-2 by intradermal injection fol-
lowed by electroporation with the CELLECTRAV R 2000
device (Inovio, CA, USA) [https://clinicaltrials.gov/ct2/

show/NCT04642638]. INO-4800 is administered in two
doses at a 28-day interval.
Vaccine based on inactivated viruses
Sinopharm vaccine (Sinopharm, Beijing, China)
The Sinopharm anti-COVID-19 vaccine is based on inac-
tivated SARS-CoV-2 and is produced in Vero cells.
According to the immunization schedule, the vaccine is
given to healthy subjects 18 years old in two doses at
a 21-day interval [https://clinicaltrials.gov/ct2/show/
NCT04510207]. Sinopharm claims a vaccine efficacy of
79% [7]. Recently, WHO granted approval to
Sinopharm’s vaccine for emergency use [8]. A clinical
trial conducted in Bahrain and United Arab Emirates
showed that the inactivated vaccine SARS-CoV-2 WIV04
and HB02 have an efficacy against COVID-19 of 72.8%
(95% CI, 58.1–82.4%) and 78.1% (95% CI, 64.8–86.3%),
respectively [9].
Chinese Academy of Medical Science Vaccine
(Chinese Academy of Medical Science,
Beijing, China)
This group is developing an inactivated SARS-CoV-2
vaccine in Vero cells. The vaccine is given in two doses
14 days apart [https://clinicaltrials.gov/ct2/show/NCT04
659239]. The efficacy as well as the storage conditions
are currently unknown.
Sinovac vaccine (Chinese Academy of Medical
Science (Sinovac, Beijing, China)
This vaccine is produced in Vero cells as well and is
given to adults 18–59 years old to prevent COVID-19.
The vaccine schedule is two doses 14 days apart. The
vaccine showed an efficacy of 50% [10].
Bharat Biotech vaccine (Bharat Biotech International
Ltd., Hyderabad, India)
This company developed the BBV152 (Covaxin) vaccine
to prevent COVID-19 [https://clinicaltrials.gov/ct2/show/
NCT04641481]. It is a whole-virion inactivated SARS-
CoV-2 Vero cell derived platform technology containing
as adjuvant Algel-imidazoquinoline. The vaccine is
administered to adults  18 years old in two doses
4 weeks apart. According to the company, after the
second dose, Covaxin showed an interim efficacy of
81% in preventing COVID-19 in participants without
prior infection [11].
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 69
Vaccines based on mRNA
Pfizer/BioNTech vaccine (Pfizer/BioNTech, Mainz,
Germany)
The vaccine developed by Pfizer/BioNTech is based on
a nucleoside-modified RNA [12] that encodes the full-
length S-protein of SARS-CoV-2 but that bears two pro-
line mutations that stabilize the protein in a prefusion
antigenically preferred conformation. mRNA is con-
tained in lipid nanoparticles that enable its delivery into
host cells where the S-antigen will be transiently
expressed. The vaccine induces both neutralizing anti-
bodies and cellular immune responses that protect
from developing COVID-19. The administration of the
vaccine in two doses 21 days apart gave 95% protection
against COVID-19 in subjects 16 years of age and older
[13]. In May 2021, the Food and Drug Administration
(FDA) and the EMA approved the Pfizer/BioNTech vac-
cine for the age range 12–15 years. The vaccine sched-
ule is the same as for adults: two injections three weeks
apart. The vaccine was studied in 2,260 children aged
12–15 years. The immune response observed in this age
group was similar to that observed in the age group
16–25 years as indicated by the antibody levels against
SARS-CoV-2. The efficacy of the vaccine was determined
in almost 2000 children aged 12–15 years who had no
signs of previous infection. Among the 1005 children
who received the vaccine, none developed COVID-19
compared to 16 out of 978 children who were adminis-
tered placebo. Thus, the vaccine was 100% effective
against COVID-19, although the true rate could range
from 75–100%.
Moderna vaccine (Moderna Inc, Cambridge,
MA, USA)
Moderna developed the mRNA-1273 vaccine to prevent
COVID-19. mRNA-1273 is a lipid nanoparticle vaccine
containing mRNA encoding for the full-length S-protein
of SARS-COV-2 in a prefusion stabilized form [14].
Efficacy and safety analysis of the phase 3 study con-
ducted on participants 18 years of age and older
showed that the mRNA-1273 vaccine has an efficacy of
94.1% at preventing COVID-19. No important safety
concerns were identified [14]. The vaccine schedule
consists of two intramuscular injections 28 days apart.
CureVac vaccine (CureVac AG, Tubingen, Germany)
CureVac developed a SARS-CoV-2 mRNA vaccine,
CVnCoV, with the goal of preventing COVID-19 in adults
18 years of age and older. The vaccine is formulated as
a lipid nanoparticle surrounding a mRNA encoding for
the full-length S-protein of SARS-CoV-2. Pre-clinical
studies showed that CVnCoV induced a robust humoral
70 M. CIOTTI ET AL.
and cellular immune responses and was effective in
rodents and non-human primates [15,16]. Phase 1 clin-
ical trial demonstrated that CVnCoV is safe and induces
complete seroconversion after the second dose with
neutralizing antibody titers comparable to that of con-
valescent sera [17]. Interim analysis of the phase 2 b/3
study (the HERALD study) conducted on 40,000 subjects
showed that the vaccine has an efficacy of 47% against
COVID-19 of any level of severity [https://www.curevac.
com/en/2021/06/16/curevac-provides-update-on-phase-
2b-3-trial-of-first-generation-covid-19-vaccine-candidate
-cvncov/].
SARS-CoV-2 variants
SARS-CoV-2 is an enveloped positive single-stranded
RNA virus belonging to the Coranaviridae family, genus
Betacoronavirus, which also includes SARS-CoV and
MERS-CoV; the latter are both respiratory viruses of zoo-
notic origin responsible of two important epidemics in
recent years, SARS and the Middle East respira-
tory syndrome.
A feature of RNA viruses is the very high frequency
of mutation that influences viral fitness, infection speed
and evolution rate [18]. However, for the virus to sur-
vive, the resulting genetic variability should not com-
promise its survival within the host and the integrity of
the genetic information [19–21]. The mutagenic process
depends on several factors, including the proofreading
activity of the viral enzymes involved in genome repli-
cation, the physical-chemical mutagens, and recombin-
ation events. In the course of the pandemic, SARS-CoV-
2 has developed mutation hotspots in different regions
of the genome [22–24]. These mutations may have
implications for viral infectivity and transmissibility and
control of the pandemic. Because variants may also
affect vaccine efficacy, it is important to monitor them.
Variants are classified as variant of interest (VOI) or vari-
ant of concern (VOC). A VOI presents genetic markers
that may affect transmission, diagnostics, therapeutics,
or antibody neutralization, while a VOC presents
increased transmissibility, more severe clinical disease,
reduced neutralization by antibodies produced during
previous infection or vaccination, reduced efficacy of
therapies or vaccines, or lack of detection by current
diagnostic methods.
In February 2020, the D614G mutation in the S-pro-
tein was identified in SARS-CoV-2 strains isolated in
southern Europe; this variant then spread rapidly and
became the most widespread genotype worldwide [25].
The N439K variant, which was uncovered in Scotland
in March 2020, affects the RBD region. Following this,
this variant was also reported in other countries. The
mutant S-protein presents a higher binding affinity to
the human ACE2 receptor but shows similar in vitro rep-
lication fitness. Furthermore, it causes infections with
similar clinical outcomes when compared to the wild
type. Importantly, the N439K mutation makes ineffect-
ive a monoclonal antibody approved by the FDA for
emergency use [26].
In September 2020, a new variant of the virus, VUI
202012/01 or B.1.1.7 (WHO Alpha variant), was identi-
fied in the United Kingdom. It is characterized by mul-
tiple mutations of the S-protein (deletion 69–70,
deletion 144, N501Y, A570D, D614G, P681H, T716I,
S982A, D1118H) and by mutations in other regions of
the genome. One of the mutations (N501Y) affects the
binding site with the cell receptor. VUI 202012/01 dif-
fers in 29 nucleotides from the strain first isolated in
Wuhan [27]. This variant is characterized by high trans-
missibility [https://www.ecdc.europa.eu/en/publications
-data/threat-assessment-brief-rapid-increase-sars-cov-2-
variant-united-kingdom].
The variant 501Y.V2 or B.1.351 (WHO Beta variant)
was identified in South Africa. Genetic data showed
that 501.V2 rapidly replaced other viral strains circulat-
ing in South Africa [28]. The variant has been also
reported in Italy, Belgium, Denmark and the
Netherlands [29,30].
The P.1 variant (WHO Gamma variant) is a branch of
the B.1.1.28 lineage. It was first discovered in some
Brazilian travelers by the National Institute of Infectious
Diseases in Japan. This variant was prevalent in
Northern Brazil [31]. The P.1 variant presents several
mutations located in the RBD S1 subunit, including
E484K, K417T, and N501Y [32,33].
Most recently, a new variant has been identified in
Maharashtra, India, in the context of a highly diffusive
epidemic. The variant, classified as lineage B.1.617, has
several mutations. Two of them, the E484Q (or the
P478K) and the L452R, are located within the RBD
region in a site critical for the binding with ACE2 [34].
Present data suggests that these mutations could
increase virus transmissibility and favor immune-eva-
sion. The lineage B.1.617 includes three subvariants,
B.1.617.1–3 (B.1.617.1 is WHO Kappa variant), where the
mutations P478K and E484Q are differently distributed
[30,35,36]. The SARS-CoV-2 VOC B.1.617.2 (WHO Delta
variant) is 40–60% more transmissible than the B.1.1.7
VOC (WHO Alpha variant), and the risk of hospitalization
may be higher with the Delta variant in individuals who
are not fully vaccinated (https://www.ecdc.europa.eu/
en/news-events/ecdc-statement-sars-cov-2-delta-variant
-eueea).

Table 2. Most prevalent SARS-CoV-2 VOCs and VOIs.
Variants Spike substitutions
B.1.1.7 69del, 70del, 144del, (E484K), (S494P),
N501Y, A570D, D614G, P681H, T716I,
S982A, D1118H (K1191N)
B.1.351 D80A, D215G, 241del, 242del, 243del,
K417N, E484K, N501Y, D614G, A701V
P.1 L18F, T20N, P26S, D138Y, R190S, K417T,
E484K, N501Y, D614G, H655Y, T1027I
B.1.617 L452R, E484Q, D614G
B.1.617.1 (T95I), G142D, E154K, L452R, E484Q,
D614G, P681R, Q1071H
B.1.617.2 T19R, (G142D), 156del, 157del, R158G,
L452R, T478K, D614G, P681R, D950N
B.1.617.3 T19R, G142D, L452R, E484Q, D614G,
P681R, D950N
Detected in some sequences; VOC: Variant of concern; VOI: Variant of interest.
Table 2 summarizes the most prevalent variants
detected to date.
Finally, different methods
Debate continues on whether the currently deployed
vaccines are effective in preventing infection, transmis-
sion and hospitalization by SARS-CoV-2 variants. Clinical
trials conducted in South Africa, where the B.1.351 vari-
ant prevails, found a lower vaccine efficacy compared
to that observed in other countries where this variant
was not prevalent. The ChAdOx1 nCoV-19 vaccine
showed an efficacy of 10.4% (95% CI, -76.8–54.8)
against the B.1.351 variant, while the NVX-CoV2373 vac-
cine was 51.0% (95% CI, -0.6–76.2) effective [37,38].
Pfizer-BioNTech and Moderna conducted their clin-
ical trials before the SARS-CoV-2 variants started circu-
lating in the USA; therefore, there is no real-life data on
the efficacy of these two mRNA vaccines against the
B.1.351 or B.1.1.7 variants. However, in vitro studies car-
ried out on sera obtained from immunized individuals
challenged with genetically engineered variants
showed that vaccines elicit a lower level of neutralizing
antibodies against SARS-CoV-2 variants compared to
the prototype strain [39]. Nonetheless, the level of neu-
tralizing antibodies should be sufficient to neutralize
the virus, because the neutralization titers induced by
vaccination are high [40]. In addition, mutations could
affect the capacity of some monoclonal antibodies to
neutralize the virus. In vitro studies on the B.1.351 vari-
ant showed that this variant can be partly or com-
pletely resistant to some monoclonal antibodies that
have been authorized for therapeutic use [41].
Experimental data has shown that the current vac-
cines are still effective against P.1 Gamma variant [33].
Thus, there is no need to redesign a vaccine against
this variant.
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 71
WHO
Earliest sample date First detected Status classification
September, 2020 United Kingdom VOC Alpha
May, 2020 South Africa VOC Beta
November, 2020 Brazil VOC Gamma
February, 2021 India VOI
October, 2020 India VOI Kappa
October, 2020 India VOC Delta
October, 2020 India VOI
A Public Health England study on vaccine efficacy
against the Delta variant, B.1.617.2, showed that the
vaccines are highly effective after the second dose
against symptomatic disease. Indeed, the Pfizer-
BioNTech vaccine was 88% effective against symptom-
atic disease due to the B.1.617.2 variant two weeks after
the second dose, and 93% effective against the B.1.1.7
variant. The AstraZeneca vaccine was 60% effective
against symptomatic disease due to the B.1.617.2 vari-
ant after two doses compared to 66% effectiveness
against the B.1.1.7 variant. Both vaccines demonstrated
33% effectiveness against symptomatic disease three
weeks after the first dose compared to 50% effective-
ness against the B.1.1.7 variant [41].
A recent study evaluated the effectiveness of the
BNT162b2 and ChAdOx1 AZD12222 vaccines against
hospital admission with the Delta variant, B.1.617.2.
Vaccine effectiveness was 94% (46–99%) after one dose
and 96% after two doses of BNT162b2. With the
ChAdOx1 vaccine, the effectiveness was 71% (51–83%)
after one dose and 92% (75–97%) after two doses.
Thus, one or two doses of both vaccines are effective
against hospitalization with the Delta variant [42].
Rare vaccine adverse reactions
Thrombotic events and thrombocytopenia have been
reported following vaccination with AstraZeneca
ChAdOx1 nCov-19 vaccine. These rare and in some
cases fatal adverse reactions were described mainly in
young women, median age 36 years old [43]. The
thrombotic events manifested 5–16 days after vaccin-
ation and included cerebral venous thrombosis, pul-
monary embolism, splanchnic-vein thrombosis, and
other thrombotic manifestations. Six patients died and
five had disseminated intravascular coagulation.
Platelet-activating antibodies against platelet factor 4
(PF4) mediates this rare complication that resembles
72 M. CIOTTI ET AL.
autoimmune heparin induced thrombocytopenia [43].
The same complication occurred in an individual who
received the Johnson & Johnson/Janssen vaccine
(Ad26.COV2. S) 14 days after vaccination. Considering
that both AstraZeneca and Janssen use vaccines based
on nonreplicating adenoviral vector suggests that this
rare event of immune thrombotic thrombocytopenia
might be triggered by adenoviral vector vaccines [44].
A hypothesis is that complexes form between poly-
anionic groups induced by the vector and PF4 or anti-
bodies generated by the inflammatory response to the
vaccine and cross-react with platelets and PF4.
According to EudraVigilance (the system for collect-
ing and analyzing information on suspected adverse
reactions to medicines that are authorized or have
been studied in clinical trials in the European Economic
Area), as of 4 April 2021, 169 cases of thrombosis of
cerebral venous sinuses and 53 of splanchnic veins,
often associated to thrombocytopenia, have occurred,
out of 34 million doses of Vaxzevria vaccine adminis-
tered within the Economic European Region and United
Kingdom; this is equal to 6.5 cases/millions of subjects
who received at least one dose [45,46].
Based on the preliminary EMA report, the frequency
of venous thrombosis in atypical sites associated with
thrombocytopenia is calculated to be about 1 case/
100,000 vaccinated individuals [47].
In the USA, 17 cases of thrombosis in atypical sites
associated to thrombocytopenia have occurred, out of
7.98 million doses of Janssen vaccine administered [48].
Cases of immune thrombocytopenia following vac-
cination with Pfizer and Moderna vaccines have been
reported, although a causal relationship with the vac-
cine remains to be demonstrated [49].
More recently, Israeli scientists hypothesized a link
between COVID-19 vaccination and myocarditis in
young men. Individuals in the age range 16–24 years
may be at higher risk of developing myocarditis. The
estimate is 1/3000 to1/6000 men. Most of the reported
cases were mild and resolved within a few weeks [50].
Further observations in other populations are needed
to confirm that a link exists.
Determination and monitoring of anti-
receptor binding domain and neutralizing
antibodies after vaccination
During this pandemic period, great efforts have been
made to develop diagnostics to determine SARS-CoV-2
infection and vaccine efficacy. Numerous different meth-
ods and technical approaches have been devised to
measure the immune response to SARS-CoV-2 and the
antibody kinetics. Given the unknown number of asymp-
tomatic infections, serological diagnosis is crucial to
determine the real number of infections and the anti-
body response in vaccinated subjects. Such information
is important for outlining the case-fatality rate and for
taking political decisions about the scale and duration of
social lockdowns [51]. In the humoral immune response,
IgM and IgA antibodies are generally produced earlier
than IgG isotypes, and they were already present in the
first 5 days of symptoms; IgA antibodies can be detected
earlier than IgM, suggesting that IgA may be more useful
than IgM for early diagnosis of SARS-CoV-2 infection [52].
The kinetics of COVID-19 IgG antibodies shows a rapid
increase after 7 days from symptom onset and the anti-
bodies had 100% sensitivity on day 12 [53].
Serological tests are often designed to measure the
presence of antibodies against the S-glycoprotein (spe-
cifically RBD) or viral nucleocapsid (N) antigens. S-pro-
teins bind target cells through its RBD, which is the
target of neutralizing antibodies. Therefore, S-based
assays may be preferable to N-based assays for assess-
ing future reinfection risk [54]. Because not all antibod-
ies against the S-protein block viral infection, these
platforms do not determine functional antibody inhib-
ition of SARS-CoV-2 infection [55] and it is currently
unknown what antibody titer in commercial assays cor-
relates with protection. N-based assays can be useful to
identify individual who have been infected or not, or if
inactivated virus vaccine was used. Table 3 summarizes
the methods used to monitor the humoral response to
the vaccines currently authorized.
New commercially available enzyme immunoassays
can detect neutralizing antibodies with a high diagnos-
tic accuracy, in contrast to current in vitro measure-
ments of neutralizing antibodies that require laborious
assays, must be performed in biosafety facilities, and
are limited to research institutions.
Receptor-blocking assays are designed to detect
functional antibodies in serum samples and can be
used to study SARS-CoV-2 infection to obtain informa-
tion on the immune status of the population and to
evaluate effectiveness of vaccines. These serological
assays measure the level of neutralizing antibodies that
Table 3. Antibody assays available for monitoring the
response to the currently authorized vaccines.
Methods Vaccines
Receptor binding domain mRNA-based: Comirnaty
(RBD) based assays and Spikevax
AdV-based: Vaxzevria and
COVID-19 Janssen vaccine
Viral nucleocapsid and receptor Modified-inactivated SARS-CoV-2
binding domain (RBD) virus: Sinopharm and Sinovac
based assays vaccines

can protect from reinfection. Neutralizing antibodies
are usually measured by the plaque reduction neutral-
ization test (PRNT), the gold standard for serological
testing and for determining immune protection.
However, PRNT is labor intensive and has low through-
put, making it unsuitable for large-scale serodiagnosis
and vaccine evaluation. This is a major drawback for
COVID-19 surveillance and vaccine development.
The S-protein-ACE2 interaction is also considered an
important starting point to detect neutralizing antibod-
ies inhibiting virus function.
Currently, several new serologic assays that are on
the market detect the presence of receptor-binding
antibodies in sera: enzyme-linked immunoassay, chemi-
luminescence immunoassay, electro-chemilumines-
cence immunoassay and most recently the surface
plasmon resonance-based assay [55,56]. All these tests
can determine both antibody binding to the SARS-CoV-
2 S-protein and measure competition to the ACE2
receptor in a single experiment. These robust and rapid
methods to detect SARS-CoV-2 neutralizing antibody
response show good correlation with PRNT [56] and
can be employed to determine SARS-CoV-2 infection
and evaluate vaccines efficacy.
In conclusion, antibodies to different antigens can
be determined. Antibodies could develop after/
between the follow up of the infection (anti-N antibod-
ies) or vaccination with a vaccine bearing a specific
antigen (S-protein for RNA vaccines or N-protein for
vaccines with modified-inactivated SARS-CoV-2 virus,
Sinopharm and Sinovac). In contrast to RNA vaccines,
anti-N antibodies could otherwise be used to identify
those who have or have not been infected.
Serological assays are also needed to identify donors
with high levels of neutralizing antibodies for convales-
cent plasma therapy, and to define the titers of protec-
tion from SARS-CoV-2.
As well, studies on functional antibody responses and
persistence in patients presenting with asymptomatic,
mild, and severe forms of SARS-CoV-2 and after vaccin-
ation will be important for improving our understanding
of the humoral responses to SARS-CoV-2.
The variety of tests on the market has led to the devel-
opment of the first WHO International Standard for anti-
SARS-CoV-2 immunoglobulin. This international standard
consists of a pool of human plasma from convalescent
patients with an assigned unit of 250 international units
(IU)/ampoule for neutralizing activity. For binding assays, a
unit of 1000 binding antibody units/mL may be used
when comparing assays that detect the same class of
immunoglobulins with the same specificity [57]. This
International Standard permits precise calibration of assays
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 73
to an arbitrary unit, thus reducing inter-laboratory variation
and allowing comparison with other laboratories.
Conclusions
The big economic investments from the pharmaceutical
industries and governments as well as great efforts by
the scientific community have allowed the develop-
ment of effective vaccines against COVID-19 in less
than one year [12,13]. From late December 2020, coun-
tries started vaccinating their citizens with vaccines
authorized by their regulatory institutions. Thanks to
the vaccination campaigns, the epidemiological situ-
ation in western countries is rapidly improving, the
number of deaths is declining and the pressures on
emergency departments and health systems have less-
ened. However, vaccinations are still concentrated in
high-income countries, leaving behind the majority of
the world population that lives in low-income countries.
COVAX is an international initiative aiming at an equit-
able access to COVID-19 vaccines under the direction of
Gavi, the Vaccine Alliance, Coalition for Epidemic
Preparedness Innovations and the WHO. To date, it has
distributed 77 million doses to 122 countries [https://
www.gavi.org/vaccineswork/world-leaders-and-private-
sector-commit-protecting-vulnerable-covid-19-vaccines].
Rapid equitable access to COVID-19 vaccines is the key
to stopping the circulation of the virus and to control-
ling the pandemic. The continuing circulation and repli-
cation of the virus favor the emergence of viral variants,
increasing the risk of selecting variants that are unre-
sponsive to the current vaccines.
The sequencing of the SARS-CoV-2 genome is the key
to identifying in real-time the origin and the spread of
emerging variants. This information may be used to
implement preventive measures. VoCs may affect mor-
bidity and mortality. They may be more transmissible,
escape the immune response, reduce response to vac-
cines or neutralizing antibodies produced upon natural
infection, reduce response to monoclonal antibodies,
and lack detection by the diagnostic methods in use.
Serological diagnosis is of paramount importance for
monitoring antibody response to vaccination and for
determining the level of neutralizing antibodies
after vaccination. Several assays are available for
this purpose, but their standardization to the
WHO International Standard is key to reducing
inter-laboratory variation and creating a common lan-
guage for reporting data.
SARS-CoV-2 vaccines also elicit T cell responses, but
knowledge about these responses is limited. Specific
epitopes recognized by CD4þ and CD8þ T cells have
74 M. CIOTTI ET AL.
been identified [58] and may play an important role in
the immune response against SARS-CoV-2. This aspect
has important implications for T cells diagnostics and
warrants further investigation.
Disclosure statement
The authors declare that they have no conflict of interest.
Funding
The author(s) reported there is no funding associated with
the work featured in this article.
ORCID
Marco Ciotti http://orcid.org/0000-0002-9943-9130
Massimo Ciccozzi http://orcid.org/0000-0003-3866-9239
Massimo Pieri http://orcid.org/0000-0002-3463-0268
Sergio Bernardini http://orcid.org/0000-0003-1984-6834
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