Article Not peer-reviewed version

Humoral immunity across the SARS-

CoV-2 Spike after Sputnik V
(Gam‑COVID‑Vac) vaccination

Alejandro Cornejo , Christopher Franco , Mariajose Rodriguez , Alexis García , Inirida Belisario ,
Soriuska Mayoría , Domingo Jose Garzaro , Jose Luis Zambrano , Rossana Celeste Jaspe , Mariana Hidalgo
, Nereida Parra-Gimenez , Franklin Ennodio Claro , Ferdinando Liprandi , Jacobus Henri de Waard ,
Hector Rafael Rangel , Flor Helene Pujol *

Posted Date: 21 March 2024

doi: 10.20944/preprints202403.1260.v1

Keywords: COVID-19; SARS-CoV-2; Antibody; ELISA; Spike; S1; S2; RBD; epitopes; Neutralization

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Article

Humoral Immunity across the SARS‐CoV‐2 Spike

after Sputnik V (Gam‐COVID‐Vac) Vaccination
Alejandro Cornejo 1, Christopher Franco 2, Mariajosé Rodríguez 3, Alexis García 4, Inirida
Belisario 4, Soriuska Mayoría 4, Domingo José Garzaro 3, José Luis Zambrano 2, Rossana Celeste
Jaspe 3, Mariana Hidalgo 5, Nereida Parra‐Giménez 6, Franklin Ennodio Claro 7, Ferdinando
Liprandi 8, Jacobus Henri de Waard 7, Héctor Rafael Rangel 3 and Flor Helene Pujol 3,*
1 Laboratorio de Bioquímica Celular, Centro de Microbiología y Biología Celular, Instituto Venezolano de
Investigaciones Científicas (IVIC), Caracas, Venezuela
2 Laboratorio de Virología Celular, Centro de Microbiología y Biología Celular, IVIC, Caracas, Venezuela

3 Laboratorio de Virología Molecular, Centro de Microbiología y Biología Celular, IVIC, Caracas, Venezuela

4 Instituto de Inmunología, Universidad Central de Venezuela (UCV), Caracas, Venezuela

5 Laboratorio de Inmunoparasitología, Centro de Microbiología y Biología Celular, IVIC, Caracas, Venezuela

6 Laboratorio de Fisiología de Parásitos, Centro Biofísica y Bioquímica, IVIC, Caracas, Venezuela

7 Departamento de Tuberculosis, Servicio Autónomo Instituto de Biomedicina “Dr. Jacinto Convit”, UCV,

Caracas, Venezuela
8 Laboratorio de Biología de Virus, Centro de Microbiología y Biología Celular, IVIC, Caracas, Venezuela

* Correspondence: fhpujol@[Link]

Abstract: SARS‐CoV‐2 vaccines have contributed to attenuate the burden of the COVID‐19 pandemic, by
promoting the development of effective immune responses, thus reducing the spread and severity of the
pandemic. A clinical trial with the Sputnik‐V vaccine was conducted in Venezuela from December 2020 to July
[Link] aim of this study was to explore the antibody reactivity of vaccinated individuals towards different
regions of the Spike protein (S). Neutralizing antibody (Nab) activity was assessed using a commercial
surrogate assay (detecting Nabs against the Receptor Binding Domain, RBD) and a Plaque Reduction
Neutralization Test. Nabs levels were correlated with the reactivity of the antibodies to the Spike regions over
time. The presence of Abs against nucleoprotein was also determined to rule out the effect of exposure to the
virus during the clinical trial in the serological response. A high serological reactivity was observed to S and
specifically to S1 and RBD. S2, although recognized with lower intensity by vaccinated individuals, was the
subunit exhibiting the highest cross‐reactivity in pre‐pandemic sera. This study is in agreement with the high
efficacy reported for the Sputnik V vaccine, and shows that this vaccine is able to induce an immunity lasting
for at least 180 days. The dissection of the Ab reactivity to different regions of S allowed to identify the relevance
of epitopes outside RBD able to induce Nabs. This research may contribute to the understanding of the vaccine
immunity against SARS‐CoV‐2, which could contribute to the design of future vaccine strategies.

Keywords: COVID‐19; SARS‐CoV‐2; antibody; ELISA; spike; S1; S2; RBD; epitopes; neutralization

1. Introduction
Four years have passed since the World Health Organization (WHO) declared the COVID‐19
pandemic due to SARS‐CoV‐2, on March 11, 2020. More than 770 million cases of COVID‐19 and over
7 million deaths have been officially reported since then, although it is estimated that these numbers
are considerably higher [1]. Nonetheless, SARS‐CoV‐2 vaccines have contributed to attenuate such a
burden by promoting the development of effective immune responses, thus reducing the spread of
the pandemic, the severity of the disease, hospitalizations and deaths [2].
SARS‐CoV‐2 belongs to the family Coronaviridae, is an enveloped virus, with a positive sense
genome of around 30,000 nt. The genome codes for four structural proteins (nucleocapsid or N, spike
or S, membrane or M and envelope or E), 15 non‐structural proteins and 8 accessory proteins [3]. The
structural homotrimeric glycoprotein S has been used as the target for many of the vaccines

© 2024 by the author(s). Distributed under a Creative Commons CC BY license.

[Link] ([Link]) | NOT PEER-REVIEWED | Posted: 21 March 2024 doi:10.20944/preprints202403.1260.v1

developed [4,5]. This protein is composed of the S1 and S2 subunits. The surface subunit S1 is
composed of 672 amino acids and is organized into four domains: an N‐terminal domain (NTD), a C‐
terminal domain (CTD, also known as the receptor‐binding domain, RBD) and two subdomains (SD1
and SD2) [5]. The highly antigenic region known as the receptor binding domain (RBD) mediates the
interaction with the receptor angiotensin converting enzyme (ACE2) and the binding of the majority
of neutralizing antibodies [6]. It is known that other epitopes outside of RBD, like NTD, are also
important in immunity and contribute to the antigenic profile of the S protein [7–9], but the effects of
antibody recognition are not yet well characterized. The transmembrane subunit S2 is composed of
588 amino acids and contains a hydrophobic N‐terminal fusion peptide (FP), two heptad repeats
(HR1 and HR2), a transmembrane domain (TM), and a cytoplasmic tail (CT), the S2 subunit being the
more conserved among all coronaviruses [10,11]. S antigen was used as immunogen in several
vaccine constructs.
Vaccine strategies included non‐replicating adenoviral vectors, nucleic acid (mRNA), whole
inactivated viruses and protein subunit‐based vaccines. The two‐component adenovirus vector
vaccine Gam‐COVID‐Vac (Sputnik‐V) was the second most distributed vaccine in Venezuela and was
also employed in other Latin American countries [12,13]. Despite being distributed in at least 35
countries and having over 1.3 billion doses administered by March 2024 (according to
[Link]), few studies are available on Sputnik‐V efficacy and immunogenicity,
compared to other vaccines. In order to deepen the understanding of its effectiveness and
immunogenicity of Sputnik‐V, the aim of this study was to explore the antibody reactivity of
individuals vaccinated with Sputnik‐V towards different regions of S. Neutralizing antibody (Nab)
activity was assessed and correlated with the reactivity of the antibodies to S over time.

2. Materials and Methods

2.1. Study Design, Participants and Serum Recollection
The samples evaluated were obtained from volunteer subjects belonging to the Gam‐COVID‐
Vac (NCT04642339): prospective, double‐blind, randomized, placebo‐controlled clinical trial in
Venezuela, from December 2020 to July 2021. Inclusion criteria included male and female volunteers
aged 18 years or older, who read and signed the informed consent form, tested negative for HIV,
hepatitis and syphilis; volunteers had no clinical history of COVID‐19 and tested negative for both
IgG and IgM antibodies to SARS‐Cov‐2 by enzyme immunoassay (PISHTAZTEB Diagnostic, Tehran,
Iran) and negative for SARS‐Cov‐2 by RT‐PCR. Exclusion criteria were any
vaccination/immunization within 30 days prior to enrollment, use of steroids (except hormonal
contraceptives) and/or immunoglobulins or other blood products not completed within 30 days
before the enrollment, completion of immunosuppressive therapy within 3 months prior to
enrollment, pregnancy and lactation. The inclusion and exclusion criteria details are shown in more
detail on the clinical trials website ([Link]/ct2/show/NCT04642339). The volunteers were
from Caracas, ranging in age from 20 to 80 years old. A total of 133 sera were obtained, being 80 male
and 53 female volunteers, divided between vaccinated (n=105) and placebo controls (n=28). All
volunteers received a first dose on day 1 and a second dose 21 days after the first dose. Samples were
taken 42 days post vaccination (dpv), counting after the first dose and 21 days after the second dose.
For a subgroup of 46 vaccinated volunteers, serum was also obtained at 180 dpv. The trial and all its
procedures were approved by the National Ethics Committee for Research in COVID‐19.

2.2. Antigens
The recombinant antigens for the enzyme‐linked immunosorbent assays (ELISA) were acquired
from MyBioSource Inc. (San Diego, CA). The ancestral S protein (MBS8574721), S1 (MBS8309640), S2
(MBS9141947), RBD (MBS8574741), and N (MBS5316649) antigens, were used. The concentration of
antigens was confirmed using Qubit™ Protein Assay according to the manufacturer’s specifications
(Thermo Fisher, catalog number Q32866).
[Link] ([Link]) | NOT PEER-REVIEWED | Posted: 21 March 2024 doi:10.20944/preprints202403.1260.v1

2.3. ELISA Reactivity

To determine the reactivity of the sera towards the different regions of S and N, the latter to rule
out a SARS‐CoV‐2 infection, the microtiter plates were sensitized with 1 μg/mL (2 μg/mL for N) of
the antigens in a final volume of 50 μL, and incubated at 4 C° overnight. The next day, the solution
was decanted, and the plates were incubated with 150 μL of 1X blocking solution (Abcam,
Cambridge, United Kingdom, ab126587) for at least 1 hour at room temperature. During this
incubation, the samples of vaccinated and controls were diluted 1/100 in 1X blocking solution. After
the blocking time, the plate was washed 6 times with 0.01% PBS‐Tween and 100 μL of the diluted
serum samples were incubated for 2 hours at 37°C and the plate was washed again. The anti‐human
IgG secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch Inc,
Pennsylvania, United States of America, 109‐035‐008) diluted 1/70,000 was added and incubated for
1 hour. The chromogenic substrate solution of peroxidase, TMB (3,3′,5,5′‐tetramethylbenzidine) was
used for the color development reaction. To stop the reaction, 50 μL of HCl [3M] was used. For the
reading and recording of the data, a spectrophotometer (SpectraMax 250) was used at 450 nm. As
negative controls, 18 sera from apparently healthy individuals obtained before the pandemic were
used. A positive control to normalize data was used, consisting of a mixture of two sera from highly
responsive individuals, vaccinated with Sputnik‐V and with two infections confirmed by PCR. An
additional 20 sera with hybrid immunity (infection(s) plus Sputnik‐V vaccination) with infections
confirmed by RT‐PCR, were included. Reactivity against SARS‐CoV‐2 antigens (ancestral: S, S1, S2
and RBD) was assessed within this group of 134 individuals. The optical densities (O.D.) of the blank
was subtracted from the O.D. of the samples. O.D. exceeding the cut off established with negative
control mean plus 3 standard deviations were considered as responders to N or S. Relative levels of
IgG antibodies were normalized as the sample‐to‐positive ratio (S/P) [12,14] using this formula with
the respective controls assayed on each ELISA plate:
S/P= ((O.D. sample‐O.D. negative controls)/(O.D. positive controls‐O.D. negative controls)) X 100.
The BAU (binding antibody units) serum international standard from the World Health
Organization (WHO) was used for some assays to establish a standard curve and measure the relative
concentration of antibodies in each sample.

2.4. Surrogate Neutralization Test (SNT) Based on ACE2 Blocking Adsorption Immunoassay
A commercial kit was used that allows the detection of neutralizing antibodies, through a
competitive ELISA (SARS‐CoV‐2 Neutralization Antibody Detection Kit, Elabscience ®, Houston,
USA), according to manufacturer instructions. Briefly, the test quantifies NAbs against RBD able to
block the interaction between S and ACE‐2. In this assay, 50 μL of the sera diluted 1/10 were incubated
with 50 μL of the solution containing the recombinant spike protein (RBD) conjugated to HRP in a
microtiter plate already sensitized with ACE2 for 1 hour. Subsequently, it was washed 3 times with
the commercial washing solution provided by the kit and 90 μL of commercial substrate from the
commercial kit were added for a 15‐minute incubation, after this time, 50 μL of the STOP solution
provided by the kit were added. For the reading and recording of the data, a spectrophotometer
(SPECTRAmax 250) was used at 450 nm. If neutralizing antibodies are present in the sample, they
will inhibit spike interaction with recombinant ACE‐2, which will result in a decrease in the optical
density signal. The commercial kit provides a titration curve to determine the titers of NAbs anti‐
RBD.

2.5. Plaque Reduction Neutralization Test

A plaque reduction neutralization test (PRNT) was conducted on 20 samples of serum collected
at 42 days post‐vaccination (dpv), and 18 samples from 180 dpv. PRNT was performed according to
a previously reported procedure [14]. VERO C1008 cells (Vero 76, clone E6, vero. ATCC, Manassas,
USA), were maintained at 37 °C and 5% CO2 in RPMI medium (Thermo Fisher Scientific, 11875093)
supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, 16000044) and 1%
[Link] ([Link]) | NOT PEER-REVIEWED | Posted: 21 March 2024 doi:10.20944/preprints202403.1260.v1

penicillin‐streptomycin (Thermo Fisher Scientific, Massachusetts, United States of America,

15140122). Infection was performed using viral seeds of a SARS‐CoV‐2 ancestral strain (B.1.1.3) in a
biosecurity cabinet within a level 3 biosecurity facility. PRNT50 of each serum was determined
through a non‐linear regression test, defining this value as the reverse of the dilution at which 50%
of the virus control (VC) is neutralized.

2.6. Statistical Analysis

All statistical tests and graphs were performed with PRISM GraphPad ©, California, USA. For
the analysis of the reactivity towards the ancestral spike protein and its different regions, the Kruskal
Wallis test with an alpha value of 0.05 was used, with its respective Dunn’s correction post‐hoc
analysis. For the analysis of reactivity towards the spike and its different regions and SNT
comparisons a Mann‐Whitney test with an alpha value of 0.05 was used. For ELISA and PRNT50,
comparisons at 42 and 180 dpv, a paired Wilcoxon test was used with an alpha value of 0.05. To
determine the degree of association between IgG antibody reactivity, SNT and PRNT50, the
correlations matrix test was performed using the Spearman correlation coefficient with a confidence
interval (CI) of 95%. For frequency comparison among groups, statistical difference was assessed
with Chi square test (Epi InfoTM, Centers for Disease Control and Prevention, Atlanta, USA).

3. Results
3.1. Seroconversion Rates in Non‐Exposed Vaccinated Individuals in the Clinical Trial
A total of 133 volunteer sera were obtained from the Gam‐COVID‐Vac clinical trial, in
Venezuela. Of them, 105 were from vaccinated individuals, and 20/106 exhibited reactivity against
N, suggestive of exposure (Figure 1). The sera of 85/105 vaccinated (with no serological evidence of
exposure) were analyzed for their reactivity against different regions of S. Figure 2 shows the levels
of Abs (BAU/mL) for S in the sera from the different groups. A total of 92% of the vaccinated non‐
exposed individuals could be classified as responders, according to their BAU titer. Some sera (52%)
from placebo individuals without reactivity to N, showed however reactivity to S in this assay (Figure
2), suggesting that some of them were also exposed during the course of the clinical trial, or before.
It is important to note, however, that some of the sera apparently reactive to N or S, exhibited very
low S/P or BAU values, near the limit of positivity, suggesting some unspecific reactivity.
[Link] ([Link]) | NOT PEER-REVIEWED | Posted: 21 March 2024 doi:10.20944/preprints202403.1260.v1

Figure 1. Sputnik V clinical trial group. The stratification of the sample set based on the reactivity
results for the SARS‐CoV‐2 N (exposure) is presented. Reactivity was quantified in a total of 133
samples (vaccinated and placebo) at 42 dpv. Then, 21 samples of vaccinated and N negative, were
selected to compare their reactivity at 42 and 18 to 180 dpv. From them, 20 sera, were selected for
neutralization assays and comparison between 42 and 180 dpv. SNT: Surrogate neutralization.
[Link] ([Link]) | NOT PEER-REVIEWED | Posted: 21 March 2024 doi:10.20944/preprints202403.1260.v1

Figure 2. BAU reactivity to S. The different groups are shown: vaccinated, exposed vaccinated,
placebos, and exposed placebos (classification made according to the reactivity to N), in addition to
hybrids and pre‐pandemics. The percentage values represent individuals whose BAU value exceeded
the cut‐off point (CP). Kruskal Wallis test (p<0.0001) with Dunn’s correction post‐hoc analysis was
used for groups comparisons. Dash lines indicate cut‐off value (119 BAU/mL).

3.2. Reactivity to Different Regions of SARS‐CoV‐2 S

In order to dissect the humoral immune response to S, the reactivity to the different regions of
this protein was evaluated (Figure 3). The reactivity of sera from apparently healthy individuals
collected before the pandemic (pre‐pandemic) was also analyzed as negative controls. S and S1
regions were recognized at a similar frequency, while fairly less sera recognized the RBD region, and
significantly less sera recognized the S2 domain (58%, p<0.001) (Figure 3A). For the vaccinated
individuals at 42 dpv, statistically significant differences were observed (p<0.0001) in the S/P average
ratio between the different antigens. Multiple comparisons revealed that the S1 region was
recognized with a greater S/P value in the sera than other regions (p=0.0054), while S and RBD
exhibited similar reactivity, and S2 was recognized with significantly lower reactivity (p<0.0001).

Figure 3. Reactivity towards different regions of S. (a) Multiple comparisons of the reactivity of sera
from vaccinated individuals at 42 dpv by the Kruskal Wallis test (p<0.0001) with Dunn’s correction
post‐hoc analysis. The Y‐axis represents percent of sample‐to‐positive ratio value. For S1, only 61
samples are plotted. Percent of responders are show. (b) Mann‐Whitney test of sera from individuals
vaccinated with Sputnik‐V and exposed. The reactivity towards the different regions of S is shown
compared to individuals who were vaccinated and had apparently exposure to the virus during the
[Link] ([Link]) | NOT PEER-REVIEWED | Posted: 21 March 2024 doi:10.20944/preprints202403.1260.v1

clinical trial (★), the increased S/P value between the vaccinated and the exposed vaccinated is shown.
(c) Reactivity of pre‐pandemic sera towards the different regions of S were analyzed by Kruskal Wallis
test (p<0.0001) with Dunn’s correction post‐hoc. (d) Paired Wilcoxon test Reactivity towards the
different regions of the Spike (ancestral) at 42 and 180 dpv in the cohort that completed the clinical
trial without N reactivity (2 samples excluded). ns: not significant.

The reactivity of the vaccinated volunteers was compared with those with serological evidence
of exposure, suggestive of hybrid immunity (Figure 3B). The average reactivity of the sera from the
group of individuals with hybrid immunity was significantly higher than that of the vaccinated only
group (Wilcoxon test), especially in the case of the S2 region, where a more pronounced difference
was observed (4.1x). Reactivity of some sera with S (n=2), S2 (n=8) and RBD (n=2) was observed in
the 20 prepandemic sera, being the S2 the most frequently recognized (p 0.0076) (Figure 3C). These
samples were excluded for the antigen cut‐off calculation. The sera at 180 dpv (in vaccinated
individuals who completed the clinical trial, n=21) exhibited a significant decrease in reactivity to S
and RBD (Wilcoxon paired test, p<0.05), while no significant differences were observed for S1 and S2.
In some cases, an increase in S/P value was observed at 180 dpv (Figure 3D).

3.3. Surrogate Neutralization Test (SNT)

The SNT based on ACE2 blocking immunoassay allowed to quantify the NAbs against SARS‐
CoV‐2 directed against RBD (Figure 4). Significant differences were observed between the placebos
and the vaccinated, as expected. No significant differences were observed between the Nabs levels at
42 and 180 dpv. The pre‐pandemic sera did not show NAbs by this test, even those that exhibited
ELISA reactivity to some S regions.

Figure 4. SNT (NAb α‐RBD) at 42‐ and 180‐days post‐vaccination. NAb titers (ng/mL) at 42 and 180
dpv. Wilcoxon was performed only in paired sera with 42 and 180 dpv sample. ★ Red: two vaccinated
individuals with evidence of exposure (excluded from the statistics). ★ Green: sera for which the SNT
[Link] ([Link]) | NOT PEER-REVIEWED | Posted: 21 March 2024 doi:10.20944/preprints202403.1260.v1

levels increased at 180 days. Kruskal Wallis test (p<0.0001) with Dunn’s correction post‐hoc was
performed for the other comparisons.

The ELISA reactivity to different regions of S was correlated with demographic parameters
(Figure 5). A low negative but significant correlation was found between reactivity to S and the level
of SNT with age. This was not observed for S1, S2 and RBD (not significant). No significant correlation
was found between the reactivity to S and its regions and sex (data not shown). Significant positive
correlations were found between reactivity to S and to S1 and RBD, but not to reactivity for S2. The
highest positive correlation was found between SNT and reactivity to RBD, as expected, with the
lowest correlation between SNT and reactivity to S2.

Figure 5. Correlation matrix between reactivity, surrogate neutralization and ages at 42 dpv.
Association between variables is represented with a heatmap where blue and red are used to
represent positive and negative correlations respectively. For S1, only 61 samples of S1 were used in
the correlations. S1, S2 and RBD doesn’t show significance correlation between age, the rest of
correlation show p values under 0.05.

3.4. Plaque Reduction Neutralization Test (PRNT50)

Figure 6 shows the PRNT50 for the sera analyzed at 42 and 180 dpv. PRNT50 titer was not
observed for any placebo sera. No significant difference was observed between the PRNT50 at 42 and
180 dpv. Some individuals even exhibited higher neutralization titers at 180 dpv. PRNT50 titers were
correlated with the other parameters analyzed in this study (Figure 7). The highest correlation was
obtained with the reactivity to S1, followed by S and RBD. A lower correlation was observed with
SNT and reactivity to S2. All comparisons showed moderate to low correlations with PRNT50.
Similar results were observed when analyzing sera at 180 dpv (Figure 8) with a reduction in the
correlation with reactivity to RBD.
[Link] ([Link]) | NOT PEER-REVIEWED | Posted: 21 March 2024 doi:10.20944/preprints202403.1260.v1

Figure 6. PRNT50 values of the sera from vaccinated individuals at 42 and 180 dpv. The Y‐axis
corresponds to the PRNT50 of each serum. ★ Red: two vaccinated individuals with evidence of
exposure (excluded from the statistics). ★ Green: sera for which the PRNT50 titer increased at 180
days. Kruskal Wallis test (p<0.0001).

Figure 7. Correlations between PRNT50 titers, SNT (NAb α‐RBD) and ELISA reactivity to S regions
in individuals vaccinated with Sputnik‐V (42 dpv). r: spearman correlation coefficient and p value
(p<0.05); 95% CI; linear regression model shown in blue line.
[Link] ([Link]) | NOT PEER-REVIEWED | Posted: 21 March 2024 doi:10.20944/preprints202403.1260.v1

10

Figure 8. Correlations between PRNT50 titers, SNT (NAb α‐RBD) and ELISA reactivity to S regions
in individuals vaccinated with Sputnik‐V (180 dpv). r: spearman correlation coefficient and p value
(p<0.05); 95% CI; linear regression model shown in blue line.

4. Discussion
The dissection of the Ab reactivity to different S regions allowed us to correlate this reactivity
with the neutralizing ability of sera from volunteers vaccinated with Sputnik V. Determining
exposure to SARS‐CoV‐2 was essential to exclude from the study those individuals who were
exposed to the virus during the course of the clinical trial, by analyzing reactivity to N. The first
observation is that a quite important proportion of volunteers exhibited evidence of SARS‐CoV‐2
exposure (anti‐N Abs) during the clinical trial. It should be noted that anti‐N Abs have low
persistence in serum compared to anti‐S ones [15]. In fact, the analysis of the reactivity to S revealed
that additional volunteers from the placebo group exhibited anti‐S Abs, without reactivity to N,
suggesting that an even higher degree of exposure in the group might have occurred. The apparent
relatively high incidence of exposure to the virus during the window of the clinical trial could be
explained by the increase in the number of cases in Venezuela during the period of time of the clinical
trial [16]. It is important to note that only 2 of the apparently exposed volunteers, from the placebo
group, reported a symptomatic COVID‐19 episode. This suggests that the Ab reactivity to SARS‐
CoV‐2 proteins might have been associated to an asymptomatic or mild exposure in most of the cases.
Of the total vaccinated, 92% of individuals responded to vaccination by generating detectable
anti‐S Abs, a frequency similar to the one reported in previous studies, and in agreement to the high
efficacy and seroconversion rate reported for the Sputnik vaccine [17–21]. It has been reported that
BAU levels between 13 and 141 BAU/ml provided only 12.4% protection against SARS‐CoV‐2
(ancestral strain), a concentration between 141 and 1700 BAU/ml 89.3% protection and a
concentration of 1700 BAU/ml and higher provided complete protection [22]. In our study, a value
less than 119 BAU/mL was considered as the threshold to define non‐responder individuals.
The analysis of Ab reactivity to the different regions of S showed that S1 was the most important
antigenic region, exhibiting higher recognition than the RBD, while S2 turned out to be the region
with the lowest reactivity. These results are consistent with previous reports describing other regions
within S1 outside RBD capable of generating an antibody response [23]. On the other hand, the fact
that S1 exhibited greater antigenicity than S in our assays, also suggests that some of these sites are
probably encrypted or less exposed when S is in its homotrimeric conformation or the pre‐fusion
states [23–25]. Most of the S in the SARS‐CoV‐2 virions are present in a metastable pre‐fusion
conformation, although some S can adopt an extended post‐fusion rod‐like conformation, due to the
premature dissociation of S1 from S2 (independent of the interaction with ACE‐2), which also releases
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11

the soluble S1s subunit. S1s can act as an independent immunogen, and probably induces a B
lymphocyte response independent of T through cross‐linking of the B cell receptor (BCR) [26] in
addition to the canonical mechanisms of B lymphocyte stimulation. This same conformational
phenomenon has been reported in the S product of the Sputnik‐V vaccine construct, which does not
have stabilization mutations [27]. The role of the dissociation of the S1 subunit in immunity or the
pathophysiology of the disease is still unclear: some data suggest that it may contribute to a higher
proportion of no NAbs compared to neutralizing ones [28]. We hypothesize that it may contribute to
the generation of Abs against regions outside of RBD, such as NTD. It has been described that
dissociated S1 subunits are capable of forming immune complexes with Abs [28] that probably can
activate Fc receptors in dendritic and follicular cells promoting the S1 antigen presentation.
Additionally, other studies have indicated that the S1 subunit, particularly the NTD, can activate cells
within the innate immune system [29].
The S2 region trigger the fusion of the viral membrane to the cellular membrane. Some authors
have suggested that it is a less immunogenic region than S1, because of its high degree of
glycosylation [30,31] In contrast to the low antigenicity exhibited by S2 in the sera of vaccinated
volunteers, this was the region of S most frequently recognized by pre‐pandemic control sera (Figure
3C). The presence of these antibodies is probably due to the seasonal prevalence of the four common
types of human coronaviruses, especially OC43 and HKU1 [32,33]. Some authors have described,
through microarray assays with recombinant S peptides, that there are immunogenic sites in S2
between amino acids 818 and 835, the region corresponding to the fusion peptide (FP) that is highly
conserved among coronaviruses [34–36]. Targeting the FP with antibodies can prevent the host
protease TMPRSS2 from cleaving the S2 subdomain, thus reducing viral entry [34]. In addition, the
amino acid sequence of FP does not vary among the variants of SARS‐CoV‐2 currently known [37].
Interestingly, when comparing the Ab reactivity to the different S regions between the sera of
vaccinated and (probably) exposed vaccinated volunteers, the highest increase in reactivity was
found for the S2 region (4.1X). This observation might suggest that natural infection induces a
stronger reactivity to S2, compared to the one elicited by the Sputnik V vaccine. The results obtained
coincide with other reports such as those of Polvere et al. who identified that natural infection by
COVID‐19 combined with vaccination results, on average, in a higher antibody titer and greater
neutralizing activity with respect to fully vaccinated people without a history of COVID‐19 [38].
No differences were observed between sex and the anti‐S Abs levels, in agreement with previous
reports [39]. The correlation between reactivities to S, SNT and age exhibited an age‐independent
response. This has also been reported in other Sputnik‐V studies [17,20,40]. While a reduction in the
total Abs was observed by ELISA, the levels of Nabs, measured by SNT or PRNT50, were maintained
on average at 180 dpv, suggesting a maturation process in the immune response. In contrast, a decline
in Nabs has been reported for mRNA vaccines [41]. Some low‐level transgene long‐term expression
has been described [42,43]. The persistence of antigen expression may be a distinctive feature of
adenovirus vector vaccines and has been proposed to contribute to the induction of sustained
immune responses and lasting immunity to S and could explain the persistence of NAbs. Another
mechanism, such as maturation of B lymphocytes in germinal centers (GC) in the lymph node [44,45]
has also been reported to contribute to the diversification of response and maturation of antibodies
affinity [46–48].
SNT levels were not significantly correlated with PRNT50 titers. Moreover, at 42 dpv, the highest
correlation with PRNT50 was observed with S1 reactivity. This observation is in agreement with the
evidence that RBD is not the only region that actively participates in inducing Nabs against SARS‐
CoV‐2 [49] like NTD. While the function of the NTD as a neutralization target is not yet fully
understood, it has been observed that other NTD‐specific neutralizing antibodies, in MERS, can
inhibit the conformational change from pre‐fusion to post‐fusion even after receptor binding occurs
[9,50].
This study has potential limitations. The number of volunteers at 180 dpv was limited, reducing
the strength of comparison for this time point. An analysis of the cellular immune response in the
participants would have been useful.
[Link] ([Link]) | NOT PEER-REVIEWED | Posted: 21 March 2024 doi:10.20944/preprints202403.1260.v1

12

In conclusion, the vaccine immunogenicity reported in this study is in agreement with the high
efficacy reported for the Sputnik V vaccine [17,21], and that this vaccine is able to induce an immunity
lasting at least for 180 days. The dissection of the Ab reactivity to different regions of S allowed to
identify the relevance of epitopes outside RBD able to induce Nabs. We suggest that polyclonal and
diverse responses of antibodies against S could confer different levels of protection including
neutralizing antibodies and a lasting response.

Author Contributions: Conceptualization, supervision, and project administration, F.H.P., A.G., H.R.R.;
investigation, A.C., C.F., M.R., D.J.G., R.C.J.; resources, I.B., S.M., M.H., F.C., J.H.W., N.P.G., writing—original
draft preparation, A.C.; writing—review and editing, A.C., C.F., F.H.P., F.L., R.C.J., J.L.Z., J.H.W.; funding
acquisition, F.H.P., H.R.R, A.G. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by Ministerio del Poder Popular de Ciencia, Tecnología e Innovación of
Venezuela.

Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki
and approval was obtained from the National Ethics Committee for Research in COVID‐19 of Venezuela.

Informed Consent Statement: Patient consent for samples used in this study was obtained in accordance with
the Declaration of Helsinki, and and approved by the National Ethics Committee for Research in COVID‐19 of
Venezuela.

Data Availability Statement: The complete genome sequence of the SARS‐CoV‐2 strain used for neutralization
assays have been deposited in the GISAID database (accession numbers EPI_ISL_6980947.

Acknowledgments: The authors are indebted to all members of the clinical trial team. We are also indebted to
the volunteers who acceded to participate in the Clinical trial. This study was supported by Ministerio del Poder
Popular de Ciencia, Tecnología e Innovación of Venezuela.

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.

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