Keywords COVID-19 vaccine - neutralizing antibodies and sVNT - SARS-CoV-2 - spike and RBD antibodies
General Message
A single or two doses of vector-based vaccine in conjunction with a natural infection
before or after the vaccination, generated high levels of severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies and may be sufficient
to provide protection against reinfection and/or severity.[1 ]
[2 ]
We recommend that this concept of hybrid immunity needs to be considered and used
in planning vaccination policies for protecting individuals at higher risk of post-vaccination
infection like older individuals and immunocompromised individuals as it is becoming
increasingly salient with emerging variants.
Background
Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is
a leading strategy to change the course of the coronavirus disease 2019 (COVID-19)
pandemic worldwide. New variants of concern increased the risk of infection in all
countries for people who are not yet vaccinated.[3 ]
[4 ] Due to potential new waves of COVID-19 and the increasing threat of new variants,[5 ]
[6 ] extensive vaccination programs should be implemented to maximize vaccine efficacy
with limited vaccine resources. The World Health Organization (WHO) is working for
equitable access to safe and effective vaccines in ending the COVID-19 pandemic, so
it is hugely encouraging to see so many vaccines being developed and tested in India.
The efficacy of vaccines against newer variants has to be assessed in the real world
to strategize the vaccination programs. Assessing vaccine efficacy periodically will
facilitate efficient implementation of the global vaccination policy. For obtaining
herd immunity globally all the countries had implemented vaccination strategy as a
game-changing tool to reduce morbidity and mortality.[7 ]
[8 ]
[9 ]
[10 ]
Most recovered patients already have some immunity against SARS-CoV-2, although it
is insufficient to protect against emerging variants. Recently, several randomized
controlled trials showed a higher risk for post-vaccination (“breakthrough”) infection
individuals who had lower neutralizing, spike, or receptor-binding domain (RBD) titers
after vaccination with (1) mRNA-1273, (2) ChAdOx1, and (3) BNT162b2 vaccines. In addition,
several studies have also reported that antibody responses begin to wane after 6 months
following vaccination and such individuals are at higher risk of breakthrough infections.[11 ] Hence, a booster dose of COVID-19 vaccine is being administered to high-risk populations
and health care workers in India. There are limited studies that have assessed the
humoral response in individuals with COVID-19 with or without vaccination. Therefore,
we evaluated and compared humoral immune response in (1) ChAdOx1 vaccinated with single
or double dose but uninfected individuals, (2) hybrid immunity, (3) break through
infection, and (4) unvaccinated individuals with only infection.
Materials and Methods
A total of 2,545 patients who were admitted to the hospital between June 2020 and
January 2022 at AIG Hospitals, Hyderabad, Telangana, India were retrospectively included
in the study. We evaluated vaccine-induced immunity, hybrid immunity, and breakthrough
along with only infection by retrieving the data pertaining to previous SARS-CoV-2
infection, vaccination status, and antibody levels from the electronic database of
the hospital and laboratory. Naive individuals were those with negative SARS-CoV reverse
transcription-polymerase chain reaction (RT-PCR) employing Taq path kits (Thermoscientific,
United States) and negative anti-SARS-CoV-2 total antibodies. The anti-SARS-CoV-2
immunoassay (electrochemiluminescence immunoassay [ECLIA]) was performed on a Cobas
e601 analyzer (Roche Diagnostics, Mannheim, Germany) and conducted according to the
manufacturer's instructions. This sandwich assay uses a SARS-CoV-2 specific recombinant
antigen representing the nucleocapsid protein. The electrochemiluminescent signal
produced is compared to the cutoff signal value previously obtained with two calibrators.
Results are expressed as cutoff index (COI) (negative COI < 1.0 or positive COI ≥
1.0) for anti-SARS CoV-2 total antibodies. COVID-19 recovered patients had been diagnosed
by RT-PCR during first, second, or third wave. Quantitative detection of immunoglobulin
G (IgG) anti-S1 and IgG anti-S2 antibodies to SARS-CoV-2 were done by anti-SARS-CoV-2 S1/S2
IgG assay by chemiluminescence immunoassay performed on LIAISON XL (DiaSorin, Saluggia,
Italy). According to the manufacturer, specificity is 98.5% (97.5–99.2) and sensitivity
is 97.4% > 15 days after diagnosis at a cutoff of > 15 AU/mL. Anti-SARS-CoV-2 S-RBD
immunoassay is an ECLIA (RBD) and measured on Cobas e601 modular analyzers (Roche
Diagnostics, Rotkreuz, Switzerland). Results are reported as the analyte concentration
of each sample in U/mL, with > 0.80 U/mL interpreted as positive. The manufacturer
states specificity of 99.98% (99.91–100), and a sensitivity ≥ 14 days after the first
positive RT-PCR of 98.8% (98.1–99.3). Neutralizing antibodies were tested by “cPass
SARS-CoV-2 Surrogate Virus Neutralization Test Kit” (GenScript Biotech, USA ELISA).
A surrogate virus neutralization test is a quick and simple assay that detects antibodies
that inhibit the RBD-angiotensin-converting enzyme 2 interaction, which is crucial
for virus entry into host cells. It was considered as positive if the inhibition was > 30%.
According to the WHO classification, COVID-19 infection was categorized into mild,
moderate, or severe infection. Mild COVID-19 defined as respiratory symptoms without
evidence of pneumonia or hypoxia, while moderate or severe infection defined as presence
of clinical and radiological evidence of pneumonia. In moderate cases, SpO2 ≥ 90%
on room air while one of the following was required to define the severe cases: respiratory
rate > 30 breaths/min or SpO2 < 90% on room air.[12 ]
Ethical Statement
All procedures were performed after acquiring necessary approvals from the institutional
ethical committee of AIG Hospitals (IRB AIG/IEC-Post BH&R 57/01.2022-01). The institutional
ethical committee had waived obtaining of the consent from the individuals.
Statistical Analysis
Data was analyzed and plotted using Graph Pad Prism 8.0 software. Quantitative data
with a normal distribution are reported as mean and standard deviations (SDs) and
qualitative data are expressed as percentages. Two independent samples were compared
using t -tests. Comparisons between two paired samples were made by the paired t -test. Multiple independent samples were compared using one-way analysis of variance
for normal distribution. A p -value of < 0.05 was regarded as statistically significant.
Results
We assessed efficacy of vaccines in previously recovered hybrid immunity, breakthrough,
only vaccinated, and only infected individuals by assessing the humoral immune response.
Based on the vaccination status, the study groups were divided into groups that comprised
of naive individuals with a single dose of vaccination (group I; n = 309), infected individuals without vaccination (group II; n = 357), individuals who were completely vaccinated (group III; n = 590), individuals who were administered a booster dose of vaccine (group IV; n = 70), individuals with hybrid immunity (single dose vaccine after previous infection)
(group V; n = 602), and individuals with breakthrough infections (infection after single or double
dose vaccination) (group VI; n = 617). The mean age was 51.8 years (SD 10.1) and individual demographic characteristics
of the study group across all the groups were similar as shown in [Table 1 ]/[Fig. 1 ]. The mean S1/S2 antibody titers were of significantly higher levels in group V (1284.5 ± 203.4)
and VI (1091 ± 234.8) as compared to the other groups (51.82 ± 8.15 in group I; 85.7 ± 12.4
in group II; 108.4 ± 15.01 in Group III, and 382.7 ± 42.96 in group IV). Likewise,
RBD antibody titers that were significantly higher were noted in group V (43610 ± 6457.5)
and VI (36042 ± 3948.9) as compared to the other groups (1307.6 ± 209.6 in group I;
2556 ± 365.65 in group II; 3779.5 ± 531.45 in group III, and 19125 ± 1882.5 in group
IV). Surrogate virus-neutralization test (sVNT) showed four to five times higher neutralization
in group V and VI followed by group III, II, and I. Baseline seropositivity was higher
in the groups V and VI as compared to groups I, II, II, and IV as shown in [Fig. 1 ]. We analyzed hybrid immunity and breakthrough by comparing individuals who received
only a single vaccine dose and double dose by S1/S2 and RBD antibody levels and sVNT
which did not show any significant difference between these two groups. Hence, we
combined them into single groups, that is, group V and VI. Groups V and VI, that is,
hybrid and breakthrough infection with a single or double vaccine dose pre- or postinfection
have good neutralizing capability in vitro. Majority of the individuals had developed
(18.2%) severe COVID-19 in group II that comprised of COVID-19 positive individuals
without vaccination as compared to other groups (V and VI; 6.5/10.8%).
Fig. 1 (A ) Log-transformed antibodies titers against the spike protein of severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) in six groups of patients with *p < 0.05. (B ) Log-transformed antibodies titers against the S-RBD (receptor-binding domain) of
SARS-CoV-2 in six groups of patients with *p < 0.05. (C ) Surrogate virus neutralization test (sVNT%) of the neutralizing antibody response
among all six groups of SARS-CoV-2 in six groups of patients.
Table 1
Cohort demographics
Characteristic
Group I
Single doseN = 309
Group II
COVID
infection N = 357
Group III
Double doseN = 590
Group IV
BoosterN = 70
Group V
Hybrid immunityN = 617
Group IV
BreakthroughN = 602
Sex
Male, N (%)
230 (74.43)
190 (53.22)
310 (52.54)
50 (71.42)
400 (64.82)
380 (63.1)
Female, N (%)
79 (25.56)
167 (44.81)
280 (47.45)
20 (28.57)
217 (45.17)
202 (33.55)
Age (y)
49.8 ± 12.6 (18–60)
50.4 ± 10.9 (21–58)
52.1 ± 9.6 (23–56)
51.2 ± 11.2 (25–54)
53.4 ± 7.9 (26–53)
55.0 ± 8.9 (28–51)
Type of vaccine
Covishield
Covishield
Covishield
Covishield
Covishield
Covishield
Critical time periods
(June 2020–January 2022)
Duration between infection and vaccination (mo)
N/A
N/A
N/A
N/A
5.0 ± 2.0
N/A
Second vaccine dose to PCR positive (mo)
N/A
N/A
N/A
N/A
N/A
5.0 ± 2.0
Latest vaccine dose to antibodies analyzed (d)
20 ± 45
N/A
20 ± 45
28
20 ± 45
20 ± 45
PCR positivity to antibody analyzed
N/A
20 ± 45
N/A
N/A
20 ± 45
20 ± 45
Duration between vaccine doses (mo)
N/A
N/A
2.0 ± 4.0
6.0 ± 3.0
N/A
N/A
S1/S2
(Antibody titers AU/mL)
51.82 (8.15)
85.7 (12.4)
108.4 (15.01)
382.7 (42.96
1284.5 (203.4)
1091 (234.8)
RBD
(Antibody titers U/mL)
1307.6 (209.6
2556 (365.65)
3779.5 (531.45)
19125 (1882.5)
43610 (6457.5)
36042 (3948.9)
sVNT > 30% neutralization
40 (17.39%)
95 (31.66%)
258 (44.10%)
46 (65.71%)
521 (84.44%)
470 (78.07%)
Baseline seropositivity (%)
(overall)
230 (74.44)
300 (84.03)
585 (99.15)
70 (100)
617 (100)
602 (100)
Male
170 (73.91%)
178 (59.33%)
307 (52.47%)
50 (71.42%)
400 (64.82%)
380 (63.1%)
Female
60 (26.08%)
122 (40.66%)
278 (47.52%)
20 (28.57%)
217 (45.17%)
202 (33.55%)
Severity
Mild
N/A
250 (70.02%)
N/A
N/A
120 (19.44%)
82 (13.61%)
Moderate
N/A
42 (11.57%)
N/A
N/A
30 (4.86%)
48 (7.97%)
Severe
N/A
65 (18.20%)
N/A
N/A
40 (6.48%)
65 (10.79%)
Abbreviations: PCR, polymerase chain reaction; RBD, receptor-binding domain; sVNT,
surrogate virus-neutralization test.
Discussion
Understanding vaccine immunity in individuals with and without exposure to the virus
is very important and aids in planning the vaccination programs world over. Assessing
neutralizing antibodies is important to assess the extent of the humoral response
in individuals. Here, we evaluated immunity between unvaccinated SARS-CoV-2 recovered
individuals and in those vaccinated with single or double dose of vaccine either before
or after infection that is in breakthrough and hybrid immunity. Our data demonstrates
that the humoral immune response in recovered SARS-CoV-2 individuals before or after
vaccination elicit a significantly higher neutralizing antibody response as compared
to primary, booster dose, single dose, and unvaccinated recovered individuals showing
natural immunity seems to be protective and elicits robust immune response by COVID-19
vaccination in SARS-CoV-2 recovered individuals. Bates et al reported that natural
infection boosts humoral response regardless of the timing of the vaccination[13 ] in SARS-CoV-2 recovered individuals.
In our study, we observed a subdued Response to the second dose after a previous strong
immune response, but it still prevented worsening of the disease and decreased the
risk of hospitalization. Krammer et al reported that individuals with previous infection
with a single dose of a messenger ribonucleic acid (mRNA) vaccine mounted strong humoral
response but muted response to the second dose.[14 ]
In particular, the seroconversion rate of the neutralizing antibodies was 84.4% in
group V and 78.07% in group VI versus 17.39% in group I, 31.66% in group II, 44.10%
in group III, and 65.71% in group IV with the ChAdOX1 vaccines. This data is consistent
with the previous findings that the individuals who received the complete mRNA vaccine
dose were all seropositive in the SARS-CoV-2 pseudovirus neutralization test, were
as response was low with Ad26.CoV2.S vaccines.[15 ] It is well demonstrated that a higher humoral response and T cell memory is elicited
with a single dose of vector-based vaccine along with previous infection.[16 ] Based on these multiple studies, the concept of hybrid immunity is being explored
as vaccination post-natural infection.[17 ]
[18 ] As immune response from natural infection alone is variable, few individuals elicit
a stronger response and few may develop a relatively lesser response. Interestingly,
ChAdOX1 induced a broad range of humoral immune responses in both hybrid immunity
and breakthrough infection compared to COVID-19-naive or only vaccinated individuals—a
similar finding was seen in the study of Jeewandara et al.[19 ] Therefore, vaccination might be more effective in COVID-19 recovered individuals
than in infection-naive vaccinated individuals.
Many other studies have shown that the T cells cellular response play an important
role in the SARS-CoV-2 vaccination and infection.[12 ] As our study focused only on the humoral response, the major limitation of our work
is that we have not looked at T cell response and follow-up studies are necessary
in order to determine the longevity of protection.
Conclusion
In summary, our study shows hybrid immunity resulted in higher antibody response compared
to vaccination alone as the primary infection will increase the potency to Prime our
immune system. This data in India demonstrates significant variation in vaccine immunogenicity
in infection-naive and SARS-CoV-2 recovered individuals.
Hybrid immunity likely provides strong protection against severe infection, provided
that the antigens of the emerging variant have same phenotypes of those of the primary
Wuhan wild strain. Existing vaccines have failed to provide the same level of increased
protection against the newer Omicron variants. As access and equitable distribution
of COVID-19 vaccine in low-income countries is still challenging, hybrid immune response
in COVID-19 recovered individuals, can also be achieved by heterologous vaccine boosters,
or by mucosal vaccination (intranasal vaccine), or by newer vaccines which include
conserved regions of viral non-spike genomic ribonucleic acid from different emerging
SARS-CoV-2 variants. Intranasal vaccines are relatively easy and quick for mass vaccination
and can be updated with emerging new variants which will induce more protection by
inducing mucosal immunity along with a strong T cell and B cell immune response.
We suggest vaccination policy should be according to vaccine availability, previous
history of infection, and by increasing the frequency of immune testing in older and
immunocompromised individuals who are at higher risk of post-vaccination infection,
which is becoming increasingly salient with emerging Omicron variants.
The hybrid immunity conferred by administrating at least a single vaccine dose to
as many individuals as possible with a confirmed history of SARS-CoV-2 infection will
result in herd immunity, and at some point in the future SARS-CoV-2 could become an
endemic infection, like a seasonal flu, instead of a pandemic.
