medRxiv preprint doi: https://doi.org/10.1101/2021.08.16.21261912; this version posted August 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .
Genomic profiles of vaccine breakthrough SARS-CoV-2 strains from Odisha, India
Arup Ghosh1#, Safal Walia1#, Roma Rattan2, Amol Kanampalliwar1, Atimukta Jha1, Shifu
Aggarwal1, Sana Fatma1, Niyati Das1, Nirupama Chayani2, Punit Prasad1, Sunil K. Raghav1*,
Ajay Parida1*
Affiliations
1Institute of Life Sciences, Autonomous Institute under Department of Biotechnology,
Government of India, Bhubaneswar, Odisha, India
2SCB medical college and hospital, Cuttack, Odisha, India
# Equal contributors.
*Correspondence:
Sunil Raghav ( [email protected]; [email protected])
Ajay Parida ([email protected]; [email protected])
1
NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2021.08.16.21261912; this version posted August 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .
Abstract
Vaccine breakthrough infections pose a vast challenge in the eradication of the COVID
pandemic situation. Emerging SARS-CoV-2 variants of concern infecting the immunized
individuals indicate an ongoing battle between host immunity and natural selection of the
pathogen. Our report sheds light on the prominent SARS-CoV-2 variations observed in the
isolates from AZD1222/Covishield and BBV152/Covaxin vaccinated subjects.
Keywords: Vaccine Breakthrough,COVID-19, SARS-CoV-2, Variants of Concern
Background
India has experienced 426,290 cumulative fatalities due to COVID-19 as of August 06, 2021
(https://covid19.who.int/table). Subsequent waves of SARS-CoV-2 infection with rapid
resurgence in transmission and mortality deployed potential vaccine candidates to generate an
effective immune response against SARS-CoV-2.
In India, five COVID-vaccines are authorized for emergency use, out of which the adenovirus-
vector based vaccine from Oxford University and AstraZeneca UK marketed as Covishield, and
the indigenous inactivated virus vaccine Covaxin by Bharat Biotech are majorly deployed
through government and private healthcare centers. Both the vaccines pose tolerable safety
outcomes and enhanced immune responses [1,2]. The emergence of novel variants of concern
(VOC), novel spike (S) protein mutations leading to higher infectivity rate and vaccine escape
poses threat to the vaccine induced protective responses. The Indian SARS-CoV-2 Genome
Consortia (INSACOG) has identified presence of 4 major VOCs i.e., B.1.1.7, B.1.1.351, P.1,
B.1.617.2 in India, that are associated with increased virulence and reduced efficacy of vaccines
[3].
2 medRxiv preprint doi: https://doi.org/10.1101/2021.08.16.21261912; this version posted August 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .
According to recent statistics from INSACOG portal (2021-08-06,
http://clingen.igib.res.in/covid19genomes/) there is a drastic increase in frequency of B.1.617.2
lineage (Delta) from 8% to ~75% after March, 2021 (Supplementary Image 1). Recent in vitro
studies showed that sera from Pfizer or the AstraZeneca vaccinated individuals is less effective
in neutralizing Delta variant in comparison to Alpha (B.1.1.7) [4]. In this study, we summarize 36
COVID vaccine breakthrough cases, which were SARS-CoV-2 RT-PCR positive despite
evidence of an antibody response following vaccination.
Methods
Data for this study were included from individuals who had been tested RT-PCR positive for
SARS-CoV-2 after vaccination for either the BBV152 (COVAXIN) indigenous Indian vaccine or
AZD1222 COVISHIELD (ChAdOx1) vaccine. Out of total 36 cases 34 received both the
recommended doses of vaccine. All the cases were tested positive between the last week of
March, 2021 and mid-June, 2021 after more than two weeks of vaccine second dose; while the
remaining two patients tested positive within two weeks of their first dose of vaccination
(Supplementary Figure 2).
Total RNA from collected nasopharyngeal swab samples was isolated using TANBead
automated RNA extraction kit. The isolated RNA was subjected to qRT-PCR using Meril Covid-
19 One Step RT-PCR Kit which includes amplification primers for viral ORF1ab and N gene for
determining viral load by Ct values. Samples with Ct values < 35 were considered for the study.
Amplicon based dual indexed paired-end libraries for viral genome sequencing were prepared
by COVIDSeq kit and sequenced using NextSeq-550 platform (Illumina). After demultiplexing,
non-Host (Human) extracted reads using Kraken2 taxonomic classifier [5], were aligned using
BWA against the Wuhan-Hu-1 (NC_045512.2) reference genome. Primer regions from the
aligned sequences were trimmed using iVar followed by removal of duplicate reads. Single
3 medRxiv preprint doi: https://doi.org/10.1101/2021.08.16.21261912; this version posted August 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .
nucleotide variants and short INDELs were called using GATK4 Haplotypecaller using ploidy 1
followed by removal of low-quality variants (Quality by depth, QD < 5) by GATK VariantFiltration.
Consensus sequences generated using BCFTOOL consensus and regions having no aligned
reads were hard-masked. Clade and lineage of the viral genomes were determined using
Nextclade (https://clades.nextstrain.org/) and PANGOLIN (pangoLEARN version 2021-07-28)
respectively [6]. Rooted (root: NC_045512.2) phylogenetic tree of 549 sequences (522 other
sequences collected in same timeframe from Odisha) was constructed using method described
in [7]. All the calculations and statistical tests were performed using base R functions.
Results
As a part of the regular COVID-19 genomic surveillance, we identified and sequenced 36
vaccine breakthrough infection cases. The study group consisted of 12 females, 24 males with
age ranging from 23 to 65 years (median = 38.50, sd = 13.58) (Supplementary table 1). All
subjects except two were fully immunized with two doses of either Covaxin/BBV1552(n=8) or
AZD1222/Covishield (n=26). The interval between two vaccine doses ranged between 27 to 49
days with a median interval of 35 days (n = 34) and the onset of infection ranged between 6 to
98 days post vaccination (median = 74, sd = 25, n = 31). Out of 36 cases, 33 reported either
single or a combination of common COVID-19 related symptoms i.e., fever, body pain, sore
throat, one individual didn’t report any symptoms during sample collection and for the rest two
samples the symptoms were unknown (Supplementary table 2). The specimens were collected
between March 29 to June 15, 2021.
Out of the 36 cases, twenty nine were identified as variants of concern Delta (80%), two
classified as Kappa (B.1.617.1) (~6%), two as B.1 (~6%), and one each in Delta (AY.3),
B.1.575, B.1.629 lineages each. In COVAXIN break-through cases 9/9 classified as Delta
variant, for COVISHIELD 20/27 were classified as Delta, 2/27 as Kappa, 2/27 as B.1, 1/27 as
4 medRxiv preprint doi: https://doi.org/10.1101/2021.08.16.21261912; this version posted August 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .
AY.3, 1/27 as B.1.575 and 1/27 as B.1.629 (Supplementary Figure 2, Supplementary table 2).
When we looked for high frequency variants (present in > 20% of samples) in these two
vaccination groups, COVAXIN cases contained an overall higher number of variants (n=42) in
comparison to the COVISHILD group (n=28) (Figure 1 A, 1B). Although two of the cases were
classified as B.1, we found both of them were classified as 21A (Delta) by Nextclade having
S:D614G and S:P681R mutations along with other lineages (Supplementary table 3). We also
found strong phylogenetic closeness of these B.1 lineages with Delta (B.1.617.2) variant cases
collected in the same time frame (Figure 1C). Looking into Spike domain specific mutations, we
observed emergence of EFR156G substitution (delE156,delF157, and R158G) in N-terminus
domain, which is becoming a common trait of recently reported Delta (B.1.617.2) variants [8].
Interestingly, most of the breakthrough cases shared presence of S:L452R, S:T478K in receptor
binding domain (RBD) along with S:D614G and S:P681R near S1-S2 furin cleavage site. All
these spike variants have been proposed to be associated with increased infectivity through
different mechanisms (Figure 1D) [9].
In the study group, 7 out of 36 patients reported comorbidities i.e., diabetes, blood pressure,
rest 26 patients did not report any complications, disease history is unknown for three cases.
Out of 36 patients only 1 patient was hospitalized with comorbidities, 23 recovered in home
isolation, for 8 individuals the treatment status is unknown. Except for three unknown cases, the
rest of the patients didn’t report any prior history of SARS-CoV-2 infection at the time of sample
collection.
Discussion
In this study we found that SARS-CoV-2 variant of concern Delta (B.1.617.2) is overrepresented
in the vaccine break-through cases, which could be due to its higher prevalence as well during
5 medRxiv preprint doi: https://doi.org/10.1101/2021.08.16.21261912; this version posted August 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .
that period. Recent studies suggested that the effectiveness of BNT162b2 (Pfizer) and
ChAdOx1 (AstraZeneca) has been reduced in comparison to Alpha (B.1.1.7) variant. Also the
same has been observed in neutralizing capability of sera from fully vaccinated individuals
[4,10]. One other in vitro study suggested that S:L452R and S:E484Q present in RDB domain of
Spike protein are responsible for immune escape and thereby 3-6 fold decrease in
neutralization capability of BNT162b2 sera [11].
Along with global reports of emerging variants of concerns and vaccine breakthrough infection
cases now there are new threats emerging in India as well. Presence of two new Delta (AY.1,
AY.2, AY.3) lineages has been observed in parallel with the Delta (B.1.617.2) that was first
observed in India. Recent reports of breakthrough infection cases from India also have pointed
out the predominance of Kappa (B.1.617.1) and Delta (B.1.617.2) variants [12].
Here we observed the co-occurrence of immune escape mutations and vaccine breakthrough
infections despite the presence of COVID-specific immune response in hosts. Due to a small
number of subjects and lack of more actionable clinically relevant information, we were not able
to associate any direct causality for the breakthrough infections. But our findings are consistent
with other recent reports showing the need for regular monitoring using genomics and
immunological methods. Although we observed thirty-six breakthrough cases in a span of three
months the number is very less in comparison to the total number of sequenced cases and the
subject group mostly consisted of healthcare workers and thus the probability of observing
breakthrough infection in a fully vaccinated population is very less. Continuing the virus genomic
surveillance is necessary for devising new and improved intervention for COVID-19.
6 medRxiv preprint doi: https://doi.org/10.1101/2021.08.16.21261912; this version posted August 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .
Acknowledgements
We would like to acknowledge the State Surveillance Officers (SSOs) of Odisha for their support
in arranging the samples. Thanks to Dr. Andrew A. Lamare, Dr. Sudeep Jena and Mr. Rahul
Biswal from SCB hospital, Cuttack for the samples and providing the metadata.
Institutional Review Board approval
The study plan was approved by the Institute of Life Sciences Institutional Biosafety Committee
(Ref no. V-122-MISC/2007-08/01) and the Institutional Human Ethical Committee (Ref no.
109/HEC/21). All necessary patient / participant consent has been obtained and the appropriate
institutional forms have been archived.
Funding
ILS provided an intramural grant to perform the study.
Conflicts of Interest
The authors declare no conflict of interest.
7 medRxiv preprint doi: https://doi.org/10.1101/2021.08.16.21261912; this version posted August 20, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license .
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Figure 1: SARS-CoV-2 variants of concern and phylogenetic analysis of vaccine
breakthrough cases; (A & B) Genome wide non-synonymous mutations present in high
fraction (>20%) of samples, grouped by the vaccines, (C) Phylogenetic tree of our study
genomes with other SARS-CoV-2 sequences (n=522) prevalent during the same time frame
from Odisha, rooted using Wuhan Hu01 sequence as outgroup, and (D) Non-synonymous
mutations observed in different domains of Spike protein grouped by vaccination type.
9
Figure 1
medRxiv preprint doi: https://doi.org/10.1101/2021.08.16.21261912; this version posted August 20, 2021. The copyright holder for this preprint A) (whichCOVAXIN was Cases not certified (n=9) by peerCODON_CHANGE_PLUS_CODON_DELETION review) is the author/funder, who has grantedCODON_DELETION medRxiv a licenseINTERGENIC to displayNON_SYNONYMOUS_CODING the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . 1.00
0.75
0.50
0.25 Fraction of samples
0.00 UTR ORF1ab S ORF3a M ORF7a ORF7b ORF8 N (.) (.) (.) (.) (T40I) (T95I) (I82T) (S26L) (K77T) (V82A) (T19R) (T120I) (D63G) (P309L) (L452R) (T478K) (DF119) (P681R) (D377Y) (T3255I) (T3750I) (D950N) (D614G) (G142D) (G261D) (G215C) 241 C>T (R203M) 210 G>T (V2930L) (P5971L) (P2046L) (P1640L) (P5401L) (P4715L) (F3138S) (T3646A) (P2287S) (A6319V) (A3209V) (A1306S) (V3718A) (G5063S) 9678 T>C 7124 C>T 9891 C>T 1191 C>T 6402 C>T 5184 C>T 9053 G>T 4181 G>T 10029 C>T 11514 C>T 18176 C>T 19220 C>T 11418 T>C 16466 C>T 14408 C>T 21846 C>T 25469 C>T 26767 T>C 27752 C>T 27638 T>C 27874 C>T 29742 G>T 21792 A>C 28916 G>T 29402 G>T 28881 G>T 22917 T>G 22995 C>A 11201 A>G 15451 G>A 23403 A>G 24410 G>A 21987 G>A 22344 G>A 28461 A>G (EFR156G) 23604 C>G 21618 C>G 28270 TA>T 28247 AGATTTC>A B) 22028 GAGTTCA>G COVISHIELD Cases (n=27) CODON_CHANGE_PLUS_CODON_DELETION CODON_DELETION INTERGENIC NON_SYNONYMOUS_CODING
1.00
0.75
0.50
0.25 Fraction of samples
0.00 UTR ORF1ab S ORF3a M ORF7a ORF8 N (.) (.) (.) (.) (I82T) (S26L) (V82A) (T19R) (T120I) (D63G) (P309L) (T478K) (L452R) (DF119) (D377Y) (P681R) (D950N) (G261D) (D614G) 241 C>T (R203M) 210 G>T (P1640L) (P5401L) (P4715L) (A3209V) (V3718A) (H2092Y) (G5063S) 6539 C>T 5184 C>T 9891 C>T 1191 C>T 25469 C>T 26767 T>C 27752 C>T 27638 T>C 11418 T>C 16466 C>T 14408 C>T 28881 G>T 29742 G>T 29402 G>T 22995 C>A 22917 T>G 15451 G>A 22344 G>A 28461 A>G 23403 A>G 24410 G>A (EFR156G) 21618 C>G 23604 C>G 28270 TA>T 28247 AGATTTC>A 22028 GAGTTCA>G
C) D)
COVAXIN (n=9) COVISHIELD (n=27) YY144Y Y145* VYY143 T95I T19R R214L M153V L176I K77T H146
G261D NTD G142D EFR156G E154K D111 A264 A263 A262T Wuhan-WH01 A222V A163
T478K L452R RBD E484Q
D950N HR1
C1247F CT
V1104L V1061 Q613H Q1071H P681R Other Lineage L5F I1130M AY.3 G669 B.1 D614G B.1.1.7 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 B.1.575 Fraction of samples B.1.617.1 Vaccination B.1.617.2 CT SNV B.1.629 COVAXIN HR1 DEL COVISHIELD Other NTD
Other
RBD