key: cord-0302199-u8r2klq3
authors: Zaveri, L.; Singh, R.; Basu, P.; Banu, S.; Mukherjee, P.; Vishwakarma, S.; Sahni, C.; Kaur, M.; Singh, N. K.; Yadav, A. K.; Ashish, A.; Mishra, S.; Tiwari, S.; Mishra, S. P.; Vodapalli, A.; Bollu, H.; Das, D.; Singh, P. P.; Chaubey, G.; Sowpati, D. T.; Tallapaka, K. B.
title: Genomic analysis of SARS-CoV-2 breakthrough infections from Varanasi, India
date: 2021-09-22
journal: nan
DOI: 10.1101/2021.09.19.21262487
sha: baf95968a74c3353fecca1b2fc0c23de3a2443f2
doc_id: 302199
cord_uid: u8r2klq3

Studies worldwide have shown that the available vaccines are highly effective against SARS-CoV-2. However, there are growing laboratory reports that the newer variants of concerns (VOCs e.g. Alpha, Beta, Delta etc) may evade vaccine induced defense. In addition to that, there are few ground reports on health workers having breakthrough infections. In order to understand VOC driven breakthrough infection we investigated 14 individuals who tested positive for SARS-CoV-2 after being administered a single or double dose of Covishield (ChAdOx1, Serum Institute of India) from the city of Varanasi, which is located in the Indian state of Uttar Pradesh. Genomic analysis revealed that 78.6% (11/14) of the patients were infected with the B.1.617.2 (Delta) variant. Notably, the frequency (37%) of this variant in the region was significantly lower (p<0.01), suggesting that the vaccinated people were asymmetrically infected with the Delta variant. Most of the patients tested displayed mild symptoms, indicating that even a single dose of the vaccine can help in reducing the severity of the disease. However, more comprehensive epidemiological studies are required to understand the effectiveness of vaccines against the newer VOCs.

With the growing knowledge on SARS-CoV-2 spread and infection, it is now clear that accomplishing community immunity by natural infections is more putative than ground reality. Therefore, in the global fight against SARS-CoV-2, vaccinating the entire population against the virus is an important step in slowing down the pandemic. By establishing community immunity with the help of vaccines, it might be possible to prevent further large outbreaks. The Government of India initiated the vaccination programme against SARS-CoV-2 in January 2021 with the aim of vaccinating the entire Indian population by the end of 2021. In the initial phase, the Government targeted frontline workers at highest risk to the disease. With greater availability of vaccines, the scope of vaccination was widened to include people above the age of 65 and then above 45. Due to the staggered nature of vaccination and the onset of a large second wave, reports emerged of people testing positive for SARS-CoV-2 despite being vaccinated [1] [2] [3] [4] . At a total population of approximately 1 billion, the time taken to vaccinate the entire Indian population would be quite long. This is further complicated by the fact that most SARS-CoV-2 vaccines approved for use are double dose vaccines with a minimum interval of four weeks between the two doses [1] . Partial immunity in the population has led to a situation where there are variants of SARS-CoV-2 emerging against which existing vaccines have decreased efficacy [6] .

Reports of breakthrough infections have impacted the public confidence in the efficacy of these vaccines. However, a majority of these infections are in healthcare workers who have repeated and high exposure to SARS-CoV-2 unlike the population at large. Also the routine nasal testing of fully vaccinated people have shown low titers of SARS-CoV-2 [7] . Therefore, it is essential to study breakthrough infections and their outcomes among general populations. In our survey in Varanasi, a city in the Indian state of Uttar Pradesh, we found 14 individuals who tested positive for SARS-CoV-2 after receiving either a single or double dose of Covishield (ChAdOx1) vaccine.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint We studied in detail the virus variants, the main symptoms and the fate of these individuals due to this infection.

This study was approved by the local institutional ethical committee. Written consent was taken from patients wherever applicable. 

Viral RNA from patients that tested positive were shipped to CCMB, Hyderabad India. The samples were sequenced using Illumina COVIDSeq (Illumina) according to the manufacturer's protocol. Briefly, first strand cDNA was synthesized in sets of 96 samples using random hexamers and reverse transcriptase. The viral first strand cDNA was then amplified using two primer pools. Post amplification, the two amplified pools were combined and the samples were . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint Tagmented. The Tagmentation process fragments the PCR products and tags the fragmented products with adapter sequences. Using paramagnetic beads, the tagmented samples were purified. An amplification step adds a 10 base pair i7 index and i5 adapter required for sequencing cluster generation. The 96 samples were then pooled into a single tube and purified using paramagnetic beads. The bead purification also serves to select for optimal sized fragments required for sequencing. The purified sample was quantified and 8 such pools were 

Basecalling was performed on raw image data using bcl2fastq v2.20.0.422 (Illumina). Quality control of FASTQ files was performed using FASTQC v0.11.9 [8] . Poor quality bases and adapters were trimmed using Trimmomatic [9] . Alignment of reads to the indexed reference genome NC_045512.2 was done using HISAT2 v2.1.0 [10] . Consensus sequences were generated using bcftools from the BAM file post alignment. Coverage across the genome was calculated using samtools depth. PANGO v3.0.5 was used to assign lineages to the consensus sequences [11] . Mutations in the sequences were identified using Nextclade v1.1.0 [12] . For sequences where lineages were not designated by PANGO, a manual assignment was provided based on the presence of key lineage defining mutations.

Datasets of 444 genomes from Uttar Pradesh, and 172 global breakthrough samples deposited on GISAID (till 15th July 2020) were used for the analysis (Supplementary Table S1 ). The metadata and submitting institutions are listed in Supplementary Data. The sequences were aligned against the WH1 reference genome using MAFFT and IQTREE was used to construct a . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 22, 2021. ; https://doi.org/10.1101/2021.09.19.21262487 doi: medRxiv preprint phylogenetic time tree using the sequence collection date [13, 14] . The trees were viewed and appropriately annotated using iTOL [15] .

The samples were collected in the month of April 2021 when the vaccine was available only for those above the age of 45 thus the median age of patients studied was 55. Of the 14 breakthrough samples, 4 patients were female while the remaining were males. The oldest female patient was in the 50 to 55 years age group while the oldest male patient was in the 80 to 85 years age group. The 'youngest' female patient was in the 50 to 55 years age group and male patient was in the 40 to 45 years age group.

The patients displayed a range of mild symptoms commonly associated with SARS-CoV-2 (Table 1) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 22, 2021. ; https://doi.org/10.1101/2021.09.19.21262487 doi: medRxiv preprint with VOCs, in light of studies revealing reduced neutralization of VOCs by vaccine sera, is a matter of concern. Achieving community immunity by rapid vaccination of populations across the world, as well as continuously monitoring the behavior of viral strains in response to existing vaccines, is imperative to prevent the evolution and spread of such VOCs.

All sequenced samples were deposited on GISAID. Accession IDs for submitted samples are available in Supplementary Table S1 . Accession IDs for samples not sequenced in this study and used in data analysis are available in Supplementary Table S1 .

This study was supported by a CSIR grant (MLP0128). GC is supported by Faculty IOE grant BHU (6031). RS is supported by funds from ICMR and DHR.

Vaccination dates, symptom onset and date of testing for each of the patients was plotted to compare the duration in days between the last vaccination dose administered, onset of the disease and confirmation of SARS-CoV-2. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 22, 2021. ; https://doi.org/10.1101/2021.09.19.21262487 doi: medRxiv preprint

Efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine against SARS-CoV-2 variant of concern 202012/01 (B.1.1.7): an exploratory analysis of a randomised controlled trial

SARS-CoV-2 infection rates of antibody-positive compared with antibodynegative health-care workers in England: a large, multicentre, prospective cohort study (SIREN). The Lancet Elsevier

COVID-19 Vaccine Breakthrough Infections Reported to CDC -United States

Breakthrough COVID19 infections after vaccinations in healthcare and other workers in a chronic care medical facility

United Nations, Department of Economic and Social Affairs, Population Division. World population prospects Highlights, 2019 revision Highlights

Epidemiological and evolutionary considerations of SARS-CoV-2 vaccine dosing regimes

Initial report of decreased SARS-CoV-2 viral load after inoculation with the BNT162b2 vaccine

Trimmomatic: a flexible trimmer for Illumina sequence data

Graph-based genome alignment and genotyping with HISAT2 and HISATgenotype

Nextstrain: real-time tracking of pathogen evolution

MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization

IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era

Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation

Dysregulation of Immune Response in Patients With Coronavirus

Contact tracing in the context of COVID-19

We thank Tulasi Nagabandi for all the help and support with sequencing.

The authors declare no conflict of interest . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted September 22, 2021. ; https://doi.org/10.1101/2021.09.19.21262487 doi: medRxiv preprint It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted September 22, 2021. ; https://doi.org/10.1101/2021.09.19.21262487 doi: medRxiv preprint It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted September 22, 2021. ; https://doi.org/10.1101/2021.09.19.21262487 doi: medRxiv preprint