key: cord-0294226-bgdx3jag authors: Kale, P.; Gupta, E.; Bihari, C.; Patel, N.; Rooge, S.; Pandey, A.; Bajpai, M.; Khillan, V.; Chattopadhyay, P. C.; Devi, P.; Maurya, R.; Jha, N.; Mehta, P.; Kumar, M.; Sharma, P.; Saifi, S.; S, A.; Alam, S.; Uppili, B.; Faruq, M.; Agrawal, A.; Pandey, R.; Sarin, S. K. title: Clinicogenomic analysis of breakthrough infections by SARS CoV2 variants after ChAdOx1 nCoV- 19 vaccination in healthcare workers date: 2021-07-03 journal: nan DOI: 10.1101/2021.06.28.21259546 sha: e79af6e4d6d304a7e5a05fe14a052daccd4109ec doc_id: 294226 cord_uid: bgdx3jag Background: India saw a massive surge and emergence of SARS CoV2 variants. We elucidated clinical and humoral immune response and genomic analysis of vaccine breakthrough (VBT) infections after ChAdOx1 nCoV-19 vaccine in healthcare workers (HCWs). Methods: The study was conducted on 1858 HCWs receiving two doses of ChAdOx1 nCoV- 19 vaccine. Serial blood samples were collected to measure SARS CoV2 IgG and neutralizing antibodies. 46 RT-PCR positive samples from VBT infections were subjected to whole genome sequencing (WGS). Results: Infection was confirmed in 219 (11.79%) HCWs of which 21.46% (47/219) were non-vaccinated, significantly more (p <0.001) than 9.52% (156/1639) vaccinated group. VBT infections were significantly higher in doctors and nurses compared to other hospital staff (p <0.001). Unvaccinated individuals had 1.57 times higher risk of infection compared to partially vaccinated (p 0.02) and 2.49 times than fully vaccinated (<0.001). Partially vaccinated were at higher risk of infection than fully vaccinated (RR 1.58,p 0.01). There were 3 (1.36%) severe cases and 1 death in unvaccinated group compared to none in the vaccinated. Non-response after 14 days of second dose was seen in 6.5% (3/46) and low antibody levels (1-4.62 S/CO) in 8.69% (4/46). Delta variant (B.1.617.2) was dominant (69.5%) and reinfection was documented in 4 (0.06%) HCWs. Conclusions: Nearly one in ten vaccinated HCWs can get infected, more so with only single dose (13.65%) than two doses (8.62%). Fully vaccinated are better protected with higher humoral immune response. Genomic analysis revealed an alarming rise of Delta variant (B.1.617.2) in VBT infections. 3 (1.36%) severe cases and 1 death in unvaccinated group compared to none in the vaccinated. Non-response after 14 days of second dose was seen in 6.5% (3/46) and low antibody levels (1) (2) (3) (4) .62 S/CO) in 8.69% (4/46) . Delta variant (B.1.617.2) was dominant (69.5%) and reinfection was documented in 4 (0.06%) HCWs. Conclusions: Nearly one in ten vaccinated HCWs can get infected, more so with only single dose (13.65%) than two doses (8.62%). Fully vaccinated are better protected with higher humoral immune response. Genomic analysis revealed an alarming rise of Delta variant (B.1.617.2) in VBT infections. . CC-BY-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. The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.28.21259546 doi: medRxiv preprint Vaccines have emerged as an effective countermeasure against the accelerating global expansion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causing coronavirus disease 2019 . Multiple vaccine rollout across the world will curb the pandemic by protecting the vulnerable population groups. 1 Vaccines are crucial for the individuals who fail to develop protective immunity, those with underlying medical or debilitating conditions, immunodeficiencies or elderly population. As the disease pathophysiology and effective immunity through vaccination is still an evolving science, we cannot predict protective immune response and breakthrough infections in vaccinated population. In India till April 30 th 2021, the cumulative vaccinations administered were 15,48,54,096, including 94,10,892 in Healthcare Workers (HCWs) as 1st dose and 62,40,077 HCWs who had taken both the doses. 2 There was a sudden surge in the COVID19 cases from mid-March to May 2021, with the worst affected states being Maharashtra and Delhi. 3 This surge was predominantly caused by highly transmissible variants with potentials of immune evasion. 4 4 Breakthrough (BT) infections after vaccination have been reported from USA at a rate of 0.01% and from a chronic care medical facility in India at a rate of 16.8%. 5, 6 In . CC-BY-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. The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06. 28.21259546 doi: medRxiv preprint India, the ChAdOx1 nCoV-19/ AZD1222 (Covishield) vaccine was the main vaccine used across the country. We studied the VBT infections, their clinical characteristics, immune response and genomic analysis in a large cohort of vaccinated HCWs. The study was conducted at the Institute of Liver and Biliary Sciences (ILBS), Delhi, India in collaboration with the CSIR-Institute of Genomics and Integrative Biology (CSIR-IGIB). The study had the approval of the Institutional Ethical Committee (IEC/2020/77/MA07) and was conducted following the Declaration of Helsinki. The study was conducted on 1858 HCWs at our university hospital from January to May 2021. Detailed demographic details and history of co-morbidities, prior history of COVID-19 infection, etc. were recorded. For the purpose of this study the following definitions were followed: the detection of SARS-CoV-2 RNA or antigen in a respiratory specimen collected from an individual who had received either one or two doses of vaccine. . CC-BY-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) The copyright holder for this preprint this version posted July 3, 2021. (Genscript, USA). SARS CoV2 infection was confirmed in 203 HCWs during the study period. Combined nasopharyngeal and oral swabs were collected in viral transport media (VTM) from all the symptomatic study subjects. Samples were tested for the presence of dual genes E and RdRP by real time reverse transcriptase polymerase chain reaction(RT-PCR) using commercial kit (Q-Line®, M/s POCT Pvt Ltd.) Samples with detection of both the genes with < 35 cycle threshold (Ct ) value were considered as positive. . CC-BY-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) The copyright holder for this preprint this version posted July 3, 2021. . CC-BY-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) The copyright holder for this preprint this version posted July 3, 2021. FastQC v0.11.9 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) was used to check the Phred quality score for all sequences. For all samples, the quality score threshold was 20 and above. Adapter trimming was performed using the Trim Galore tool v0.6.1 (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and alignment of sequences was performed using the HISAT2 [7] algorithm on human data build GRCh38. To remove any human sequences from the dataset, samtools v1.12 [8] were used to remove aligned sequences. Henceforth, only unaligned sequences were taken into consideration. BCFTools v1.12 was used to generate consensus fasta and variant calling. We sequenced 46 vaccinated COVID-19 positive samples that were aligned to NC_045512 reference genome using MAFFT v7.475 [9] multiple alignment tool. The aligned sequences were trimmed to remove gaps and a phylogenetic tree was generated using the default model of the IQ-TREE tool v2.0.3 [10] . The tree was visualized using FigTree v1.4.4 [11] . Further, the assembled SARS-CoV-2 genomes were assigned lineages using the package Phylogenetic Assignment of Named Global Outbreak LINeages (PANGOLIN) [12] . The lollipop plot is generated in RStudio using g3viz, rtracklayer, and trackViewer packages followed by data visualization using the ggplot2 package. All the figures were updated using Inkscape software [13] . The consensus fasta generated for this study has been submitted in GISAID under the accessions: EPI_ISL_2424135 = 1, EPI_ISL_2426145 to EPI_ISL_2426189 = 45 . CC-BY-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. The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.28.21259546 doi: medRxiv preprint Data analysis: The collected data was entered in excel spreadsheet and expressed as median or as percentage. The categorical data were analysed using Chi-Square or Fisher's exact test. The statistical analysis was done using SPSS software version 22. A total of 1858 HCWs were enrolled in the study. The HCWs were divided in two groups . CC-BY-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. The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.28.21259546 doi: medRxiv preprint (17/553) in GDA. (Table 1 ) (Figure1). Doctors and nurses in the fully vaccinated group were significantly more prone to acquire BT as compared to technical staff, non-medical staff and GDA (P<0.001). Reinfection was documented in 4 HCWs, 6-8 months after the previous infection in the vaccinated group. These included doctors (2), who had received 2 doses of vaccine and nursing staff (1) and non-medical staff (1) who had received single dose of vaccine. The viral load was low in first 3 (Ct value 33.10, 29.92, 29.52) and high in the remainder (Ct value 20.52). All had mild symptoms of fever and body ache and no significant lung involvement. The median age was 34 (IQR: 21- . CC-BY-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. The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.28.21259546 doi: medRxiv preprint (1.36%) severe cases and 1 death in the unvaccinated group. There was no significant association seen in co-morbidities with the BT. Genomic sequencing and analysis were available in 46 samples. We analysed the humoral immune response post vaccination in them (Table 3) . Thirty-two (78.26%) had received 2 doses of vaccine and 14 (30.4%) received single dose. The approximate burden of viral load as measured indirectly by Ct value was almost similar in both the groups and did not differ with the vaccination status. The median Ct value was During the initial period of vaccination drive from January to March, the recommended gap between two doses was 4-6 weeks. Hence the study protocol included sample collection at 28 . CC-BY-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. The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.28.21259546 doi: medRxiv preprint days. At this time point, rise in antibodies was significantly higher in partially vaccinated group (7.61 + 4.06 vs 4.41 + 3.27, p= 0.001 (S/CO, mean + SD) (Fig 2) . The antibody levels were tested 14 days after the second dose and showed; non-response in Table 4 ). The Kappa variant was significantly associated with moderate cases in partially vaccinated HCWs (p=0.04) ( Table 5) . CC-BY-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. The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.28.21259546 doi: medRxiv preprint The COVID-19 cases reported in Delhi during the study period were retrieved. The clustering of cases in the HCWs overlapped with the surge in the cases in the community. The number of cases, viral load and the antibody response in these cases is depicted in figure 7 . Our prospective observational study documents the clinicogenomic analyses of largest is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.28.21259546 doi: medRxiv preprint 66·7%. 19 The findings in our study shows similar efficacy 14 days after the second dose (60%). There was predominance of BT among the doctors and nurses as compared to the technicians, non-medical staff and GDA. There was no difference in the infection rates in these is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 3, 2021. was also reported from another tertiary care centre in Delhi during the surge of infections during March and April. 6 The variants of concern B.1.617.1 partially impairs neutralizing antibodies elicited by BNT162b2 vaccine. 24 Despite the existence of several RBD mutations, studies to examine the capability of vaccinee sera to neutralise circulating SARS-CoV-2 variations showed that strains such as B.1.1.7 remain potently neutralised. Several RBD-specific antibodies can bind only the open spike protein 24 and it has been discovered that D614G renders the spike protein more sensitive to neutralising antibodies by increasing the likelihood of the open conformation occurring. 25 Nevertheless, other circulating SARS-COV-2 variations escape vaccine-induced humoral immunity. 26 The L452R mutation was found in roughly 93 percent of the vaccinated COVID-19 positive samples, indicating that this mutation was positively selected. Leucine-452 is located in the RBD receptor-binding motif, at the point of direct interaction with the ACE2 receptor. Its substitution with arginine is expected to result in substantially greater receptor affinity as well as escape from neutralising antibodies. 27 The structural investigation of RBD mutations L452R and E484Q, as well as P681R at the furin cleavage region, revealed . CC-BY-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. The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.28.21259546 doi: medRxiv preprint the possibility of enhanced ACE2 binding and the rate of S1-S2 cleavage, resulting in improved transmissibility. The same two RBD mutations resulted in reduced binding to selected monoclonal antibodies (mAbs), decreasing mAb neutralising capacity, according to an Indian study. 28 Considering the immune escape conferred by these RBD mutations, needs further survey. Here we report the alarming rise in the SARS Cov2 variants causing breakthrough infections in vaccinated healthcare workers. Further immune surveillance and characterization of the variants is warranted to understand the pathophysiology and design measures to curb the spread of the variants. . CC-BY-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. The copyright holder for this preprint this version posted July 3, 2021. . CC-BY-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. The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.28.21259546 doi: medRxiv preprint . CC-BY-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. The copyright holder for this preprint this version posted July 3, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 3, 2021. . CC-BY-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. The copyright holder for this preprint this version posted July 3, 2021. RR-Relative risk, ICU-Intensive care unit, HCW-healthcare worker . CC-BY-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. The copyright holder for this preprint this version posted July 3, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 3, 2021. . CC-BY-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) The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.28.21259546 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.28.21259546 doi: medRxiv preprint . CC-BY-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) The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.28.21259546 doi: medRxiv preprint . CC-BY-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) The copyright holder for this preprint this version posted July 3, 2021. ; https://doi.org/10.1101/2021.06.28.21259546 doi: medRxiv preprint . CC-BY-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. 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