Sex: M
Vaccine: COVID19
Symptoms: COVID-19, Gene sequencing, SARS-CoV-2 test, Vaccination failure
Clinical narrative: tested positive for SARS-CoV-2/Fever/ cough/ fatigue; tested positive for SARS-CoV-2/Fever/ cough/ fatigue; This is a literature report from an unspecified journal in 2021, entitled Transmission event of SARS-CoV-2 Delta variant reveals 1 multiple vaccine breakthrough infections. A contactable other hcp reported similar events for 3 patients. This is the second of three reports for patient 2.   Question: Which SARS-CoV-2 variant is responsible for 6 cases of vaccine breakthrough, one interventional monoclonal antibody treatment, and one death? Findings: Viral sequencing revealed 6 vaccinated patients were infected with the Delta SARS CoV-2 variant. With no histories of vaccine breakthrough, this suggests Delta variant may possess immune evasion in patients that received the Pfizer BNT162b2, Moderna mRNA-1273, and Covaxin BBV152. Meaning: Delta variant may pose the highest risk out of any currently circulating SARS-CoV-2 variants, with increased transmissibility over Alpha variant and possible vaccine breakthrough. Abstract is as follows: Importance: Vaccine breakthrough by an emergent SARS-CoV-2 variant poses a great risk to global public health. Objective: To determine the SARS-CoV-2 variant responsible for 6 cases of vaccine breakthrough. Design: Nasopharyngeal swabs from suspected vaccine breakthrough cases were tested for SARS-CoV-2 by qPCR for (place name)-Hu1 and Alpha variant. Positive samples were then sequenced by Swift Normalase Amplicon Panels to determine the causal variant. Setting: Transmission event occurred at events surrounding a wedding outside of (city, state). Two patients from another country, likely transmitted the Delta variant to other guests. Participants: Following a positive SARS-CoV-2 qPCR test at a third-party site, six fully vaccinated patients were investigated. Three males and three females ranged from 53 to 69 years old. One patient suffered from diabetes while three others were classified as overweight. No significant other comorbidities were identified. None of the patients had a history of failed vaccination. Introduction: High numbers of global SARS-CoV-2 infections have led to the emergence of variants, notably Alpha variant (B.1.1.7 UK), Beta (B.1.351 S. Africa), Gamma (P.1 Brazil), Epsilon (B.1.429 (state)), Iota (B.1.526 (state)) and now, Delta and Kappa (B.1.617.2 and B.1.617.1). Each of these strains gained advantageous mutations to become a dominant strain, e.g., Iota first discovered 23Nov2020, represented 45 percentage of new cases as of 07Feb20211. Increased transmissibility results from genomic changes such as nonsynonymous mutations in the receptor-binding domain (RBD) of the S-gene (encodes the spike protein) conferring higher binding affinity to host angiotensin-converting enzyme 2 (ACE2) receptors or more efficient cleavage by Transmembrane Serine Protease 2 (TMPRSS2) and subsequently, viral entry. Mutations could also lead to vaccine breakthrough. The spike protein's RBD is immune dominant, targeted by convalescent sera and vaccine-elicited antibodies (Pfizer BNT162b26), though evidence suggest substantial role of the amino-terminal domain (NTD). Mutations in the RBD therefore pose a risk of allowing immune evasion to one or more of the current vaccines . The Kappa and Delta variants emerged from this countries state of (state) in Dec2020, contributing to a resurgence of cases in the country, representing 70 percentage of daily new cases on 02May2021 The B.1.617 lineage is now widely circulating in over 50 countries based on viral sequence data and is classified as a variant of concern by the disease control. The Kappa and Delta variant lineages are defined by 7 and 8 nonsynonymous mutations in the S protein respectively. Emergent data suggests partial immunity to the Kappa variant, as convalescent sera and vaccine-elicited (Pfizer BNT162b2 and Moderna mRNA-1273) antibodies show a 2.3- and 4-fold reduction in neutralization in vitro respectively (noting that this study used protein-pseudo typed lentiviruses lacking the T478K mutation found in Delta variant) . A test negative case control study estimated the effectiveness of vaccination (two weeks post second vaccination) against symptomatic disease by Delta variant to be as high as 88 percentage for Pfizer BNT162b2 (compared to 93 percentage for Alpha variant). Here we describe a transmission of a Delta variant containing SARS-CoV-2 strain, between family members associated with events surrounding a wedding with 92 attendees, near (city, state). Attendance required guests be fully vaccinated and took place outdoors in a large, open-air tent. To date, 6 individuals have tested positive for SARS-CoV-2, all patients were symptomatic, one patient severely enough to receive monoclonal antibody infusion treatment and one patient has died. Encounter timings and viral sequence similarities suggest the strain containing the Delta variant was transmitted to wedding guests from two patients travelling from another country. With no history of vaccine failure in these patients, our observations suggest these are true cases of vaccine breakthrough, mediated by the Delta variant. Results: In early Apr2021, Patient 0a, a man with no comorbidities, and Patient 0b, a woman with diabetes, travelled from another country to attend a wedding in (state) (designated 0a and 0b due to difficulty establishing true patient 0). Both tested negative for SARS-CoV-2 by qPCR as part of the pre-flight criteria. Formal wedding events were held outdoors and in a large open-air tent and attendance required full vaccination (Patient 0a and 0b travelled to (city) 10 days after their second doses of Covaxin BBV152, Table 1). Patients 1-5 confirmed having close encounters with Patient 0a and 0b at the wedding. Events were attended by fewer than 100 guests. On the evening of the first night of the wedding, Patient 0b complained of fatigue but associated it with diabetes and jet lag. Patient 0a developed a cough two days after the wedding and both him and Patient 0b developed a fever three days after. Patient 0a and 0b tested positive for SARS-CoV-2 by nasal swab qPCR 4 days after the wedding at a third-party site. Patient 0a's symptoms progressed over the following days and was admitted to the hospital on day 6 post wedding. He was transferred to (hospital name) hospital in the (state) Medical Center with worsening symptoms. A month after the wedding, Patient 0a died from complications of COVID-19. Five other guests have tested positive for SARS-CoV-2 following confirmed interactions with Patient 0a and 0b. All positive patients received Pfizer BNT162b2, Moderna mRNA-1273, or Covaxin BBV152 (Table 1). Six of these have experienced symptoms of COVID-19 (Table 1). Patient 1, who received the Pfizer BNT162b2 vaccine developed severe symptoms and was admitted to (hospital name) for monoclonal antibody infusion treatment (company name.) on ten days after the wedding. The density of vaccine breakthrough resulting in COVID-19 symptoms suggested the patients were carrying a SARS-CoV-2 variant. To characterize the variant, total RNA was extracted from nasopharyngeal swabs of each of the 6 patients. All positive for SARS-CoV-2 Wuhan-Hu-1 and negative for Alpha variant by qPCR (Table 1). Human RNase P (RP) gene control values suggested sampling of patients and RNA isolation were performed optimally. Amplicon libraries were successfully prepared from all 6 qPCR positive samples (N1 Ct value 17-29), with 900,754-2,381,756 pass-filter reads generated using (organization name) Sarscov2 analysis pipeline (Table 1). Median sequence coverage ranged from 2085x to 12,932x with less than 99.7 percentage of the genome covered at 40x or greater. All 6 samples were identified as the SARS-CoV-2 Delta variant based on the presence of the 10 mutations listed by the disease control's Selected Characteristics of SARS-CoV-2 Variants of Interest. These mutations, located on the S protein were T19R, (G142D), 156del, 157del, R158G, L452R, T478K, D614G, P681R, D950N. 156del, 157del, R158G are annotated as a single mutation (S:GAGTTCA22028G:Glu156_Arg158delinsGly) due to their proximity (Figure 1 Spike protein mutation prevalence in SARS-CoV-2 variant lineages. Only mutations found in greater than 50 percentage in at least one lineage are displayed. Green text denotes a mutation found in all patients in this report. Figure modified from Outbreak.info). Phylogenetic analysis places each patient sample in a subclade of the Delta variant (white box, Figure 2 Phylogenetic analysis of SARS-CoV-2 variants. All patients (white box) cluster in a sub clade of the Delta variant (red). Sequences obtained from GISAID). Discussion: Ending the current SARS-CoV-2 pandemic requires limiting the spread through continued vigilance of masking, social distancing, and vaccination. Variants emerge from areas experiencing uncontrolled viral spread and display increased transmissibility due to mutations in the spike protein. These mutations may occur in the antigenic region of the RBD, altering binding sites for vaccine-elicited antibodies. Mutations such as the ones found in the Kappa variant, provide partial resistance to antibody neutralization (Pfizer BNT162b2, Moderna Mrna 1273, Regeneron8 and Covaxin BBV152), likely due to changes in epitope sequence. Vaccine breakthrough by highly transmissible variants (Delta variant up to 50 percentage more transmissible than Alpha variant, SAGE) could lead to significant setbacks ina pandemic control efforts, requiring renewed social distancing and masking efforts. Significant vaccine breakthrough could necessitate vaccine boosters or targeted lockdowns to reduce spread of infection. An analysis of spike protein epitopes found several antigenic regions (IDa-IDi, (name).). Delta variant spike protein contains mutations in three of these regions (450-469 IDf, 480-499 IDg, and 522-646 IDh, Figure 1) possibly resulting in decreased neutralization by vaccine-elicited antibodies. According to the cases presented in this study, antibodies elicited in patients receiving Pfizer BNT162b2, Moderna mRNA-1273, and Covaxin BBV152 may provide decreased immunity to the Delta variant. It is possible that some individuals in this study failed to produce an effective immune response to their immunization, however, none of the patients had a history of vaccine failure. Our observations support continued efforts to generate SARS-CoV-2 genomic sequences from positive patient samples, in order to identify possible vaccine breakthrough mutations. The continued effectiveness of vaccine-elicited antibodies towards SARS-CoV-2 variants highlights the importance of vaccination efforts. Partial efficacy of current vaccines (70percentage at 75percentage coverage) is theoretically sufficient to stop a pandemic 11. Slowing the spread could prevent emergence of future variants, hastening the end of this pandemic. Materials and Methods: Specimen collection and ethical considerations, All individuals were initially tested by third party SARS-CoV-2 testing sites. To confirm, nasopharyngeal swabs were collected by a physician or nurse as close to the first positive test as possible. Samples were submitted to the (Research Centre name) for RNA extraction and the Human Genome Sequencing Center for qPCR confirmation. Protocols for collection, qPCR testing, and whole genome sequencing were approved by the (college name) Institutional Review Board (H-#). cDNA synthesis and amplicon libraries. RNA extracted from nasopharyngeal swabs of six individuals that tested positive for SARS- COV-2 using qPCR was converted to 1st strand cDNA using SuperScript IV First-Strand Synthesis System (company), Cat. No. #). The 1st strand cDNA reaction was performed starting with 10 l of total RNA in 25 l reaction mix, which was incubated at 23-degree C for 10 minutes followed by 50 degree C for 50 minutes. The resulting 1st strand cDNA was then diluted with DEPC treated water, where for two samples with Ct remaining four samples, with Ct less than 24, cDNA was diluted 2 times, respectively. This diluted 1st strand cDNA (10 ul) was used as input for amplification of the SARS-CoV-2 viral genome, using the SARS-CoV-2 Additional Genome Coverage Panel (Cat#COVG1V2-96). This panel was designed against SARS-CoV-2 Wuhan-Hu-1 strain (NC 045512.2) and has 345 amplicons of 116-255 bp (average 150 bp) that cover 99.7 percentage (29,828 of 29,903 total bases) of the genome. These amplicons come in a single tube, and the workflow involves two rounds of PCR, a multiplex PCR (4 plus 18 cycles) and the indexing PCR (9 cycles) to generate sequence ready libraries. The reaction mixes and the thermocycler conditions were performed according to the Swift Normalase Amplicon Panels (SNAP) Workflow. Libraries were barcoded with 8bp unique dual indices at the Indexing PCR. For library normalization, the 2 nM Normalase I protocol was performed on libraries individually, followed by pooling 5 l of each post-Normalase I library to perform Normalase II reaction, which results in sequence ready library pool. Before sequencing, he normalized library pool concentration was measured using qPCR with KAPA Library Quantification Kits (company), KK#, #) Illumina Sequencing. The pooled SARS-CoV-2 amplicon libraries were sequenced on Illumina NovaSeq 6000 S4 flowcell to generate 2x150bp reads. Swift amplicon data analysis. Swift amplicon data were analyzed using Swift Biosciences Sarscov2 analysis pipeline (website) with minimum read coverage depth of 3. The GATK variants were next filtered with allele fraction less than equal to 80, and min read depth 30x12,13 . Swift analysis pipeline produced variant vcf file, consensus genome, pangolin lineage and Nextclade assignment (website). Variant vcf from Swift amplicon data was also annotated using SnpEff13. Phylogenetic Analysis. Sequences for the designated variants of concern and variants of interest by disease control, was downloaded from GISAID on 02Jun2021 . All samples downloaded from GISAID, were analyzed using Pangolin V3.0.3 with pangoLEARN 27Jun2015 to ensure that the variant designation assigned by GISAID is accurate. Global alignment of 334 sequences including the sequence from the current study was done using MAFFT v7.480 . Maximum likelihood phylogenetic tree with boot strap (5,000) was generated using IQ-Tree V2.1.2 . Annotation and visualization of the tree was carried out by using Fig Tree v1.4.4 (website) Clades were labeled with the WHO nomenclature.   Table 1: Patient 2, Sex: Male, Age: 59-64, Height(cm): 170.1, Weight(kg): 77, Vaccine received: Pfizer BNT162B2, Symptoms: Fever, cough, fatigue, Comorbidities (As defined by the Centers for Disease Control and Prevention): Hypertension, N1 qPCR Ct: 24.  No follow-up attempts are needed; Information on lot/batch number cannot be obtained.; Sender's Comments: Based on the information currently available, a lack of efficacy with BNT162b2 in this patient cannot be completely excluded.,Linked Report(s) : US-PFIZER INC-202100911825 same reporter/drug/event, different patient;US-PFIZER INC-202100913182 same reporter/drug/event, different patient