key: cord-0266033-3dmcseuh authors: Dingemans, J.; van der Veer, B. M. J. W.; Gorgels, K. M. F.; Hackert, V.; Hensels, A. Y. J.; den Heijer, C. D. J.; Hoebe, C. J. P. A.; Savelkoul, P. H. M.; van Alphen, L. B. title: Investigating SARS-CoV-2 breakthrough infections per variant and vaccine type date: 2021-11-24 journal: nan DOI: 10.1101/2021.11.22.21266676 sha: ab72cc9db1dd9c1eae7f3f1d70684fcc5f924f3a doc_id: 266033 cord_uid: 3dmcseuh Breakthrough SARS-CoV-2 infections have been reported in fully vaccinated individuals, in spite of the high efficacy of the currently available vaccines, proven in trials and real-world studies. Several variants of concern (VOC) have been proffered to be associated with breakthrough infections following immunization. In this study, we investigated 378 breakthrough infections recorded between January and July 2021 and compared the distribution of SARS-CoV-2 genotypes identified in 225 fully vaccinated individuals to the frequency of circulating community lineages in the region of South Limburg (The Netherlands) in a week-by-week comparison. Although the proportion of breakthrough infections was relatively low and stable when the Alpha variant was predominant, the rapid emergence of the Delta variant lead to a strong increase in breakthrough infections, with a higher relative proportion of individuals vaccinated with Oxford-AstraZeneca or J&J/Janssen being infected compared to those immunized with mRNA-based vaccines. A significant difference in median age was observed when comparing fully vaccinated individuals with severe symptoms (83 years) to asymptomatic cases (46.5 years) or individuals with mild-to-moderate symptoms (42 years). There was no association between SARS-CoV-2 genotype or vaccine type and disease symptoms. Interestingly, symptomatic individuals harbored significantly higher SARS-CoV-2 loads than asymptomatic vaccinated individuals and breakthrough infections caused by the Delta variant are associated with increased viral loads compared to those caused by the Alpha variant. Altogether, these results indicate that the emergence of the Delta variant might have lowered the efficiency of particular vaccine types to prevent SARS-CoV-2 infections and that, although rare, the elderly are particularly at risk of becoming severely infected as the consequence of a breakthrough infection. Breakthrough SARS-CoV-2 infections have been reported in fully vaccinated individuals, in spite of 17 the high efficacy of the currently available vaccines, proven in trials and real-world studies. Several 18 variants of concern (VOC) have been proffered to be associated with breakthrough infections 19 following immunization. In this study, we investigated 378 breakthrough infections recorded 20 between January and July 2021 and compared the distribution of SARS-CoV-2 genotypes identified in 21 225 fully vaccinated individuals to the frequency of circulating community lineages in the region of 22 South Limburg (The Netherlands) in a week-by-week comparison. Although the proportion of 23 breakthrough infections was relatively low and stable when the Alpha variant was predominant, the 24 rapid emergence of the Delta variant lead to a strong increase in breakthrough infections, with a 25 higher relative proportion of individuals vaccinated with Oxford-AstraZeneca or J&J/Janssen being 26 infected compared to those immunized with mRNA-based vaccines. A significant difference in 27 median age was observed when comparing fully vaccinated individuals with severe symptoms (83 28 years) to asymptomatic cases (46.5 years) or individuals with mild-to-moderate symptoms (42 29 years). There was no association between SARS-CoV-2 genotype or vaccine type and disease 30 symptoms. Interestingly, symptomatic individuals harbored significantly higher SARS-CoV-2 loads 31 than asymptomatic vaccinated individuals and breakthrough infections caused by the Delta variant 32 are associated with increased viral loads compared to those caused by the Alpha variant. Altogether, 33 these results indicate that the emergence of the Delta variant might have lowered the efficiency of 34 particular vaccine types to prevent SARS-CoV-2 infections and that, although rare, the elderly are 35 particularly at risk of becoming severely infected as the consequence of a breakthrough infection. 36 37 Introduction 38 Since its discovery in December 2019 1 , SARS-CoV-2 has infected more than 250 million people and 39 caused about 5 million deaths 2 . Although SARS-CoV-2 has a limited mutation rate of about 2 40 mutations per month 3 , which can be attributed to its 3' to 5' exonuclease proofreading activity 4 , its 41 rapid expansion across the globe has led to the emergence of numerous variants 5,6 . Whereas the 42 majority of variants initially harbored mutations outside the gene encoding the spike protein, a 43 number of variants have emerged with mutations in the spike protein that affect infectivity or 44 immune evasion. For example, the mutation leading to the D614G substitution in the spike protein 45 emerged soon after SARS-CoV-2 spread to Europe at the beginning of the first wave 7 . This mutation 46 greatly affected the fitness of SARS-CoV-2 by stabilizing the open conformation of the spike protein, 47 enhancing viral infectivity and leading to dominance of this variant across the globe 7-9 . More 48 recently, a number of variants of concern (VOC) have emerged that harbor mutations in the receptor 49 binding domain (RBD) of the spike protein, enhancing the affinity for the human angiotensin-50 converting enzyme 2 (ACE2) receptor 10 or leading to evasion of the immune system 11 . In November 51 2020, the B.1.1.7 variant was first reported as it quickly gained dominance in the UK, imposing a 52 great burden on the healthcare system due to increased infectivity and slightly heightened 53 mortality [12] [13] [14] . Both the N501Y and V69/H70del mutations in the spike protein of B.1.1.7 have been 54 shown to enhance its infectivity 10,14,15 , while the Y144del mutation was found to be in the antigenic 55 supersite 16 . Although the B. The first individuals in the region of South Limburg received their 2 nd dose of the Pfizer-BioNTech 175 vaccine in week 4 and the number of people who were fully vaccinated steadily increased starting 176 from week 6 ( Figure 1A ). The majority of people received an mRNA-based vaccine (mostly Pfizer-177 BioNTech) and only starting from week 9, the first individuals were fully vaccinated with Oxford-178 AstraZeneca, while the first fully vaccinated individuals who received the J&J/Janssen vaccine started 179 accumulating in week 21. 180 The first breakthrough infections were reported in week 8 and continually occurred starting from 181 week 11, with a frequency ranging between 0.3 to 13.7% relative to the total number of infections 182 ( Figure 1A ). The number of breakthrough cases steeply increased in week 27, after most COVID-19 183 measures were relaxed in The Netherlands and comprised disproportionally higher numbers of 184 individuals who received Oxford-AstraZeneca or J&J/Janssen compared to the mRNA-based vaccines 185 Pfizer-BioNTech/Moderna ( Figure 1B ). This was reflected by a considerably lower number of 186 breakthrough cases relative to the number of administered vaccines for Pfizer-BioNTech/Moderna 187 (0.14%) compared to Oxford-AstraZeneca (0.89%) or J&J/Janssen (0.46%) ( Table 2) . 188 The distribution of SARS-CoV-2 variants in fully vaccinated individuals generally coincides with 190 their regional prevalence 191 From the 378 breakthrough cases that were identified from week 2 until week 28, 225 were 192 successfully genotyped via whole-genome sequencing. The remaining samples had a Ct-value >32 193 (n=19), were unavailable for sequencing (n=83), or did not comply to quality requirements of 194 sequencing (n=51). 195 At the same time, regional surveillance of SARS-CoV-2 variants was performed on a weekly base. The 196 non-VOC genotypes, which mainly consisted of the B.1.177 and B.1.221 lineages that have been 197 prominent in the region since the summer of 2020 30 , were responsible for more than 50% of CoV2 infections up to week 7, after which the Alpha variant became dominant ( Figure 1B ). From this 199 point, the frequency of Alpha infections rose quickly, constituting >90% of SARS-CoV-2 cases in week 200 11. In week 8 the first two breakthrough cases were identified, belonging to the Alpha variant 201 ( Figure 1B ). Starting from week 11, the great majority of SARS-CoV-2 breakthrough infections were 202 caused by the Alpha variant, with the exception of an occasional Gamma or a non-VOC infection 203 ( Figure 1C ). The first breakthrough infection caused by the Delta variant was detected in week 25, 204 when this variant was responsible for 50% of cases in the region and became dominant. As COVID-19 205 measures were relaxed in The Netherlands by the end of week 25, the number of breakthrough 206 cases peaked during week 27 and 28, of which the great majority was caused by the Delta variant 207 that was also dominant at that moment as determined via regional surveillance. The proportion of 208 breakthrough infections caused by the Delta variant seems to be slightly higher than its share in 209 genomic surveillance (100% vs 64% in week 26, 95% vs 84% in week 27, and 100% vs 95% in week 210 28). 211 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. ; is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. ; Of the 225 isolates that were sequenced, 4 were non-variants of concern (non-VOC), 112 Alpha 237 variant, 3 Gamma, and 106 Delta variant. In total, 46 non-lineage defining mutations were identified 238 in the spike protein of the 225 isolates that were sequenced (Table 2) is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. ; Table 3 . Non-lineage-defining mutations in the spike protein found in this study. 245 L5F 8 3.6% 0 (0%) 8 (7%) 0 (0%) 0 (0%) S12F 1 0.4% 0 (0%) 1 (1%) 0 (0%) 0 (0%) 1.3% 0 (0%) 3 (3%) 0 (0%) 0 (0%) V1228L 1 0.4% 0 (0%) 1 (1%) 0 (0%) 0 (0%) I1232L 4 1.8% 0 (0%) 4 (4%) 0 (0%) 0 (0%) M1237V 1 0.4% 0 (0%) 0 (0%) 0 (0%) 1 (1%) V1264L 2 0.9% 0 (0%) 1 (1%) 0 (0%) 1 (1%) L1265I 8 3.5% 0 (0%) 0 (0%) 0 (0%) 8 (8%) a N=225, consisting of 4 non-VOC, 112 Alpha, 3 Gamma, and 106 Delta isolates. b Not applicable, this 246 mutation is a defining mutation of the Gamma variant. 247 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint People experiencing severe symptoms were significantly older than those who remained 256 asymptomatic or experienced mild to moderate symptoms 257 A significant difference in median age was found between asymptomatic (46.5 years old) and severe 258 symptomatic cases (83 years old) on one hand, and mild-moderate symptomatic (42 years old) and 259 severe COVID-19 cases on the other hand ( Figure 4A ). Two out of seven severe SARS-CoV-2 260 infections had a fatal outcome. Nevertheless, the great majority of cases (75.4%) belonged to the 261 mild-moderate category. There was no association between SARS-CoV-2 genotype and disease 262 severity (Table S3) . 263 When the time of the first positive test after receiving the 2 nd dose was plotted against disease 264 severity for each case, no significant difference could be observed ( Figure 4B) . 265 However, a significant difference in median CT value was observed between the three different 266 groups of symptoms, as lower median CT values were observed in the severe and mild-moderate 267 categories compared to asymptomatic cases ( Figure 4C&D ). 268 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. ; https://doi.org/10.1101/2021.11.22.21266676 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. ; https://doi.org/10.1101/2021.11.22.21266676 doi: medRxiv preprint Finally, individuals who received the Pfizer-BioNTech vaccine were significantly older (median age 51 287 years old) than those who received the Oxford-AstraZeneca (median age 35.5 years old) or the 288 J&J/Janssen vaccine (median age 22 years old) ( Figure 5D ). 289 There was no association between vaccine type and disease severity (Table S4) is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint When comparing the distribution of SARS-CoV-2 variants retrieved from fully vaccinated individuals 341 compared to the frequency of variants that were circulating in the entire population in the region of 342 South Limburg, we found that breakthrough infections were caused by SARS-CoV-2 genotypes that 343 were present in similar proportions during genomic regional surveillance. Before the Delta variant 344 emerged, the great majority of breakthrough infections were caused by the Alpha variant with the 345 exception of an occasional Gamma or a non-VOC infection. This is in line with data from the CDC in 346 which 555 SARS-CoV-2 isolates from a total of 10,262 SARS-CoV-2 vaccine breakthrough infections 347 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. ; were sequenced and the proportion of VOCs identified in breakthrough infections (64%) 348 corresponded with their prevalence in the national genomic surveillance (70%) during the period 349 between January 1 and April 30, 2021 32 . 350 When the Delta variant emerged in the South Limburg region in week 23, the number of positive 351 cases was low and this variant quickly became dominant in the following weeks when there was a 352 surge in the number of positive cases, making it difficult to estimate what proportion of 353 breakthrough infections are attributable to the Delta variant as compared to regionally surveillance 354 data. Nevertheless, the proportion of breakthrough infections caused by the Delta variant seems to 355 be slightly higher than its share in genomic surveillance (100% vs 64% in week 26, 95% vs 84% in 356 week 27, and 100% vs 95% in week 28), although still being in line with our observations for the 357 Alpha variant and other VOCs. and T95I mutations are in close proximity to the antigenic supersite residues, they have no effect on 372 neutralization 16 . Nevertheless, the effect of these mutation in the Delta background on 373 neutralization has not been determined yet and the T95I mutations is found in many VUM/VOI, 374 including the B. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted November 24, 2021. ; the elderly and healthcare workers, whereas the J&J/Janssen vaccine was administered to young 435 adults who received their second dose right before relaxation of COVID-19 measures when there 436 was a spike in positive cases. 437 Strengths of this study were the ability to monitor and compare breakthrough infections against the 438 regional genomic surveillance over an extensive period, even before the first vaccines were 439 administered as well as the regional follow-up of cases, allowing monitoring of the amount of 440 breakthrough infections at a high resolution. A high proportion (60%) of all breakthrough cases were 441 successfully sequenced further enhancing the resolution. Additionally, analysis were conducted on 442 cases one day after vaccination giving an overall picture of susceptibility directly post vaccination. 443 Limitations were the fact that the number of reported breakthrough infections are an 444 underestimation of the effective number, due to asymptomatic or mild cases or because of severe 445 cases in local hospitals that were not reported to the regional public health service. Furthermore, 446 asymptomatic cases were only followed up for a limited amount of time, possibly leading to the 447 underestimation of symptomatic cases. Thirdly, only samples from cases with sufficiently high viral 448 loads could be sequenced (5' exoribonuclease that is critically involved 500 in coronavirus RNA synthesis SARS-CoV-2 one year on: 503 evidence for ongoing viral adaptation SARS-CoV-2 variants, spike mutations and immune escape Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases 507 Infectivity of the COVID-19 Virus Spike mutation D614G alters SARS-CoV-2 fitness SARS-CoV-2 spike-protein D614G mutation increases virion spike density and 512 infectivity SARS-CoV-2 B.1.1.7 and B.1.351 514 spike variants bind human ACE2 with increased affinity SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing 517 antibodies Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in 519 Increased mortality in community-tested cases of SARS-CoV-2 lineage 521 B.1.1.7 Genomic characteristics and clinical effect of the emergent SARS-CoV-2 UK: a whole-genome sequencing and hospital-based cohort study Recurrent emergence and transmission of a SARS-CoV-2 spike deletion 526 N-terminal domain antigenic mapping reveals a site of vulnerability for 528 SARS-CoV-2 Sensitivity of infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants to 530 neutralizing antibodies Resistance of SARS-CoV-2 variants to neutralization by monoclonal and 532 serum-derived polyclonal antibodies SARS-CoV-2 variants of concern partially escape humoral but not T-cell 535 responses in COVID-19 convalescent donors and vaccinees Neutralising antibody activity against SARS-CoV-2 VOCs B.1.617.2 and 538 B.1.351 by BNT162b2 vaccination mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants. 540 Escape from neutralizing antibodies by SARS-CoV-2 spike protein 542 variants Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-544 induced sera Effectiveness of Covid-19 Vaccines against the B.1.617.2 (Delta) Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent 550 serum Rapid SARS-CoV-2 whole-genome sequencing and analysis for 552 informed public health decision-making in the Netherlands Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex 555 with receptor ACE2 revealed by cryo-EM UCSF Chimera--a visualization system for exploratory research and 557 analysis Spread of a SARS-CoV-2 variant through Europe in the summer of 2020 SARS-CoV-2 variants associated with vaccine breakthrough in the 561 Delaware Valley through summer 2021. medRxiv COVID-19 Vaccine Breakthrough Infections Reported to CDC -United 564 Effect of Delta variant on viral burden and vaccine effectiveness against 567 new SARS-CoV-2 infections in the UK SARS-CoV-2 Delta VOC in Scotland: demographics, risk of hospital admission, 569 and vaccine effectiveness SARS-CoV-2 572 vaccine protection and deaths among US veterans during 2021 DC COVID-19 vaccination data, 575 A novel B.1.1.523 SARS-CoV-2 variant that combines many spike mutations linked to 578 immune evasion with current variants of concern. bioRxiv Ineffective neutralization of the SARS-CoV-2 Mu variant by convalescent and 581 vaccine sera. bioRxiv Variant-driven multi-wave pattern of COVID-19 via a Machine Learning 583 analysis of spike protein mutations. medRxiv Clinical Characterization and Genomic Analysis of Samples from COVID-19 Breakthrough Infections during the Second Wave among the Various States of India Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 589 Delta variant Infections caused by the Delta variant (B.1.617.2) of SARS-CoV-2 591 are associated with increased viral loads compared to infections with the Alpha variant 592 (B.1.1.7) or non-Variants of Concern Characterisation of vaccine breakthrough infections of SARS-CoV-2 Delta 595 and Alpha variants and within-host viral load dynamics in the community Viral loads of Delta-variant SARS-CoV-2 breakthrough infections 598 after vaccination and booster with BNT162b2 Occurence and Patient Characteristics of COVID-19 Breakthrough 601 Infections Hospitalisation among vaccine breakthrough COVID-19 infections SARS -CoV-2 T-cell immunity to variants of concern following 605 vaccination. bioRxiv Thymic Aging May Be Associated with COVID-19 607 Virological characteristics of SARS-CoV-2 vaccine breakthrough 609 infections in health care workers. medRxiv Covid-19 Breakthrough Infections in Vaccinated Health Care Workers Antibody Response Following Vaccination With BNT162b2 and mRNA-1273 Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR US CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute 619 Respiratory Syndrome Coronavirus 2 We would like to thank Edou Heddema from Zuyderland MC and Boris Vlaemynck from Synlab 470Belgium for providing samples and data. 471