key: cord-0279440-gbgcfa2o
authors: Shamier, M. C.; Tostmann, A.; Bogers, S.; De Wilde, J.; IJpelaar, J.; Van Der Kleij, W.; De Jager, H.; Haagmans, B.; Molenkamp, R.; Oude Munnink, B.; van Rossum, C.; Rahamat-Langendoen, J.; Van Der Geest, N.; Bleeker-Rovers, C.; Wertheim, H.; Koopmans, M.; GeurtsvanKessel, C.
title: Virological characteristics of SARS-CoV-2 vaccine breakthrough infections in health care workers
date: 2021-08-21
journal: nan
DOI: 10.1101/2021.08.20.21262158
sha: 67cc11d7ae1e52a3201a71a4d0e5727776e1f064
doc_id: 279440
cord_uid: gbgcfa2o

SARS-CoV-2 vaccines are highly effective at preventing COVID-19-related morbidity and mortality. As no vaccine is 100% effective, breakthrough infections are expected to occur. We analyzed the virological characteristics of 161 vaccine breakthrough infections in a population of 24,706 vaccinated healthcare workers (HCWs), using RT-PCR and virus culture. The delta variant (B.1.617.2) was identified in the majority of cases. Despite similar Ct-values, we demonstrate lower probability of infectious virus detection in respiratory samples of vaccinated HCWs with breakthrough infections compared to unvaccinated HCWs with primary SARS-CoV-2 infections. Nevertheless, infectious virus was found in 68.6% of breakthrough infections and Ct-values decreased throughout the first 3 days of illness. We conclude that rare vaccine breakthrough infections occur, but infectious virus shedding is reduced in these cases.

Once COVID-19 vaccines became available, health care workers (HCWs) were among the first groups to 36 be vaccinated and reach high vaccine coverage. Registered vaccines have been highly effective in 37 preventing clinically significant coronavirus disease 2019 , caused by severe acute respiratory 38 syndrome coronavirus 2 (SARS-CoV-2) 1 , and have shown to reduce the incidence of 39 infections 2,3 . However, mild breakthrough infections in a small percentage of vaccine recipients have been 40 described [4] [5] [6] [7] [8] [9] which is not surprising as none of the registered vaccines will provide sterile immunity against 41 infection [10] [11] [12] [13] . In a setting of mass vaccination, the BNT162b2 vaccine was highly effective (92%) at 42 preventing infection from 7 days after the second dose 14 , but a recent study from Israel described vaccine 43 breakthrough infections in 39 health care workers vaccinated with the BNT162b2 mRNA vaccine. The 44 alpha variant was identified as the main causative strain and a majority of cases presented low Ct-values 45 (<30), indicating probable infectivity 4 . For the single dose Ad26.COV2.S adenoviral vector vaccine, a phase 46 IV study reported a 76.1% effectiveness to prevent infection from 14 days after vaccination 15 . The 47 effectiveness against infection with the delta (B.1.617.2) variant was 88% for the BNT162B2 vaccine and 48 67% for the ChAdOx1 vaccine, moderately lower than against infection with the alpha (B.1.1.7) variant 16 . 49 Up to present, little is known about the virological kinetics of SARS-CoV-2 breakthrough infections, and 50 the role of the vaccinated host in the transmission cycle. Better understanding of the dynamics of 51 breakthrough infections is essential to define infection prevention and (public) health policies during the 52 next phase of the pandemic. In this study, we report the virological findings of 161 vaccine breakthrough 53 infections occurring from April to July 2021 in the Netherlands. The infections occurred in HCWs working 54 in two tertiary care hospitals, who were immunized with various mRNA and viral vector vaccines. 55

Infections were caused predominantly by the SARS-CoV-2 delta variant and virus culture was performed 56 as a proxy for infectivity. 57 58 . 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 August 21, 2021.

A total of 161 fully vaccinated HCWs diagnosed with COVID-19 by PCR were included in this study. In 60   accordance with case definitions defined by the Centers for Disease Control and Prevention, infections  61 were classified as breakthrough infections if the date of the first positive SARS-CoV-2 RT-PCR was more 62 than 14 days after completion of all recommended vaccine doses 17 . Cases with symptom onset <14 days 63 after the last vaccine dose and cases with a previous positive test <45 days prior were not considered 64 breakthrough infections. In parallel with a surge in cases in the general Dutch population 18 , an increased 65 incidence of breakthrough infections in HCWs was observed in July 2021. In 126 samples a SARS-CoV-2 66 lineage could be identified, 90.5% of these showed presence of the delta variant. The mean age of the 67 HCWs with a breakthrough infection was 25.5 years and 91% were less than 50 years old ( Table 1 ). All 68 infections were mild and did not require hospital admission. The individuals were vaccinated between 69 January and May 2021 with either an mRNA vaccine or a viral vector vaccine. Table 1 shows the 70 distribution of vaccines among all HCWs and among the HCWs with breakthrough infections. Although 71 the data may imply an overrepresentation of Ad26.COV2.S and BNT162b2 vaccine recipients among the 72 cases, this study was not designed to compare vaccine effectivity. The indication to receive a certain 73 vaccine was not random and data on risk factors for exposure were not recorded, therefore potential 74 confounders could not be adjusted for. 75 Table 1 shows the distribution of Ct-values of the breakthrough infections, as a proxy for the 76 nasopharyngeal viral load. Ct-values were significantly lower in symptomatic breakthrough infections (μ 77 = 23.2) than in asymptomatic breakthrough infections (μ = 26.7), corresponding to higher viral loads (p = 78 0.022, t-test). In symptomatic vaccinated HCWs, the Ct-values decreased significantly throughout the first 79 days from symptom onset and were lowest on the third day of illness ( Figure 1A ). There were no 80 statistically significant differences in Ct-values between HCWs immunized with the 4 different vaccines. 81 . 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 August 21, 2021. ;  Furthermore, the time since the administration of the last vaccine dose showed no clear relationship with 82

Ct-values (R 2 = 0.02, p = 0.13, linear regression). 83

Subsequently, RT-PCR positive swabs were tested for the presence of infectious virus using cell culture. 84

As a reference, we used the (first positive) samples from mild primary infections that occurred in the same 85 cohort of HCWs prior to the onset of vaccination, these infections were primarily caused by SARS-CoV-2 86 D614G. The mean Ct-value upon diagnosis was similar between these two groups: 24.6 (15.3 -33.9) for 87 vaccinated HCWs and 24.2 (14.53 -33.8) for unvaccinated HCWs (p = 0.53, t-test) ( Figure 1B ). The SARS-88

CoV-2 culture of nasopharyngeal swabs was positive in 68.6% of vaccinated HCWs versus 84.9% of 89 unvaccinated HCWs with primary infections (p = 0.005, t-test). As the probability of culture positivity 90 depends on viral load 19 , this was corrected for using a probit regression model with both viral load and 91 vaccination status as predictors. Figure 1C It remains a challenge to assess infectivity of an individual based on clinical sampling. Although RT-PCR is 103 a highly sensitive method to diagnose SARS-CoV-2 infection, it detects RNA produced in infected cells 104 . 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 August 21, 2021. ; https://doi.org/10.1101/2021.08.20.21262158 doi: medRxiv preprint rather than whole (infectious) virions and is therefore not an optimal indicator of infectivity 19 . Ct-values 105 are sometimes used to differentiate between phases of infection, but the definition of a cut-off value is 106 complicated, due to the large variety of assays and clinical samples. Considering these limitations of RT-107 PCR, demonstrating viral viability through replication in cell culture is currently considered the best proxy 108 to demonstrate infectious virus in a clinical specimen 20, 21 . We and others previously showed that the 109 viability of SARS-CoV-2 depends on several factors among which the severity of disease, timing of 110 sampling, the type of specimen and presence of antibodies 19, 22 . To our knowledge this is the first study to 111 report on virus cultures in COVID-19 vaccine breakthrough infections. Although reduced immune 112 responses may likely account for breakthrough infections, further studies are needed to investigate 113 whether these are still able to reduce infectious virus shedding. 114

Obviously, the use of virus culture has its limitations as well: it is a laborious method only performed in 115 specialized BSL-3 laboratories and therefore not widely applicable. In addition, lack of standardization of 116 methods (e.g. the cell line used) still hampers interchangeability of results between laboratories. 117

Nevertheless, an experimental animal study on SARS-CoV-2 transmission recently confirmed a strong 118 correlation between transmission and virus culture 23 . 119

To study the effect of vaccination on infectivity, it would be preferable to compare infections occurring in 120 vaccinated and unvaccinated individuals during the same time period, to minimize the impact of different 121 SARS-CoV-2 variants. Due to the high vaccine coverage in HCW, we diagnosed very few infections in 122 unvaccinated HCWs. For this reason, we used infections prior to the onset of vaccination as a reference. 123

Although the predominant SARS-CoV-2 variant differed between groups, the groups were similar with 124 respect to demographic characteristics, severity of disease, testing algorithms and Ct-values upon 125 diagnosis. The study participants comprise a population immunized with several vaccines, which reflects 126 the current situation in many countries. This study was not designed to detect differences in vaccine 127 effectiveness, as HCWs who received different vaccines also differed with respect to demographic 128 . 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 August 21, 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 August 21, 2021. ;

SARS-CoV-2 RT-PCR tests were performed on nasopharyngeal swabs, using the SARS-CoV-2 test on a 163

Cobas® 6800 system (Roche Diagnostics) in Erasmus MC and using the Aurora Flow (Roche Diagnostics) in 164 Radboud Medical Center. Using a formula based on E-gene calibration curves, cycle threshold (Ct) values 165 were converted to viral load in Log 10 RNA copies/mL. This conversion method was previously validated 25 . 166

Typing of SARS-CoV-2 variants using RT-PCR or next generation sequencing 167 

Virus culture was performed on all samples collected in the Erasmus Medical Center, by inoculating Vero 177 cells (clone 118) as previously described 19 . All cultures were performed in twofold, with one replicate for 178 immunofluorescence analysis after acetone fixation at 48h of incubation. The second replicate was 179 microscopically examined for the presence of cytopathic effect daily for 2 weeks. Viral culture was 180 considered negative if no cytopathic effect was observed after 14 days of incubation. To investigate how 181 the probability of the binary outcome (culture positivity) depends on viral load and vaccination, the 182 culture results were analyzed using probit regression. 183 . 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) . 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 August 21, 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 August 21, 2021. ; 

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David van de Vijver, Jolanda Kreeft-Voermans, Amber Weevers, Anoushka Comvalius, Djenolan van Mourik and Michael van der Voorden are gratefully acknowledged for their technical and analytical contributions.