key: cord-0282087-nlwpv0z5
authors: Garcia-Knight, M. A.; Anglin, K.; Tassetto, M.; Lu, S.; Zhang, A.; Goldberg, S. A.; Catching, A.; Davidson, M. C.; Shak, J. R.; Romero, M.; Pineda-Ramirez, J.; Diaz-Sanchez, R.; Rugart, P.; Donohue, K.; Massachi, J.; Sans, H. M.; Djomaleu, M.; Mathur, S.; Servellita, V.; McIlwain, D.; Gaudiliere, B.; Chen, J.; Martinez, E. O.; Tavs, J. M.; Bronstone, G.; Weiss, J.; Watson, J. T.; Briggs-Hagen, M.; Abedi, G. R.; Rutherford, G. W.; Deeks, S. G.; Chiu, C.; Saydah, S.; Peluso, M. J.; Midgley, C. M.; Martin, J. N.; Andino, R.; Kelly, J. D.
title: Infectious viral shedding of SARS-CoV-2 Delta following vaccination: a longitudinal cohort study
date: 2022-05-19
journal: nan
DOI: 10.1101/2022.05.15.22275051
sha: 004dfd03a773293e43d2f4d111f6f4f574f97f14
doc_id: 282087
cord_uid: nlwpv0z5

The impact of vaccination on SARS-CoV-2 infectiousness is not well understood. We compared longitudinal viral shedding dynamics in unvaccinated and fully vaccinated adults. SARS-CoV-2-infected adults were enrolled within 5 days of symptom onset and nasal specimens were self-collected daily for two weeks and intermittently for an additional two weeks. SARS-CoV-2 RNA load and infectious virus were analyzed relative to symptom onset stratified by vaccination status. We tested 1080 nasal specimens from 52 unvaccinated adults enrolled in the pre-Delta period and 32 fully vaccinated adults with predominantly Delta infections. While we observed no differences by vaccination status in maximum RNA levels, maximum infectious titers and the median duration of viral RNA shedding, the rate of decay from the maximum RNA load was faster among vaccinated; maximum infectious titers and maximum RNA levels were highly correlated. Furthermore, amongst participants with infectious virus, median duration of infectious virus detection was reduced from 7.5 days (IQR: 6.0-9.0) in unvaccinated participants to 6 days (IQR: 5.0-8.0) in those vaccinated (P=0.02). Accordingly, the odds of shedding infectious virus from days 6 to 12 post-onset were lower among vaccinated participants than unvaccinated participants (OR 0.42 95% CI 0.19-0.89). These results indicate that vaccination had reduced the probability of shedding infectious virus after 5 days from symptom onset.

having infectious virus after 5 days of symptoms compared to unvaccinated participants (infected with mostly pre-delta viral lineages), even though both groups had a similar magnitude of infectious virus at or near the peak. These data help improve our understanding of the duration of the infectious period when infection occurs following vaccination and serves as a reference for future studies of shedding dynamics following infections with novel variants of concern.

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Understanding the impact of vaccination on viral shedding and infectiousness of SARS-CoV-2 is key to the public health response against the COVID-19

pandemic. COVID-19 vaccines are highly effective against severe COVID-19 illness including hospitalization and death 1 and reduce transmission [2] [3] [4] . However, effectiveness against infection is variable, in part due to the emergence of viral variants of concern (VOC) which are able to evade neutralizing antibody responses 5 .

Clinical studies have shown a reduction in vaccine effectiveness against symptomatic infection with the VOCs Delta 6,7 , Beta 8 , Gamma 9 and Omicron 10, 11 .

Prior to December 2021, the Delta and Gamma variants were associated with a higher proportion of infections in the United States (US) following vaccination compared to other variants that were dominant 12, 13 and vaccine breakthrough infections with these variants were documented in diverse settings 14, 15 . How vaccination modulates viral shedding and the infectious period of highly transmissible viral variants is not well understood.

Most investigations of SARS-CoV-2 shedding by vaccination status have focused on viral RNA [16] [17] [18] [19] [20] . Comparisons of vaccinated and unvaccinated individuals show limited effects of vaccination on peak viral RNA loads 19, 20 , particularly as time since vaccination increases 17 . By contrast, faster viral RNA clearance has been shown following vaccination [18] [19] [20] , suggesting a potential shortening of the infectious period. However, as viral RNA is not a direct measure of infectious virus and viral RNA is detected for substantially longer than infectious virus among unvaccinated individuals 21 , a critical gap exists when inferring potential periods of infectiousness following vaccination.

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The copyright holder for this preprint this version posted May 19, 2022. ;  https://doi.org/10.1101/2022.05. 15.22275051 doi: medRxiv preprint To address this, we analyzed specimens from SARS-CoV-2-infected adults who formed part of a longitudinal cohort established to characterize household transmission dynamics and the natural history of SARS-CoV-2 infection among nonhospitalized persons. Participants were recruited from September 2020 to October 2021, a period that spanned successive waves of the pandemic, and the introduction of vaccines against COVID-19. We compared key shedding outcomes between participants stratified by vaccination status.

Our analysis included 84 non-hospitalized participants with acute SARS-CoV-2 infection (based on health provider-ordered molecular testing) who were >18 years of age, immunocompetent, and did not receive monoclonal antibody therapy ( Figure   1 viral genomes from unvaccinated and vaccinated individuals, respectively. In accordance with the circulation of viral lineages at the time of recruitment, among unvaccinated individuals, 30/52 (58%) were infected with lineages not classified as variants of concern and 5/52(10%) and 1/52 (2%) were Epsilon and Alpha lineage, respectively. By contrast, 26/32 (81%) infections following vaccination were Delta lineage and sub-lineages.

The quantity of viral RNA was assessed in a total of 1080 nasal samples from 84 participants (Supp. Figure 1) . Overall, copies of nucleocapsid (N) and envelope (E) genes in each nasal specimen were of similar magnitudes and highly correlated with each other (R = 0.98, P <0.001) (Supp. Table 2) . However, we observed a significantly faster . 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) (Figure 2) .

To define the infectious period at the individual level, viral culture was done in a targeted manner on 416/650 (64%) specimens from unvaccinated participants and 242/430 (56%) specimens from vaccinated participants (Supp. Figure 1 ). In addition, infectious titers in specimens from each participant with the maximum quantity of viral RNA were determined. The proportion of unvaccinated ( Table 2 ).

However, this comparison was not statistically significant when including participants who had no detectable infectious virus in any sample (P=0.12). The duration of infectious virus shedding was not found to be modulated by mRNA vaccine type or time between vaccination and infection (Supp. Table 2) .

Overall, the duration of infectious virus shedding was significantly correlated with the maximum viral RNA load (P <0.01; Supp. Figure 3 ). In addition, maximum viral RNA load was strongly correlated with the infectious viral titer in the same specimen (R 2 = 0.78, P=<0.001; Figure 3A ). No differences in maximum infectious titers were observed between vaccination groups (P=0.60, Table 2 ).

Throughout the infectious period, the proportion of specimens with infectious virus was lower in fully vaccinated participants compared to unvaccinated participants ( Figure 3B ). The odds of detecting infectious virus over the first 5 days post symptom onset did not differ for vaccinated compared to unvaccinated participants (OR 0.76, 95% CI 0.29-1.95). However, from days 6 -12, the odds were reduced for vaccinated compared to unvaccinated participants (OR 0.42 95% CI 0.19-0.89, P=0.02). In addition, a lower proportion of vaccinated participants had infectious virus after 5 days of symptom onset compared to unvaccinated participants ( Table 2 , P=0.03). Symptom resolution preceded the cessation of infectious viral shedding in most participants; however, amongst the 44 symptomatic participants with infectious virus shedding for > 5 days after symptom onset, 3/32 (9.4%) of unvaccinated and 0/12 (0%) of fully vaccinated participants had infectious virus after symptoms resolved.

Our analysis indicates that when infected with SARS-CoV-2, individuals with a full primary vaccination course have a reduced likelihood of shedding infectious virus from the nose from 5 days after symptom onset, have a more rapid decline of viral RNA from day of maximal load, and, when infectious, exhibit a shorter duration of infectious virus detection, as compared to unvaccinated individuals. However, we observed no impact on the magnitude of infectious virus at or near the peak of infectivity. These combined data suggest that although full vaccination may be inefficient at reducing infectiousness in early infection, it likely leads to a reduction in the duration of infectiousness. These findings are consistent with previous reports that fully vaccinated adults are less likely to transmit SARS-CoV-2 to others 20,22-25 . 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.

The copyright holder for this preprint this version posted May 19, 2022. and underscore the need for novel vaccination strategies that reduce viral titers during peak infectivity. suggest that the rate of infectious decline may also be impacted by vaccination.

We found significant heterogeneity in both infectious virus and viral RNA shedding dynamics in both vaccinated and unvaccinated groups with the IQR of . 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.

The copyright holder for this preprint this version posted May 19, 2022 32 . The mechanisms that underpin heterogeneity of viral shedding of SARS-CoV-2, which may involve infectious dose, pre-existing adaptive immunity 33 , host genetics 34 and innate immune responses 35 , among others, are unclear and merit targeted studies.

We identified infectious viral shedding beyond day 10 from symptom onset among both unvaccinated and vaccinated non-hospitalized adults. Prior studies have suggested that infectious virus is no longer detectable beyond 10 days after symptom onset in most mild-to-moderately ill individuals 21 , though a longer infectious period has been observed in severe infections 36 . The prolonged shedding in our study may be related to the cell line used for virus culture; Vero TMPRSS2/hACE-2 cells were engineered to prevent furin cleavage site mutations in spike following serial passage 37 and may provide enhanced sensitivity to detect infectious virus compared to other commonly used cell lines. To our knowledge, this is the first infectious viral shedding study to use cells co-expressing TMPRSS2/hACE-2.

Despite epidemiological evidence that most transmission occurs early in the infectious period 38 , our findings cannot rule out the possibility of transmission in mild infection in vaccinated and unvaccinated adults beyond 10 days, particularly in those with high peak viral loads (Supp. Figure 3) . Additional studies are needed to establish the thresholds of infectious virus titers in late infection required for community transmission.

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The copyright holder for this preprint this version posted May 19, 2022. Our study has some limitations. Unvaccinated individuals in our analysis were mostly enrolled prior to the emergence and global spread of the Delta lineage, whereas fully vaccinated individuals were almost exclusively enrolled once Delta was the dominant circulating lineage. Thus, we were unable to control for this as a potential confounder. However, previous studies have indicated that infections with Delta lineage viruses have higher peak viral loads 30, 39 and longer duration of shedding than pre-Delta lineages 40, 41 . This suggests that we may have Figure 1) . Lastly, our study was likely underpowered to detect differences in overall infectious virus shedding duration between groups, though our comparison when restricted to participants with infectious virus ( Table 2 ) is in line with the regression analysis in Figure 3 .

In summary, in addition to the protective effect of COVID-19 vaccines against severe disease, we provide evidence that full primary vaccination may reduce infectiousness during the late stage of acute infection. The impact of this finding on the transmission dynamics in households and in the community is a key question that needs further investigation. As the SARS-CoV-2 pandemic continues and the landscape of viral variants and vaccine formulations change, monitoring periods of infectiousness and their association with transmission will be key to developing . 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.

The copyright holder for this preprint this version posted May 19, 2022. ; https://doi.org/10.1101/2022.05.15.22275051 doi: medRxiv preprint public health policies that maintain transmission of SARS-CoV-2 at levels that limit morbidity, mortality, and societal disruption.

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All anterior nasal specimens were self-collected by participants. We instructed participants to rotate flocked swabs 5 times in each nostril and place in conical tubes containing 3mL of Viral Transport Medium (CDC SOP# DSR-052-03). Samples were stored in study-dedicated -20°C freezers at the participant's homes until weekly collection by the study team, at which time they were transported on dry ice for storage at -80°C. For study assays, the samples were thawed and aliquoted into screw cap microtubes. RNA extraction was done after this initial thaw cycle and virus culture was attempted following a second freeze-thaw cycle. We performed additional testing to evaluate for infectious viral degradation between these freezethaw cycles and did not observe any evidence of degradation.

Automated RNA extraction was done using the KingFisher (Thermo Scientific) automated extraction instrument and compatible extraction kits in a 96 well format. . 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.

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For each RT-qPCR reaction, 4µL of RNA sample were mixed with 5µL 2x Luna Universal Probe One-Step Reaction Mix, 0.5µL 20x WarmStart RT Enzyme Mix (NEB), 0.5µL of target gene specific forward and reverse primers and probe mix (Supp. Table 1 ). RT-qPCR were run for SARS-CoV2 N and E and for host mRNA, RNaseP, as a control for RNA extraction. Primers (forward and reverse) and probe concentrations in each mix used per RT-qPCR reaction were as follows: 8µM forward/reverse each and 4µM probe for E, 5.6µM forward/reverse each and 1.4µM probe for N and 4µM forward/reverse each and 1µM probe for RNaseP. Each 96 well RT-qPCR plate was run with a 10-fold serial dilution of an equal mix of plasmids containing a full copy of nucleocapsid (N) and envelope (E) genes (IDT) as an absolute standard for RNA copies calculation and primer efficiency assessment. RT-qPCR were run on a CFX Connect Real-Time PCR detection system (Biorad) with the following settings: 55˚C for 10 min, 95˚C for 1 min, and then cycled 40 times at 95˚C for 10s followed by 60˚C for 30s. Probe fluorescence was measured at the end of each cycle. All probes, primers and standards were purchased from IDT. We defined a sample as being RNA positive if both N and E were detected at Ct ≤40.

These two RNA targets were selected to optimize specificity of the PCR platform, excluding spurious RT-qPCR signals that would have been false positives. To control for the quality of self-sampling, RNAse P Ct values 2 standard deviations from the mean of all samples were repeated or excluded.

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The copyright holder for this preprint this version posted May 19, 2022 and 5% CO2 and checked for CPE from day 2 to 5. Vero-hACE2-TMPRSS2 cells form characteristic syncytia upon infection with SARS-CoV-2, enabling rapid and specific visual evaluation for CPE. After 5 days of incubation, the supernatant (200uL) from one well from each dilution series was mixed 1:1 with 2x RNA/DNA Shield (Zymo) for viral inactivation and RNA extraction as described above. Among specimens with visible CPE, the presence of infectious SARS-CoV-2 was confirmed by RT-qPCR using N primers as described above. All assays were done in the BSL3 facility at Genentech Hall, UCSF, following the study protocol that had received Biosafety Use Authorization.

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The copyright holder for this preprint this version posted May 19, 2022. ; https://doi.org/10.1101/2022.05.15.22275051 doi: medRxiv preprint

Viral titers were determined by conventional plaque assay. Briefly, nasal specimens were diluted at 10-fold or 4-fold serial dilutions six times in DMEM (Gibco) supplemented with 100ug/mL penicillin and streptomycin (Gibco). 250uL of each sample dilutions were added the wells of 6-well plate seeded with confluent Vero-hAce2-TMPRSS2. Cells were cultured for 1hr in a humidified incubator at 37°C in 5% CO2. After incubation, 3 mL of a mixture of MEM containing a final concentration of 2% FCS, 1x penicillin-streptomycin-glutamine and 1% melted agarose, maintained at 56°C, was added to the wells. After 72 h of culture as above, the wells were fixed with 4% paraformaldehyde for 2 hrs, agarose plugs were removed, and wells were stained with 0.1% crystal violet solution. Plaques were counted and titres were expressed as plaque forming units (pfu)/mL.

For each participant, the nasal specimen with the highest detectable RNA level, as previously determined by RT-qPCR targeting the N gene, was selected for sequencing. Whole genome sequencing was done by following the ARTIC Network amplicon-based sequencing protocol for SARS-CoV-2 43 . Briefly, specimens were thawed and converted to cDNA using the Luna RT mix (NEB). Arctic V3 multiplex PCR primer pools (IDT) were used to generate amplicons that were barcoded using the Native Barcode expansion kits 1-24 (Nanopore), pooled and used for adaptor ligation. Libraries were run on a MinION sequencer (Oxford Nanopore Technologies) for 12-16 hours. Consensus sequences were generated using the nCoV-2019 novel coronavirus bioinformatics protocol using the MinIon Pipeline. 43 Lineage determination was done using the online Pangolin COVID-19 Lineage Assigner.

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Baseline characteristics were summarized using medians and interquartile ranges or counts and frequencies. Symptom onset (day 0) was defined as either the first day of reported symptoms or, for those who did not report symptoms, the day of first positive SARS-CoV-2 RT-qPCR. RT-qPCR viral culture data were used to define . 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.

The copyright holder for this preprint this version posted May 19, 2022 . 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) . 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)

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We thank the participants for making this study possible while acutely infected with SARS-CoV-2. We appreciate the input and support of Thomas M. Lietman, Will Brett, Eric Talbert, Will Brannen, Theresa Brady, Natalie Thornburg, Ian Plumb, Holly Biggs, and others in the CDC COVID-19 response who contributed to this study.Vero TMPRSS2 hAce2 cells were a kind gift from Barney Graham (NIH).