key: cord-0981565-la2wvnb0
authors: Ke, R.; Martinez, P.; Smith, R. L.; Gibson, L.; Achenbach, C.; McFall, S.; Qi, C.; Jacob, J.; Dembele, E.; Bundy, C.; Simons, L. M.; Ozer, E. A.; Hultquist, J. F.; Lorenzo-Redondo, R.; Opdycke, A.; Hawkins, C.; Murphy, R.; Mirza, A.; Conte, M.; Gallagher, N.; Luo, C. H.; Jarrett, J.; Conte, A.; Zhou, R.; Farjo, M.; Rendon, G.; Fields, C. J.; Wang, L.; Fredrickson, R.; Baughman, M.; Chiu, K.; Choi, H.; Scardina, K.; Owens, A.; Broach, J.; Barton, B.; Lazar, P.; Robinson, M. L.; Mostafa, H.; Manabe, Y. C.; Pekosz, A.; McManus, D.; Brooke, C. B.
title: Longitudinal analysis of SARS-CoV-2 vaccine breakthrough infections reveal limited infectious virus shedding and restricted tissue distribution
date: 2021-09-02
journal: medRxiv : the preprint server for health sciences
DOI: 10.1101/2021.08.30.21262701
sha: 33116ddf2070d5ec0fae753e9b8f4b4c3af52898
doc_id: 981565
cord_uid: la2wvnb0

The global effort to vaccinate people against SARS-CoV-2 in the midst of an ongoing pandemic has raised questions about the nature of vaccine breakthrough infections and the potential for vaccinated individuals to transmit the virus. These questions have become even more urgent as new variants of concern with enhanced transmissibility, such as Delta, continue to emerge. To shed light on how vaccine breakthrough infections compare with infections in immunologically naive individuals, we examined viral dynamics and infectious virus shedding through daily longitudinal sampling in a small cohort of adults infected with SARS-CoV-2 at varying stages of vaccination. The durations of both infectious virus shedding and symptoms were significantly reduced in vaccinated individuals compared with unvaccinated individuals. We also observed that breakthrough infections are associated with strong tissue compartmentalization and are only detectable in saliva in some cases. These data indicate that vaccination shortens the duration of time of high transmission potential, minimizes symptom duration, and may restrict tissue dissemination.

the potential for vaccinated individuals to transmit the virus. These questions have 66 become even more urgent as new variants of concern with enhanced transmissibility, 67

such as Delta, continue to emerge. To shed light on how vaccine breakthrough 68

infections compare with infections in immunologically naive individuals, we examined 69 viral dynamics and infectious virus shedding through daily longitudinal sampling in a 70

small cohort of adults infected with SARS-CoV-2 at varying stages of vaccination. The 71

durations of both infectious virus shedding and symptoms were significantly reduced in 72 vaccinated individuals compared with unvaccinated individuals. We also observed that 73

breakthrough infections are associated with strong tissue compartmentalization and are 74 only detectable in saliva in some cases. i.e. not fully vaccinated but enrolled at least 14 days past first mRNA vaccine dose; and 110 11 newly vaccinated individuals that enrolled less than 14 days after first vaccine dose 111

(either mRNA or J&J)), captured at two study sites through daily nasal swab and saliva 112 collection, along with symptom reporting (Fig 1A-C) . These individuals were primarily 113 infected with B.1.1.7 (Alpha) and P.1 (Gamma) variants, as enrollment in this study 114

concluded before the widespread circulation of Delta at the study sites (Table 1) 

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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) 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 September 2, 2021. ; Interestingly, in 2 (487941 and 487250) of the 3 viral culture negative individuals for 150 which we collected both saliva and nasal samples, viral RNA was detectable in saliva 151

for 5 to 10 days while remaining either undetectable (487941) or detectable at very low 152 level for 2 days (487250) in nasal swabs. These data suggest that in 2 of the 4 fully 153 vaccinated individuals for which both saliva and nasal swabs were collected, infection 154 was initially established within the oral cavity or other saliva-exposed tissue site and 155

was restricted from disseminating to the nasal passages. We did not observe a similar 156 restriction of virus to saliva across 60 non-vaccinated individuals that we examined in a 157 previous report 7 , suggesting that severe compartmentalization and tissue-restriction of 158

virus may be a unique feature of vaccine breakthrough infections. 159 160

The one fully vaccinated individual (475670) that did test viral culture positive exhibited 161

highly discordant patterns of viral shedding between saliva and nasal swabs. Viral 162 genome loads expanded and declined in saliva samples over the first week of sample 163 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Patterns of viral shedding in partially vaccinated individuals were more variable. Of the 6 170 individuals that were not considered fully vaccinated but enrolled ³14 days after 171

receiving the first dose of mRNA vaccine, 2 only tested positive by RT-qPCR in a single 172 sample (out of 13 or 15 total samples) (Fig. 1B) , suggesting highly restricted infection 173

with minimal transmission risk. In the other 3 individuals, viral shedding dynamics were 174

indistinguishable from what we previously observed in unvaccinated individuals 7 , and 2 175 of these 3 tested viral culture positive on at least one day (Fig. 1B) . Of the 11 176

individuals that enrolled within 14 days of receiving their first vaccine dose ("newly 177 vaccinated"), most appeared similar to unvaccinated individuals with the exception of 178

three that appeared to exhibit restricted shedding (Fig. 1C) . These data are consistent 179

with individual variation in the onset and magnitude of vaccine-mediated protection.

We directly compared duration of infectious virus shedding between fully and partially 182

vaccinated individuals (combined here as "vaccinated" due to low numbers), newly 183 vaccinated individuals, and unvaccinated individuals from our previous study (Fig. 1D) 

The total numbers of days that vaccinated individuals tested viral culture positive was 185 significantly fewer than both newly vaccinated and unvaccinated groups, indicating that 186 vaccination significantly reduces infectious virus shedding.

We also examined whether the relationship between nasal swab Cn value and viral 189 culture status differed in vaccinated (both fully and partially), newly vaccinated, and 190 unvaccinated individuals (from Ke et al 7 ) (Fig. 1E) . For samples with Cn values below 191 27, we found that the probability of being viral culture positive was lower for samples 192 coming from vaccinated individuals versus newly vaccinated and unvaccinated 193

individuals. These data suggest that for a given viral genome load (as measured by 194

RTqPCR), vaccinated individuals may be less infectious than unvaccinated individuals, 195 consistent with a recent report examining Delta breakthrough infections 8 . However, we 196 must emphasize that this difference is not statistically significant, potentially due to both 197 the relatively small number of samples from vaccinees and the fact that only 6 out of 12 198

individuals included in the vaccinated group were fully vaccinated at the time of 199 enrollment. Regardless, these data further illustrate that Ct/Cn values cannot be used 200 as a simple surrogate for infectious potential.

We next examined whether there were any differences in self-reported symptoms 203 between vaccinated and unvaccinated individuals (using the 60 unvaccinated 204 individuals previously reported 7 ) (Fig. 1F) . A Poisson regression shows that those who 205 received at least one vaccine dose had significantly more days with no reported 206 symptoms than the unvaccinated (p<0.0001). The mean proportion of study days with 207 no symptoms was 0.74 in the vaccinated group dose compared with 0.37 in the 208 unvaccinated group (range: 0 to 1 for both groups). 209 210 Finally, we examined the relationship between viral culture and antigen FIA results in 211 vaccinated (fully plus partially) and newly vaccinated individuals (Fig 1G) . We observed 212 that vaccinated and newly vaccinated participants tested positive by antigen FIA on 213 78% and 85% of the days on which they also tested positive by viral culture, suggesting 214 that antigen FIA can be used to identify vaccine breakthrough infections with high 215 transmission risk, especially if used as part of a serial screening program 9 . These 216 results are consistent with our previous results in unvaccinated individuals as well as 217 earlier cross-sectional studies examining the relationship between antigen tests 218 positivity and infectious virus shedding 7,10,11 . 219 220

This study has several limitations that must be considered. underestimate the true number of breakthrough infections, and that an important role of 237 vaccine-elicited immunity may be restricting viral dissemination and thus limiting 238 symptom severity and transmission potential. These data also further support a role for 239 the oral cavity or other saliva-associated tissue sites as an initial site for SARS-CoV-2 240 infection prior to dissemination and replication of the virus in nasal passages in some 241

individuals. Altogether, this study provides a set of high resolution data that ratify the 242 role of the current SARS-CoV-2 vaccines not only in reducing severity of the disease, 243

but also the infectiousness of individuals with breakthrough infections.

This study was approved by the Western Institutional Review Board, and all participants 247 provided informed consent.

Participants 250

University of Illinois at Urbana-Champaign (UIUC) enrollment site: All on-campus 251 students and employees of the University of Illinois at Urbana-Champaign were required 252

to submit saliva for RTqPCR testing every 2-4 days as part of the SHIELD campus 253

surveillance testing program 14 . Those testing positive were instructed to isolate and 254

were eligible to enroll in this study for a period of 24 hours following receipt of their 255 positive test result. Close contacts of individuals who test positive (particularly those co-256 housed with them) were instructed to quarantine and were eligible to enroll for up to 5 257 days after their last known exposure to an infected individual. All participants were also 258 required to have received a negative saliva RTqPCR result 7 days prior to enrollment.

Northwestern University (NU) enrollment site: All NU on-campus students were 261 required to have nasal swab samples collected for LAMP testing once per week as part 262

of the campus surveillance program. Those testing positive were required to go in the 263

Health Service Quarantine and Isolation (QI) program for isolation. They were eligible 264

for enrollment in this study within 24 hours of going into isolation. Close contacts of 265 individuals who tested positive (particularly those co-housed with them) were also 266 entered in the NU QI program. They were instructed to quarantine and were eligible to 267 enroll in this study for up to 5 days after their last known exposure to an infected 268

individual. All participants were also required to have received a negative nasal swab 269 LAMP assay result 7 days prior to enrollment.

Individuals were recruited via either a link shared in an automated text message 272

providing isolation information sent within 30 minutes of a positive test result, a call from 273 a study recruiter, or a link shared by an enrolled study participant or included in 274

information provided to all quarantining close contacts. In addition, signs/flyers were 275 used at each testing location and a website was available to inform the community 276 about the study.

Participants were required to be at least 18 years of age, have a valid university ID, 279

speak English, have internet access, and live within 8 miles of the university campus. 280

After enrollment and consent, participants completed an initial survey to collect 281 information on demographics, vaccination status, prior infection history, and health 282 history and were provided with sample collection supplies. Participants who tested 283 positive prior to enrollment or during quarantine were followed for up to 14 days. 284

Quarantining participants who continued to test negative by saliva RTqPCR (UIUC) or 285 nasal swab RTqPCR (NU) were followed for up to 7 days after their last exposure. All 286

participants' data and survey responses were collected in the Eureka digital study 287

platform.

Sample collection 290

Each day, participants were remotely observed by trained study staff collecting: 291

1. 2 mL of saliva into a 50mL conical tube (UIUC study site only). 292

2. 1 nasal swab from a single nostril using a foam-tipped swab that was placed 293 within a dry collection tube. 294

3. 1 nasal swab from the other nostril using a flocked swab that was subsequently 295 placed in a collection vial containing viral transport media (VTM).

The order of nostrils (left vs. right) used for the two different swabs was randomized. For 298 nasal swabs, participants were instructed to insert the soft tip of the swab at least 1 cm 299

into the indicated nostril until they encountered mild resistance, rotate the swab around 300 the nostril 5 times, leaving it in place for 10-15 seconds. After daily sample collection, 301 participants completed a symptom survey. A courier collected all participant samples 302 within 1 hour of collection using a no-contact pickup protocol designed to minimize 303 courier exposure to infected participants.

Saliva RTqPCR 306

After collection, saliva samples were stored at room temperature and RTqPCR was run 307

within 12 hours of initial collection. The protocol for the covidSHIELD direct saliva-to-308

RTqPCR assay used has been detailed previously 14 We also thank Jeffrey Olgin, Noah Peyser, and Xochitl Butcher for assistance with the 381

Eureka platform, Michelle Lore for assistance with REDcap, Melanie Loots for 382 assistance with administration, Gillian Snyder for assistance in development of study 383 protocols and logistics, and Erin Iturriaga and Jue Chen for study protocol development.

Finally, we are grateful to Dr. Alvaro Hernandez and Ms. Chris Wright of the DNA 385

Services Lab within the Roy J. Carver Biotechnology Center for expert assistance in 386 establishment of a SARS-CoV-2 genomic sequencing protocol. Vero-TMPRSS2 cells 387

were kindly provided by the National Institute of Infectious Diseases, Japan. Sofia 2 388 devices and associated supplies were provided to Carle Foundation Hospital by Quidel, 389

however Quidel played no role in the design of the study or the interpretation or 390

presentation of the data. 391 392

Jagadeesh Yedetore, and 368 Todd Young for their efforts supporting recruitment, enrollment, logistics, and/or sample 369 collection and processing at the UIUC study site

We also wish to thank Kate Klein

Hiba Abbas, Rana 375 Saber, Eric Fickes

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Mitigation of SARS-CoV-2 Transmission at a Large Public 453 University

Saliva-Based Molecular Testing for SARS-CoV-2 that

Repeat COVID-19 Molecular Testing: Correlation of SARS

CoV-2 Culture with Molecular Assays and Cycle Thresholds

Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing 462 cells

Triplex Real-Time RT-PCR for Severe Acute Respiratory 464 Syndrome Coronavirus 2. Emerg

Acknowledgments. We wish to thank Shumon Ahmed, Carly Bell, Nate Bouton, 358