Transmission from vaccinated individuals in a large SARS-CoV-2 Delta variant outbreak.

An outbreak of over 1,000 COVID-19 cases in Provincetown, Massachusetts (MA), in July 2021-the first large outbreak mostly in vaccinated individuals in the US-prompted a comprehensive public health response, motivating changes to national masking recommendations and raising questions about infection and transmission among vaccinated individuals. To address these questions, we combined viral genomic and epidemiological data from 467 individuals, including 40% of outbreak-associated cases. The Delta variant accounted for 99% of cases in this dataset; it was introduced from at least 40 sources, but 83% of cases derived from a single source, likely through transmission across multiple settings over a short time rather than a single event. Genomic and epidemiological data supported multiple transmissions of Delta from and between fully vaccinated individuals. However, despite its magnitude, the outbreak had limited onward impact in MA and the US overall, likely due to high vaccination rates and a robust public health response.

Acute SARS-CoV-2 infections harbor limited within-host diversity and transmit via tight transmission bottlenecks.

The emergence of divergent SARS-CoV-2 lineages has raised concern that novel variants eliciting immune escape or the ability to displace circulating lineages could emerge within individual hosts. Though growing evidence suggests that novel variants arise during prolonged infections, most infections are acute. Understanding how efficiently variants emerge and transmit among acutely-infected hosts is therefore critical for predicting the pace of long-term SARS-CoV-2 evolution. To characterize how within-host diversity is generated and propagated, we combine extensive laboratory and bioinformatic controls with metrics of within- and between-host diversity to 133 SARS-CoV-2 genomes from acutely-infected individuals. We find that within-host diversity is low and transmission bottlenecks are narrow, with very few viruses founding most infections. Within-host variants are rarely transmitted, even among individuals within the same household, and are rarely detected along phylogenetically linked infections in the broader community. These findings suggest that most variation generated within-host is lost during transmission.

Transmission of SARS-CoV-2 in domestic cats imposes a narrow bottleneck.

The evolutionary mechanisms by which SARS-CoV-2 viruses adapt to mammalian hosts and, potentially, undergo antigenic evolution depend on the ways genetic variation is generated and selected within and between individual hosts. Using domestic cats as a model, we show that SARS-CoV-2 consensus sequences remain largely unchanged over time within hosts, while dynamic sub-consensus diversity reveals processes of genetic drift and weak purifying selection. We further identify a notable variant at amino acid position 655 in Spike (H655Y), which was previously shown to confer escape from human monoclonal antibodies. This variant arises rapidly and persists at intermediate frequencies in index cats. It also becomes fixed following transmission in two of three pairs. These dynamics suggest this site may be under positive selection in this system and illustrate how a variant can quickly arise and become fixed in parallel across multiple transmission pairs. Transmission of SARS-CoV-2 in cats involved a narrow bottleneck, with new infections founded by fewer than ten viruses. In RNA virus evolution, stochastic processes like narrow transmission bottlenecks and genetic drift typically act to constrain the overall pace of adaptive evolution. Our data suggest that here, positive selection in index cats followed by a narrow transmission bottleneck may have instead accelerated the fixation of S H655Y, a potentially beneficial SARS-CoV-2 variant. Overall, our study suggests species- and context-specific adaptations are likely to continue to emerge. This underscores the importance of continued genomic surveillance for new SARS-CoV-2 variants as well as heightened scrutiny for signatures of SARS-CoV-2 positive selection in humans and mammalian model systems.

Anti-membrane Antibodies Persist at Least One Year and Discriminate Between Past Coronavirus Disease 2019 Infection and Vaccination.

BACKGROUND: The consequences of past coronavirus disease 2019 (COVID-19) infection for personal and population health are emerging, but accurately identifying distant infection is a challenge. Anti-spike antibodies rise after both vaccination and infection and anti-nucleocapsid antibodies rapidly decline. METHODS: We evaluated anti-membrane antibodies in COVID-19 naive, vaccinated, and convalescent subjects to determine if they persist and accurately detect distant infection. RESULTS: We found that anti-membrane antibodies persist for at least 1 year and are a sensitive and specific marker of past COVID-19 infection. CONCLUSIONS: Thus, anti-membrane and anti-spike antibodies together can differentiate between COVID-19 convalescent, vaccinated, and naive states to advance public health and research.

High-Throughput Identification of MHC Class I Binding Peptides Using an Ultradense Peptide Array.

Rational vaccine development and evaluation requires identifying and measuring the magnitude of epitope-specific CD8 T cell responses. However, conventional CD8 T cell epitope discovery methods are labor intensive and do not scale well. In this study, we accelerate this process by using an ultradense peptide array as a high-throughput tool for screening peptides to identify putative novel epitopes. In a single experiment, we directly assess the binding of four common Indian rhesus macaque MHC class I molecules (Mamu-A1*001, -A1*002, -B*008, and -B*017) to ∼61,000 8-mer, 9-mer, and 10-mer peptides derived from the full proteomes of 82 SIV and simian HIV isolates. Many epitope-specific CD8 T cell responses restricted by these four MHC molecules have already been identified in SIVmac239, providing an ideal dataset for validating the array; up to 64% of these known epitopes are found in the top 192 SIVmac239 peptides with the most intense MHC binding signals in our experiment. To assess whether the peptide array identified putative novel CD8 T cell epitopes, we validated the method by IFN-γ ELISPOT assay and found three novel peptides that induced CD8 T cell responses in at least two Mamu-A1*001-positive animals; two of these were validated by ex vivo tetramer staining. This high-throughput identification of peptides that bind class I MHC will enable more efficient CD8 T cell response profiling for vaccine development, particularly for pathogens with complex proteomes for which few epitope-specific responses have been defined.

Severe Acute Respiratory Syndrome Coronavirus 2 Transmission in Intercollegiate Athletics Not Fully Mitigated With Daily Antigen Testing.

BACKGROUND: High-frequency, rapid-turnaround severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing continues to be proposed as a way of efficiently identifying and mitigating transmission in congregate settings. However, 2 SARS-CoV-2 outbreaks occurred among intercollegiate university athletic programs during the fall 2020 semester, despite mandatory directly observed daily antigen testing. METHODS: During the fall 2020 semester, athletes and staff in both programs were tested daily using Quidel's Sofia SARS Antigen Fluorescent Immunoassay, with positive antigen results requiring confirmatory testing with real-time reverse-transcription polymerase chain reaction. We used genomic sequencing to investigate transmission dynamics in these 2 outbreaks. RESULTS: In the first outbreak, 32 confirmed cases occurred within a university athletics program after the index patient attended a meeting while infectious, despite a negative antigen test on the day of the meeting. Among isolates sequenced from that outbreak, 24 (92%) of 26 were closely related, suggesting sustained transmission following an initial introduction event. In the second outbreak, 12 confirmed cases occurred among athletes from 2 university programs that faced each other in an athletic competition, despite receipt of negative antigen test results on the day of the competition. Sequences from both teams were closely related and distinct from viruses circulating in the community for team 1, suggesting transmission during intercollegiate competition in the community for team 2. CONCLUSIONS: These findings suggest that antigen testing alone, even when mandated and directly observed, may not be sufficient as an intervention to prevent SARS-CoV-2 outbreaks in congregate settings, and they highlight the importance of vaccination to prevent SARS-CoV-2 outbreak in congregate settings.

Viral Sequencing to Investigate Sources of SARS-CoV-2 Infection in US Healthcare Personnel.

BACKGROUND: Healthcare personnel (HCP) are at increased risk of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We posit that current infection control guidelines generally protect HCP from SARS-CoV-2 infection in a healthcare setting. METHODS: In this retrospective case series, we used viral genomics to investigate the likely source of SARS-CoV-2 infection in HCP at a major academic medical institution in the Upper Midwest of the United States between 25 March and 27 December 2020. We obtained limited epidemiological data through informal interviews and review of the electronic health record and combined this information with healthcare-associated viral sequences and viral sequences collected in the broader community to infer the most likely source of infection in HCP. RESULTS: We investigated SARS-CoV-2 infection clusters involving 95 HCP and 137 possible patient contact sequences. The majority of HCP infections could not be linked to a patient or coworker (55 of 95 [57.9%]) and were genetically similar to viruses circulating concurrently in the community. We found that 10.5% of HCP infections (10 of 95) could be traced to a coworker. Strikingly, only 4.2% (4 of 95) could be traced to a patient source. CONCLUSIONS: Infections among HCP add further strain to the healthcare system and put patients, HCP, and communities at risk. We found no evidence for healthcare-associated transmission in the majority of HCP infections evaluated. Although we cannot rule out the possibility of cryptic healthcare-associated transmission, it appears that HCP most commonly become infected with SARS-CoV-2 via community exposure. This emphasizes the ongoing importance of mask wearing, physical distancing, robust testing programs, and rapid distribution of vaccines.

Interventions to Disrupt Coronavirus Disease Transmission at a University, Wisconsin, USA, August-October 2020.

University settings have demonstrated potential for coronavirus disease (COVID-19) outbreaks; they combine congregate living, substantial social activity, and a young population predisposed to mild illness. Using genomic and epidemiologic data, we describe a COVID-19 outbreak at the University of Wisconsin-Madison, Madison, Wisconsin, USA. During August-October 2020, a total of 3,485 students, including 856/6,162 students living in dormitories, tested positive. Case counts began rising during move-in week, August 25-31, 2020, then rose rapidly during September 1-11, 2020. The university initiated multiple prevention efforts, including quarantining 2 dormitories; a subsequent decline in cases was observed. Genomic surveillance of cases from Dane County, in which the university is located, did not find evidence of transmission from a large cluster of cases in the 2 quarantined dorms during the outbreak. Coordinated implementation of prevention measures can reduce COVID-19 spread in university settings and may limit spillover to the surrounding community.

Long-Term Protection of Rhesus Macaques from Zika Virus Reinfection.

By the end of the 2016 Zika virus (ZIKV) outbreak, it is estimated that there were up to 100 million infections in the Americas. In approximately one in seven infants born to mothers infected during pregnancy, ZIKV has been linked to microcephaly, developmental delays, or other congenital disorders collectively known as congenital Zika syndrome, as well as Guillain-Barré syndrome, in ZIKV-infected adults. It is a global health priority to develop a vaccine against ZIKV that elicits long-lasting immunity; however, the durability of immunity to ZIKV is unknown. Previous studies in mice and nonhuman primates have been crucial in vaccine development but have not defined the duration of immunity generated by ZIKV infection. In this study, we rechallenged five rhesus macaques with ZIKV 22 to 28 months after a primary ZIKV infection. We show that primary ZIKV infection generates high titers of neutralizing antibodies that protect from detectable plasma viremia following rechallenge and persist for at least 22 to 28 months. While additional longitudinal studies are necessary with longer time frames, this study establishes a new experimentally defined minimal length of protective ZIKV immunity.IMPORTANCE ZIKV emerged as a vector-borne pathogen capable of causing illness in infected adults and congenital birth defects in infants born to mothers infected during pregnancy. Despite the decrease in ZIKV cases since the 2015-2016 epidemic, questions concerning the prevalence and longevity of protective immunity have left vulnerable communities fearful that they may become the center of next ZIKV outbreak. Although preexisting herd immunity in regions of past outbreaks may dampen the potential for future outbreaks to occur, we currently do not know the longevity of protective immunity to ZIKV after a person becomes infected. Here, we establish a new experimentally defined minimal length of protective ZIKV immunity. We show that five rhesus macaques initially infected with ZIKV 22 to 28 months prior to rechallenge elicit a durable immune response that protected from detectable plasma viremia. This study establishes a new minimal length of protective immunity.

Using Virus Sequencing to Determine Source of SARS-CoV-2 Transmission for Healthcare Worker.

Whether a healthcare worker's severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is community or hospital acquired affects prevention practices. We used virus sequencing to determine that infection of a healthcare worker who cared for 2 SARS-CoV-2-infected patients was probably community acquired. Appropriate personal protective equipment may have protected against hospital-acquired infection.

Characterization of 100 extended major histocompatibility complex haplotypes in Indonesian cynomolgus macaques.

Many medical advancements-including improvements to anti-rejection therapies in transplantation and vaccine development-rely on preclinical studies conducted in cynomolgus macaques (Macaca fascicularis). Major histocompatibility complex (MHC) class I and class II genes of cynomolgus macaques are orthologous to human leukocyte antigen complex (HLA) class I and class II genes, respectively. Both encode cell-surface proteins involved in cell recognition and rejection of non-host tissues. MHC class I and class II genes are highly polymorphic, so comprehensive genotyping requires the development of complete databases of allelic variants. Our group used PacBio circular consensus sequencing of full-length cDNA amplicons to characterize MHC class I and class II transcript sequences for a cohort of 293 Indonesian cynomolgus macaques (ICM) in a large, pedigreed breeding colony. These studies allowed us to expand the existing database of Macaca fascicularis (Mafa) alleles by identifying an additional 141 MHC class I and 61 class II transcript sequences. In addition, we defined co-segregating combinations of allelic variants as regional haplotypes for 70 Mafa-A, 78 Mafa-B, and 45 Mafa-DRB gene clusters. Finally, we defined class I and class II transcripts that are associated with 100 extended MHC haplotypes in this breeding colony by combining our genotyping analyses with short tandem repeat (STR) patterns across the MHC region. Our sequencing analyses and haplotype definitions improve the utility of these ICM for transplantation studies as well as infectious disease and vaccine research.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Household Transmission during the Omicron Era in Massachusetts: A Prospective, Case-Ascertained Study using Genomic Epidemiology.

Background: Households are a major setting for SARS-CoV-2 infections, but there remains a lack of knowledge regarding the dynamics of viral transmission, particularly in the setting of widespread pre-existing SARS-CoV-2 immunity and evolving variants. Methods: We conducted a prospective, case-ascertained household transmission study in the greater Boston area in March-July 2022. Anterior nasal swabs, along with clinical and demographic data, were collected for 14 days. Nasal swabs were tested for SARS-CoV-2 by PCR. Whole genome sequencing was performed on high-titer samples. Results: We enrolled 33 households in a primary analysis set, with a median age of participants of 25 years old (range 2-66); 98% of whom had received at least 2 doses of a COVID-19 vaccine. 58% of households had a secondary case during follow up and the secondary attack rate (SAR) for contacts infected was 39%. We further examined a strict analysis set of 21 households that had only 1 PCR+ case at baseline, finding an SAR of 22.5%. Genomic epidemiology further determined that there were multiple sources of infection for household contacts, including the index case and outside introductions. When limiting estimates to only highly probable transmissions given epidemiologic and genomic data, the SAR was 18.4%. Conclusions: Household contacts of a person newly diagnosed with COVID-19 are at high risk for SARS-CoV-2 infection in the following 2 weeks. This is, however, not only due to infection from the household index case, but also because the presence of an infected household member implies increased SARS-CoV-2 community transmission. Further studies to understand and mitigate household transmission are needed.

Inferring Viral Transmission Pathways from Within-Host Variation.

Genome sequencing can offer critical insight into pathogen spread in viral outbreaks, but existing transmission inference methods use simplistic evolutionary models and only incorporate a portion of available genetic data. Here, we develop a robust evolutionary model for transmission reconstruction that tracks the genetic composition of within-host viral populations over time and the lineages transmitted between hosts. We confirm that our model reliably describes within-host variant frequencies in a dataset of 134,682 SARS-CoV-2 deep-sequenced genomes from Massachusetts, USA. We then demonstrate that our reconstruction approach infers transmissions more accurately than two leading methods on synthetic data, as well as in a controlled outbreak of bovine respiratory syncytial virus and an epidemiologically-investigated SARS-CoV-2 outbreak in South Africa. Finally, we apply our transmission reconstruction tool to 5,692 outbreaks among the 134,682 Massachusetts genomes. Our methods and results demonstrate the utility of within-host variation for transmission inference of SARS-CoV-2 and other pathogens, and provide an adaptable mathematical framework for tracking within-host evolution.

The 2022 RSV surge was driven by multiple viral lineages.

The US experienced an early and severe respiratory syncytial virus (RSV) surge in autumn 2022. Despite the pressure this has put on hospitals and care centers, the factors promoting the surge in cases are unknown. To investigate whether viral characteristics contributed to the extent or severity of the surge, we sequenced 105 RSV-positive specimens from symptomatic patients diagnosed with RSV who presented to the Massachusetts General Hospital (MGH) and its outpatient practices in the Greater Boston Area. Genomic analysis of the resulting 77 genomes (54 with >80% coverage, and 23 with >5% coverage) demonstrated that the surge was driven by multiple lineages of RSV-A (91%; 70/77) and RSV-B (9%; 7/77). Phylogenetic analysis of all US RSV-A revealed 12 clades, 4 of which contained Massachusetts and Washington genomes. These clades individually had times to most recent common ancestor (tMRCA) between 2014 and 2017, and together had a tMRCA of 2009, suggesting that they emerged well before the COVID-19 pandemic. Similarly, the RSV-B genomes had a tMRCA between 2016 and 2019. We found that the RSV-A and RSV-B genomes in our sample did not differ statistically from the estimated clock rate of the larger phylogenetic tree (10.6 and 12.4 substitutions per year, respectively). In summary, the polyphyletic nature of viral genomes sequenced in the US during the autumn 2022 surge is inconsistent with the emergence of a single, highly transmissible causal RSV lineage.

Evolution of a globally unique SARS-CoV-2 Spike E484T monoclonal antibody escape mutation in a persistently infected, immunocompromised individual.

Prolonged infections in immunocompromised individuals may be a source for novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants, particularly when both the immune system and antiviral therapy fail to clear the infection and enable within-host evolution. Here we describe a 486-day case of SARS-CoV-2 infection in an immunocompromised individual. Following monotherapy with the monoclonal antibody Bamlanivimab, the individual's virus acquired resistance, likely via the earliest known occurrence of Spike amino acid variant E484T. Recently, E484T has arisen again as a derivative of E484A in the Omicron Variant of Concern, supporting the hypothesis that prolonged infections can give rise to novel variants long before they become prevalent in the human population.

Characterization of the SARS-CoV-2 B.1.621 (Mu) variant.

The SARS-CoV-2 B.1.621 (Mu) variant emerged in January 2021 and was categorized as a variant of interest by the World Health Organization in August 2021. This designation prompted us to study the sensitivity of this variant to antibody neutralization. In a live virus neutralization assay with serum samples from individuals vaccinated with the Pfizer/BioNTech or Moderna mRNA vaccines, we measured neutralization antibody titers against B.1.621, an early isolate (spike 614D), and a variant of concern (B.1.351, Beta variant). We observed reduced neutralizing antibody titers against the B.1.621 variant (3.4- to 7-fold reduction, depending on the serum sample and time after the second vaccination) compared to the early isolate and a similar reduction when compared to B.1.351. Likewise, convalescent serum from hamsters previously infected with an early isolate neutralized B.1.621 to a lower degree. Despite this antibody titer reduction, hamsters could not be efficiently rechallenged with the B.1.621 variant, suggesting that the immune response to the first infection is adequate to provide protection against a subsequent infection with the B.1.621 variant.

Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and identification of SARS-CoV-2 variants.

The coronavirus disease 2019 (COVID-19) pandemic has demonstrated a clear need for high-throughput, multiplexed and sensitive assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses and their emerging variants. Here, we present a cost-effective virus and variant detection platform, called microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN), which combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel to test for up to 21 viruses, including SARS-CoV-2, other coronaviruses and both influenza strains, and demonstrated its diagnostic-grade performance on 525 patient specimens in an academic setting and 166 specimens in a clinical setting. We further developed an mCARMEN panel to enable the identification of 6 SARS-CoV-2 variant lineages, including Delta and Omicron, and evaluated it on 2,088 patient specimens with near-perfect concordance to sequencing-based variant classification. Lastly, we implemented a combined Cas13 and Cas12 approach that enables quantitative measurement of SARS-CoV-2 and influenza A viral copies in samples. The mCARMEN platform enables high-throughput surveillance of multiple viruses and variants simultaneously, enabling rapid detection of SARS-CoV-2 variants.

Multimodal surveillance of SARS-CoV-2 at a university enables development of a robust outbreak response framework.

BACKGROUND: Universities are vulnerable to infectious disease outbreaks, making them ideal environments to study transmission dynamics and evaluate mitigation and surveillance measures. Here, we analyze multimodal COVID-19-associated data collected during the 2020-2021 academic year at Colorado Mesa University and introduce a SARS-CoV-2 surveillance and response framework. METHODS: We analyzed epidemiological and sociobehavioral data (demographics, contact tracing, and WiFi-based co-location data) alongside pathogen surveillance data (wastewater and diagnostic testing, and viral genomic sequencing of wastewater and clinical specimens) to characterize outbreak dynamics and inform policy. We applied relative risk, multiple linear regression, and social network assortativity to identify attributes or behaviors associated with contracting SARS-CoV-2. To characterize SARS-CoV-2 transmission, we used viral sequencing, phylogenomic tools, and functional assays. FINDINGS: Athletes, particularly those on high-contact teams, had the highest risk of testing positive. On average, individuals who tested positive had more contacts and longer interaction durations than individuals who never tested positive. The distribution of contacts per individual was overdispersed, although not as overdispersed as the distribution of phylogenomic descendants. Corroboration via technical replicates was essential for identification of wastewater mutations. CONCLUSIONS: Based on our findings, we formulate a framework that combines tools into an integrated disease surveillance program that can be implemented in other congregate settings with limited resources. FUNDING: This work was supported by the National Science Foundation, the Hertz Foundation, the National Institutes of Health, the Centers for Disease Control and Prevention, the Massachusetts Consortium on Pathogen Readiness, the Howard Hughes Medical Institute, the Flu Lab, and the Audacious Project.

SARS-CoV-2 transmission in intercollegiate athletics not fully mitigated with daily antigen testing.

BACKGROUND: High frequency, rapid turnaround SARS-CoV-2 testing continues to be proposed as a way of efficiently identifying and mitigating transmission in congregate settings. However, two SARS-CoV-2 outbreaks occurred among intercollegiate university athletic programs during the fall 2020 semester despite mandatory directly observed daily antigen testing. METHODS: During the fall 2020 semester, athletes and staff in both programs were tested daily using Quidel's Sofia SARS Antigen Fluorescent Immunoassay (FIA), with positive antigen results requiring confirmatory testing with real-time reverse transcription polymerase chain reaction (RT-PCR). We used genomic sequencing to investigate transmission dynamics in these two outbreaks. RESULTS: In Outbreak 1, 32 confirmed cases occurred within a university athletics program after the index patient attended a meeting while infectious despite a negative antigen test on the day of the meeting. Among isolates sequenced from Outbreak 1, 24 (92%) of 26 were closely related, suggesting sustained transmission following an initial introduction event. In Outbreak 2, 12 confirmed cases occurred among athletes from two university programs that faced each other in an athletic competition despite receiving negative antigen test results on the day of the competition. Sequences from both teams were closely related and unique from strains circulating in the community, suggesting transmission during intercollegiate competition. CONCLUSIONS: These findings suggest that antigen testing alone, even when mandated and directly observed, may not be sufficient as an intervention to prevent SARS-CoV-2 outbreaks in congregate settings, and highlights the importance of supplementing serial antigen testing with appropriate mitigation strategies to prevent SARS-CoV-2 outbreak in congregate settings. SUMMARY: High frequency, rapid turnaround SARS-CoV-2 testing continues to be proposed as a way of efficiently identifying and mitigating transmission in congregate settings. However, here we describe two SARS-CoV-2 outbreaks occurred among intercollegiate university athletic programs during the fall 2020 semester.