SARS-CoV-2 mRNA vaccination induces functionally diverse antibodies to NTD, RBD, and S2.

In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast-derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to spikes of seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine-induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations.

SARS-CoV-2-infection- and vaccine-induced antibody responses are long lasting with an initial waning phase followed by a stabilization phase.

It is thought that mRNA-based vaccine-induced immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wanes quickly, based mostly on short-term studies. Here, we analyzed the kinetics and durability of the humoral responses to SARS-CoV-2 infection and vaccination using >8,000 longitudinal samples collected over a 3-year period in New York City. Upon primary immunization, participants with pre-existing immunity mounted higher antibody responses faster and achieved higher steady-state antibody titers than naive individuals. Antibody kinetics were characterized by two phases: an initial rapid decay, followed by a stabilization phase with very slow decay. Booster vaccination equalized the differences in antibody concentration between participants with and without hybrid immunity, but the peak antibody titers decreased with each successive antigen exposure. Breakthrough infections increased antibodies to similar titers as an additional vaccine dose in naive individuals. Our study provides strong evidence that SARS-CoV-2 antibody responses are long lasting, with initial waning followed by stabilization.

Activity of convalescent and vaccine serum against SARS-CoV-2 Omicron.

The Omicron (B.1.1.529) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially identified in November 2021 in South Africa and Botswana, as well as in a sample from a traveller from South Africa in Hong Kong1,2. Since then, Omicron has been detected globally. This variant appears to be at least as infectious as Delta (B.1.617.2), has already caused superspreader events3, and has outcompeted Delta within weeks in several countries and metropolitan areas. Omicron hosts an unprecedented number of mutations in its spike gene and early reports have provided evidence for extensive immune escape and reduced vaccine effectiveness2,4-6. Here we investigated the virus-neutralizing and spike protein-binding activity of sera from convalescent, double mRNA-vaccinated, mRNA-boosted, convalescent double-vaccinated and convalescent boosted individuals against wild-type, Beta (B.1.351) and Omicron SARS-CoV-2 isolates and spike proteins. Neutralizing activity of sera from convalescent and double-vaccinated participants was undetectable or very low against Omicron compared with the wild-type virus, whereas neutralizing activity of sera from individuals who had been exposed to spike three or four times through infection and vaccination was maintained, although at significantly reduced levels. Binding to the receptor-binding and N-terminal domains of the Omicron spike protein was reduced compared with binding to the wild type in convalescent unvaccinated individuals, but was mostly retained in vaccinated individuals.

SARS-CoV-2 Omicron virus causes attenuated disease in mice and hamsters.

The recent emergence of B.1.1.529, the Omicron variant1,2, has raised concerns of escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in preclinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of several B.1.1.529 isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2)-expressing mice and hamsters. Despite modelling data indicating that B.1.1.529 spike can bind more avidly to mouse ACE2 (refs. 3,4), we observed less infection by B.1.1.529 in 129, C57BL/6, BALB/c and K18-hACE2 transgenic mice than by previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease and pathology with B.1.1.529 were also milder than with historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from the SAVE/NIAID network with several B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.

Repeated cross-sectional sero-monitoring of SARS-CoV-2 in New York City.

In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first detected in China and has since caused a pandemic of coronavirus disease 2019 (COVID-19). The first case of COVID-19 in New York City was officially confirmed on 1 March 2020 followed by a severe local epidemic1. Here, to understand seroprevalence dynamics, we conduct a retrospective, repeated cross-sectional analysis of anti-SARS-CoV-2 spike antibodies in weekly intervals from the beginning of February to July 2020 using more than 10,000 plasma samples from patients at Mount Sinai Hospital in New York City. We describe the dynamics of seroprevalence in an 'urgent care' group, which is enriched in cases of COVID-19 during the epidemic, and a 'routine care' group, which more closely represents the general population. Seroprevalence increased at different rates in both groups; seropositive samples were found as early as mid-February, and levelled out at slightly above 20% in both groups after the epidemic wave subsided by the end of May. From May to July, seroprevalence remained stable, suggesting lasting antibody levels in the population. Our data suggest that SARS-CoV-2 was introduced in New York City earlier than previously documented and describe the dynamics of seroconversion over the full course of the first wave of the pandemic in a major metropolitan area.

Human Anti-neuraminidase Antibodies Reduce Airborne Transmission of Clinical Influenza Virus Isolates in the Guinea Pig Model.

The public health burden caused by influenza virus infections is not adequately addressed with existing vaccines and antivirals. Identifying approaches that interfere with human-to-human transmission of influenza viruses remains a pressing need. The importance of neuraminidase (NA) activity for the replication and spread of influenza viruses led us to investigate whether broadly reactive human anti-NA monoclonal antibodies (MAbs) could affect airborne transmission of the virus using the guinea pig model. In that model, infection with recent influenza virus clinical isolates resulted in 100% transmission from inoculated donors to recipients in an airborne transmission setting. Anti-NA MAbs were administered either to the inoculated animals on days 1, 2, and 4 after infection or to the naive contacts on days 2 and 4 after donor infection. Administration of NA-1G01, a broadly cross-reactive anti-NA MAb, to either the donor or recipient reduced transmission of the A/New York City/PV02669/2019 (H1N1) and A/New York City/PV01148/2018 (H3N2) viruses. Administration of 1000-3C05, an anti-N1 MAb, to either the donor or recipient reduced transmission of A/New York City/PV02669/2019 (H1N1) virus but did not reduce transmission of A/New York City/PV01148 (H3N2) virus. Conversely, 229-2C06, an anti-N2 MAb, reduced transmission of A/New York City/PV01148 (H3N2) but did not impact transmission of A/New York City/PV02669/2019 (H1N1) virus. Our work demonstrates that anti-NA MAbs could be further developed into prophylactic or therapeutic agents to prevent influenza virus transmission to control viral spread. IMPORTANCE The burden of influenza remains substantial despite unremitting efforts to reduce the magnitude of seasonal influenza epidemics and prepare for pandemics. Although vaccination remains the mainstay of these efforts, current vaccines are designed to stimulate an immune response against the viral hemagglutinin. Interest in the role immunity against neuraminidase plays in influenza virus infection and transmission has recently surged. Human antibodies that bind broadly to neuraminidases of diverse influenza viruses and protect mice against lethal viral challenge have previously been characterized. Here, we show that three such antibodies inhibit the neuraminidase activity of recent isolates and reduce their airborne transmission in a guinea pig model. In addition to contributing to the accumulating support for incorporating neuraminidase as a vaccine antigen, these findings also demonstrate the potential of direct administration of anti-neuraminidase antibodies to individuals infected with influenza virus and to individuals for postexposure prophylaxis to prevent the spread of influenza virus.

Food for thought: Eating before saliva collection and interference with SARS-CoV-2 detection.

Saliva is a promising specimen for the detection of viruses that cause upper respiratory infections including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to its cost-effectiveness and noninvasive collection. However, together with intrinsic enzymes and oral microbiota, children's unique dietary habits may introduce substances that interfere with diagnostic testing. To determine whether children's dietary choices impact SARS-CoV-2 molecular detection in saliva, we performed a diagnostic study that simulates testing of real-life specimens provided from healthy children (n = 5) who self-collected saliva at home before and at 0, 20, and 60 min after eating 20 foods they selected. Each of 72 specimens was split into two volumes and spiked with SARS-CoV-2-negative or SARS-CoV-2-positive clinical standards before side-by-side testing by reverse-transcription polymerase chain reaction matrix-assisted laser desorption ionization time-of-flight (RT-PCR/MALDI-TOF) assay. Detection of internal extraction control and SARS-CoV-2 nucleic acids was reduced in replicates of saliva collected at 0 min after eating 11 of 20 foods. Interference resolved at 20 and 60 min after eating all foods except hot dogs in one participant. This represented a significant improvement in the detection of nucleic acids compared to saliva collected at 0 min after eating (p = 0.0005). We demonstrate successful detection of viral nucleic acids in saliva self-collected by children before and after eating a variety of foods. Fasting is not required before saliva collection for SARS-CoV-2 testing by RT-PCR/MALDI-TOF, but waiting for 20 min after eating is sufficient for accurate testing. These findings should be considered for SARS-CoV-2 testing and broader viral diagnostics in saliva specimens.

Robust clinical detection of SARS-CoV-2 variants by RT-PCR/MALDI-TOF multitarget approach.

The coronavirus disease 2019 (COVID-19) pandemic has sparked the rapid development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics. However, emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY® SARS-CoV-2 Panel combines reverse-transcription polymerase chain reaction and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified data set of 1262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 to April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly specific for the Alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS-CoV-2 variants.

Real-Time Investigation of a Large Nosocomial Influenza A Outbreak Informed by Genomic Epidemiology.

BACKGROUND: Nosocomial respiratory virus outbreaks represent serious public health challenges. Rapid and precise identification of cases and tracing of transmission chains is critical to end outbreaks and to inform prevention measures. METHODS: We combined conventional surveillance with influenza A virus (IAV) genome sequencing to identify and contain a large IAV outbreak in a metropolitan healthcare system. A total of 381 individuals, including 91 inpatients and 290 healthcare workers (HCWs), were included in the investigation. RESULTS: During a 12-day period in early 2019, infection preventionists identified 89 HCWs and 18 inpatients as cases of influenza-like illness (ILI), using an amended definition without the requirement for fever. Sequencing of IAV genomes from available nasopharyngeal specimens identified 66 individuals infected with a nearly identical strain of influenza A H1N1pdm09 (43 HCWs, 17 inpatients, and 6 with unspecified affiliation). All HCWs infected with the outbreak strain had received the seasonal influenza virus vaccination. Characterization of 5 representative outbreak viral isolates did not show antigenic drift. In conjunction with IAV genome sequencing, mining of electronic records pinpointed the origin of the outbreak as a single patient and a few interactions in the emergency department that occurred 1 day prior to the index ILI cluster. CONCLUSIONS: We used precision surveillance to delineate a large nosocomial IAV outbreak, mapping the source of the outbreak to a single patient rather than HCWs as initially assumed based on conventional epidemiology. These findings have important ramifications for more-effective prevention strategies to curb nosocomial respiratory virus outbreaks.

Modified methicillin-resistant Staphylococcus aureus detected in neonatal intensive care patients.

OBJECTIVES: As part of an active MRSA surveillance programme in our neonatal ICU, we identified nares surveillance cultures from two infants that displayed heterogeneity in methicillin resistance between isolated subclones that lacked mecA and mecC. METHODS: The underlying mechanism for the modified Staphylococcus aureus (MODSA) methicillin-resistance phenotype was investigated by WGS. RESULTS: Comparison of finished-quality genomes of four MODSA and four MSSA subclones demonstrated that the resistance changes were associated with unique truncating mutations in the gene encoding the cyclic diadenosine monophosphate phosphodiesterase enzyme GdpP or a non-synonymous substitution in the gene encoding PBP2. CONCLUSIONS: These two cases highlight the difficulty in identifying non-mecA, non-mecC-mediated MRSA isolates in the clinical microbiology laboratory, which leads to difficulties in implementing appropriate therapy and infection control measures.

Multisystem inflammatory syndrome in children related to COVID-19: A New York City experience.

In December 2019, the 2019, a novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) first emerged in Wuhan, China. This has now spread worldwide and was declared a pandemic by March 2020. Initially, the pediatric population was described as a low risk for severe COVID-19. However, reports have emerged recently of cases of COVID-19 in children with a systemic inflammatory disease, with features that overlap with Kawasaki disease (KD). We describe the first 15 cases with the multi-systeminflammatory syndrome in children (MIS-C), temporally related to COVID-19, who presented for care to a tertiary pediatric referral center in New York City. We discuss the disproportionate burden of disease among Hispanic/Latino and Black/African American ancestry, the distinct cytokine signature across the disease spectrum (IL-1/IL-6), and the potential role and pathogenesis of SARS-CoV-2 in this new clinical entity.

RT-PCR/MALDI-TOF mass spectrometry-based detection of SARS-CoV-2 in saliva specimens.

As severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections continue, there is a substantial need for cost-effective and large-scale testing that utilizes specimens that can be readily collected from both symptomatic and asymptomatic individuals in various community settings. Although multiple diagnostic methods utilize nasopharyngeal specimens, saliva specimens represent an attractive alternative as they can rapidly and safely be collected from different populations. While saliva has been described as an acceptable clinical matrix for the detection of SARS-CoV-2, evaluations of analytic performance across platforms for this specimen type are limited. Here, we used a novel sensitive RT-PCR/MALDI-TOF mass spectrometry-based assay (Agena MassARRAY®) to detect SARS-CoV-2 in saliva specimens. The platform demonstrated high diagnostic sensitivity and specificity when compared to matched patient upper respiratory specimens. We also evaluated the analytical sensitivity of the platform and determined the limit of detection of the assay to be 1562.5 copies/ml. Furthermore, across the five individual target components of this assay, there was a range in analytic sensitivities for each target with the N2 target being the most sensitive. Overall, this system also demonstrated comparable performance when compared to the detection of SARS-CoV-2 RNA in saliva by the cobas® 6800/8800 SARS-CoV-2 real-time RT-PCR Test (Roche). Together, we demonstrate that saliva represents an appropriate matrix for SARS-CoV-2 detection on the novel Agena system as well as on a conventional real-time RT-PCR assay. We conclude that the MassARRAY® system is a sensitive and reliable platform for SARS-CoV-2 detection in saliva, offering scalable throughput in a large variety of clinical laboratory settings.

The arrival and spread of SARS-CoV-2 in Colombia.

We performed phylogenomic analysis of severe acute respiratory syndrome coronavirus-2 from 88 infected individuals across different regions of Colombia. Eleven different lineages were detected, suggesting multiple introduction events. Pangolin lineages B.1 and B.1.5 were the most frequent, with B.1 being associated with prior travel to high-risk areas.

Brucella Exposure Risk Events in 10 Clinical Laboratories, New York City, USA, 2015 to 2017.

From 2015 to 2017, 11 confirmed brucellosis cases were reported in New York City, leading to 10 Brucella exposure risk events (Brucella events) in 7 clinical laboratories (CLs). Most patients had traveled to countries where brucellosis is endemic and presented with histories and findings consistent with brucellosis. CLs were not notified that specimens might yield a hazardous organism, as the clinicians did not consider brucellosis until they were notified that bacteremia with Brucella was suspected. In 3 Brucella events, the CLs did not suspect that slow-growing, small Gram-negative bacteria might be harmful. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), which has a limited capacity to identify biological threat agents (BTAs), was used during 4 Brucella events, which accounted for 84% of exposures. In 3 of these incidents, initial staining of liquid media showed Gram-positive rods or cocci, including some cocci in chains, suggesting streptococci. Over 200 occupational exposures occurred when the unknown isolates were manipulated and/or tested on open benches, including by procedures that could generate infectious aerosols. During 3 Brucella events, the CLs examined and/or manipulated isolates in a biological safety cabinet (BSC); in each CL, the CL had previously isolated Brucella Centers for Disease Control and Prevention recommendations to prevent laboratory-acquired brucellosis (LAB) were followed; no seroconversions or LAB cases occurred. Laboratory assessments were conducted after the Brucella events to identify facility-specific risks and mitigations. With increasing MALDI-TOF MS use, CLs are well-advised to adhere strictly to safe work practices, such as handling and manipulating all slow-growing organisms in BSCs and not using MALDI-TOF MS for identification until BTAs have been ruled out.

Central nervous system involvement by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

Neurologic sequelae can be devastating complications of respiratory viral infections. We report the presence of virus in neural and capillary endothelial cells in frontal lobe tissue obtained at postmortem examination from a patient infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Our observations of virus in neural tissue, in conjunction with clinical correlates of worsening neurologic symptoms, pave the way to a closer understanding of the pathogenic mechanisms underlying central nervous system involvement by SARS-CoV-2.

Clinical performance of a quantitative pan-genus Leishmania Real-time PCR assay for diagnosis of cutaneous and visceral leishmaniasis.

Leishmaniasis is a complex vector-borne disease caused by various Leishmania species, affecting humans and animals. Current diagnostic methods have limitations, leading to potential misdiagnosis. Therefore, there is an urgent need for specific and sensitive diagnostic tools. We evaluated the sensitivity of a quantitative real-time PCR (qPCR) assay targeting the 18S gene in diverse clinical sample matrices. The assay showed a wide dynamic range and a limit of detection (LoD) of 1 parasite equivalent per milliliter (eq-p/mL) for all tested species. It exhibited high specificity for Leishmania DNA, with no amplification against other microorganisms. When applied to samples from patients with visceral and cutaneous leishmaniasis, the qPCR assay provided results that matched the reference methods and allowed estimation of parasite burdens. This assay holds promise for diagnosing and monitoring leishmaniasis by offering high sensitivity, specificity, and the ability to estimate parasitemia. Further studies are needed to enhance Leishmania molecular diagnostics and expand their coverage for improved clinical impact.

Molecular Detection of Candida auris Using DiaSorin Molecular Simplexa® Detection Kit: A Diagnostic Performance Evaluation.

Candida auris is a globally emerging fungal pathogen that is associated with healthcare-related infections. The accurate and rapid detection of C. auris is crucial for effective infection prevention, control, and patient management. This study aimed to validate the analytical and diagnostic performance of the DiaSorin Molecular C. auris Detection Kit. The analytical specificity, sensitivity, and reproducibility of the assay were evaluated. The limit of detection (LOD) was determined to be 266 CFU/µL using the ZeptoMetrix Candida auris Z485 strain and standard calibration curves. The assay demonstrated high analytical specificity and showed no amplification against a diverse panel of bacteria and fungi. Clinical validation was conducted using deidentified residual axillary/groin surveillance culture specimens from C. auris culture-positive and culture-negative patients. The DiaSorin Molecular Detection Kit exhibited 100% agreement in sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) when compared to cultures coupled with MALDI-TOF identification. Intra- and inter-reproducibility testing demonstrated consistent and reliable diagnostic performance. This validated assay offers rapid and accurate detection of C. auris, facilitating timely implementation of infection control measures and appropriate patient care. The DiaSorin Molecular C. auris Detection Kit has the potential to aid in controlling the outbreaks caused by this emerging fungal pathogen. Providing a reliable diagnostic tool can contribute to the effective management and containment of C. auris infections in healthcare settings and ultimately improve patient outcomes.

Phylogenetic landscape of Monkeypox Virus (MPV) during the early outbreak in New York City, 2022.

Monkeypox (MPOX) is a zoonotic disease endemic to regions of Central/Western Africa. The geographic endemicity of MPV has expanded, broadening the human-monkeypox virus interface and its potential for spillover. Since May 2022, a large multi-country MPV outbreak with no proven links to endemic countries has originated in Europe and has rapidly expanded around the globe, setting off genomic surveillance efforts. Here, we conducted a genomic analysis of 23 MPV-infected patients from New York City during the early outbreak, assessing the phylogenetic relationship of these strains against publicly available MPV genomes. Additionally, we compared the genomic sequences of clinical isolates versus culture-passaged samples from a subset of samples. Phylogenetic analysis revealed that MPV genomes included in this study cluster within the B.1 lineage (Clade IIb), with some of the samples displaying further differentiation into five different sub-lineages of B.1. Mutational analysis revealed 55 non-synonymous polymorphisms throughout the genome, with some of these mutations located in critical regions required for viral multiplication, structural and assembly functions, as well as the target region for antiviral treatment. In addition, we identified a large majority of polymorphisms associated with GA > AA and TC > TT nucleotide replacements, suggesting the action of human APOBEC3 enzyme. A comparison between clinical isolates and cell culture-passaged samples failed to reveal any difference. Our results provide a first glance at the mutational landscape of early MPV-2022 (B.1) circulating strains in NYC.

Cellular mechanisms associated with sub-optimal immune responses to SARS-CoV-2 bivalent booster vaccination in patients with Multiple Myeloma.

BACKGROUND: The real-world impact of bivalent vaccines for wild type (WA.1) and Omicron variant (BA.5) is largely unknown in immunocompromised patients with Multiple Myeloma (MM). We characterize the humoral and cellular immune responses in patients with MM before and after receiving the bivalent booster, including neutralizing assays to identify patterns associated with continuing vulnerability to current variants (XBB1.16, EG5) in the current post-pandemic era. METHODS: We studied the humoral and cellular immune responses before and after bivalent booster immunization in 48 MM patients. Spike binding IgG antibody levels were measured by SARS-CoV-2 spike binding ELISA and neutralization capacity was assessed by a SARS-CoV-2 multi-cycle microneutralization assays to assess inhibition of live virus. We measured spike specific T-cell function using the QuantiFERON SARS-CoV-2 (Qiagen) assay as well as flow-cytometry based T-cell. In a subset of 38 patients, high-dimensional flow cytometry was performed to identify immune cell subsets associated with lack of humoral antibodies. FINDINGS: We find that bivalent vaccination provides significant boost in protection to the omicron variant in our MM patients, in a treatment specific manner. MM patients remain vulnerable to newer variants with mutations in the spike portion. Anti-CD38 and anti-BCMA therapies affect the immune machinery needed to produce antibodies. INTERPRETATION: Our study highlights varying immune responses observed in MM patients after receiving bivalent COVID-19 vaccination. Specifically, a subgroup of MM patients undergoing anti-CD38 and anti-BCMA therapy experience impairment in immune cells such DCs, B cells, NK cells and TFH cells, leading to an inability to generate adequate humoral and cellular responses to vaccination. FUNDING: National Cancer Institute (National Institutes of Health), National Institute of Allergy and Infectious Diseases (National Institutes of Health), NCI Serological Sciences Network for COVID-19 (SeroNet) and The Icahn School of Medicine at Mount Sinai.

Sequential intrahost evolution and onward transmission of SARS-CoV-2 variants.

Persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been reported in immune-compromised individuals and people undergoing immune-modulatory treatments. Although intrahost evolution has been documented, direct evidence of subsequent transmission and continued stepwise adaptation is lacking. Here we describe sequential persistent SARS-CoV-2 infections in three individuals that led to the emergence, forward transmission, and continued evolution of a new Omicron sublineage, BA.1.23, over an eight-month period. The initially transmitted BA.1.23 variant encoded seven additional amino acid substitutions within the spike protein (E96D, R346T, L455W, K458M, A484V, H681R, A688V), and displayed substantial resistance to neutralization by sera from boosted and/or Omicron BA.1-infected study participants. Subsequent continued BA.1.23 replication resulted in additional substitutions in the spike protein (S254F, N448S, F456L, M458K, F981L, S982L) as well as in five other virus proteins. Our findings demonstrate not only that the Omicron BA.1 lineage can diverge further from its already exceptionally mutated genome but also that patients with persistent infections can transmit these viral variants. Thus, there is, an urgent need to implement strategies to prevent prolonged SARS-CoV-2 replication and to limit the spread of newly emerging, neutralization-resistant variants in vulnerable patients.