Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization.

The emergence of the SARS-CoV-2 variant of concern Omicron (Pango lineage B.1.1.529), first identified in Botswana and South Africa, may compromise vaccine effectiveness and lead to re-infections1. Here we investigated Omicron escape from neutralization by antibodies from South African individuals vaccinated with Pfizer BNT162b2. We used blood samples taken soon after vaccination from individuals who were vaccinated and previously infected with SARS-CoV-2 or vaccinated with no evidence of previous infection. We isolated and sequence-confirmed live Omicron virus from an infected person and observed that Omicron requires the angiotensin-converting enzyme 2 (ACE2) receptor to infect cells. We compared plasma neutralization of Omicron relative to an ancestral SARS-CoV-2 strain and found that neutralization of ancestral virus was much higher in infected and vaccinated individuals compared with the vaccinated-only participants. However, both groups showed a 22-fold reduction in vaccine-elicited neutralization by the Omicron variant. Participants who were vaccinated and had previously been infected exhibited residual neutralization of Omicron similar to the level of neutralization of the ancestral virus observed in the vaccination-only group. These data support the notion that reasonable protection against Omicron may be maintained using vaccination approaches.

Omicron infection enhances Delta antibody immunity in vaccinated persons.

The extent to which Omicron infection1-9, with or without previous vaccination, elicits protection against the previously dominant Delta (B.1.617.2) variant is unclear. Here we measured the neutralization capacity against variants of severe acute respiratory syndrome coronavirus 2 in 39 individuals in South Africa infected with the Omicron sublineage BA.1 starting at a median of 6 (interquartile range 3-9) days post symptom onset and continuing until last follow-up sample available, a median of 23 (interquartile range 19-27) days post symptoms to allow BA.1-elicited neutralizing immunity time to develop. Fifteen participants were vaccinated with Pfizer's BNT162b2 or Johnson & Johnson's Ad26.CoV2.S and had BA.1 breakthrough infections, and 24 were unvaccinated. BA.1 neutralization increased from a geometric mean 50% focus reduction neutralization test titre of 42 at enrolment to 575 at the last follow-up time point (13.6-fold) in vaccinated participants and from 46 to 272 (6.0-fold) in unvaccinated participants. Delta virus neutralization also increased, from 192 to 1,091 (5.7-fold) in vaccinated participants and from 28 to 91 (3.0-fold) in unvaccinated participants. At the last time point, unvaccinated individuals infected with BA.1 had low absolute levels of neutralization for the non-BA.1 viruses and 2.2-fold lower BA.1 neutralization, 12.0-fold lower Delta neutralization, 9.6-fold lower Beta variant neutralization, 17.9-fold lower ancestral virus neutralization and 4.8-fold lower Omicron sublineage BA.2 neutralization relative to vaccinated individuals infected with BA.1. These results indicate that hybrid immunity formed by vaccination and Omicron BA.1 infection should be protective against Delta and other variants. By contrast, infection with Omicron BA.1 alone offers limited cross-protection despite moderate enhancement.

T cell responses to SARS-CoV-2 spike cross-recognize Omicron.

The SARS-CoV-2 Omicron variant (B.1.1.529) has multiple spike protein mutations1,2 that contribute to viral escape from antibody neutralization3-6 and reduce vaccine protection from infection7,8. The extent to which other components of the adaptive response such as T cells may still target Omicron and contribute to protection from severe outcomes is unknown. Here we assessed the ability of T cells to react to Omicron spike protein in participants who were vaccinated with Ad26.CoV2.S or BNT162b2, or unvaccinated convalescent COVID-19 patients (n = 70). Between 70% and 80% of the CD4+ and CD8+ T cell response to spike was maintained across study groups. Moreover, the magnitude of Omicron cross-reactive T cells was similar for Beta (B.1.351) and Delta (B.1.617.2) variants, despite Omicron harbouring considerably more mutations. In patients who were hospitalized with Omicron infections (n = 19), there were comparable T cell responses to ancestral spike, nucleocapsid and membrane proteins to those in patients hospitalized in previous waves dominated by the ancestral, Beta or Delta variants (n = 49). Thus, despite extensive mutations and reduced susceptibility to neutralizing antibodies of Omicron, the majority of T cell responses induced by vaccination or infection cross-recognize the variant. It remains to be determined whether well-preserved T cell immunity to Omicron contributes to protection from severe COVID-19 and is linked to early clinical observations from South Africa and elsewhere9-12.

Escape of SARS-CoV-2 501Y.V2 from neutralization by convalescent plasma.

SARS-CoV-2 variants of concern (VOC) have arisen independently at multiple locations1,2 and may reduce the efficacy of current vaccines that target the spike glycoprotein of SARS-CoV-23. Here, using a live-virus neutralization assay, we compared the neutralization of a non-VOC variant with the 501Y.V2 VOC (also known as B.1.351) using plasma collected from adults who were hospitalized with COVID-19 during the two waves of infection in South Africa, the second wave of which was dominated by infections with the 501Y.V2 variant. Sequencing demonstrated that infections of plasma donors from the first wave were with viruses that did not contain the mutations associated with 501Y.V2, except for one infection that contained the E484K substitution in the receptor-binding domain. The 501Y.V2 virus variant was effectively neutralized by plasma from individuals who were infected during the second wave. The first-wave virus variant was effectively neutralized by plasma from first-wave infections. However, the 501Y.V2 variant was poorly cross-neutralized by plasma from individuals with first-wave infections; the efficacy was reduced by 15.1-fold relative to neutralization of 501Y.V2 by plasma from individuals infected in the second wave. By contrast, cross-neutralization of first-wave virus variants using plasma from individuals with second-wave infections was more effective, showing only a 2.3-fold decrease relative to neutralization of first-wave virus variants by plasma from individuals infected in the first wave. Although we tested only one plasma sample from an individual infected with a SARS-CoV-2 variant with only the E484K substitution, this plasma sample potently neutralized both variants. The observed effective neutralization of first-wave virus by plasma from individuals infected with 501Y.V2 provides preliminary evidence that vaccines based on VOC sequences could retain activity against other circulating SARS-CoV-2 lineages.

Group 3 innate lymphoid cells mediate early protective immunity against tuberculosis.

Tuberculosis is the leading cause of death by an infectious disease worldwide1. However, the involvement of innate lymphoid cells (ILCs) in immune responses to infection with Mycobacterium tuberculosis (Mtb) is unknown. Here we show that circulating subsets of ILCs are depleted from the blood of participants with pulmonary tuberculosis and restored upon treatment. Tuberculosis increased accumulation of ILC subsets in the human lung, coinciding with a robust transcriptional response to infection, including a role in orchestrating the recruitment of immune subsets. Using mouse models, we show that group 3 ILCs (ILC3s) accumulated rapidly in Mtb-infected lungs and coincided with the accumulation of alveolar macrophages. Notably, mice that lacked ILC3s exhibited a reduction in the accumulation of early alveolar macrophages and decreased Mtb control. We show that the C-X-C motif chemokine receptor 5 (CXCR5)-C-X-C motif chemokine ligand 13 (CXCL13) axis is involved in Mtb control, as infection upregulates CXCR5 on circulating ILC3s and increases plasma levels of its ligand, CXCL13, in humans. Moreover, interleukin-23-dependent expansion of ILC3s in mice and production of interleukin-17 and interleukin-22 were found to be critical inducers of lung CXCL13, early innate immunity and the formation of protective lymphoid follicles within granulomas. Thus, we demonstrate an early protective role for ILC3s in immunity to Mtb infection.

Publisher Correction: Group 3 innate lymphoid cells mediate early protective immunity against tuberculosis.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

A SARS-CoV-2 variant elicits an antibody response with a shifted immunodominance hierarchy.

Many SARS-CoV-2 variants have mutations at key sites targeted by antibodies. However, it is unknown if antibodies elicited by infection with these variants target the same or different regions of the viral spike as antibodies elicited by earlier viral isolates. Here we compare the specificities of polyclonal antibodies produced by humans infected with early 2020 isolates versus the B.1.351 variant of concern (also known as Beta or 20H/501Y.V2), which contains mutations in multiple key spike epitopes. The serum neutralizing activity of antibodies elicited by infection with both early 2020 viruses and B.1.351 is heavily focused on the spike receptor-binding domain (RBD). However, within the RBD, B.1.351-elicited antibodies are more focused on the "class 3" epitope spanning sites 443 to 452, and neutralization by these antibodies is notably less affected by mutations at residue 484. Our results show that SARS-CoV-2 variants can elicit polyclonal antibodies with different immunodominance hierarchies.

Association Between Human Immunodeficiency Virus Viremia and Compromised Neutralization of Severe Acute Respiratory Syndrome Coronavirus 2 Beta Variant.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may be associated with worse clinical outcomes in people with human immunodeficiency virus (HIV) (PWH). We report anti-SARS-CoV-2 antibody responses in patients hospitalized with coronavirus disease 2019 in Durban, South Africa, during the second SARS-CoV-2 infection wave dominated by the Beta (B.1.351) variant. METHODS: Thirty-four participants with confirmed SARS-CoV-2 infection were followed up with weekly blood sampling to examine antibody levels and neutralization potency against SARS-CoV-2 variants. Participants included 18 PWH, of whom 11 were HIV viremic. RESULTS: SARS-CoV-2-specific antibody concentrations were generally lower in viremic PWH than in virologically suppressed PWH and HIV-negative participants, and neutralization of the Beta variant was 4.9-fold lower in viremic PWH. Most HIV-negative participants and antiretroviral therapy-suppressed PWH also neutralized the Delta (B.1.617.2) variant, whereas the majority of viremic PWH did not. CD4 cell counts <500/µL were associated with lower frequencies of immunoglobulin G and A seroconversion. In addition, there was a high correlation between a surrogate virus neutralization test and live virus neutralization against ancestral SARS-CoV-2 virus in both PWH and HIV-negative individuals, but correlation decreased for the Beta variant neutralization in PWH. CONCLUSIONS: HIV viremia was associated with reduced Beta variant neutralization. This highlights the importance of HIV suppression in maintaining an effective SARS-CoV-2 neutralization response.

T cell derived HIV-1 is present in the CSF in the face of suppressive antiretroviral therapy.

HIV cerebrospinal fluid (CSF) escape, where HIV is suppressed in blood but detectable in CSF, occurs when HIV persists in the CNS despite antiretroviral therapy (ART). To determine the virus producing cell type and whether lowered CSF ART levels are responsible for CSF escape, we collected blood and CSF from 156 neurosymptomatic participants from Durban, South Africa. We observed that 28% of participants with an undetectable HIV blood viral load showed CSF escape. We detected host cell surface markers on the HIV envelope to determine the cellular source of HIV in participants on the first line regimen of efavirenz, emtricitabine, and tenofovir. We confirmed CD26 as a marker which could differentiate between T cells and macrophages and microglia, and quantified CD26 levels on the virion surface, comparing the result to virus from in vitro infected T cells or macrophages. The measured CD26 level was consistent with the presence of T cell produced virus. We found no significant differences in ART concentrations between CSF escape and fully suppressed individuals in CSF or blood, and did not observe a clear association with drug resistance mutations in CSF virus which would allow HIV to replicate. Hence, CSF HIV in the face of ART may at least partly originate in CD4+ T cell populations.

Similar Antibody Responses Against Severe Acute Respiratory Syndrome Coronavirus 2 in Individuals Living Without and With Human Immunodeficiency Virus on Antiretroviral Therapy During the First South African Infection Wave.

BACKGROUND: There is limited understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis in African populations with a high burden of infectious disease comorbidities such as human immunodeficiency virus (HIV). The kinetics, magnitude, and duration of virus-specific antibodies and B-cell responses in people living with HIV (PLWH) in sub-Saharan Africa have not been fully characterized. METHODS: We longitudinally followed SARS-CoV-2-infected individuals in Durban, KwaZulu-Natal, South Africa, and characterized SARS-CoV-2 receptor-binding domain-specific immunoglobulin (Ig) M, IgG, and IgA weekly for 1 month and at 3 months post-diagnosis. Thirty of 72 (41.7%) were PLWH, 25/30 (83%) of whom were on antiretroviral therapy (ART) with full HIV suppression. Plasma neutralization was determined using a live virus neutralization assay, and antibody-secreting cell population frequencies were determined by flow cytometry. RESULTS: Similar seroconversion rates, time to peak antibody titer, peak magnitude, and durability of anti-SARS-CoV-2 IgM, IgG, and IgA were observed in people not living with HIV and PLWH with complete HIV suppression on ART. In addition, similar potency in a live virus neutralization assay was observed in both groups. Loss of IgA was significantly associated with age (P = .023) and a previous diagnosis of tuberculosis (P = .018). CONCLUSIONS: Similar antibody responses and neutralization potency in people not living with HIV and PLWH on stable ART in an African setting suggest that coronavirus disease 2019 (COVID-19) natural infections may confer comparable antibody immunity in these groups. This provides hope that COVID-19 vaccines will be effective in PLWH on stable ART.

Immunogenicity of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection and Ad26.CoV2.S Vaccination in People Living With Human Immunodeficiency Virus (HIV).

BACKGROUND: People living with HIV (PLWH) have been reported to have a higher risk of more severe COVID-19 disease and death. We assessed the ability of the Ad26.CoV2.S vaccine to elicit neutralizing activity against the Delta variant in PLWH relative to HIV-negative individuals. We also examined effects of HIV status and suppression on Delta neutralization response in SARS-CoV-2-infected unvaccinated participants. METHODS: We enrolled participants who were vaccinated through the SISONKE South African clinical trial of the Ad26.CoV2.S vaccine in healthcare workers (HCWs). PLWH in this group had well-controlled HIV infection. We also enrolled unvaccinated participants previously infected with SARS-CoV-2. Neutralization capacity was assessed by a live virus neutralization assay of the Delta variant. RESULTS: Most Ad26.CoV2.S vaccinated HCWs were previously infected with SARS-CoV-2. In this group, Delta variant neutralization was 9-fold higher compared with the infected-only group and 26-fold higher relative to the vaccinated-only group. No decrease in Delta variant neutralization was observed in PLWH relative to HIV-negative participants. In contrast, SARS-CoV-2-infected, unvaccinated PLWH showed 7-fold lower neutralization and a higher frequency of nonresponders, with the highest frequency of nonresponders in people with HIV viremia. Vaccinated-only participants showed low neutralization capacity. CONCLUSIONS: The neutralization response of the Delta variant following Ad26.CoV2.S vaccination in PLWH with well-controlled HIV was not inferior to HIV-negative participants, irrespective of past SARS-CoV-2 infection. In SARS-CoV-2-infected and nonvaccinated participants, HIV infection reduced the neutralization response to SARS-CoV-2, with the strongest reduction in HIV viremic individuals.

Early Detection of Emergent Extensively Drug-Resistant Tuberculosis by Flow Cytometry-Based Phenotyping and Whole-Genome Sequencing.

A critical gap in tuberculosis (TB) treatment is detection of emergent drug resistance. We hypothesized that advanced phenotyping with whole-genome sequencing (WGS) will detect low-frequency Mycobacterium tuberculosis drug resistance. We assessed a reporter mycobacteriophage (Φ2GFP10) in vitro to detect drug-resistant subpopulations and predict M. tuberculosis bactericidal activity in this pilot study. Subsequently, we prospectively studied 20 TB patients with serial Φ2GFP10, Xpert MTB/RIF, and M. tuberculosis culture through end of treatment. WGS was performed, and single nucleotide polymorphisms (SNPs) were examined to detect mixed infection in selected M. tuberculosis isolates. Resistant M. tuberculosis isolates were detected at 1:100,000, and changes in cytometry-gated events were predictive of in vitroM. tuberculosis bactericidal activity using the Φ2GFP10 assay. Emergent drug resistance was detected in one patient by Φ2GFP10 at 3 weeks but not by conventional testing (M. tuberculosis culture and GeneXpert). WGS revealed a phylogeographically distinct extensively drug-resistant tuberculosis (XDR-TB) genome, identical to an XDR-TB isolate from the patient's spouse. Variant lineage-specific SNPs were present early, suggesting mixed infection as the etiology of emergent resistance with temporal trends providing evidence for selection during treatment. Φ2GFP10 can detect low-frequency drug-resistant M. tuberculosis and with WGS characterize emergent M. tuberculosis resistance. In areas of high TB transmission and drug resistance, rapid screening for heteroresistance should be considered.

A Novel Reporter Phage To Detect Tuberculosis and Rifampin Resistance in a High-HIV-Burden Population.

Improved diagnostics and drug susceptibility testing for Mycobacterium tuberculosis are urgently needed. We developed a more powerful mycobacteriophage (Φ(2)GFP10) with a fluorescent reporter. Fluorescence-activated cell sorting (FACS) allows for rapid enumeration of metabolically active bacilli after phage infection. We compared the reporter phage assay to GeneXpert MTB/RIF for detection of M. tuberculosis and rifampin (RIF) resistance in sputum. Patients suspected to have tuberculosis were prospectively enrolled in Durban, South Africa. Sputum was incubated with Φ(2)GFP10, in the presence and absence of RIF, and bacilli were enumerated using FACS. Sensitivity and specificity were compared to those of GeneXpert MTB/RIF with an M. tuberculosis culture as the reference standard. A total of 158 patients were prospectively enrolled. Overall sensitivity for M. tuberculosis was 95.90% (95% confidence interval (CI), 90.69% to 98.64%), and specificity was 83.33% (95% CI, 67.18% to 93.59%). In acid-fast bacillus (AFB)-negative sputum, sensitivity was 88.89% (95% CI, 73.92% to 96.82%), and specificity was 83.33% (95% CI, 67.18% to 93.59%). Sensitivity for RIF-resistant M. tuberculosis in AFB-negative sputum was 90.00% (95% CI, 55.46% to 98.34%), and specificity was 91.94% (95% CI, 82.16% to 97.30%). Compared to GeneXpert, the reporter phage was more sensitive in AFB smear-negative sputum, but specificity was lower. The Φ(2)GFP10 reporter phage showed high sensitivity for detection of M. tuberculosis and RIF resistance, including in AFB-negative sputum, and has the potential to improve phenotypic testing for complex drug resistance, paucibacillary sputum, response to treatment, and detection of mixed infection in clinical specimens.

Subtherapeutic concentrations of first-line anti-TB drugs in South African children treated according to current guidelines: the PHATISA study.

OBJECTIVES: There is a paucity of evidence regarding the optimal dosing of anti-TB drugs in children. The aim of this study was to identify the pharmacokinetic parameters of first-line anti-TB drugs and the concentrations achieved after implementation of the 2010 WHO-recommended paediatric dosages. METHODS: We conducted a prospective, observational pharmacokinetic study in children 10 years old or younger who were on isoniazid, rifampicin, pyrazinamide and ethambutol therapy in Durban, KwaZulu-Natal, South Africa. Blood was collected at six timepoints over a 24 h period, chosen using optimal sampling theory. The drug concentrations were simultaneously modelled to identify the compartmental pharmacokinetics of each drug in each child, using the ADAPT program. RESULTS: The best six sampling timepoints in children were identified as 0 (pre-dose) and 0.42, 1.76, 3.37, 10.31 and 24 h post-dose. Thirty-one children were recruited and blood was drawn at these timepoints. Rifampicin, ethambutol and pyrazinamide were best described using a one-compartment model, while isoniazid was best described with a two-compartment model. Only 2/31 (6%), 20/31 (65%), 17/31 (55%) and 2/13 (15%) of children attained the WHO 2 h target therapeutic concentrations of rifampicin, isoniazid, pyrazinamide and ethambutol, respectively. Moreover, only 24/31 (77%), 6/31 (19%) and 8/31 (26%) achieved the AUCs associated with an optimal clinical response to rifampicin, pyrazinamide and isoniazid, respectively. No single risk factor was significantly associated with below-normal drug levels. CONCLUSIONS: The drug concentrations of all first-line anti-TB drugs were markedly below the target therapeutic concentrations in most South African children who received the revised WHO-recommended paediatric weight-based dosages.

Detailed phenotyping reveals diverse and highly skewed neutrophil subsets in both the blood and airways during active tuberculosis infection.

Introduction: Neutrophils play a complex and important role in the immunopathology of TB. Data suggest they are protective during early infection but become a main driver of immunopathology if infection progresses to active disease. Neutrophils are now recognized to exist in functionally diverse states, but little work has been done on how neutrophil states or subsets are skewed in TB disease. Methods: To address this, we carried out comprehensive phenotyping by flow cytometry of neutrophils in the blood and airways of individuals with active pulmonary TB with and without HIV co-infection recruited in Durban, South Africa. Results: Active TB was associated with a profound skewing of neutrophils in the blood toward phenotypes associated with activation and apoptosis, reduced phagocytosis, reverse transmigration, and immune regulation. This skewing was also apparently in airway neutrophils, particularly the regulatory subsets expressing PDL-1 and LOX-1. HIV co-infection did not impact neutrophil subsets in the blood but was associated with a phenotypic change in the airways and a reduction in key neutrophil functional proteins cathelicidin and arginase 1. Discussion: Active TB is associated with profound skewing of blood and airway neutrophils and suggests multiple mechanisms by which neutrophils may exacerbate the immunopathology of TB. These data indicate potential avenues for reducing neutrophil-mediated lung pathology at the point of diagnosis.

B cell heterogeneity in human tuberculosis highlights compartment-specific phenotype and functional roles.

B cells are important in tuberculosis (TB) immunity, but their role in the human lung is understudied. Here, we characterize B cells from lung tissue and matched blood of patients with TB and found they are decreased in the blood and increased in the lungs, consistent with recruitment to infected tissue, where they are located in granuloma associated lymphoid tissue. Flow cytometry and transcriptomics identify multiple B cell populations in the lung, including those associated with tissue resident memory, germinal centers, antibody secretion, proinflammatory atypical B cells, and regulatory B cells, some of which are expanded in TB disease. Additionally, TB lungs contain high levels of Mtb-reactive antibodies, specifically IgM, which promotes Mtb phagocytosis. Overall, these data reveal the presence of functionally diverse B cell subsets in the lungs of patients with TB and suggest several potential localized roles that may represent a target for interventions to promote immunity or mitigate immunopathology.

Myeloid cell expression of CD200R is modulated in active TB disease and regulates Mycobacterium tuberculosis infection in a biomimetic model.

A robust immune response is required for resistance to pulmonary tuberculosis (TB), the primary disease caused by Mycobacterium tuberculosis (Mtb). However, pharmaceutical inhibition of T cell immune checkpoint molecules can result in the rapid development of active disease in latently infected individuals, indicating the importance of T cell immune regulation. In this study, we investigated the potential role of CD200R during Mtb infection, a key immune checkpoint for myeloid cells. Expression of CD200R was consistently downregulated on CD14+ monocytes in the blood of subjects with active TB compared to healthy controls, suggesting potential modulation of this important anti-inflammatory pathway. In homogenized TB-diseased lung tissue, CD200R expression was highly variable on monocytes and CD11b+HLA-DR+ macrophages but tended to be lowest in the most diseased lung tissue sections. This observation was confirmed by fluorescent microscopy, which showed the expression of CD200R on CD68+ macrophages surrounding TB lung granuloma and found expression levels tended to be lower in macrophages closest to the granuloma core and inversely correlated with lesion size. Antibody blockade of CD200R in a biomimetic 3D granuloma-like tissue culture system led to significantly increased Mtb growth. In addition, Mtb infection in this system reduced gene expression of CD200R. These findings indicate that regulation of myeloid cells via CD200R is likely to play an important part in the immune response to TB and may represent a potential target for novel therapeutic intervention.

SARS-CoV-2 infection in immunosuppression evolves sub-lineages which independently accumulate neutralization escape mutations.

One mechanism of variant formation may be evolution during long-term infection in immunosuppressed people. To understand the viral phenotypes evolved during such infection, we tested SARS-CoV-2 viruses evolved from an ancestral B.1 lineage infection lasting over 190 days post-diagnosis in an advanced HIV disease immunosuppressed individual. Sequence and phylogenetic analysis showed two evolving sub-lineages, with the second sub-lineage replacing the first sub-lineage in a seeming evolutionary sweep. Each sub-lineage independently evolved escape from neutralizing antibodies. The most evolved virus for the first sub-lineage (isolated day 34) and the second sub-lineage (isolated day 190) showed similar escape from ancestral SARS-CoV-2 and Delta-variant infection elicited neutralizing immunity despite having no spike mutations in common relative to the B.1 lineage. The day 190 isolate also evolved higher cell-cell fusion and faster viral replication and caused more cell death relative to virus isolated soon after diagnosis, though cell death was similar to day 34 first sub-lineage virus. These data show that SARS-CoV-2 strains in prolonged infection in a single individual can follow independent evolutionary trajectories which lead to neutralization escape and other changes in viral properties.