Multi-compartmental diversification of neutralizing antibody lineages dissected in SARS-CoV-2 spike-immunized macaques.

The continued evolution of SARS-CoV-2 underscores the need to understand qualitative aspects of the humoral immune response elicited by spike immunization. Here, we combine monoclonal antibody (mAb) isolation with deep B cell receptor (BCR) repertoire sequencing of rhesus macaques immunized with prefusion-stabilized spike glycoprotein. Longitudinal tracing of spike-sorted B cell lineages in multiple immune compartments demonstrates increasing somatic hypermutation and broad dissemination of vaccine-elicited B cells in draining and non-draining lymphoid compartments, including the bone marrow, spleen and, most notably, periaortic lymph nodes. Phylogenetic analysis of spike-specific monoclonal antibody lineages identified through deep repertoire sequencing delineates extensive intra-clonal diversification that shaped neutralizing activity. Structural analysis of the spike in complex with a broadly neutralizing mAb provides a molecular basis for the observed differences in neutralization breadth between clonally related antibodies. Our findings highlight that immunization leads to extensive intra-clonal B cell evolution where members of the same lineage can both retain the original epitope specificity and evolve to recognize additional spike variants not previously encountered.

Spike deep mutational scanning helps predict success of SARS-CoV-2 clades.

SARS-CoV-2 variants acquire mutations in the spike protein that promote immune evasion1 and affect other properties that contribute to viral fitness, such as ACE2 receptor binding and cell entry2,3. Knowledge of how mutations affect these spike phenotypes can provide insight into the current and potential future evolution of the virus. Here we use pseudovirus deep mutational scanning4 to measure how more than 9,000 mutations across the full XBB.1.5 and BA.2 spikes affect ACE2 binding, cell entry or escape from human sera. We find that mutations outside the receptor-binding domain (RBD) have meaningfully affected ACE2 binding during SARS-CoV-2 evolution. We also measure how mutations to the XBB.1.5 spike affect neutralization by serum from individuals who recently had SARS-CoV-2 infections. The strongest serum escape mutations are in the RBD at sites 357, 420, 440, 456 and 473; however, the antigenic effects of these mutations vary across individuals. We also identify strong escape mutations outside the RBD; however, many of them decrease ACE2 binding, suggesting they act by modulating RBD conformation. Notably, the growth rates of human SARS-CoV-2 clades can be explained in substantial part by the measured effects of mutations on spike phenotypes, suggesting our data could enable better prediction of viral evolution.

Optimized SMRT-UMI protocol produces highly accurate sequence datasets from diverse populations-Application to HIV-1 quasispecies.

Pathogen diversity resulting in quasispecies can enable persistence and adaptation to host defenses and therapies. However, accurate quasispecies characterization can be impeded by errors introduced during sample handling and sequencing, which can require extensive optimizations to overcome. We present complete laboratory and bioinformatics workflows to overcome many of these hurdles. The Pacific Biosciences single molecule real-time platform was used to sequence polymerase-chain reaction (PCR) amplicons derived from cDNA templates tagged with unique molecular identifiers (SMRT-UMI). Optimized laboratory protocols were developed through extensive testing of different sample preparation conditions to minimize between-template recombination during PCR. The use of UMI allowed accurate template quantitation as well as removal of point mutations introduced during PCR and sequencing to produce a highly accurate consensus sequence from each template. Production of highly accurate sequences from the large datasets produced from SMRT-UMI sequencing is facilitated by a novel bioinformatic pipeline, Probabilistic Offspring Resolver for Primer IDs (PORPIDpipeline). PORPIDpipeline automatically filters and parses circular consensus reads by sample, identifies and discards reads with UMIs likely created from PCR and sequencing errors, generates consensus sequences, checks for contamination within the dataset, and removes any sequence with evidence of PCR recombination, heteroduplex formation, or early cycle PCR errors. The optimized SMRT-UMI sequencing and PORPIDpipeline methods presented here represent a highly adaptable and established starting point for accurate sequencing of diverse pathogens. These methods are illustrated through characterization of human immunodeficiency virus quasispecies in a virus transmitter-recipient pair of individuals.

Structural basis of broad SARS-CoV-2 cross-neutralization by affinity-matured public antibodies.

Descendants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant now account for almost all SARS-CoV-2 infections. The Omicron variant and its sublineages have spike glycoproteins that are highly diverged from the pandemic founder and first-generation vaccine strain, resulting in significant evasion from monoclonal antibody therapeutics and vaccines. Understanding how commonly elicited antibodies can broaden to cross-neutralize escape variants is crucial. We isolate IGHV3-53, using "public" monoclonal antibodies (mAbs) from an individual 7 months post infection with the ancestral virus and identify antibodies that exhibit potent and broad cross-neutralization, extending to the BA.1, BA.2, and BA.4/BA.5 sublineages of Omicron. Deep mutational scanning reveals these mAbs' high resistance to viral escape. Structural analysis via cryoelectron microscopy of a representative broadly neutralizing antibody, CAB-A17, in complex with the Omicron BA.1 spike highlights the structural underpinnings of this broad neutralization. By reintroducing somatic hypermutations into a germline-reverted CAB-A17, we delineate the role of affinity maturation in the development of cross-neutralization by a public class of antibodies.

Maintenance of caecal homeostasis by diverse adaptive immune cells in the rhesus macaque.

Objectives: The caecum bridges the small and large intestine and plays a front-line role in discriminating gastrointestinal antigens. Although dysregulated in acute and chronic conditions, the tissue is often overlooked immunologically. Methods: To address this issue, we applied single-cell transcriptomic-V(D)J sequencing to FACS-isolated CD45+ caecal patch/lamina propria leukocytes from a healthy (5-year-old) female rhesus macaque ex vivo and coupled these data to VDJ deep sequencing reads from haematopoietic tissues. Results: We found caecal NK cells and ILC3s to co-exist with a spectrum of effector T cells partially derived from SOX4 + recent thymic emigrants. Tolerogenic Vγ8Vδ1-T cells, plastic CD4+ T helper cells and GZMK + EOMES + and TMIGD2 + tissue-resident memory CD8+ T cells were present and differed metabolically. An IL13 + GATA3 + Th2 subset expressing eicosanoid pathway enzymes was accompanied by IL1RL1 + GATA3 + regulatory T cells and a minor proportion of IgE+ plasma cells (PCs), illustrating tightly regulated type 2 immunity devoid of ILC2s. In terms of B lymphocyte lineages, caecal patch antigen-presenting memory B cells sat alongside germinal centre cells undergoing somatic hypermutation and differentiation into IGF1 + PCs. Prototypic gene expression signatures decreased across PC clusters, and notably, expanded IgA clonotypes could be traced in VDJ deep sequencing reads from additional compartments, including the bone marrow, supporting that these cells contribute a steady stream of systemic antibodies. Conclusions: The data advance our understanding of caecal immunological function, revealing processes involved in barrier maintenance and molecular networks relevant to disease.

Prevention efficacy of the broadly neutralizing antibody VRC01 depends on HIV-1 envelope sequence features.

In the Antibody Mediated Prevention (AMP) trials (HVTN 704/HPTN 085 and HVTN 703/HPTN 081), prevention efficacy (PE) of the monoclonal broadly neutralizing antibody (bnAb) VRC01 (vs. placebo) against HIV-1 acquisition diagnosis varied according to the HIV-1 Envelope (Env) neutralization sensitivity to VRC01, as measured by 80% inhibitory concentration (IC80). Here, we performed a genotypic sieve analysis, a complementary approach to gaining insight into correlates of protection that assesses how PE varies with HIV-1 sequence features. We analyzed HIV-1 Env amino acid (AA) sequences from the earliest available HIV-1 RNA-positive plasma samples from AMP participants diagnosed with HIV-1 and identified Env sequence features that associated with PE. The strongest Env AA sequence correlate in both trials was VRC01 epitope distance that quantifies the divergence of the VRC01 epitope in an acquired HIV-1 isolate from the VRC01 epitope of reference HIV-1 strains that were most sensitive to VRC01-mediated neutralization. In HVTN 704/HPTN 085, the Env sequence-based predicted probability that VRC01 IC80 against the acquired isolate exceeded 1 µg/mL also significantly associated with PE. In HVTN 703/HPTN 081, a physicochemical-weighted Hamming distance across 50 VRC01 binding-associated Env AA positions of the acquired isolate from the most VRC01-sensitive HIV-1 strain significantly associated with PE. These results suggest that incorporating mutation scoring by BLOSUM62 and weighting by the strength of interactions at AA positions in the epitope:VRC01 interface can optimize performance of an Env sequence-based biomarker of VRC01 prevention efficacy. Future work could determine whether these results extend to other bnAbs and bnAb combinations.

Three immunizations with Novavax's protein vaccines increase antibody breadth and provide durable protection from SARS-CoV-2.

The immune responses to Novavax's licensed NVX-CoV2373 nanoparticle Spike protein vaccine against SARS-CoV-2 remain incompletely understood. Here, we show in rhesus macaques that immunization with Matrix-MTM adjuvanted vaccines predominantly elicits immune events in local tissues with little spillover to the periphery. A third dose of an updated vaccine based on the Gamma (P.1) variant 7 months after two immunizations with licensed NVX-CoV2373 resulted in significant enhancement of anti-spike antibody titers and antibody breadth including neutralization of forward drift Omicron variants. The third immunization expanded the Spike-specific memory B cell pool, induced significant somatic hypermutation, and increased serum antibody avidity, indicating considerable affinity maturation. Seven months after immunization, vaccinated animals controlled infection by either WA-1 or P.1 strain, mediated by rapid anamnestic antibody and T cell responses in the lungs. In conclusion, a third immunization with an adjuvanted, low-dose recombinant protein vaccine significantly improved the quality of B cell responses, enhanced antibody breadth, and provided durable protection against SARS-CoV-2 challenge.

Full-spike deep mutational scanning helps predict the evolutionary success of SARS-CoV-2 clades.

SARS-CoV-2 variants acquire mutations in spike that promote immune evasion and impact other properties that contribute to viral fitness such as ACE2 receptor binding and cell entry. Knowledge of how mutations affect these spike phenotypes can provide insight into the current and potential future evolution of the virus. Here we use pseudovirus deep mutational scanning to measure how >9,000 mutations across the full XBB.1.5 and BA.2 spikes affect ACE2 binding, cell entry, or escape from human sera. We find that mutations outside the receptor-binding domain (RBD) have meaningfully impacted ACE2 binding during SARS-CoV-2 evolution. We also measure how mutations to the XBB.1.5 spike affect neutralization by serum from individuals who recently had SARS-CoV-2 infections. The strongest serum escape mutations are in the RBD at sites 357, 420, 440, 456, and 473-however, the antigenic impacts of these mutations vary across individuals. We also identify strong escape mutations outside the RBD; however many of them decrease ACE2 binding, suggesting they act by modulating RBD conformation. Notably, the growth rates of human SARS-CoV-2 clades can be explained in substantial part by the measured effects of mutations on spike phenotypes, suggesting our data could enable better prediction of viral evolution.

SARS-CoV-2 evolution in the Omicron era.

Since SARS-CoV-2 BA.5 (Omicron) emerged and spread in 2022, Omicron lineages have markedly diversified. Here we review the evolutionary trajectories and processes that underpin the emergence of these lineages, and identify the most prevalent sublineages. We discuss the potential origins of second-generation BA.2 lineages. Simple and complex recombination, antigenic drift and convergent evolution have enabled SARS-CoV-2 to accumulate mutations that alter its antigenicity. We also discuss the potential evolutionary trajectories of SARS-CoV-2 in the future.

Antigen receptor stimulation induces purifying selection against pathogenic mitochondrial tRNA mutations.

Pathogenic mutations in mitochondrial (mt) tRNA genes that compromise oxidative phosphorylation (OXPHOS) exhibit heteroplasmy and cause a range of multisyndromic conditions. Although mitochondrial disease patients are known to suffer from abnormal immune responses, how heteroplasmic mtDNA mutations affect the immune system at the molecular level is largely unknown. Here, in mice carrying pathogenic C5024T in mt-tRNAAla and in patients with mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes (MELAS) syndrome carrying A3243G in mt-tRNALeu, we found memory T and B cells to have lower pathogenic mtDNA mutation burdens than their antigen-inexperienced naive counterparts, including after vaccination. Pathogenic burden reduction was less pronounced in myeloid compared with lymphoid lineages, despite C5024T compromising macrophage OXPHOS capacity. Rapid dilution of the C5024T mutation in T and B cell cultures could be induced by antigen receptor-triggered proliferation and was accelerated by metabolic stress conditions. Furthermore, we found C5024T to dysregulate CD8+ T cell metabolic remodeling and IFN-γ production after activation. Together, our data illustrate that the generation of memory lymphocytes shapes the mtDNA landscape, wherein pathogenic variants dysregulate the immune response.

Vaccine-induced correlate of protection against fatal COVID-19 in older and frail adults during waves of neutralization-resistant variants of concern: an observational study.

Background: To inform future preventive measures including repeated vaccinations, we have searched for a clinically useful immune correlate of protection against fatal COVID-19 among nursing homes residents. Methods: We performed repeated capillary blood sampling with analysis of S-binding IgG in an open cohort of nursing home residents in Sweden. We analyzed immunological and registry data from 16 September 2021 to 31 August 2022 with follow-up of deaths to 30 September 2022. The study period included implementation of the 3rd and 4th mRNA monovalent vaccine doses and Omicron virus waves. Findings: A total of 3012 nursing home residents with median age 86 were enrolled. The 3rd mRNA dose elicited a 99-fold relative increase of S-binding IgG in blood and corresponding increase of neutralizing antibodies. The 4th mRNA vaccine dose boosted levels 3.8-fold. Half-life of S-binding IgG was 72 days. A total 528 residents acquired their first SARS-CoV-2 infection after the 3rd or the 4th vaccine dose and the associated 30-day mortality was 9.1%. We found no indication that levels of vaccine-induced antibodies protected against infection with Omicron VOCs. In contrast, the risk of death was inversely correlated to levels of S-directed IgG below the 20th percentile. The death risk plateaued at population average above the lower 35th percentile of S-binding IgG. Interpretation: In the absence of neutralizing antibodies that protect from infection, quantification of S-binding IgG post vaccination may be useful to identify the most vulnerable for fatal COVID-19 among the oldest and frailest. This information is of importance for future strategies to protect vulnerable populations against neutralization resistant variants of concern. Funding: Swedish Research Council, SciLifeLab via Knut and Alice Wallenberg Foundation, VINNOVA. Swedish Healthcare Regions, and Erling Persson Foundation.

Antigen pressure from two founder viruses induces multiple insertions at a single antibody position to generate broadly neutralizing HIV antibodies.

Vaccination strategies aimed at maturing broadly neutralizing antibodies (bnAbs) from naïve precursors are hindered by unusual features that characterize these Abs, including insertions and deletions (indels). Longitudinal studies of natural HIV infection cases shed light on the complex processes underlying bnAb development and have suggested a role for superinfection as a potential enhancer of neutralization breadth. Here we describe the development of a potent bnAb lineage that was elicited by two founder viruses to inform vaccine design. The V3-glycan targeting bnAb lineage (PC39-1) was isolated from subtype C-infected IAVI Protocol C elite neutralizer, donor PC39, and is defined by the presence of multiple independent insertions in CDRH1 that range from 1-11 amino acids in length. Memory B cell members of this lineage are predominantly atypical in phenotype yet also span the class-switched and antibody-secreting cell compartments. Development of neutralization breadth occurred concomitantly with extensive recombination between founder viruses before each virus separated into two distinct population "arms" that evolved independently to escape the PC39-1 lineage. Ab crystal structures show an extended CDRH1 that can help stabilize the CDRH3. Overall, these findings suggest that early exposure of the humoral system to multiple related Env molecules could promote the induction of bnAbs by focusing Ab responses to conserved epitopes.

Evaluation of Archival HIV DNA in Brain and Lymphoid Tissues.

HIV reservoirs persist in anatomic compartments despite antiretroviral therapy (ART). Characterizing archival HIV DNA in the central nervous system (CNS) and other tissues is crucial to inform cure strategies. We evaluated paired autopsy brain-frontal cortex (FC), occipital cortex (OCC), and basal ganglia (BG)-and peripheral lymphoid tissues from 63 people with HIV. Participants passed away while virally suppressed on ART at the last visit and without evidence of CNS opportunistic disease. We quantified total HIV DNA in all participants and obtained full-length HIV-envelope (FL HIV-env) sequences from a subset of 14 participants. We detected HIV DNA (gag) in most brain (65.1%) and all lymphoid tissues. Lymphoid tissues had higher HIV DNA levels than the brain (P < 0.01). Levels of HIV gag between BG and FC were similar (P > 0.2), while OCC had the lowest levels (P = 0.01). Females had higher HIV DNA levels in tissues than males (gag, P = 0.03; 2-LTR, P = 0.05), suggesting possible sex-associated mechanisms for HIV reservoir persistence. Most FL HIV-env sequences (n = 143) were intact, while 42 were defective. Clonal sequences were found in 8 out of 14 participants, and 1 participant had clonal defective sequences in the brain and spleen, suggestive of cell migration. From 10 donors with paired brain and lymphoid sequences, we observed evidence of compartmentalized sequences in 2 donors. Our data further the idea that the brain is a site for archival HIV DNA during ART where compartmentalized provirus may occur in a subset of people. Future studies assessing FL HIV-provirus and replication competence are needed to further evaluate the HIV reservoirs in tissues. IMPORTANCE HIV infection of the brain is associated with adverse neuropsychiatric outcomes, despite efficient antiretroviral treatment. HIV may persist in reservoirs in the brain and other tissues, which can seed virus replication if treatment is interrupted, representing a major challenge to cure HIV. We evaluated reservoirs and genetic features in postmortem brain and lymphoid tissues from people with HIV who passed away during suppressed HIV replication. We found a differential distribution of HIV reservoirs across brain regions which was lower than that in lymphoid tissues. We observed that most HIV reservoirs in tissues had intact envelope sequences, suggesting they could potentially generate replicative viruses. We found that women had higher HIV reservoir levels in brain and lymphoid tissues than men, suggesting possible sex-based mechanisms of maintenance of HIV reservoirs in tissues, warranting further investigation. Characterizing the archival HIV DNA in tissues is important to inform future HIV cure strategies.

Emergence and antibody evasion of BQ, BA.2.75 and SARS-CoV-2 recombinant sub-lineages in the face of maturing antibody breadth at the population level.

BACKGROUND: The Omicron era of the COVID-19 pandemic commenced at the beginning of 2022 and whilst it started with primarily BA.1, it was latter dominated by BA.2 and the related sub-lineage BA.5. Following resolution of the global BA.5 wave, a diverse grouping of Omicron sub-lineages emerged derived from BA.2, BA.5 and recombinants thereof. Whilst emerging from distinct lineages, all shared similar changes in the Spike glycoprotein affording them an outgrowth advantage through evasion of neutralising antibodies. METHODS: Over the course of 2022, we monitored the potency and breadth of antibody neutralization responses to many emerging variants in the Australian community at three levels: (i) we tracked over 420,000 U.S. plasma donors over time through various vaccine booster roll outs and Omicron waves using sequentially collected IgG pools; (ii) we mapped the antibody response in individuals using blood from stringently curated vaccine and convalescent cohorts. (iii) finally we determine the in vitro efficacy of clinically approved therapies Evusheld and Sotrovimab. FINDINGS: In pooled IgG samples, we observed the maturation of neutralization breadth to Omicron variants over time through continuing vaccine and infection waves. Importantly, in many cases, we observed increased antibody breadth to variants that were yet to be in circulation. Determination of viral neutralization at the cohort level supported equivalent coverage across prior and emerging variants with isolates BQ.1.1, XBB.1, BR.2.1 and XBF the most evasive. Further, these emerging variants were resistant to Evusheld, whilst increasing neutralization resistance to Sotrovimab was restricted to BQ.1.1 and XBF. We conclude at this current point in time that dominant variants can evade antibodies at levels equivalent to their most evasive lineage counterparts but sustain an entry phenotype that continues to promote an additional outgrowth advantage. In Australia, BR.2.1 and XBF share this phenotype and, in contrast to global variants, are uniquely dominant in this region in the later months of 2022. INTERPRETATION: Whilst the appearance of a diverse range of omicron lineages has led to primary or partial resistance to clinically approved monoclonal antibodies, the maturation of the antibody response across both cohorts and a large donor pools importantly observes increasing breadth in the antibody neutralisation responses over time with a trajectory that covers both current and known emerging variants. FUNDING: This work was primarily supported by Australian Medical Foundation research grants MRF2005760 (SGT, GM & WDR), Medical Research Future Fund Antiviral Development Call grant (WDR), the New South Wales Health COVID-19 Research Grants Round 2 (SGT & FB) and the NSW Vaccine Infection and Immunology Collaborative (VIIM) (ALC). Variant modeling was supported by funding from SciLifeLab's Pandemic Laboratory Preparedness program to B.M. (VC-2022-0028) and by the European Union's Horizon 2020 research and innovation programme under grant agreement no. 101003653 (CoroNAb) to B.M.

Vaccination of SARS-CoV-2-infected individuals expands a broad range of clonally diverse affinity-matured B cell lineages.

Vaccination of SARS-CoV-2 convalescent individuals generates broad and potent antibody responses. Here, we isolate 459 spike-specific monoclonal antibodies (mAbs) from two individuals who were infected with the index variant of SARS-CoV-2 and later boosted with mRNA-1273. We characterize mAb genetic features by sequence assignments to the donors' personal immunoglobulin genotypes and assess antibody neutralizing activities against index SARS-CoV-2, Beta, Delta, and Omicron variants. The mAbs used a broad range of immunoglobulin heavy chain (IGH) V genes in the response to all sub-determinants of the spike examined, with similar characteristics observed in both donors. IGH repertoire sequencing and B cell lineage tracing at longitudinal time points reveals extensive evolution of SARS-CoV-2 spike-binding antibodies from acute infection until vaccination five months later. These results demonstrate that highly polyclonal repertoires of affinity-matured memory B cells are efficiently recalled by vaccination, providing a basis for the potent antibody responses observed in convalescent persons following vaccination.

Frequent use of IGHV3-30-3 in SARS-CoV-2 neutralizing antibody responses.

The antibody response to SARS-CoV-2 shows biased immunoglobulin heavy chain variable (IGHV) gene usage, allowing definition of genetic signatures for some classes of neutralizing antibodies. We investigated IGHV gene usage frequencies by sorting spike-specific single memory B cells from individuals infected with SARS-CoV-2 early in the pandemic. From two study participants and 703 spikespecific B cells, the most used genes were IGHV1-69, IGHV3-30-3, and IGHV3-30. Here, we focused on the IGHV3-30 group of genes and an IGHV3-30-3-using ultrapotent neutralizing monoclonal antibody, CAB-F52, which displayed broad neutralizing activity also in its germline-reverted form. IGHV3-30-3 is encoded by a region of the IGH locus that is highly variable at both the allelic and structural levels. Using personalized IG genotyping, we found that 4 of 14 study participants lacked the IGHV3-30-3 gene on both chromosomes, raising the question if other, highly similar IGHV genes could substitute for IGHV3-30-3 in persons lacking this gene. In the context of CAB-F52, we found that none of the tested IGHV3-33 alleles, but several IGHV3-30 alleles could substitute for IGHV3-30-3, suggesting functional redundancy between the highly homologous IGHV3-30 and IGHV3-30-3 genes for this antibody.

A Multiparametric and High-Throughput Platform for Host-Virus Binding Screens.

Speed is key during infectious disease outbreaks. It is essential, for example, to identify critical host binding factors to pathogens as fast as possible. The complexity of host plasma membrane is often a limiting factor hindering fast and accurate determination of host binding factors as well as high-throughput screening for neutralizing antimicrobial drug targets. Here, we describe a multiparametric and high-throughput platform tackling this bottleneck and enabling fast screens for host binding factors as well as new antiviral drug targets. The sensitivity and robustness of our platform were validated by blocking SARS-CoV-2 particles with nanobodies and IgGs from human serum samples.

Optimized SMRT-UMI protocol produces highly accurate sequence datasets from diverse populations - application to HIV-1 quasispecies.

Pathogen diversity resulting in quasispecies can enable persistence and adaptation to host defenses and therapies. However, accurate quasispecies characterization can be impeded by errors introduced during sample handling and sequencing which can require extensive optimizations to overcome. We present complete laboratory and bioinformatics workflows to overcome many of these hurdles. The Pacific Biosciences single molecule real-time platform was used to sequence PCR amplicons derived from cDNA templates tagged with universal molecular identifiers (SMRT-UMI). Optimized laboratory protocols were developed through extensive testing of different sample preparation conditions to minimize between-template recombination during PCR and the use of UMI allowed accurate template quantitation as well as removal of point mutations introduced during PCR and sequencing to produce a highly accurate consensus sequence from each template. Handling of the large datasets produced from SMRT-UMI sequencing was facilitated by a novel bioinformatic pipeline, Probabilistic Offspring Resolver for Primer IDs (PORPIDpipeline), that automatically filters and parses reads by sample, identifies and discards reads with UMIs likely created from PCR and sequencing errors, generates consensus sequences, checks for contamination within the dataset, and removes any sequence with evidence of PCR recombination or early cycle PCR errors, resulting in highly accurate sequence datasets. The optimized SMRT-UMI sequencing method presented here represents a highly adaptable and established starting point for accurate sequencing of diverse pathogens. These methods are illustrated through characterization of human immunodeficiency virus (HIV) quasispecies.