High serum neutralization following BG505 SOSIP.664 envelope trimer immunization was associated with protection against BG505.SHIV challenge in rhesus macaques in a previous study. In an animal that developed high titer, durable neutralization against a glycan hole on envelope gp120, high throughput, longitudinal, antigen-specific B cell receptor sequencing was conducted. This analysis of more than 4,700 antigen-specific B cells revealed marked intra-clonal expansion and divergence from germline, including three abundant clonotypes that produced autologous neutralizing monoclonal antibodies. Monoclonal antibodies from the neutralizing clonotypes and two other expanded non-neutralizing clonotypes targeted epitopes in the same glycan hole, with neutralizers also demonstrating different capacities to obstruct CD4 binding. Cryo-electron microscopy structures of four neutralizing monoclonal antibodies revealed that they bound to glycan hole epitopes using distinct binding modes. One neutralizing antibody displaced a glycan in the loop V5 upon binding and its footprint includes the CD4 binding loop. The findings provide insight into how antibody recognition of a prominent glycan hole could facilitate different mechanisms of neutralization while underscoring how intra-clonal expansion and maturation with repeated BG505 SOSIP.664 immunization drove high serum neutralization.

Determining the sequence of the transmitted founder virus, the virus that establishes infection in a new host, is critical for understanding early viral dynamics and evolution. Methods to estimate transmitted founder virus sequences using ancestral sequence reconstruction require either sequences collected early in infection or longitudinal samples that can capture the evolutionary history of the viral sequences, which can be challenging to collect. In human immunodeficiency virus infection, viral genomes are integrated into host cells which can persist, creating a proviral archive of the evolutionary history of the virus. We can potentially utilize these proviral sequences, which can be collected later during infection and while the individual is on therapy, to perform ancestral sequence reconstruction to estimate founder virus sequences. We analysed a previously described data set of 12 participants from Zambia who had human immunodeficiency virus sequences collected within months of infection and proviral sequences collected before and after suppressive therapy initiation. We investigated the accuracy of root placement and founder virus sequence reconstruction in these individuals from their proviral sequences using a variety of phylogenetic methods. We had limited success in reconstructing founder virus sequences across all ancestral sequence reconstruction and rooting methods. However, we observed lower error in founder virus sequence reconstruction with participants that had proviral sequences similar to their founder sequence. Our results highlight a need for new methods to be developed in order to effectively reconstruct founder virus sequences from proviral sequences.

CD8+ resident memory T (Trm) cells comprise a small population of frontline sentinels compared with the large tissues they surveil, making outsized contributions to immune protection from infection. Here, we interrogated mechanisms of Trm cell function in primates. Intravenous immunization of macaques with a simian immunodeficiency virus (SIV)-gag-containing heterologous prime-boost-boost vaccine established memory T cells in >30 tissues, including visceral and mucosal compartments. Upon in vivo reactivation in the reproductive tract, antigen-sensing CD8+ Trm activated local stromal, parenchymal, and innate and adaptive immune cells. Stromal and parenchymal cells accentuated leukocyte migration and antiviral defenses. B and plasma cells mobilized into the vaginal mucosa, and bloodborne CD4+ T cells were recruited and adopted a host-defense program. Our findings demonstrate that systemic vaccination promotes a Trm cell response in barrier compartments and that Trm cells repurpose abundant neighboring stromal, parenchymal, and immune cells to amplify alarm signals and activate diverse host defenses.

Sex differences play a role in the pathogenesis of a number of viral diseases. In HIV-1, several studies have reported that chronically infected women have significantly lower plasma viremia than men, although the exact mechanism by which this occurs has yet to be identified. We have performed bulk RNA-seq experiments comparing gene expression between CD4+ T cells from acutely HIV-1–infected men and women in Zambia, because we observe lower viral load (VL) despite higher CD4+ T-cell activation in these women during acute/early infection. In a univariate analysis, we have identified a number of differentially expressed genes in naïve, central memory, and effector memory CD4 T cells of women with consistent elevated expression of genes linked to type 1 interferon (IFN) signaling. Moreover, after controlling for differences in VL and CD4+ T-cell count, genes within the type I IFN signaling pathway were further shown to be more highly expressed in women, whereas those genes more highly expressed in men showed no such enrichment. A subset of the genes highly expressed in women was further identified, including several involved in type I IFN signaling in response to viral infections (IRF7, DDX58, SAMHD1, OAS2, and TRIM14), that both are more highly expressed in CD4+ T cells from women and negatively correlated with VL, suggesting that they play a role in the comparative control of VL observed in women.

OBJECTIVE:To compare HIV incidence among female sex workers (FSW) and single mothers, and to determine the factors associated with seroconversion among both populations. DESIGN:Prospective cohort conducted in Lusaka and Ndola, Zambia between 2012 and 2022. METHODS:Study staff recruited FSW from common sex work locales and recruited single mothers from postnatal infant vaccination clinics. Enrolled participants were HIV-negative, aged 18-45, and identified as either a FSW or single mother. We measured HIV incidence and assessed associated factors using Poisson regression with adjusted rate ratios (aRRs) and 95% confidence intervals (CIs). RESULTS:The study enrolled 2539 women (1533 FSW and 1006 single mothers). HIV incidence was not statistically different for FSW (3.24 per 100 person-years; 95% CI: 2.63-3.95) and single mothers (2.64 per 100 person-years; 95% CI: 2.00-3.43). Factors associated with HIV seroconversion were positive syphilis (aRR: 2.03; 95% CI: 1.46-2.83) and trichomonas (aRR: 1.48; 95% CI: 1.06-2.06) diagnoses, inconsistent condom use (aRR: 1.60; 95% CI: 1.06-2.40), and greater than 6months follow-up time in the study (aRR: 2.45; 95% CI: 1.52-3.94). CONCLUSIONS:Single mothers share similar HIV risk to FSW, and both populations require targeted interventions. For single mothers, government postnatal clinics should combine comprehensive sexual education with screening and treatment for syphilis and trichomoniasis. For FSW, we recommend integrated and accessible interventions to prevent HIV and sexually transmitted infections. Future studies should investigate the social determinants of condom use among both FSW and single mothers.

Antigen-experienced lymphocytes can be equilibrating (continuously migratory between blood and peripheral tissues) or resident (stably surveilling within tissues). Because migration is difficult to measure outside of mice, it is common to extrapolate phenotypic proxies of residence derived from mouse studies to other species. We wished to more rigorously assess the differentiation state and function of equilibrating vs. resident memory CD8+ T cells (TRM) in nonhuman primates (NHP). To this end, we delivered a heterologous prime-boost-boost (HPBB) vaccine to both mice and Indian rhesus macaques that resulted in preternaturally abundant memory CD8 T cells that were distributed in over 30 anatomical sites. We then conducted a constellation of assays to infer migration properties of primate T cells: through single cell genomic analyses of paired mouse vaccinees that underwent parabiotic migration tests, through staged intravascular staining in NHP, and through comparisons of systemic and local routes of immunization. These data informed a cross-species signature that correlated with T cell migration properties. Functional assays were performed in NHP by reactivating memory CD8 T cells in situ and ex vivo, revealing that Trm are uniquely poised to communicate reactivation events to neighboring immune and stromal cells. Bill and Melinda Gates Foundation (OPP1116224, D.M.) NIH grants AI090732 and 5R01AI084913-14 (D.M.) U19AI096187, UM1AI124436 and UM1AI169662 (E.H and R.R.A) NCRR/NIH base grant P51 OD011132 to Emory National Primate Research Center. Lymphocyte Differentiation and Peripheral Maintenance (LYM)

IntroductionHIV-1 subtype A and subtype C infections have different rates of clinical disease progression, with subtype C infected individuals in the IAVI Protocol C multisite acute infection cohort having a 60% faster CD4 loss compared to subtype A.MethodsIn order to investigate whether differences were due to the phenotype of the transmitted founder virus (TFV), or inflammatory cytokines and chemokines, known to drive pathogenesis, we PCR amplified, sequenced and constructed infectious molecular HIV-1 clones from the plasma of 30 acutely infected individuals in Rwanda and Zambia. We next compared the inflammatory plasma cytokine/chemokine profiles of individuals pre- and post-the estimated date of infection of 20 Rwandan individuals infected with subtype A and 34 Zambians infected with subtype C HIV-1.ResultsA comparison of the replicative capacity of 14 subtype A and 16 subtype C TFV showed that they had similar replicative capacity (RC) scores. Nevertheless, high TFV RC scores were linked to more rapid CD4+ T cell loss, and higher inflammatory cytokine levels irrespective of subtype. Multivariable analyses showed that individuals infected with subtype C exhibited a significant increase in the levels of eleven pro-inflammatory cytokines/chemokines after infection, while, in subtype A infections only six cytokines were significantly elevated postinfection. Despite these differences, at 3-months post infection, similar overall biomarker profiles were observed in individuals infected with subtype A or subtype C viruses, primarily due to higher pre-infection baseline biomarker levels in Rwanda. In the combined cohort, we found a highly significant association between faster CD4+ T cell decline and higher levels of ITAC (CXCL11), which in turn was linked to higher TFV RC.DiscussionOverall, the data presented here argue against TFV RC as the basis for different pathogenic outcomes in the subtypes A and C. Moreover, levels of inflammatory cytokines that might drive disease progression were similar during acute infection indicating that additional studies are required to understand the mechanism underlying differences in disease progression between the two subtypes. For both subtypes, high levels of ITAC during acute HIV-1 infection are linked to rapid disease progression.

Background:Human immunodeficiency virus type 1 (HIV-1) is one of the fastest-evolving human pathogens. Understanding HIV-1 transmission, within-host adaptation, and evolutionary dynamics is pivotal for development of interventions and vaccines. HIV-1 infection is generally caused by a single transmitted founder virus (TFV), and TFV sequences are typically obtained using single genome amplification (SGA). However, suboptimal sample quality can cause sequencing failures, representing considerable losses considering the scarcity of acute HIV-1 infection (AHI) samples. Sequencing failures may be mitigated by molecular cloning (MC), which can be less vulnerable to sample quality but more susceptible to polymerase chain reaction (PCR) errors. Here, we explore the feasibility of supplementing SGA with MC data using samples from clinical and research cohorts to determine whether sequence diversity and evolutionary rate estimates are comparable between the techniques. Methods:Plasma samples were selected from participants with documented AHI from an East African research cohort (the International AIDS Vaccine Initiative, 2006-2011) and a clinical cohort from Sweden (1983-2011). SGA and MC sequencing were done on the HIV-1 env V1-V3 region (~940 base pairs). Within-host sequence diversity was determined from maximum likelihood phylogenetic trees, and evolutionary rate by Bayesian phylogenetic analysis. Highlighter plots, Hamming distances, and assessment of star phylogenies were used to quantify TFVs. Results:One hundred participants (median age 30.3 years, 15% female), contributing 350 samples from four longitudinal time points, 10-540 days post-infection, met the inclusion criteria. SGA succeeded on 90% of research cohort and 48% of clinical cohort samples. Comparative analysis of linked SGA and MC data from 10 samples indicated that approximately eight sequences were necessary for diversity estimates. Consistently higher sequence diversity was observed among MC relative to SGA sequences (median [IQR]: 0.009 [0.003, 0.015] and 0.004 [0.001, 0.012] substitutions/site, P = .002), whereas evolutionary rates were comparable between the two methods (0.016 [0.012, 0.019] and 0.011 [0.008, 0.020] substitutions/site/year, P = .232). Five participants with samples obtained within 45 days post-infection were eligible for TFV quantification, and all found to have one TFV using both techniques. Conclusion:MC data is a suitable supplement for SGA-based HIV-1 studies to preserve the value of precious samples for analysis of evolutionary rate, but not for sequence diversity.

Initial interactions between HIV-1 and the immune system at mucosal exposure sites play a critical role in determining whether the virus is eliminated or progresses to establish systemic infection. The virus that successfully crosses the mucosal barrier to establish infection in the new host is referred to as the transmitted/founder (TF) virus. Following mucosal HIV-1 transmission, type 1 interferons (IFN-I) are rapidly induced at sites of initial virus replication. The resistance of TF variants to these antiviral effects of the IFN-I has been studied among HIV-1 subtypes B and C. However, their role in restricting HIV-1 replication among subtypes D and AD recombinant remains unexplored. This study assessed the sensitivity of HIV-1 subtype D and AD recombinant TF viruses to IFN-I by infecting peripheral blood mononuclear cells in vitro with infectious molecular clones of these viruses. Cells were exposed to varying concentrations of interferon-α and interferon-β, and viral replicative capacity was measured using HIV-1 p24 antigen ELISA from culture supernatants. Sensitivity to IFN-I was quantified based on viral replication levels. The results showed that interferon-α was more effective in inhibiting viral replication than interferon-β, regardless of the varying amounts of IFN-I used. However, recombinant AD viruses were found to be more resistant to the antiviral effects of IFN-I compared to subtype D viruses. These findings highlight the differential sensitivity of HIV-1 subtypes AD recombinant and D TF viruses to IFN-I and underscore the potential of IFN-I as a therapeutic strategy to target TF viruses and reduce HIV-1 transmission, particularly in populations where subtype D is prevalent.

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BACKGROUND:CFTR modulators are approved for approximately 90% of people with cystic fibrosis in the USA and provide substantial clinical benefit. N1303K (Asn1303Lys), one of the most common class 2 CFTR defects, has not been approved for these therapies by any regulatory agency. Preclinical investigation by our laboratories showed N1303K CFTR activation with elexacaftor-tezacaftor-ivacaftor (ETI). In this trial, we evaluate whether ETI improves CFTR function, measured by sweat chloride and other clinical outcomes, in people with cystic fibrosis and CFTRN1303K. METHODS:In this prospective, open-label, single-arm trial, participants aged 12 years or older with cystic fibrosis encoding at least one N1303K variant and at least one CFTRN1303K allele who were ineligible for modulator therapy by US Food and Drug Administration labelling were given ETI for 28 days followed by a 28-day washout period at two cystic fibrosis centres in the USA. Participants received two orally administered pills of 100 mg elexacaftor, 50 mg tezacaftor, and 75 mg ivacaftor once daily in the morning, and 150 mg ivacaftor once daily in the evening. The primary endpoint was mean change in sweat chloride from baseline up to day 28 compared with mixed-effects models. Secondary endpoints were changes in percentage of predicted FEV1 (ppFEV1), Cystic Fibrosis Questionnaire-Revised (CFQ-R) respiratory domain, BMI, and weight after ETI therapy. Safety was assessed in all participants who received at least one dose of the study drug and primary and secondary analyses were performed in all participants who took the study drug per protocol. The trial was registered at ClinicalTrials.gov (NCT03506061) and remains open for reporting purposes. FINDINGS:Between June 7, 2022, and Oct 20, 2023, 20 participants (ten male and ten female) were enrolled and received ETI treatment. One participant was lost to follow-up but was included in intention-to-treat analyses. At 28 days, the mean sweat chloride reduction was -1·1 mmol/L (95% CI -5·3 to 3·1; p=0·61) with only one participant showing a sweat chloride decrease greater than 15 mmol/L. There was a mean increase in ppFEV1 from baseline at day 28 of 9·5 percentage points (6·7-12·3; p<0·0001) with 15 (75%) participants showing at least a 5% increase in ppFEV1. Improvements were also identified in mean CFQ-R respiratory domain score (20·8 increase [95% CI 11·9-29·8]; p<0·0001), BMI (0·4 kg/m2 increase [0·2-0·7]; p=0·0017), and weight (1·0 kg increase [0·4-1·7]; p=0·0020) after 28 days of ETI treatment. 14 (70%) of 20 participants had adverse events (12 [60%] mild, one [5%] moderate), with one (5%) serious adverse event of hospitalisation attributed to pneumonia. No deaths were recorded in the study. INTERPRETATION:Individuals with CFTRN1303K showed no change in sweat chloride after 28 days of treatment with ETI. However, there were improvements in secondary clinical endpoints, which suggest clinical efficacy. Our approach provides support for the use of in vitro model systems to inform clinical trials for rare CFTR variants. FUNDING:The Cystic Fibrosis Foundation and the US National Institutes of Health.

The biological characteristics of early transmitted/founder (T/F) variants are crucial factors for viral transmission and constitute key determinants for the development of better therapeutics and vaccine strategies. The present study aimed to generate T/F viruses and to characterize their biological properties. For this purpose, we constructed 18 full-length infectious molecular clones (IMCs) of HIV from recently infected infants. All the clones were characterized genotypically through whole genome sequencing and phenotypically for infectivity, replication kinetics, co-receptor usage, as well as their susceptibility to neutralizing antibodies and entry inhibitors using standard virological assays. Genotypic analysis revealed that all the T/F clones were of non-recombinant subtype C, but some of them harboured the Y181C drug resistance mutation associated with resistance to the non-nucleoside reverse transcriptase inhibitor (NNRTI) class of antiretroviral drugs. In vitro studies showed that while all the IMCs were capable of replicating in PBMCs and utilized the CCR5 co-receptor for cellular entry, the drug-resistant variants had significantly lower replicative capacity and per particle infectivity than the drug-sensitive viruses. Both exhibited similar sensitivities to a standard panel of broadly neutralizing monoclonal antibodies and viral entry inhibitors. These findings suggest that despite their diminished replicative fitness, the drug-resistant T/F variants retain transmission fitness and remain susceptible to neutralizing antibody-based interventions and viral entry inhibitors.

HIV-1 subtypes have distinct geographical distributions, with subtypes A, C, and D and inter-subtype recombinants circulating in sub-Saharan Africa. Historically, individuals living with subtype A viruses exhibit slower CD4 decline and progression to AIDS diagnosis. Despite this, there are few authentic infectious molecular clones (IMCs) of subtype A or AC recombinant transmitted founder (TF) viruses with which to investigate viral impacts on pathogenesis. In this study, we constructed 16 authentic subtype A1 and 4 A1C recombinant IMCs from the IAVI Rwandan Protocol C acute infection cohort and characterized these viruses phenotypically. The virus replicative capacity (RC) scores varied over 50-fold, but the natural substitution of non-consensus amino acids in the p17(MA) domain of Gag was generally linked to higher RC levels. Sensitivity to a panel of broadly neutralizing antibodies (bNAbs) showed that all but one TF was sensitive to N6, which targets the CD4 binding site, while bNAbs PG16 and PGT 128 had a similar level of potency but reduced breadth against our panel of viruses. In contrast, bNAb 10E8V4 revealed high breadth but much lower potency. This panel of well-characterized, authentic subtype A and AC recombinant IMCs provides a resource for studies on the role of the virus subtype in HIV-1 transmission, pathogenesis, and vaccine design.

Background: A goal of mucosal human immunodeficiency virus type 1 (HIV-1) vaccines is to generate mucosal plasma cells producing polymeric IgA (pIgA)-neutralizing antibodies at sites of viral entry. However, vaccine immunogens capable of eliciting IgA neutralizing antibodies (nAbs) that recognize tier 2 viral isolates have not yet been identified. Methods: To determine if stabilized native-like HIV-1 envelope (Env) trimers could generate IgA nAbs, we purified total IgA and IgG from the banked sera of six rhesus macaques that had been found in a previous study to develop serum nAbs after subcutaneous immunization with BG505.664 SOSIP and 3M-052 adjuvant, which is a TLR7/8 agonist. The neutralization of autologous tier 2 BG505 T332N pseudovirus by the IgA and IgG preparations was measured using the TZM-bl assay. Anti-SOSIP binding antibodies (bAbs) were measured by ELISA. Results: The IgG samples were found to have significantly greater levels of both nAb and bAb. However, after normalizing the nAb titer relative to the concentration of bAb, SOSIP-specific IgA purified from 2/6 animals was found to neutralize just as effectively as SOSIP-specific IgG, and in 3/6 animals, neutralization by the specific IgA was significantly greater. The more potent neutralization by IgA in these three animals was associated with a higher percentage of anti-SOSIP J chain-bound (polymeric) antibody. Conclusions: The parenteral vaccination of nonhuman primates with BG505.664 SOSIP generates HIV-1 tier 2 IgA nAbs in serum, including SOSIP-specific polymeric IgA, which appears to neutralize more efficiently than monomeric IgA or IgG. Mucosal delivery of this SOSIP or other stable Env trimers could generate locally synthesized polymeric IgA nAbs in mucosal tissues and secretions.

The complex dynamics of protein expression in plasma during hyperacute HIV-1 infection and its relation to acute retroviral syndrome, viral control, and disease progression are largely unknown. Here, we quantify 1293 blood plasma proteins from 157 longitudinally linked plasma samples collected before, during, and after hyperacute HIV-1 infection of 54 participants from four sub-Saharan African countries. Six distinct longitudinal expression profiles are identified, of which four demonstrate a consistent decrease in protein levels following HIV-1 infection. Proteins involved in inflammatory responses, immune regulation, and cell motility are significantly altered during the transition from pre-infection to one month post-infection. Specifically, decreased ZYX and SCGB1A1 levels, and increased LILRA3 levels are associated with increased risk of acute retroviral syndrome; increased NAPA and RAN levels, and decreased ITIH4 levels with viral control; and increased HPN, PRKCB, and ITGB3 levels with increased risk of disease progression. Overall, this study provides insight into early host responses in hyperacute HIV-1 infection, and present potential biomarkers and mechanisms linked to HIV-1 disease progression and viral load.

The role of human leukocyte antigen (HLA) class I and killer immunoglobulin-like receptor molecules in mediating acute retroviral syndrome (ARS) during human immunodeficiency virus type 1 (HIV-1) infection is unclear. Among 72 sub-Saharan African adults, HLA-A*23 was associated with lower odds of ARS (adjusted odds ratio, 0.10 [95% confidence interval, .01-.48]; P = .009), which warrants further studies to explore its role on HIV-1-specific immunopathogenesis.

Aims. (1) Determine the difference in vocal fry phonation in English and Spanish productions among bilingual young adults, (2) Characterize the effect of spoken language and native language on vocal fry production among English-Spanish bilingual speakers, (3) Identify the effect of first and second language knowl-edge of the listener in the voice perceptual assessment, and (4) Define the effect of the environment of the assess-ment (in situ vs. online), in the voice perceptual assessment. Method. Exploratory cross-sectional study of 34 bilingual (Spanish-English) speakers and six inexperienced lis-teners. Participating speakers produced two speech samples (one in English and one in Spanish). Six inexperi-enced monolingual and bilingual listeners performed the voice perceptual assessment of vocal fry, General grade of hoarseness, and Roughness using a 4-point rating scale. Results. Bilingual speakers used vocal fry more often when they were speaking in English (around 3%) com-pared with their production in Spanish (around 2%). Bilingual native English speakers used vocal fry more often during their productions in both languages compared with bilingual native Spanish speakers. Bilingual listeners had the highest agreement when identifying vocal fry in both languages. Conclusions. Differences in production of vocal fry between native speakers of American English and native speakers of Spanish may be evidence of transferring of vocal behavior (such as vocal fry) from one language to the second one. In addition, being a bilingual listener may have an important effect on the perceptual identifica-tion of voice quality in English and Spanish, as well as vocal fry in English.

Route of immunization can markedly influence the quality of immune response. Here, we show that intradermal (ID) but not intramuscular (IM) modified vaccinia Ankara (MVA) vaccinations provide protection from acquisition of intravaginal tier2 simian-human immunodeficiency virus (SHIV) challenges in female macaques. Both routes of vaccination induce comparable levels of serum IgG with neutralizing and non-neutralizing activities. The protection in MVA-ID group correlates positively with serum neutralizing and antibody-dependent phagocytic activities, and envelope-specific vaginal IgA; while the limited protection in MVA-IM group correlates only with serum neutralizing activity. MVA-ID immunizations induce greater germinal center Tfh and B cell responses, reduced the ratio of Th1 to Tfh cells in blood and showed lower activation of intermediate monocytes and inflammasome compared to MVA-IM immunizations. This lower innate activation correlates negatively with induction of Tfh responses. These data demonstrate that the MVA-ID vaccinations protect against intravaginal SHIV challenges by modulating the innate and T helper responses.

Methods: Primary hNECs as described above were electroporated with ABE8e/sgRNA and allowed to differentiate for 21 days on air-liquid interface culture before being dissociated and prepared for single-cell library preparation performed using 10X Genomics v3.1 3 Library Preparation chemistry with a dual indexing system.Sequencing was performed at the Johns Hopkins Genomic Resources Core Facility via paired-end sequencing 2 × 100 cycles using a NovaSeq 6000 Illumina sequencing machine.Analysis of scRNA-seq results was performed using 10X Genomics Cell Ranger 3.1.0followed by the Seurat package (version 4.1) in R created by the Satija Lab.Subsetting was determined based on comparisons of the number of genes expressed, unique molecular identifier count, and percentage of reads mapped to mitochondrial RNA.Log normalization with a scale factor of 10,000 was used.Results: Analysis of scRNA-seq of 8,791 cells across two samples of primary hNECs (unedited Control, edited 4L) compared CFTR-positive cells and expression levels.Although these cells are from an individual harboring missense variant G480S that produces stable CFTR RNA, an increase in RNA expression can be attributed to the edited 3120+1 allele that generates stable full-length wt RNA.There was a consistent difference in CFTR+ cells between unedited and edited cells across all defined cell type clusters, most noticeably in ciliated/deuterosomal (500% more in edited cells) and cycling basal cells (175% more) (Figure 1A).Cells expressing CFTR increase from 2.5% of total cells in unedited samples to 3.96% in edited samples, and normalized read count per sample increased from 0.049 to 0.081.Editing increased the percentage of basal and cycling basal cells expressing CFTR, indicating that correction was achieved in airway progenitor cells (Figure 1B). Figure 1.(A) Percentage increase in CFTR+ cells between unedited and edited samples.(B) Comparison of percentage of CFTR+ cells in cell type clusters between unedited and edited samples.Conclusions: Base editing of primary hNECs carrying the 3120+1G >A variant show levels of CFTR function after editing equivalent to more than 50% of wt, exceeding the established clinically significant threshold of approximately 10%.We demonstrate that editing is occurring in progenitor cells after delivery of RNA encoding CRISPR along with guide RNAs by electroporation to undifferentiated primary hNECs.In ongoing work, we are repeating single-cell analysis and investigating whether delivery of this technology using nanoparticles corrects splicing of the 3120+1G >A variant in progenitor airway cells as a prelude to in vivo delivery.