Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines.

Understanding vaccine-elicited protection against SARS-CoV-2 variants and other sarbecoviruses is key for guiding public health policies. We show that a clinical stage multivalent SARS-CoV-2 spike receptor-binding domain nanoparticle (RBD-NP) vaccine protects mice from SARS-CoV-2 challenge after a single immunization, indicating a potential dose-sparing strategy. We benchmarked serum neutralizing activity elicited by RBD-NPs in non-human primates against a lead prefusion-stabilized SARS-CoV-2 spike (HexaPro) using a panel of circulating mutants. Polyclonal antibodies elicited by both vaccines are similarly resilient to many RBD residue substitutions tested, although mutations at and surrounding position 484 have negative consequences for neutralization. Mosaic and cocktail nanoparticle immunogens displaying multiple sarbecovirus RBDs elicit broad neutralizing activity in mice and protect mice against SARS-CoV challenge even in the absence of SARS-CoV RBD in the vaccine. This study provides proof of principle that multivalent sarbecovirus RBD-NPs induce heterotypic protection and motivates advancing such broadly protective sarbecovirus vaccines to the clinic.

Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2.

A safe, effective, and scalable vaccine is needed to halt the ongoing SARS-CoV-2 pandemic. We describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 SARS-CoV-2 spike receptor-binding domains (RBDs) in a highly immunogenic array and induce neutralizing antibody titers 10-fold higher than the prefusion-stabilized spike despite a 5-fold lower dose. Antibodies elicited by the RBD nanoparticles target multiple distinct epitopes, suggesting they may not be easily susceptible to escape mutations, and exhibit a lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the assembled nanoparticles suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms and have launched cGMP manufacturing efforts to advance the SARS-CoV-2-RBD nanoparticle vaccine into the clinic.

The adverse association between stimulant use for attention deficit hyperactivity disorder (ADHD) and semen parameters.

This study examined the relationship between stimulant medications used for the treatment of attention deficit hyperactivity disorder and semen parameters. We performed a retrospective cohort study at a large, academic institution between 2002 and 2020. We included men with a semen analysis without prior spermatotoxic medication use, empiric medical therapy exposure or confounding medical diagnoses (varicocele, Klinefelter's syndrome, cryptorchidism, cystic fibrosis, diabetes, cancer or cancer-related treatment, and azoospermia). Men were stratified by stimulant exposure (methylphenidate or amphetamines). A multivariable linear regression was fit to assess the association between individual semen parameters, age, stimulant exposure and non-stimulant medication use. Of 8,861 men identified, 106 men had active prescriptions for stimulants within 90 days prior to semen testing. After controlling for age and exposure to non-stimulant medications, stimulant use was associated with decreased total motile sperm count (ß: -18.00 mil/ejaculate and standard error: 8.44, p = 0.033) in the setting of decreased semen volume (ß: -0.35 ml, and standard error: 0.16, p = 0.035), but not sperm concentration, motility and morphology. These findings suggest a role for reproductive physicians and mental health providers to consider counselling men on the potential negative impact of stimulants prescribed for attention deficit hyperactivity disorder on semen volume during fertility planning.

Predictors of use and overall survival for patients undergoing metastasectomy for bladder cancer in a national cohort.

OBJECTIVES: To determine the use of surgical resection of metastatic disease in a large national sample and its association with overall survival. METHODS: The National Cancer Database was queried for patients with metastatic bladder cancer (2004-2016). Overall survival was assessed using Kaplan-Meier and multivariable Cox analyses. The associations between covariates and use of metastasectomy were assessed with multivariable logistic regression. RESULTS: Of the 16 382 patients with metastatic bladder cancer included, 6.8% underwent metastasectomy. Its use increased over time (4.7% in 2004 to 6.6% in 2016; per year odds ratio 1.02, 95% confidence interval 1.00-1.04, P = 0.019). Median survival was 7.0 months for patients who received metastasectomy and 5.1 months for those who did not (hazard ratio 0.85, 95% confidence interval 0.79-0.91, P < 0.001). In subgroup analyses, metastasectomy predicted longer survival in patients with lung (hazard ratio 0.73, 95% confidence interval 0.61-0.88, P = 0.001) or brain metastases (hazard ratio 0.58, 95% confidence interval 0.35-0.96, P = 0.035) and in patients with variant histology (hazard ratio 0.80, 95% confidence interval 0.69-0.93, P = 0.003). CONCLUSIONS: In a national sample, the use of metastasectomy for bladder cancer is low. Furthermore, metastasectomy is associated with longer survival overall and in multiple subgroups. However, these results should be validated in future studies.

Metabolically inactive insulin analogue does not prevent autoimmune diabetes in NOD mice.

AIMS/HYPOTHESIS: Insulin is widely considered to be a driver antigen in type 1 diabetes in humans and in mouse models of the disease. Therefore, insulin or insulin analogues are candidates for tolerogenic drugs to prevent disease onset in individuals with risk of diabetes. Previous experiments have shown that autoimmune diabetes can be prevented in NOD mice by repeated doses of insulin administered via an oral, nasal or parenteral route, but clinical trials in humans have not succeeded. The hypoglycaemic activity of insulin is dose-limiting in clinical studies attempting tolerance and disease prevention. Here, we aimed to investigate the therapeutic potential of metabolically inactive insulin analogue (MII) in NOD mice. METHODS: The tolerogenic potential of MII to prevent autoimmune diabetes was studied by administering multiple i.v. or s.c. injections of MII to non-diabetic 7-12-week-old female NOD mice in three geographical colony locations. The incidence of diabetes was assessed from daily or weekly blood glucose measurements. The effect of MII on insulin autoantibody levels was studied using an electrochemiluminescence-based insulin autoantibody assay. The effect on the number of insulin-reactive CD8+ and CD4+ T lymphocytes in peripheral lymphoid tissue was studied with MHC class I and MHC class II tetramers, respectively. RESULTS: We found that twice-weekly s.c. administration of MII accelerates rather than prevents diabetes. High-dose i.v. treatment did not prevent disease or affect insulin autoantibody levels, but it increased the amount of insulin-reactive CD4+ T lymphocytes in peripheral lymphoid tissue. CONCLUSIONS/INTERPRETATION: Our data suggest that parenteral MII, even when used in high doses, has little or no therapeutic potential in NOD mice and may exacerbate disease.

Oral insulin (human, murine, or porcine) does not prevent diabetes in the non-obese diabetic mouse.

Studies have shown oral insulin prevents type 1 diabetes (T1D) in mouse models, however human trials were inconclusive. We tested the ability of different insulins to prevent T1D in non-obese diabetic mice. Mice received oral insulin or PBS twice weekly and disease was monitored. Contrary to previous studies, no insulin tested showed significant ability to prevent T1D, nor did testing of linked suppression in a delayed type hypersensitivity model have reproducible effect. To investigate delivery of antigen within the GI tract, blue dye was fed to mice. Dye traveled 5-8 cm from stomach to small intestine within 10s, suggesting orally administered antigen may not get digested in the stomach in mice. Insulin incubated with jejunum extracts was instantly digested. Thus, in humans large doses of insulin may be required to achieve tolerance as antigen may be more vulnerable to digestion in the stomach even before reaching the small intestine.

Unique case of vascularization: superficial brachial artery and radial persistent median artery.

During a routine cadaveric dissection of a 93-year-old male donor, unique arterial variations were observed in the right upper extremity. This rare arterial branching pattern began at the third part of the axillary artery (AA), where it gave off a large superficial brachial artery (SBA) before bifurcating into the subscapular artery and a common stem. The common stem then gave off a division for the anterior and posterior circumflex humeral arteries, before continuing as a small brachial artery (BA). The BA terminated as a muscular branch to the brachialis muscle. The SBA bifurcated into a large radial artery (RA) and small ulnar artery (UA) in the cubital fossa. The UA branching pattern was atypical, giving off only muscular branches in the forearm and a deep UA before contributing to the superficial palmar arch (SPA). The RA provided the radial recurrent artery and a common trunk (CT) proximally before continuing its course to the hand. The CT from the RA gave off a branch that divided into anterior and posterior ulnar recurrent arteries, as well as muscular branches, before it bifurcated into the persistent median artery (PMA) and the common interosseous artery. The PMA anastomosed with the UA before entering the carpal tunnel and contributed to the SPA. This case presents a unique combination of arterial variations in the upper extremity and is clinically and pathologically relevant.

Macromolecular Cargo Encapsulation via In Vitro Assembly of Two-Component Protein Nanoparticles.

Self-assembling protein nanoparticles are a promising class of materials for targeted drug delivery. Here, the use of a computationally designed, two-component, icosahedral protein nanoparticle is reported to encapsulate multiple macromolecular cargoes via simple and controlled self-assembly in vitro. Single-stranded RNA molecules between 200 and 2500 nucleotides in length are encapsulated and protected from enzymatic degradation for up to a month with length-dependent decay rates. Immunogenicity studies of nanoparticles packaging synthetic polymers carrying a small-molecule TLR7/8 agonist show that co-delivery of antigen and adjuvant results in a more than 20-fold increase in humoral immune responses while minimizing systemic cytokine secretion associated with free adjuvant. Coupled with the precise control over nanoparticle structure offered by computational design, robust and versatile encapsulation via in vitro assembly opens the door to a new generation of cargo-loaded protein nanoparticles that can combine the therapeutic effects of multiple drug classes.

Factors associated with undergoing microdissection testicular sperm extraction among men with non-obstructive azoospermia following evaluation by a reproductive urologist.

Background: Microdissection testicular sperm extraction (mTESE) is the gold standard treatment for men with non-obstructive azoospermia (NOA). However, many men do not elect to pursue this surgical intervention. We aimed to identify factors associated with NOA patients undergoing mTESE after initial evaluation by a reproductive urologist (RU) through a retrospective cohort study. Methods: We retrospectively reviewed NOA patient who underwent evaluation by a RU between 2002-2018. Demographic and clinical data were collected. Our primary outcome was electing to undergo mTESE. Results: 44.4% (75/169) of NOA men underwent mTESE. These patients earned significantly higher median neighborhood income ($133,000 vs. $97,000, P<0.001), spent fewer years trying to conceive before seeking care {1.3 [interquartile range (IQR): 1-3] vs. 2.3 (IQR: 1-5), P=0.012}, and were more likely to be married (79.7% vs. 53.9%, P=0.001). On univariate analysis, married men [odds ratio (OR) 3.37, 95% confidence interval (CI): 1.67-6.79, P=0.001] and men with higher neighborhood income (OR 1.14, 95% CI: 1.06-1.21, P<0.001) were more likely to undergo mTESE, while couples attempting to conceive for a longer period of time prior to initial evaluation were less likely to undergo mTESE (OR 0.79, 95% CI: 0.68-0.92, P=0.003). On multivariable regression analysis, marital status and years attempting to conceive remained significantly associated with NOA patients undergoing mTESE (OR 4.61, 95% CI: 1.16-18.25, P=0.03; OR 0.67, 95% CI: 0.52-0.88, P=0.003, respectively). Conclusions: Higher neighborhood income and marital status were positively associated with patients undergoing mTESE, while couples who attempted to conceive for a longer period of time before seeking infertility care were less likely to undergo mTESE.

Characteristics of men who use direct-to-consumer men's health telemedicine services.

The characteristics of men who use direct-to-consumer (DTC) men's health services are not well understood. We conducted an online survey of adult men via ResearchMatch, assessing sociodemographic data, health behaviors, and concern for low testosterone and infertility. Logistic regression estimated the association between participant characteristics and familiarity with and reported use of DTC services such as Hims® and Roman®. Among 1276 men surveyed, 62.2% were concerned about low testosterone. While almost half (48.5%) were familiar with men's DTC health services, only 37 (2.9%) reported using these services. On multivariable analysis, men who used DTC men's health services were more likely to be younger (age 18-39: odds ratio [OR] 2.94, 95% confidence interval [CI] 1.03-8.38, p = 0.04; age 40-59: OR 3.26, CI 1.17-9.10, p = 0.02; referent age ≥60), have annual income between $75k and $100k (OR 5.25, CI 1.39-19.87.45, p = 0.02), and be concerned about low testosterone (OR 3.81, CI 1.46-9.96, p = 0.01). In conclusion, younger men and those with mid-range incomes were more likely to use online DTC men's health services compared to older or wealthier men. Likewise, men with concerns about low testosterone were more likely to use DTC services, but other health-conscious behaviors and frequency of doctor visits did not predict use.

Combinatorial immune refocusing within the influenza hemagglutinin RBD improves cross-neutralizing antibody responses.

The receptor-binding domain (RBD) of influenza virus hemagglutinin (HA) elicits potently neutralizing yet mostly strain-specific antibodies. Here, we evaluate the ability of several immunofocusing techniques to enhance the functional breadth of vaccine-elicited immune responses against the HA RBD. We present a series of "trihead" nanoparticle immunogens that display native-like closed trimeric RBDs from the HAs of several H1N1 influenza viruses. The series includes hyperglycosylated and hypervariable variants that incorporate natural and designed sequence diversity at key positions in the receptor-binding site periphery. Nanoparticle immunogens displaying triheads or hyperglycosylated triheads elicit higher hemagglutination inhibition (HAI) and neutralizing activity than the corresponding immunogens lacking either trimer-stabilizing mutations or hyperglycosylation. By contrast, mosaic nanoparticle display and antigen hypervariation do not significantly alter the magnitude or breadth of vaccine-elicited antibodies. Our results yield important insights into antibody responses against the RBD and the ability of several structure-based immunofocusing techniques to influence vaccine-elicited antibody responses.

Combinatorial immune refocusing within the influenza hemagglutinin head elicits cross-neutralizing antibody responses.

The head domain of influenza hemagglutinin (HA) elicits potently neutralizing yet mostly strain-specific antibodies during infection and vaccination. Here we evaluated a series of immunogens that combined several immunofocusing techniques for their ability to enhance the functional breadth of vaccine-elicited immune responses. We designed a series of "trihead" nanoparticle immunogens that display native-like closed trimeric heads from the HAs of several H1N1 influenza viruses, including hyperglycosylated variants and hypervariable variants that incorporate natural and designed sequence diversity at key positions in the periphery of the receptor binding site (RBS). Nanoparticle immunogens displaying triheads or hyperglycosylated triheads elicited higher HAI and neutralizing activity against vaccine-matched and -mismatched H1 viruses than corresponding immunogens lacking either trimer-stabilizing mutations or hyperglycosylation, indicating that both of these engineering strategies contributed to improved immunogenicity. By contrast, mosaic nanoparticle display and antigen hypervariation did not significantly alter the magnitude or breadth of vaccine-elicited antibodies. Serum competition assays and electron microscopy polyclonal epitope mapping revealed that the trihead immunogens, especially when hyperglycosylated, elicited a high proportion of antibodies targeting the RBS, as well as cross-reactive antibodies targeting a conserved epitope on the side of the head. Our results yield important insights into antibody responses against the HA head and the ability of several structure-based immunofocusing techniques to influence vaccine-elicited antibody responses.

The broad reach and inaccuracy of men's health information on social media: analysis of TikTok and Instagram.

Social media (SoMe) offers great potential to expand access to health information, but a significant proportion of users consume its content instead of consulting a physician. We sought to quantify the volume and characterize the accuracy of men's health-related content on TikTok and Instagram. We searched TikTok and Instagram for the terms: testosterone, erectile dysfunction, male infertility, semen retention, Peyronie's disease, and vasectomy. The top 10 hashtags for each term were used to estimate the total impressions for each term on each platform, and posts were then characterized by creator type, content type, and accuracy (1 to 5 scale). TikTok had 2,312,407,100 impressions and Instagram had 3,107,300 posts across all topics. Semen retention had the most impressions on TikTok (1,216,074,000) and posts on Instagram (1,077,000). Physicians created only a small portion of total TikTok and Instagram posts (10.3% and 12.9%, respectively). Across all topics, the accuracy of content was poor (2.6 ± 1.7), however, physician posts were more accurate than non-physician posts (mean 4.2 ± 1.2 vs 2.3 ± 1.6, p < 0.001, respectively). Men's health content is popular on TikTok and Instagram but is not accurate. We recommend that physicians actively engage in SoMe to address misinformation.

Reproductive urologic consultation in subfertile men: predictors of establishing care and patient perceptions after abnormal semen testing.

OBJECTIVE: To evaluate the predictors of establishing care with a reproductive urologist (RU) among men with abnormal semen analyses (SAs) ordered by nonurologists and examine patient perceptions of abnormal SAs in the absence of RU consultation. DESIGN: Retrospective cohort study with cross-sectional survey. SETTING: Large, integrated academic healthcare system during 2002-2019. PATIENT(S): We identified adult men undergoing initial SAs with nonurologists who had abnormalities. Patients with index SAs during 2002-2018 were included for the analysis of RU consultation. Men tested in 2019 were recruited for cross-sectional survey. INTERVENTION(S): Cross-sectional survey. MAIN OUTCOME MEASURE(S): RU consultation and accurate perception of abnormal SAs. RESULT(S): A total of 2,283 men had abnormal SAs ordered by nonurologists, among whom 20.5% underwent RU consultation. Mixed-effect logistic regression modeling identified oligospermia as the strongest predictor of RU care (odds ratio, 3.08; 95% confidence interval, 2.43-3.90) with a significant provider-level random intercept. We observed substantial provider-level heterogeneity among nonurologists with provider-specific rates of RU evaluation ranging from 3.7% to 35.8%. We contacted 310 men who did not undergo RU consultation with a 27.2% survey response rate. Of respondents, 6.7% reported receiving an RU referral. Among men with abnormal SAs not evaluated by RU, 22.7% appropriately perceived an abnormal SA. CONCLUSION(S): In men with abnormal SAs diagnosed by nonurologists, the rate of RU consultation was low and associated with substantial provider-level variation among ordering providers. Patients without RU consultation reported inaccurate perceptions of their SA. Multidisciplinary efforts are needed to ensure that subfertile men receive appropriate RU evaluation.

Selective Serotonin Reuptake Inhibitor (SSRI) Use is Not Associated With Impaired Semen Parameters.

OBJECTIVE: To examine the association between selective serotonin reuptake inhibitor (SSRI) use and semen quality. METHODS: We performed a retrospective review of all men undergoing semen analysis (SA) for fertility evaluation from 2002-2020 at a single academic medical center. Men were excluded if they had prior exposure to spermatotoxic medications, clomiphene citrate, gonadotropins, selective estrogen receptor modulators, or medical conditions known to impact male fertility. SSRI exposure was defined by an outpatient prescription within 90 days prior to any semen test. Differences between men with and without SSRI exposure were assessed with Wilcoxon rank sum for continuous variables and chi-squared testing for proportions. Univariable and multivariable linear regression models were fit to evaluate the relationship between SSRI use and individual semen parameters, controlling for age at the time of the semen analysis and non-SSRI drug use. RESULTS: A total of 8861 men were identified, of whom 153 men (1.7%) were exposed to SSRIs prior to SA. Median age was 35 years (interquartile range: 32-39) and was similar between groups (P = .999). Non-SSRI medication use was significantly higher in men taking SSRIs (78.4% vs 23.3%, < .001). On univariable and multivariable analyses, SSRI exposure was not associated with differences in semen volume, sperm concentration, motility, total motile sperm count, or normal morphology. CONCLUSION: In adult men undergoing fertility evaluation, SSRI exposure was not associated with impaired semen parameters. These data may help inform reproductive counseling and medical decision-making regarding SSRI use in men seeking paternity.

An Evaluation of Peer-Rated Surgical Skill and its Relationship With Detrusor Muscle Sampling in Transurethral Resection of Bladder Tumor.

OBJECTIVE: To assess the reliability of peer-review of TURBT videos as a means to evaluate surgeon skill and its relationship to detrusor sampling. METHODS: Urologists from an academic health system submitted TURBT videos in 2019. Ten blinded peers evaluated each surgeon's performance using a 10-item scoring instrument to quantify surgeon skill. Normalized composite skill scores for each surgeon were calculated using peer ratings. For surgeons submitting videos, we retrospectively reviewed all TURBT pathology results (2018-2019) to assess surgeon-specific detrusor sampling. A hierarchical logistic regression model was fit to evaluate the association between skill and detrusor sampling, adjusting for patient and surgeon factors. RESULTS: Surgeon skill scores and detrusor sampling rates were determined for 13 surgeons performing 245 TURBTs. Skill scores varied from -6.0 to 5.1 [mean: 0; standard deviation (SD): 2.40]. Muscle was sampled in 72% of cases, varying considerably across surgeons (mean: 64.5%; SD: 30.7%). Among 8 surgeons performing >5 TURBTs during the study period, adjusted detrusor sampling rate was associated with sending separate deep specimens (odds ratio [OR]: 1.97; 95% confidence interval [CI]: 1.02-3.81, P = .045) but not skill (OR: 0.81; 95% CI: 0.57-1.17, P = .191). CONCLUSION: Surgeon skill was not associated with detrusor sampling, suggesting there may be other drivers of variability of detrusor sampling in TURBT.

Thermodynamically coupled biosensors for detecting neutralizing antibodies against SARS-CoV-2 variants.

We designed a protein biosensor that uses thermodynamic coupling for sensitive and rapid detection of neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in serum. The biosensor is a switchable, caged luciferase-receptor-binding domain (RBD) construct that detects serum-antibody interference with the binding of virus RBD to angiotensin-converting enzyme 2 (ACE-2) as a proxy for neutralization. Our coupling approach does not require target modification and can better distinguish sample-to-sample differences in analyte binding affinity and abundance than traditional competition-based assays.

Antigen- and scaffold-specific antibody responses to protein nanoparticle immunogens.

Protein nanoparticle scaffolds are increasingly used in next-generation vaccine designs, and several have established records of clinical safety and efficacy. Yet the rules for how immune responses specific to nanoparticle scaffolds affect the immunogenicity of displayed antigens have not been established. Here we define relationships between anti-scaffold and antigen-specific antibody responses elicited by protein nanoparticle immunogens. We report that dampening anti-scaffold responses by physical masking does not enhance antigen-specific antibody responses. In a series of immunogens that all use the same nanoparticle scaffold but display four different antigens, only HIV-1 envelope glycoprotein (Env) is subdominant to the scaffold. However, we also demonstrate that scaffold-specific antibody responses can competitively inhibit antigen-specific responses when the scaffold is provided in excess. Overall, our results suggest that anti-scaffold antibody responses are unlikely to suppress antigen-specific antibody responses for protein nanoparticle immunogens in which the antigen is immunodominant over the scaffold.

Distinct sensitivities to SARS-CoV-2 variants in vaccinated humans and mice.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has led to the development of a large number of vaccines, several of which are now approved for use in humans. Understanding vaccine-elicited antibody responses against emerging SARS-CoV-2 variants of concern (VOCs) in real time is key to inform public health policies. Serum neutralizing antibody titers are the current best correlate of protection from SARS-CoV-2 challenge in non-human primates and a key metric to understand immune evasion of VOCs. We report that vaccinated BALB/c mice do not recapitulate faithfully the breadth and potency of neutralizing antibody responses elicited by various vaccine platforms against VOCs, compared with non-human primates or humans, suggesting caution should be exercised when interpreting data obtained with this animal model.

In Vivo Expansion of Antigen-Specific Regulatory T Cells through Staggered Fc.IL-2 Mutein Dosing and Antigen-Specific Immunotherapy.

In mice, Ag administration in the absence of adjuvant typically elicits tolerogenic immune responses through the deletion or inactivation of conventional CD4 T cells and the formation or expansion of regulatory CD4 T cells (Treg). Although these "Ag-specific immunotherapy" (ASI) approaches are currently under clinical development to treat autoinflammatory conditions, efficacy and safety may be variable and unpredictable because of the diverse activation states of immune cells in subjects with autoimmune and allergic diseases. To reliably induce Ag-specific tolerance in patients, novel methods to control T cell responses during ASI are needed, and strategies that permanently increase Treg frequencies among Ag-specific CD4 T cells may provide long-lasting immunosuppression between treatments. In this study, we present an approach to durably increase the frequency of Ag-specific Treg in mice by administering ASI when Treg numbers are transiently increased with individual doses of a half-life-extended Treg-selective IL-2 mutein. Repeated weekly cycles of IL-2 mutein doses (day 0) followed by ASI (day 3) resulted in a 3- to 5-fold enrichment in Treg among Ag-responsive CD4 T cells. Expanded Ag-specific Treg persisted for more than 3 wk following treatment cessation, as well as through an inflammatory T cell response to an Ag-expressing virus. Combining Treg enrichment with ASI has the potential to durably treat autoimmune disease or allergy by increasing the Treg/conventional CD4 T cell ratio among autoantigen- or allergen-specific T cells.