Keratinocyte-associated protein 3 plays a role in body weight and adiposity with differential effects in males and females.

Despite the obesity crisis in the United States, the underlying genetics are poorly understood. Our lab previously identified Keratinocyte-associated protein 3, Krtcap3, as a candidate gene for adiposity through a genome-wide association study in outbred rats, where increased liver expression of Krtcap3 correlated with decreased fat mass. Here we seek to confirm that Krtcap3 expression affects adiposity traits. To do so, we developed an in vivo whole-body Krtcap3 knock-out (KO) rat model. Wild-type (WT) and KO rats were placed onto a high-fat (HFD) or low-fat diet (LFD) at 6 weeks of age and were maintained on diet for 13 weeks, followed by assessments of metabolic health. We hypothesized that Krtcap3-KO rats will have increased adiposity and a worsened metabolic phenotype relative to WT. We found that KO male and female rats have significantly increased body weight versus WT, with the largest effect in females on a HFD. KO females also ate more and had greater adiposity, but were more insulin sensitive than WT regardless of diet condition. Although KO males weighed more than WT under both diet conditions, there were no differences in eating behavior or fat mass. Interestingly, KO males on a HFD were more insulin resistant than WT. This study confirms that Krtcap3 plays a role in body weight regulation and demonstrates genotype- and sex-specific effects on food intake, adiposity, and insulin sensitivity. Future studies will seek to better understand these sex differences, the role of diet, and establish a mechanism for Krtcap3 in obesity.

Meta-analysis of HIV-1 vaccine elicited mucosal antibodies in humans.

We studied mucosal immune responses in six HIV-1 vaccine trials investigating different envelope (Env)-containing immunogens. Regimens were classified into four categories: DNA/vector, DNA/vector plus protein, protein alone, and vector alone. We measured HIV-1-specific IgG and IgA in secretions from cervical (n = 111) and rectal swabs (n = 154), saliva (n = 141), and seminal plasma (n = 124) and compared to corresponding blood levels. Protein-containing regimens had up to 100% response rates and the highest Env-specific IgG response rates. DNA/vector groups elicited mucosal Env-specific IgG response rates of up to 67% that varied across specimen types. Little to no mucosal IgA responses were observed. Overall, gp41- and gp140-specific antibodies dominated gp120 mucosal responses. In one trial, prior vaccination with a protein-containing immunogen maintained durability of cervical and rectal IgG for up to 17 years. Mucosal IgG responses were boosted after revaccination. These findings highlight a role for protein immunization in eliciting HIV-1-specific mucosal antibodies and the ability of HIV-1 vaccines to elicit durable HIV-1-specific mucosal IgG.

Sept8/SEPTIN8 involvement in cellular structure and kidney damage is identified by genetic mapping and a novel human tubule hypoxic model.

Chronic kidney disease (CKD), which can ultimately progress to kidney failure, is influenced by genetics and the environment. Genes identified in human genome wide association studies (GWAS) explain only a small proportion of the heritable variation and lack functional validation, indicating the need for additional model systems. Outbred heterogeneous stock (HS) rats have been used for genetic fine-mapping of complex traits, but have not previously been used for CKD traits. We performed GWAS for urinary protein excretion (UPE) and CKD related serum biochemistries in 245 male HS rats. Quantitative trait loci (QTL) were identified using a linear mixed effect model that tested for association with imputed genotypes. Candidate genes were identified using bioinformatics tools and targeted RNAseq followed by testing in a novel in vitro model of human tubule, hypoxia-induced damage. We identified two QTL for UPE and five for serum biochemistries. Protein modeling identified a missense variant within Septin 8 (Sept8) as a candidate for UPE. Sept8/SEPTIN8 expression increased in HS rats with elevated UPE and tubulointerstitial injury and in the in vitro hypoxia model. SEPTIN8 is detected within proximal tubule cells in human kidney samples and localizes with acetyl-alpha tubulin in the culture system. After hypoxia, SEPTIN8 staining becomes diffuse and appears to relocalize with actin. These data suggest a role of SEPTIN8 in cellular organization and structure in response to environmental stress. This study demonstrates that integration of a rat genetic model with an environmentally induced tubule damage system identifies Sept8/SEPTIN8 and informs novel aspects of the complex gene by environmental interactions contributing to CKD risk.

Early life stress induces hyperactivity but not increased anxiety-like behavior or ethanol drinking in outbred heterogeneous stock rats.

Early life stress is known to impact vulnerability to psychopathological disorders in adulthood, including anxiety and alcohol use disorder (AUD), but the mechanisms underlying susceptibility to these outcomes are not fully understood. In the current study, we used adolescent social isolation (ASI) to determine whether Heterogeneous Stock (HS) rats, an outbred model used for genetic fine-mapping, could be used to study the genetics contributing to ASI-induced anxiety- and AUD-like behavior. We isolated (ASI) or group-housed (adolescent group-housed; AGH) 64 male HS rats at 4 weeks of age. After 5 weeks in these housing conditions, multiple anxiety and coping/despair-like behaviors were measured. All rats were then individually housed and assessed for voluntary ethanol self-administration. At euthanasia, synaptoneurosomes were isolated from a subset of brains to examine the expression of two proteins associated with alcohol drinking-related behaviors, GluA1 and SK2, in the dorsal (dHC) and ventral hippocampus (vHC). We found that ASI increased hyperactivity in the open field test relative to AGH, with no changes in other anxiety-like behaviors. Surprisingly, ASI rats demonstrated decreased immobility and increased climbing in the forced swim test relative to AGH. In contrast to prior studies by us and others, we found no difference in self-administration of 20% ethanol, with decreased ethanol self-administration in ASI relative to AGH rats at higher ethanol concentrations. Furthermore, while ASI in Long-Evans rats resulted in decreased SK2 expression in vHC synaptosomes, no differences were seen in vHC synaptosomes for SK2 or GluA1 in HS rats. These results demonstrate that HS rats are protected against many of the negative effects previously seen in response to ASI, namely anxiety-like behavior and increased ethanol self-administration. The current work suggests that a lack of change in SK2 and GluA1 expression levels in the vHC may play a role in conferring this protection.

High-fat diet negatively impacts both metabolic and behavioral health in outbred heterogeneous stock rats.

Obesity is influenced by genetics and diet and has wide ranging comorbidities, including anxiety and depressive disorders. Outbred heterogeneous stock (HS) rats are used for fine-genetic mapping of complex traits and may be useful for understanding gene by diet interactions. In this study, HS rats were fed diets containing 60% kcal from fat (high-fat diet, HFD) or 10% kcal from fat (low-fat diet, LFD) and tested for metabolic (study 1) and behavioral (study 2) outcomes. In study 1, we measured glucose tolerance, fasting glucose and insulin, fat pad weights and despair-like behavior in the forced swim test (FST). In study 2, we assessed anxiety-like (elevated plus maze, EPM; open field test, OFT) and despair-like/coping (splash test, SpT; and FST) behaviors. Body weight and food intake were measured weekly in both studies. We found negative effects of HFD on metabolic outcomes, including increased body weight and fat pad weights, decreased glucose tolerance, and increased fasting insulin. We also found negative effects of HFD on despair-like/coping and anxiety-like behaviors. These include increased immobility in the FST, decreased open arm time in the EPM, and increased movement and rest episodes and decreased rearing in the OFT. The diet-induced changes in EPM and OFT were independent of overall locomotion. Additionally, diet-induced changes in OFT behaviors were independent of adiposity, while adiposity was a confounding factor for EPM and FST behavior. This work establishes the HS as a model to study gene by diet interactions affecting metabolic and behavioral health.

Rare Detection of Antiviral Functions of Polyclonal IgA Isolated from Plasma and Breast Milk Compartments in Women Chronically Infected with HIV-1.

The humoral response to invading mucosal pathogens comprises multiple antibody isotypes derived from systemic and mucosal compartments. To understand the contribution of each antibody isotype/source to the mucosal humoral response, parallel investigation of the specificities and functions of antibodies within and across isotypes and compartments is required. The role of IgA against HIV-1 is complex, with studies supporting a protective role as well as a role for serum IgA in blocking effector functions. Thus, we explored the fine specificity and function of IgA in both plasma and mucosal secretions important to infant HIV-1 infection, i.e., breast milk. IgA and IgG were isolated from milk and plasma from 20 HIV-1-infected lactating Malawian women. HIV-1 binding specificities, neutralization potency, inhibition of virus-epithelial cell binding, and antibody-mediated phagocytosis were measured. Fine-specificity mapping showed IgA and IgG responses to multiple HIV-1 Env epitopes, including conformational V1/V2 and linear V2, V3, and constant region 5 (C5). Env IgA was heterogeneous between the milk and systemic compartments (Env IgA, τ = 0.00 to 0.63, P = 0.0046 to 1.00). Furthermore, IgA and IgG appeared compartmentalized as there was a lack of correlation between the specificities of Env-specific IgA and IgG (in milk, τ = -0.07 to 0.26, P = 0.35 to 0.83). IgA and IgG also differed in functions: while neutralization and phagocytosis were consistently mediated by milk and plasma IgG, they were rarely detected in IgA from both milk and plasma. Understanding the ontogeny of the divergent IgG and IgA antigen specificity repertoires and their effects on antibody function will inform vaccination approaches targeted toward mucosal pathogens.IMPORTANCE Antibodies within the mucosa are part of the first line of defense against mucosal pathogens. Evaluating mucosal antibody isotypes, specificities, and antiviral functions in relationship to the systemic antibody profile can provide insights into whether the antibody response is coordinated in response to mucosal pathogens. In a natural immunity cohort of HIV-infected lactating women, we mapped the fine specificity and function of IgA in breast milk and plasma and compared these with the autologous IgG responses. Antigen specificities and functions differed between IgG and IgA, with antiviral functions (neutralization and phagocytosis) predominantly mediated by the IgG fraction in both milk and plasma. Furthermore, the specificity of milk IgA differed from that of systemic IgA. Our data suggest that milk IgA and systemic IgA should be separately examined as potential correlates of risk. Preventive vaccines may need to employ different strategies to elicit functional antiviral immunity by both antibody isotypes in the mucosa.

Modification of the Association Between T-Cell Immune Responses and Human Immunodeficiency Virus Type 1 Infection Risk by Vaccine-Induced Antibody Responses in the HVTN 505 Trial.

Background: HVTN 505 was a human immunodeficiency virus type 1 (HIV-1) preventive vaccine efficacy trial of a DNA/recombinant adenovirus serotype 5 (rAd5) vaccine regimen. We assessed antibody responses measured 1 month after final vaccination (month 7) as correlates of HIV-1 acquisition risk. Methods: Binding antibody responses were quantified in serum samples from 25 primary endpoint vaccine cases (diagnosed with HIV-1 infection between month 7 and month 24) and 125 randomly sampled frequency-matched vaccine controls (HIV-1 negative at month 24). We prespecified for a primary analysis tier 6 antibody response biomarkers that measure immunoglobulin G (IgG) and immunoglobulin A (IgA) binding to Env proteins and 2 previously assessed T-cell response biomarkers. Results: Envelope-specific IgG responses were significantly correlated with decreased HIV-1 risk. Moreover, the interaction of IgG responses and Env-specific CD8+ T-cell polyfunctionality score had a highly significant association with HIV-1 risk after adjustment for multiple comparisons. Conclusions: Vaccinees with higher levels of Env IgG have significantly decreased HIV-1 risk when CD8+ T-cell responses are low. Moreover, vaccinees with high CD8+ T-cell responses generally have low risk, and those with low CD8+ T-cell and low Env antibody responses have high risk. These findings suggest the critical importance of inducing a robust IgG Env response when the CD8+ T-cell response is low.

Genetic Fine-Mapping and Identification of Candidate Genes and Variants for Adiposity Traits in Outbred Rats.

OBJECTIVE: Obesity is a major risk factor for multiple diseases and is in part heritable, yet the majority of causative genetic variants that drive excessive adiposity remain unknown. Here, outbred heterogeneous stock (HS) rats were used in controlled environmental conditions to fine-map novel genetic modifiers of adiposity. METHODS: Body weight and visceral fat pad weights were measured in male HS rats that were also genotyped genome-wide. Quantitative trait loci (QTL) were identified by genome-wide association of imputed single-nucleotide polymorphism (SNP) genotypes using a linear mixed effect model that accounts for unequal relatedness between the HS rats. Candidate genes were assessed by protein modeling and mediation analysis of expression for coding and noncoding variants, respectively. RESULTS: HS rats exhibited large variation in adiposity traits, which were highly heritable and correlated with metabolic health. Fine-mapping of fat pad weight and body weight revealed three QTL and prioritized five candidate genes. Fat pad weight was associated with missense SNPs in Adcy3 and Prlhr and altered expression of Krtcap3 and Slc30a3, whereas Grid2 was identified as a candidate within the body weight locus. CONCLUSIONS: These data demonstrate the power of HS rats for identification of known and novel heritable mediators of obesity traits.

Computational analysis of antibody dynamics identifies recent HIV-1 infection.

Accurate HIV-1 incidence estimation is critical to the success of HIV-1 prevention strategies. Current assays are limited by high false recent rates (FRRs) in certain populations and a short mean duration of recent infection (MDRI). Dynamic early HIV-1 antibody response kinetics were harnessed to identify biomarkers for improved incidence assays. We conducted retrospective analyses on circulating antibodies from known recent and longstanding infections and evaluated binding and avidity measurements of Env and non-Env antigens and multiple antibody forms (i.e., IgG, IgA, IgG3, IgG4, dIgA, and IgM) in a diverse panel of 164 HIV-1-infected participants (clades A, B, C). Discriminant function analysis identified an optimal set of measurements that were subsequently evaluated in a 324-specimen blinded biomarker validation panel. These biomarkers included clade C gp140 IgG3, transmitted/founder clade C gp140 IgG4 avidity, clade B gp140 IgG4 avidity, and gp41 immunodominant region IgG avidity. MDRI was estimated at 215 day or alternatively, 267 days. FRRs in untreated and treated subjects were 5.0% and 3.6%, respectively. Thus, computational analysis of dynamic HIV-1 antibody isotype and antigen interactions during infection enabled design of a promising HIV-1 recency assay for improved cross-sectional incidence estimation.

Safety, pharmacokinetics, and immunological activities of multiple intravenous or subcutaneous doses of an anti-HIV monoclonal antibody, VRC01, administered to HIV-uninfected adults: Results of a phase 1 randomized trial.

BACKGROUND: VRC01 is an HIV-1 CD4 binding site broadly neutralizing antibody (bnAb) that is active against a broad range of HIV-1 primary isolates in vitro and protects against simian-human immunodeficiency virus (SHIV) when delivered parenterally to nonhuman primates. It has been shown to be safe and well tolerated after short-term administration in humans; however, its clinical and functional activity after longer-term administration has not been previously assessed. METHODS AND FINDINGS: HIV Vaccine Trials Network (HVTN) 104 was designed to evaluate the safety and tolerability of multiple doses of VRC01 administered either subcutaneously or by intravenous (IV) infusion and to assess the pharmacokinetics and in vitro immunologic activity of the different dosing regimens. Additionally, this study aimed to assess the effect that the human body has on the functional activities of VRC01 as measured by several in vitro assays. Eighty-eight healthy, HIV-uninfected, low-risk participants were enrolled in 6 United States clinical research sites affiliated with the HVTN between September 9, 2014, and July 15, 2015. The median age of enrollees was 27 years (range, 18-50); 52% were White (non-Hispanic), 25% identified as Black (non-Hispanic), 11% were Hispanic, and 11% were non-Hispanic people of diverse origins. Participants were randomized to receive the following: a 40 mg/kg IV VRC01 loading dose followed by five 20 mg/kg IV VRC01 doses every 4 weeks (treatment group 1 [T1], n = 20); eleven 5 mg/kg subcutaneous (SC) VRC01 (treatment group 3 [T3], n = 20); placebo (placebo group 3 [P3], n = 4) doses every 2 weeks; or three 40 mg/kg IV VRC01 doses every 8 weeks (treatment group 2 [T2], n = 20). Treatment groups T4 and T5 (n = 12 each) received three 10 or 30 mg/kg IV VRC01 doses every 8 weeks, respectively. Participants were followed for 32 weeks after their first VRC01 administration and received a total of 249 IV infusions and 208 SC injections, with no serious adverse events, dose-limiting toxicities, nor evidence for anti-VRC01 antibodies observed. Serum VRC01 levels were detected through 12 weeks after final administration in all participants who received all scheduled doses. Mean peak serum VRC01 levels of 1,177 µg/ml (95% CI: 1,033, 1,340) and 420 µg/ml (95% CI: 356, 494) were achieved 1 hour after the IV infusion series of 30 mg/kg and 10 mg/kg doses, respectively. Mean trough levels at week 24 in the IV infusion series of 30 mg/kg and 10 mg/kg doses, respectively, were 16 µg/ml (95% CI: 10, 27) and 6 µg/ml (95% CI: 5, 9) levels, which neutralize a majority of circulating strains in vitro (50% inhibitory concentration [IC50] > 5 µg/ml). Post-infusion/injection serum VRC01 retained expected functional activity (virus neutralization, antibody-dependent cellular cytotoxicity, phagocytosis, and virion capture). The limitations of this study include the relatively small sample size of each VRC01 administration regimen and missing data from participants who were unable to complete all study visits. CONCLUSIONS: VRC01 administered as either an IV infusion (10-40 mg/kg) given monthly or bimonthly, or as an SC injection (5 mg/kg) every 2 weeks, was found to be safe and well tolerated. In addition to maintaining drug concentrations consistent with neutralization of the majority of tested HIV strains, VRC01 concentrations from participants' sera were found to avidly capture HIV virions and to mediate antibody-dependent cellular phagocytosis, suggesting a range of anti-HIV immunological activities, warranting further clinical trials. TRIAL REGISTRATION: Clinical Trials Registration: NCT02165267.

Association of HIV-1 Envelope-Specific Breast Milk IgA Responses with Reduced Risk of Postnatal Mother-to-Child Transmission of HIV-1.

UNLABELLED: Infants born to HIV-1-infected mothers in resource-limited areas where replacement feeding is unsafe and impractical are repeatedly exposed to HIV-1 throughout breastfeeding. Despite this, the majority of infants do not contract HIV-1 postnatally, even in the absence of maternal antiretroviral therapy. This suggests that immune factors in breast milk of HIV-1-infected mothers help to limit vertical transmission. We compared the HIV-1 envelope-specific breast milk and plasma antibody responses of clade C HIV-1-infected postnatally transmitting and nontransmitting mothers in the control arm of the Malawi-based Breastfeeding Antiretrovirals and Nutrition Study using multivariable logistic regression modeling. We found no association between milk or plasma neutralization activity, antibody-dependent cell-mediated cytotoxicity, or HIV-1 envelope-specific IgG responses and postnatal transmission risk. While the envelope-specific breast milk and plasma IgA responses also did not reach significance in predicting postnatal transmission risk in the primary model after correction for multiple comparisons, subsequent exploratory analysis using two distinct assay methodologies demonstrated that the magnitudes of breast milk total and secretory IgA responses against a consensus HIV-1 envelope gp140 (B.con env03) were associated with reduced postnatal transmission risk. These results suggest a protective role for mucosal HIV-1 envelope-specific IgA responses in the context of postnatal virus transmission. This finding supports further investigations into the mechanisms by which mucosal IgA reduces risk of HIV-1 transmission via breast milk and into immune interventions aimed at enhancing this response. IMPORTANCE: Infants born to HIV-1-infected mothers are repeatedly exposed to the virus in breast milk. Remarkably, the transmission rate is low, suggesting that immune factors in the breast milk of HIV-1-infected mothers help to limit transmission. We compared the antibody responses in plasma and breast milk of HIV-1-transmitting and -nontransmitting mothers to identify responses that correlated with reduced risk of postnatal HIV-1 transmission. We found that neither plasma nor breast milk IgG antibody responses were associated with risk of HIV-1 transmission. In contrast, the magnitudes of the breast milk IgA and secretory IgA responses against HIV-1 envelope proteins were associated with reduced risk of postnatal HIV-1 transmission. The results of this study support further investigations of the mechanisms by which mucosal IgA may reduce the risk of HIV-1 transmission via breastfeeding and the development of strategies to enhance milk envelope-specific IgA responses to reduce mother-to-child HIV transmission and promote an HIV-free generation.