Red blood cell (RBC) traits are important heritable clinical biomarkers and modifiers of disease severity. To identify coding genetic variants associated with these traits, we conducted meta-analyses of seven RBC phenotypes in 130,273 multi-ethnic individuals from studies genotyped on an exome array. After conditional analyses and replication in 27,480 independent individuals, we identified 16 new RBC variants. We found low-frequency missense variants in MAP1A (rs55707100, minor allele frequency [MAF] = 3.3%, p = 2 × 10(-10) for hemoglobin [HGB]) and HNF4A (rs1800961, MAF = 2.4%, p < 3 × 10(-8) for hematocrit [HCT] and HGB). In African Americans, we identified a nonsense variant in CD36 associated with higher RBC distribution width (rs3211938, MAF = 8.7%, p = 7 × 10(-11)) and showed that it is associated with lower CD36 expression and strong allelic imbalance in ex vivo differentiated human erythroblasts. We also identified a rare missense variant in ALAS2 (rs201062903, MAF = 0.2%) associated with lower mean corpuscular volume and mean corpuscular hemoglobin (p < 8 × 10(-9)). Mendelian mutations in ALAS2 are a cause of sideroblastic anemia and erythropoietic protoporphyria. Gene-based testing highlighted three rare missense variants in PKLR, a gene mutated in Mendelian non-spherocytic hemolytic anemia, associated with HGB and HCT (SKAT p < 8 × 10(-7)). These rare, low-frequency, and common RBC variants showed pleiotropy, being also associated with platelet, white blood cell, and lipid traits. Our association results and functional annotation suggest the involvement of new genes in human erythropoiesis. We also confirm that rare and low-frequency variants play a role in the architecture of complex human traits, although their phenotypic effect is generally smaller than originally anticipated.

Broadly neutralizing antibodies to HIV-1 elicited in infected individuals evolves through shifts in their molecular specificities to viral envelope (Env) in the disease course. Recently, we showed that resistance of circulating HIV-1 clade C to the autologous plasma obtained from one Indian elite neutralizer is associated with mutations in V1 loop. In the present study, we examined the genetic attributes associated with exceptional sensitivity of pseudoviruses expressing an env gene obtained from the follow up visit contemporaneous plasma of the same donor.

Whole exome sequencing has proven to be a powerful tool for understanding the genetic architecture of human disease. Here we apply it to more than 2,500 simplex families, each having a child with an autistic spectrum disorder. By comparing affected to unaffected siblings, we show that 13% of de novo missense mutations and 43% of de novo likely gene-disrupting (LGD) mutations contribute to 12% and 9% of diagnoses, respectively. Including copy number variants, coding de novo mutations contribute to about 30% of all simplex and 45% of female diagnoses. Almost all LGD mutations occur opposite wild-type alleles. LGD targets in affected females significantly overlap the targets in males of lower intelligence quotient (IQ), but neither overlaps significantly with targets in males of higher IQ. We estimate that LGD mutation in about 400 genes can contribute to the joint class of affected females and males of lower IQ, with an overlapping and similar number of genes vulnerable to contributory missense mutation. LGD targets in the joint class overlap with published targets for intellectual disability and schizophrenia, and are enriched for chromatin modifiers, FMRP-associated genes and embryonically expressed genes. Most of the significance for the latter comes from affected females.

Next-generation sequencing (NGS) of exomes and genomes has accelerated the identification of genes involved in Mendelian phenotypes. However, many NGS studies fall short of identifying causal variants, with estimates for success rates as low as 25% for uncovering the pathological variant underlying disease etiology. An important reason for such failures is familial locus heterogeneity, where within a single pedigree causal variants in two or more genes underlie Mendelian trait etiology. As examples of intra- and inter-sibship familial locus heterogeneity, we present 10 consanguineous Pakistani families segregating hearing impairment due to homozygous variants in two different hearing impairment genes and a European-American pedigree in which hearing impairment is caused by four variants in three different genes. We have identified 41 additional pedigrees with syndromic and nonsyndromic hearing impairment for which a single previously reported hearing impairment gene has been identified but only segregates with the phenotype in a subset of affected pedigree members. We estimate that locus heterogeneity occurs in 15.3% (95% confidence interval: 11.9%, 19.9%) of the families in our collection. We demonstrate novel approaches to apply linkage analysis and homozygosity mapping (for autosomal recessive consanguineous pedigrees), which can be used to detect locus heterogeneity using either NGS or SNP array data. Results from linkage analysis and homozygosity mapping can also be used to group sibships or individuals most likely to be segregating the same causal variants and thereby increase the success rate of gene identification.

Discovering the genetic basis of a Mendelian phenotype establishes a causal link between genotype and phenotype, making possible carrier and population screening and direct diagnosis. Such discoveries also contribute to our knowledge of gene function, gene regulation, development, and biological mechanisms that can be used for developing new therapeutics. As of February 2015, 2,937 genes underlying 4,163 Mendelian phenotypes have been discovered, but the genes underlying ∼50% (i.e., 3,152) of all known Mendelian phenotypes are still unknown, and many more Mendelian conditions have yet to be recognized. This is a formidable gap in biomedical knowledge. Accordingly, in December 2011, the NIH established the Centers for Mendelian Genomics (CMGs) to provide the collaborative framework and infrastructure necessary for undertaking large-scale whole-exome sequencing and discovery of the genetic variants responsible for Mendelian phenotypes. In partnership with 529 investigators from 261 institutions in 36 countries, the CMGs assessed 18,863 samples from 8,838 families representing 579 known and 470 novel Mendelian phenotypes as of January 2015. This collaborative effort has identified 956 genes, including 375 not previously associated with human health, that underlie a Mendelian phenotype. These results provide insight into study design and analytical strategies, identify novel mechanisms of disease, and reveal the extensive clinical variability of Mendelian phenotypes. Discovering the gene underlying every Mendelian phenotype will require tackling challenges such as worldwide ascertainment and phenotypic characterization of families affected by Mendelian conditions, improvement in sequencing and analytical techniques, and pervasive sharing of phenotypic and genomic data among researchers, clinicians, and families.

The success of highly active antiretroviral therapy (HAART) is challenged by the emergence of resistance-associated mutations in human immunodeficiency virus-1 (HIV-1). In this study, resistance associated mutations in the reverse transcriptase (RT) and protease (PR) genes in antiretroviral therapy (ART) naïve and treated HIV-1 infected pediatric patients from North India were evaluated. Genotyping was successfully performed in 46 patients (30 ART naive and 16 treated) for the RT gene and in 53 patients (27 ART naive and 26 treated) for PR gene and mutations were identified using Stanford HIV Drug Resistance Database. A major drug resistant mutation in RT gene, L74I (NRTI), and two such mutations, K101E and G190A (NNRTI), were observed in two ART naïve patients, while M184V was detected in two ART treated patients. Overall, major resistance associated mutations in RT gene were observed in nine (30%) and seven (36%) of ART naïve and treated children respectively. Minor mutations were identified in PR gene in five children. Few non-clade C viral strains (≈30%) were detected, although subtype C was most predominant. The screening of ART naïve children for mutations in HIV-1 RT and protease genes, before and after initiation of ART is desirable for drug efficacy and good prognosis.

Non-secretor status due to homozygosity for the common FUT2 variant c.461G>A (p.Trp154∗) is associated with either risk for autoimmune diseases or protection against viral diarrhea and HIV. We determined the role of FUT2 in otitis media susceptibility by obtaining DNA samples from 609 multi-ethnic families and simplex case subjects with otitis media. Exome and Sanger sequencing, linkage analysis, and Fisher exact and transmission disequilibrium tests (TDT) were performed. The common FUT2 c.604C>T (p.Arg202∗) variant co-segregates with otitis media in a Filipino pedigree (LOD = 4.0). Additionally, a rare variant, c.412C>T (p.Arg138Cys), is associated with recurrent/chronic otitis media in European-American children (p = 1.2 × 10-5) and US trios (TDT p = 0.01). The c.461G>A (p.Trp154∗) variant was also over-transmitted in US trios (TDT p = 0.01) and was associated with shifts in middle ear microbiota composition (PERMANOVA p < 10-7) and increased biodiversity. When all missense and nonsense variants identified in multi-ethnic US trios with CADD > 20 were combined, FUT2 variants were over-transmitted in trios (TDT p = 0.001). Fut2 is transiently upregulated in mouse middle ear after inoculation with non-typeable Haemophilus influenzae. Four FUT2 variants-namely p.Ala104Val, p.Arg138Cys, p.Trp154∗, and p.Arg202∗-reduced A antigen in mutant-transfected COS-7 cells, while the nonsense variants also reduced FUT2 protein levels. Common and rare FUT2 variants confer susceptibility to otitis media, likely by modifying the middle ear microbiome through regulation of A antigen levels in epithelial cells. Our families demonstrate marked intra-familial genetic heterogeneity, suggesting that multiple combinations of common and rare variants plus environmental factors influence the individual otitis media phenotype as a complex trait.

Genome-wide association studies have reported 56 independently associated colorectal cancer (CRC) risk variants, most of which are non-coding and believed to exert their effects by modulating gene expression. The computational method PrediXcan uses cis-regulatory variant predictors to impute expression and perform gene-level association tests in GWAS without directly measured transcriptomes. In this study, we used reference datasets from colon (n = 169) and whole blood (n = 922) transcriptomes to test CRC association with genetically determined expression levels in a genome-wide analysis of 12,186 cases and 14,718 controls. Three novel associations were discovered from colon transverse models at FDR ≤ 0.2 and further evaluated in an independent replication including 32,825 cases and 39,933 controls. After adjusting for multiple comparisons, we found statistically significant associations using colon transcriptome models with TRIM4 (discovery P = 2.2 × 10- 4, replication P = 0.01), and PYGL (discovery P = 2.3 × 10- 4, replication P = 6.7 × 10- 4). Interestingly, both genes encode proteins that influence redox homeostasis and are related to cellular metabolic reprogramming in tumors, implicating a novel CRC pathway linked to cell growth and proliferation. Defining CRC risk regions as one megabase up- and downstream of one of the 56 independent risk variants, we defined 44 non-overlapping CRC-risk regions. Among these risk regions, we identified genes associated with CRC (P < 0.05) in 34/44 CRC-risk regions. Importantly, CRC association was found for two genes in the previously reported 2q25 locus, CXCR1 and CXCR2, which are potential cancer therapeutic targets. These findings provide strong candidate genes to prioritize for subsequent laboratory follow-up of GWAS loci. This study is the first to implement PrediXcan in a large colorectal cancer study and findings highlight the utility of integrating transcriptome data in GWAS for discovery of, and biological insight into, risk loci.

Obstructive sleep apnea (OSA) affects approximately 10% of adults, and alters brain gray and white matter. Psychological and physiological symptoms of the disorder are sex-specific, perhaps related to greater injury occurs in female than male patients in white matter. Our objective was to identify influences of OSA separated by sex on cortical gray matter.

Introduction: Obesity is associated with an increased risk of nonalcoholic fatty liver disease (NAFLD). While Magnetic Resonance Imaging (MRI) is a non-invasive gold standard to detect fatty liver, we demonstrate a low-cost and portable electrical impedance tomography (EIT) approach with circumferential abdominal electrodes for liver conductivity measurements. Methods and Results: A finite element model (FEM) was established to simulate decremental liver conductivity in response to incremental liver lipid content. To validate the FEM simulation, we performed EIT imaging on an ex vivo porcine liver in a non-conductive tank with 32 circumferentially-embedded electrodes, demonstrating a high-resolution output given a priori information on location and geometry. To further examine EIT capacity in fatty liver detection, we performed EIT measurements in age- and gender-matched New Zealand White rabbits (3 on normal, 3 on high-fat diets). Liver conductivity values were significantly distinct following the high-fat diet (p = 0.003 vs. normal diet, n=3), accompanied by histopathological evidence of hepatic fat accumulation. We further assessed EIT imaging in human subjects with MRI quantification for fat volume fraction based on Dixon procedures, demonstrating average liver conductivity of 0.331 S/m for subjects with low Body-Mass Index (BMI < 25 kg/m²) and 0.286 S/m for high BMI (> 25 kg/m²). Conclusion: We provide both the theoretical and experimental framework for a multi-scale EIT strategy to detect liver lipid content. Our preliminary studies pave the way to enhance the spatial resolution of EIT as a marker for fatty liver disease and metabolic syndrome.

Motile cilia are essential components of the mucociliary escalator and are central to respiratory-tract host defenses. Abnormalities in these evolutionarily conserved organelles cause primary ciliary dyskinesia (PCD). Despite recent strides characterizing the ciliome and sensory ciliopathies through exploration of the phenotype-genotype associations in model organisms, the genetic bases of most cases of PCD remain elusive. We identified nine related subjects with PCD from geographically dispersed Amish communities and performed exome sequencing of two affected individuals and their unaffected parents. A single autosomal-recessive nonsynonymous missense mutation was identified in HEATR2, an uncharacterized gene that belongs to a family not previously associated with ciliary assembly or function. Airway epithelial cells isolated from PCD-affected individuals had markedly reduced HEATR2 levels, absent dynein arms, and loss of ciliary beating. MicroRNA-mediated silencing of the orthologous gene in Chlamydomonas reinhardtii resulted in absent outer dynein arms, reduced flagellar beat frequency, and decreased cell velocity. These findings were recapitulated by small hairpin RNA-mediated knockdown of HEATR2 in airway epithelial cells from unaffected donors. Moreover, immunohistochemistry studies in human airway epithelial cells showed that HEATR2 was localized to the cytoplasm and not in cilia, which suggests a role in either dynein arm transport or assembly. The identification of HEATR2 contributes to the growing number of genes associated with PCD identified in both individuals and model organisms and shows that exome sequencing in family studies facilitates the discovery of novel disease-causing gene mutations.

Broadly cross neutralizing antibodies (NAbs) are generated in a group of HIV-1 infected individuals during the natural infection, but little is known about their prevalence in patients infected with viral subtypes from different geographical regions. We tested here the neutralizing efficiency of plasma antibodies from 80 HIV-1 infected antiretroviral drug naive patients against a panel of subtype-B and C tier 2 viruses. We detected cross-neutralizing antibodies in approximately 19-27% of the plasma, however the subtype-C specific neutralization efficiency predominated (p = 0.004). The neutralizing activity was shown to be exclusively mediated by the immunoglobulin G (IgG) fraction in the representative plasma samples. Epitope mapping of three, the most cross-neutralizing plasma (CNP) AIIMS206, AIIMS239 and AIIMS249 with consensus-C overlapping envelope peptides revealed ten different binding specificities with only V3 and IDR being common. The V3 and IDR were highly antigenic regions but no correlation between their reciprocal Max50 binding titers and neutralization was observed. In addition, the neutralizing activity of CNP was not substantially reduced by V3 and gp41 peptides except a modest contribution of MPER peptide. The MPER was rarely recognized by plasma antibodies though antibody depletion and competition experiments demonstrated MPER dependent neutralization in two out of three CNP. Interestingly, the binding specificity of one of the CNP (AIIMS206) overlapped with broadly neutralizing mAb 2F5 epitope. Overall, the data suggest that, despite the low immunogenicity of HIV-1 MPER, the antibodies directed to this region may serve as crucial reagents for HIV-1 vaccine design.

Inherited deafness is clinically and genetically heterogeneous. We recently mapped DFNB86, a locus associated with nonsyndromic deafness, to chromosome 16p. In this study, whole-exome sequencing was performed with genomic DNA from affected individuals from three large consanguineous families in which markers linked to DFNB86 segregate with profound deafness. Analyses of these data revealed homozygous mutation c.208G>T (p.Asp70Tyr) or c.878G>C (p.Arg293Pro) in TBC1D24 as the underlying cause of deafness in the three families. Sanger sequence analysis of TBC1D24 in an additional large family in which deafness segregates with DFNB86 identified the c.208G>T (p.Asp70Tyr) substitution. These mutations affect TBC1D24 amino acid residues that are conserved in orthologs ranging from fruit fly to human. Neither variant was observed in databases of single-nucleotide variants or in 634 chromosomes from ethnically matched control subjects. TBC1D24 in the mouse inner ear was immunolocalized predominantly to spiral ganglion neurons, indicating that DFNB86 deafness might be an auditory neuropathy spectrum disorder. Previously, six recessive mutations in TBC1D24 were reported to cause seizures (hearing loss was not reported) ranging in severity from epilepsy with otherwise normal development to epileptic encephalopathy resulting in childhood death. Two of our four families in which deafness segregates with mutant alleles of TBC1D24 were available for neurological examination. Cosegregation of epilepsy and deafness was not observed in these two families. Although the causal relationship between genotype and phenotype is not presently understood, our findings, combined with published data, indicate that recessive alleles of TBC1D24 can cause either epilepsy or nonsyndromic deafness.

Acromelic frontonasal dysostosis (AFND) is a rare disorder characterized by distinct craniofacial, brain, and limb malformations, including frontonasal dysplasia, interhemispheric lipoma, agenesis of the corpus callosum, tibial hemimelia, preaxial polydactyly of the feet, and intellectual disability. Exome sequencing of one trio and two unrelated probands revealed the same heterozygous variant (c.3487C>T [p. Arg1163Trp]) in a highly conserved protein domain of ZSWIM6; this variant has not been seen in the 1000 Genomes data, dbSNP, or the Exome Sequencing Project. Sanger validation of the three trios confirmed that the variant was de novo and was also present in a fourth isolated proband. In situ hybridization of early zebrafish embryos at 24 hr postfertilization (hpf) demonstrated telencephalic expression of zswim6 and onset of midbrain, hindbrain, and retinal expression at 48 hpf. Immunohistochemistry of later-stage mouse embryos demonstrated tissue-specific expression in the derivatives of all three germ layers. qRT-PCR expression analysis of osteoblast and fibroblast cell lines available from two probands was suggestive of Hedgehog pathway activation, indicating that the ZSWIM6 mutation associated with AFND may lead to the craniofacial, brain and limb malformations through the disruption of Hedgehog signaling.

The in vitro blood stage antiplasmodial activity of a series of allylated chalcones based on the licochalcone A as lead molecule was investigated against chloroquine (CQ) sensitive Pf3D7 and CQ resistant PfINDO strains of Plasmodium falciparum using SYBR Green I assay. Of the forty two chalcones tested, eight showed IC50 ≤ 5 μM. Structure-activity relationship (SAR) studies revealed 9 {1-(4-Chlorophenyl)-3-[3-methoxy-4-(prop-2-en-1-yloxy)phenyl]-prop-2-en-1-one} as the most potent (IC50: 2.5 μM) against Pf3D7 with resistance indices of 1.2 and 6.6 against PfDd2 and PfINDO strains, respectively. Later on, the synergistic effects 9 with standard antimalarials {artemisinin (ART) and chloroquine (CQ)} were studied in order to provide the basis for the selection of the best partner drug. In vitro combinations of 9 with ART showed strong synergy against PfINDO (ΣFIC50: 0.31-0.72) but additive to slight antagonistic effects (ΣFIC50: 1.97-2.64) against Pf3D7. ΣFIC50 0.31 of ART+9 combination corresponded to a 320 fold and 3 fold reduction in IC50 of 9 and ART, respectively. Similar combinations of 9 with CQ showed synergy to additivity to mild antagonism against the two strains {ΣFIC50: 0.668-2.269 (PfINDO); 1.45-2.83 (Pf3D7)}. Drug exposure followed by drug withdrawal indicated that 9 taken alone at IC100 killed rings, trophozoites and schizonts of P. falciparum. The combination of ART and 9 (1X ΣFIC100) selectively inhibited the growth of rings while the 2X ΣFIC100 combination of the same caused killing of rings without affecting trophozoites and schizonts. In contrast, the 1X combination of CQ and 9 (ΣFIC100: 0.5) killed rings and trophozoites. DNA fragmentation and loss of mitochondrial membrane potential (ΔΨm) in the 9 treated P. falciparum culture indicated apoptotic death in malaria parasites. Prediction of ADME properties revealed that most of the molecules did not violate Lipinski's parameters and have low TPSA value suggesting good absorption. The results suggest the promising drug-like properties of 9 against CQ resistant Pf and propensity for synergy with classical antimalarial drugs together with easy and economical synthesis.

Gordon syndrome (GS), or distal arthrogryposis type 3, is a rare, autosomal-dominant disorder characterized by cleft palate and congenital contractures of the hands and feet. Exome sequencing of five GS-affected families identified mutations in piezo-type mechanosensitive ion channel component 2 (PIEZO2) in each family. Sanger sequencing revealed PIEZO2 mutations in five of seven additional families studied (for a total of 10/12 [83%] individuals), and nine families had an identical c.8057G>A (p.Arg2686His) mutation. The phenotype of GS overlaps with distal arthrogryposis type 5 (DA5) and Marden-Walker syndrome (MWS). Using molecular inversion probes for targeted sequencing to screen PIEZO2, we found mutations in 24/29 (82%) DA5-affected families and one of two MWS-affected families. The presence of cleft palate was significantly associated with c.8057G>A (Fisher's exact test, adjusted p value < 0.0001). Collectively, although GS, DA5, and MWS have traditionally been considered separate disorders, our findings indicate that they are etiologically related and perhaps represent variable expressivity of the same condition.

Massively parallel whole-genome sequencing (WGS) data have ushered in a new era in human genetics. These data are now being used to understand the role of rare variants in complex traits and to advance the goals of precision medicine. The technological and computing advances that have enabled us to generate WGS data on thousands of individuals have also outpaced our ability to perform analyses in scientifically and statistically rigorous and thoughtful ways. The past several years have witnessed the application of whole-exome sequencing (WES) to complex traits and diseases. From our analysis of NHLBI Exome Sequencing Project (ESP) data, not only have a number of important disease and complex trait association findings emerged, but our collective experience offers some valuable lessons for WGS initiatives. These include caveats associated with generating automated pipelines for quality control and analysis of rare variants; the importance of studying minority populations; sample size requirements and efficient study designs for identifying rare-variant associations; and the significance of incidental findings in population-based genetic research. With the ESP as an example, we offer guidance and a framework on how to conduct a large-scale association study in the era of WGS.

More than 50% of HIV-1 infection globally is caused by subtype_C viruses. Majority of the broadly neutralizing antibodies (bnAbs) targeting HIV-1 have been isolated from non-subtype_C infected donors. Mapping the epitope specificities of bnAbs provides useful information for vaccine design. Recombinant antibody technology enables generation of a large repertoire of monoclonals with diverse specificities. We constructed a phage recombinant single chain variable fragment (scFv) library with a diversity of 7.8 × 108 clones, using a novel strategy of pooling peripheral blood mononuclear cells (PBMCs) of six select HIV-1 chronically infected Indian donors whose plasma antibodies exhibited potent cross neutralization efficiency. The library was panned and screened by phage ELISA using trimeric recombinant proteins to identify viral envelope specific clones. Three scFv monoclonals D11, C11 and 1F6 selected from the library cross neutralized subtypes A, B and C viruses at concentrations ranging from 0.09 μg/mL to 100 μg/mL. The D11 and 1F6 scFvs competed with mAbs b12 and VRC01 demonstrating CD4bs specificity, while C11 demonstrated N332 specificity. This is the first study to identify cross neutralizing scFv monoclonals with CD4bs and N332 glycan specificities from India. Cross neutralizing anti-HIV-1 human scFv monoclonals can be potential candidates for passive immunotherapy and for guiding immunogen design.

Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterized by hypotonia, ataxia, abnormal eye movements, and variable cognitive impairment. It is defined by a distinctive brain malformation known as the "molar tooth sign" on axial MRI. Subsets of affected individuals have malformations such as coloboma, polydactyly, and encephalocele, as well as progressive retinal dystrophy, fibrocystic kidney disease, and liver fibrosis. More than 35 genes have been associated with JS, but in a subset of families the genetic cause remains unknown. All of the gene products localize in and around the primary cilium, making JS a canonical ciliopathy. Ciliopathies are unified by their overlapping clinical features and underlying mechanisms involving ciliary dysfunction. In this work, we identify biallelic rare, predicted-deleterious ARMC9 variants (stop-gain, missense, splice-site, and single-exon deletion) in 11 individuals with JS from 8 families, accounting for approximately 1% of the disorder. The associated phenotypes range from isolated neurological involvement to JS with retinal dystrophy, additional brain abnormalities (e.g., heterotopia, Dandy-Walker malformation), pituitary insufficiency, and/or synpolydactyly. We show that ARMC9 localizes to the basal body of the cilium and is upregulated during ciliogenesis. Typical ciliopathy phenotypes (curved body shape, retinal dystrophy, coloboma, and decreased cilia) in a CRISPR/Cas9-engineered zebrafish mutant model provide additional support for ARMC9 as a ciliopathy-associated gene. Identifying ARMC9 mutations as a cause of JS takes us one step closer to a full genetic understanding of this important disorder and enables future functional work to define the central biological mechanisms underlying JS and other ciliopathies.

The HIV-1 envelope (Env) is a glycoprotein consisting of a trimer of heterodimers containing gp120 and gp41 subunits that mediates virus entry and is a major target of broadly neutralizing antibodies (bnAbs) developed during infection in some individuals. The engagement of the HIV-1 gp120 glycoprotein to the host CD4 protein triggers conformational changes in gp120 that allow its binding to co-receptors and is necessary for virus entry to establish infection. Native-like HIV-1 Env immunogens representing distinct clades have been proposed to improve immunogenicity. In the present study, we examined the basis of resistance of an HIV-1 B/C recombinant Env (LT5.J4b12C) to non-neutralizing antibodies targeting CD4-induced Env epitopes in the presence of soluble CD4 (sCD4). Using native polyacrylamide gel shift assay and negative-stain EM, we found that the prefusion conformational state of LT5.J4b12C trimeric Env was largely unaffected in the presence of excess sCD4 with most Env trimers appearing to be in a ligand-free state. This resistance to CD4-induced conformational changes was associated with a lower affinity for CD4. Moreover, the LT5.J4b12C trimeric Env preferentially bound to the neutralizing antibodies compared with non-neutralizing antibodies. Taken together, we report on an HIV-1 B/C recombinant, native-like trimeric Env protein that is highly resistant to CD4-induced conformational changes but displays epitopes recognized by a diverse array of bnAbs. Such features make this B/C recombinant trimeric Env a useful addition to the pool of other recently identified native-like HIV-1 Env trimers suitable for use as antigenic bait for bnAb isolation, structural studies, and use as potential immunogens.