High-plex imaging of RNA and proteins at subcellular resolution in fixed tissue by spatial molecular imaging.

Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .

Comparisons of Human Immunodeficiency Virus Type 1 Envelope Variants in Blood and Genital Fluids near the Time of Male-to-Female Transmission.

To better understand the transmission of human immunodeficiency virus type 1 (HIV-1), the genetic characteristics of blood and genital viruses from males were compared to those of the imputed founding virus population in their female partners. Initially serodiscordant heterosexual African couples with sequence-confirmed male-to-female HIV-1 transmission and blood and genital specimens collected near the time of transmission were studied. Single viral templates from blood plasma and genital tract RNA and DNA were sequenced across HIV-1 env gp160. Eight of 29 couples examined yielded viral sequences from both tissues. Analysis of these couples' sequences demonstrated, with one exception, that the women's founding viral populations arose from a single viral variant and were CCR5 tropic, even though CXCR4 variants were detected within four males. The median genetic distance of the imputed most recent common ancestor of the women's founder viruses showed that they were closer to the semen viruses than to the blood viruses of their transmitting male partner, but this finding was biased by detection of a greater number of viral clades in the blood. Using multiple assays, the blood and genital viruses were consistently found to be compartmentalized in only two of eight men. No distinct amino acid signatures in the men's viruses were found to link to the women's founders, nor did the women's env sequences have shorter variable loops or fewer N-linked glycosylation sites. The lack of selective factors, except for coreceptor tropism, is consistent with others' findings in male-to-female and high-risk transmissions. The infrequent compartmentalization between the transmitters' blood and semen viruses suggests that cell-free blood virus likely includes HIV-1 sequences representative of those of viruses in semen.IMPORTANCE Mucosal transmissions account for the majority of HIV-1 infections. Identification of the viral characteristics associated with transmission would facilitate vaccine design. This study of HIV strains from transmitting males and their seroconverting female partners found that the males' genital tract viruses were rarely distinct from the blood variants. The imputed founder viruses in women were genetically similar to both the blood and genital tract variants of their male partners, indicating a lack of evidence for genital tract-specific lineages. These findings suggest that targeting vaccine responses to variants found in blood are likely to also protect from genital tract variants.

Genetic analyses of HIV env associated with uveitis in antiretroviral-naive individuals.

OBJECTIVE: To analyze and compare HIV-1 env sequences from the eye to those from the blood of individuals with uveitis attributed to HIV with the goal of gaining insight into the pathogenesis of HIV-associated eye disease. DESIGN: A prospective case series of five HIV-infected antiretroviral-naive individuals with uveitis negative for other pathogens. METHODS: RNA from blood plasma and ocular aqueous humor was reverse transcribed using random hexamers. HIV env C2-V5 (HXB2: 6990-7668) sequences were generated by single-genome amplification using nested polymerase chain reaction followed by bidirectional Sanger sequencing. Sequence analyses by Geneious, Geno2Pheno, N-GLYCOSITE, DIVEIN, and HyPhy evaluated relationships between HIV in plasma and aqueous humor. RESULTS: A median of 20 (range: 13-22) plasma and 15 (range: 9-18) aqueous humor sequences were generated from each individual. The frequencies of sequences with predicted-N-linked-glycosylation sites and C-X-C chemokine receptor type 4 were comparable in aqueous humor and plasma of all five patients. Aqueous humor sequences had lower median genetic diversity compared with plasma across all patients, but similar divergence, in four of five patients. Aqueous humor HIV sequences were compartmentalized from plasma across subjects by Critchlow correlation coefficient, Slatkin and Maddison, nearest-neighbor statistic, and Fixation index. CONCLUSION: Among antiretroviral-naive individuals with uveitis attributed to HIV, the universal compartmentalization and decreased diversity of eye compared with blood sequences suggests time-limited passage of a small subset of variants from each patient's viral population into the eye tissues, followed by limited immune selection despite the inflammatory uveitis.