No abstract

Spatial Molecular Imager (SMI) is an automated microscope imaging system with microfluidic reagent cycling, for high-plex, spatial in-situ detection of multiomic targets (RNA and protein) on FFPE and other intact samples with subcellular resolution. The key attributes of the CosMxTM SMI platform (NanoString®, Seattle, WA) include: 1) high-plex and high-sensitivity imaging chemistry that works for both RNA and protein detection, 2) three-dimensional subcellular-resolution image analysis with a target localization accuracy of ∼50 nm in the XY plane, 3) large Hamming-distance encoding scheme with low error rate (0.0092 false calls per cell per gene) and low background (∼ 0.04 counts per cell per gene), 4) high-throughput (up to 1 million cells per sample, four samples per run), 5) antibody-based cell segmentation methods, and 6) compatibility with formalin-fixed, paraffin-embedded (FFPE) samples.In this study, 980 RNAs and 80 proteins were measured at subcellular resolution in FFPE cultured cell pellets, as well as FFPE tissues from biobanked samples of non-small cell lung cancer (NSCLC) and breast cancer. Cross-platform analysis using 16 cancer cell lines validated high-correlation (R2 ∼0.77) and high sensitivity (∼1.44 FPKM/TPM; roughly 1 to 2 copies of RNA per cell) when compared to RNA-seq. Real-world archived NSCLC FFPE tumor sections revealed greater than 94% cell detection efficiency for RNA, despite the low RNA quality QV200 20% to the medium quality 65%. The accuracy of protein expression measurements was independent of the level of multiplexing, as demonstrated by the linear behavior of nested multiplexing panels (R2 > 0.9). At 980-plex RNA detection, data analysis allowed identification of over 18 distinct cell types, at least 10 unique tumor microenvironment neighborhoods, and over 100 pairwise ligand-receptor interactions. Data from 8 NSCLC samples comprising over 800,000 single cells and ∼260 million transcripts are released into the public domain (www.nanostring.com) to allow for extended data analysis by the entire spatial biology research community.

Objective:During effective antiretroviral therapy (ART), low-level plasma viremias (LLV) (HIV RNA >30–1000 copies/ml) can be detected intermittently. We hypothesized that systemic inflammation is associated with LLV either as the cause or result of the production of virions from clonally expanded cells.Methods:Prospective cohort study of HIV-infected ART-naive Peruvians enrolled prior to ART and followed for 2 years. Plasma HIV RNA and peripheral blood mononuclear cell (PBMC) HIV DNA concentrations were quantified pre-ART from individuals whose plasma HIV RNA was ART-suppressed. Inflammatory biomarker concentrations were measured pre and during ART. Single-genome amplification (SGA) derived HIV env and pol genotypes from pre-ART and LLV specimens. Antiretroviral levels during ART assessed adherence. Statistical associations and phylogenetic relationships were examined.Results:Among 82 participants with median plasma HIV RNA less than 30 copies/ml, LLV were detected in 33 of 82 (40%), with a LLV median HIV RNA of 73 copies/ml. Participants with vs. without LLV had significantly higher pre-ART plasma HIV RNA (P < 0.001) and PBMC HIV DNA (P < 0.007); but, during ART, their antiretroviral drug levels were similar. LLV env sequences were monotypic in 17 of 28 (61%) and diverse in 11 of 28 (39%) participants. Those with the monotypic vs. diverse LLV pattern had elevated hsCRP and sCD163 (P = 0.004) and LLV with more X4 variants (P = 0.02).Conclusion:In individuals with monotypic LLV sequences, higher levels of pre-ART HIV DNA and RNA, systemic inflammation and X4 viruses suggest an interaction between inflammation and the production of virions from proliferating infected cells, and that naïve T cells may be a source of LLV.

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.