Single Genome Amplification Research Articles

Development of a Versatile, Near Full Genome Amplification and Sequencing Approach for a Broad Variety of HIV-1 Group M Variants.

Near full genome sequencing (NFGS) of HIV-1 is required to assess the genetic composition of HIV-1 strains comprehensively. Population-wide, it enables a determination of the heterogeneity of HIV-1 and the emergence of novel/recombinant strains, while for each individual it constitutes a diagnostic instrument to assist targeted therapeutic measures against viral components. There is still a lack of robust and adaptable techniques for efficient NFGS from miscellaneous HIV-1 subtypes. Using rational primer design, a broad primer set was developed for the amplification and sequencing of diverse HIV-1 group M variants from plasma. Using pure subtypes as well as diverse, unique recombinant forms (URF), variable amplicon approaches were developed for NFGS comprising all functional genes. Twenty-three different genomes composed of subtypes A (A1), B, F (F2), G, CRF01_AE, CRF02_AG, and CRF22_01A1 were successfully determined. The NFGS approach was robust irrespective of viral loads (≥306 copies/mL) and amplification method. Third-generation sequencing (TGS), single genome amplification (SGA), cloning, and bulk sequencing yielded similar outcomes concerning subtype composition and recombinant breakpoint patterns. The introduction of a simple and versatile near full genome amplification, sequencing, and cloning method enables broad application in phylogenetic studies of diverse HIV-1 subtypes and can contribute to personalized HIV therapy and diagnosis.

HIV controllers suppress viral replication and evolution and prevent disease progression following intersubtype HIV-1 superinfection.

The aim of this study was to investigate the impact of intersubtype HIV-1 superinfection on viremia, reservoir reseeding, viral evolution and disease progression in HIV controllers (HIC). A longitudinal analysis of two Brazilian HIC individuals (EEC09 and VC32) previously identified as dually infected with subtypes B and F1 viruses. Changes in plasma viremia, total HIV-1 DNA levels, CD4+ T-cell counts and HIV-1 quasispecies composition were measured over time. HIV-1 env diversity in peripheral blood mononuclear cell (PBMC) and plasma samples was accessed by single genome amplification and next-generation sequencing approaches, respectively. Viral evolution was evaluated by estimating nucleotide diversity and divergence. Individual EEC09 was probably initially infected with a CCR5-tropic subtype B strain and sequentially superinfected with a CXCR4-tropic subtype B strain and with a subtype F1 variant. Individual VC32 was infected with a subtype B strain and superinfected with a subtype F1 variant. The intersubtype superinfection events lead to a moderate increase in viremia and extensive turnover of viral population in plasma but exhibited divergent impact on the size and composition of cell-associated HIV DNA population. Both individuals maintained virologic control (<2000 copies/ml) and presented no evidence of viral evolution or immunologic progression for at least 2 years after the intersubtype superinfection event. These data revealed that some HIC are able to repeatedly limit replication and evolution of superinfecting viral strains of a different subtype with no signs of disease progression.

Next-generation sequencing of HIV-1 single genome amplicons

The analysis of HIV-1 sequences has helped understand the viral molecular epidemiology, monitor the development of antiretroviral drug resistance, and design candidate vaccines. The introduction of single genome amplification (SGA) has been a major advancement in the field, allowing for the characterization of multiple sequences per patient while preserving linkage among polymorphisms in the same viral genome copy. Sequencing of SGA amplicons is performed by capillary Sanger sequencing, which presents low throughput, requires a high amount of template, and is highly sensitive to template/primer mismatching. In order to meet the increasing demand for HIV-1 SGA amplicon sequencing, we have developed a platform based on benchtop next-generation sequencing (NGS) (IonTorrent) accompanied by a bioinformatics pipeline capable of running on computer resources commonly available at research laboratories. During assay validation, the NGS-based sequencing of 10 HIV-1 env SGA amplicons was fully concordant with Sanger sequencing. The field test was conducted on plasma samples from 10 US Navy and Marine service members with recent HIV-1 infection (sampling interval: 2005–2010; plasma viral load: 5,884–194,984 copies/ml). The NGS analysis of 101 SGA amplicons (median: 10 amplicons/individual) showed within-individual viral sequence profiles expected in individuals at this disease stage, including individuals with highly homogeneous quasispecies, individuals with two highly homogeneous viral lineages, and individuals with heterogeneous viral populations. In a scalability assessment using the Ion Chef automated system, 41/43 tested env SGA amplicons (95%) multiplexed on a single Ion 318 chip showed consistent gene-wide coverage >50×. With lower sample requirements and higher throughput, this approach is suitable to support the increasing demand for high-quality and cost-effective HIV-1 sequences in fields such as molecular epidemiology, and development of preventive and therapeutic strategies.

3296 Endometrial cancer microbiome biomarker for disease detection and microbial role in the disease

OBJECTIVES/SPECIFIC AIMS: Our primary objective is to determine whether the bacteria exerts its effect intra- or extra-cellularly. We have genomic and microscopy preliminary evidence indicating that the bacteria is capable of invading endometrial cells. Our secondary objective is to identify what type of impact the bacteria have on the host cells and whether they are capable of transforming the host cells from a benign into a malignant phenotype. We are currently testing a putative mechanism by which the bacteria may cause the overexpression of the hypoxia inducible factor (HIF), a hallmark of endometrial cancer. METHODS/STUDY POPULATION: We are utilizing our custom built optofluidics platform (microfluidics platform incorporated into an advanced microscope with optical laser tweezers) to isolate single cells from the endometrial tissues of 150 patients with and without endometrial cancer. We are utilizing single cell whole genome amplification followed by qPCR to identify if the bacteria is present intracellularly. We are coupling this procedure with standard microbiological invasion assays with endometrial cell line cultures and P.somerae. We are also utilizing our optofluidics platform to perform single cell transcriptomic amplification, followed by sequencing of cells invaded or in the presence of the bacteria to determine the impact in the transcriptome of the host cell. We are coupling this with western blots of factors hypothesized to be impacted by the bacteria in the overexpression of HIF. RESULTS/ANTICIPATED RESULTS: Based on our preliminary data we anticipate to find evidence that P.somerae is invading the host cells, in particular the cells in tumor tissues. We also expect to find that the intracellular presence of the bacteria is causing the overexpression of the HIF pathway, hence resulting in a cancerous phenotype. DISCUSSION/SIGNIFICANCE OF IMPACT: Our long-term goal is to develop primary prevention strategies that will reduce endometrial cancer incidence rates. A confirmation of our hypothesis could suggest that it is sufficient for endometrial cancer prevention efforts to eliminate P.somerae, in line with gastric and cervical cancer efforts. It could also mean that targeting P.somerae in cancer treatment is necessary to contain the disease and prevent recurrence.

Reduction of HIV-1 Reservoir Size and Diversity After 1 Year of cART Among Brazilian Individuals Starting Treatment During Early Stages of Acute Infection.

The aim of early combined antiretroviral therapy (cART) of HIV is to limit the seeding of the viral reservoir during the initial phase of infection and, consequently, decrease intrahost viral diversity. Here, we assessed the effect of early cART on size and complexity of the proviral reservoir. Peripheral blood mononuclear cell (PBMC) and plasma samples were obtained from ten HIV-infected Brazilian individuals diagnosed at the acute phase of infection, before (PREART) and 12 months (M12ART) after suppressive cART. HIV proviral reservoir size was determined by quantitative real-time PCR; intrahost viral diversity of the env C2-V3 region was assessed by single genome amplification or next-generation sequencing in PBMC and plasma, respectively. Mean nucleotide diversity (π) and normalized Shannon entropy (HSN) were used to infer the complexity of the viral population. Compared to PREART, M12ART saw an immunological recovery with a gain of ∼200 CD4+ T cells (P = 0.008) and a normalization of the CD4/CD8 ratio [1.0 (IQR: 0.88–1.18), P = 0.016], as well as a significant decrease in HIV-1 RNA (∼4 log, P = 0.004) and DNA (∼1 log, P = 0.002) levels. The median time to achieve viral suppression was 3 months (IQR: 2.8–5.8 months). The high intermixing between sequences from both visits suggests that the HIV-1 DNA reservoir remained remarkably stable under cART. After 1 year of cART, there was a minor reduction in proviral π (PreART = 0.20 vs. M12ART = 0.10; P = 0.156) but a significant decrease in HSN (PreART = 0.41 vs. M12ART = 0.25; P = 0.019). We found no correlation between π or HSN at PreART and the rate of HIV DNA decay, T CD4+ counts, or CD4/CD8 ratio at M12ART. Based on a small cohort of Brazilian infected individuals under early cART and analyses of the env region, 1 year of follow-up suggested a reservoir size reduction, allowed a significant decrease of HIV-1 complexity, and achieved immunological restoration regardless of the initial HIV-1 plasma viral load, CD4+ T cell counts, or HIV-1 subtype. However, further studies in the Brazilian setting aiming a longer follow-up and larger cohort are required in this field.

Persistent Viral Reservoirs in Lymphoid Tissues in SIV-Infected Rhesus Macaques of Chinese-Origin on Suppressive Antiretroviral Therapy

Understanding HIV latent reservoirs in tissues is essential for the development of new strategies targeting these sites for eradication. Here, we assessed the size of latent reservoirs and the source of residual viruses in multiple lymphoid tissues of SIV-infected and fully suppressed rhesus macaques of Chinese-origin (cRMs). Eight cRMs were infected with SIVmac251 and treated with tenofovir and emtricitabine daily for 24 weeks initiated 4 weeks post-infection. Four of the eight animals reached sustained full viral suppression with undetectable viremia. The levels of cell-associated SIV DNA varied in peripheral blood mononuclear cells (PBMCs) and multiple lymphoid tissues, but with higher levels in the mesenteric lymph nodes (MesLNs). The levels of cell-associated SIV RNA also varied in different tissues. The higher frequency of viral RNA detection in the MesLNs was also observed by in situ hybridization. Consistently, the infection unit per million cells (IUPM) in the MesLNs was higher than in PBMCs and other tested lymphoid tissues by quantitative viral outgrowth assay (QVOA). Furthermore, env gp120 from tissue SIV RNA was amplified by single genome amplification. Phylogenetic analysis revealed diverse variants from tissues parallel to the viral inoculum in all viral suppressed animals. These results demonstrate that the latency and viral reservoirs in the lymphoid tissues still exist in aviremic macaques under full suppressive therapy. Moreover, the size of viral latent reservoirs differs in various lymphoid tissues with a relatively larger size in the MesLNs.

DIVERSITY OF HEPATITIS C VIRUS

Objective: (1)To understand the diversity in HCV glycoprotein E1 and E2sequences at different stages of infection. (2) Method widely used in evolutionary studies of HIV,as a new gold standard in HCV research.Setting: Department of Biochemistry Sir Syed Institutest of Medical Sciences Karachi. Period. 1 Jan 2012 to 31 Dec 2012. Design: Experimentalanalysis. Methods: The samples derived from a xenomouse model of transmission and severalsamples from naturally occurring transmission as well as sequences from acute stage ofinfection. we utilized single genome amplification (SGA) technique to recover full- length E1sequences. SGA of full-length E1 glycoprotein sequences, followed by direct sequencingminimizes in vitro generated artifacts and experimental biases associated with the standard bulkamplification and cloning approach, giving an accurate representation of investigated intrahostpopulation. Analysis and results: Comparative analyses of sequences derived from threedifferent settings were performed, in conjunction with a range of phylogenetic tests. As full-lengthE1 sequences were utilised. Conclusions: The role of glycoprotein E1 and E2 during theinfection of HCV was Known. The new gold standard in HCV research prove to be of greatimportance.

A Patient Derived Xenograft As a Preclinical Model for Monomorphic Epitheliotropic Intestinal T-Cell Lymphoma

Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) is a type of rare aggressive lymphoma accounting for 5.4% of primary intestinal peripheral T-cell lymphoma and 10-25% of all primary intestinal lymphoma. MEITL also has an extremely poor prognosis with the median overall survival of only seven months. No effective treatment or targeted therapies are currently available to manage this disease. Therefore, there is an urgent need to devise and establish an appropriate preclinical model for a better understanding of this disease and for new therapeutic strategies to be developed on it.Here, we present the first MEITL patient derived xenograft (PDX) as both orthotropic intestinal and as a subcutaneous model. The histological analysis demonstrated high similarity in the overall immunomorphologic features between the primary tumour and PDX tumours. Importantly, all the PDX tumors carried the distinctive MEITL immunophenotype; CD3+CD4-CD8+CD56+ and extensive nuclear expression of MATK. We also had Sanger sequenced 83 somatic mutations in the original tumour and rediscovered them in the 6th passage of our xenografts, which suggest that the generated MEITL PDX tumours were genetically stable.Moreover, whole exome sequencing results showed that the clonal architecture in the primary tumour was well preserved in the PDX tumours. The clonal architecture was further analysed with single cell whole genome amplification following by Sanger sequencing. We found that the most of heterozygous mutations in the primary tumour were a mixture of wild type alleles and heterozygous/homozygous mutations from separated clones, including DUPS14 heterozygous mutation (p.R300W) which was known to activate MEK pathway, and SETD2 mutations, (g.chr3:47061285_ 47061286insA and g.chr3:47127805C>A) which were the synthetic lethal partner of Wee1. Interestingly, we found that the two SETD2 mutations alone were able to differentiate three subclones. This result indicates that the tumour was highly subclonal, which could have a strong impact on the selection of drug resistant clones in the drug treatment.In order to verify this hypothesis, we proceeded to test the efficacy of MEK inhibitor, Trametinib and Wee1 inhibitor, AZD1775 both separately and combined treatment, in our PDX subcutaneous model. The results showed that both 1mg/kg/day of Trametinib and 60mg/kg/day of AZD1775 treatments significantly delayed 60-70% of tumour growth as compared with the vehicle group. The tumour growth was completely arrested in the combined treatment group. In addition, 20% and 40% of the mice exhibited Trametinib and AZD1775 resistance, respectively. From our genomic analysis, all DUPS14 and SETD2 mutations from the pretreated tumours were preserved in the treatment-resistant tumours but absent in the sensitive post-treated residual tumours. The clonality analysis from the sequencing data of the pretreated tumour supports and suggests that single-agent regimes will often not be able to induce a complete response in MEITL patients.Altogether, the current results demonstrated that our MEITL PDX model preserved the principal histological and genetic characteristics of its original tumour and is genomic stable across passages. The model also showed that MEITL could be highly subclonal and combined targeted therapy might achieve a higher efficacy for MEITL treatment. DisclosuresNo relevant conflicts of interest to declare.

Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing

BackgroundThe current literature on single cell genomic analyses on the DNA level is conflicting regarding requirements for cell quality, amplification success rates, allelic dropouts and resolution, lacking a systematic comparison of multiple cell input down to the single cell. We hypothesized that such a correlation assay would provide an approach to address the latter issues, utilizing the leukemic cell line OCI-AML3 with a known set of genetic aberrations.ResultsBy analyzing single and multiple cell replicates (2 to 50 cells) purified by micromanipulation and serial dilution we stringently assessed the signal-to-noise ratio (SNR) from single as well as a discrete number of cells based on a multiple displacement amplification method, with whole exome sequencing as signal readout. In this setting, known OCI-AML3 mutations as well as large copy number alterations could be identified, adding to the current knowledge of cytogenetic status. The presence of DNMT3A R882C, NPM1 W288 fs and NRAS Q61L was consistent, in spite of uneven allelic read depths. In contrast, at the level of single cells, we observed that one-third to half of all variants were not reproduced in the replicate sample, and this allelic mismatch displayed an exponential function of cell input. Large signature duplications were discernible from 5 cells, whereas deletions were visible down to the single cell. Thus, even under highly optimized conditions, single cell whole genome amplification and interpretation must be taken with considerable caution, given that allelic change is frequent and displays low SNR. Allelic noise is rapidly alleviated with increased cell input, and the SNR is doubled from 2 to 50 cells.ConclusionsIn conclusion, we demonstrate noisy allele distributions, when analyzing genetic aberrations within single cells relative to multiple cells. Based on the presented data we recommend that single cell analyses should include replicate cell dilution assays for a given setup for relative assessment of procedure-specific SNR to ensure that the resolution supports the specific hypotheses.

Monotypic low-level HIV viremias during antiretroviral therapy are associated with disproportionate production of X4 virions and systemic immune activation.

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.

Abstract 219: Development of TEAPOT algorithm to reconstruct individual ovarian tumors' evolutionary history based upon bulk and single cell whole exome sequencing data

Abstract Background. Mutation detection through genetic testing is playing an increasingly important role in personalized precision medicine in cancer. However, current tests identifying driver mutations as therapeutic targets are based on detection of common mutations in cancer genes. These tests are not patient specific and do not address intra-tumor heterogeneity. Ubiquitous intra-tumor genetic heterogeneity is a mechanism of drug resistance and cancer recurrence. Methods. Approximately 16-24 microsamples are acquired to represent the entire cancer cell population for every ovarian tumor. Each microsample consists of a few cells within a clone and is selected to substitute for a single cell and overcome the large allele dropout rate commonly seen in single genome amplification and sequencing. TEAPOT (Tumor Evolution Assay for Personalized Oncology Therapy) algorithm has been developed to reconstruct a tumor's evolutionary history through integration of whole exome sequencing data from the bulk primary tumor and 16-24 microsamples taken from the bulk tumor. The evolutionary history for an individual tumor is expressed as a rooted and binary tumor developmental tree representing the mitotic process starting from an ancestral cancer cell. Individual mutations are assigned to the cells where they originally occur. The offspring size carrying a mutation was estimated based on tumor purity, variant allele frequency and the variant's copy number. Results. TEAPOT algorithm builds a tumor's evolutionary history with the following features: 1) a tumor's evolutionary history is unique for each ovarian cancer patient; 2) the size of a tree is proportional to the number of microsamples selected; 3) 16-24 microsamples builds a tree with 5 or more generations; 4) TEAPOT detects a driver mutation's occurrence at a specific developmental stage such as 1-cell, 2-cell, 4-cell, etc; 5) The size of offspring carrying a mutation thus the intra-tumor prevalence of the mutation can be estimated; 6) multiple driver mutations can be located separately in different clones. Therefore, TEAPOT provides a quantitative description of intra-tumor genetic heterogeneity and identifies sub-clonal driver mutations in a tumor. Conclusion. TEAPOT reconstructs a tumor's developmental process thus providing a patient-specific evolutionary history. Quantitation of intra-tumor prevalence of driver mutations may inform selection of an effective targeted agent and may provide rationale for cocktail treatment targeting multiple driver mutations simultaneously. TEAPOT can be also used for other solid and liquid cancers. A driver mutation's role in a patient may be functionally defined and quantitated based upon the growth advantage (fitness) it confers on its host cells in the reconstructed tumor evolutionary history. Citation Format: Jianshu Zhang, Helaman Escobar, Harshmi Shah, Mickey Miller, Yang Wei, Kristen Schneider, Michelle Knirr, Kenny Day, Christopher Johnson, Baoli Yang, Eric Devor, Kristina Thiel, Lincoln Nadauld, Kimberly Leslie, Donghai Dai. Development of TEAPOT algorithm to reconstruct individual ovarian tumors' evolutionary history based upon bulk and single cell whole exome sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 219.

Seminal Simian Immunodeficiency Virus in Chronically Infected Cynomolgus Macaques Is Dominated by Virus Originating from Multiple Genital Organs.

ABSTRACTThe sexual transmission of viruses is responsible for the spread of multiple infectious diseases. Although the human immunodeficiency virus (HIV)/AIDS pandemic remains fueled by sexual contacts with infected semen, the origin of virus in semen is still unknown. In a substantial number of HIV-infected men, viral strains present in semen differ from the ones in blood, suggesting that HIV is locally produced within the genital tract. Such local production may be responsible for the persistence of HIV in semen despite effective antiretroviral therapy. In this study, we used single-genome amplification, amplicon sequencing (env gene), and phylogenetic analyses to compare the genetic structures of simian immunodeficiency virus (SIV) populations across all the male genital organs and blood in intravenously inoculated cynomolgus macaques in the chronic stage of infection. Examination of the virus populations present in the male genital tissues of the macaques revealed compartmentalized SIV populations in testis, epididymis, vas deferens, seminal vesicles, and urethra. We found genetic similarities between the viral strains present in semen and those in epididymis, vas deferens, and seminal vesicles. The contribution of male genital organs to virus shedding in semen varied among individuals and could not be predicted based on their infection or proinflammatory cytokine mRNA levels. These data indicate that rather than a single source, multiple genital organs are involved in the release of free virus and infected cells into semen. These findings have important implications for our understanding of systemic virus shedding and persistence in semen and for the design of eradication strategies to access viral reservoirs.IMPORTANCE Semen is instrumental for the dissemination of viruses through sexual contacts. Worryingly, a number of systemic viruses, such as HIV, can persist in this body fluid in the absence of viremia. The local source(s) of virus in semen, however, remains unknown. To elucidate the anatomic origin(s) of the virus released in semen, we compared viral populations present in semen with those in the male genital organs and blood of the Asian macaque model, using single-genome amplification, amplicon sequencing (env gene), and phylogenetic analysis. Our results show that multiple genital tissues harbor compartmentalized strains, some of them (i.e., from epididymis, vas deferens, and seminal vesicles) displaying genetic similarities with the viral populations present in semen. This study is the first to uncover local genital sources of viral populations in semen, providing a new basis for innovative targeted strategies to prevent and eradicate HIV in the male genital tract.

Individuals with HIV-1 Subtype C Infection and Cryptococcal Meningitis Exhibit Viral Genetic Intermixing of HIV-1 Between Plasma and Cerebrospinal Fluid and a High Prevalence of CXCR4-Using Variants.

The genotypic properties of human immunodeficiency virus type 1 (HIV-1) subtype C in individuals presenting with cryptococcal meningitis (CM) are not well established. Employing single-genome amplification as well as bulk PCR, cloning and sequencing strategies, we evaluated the genetic properties of HIV-1 subtype C env in 16 antiretroviral therapy-naive study participants with CM. Eleven of the 16 participants had matched blood plasma and cerebrospinal fluid (CSF) evaluated, with the rest having either a plasma or CSF sample evaluated. Before antiretroviral therapy initiation, matched plasma and CSF-derived env sequences of all 11 participants displayed genetic intermixing between the two compartments. Overall, 7 of the 16 (∼43.8%) participants harbored CXCR4-using variants in plasma and/or CSF, according to coreceptor usage prediction algorithms. This study suggests that HIV-1 subtype C genetic intermixing between peripheral blood and the central nervous system is common in individuals presenting with CM, and that CXCR4 usage is present in one or both compartments in approximately 44% of individuals.

Infant transmitted/founder HIV-1 viruses from peripartum transmission are neutralization resistant to paired maternal plasma

Despite extensive genetic diversity of HIV-1 in chronic infection, a single or few maternal virus variants become the founders of an infant’s infection. These transmitted/founder (T/F) variants are of particular interest, as a maternal or infant HIV vaccine should raise envelope (Env) specific IgG responses capable of blocking this group of viruses. However, the maternal or infant factors that contribute to selection of infant T/F viruses are not well understood. In this study, we amplified HIV-1 env genes by single genome amplification from 16 mother-infant transmitting pairs from the U.S. pre-antiretroviral era Women Infant Transmission Study (WITS). Infant T/F and representative maternal non-transmitted Env variants from plasma were identified and used to generate pseudoviruses for paired maternal plasma neutralization sensitivity analysis. Eighteen out of 21 (85%) infant T/F Env pseudoviruses were neutralization resistant to paired maternal plasma. Yet, all infant T/F viruses were neutralization sensitive to a panel of HIV-1 broadly neutralizing antibodies and variably sensitive to heterologous plasma neutralizing antibodies. Also, these infant T/F pseudoviruses were overall more neutralization resistant to paired maternal plasma in comparison to pseudoviruses from maternal non-transmitted variants (p = 0.012). Altogether, our findings suggest that autologous neutralization of circulating viruses by maternal plasma antibodies select for neutralization-resistant viruses that initiate peripartum transmission, raising the speculation that enhancement of this response at the end of pregnancy could further reduce infant HIV-1 infection risk.

I-108 Assessing the contribution of myeloid cells to HIV-1 persistence under ART

Assessing whether myeloid cells support HIV-1 persistence in the face of effective ART represents a significant technical challenge. As a result, there is as yet, no direct evidence that myeloid cells play any role in viral persistence in ART-suppressed individuals. As a consequence, research on myeloid cell reservoirs is falling into obscurity. Infection of macrophages can only be initiated by HIV-1 variants that have the ability to use low levels of CD4 on the cell surface. Therefore, if a functional myeloid reservoir contributes to viral persistence under effective ART, we would predict that viremia that rebounds following analytic treatment interruption (ATI), would contain viral variants that have a high affinity for CD4. We have developed an approach that allows identification of low frequency macrophage-tropic variants in rebounding viremia post ATI. Through single genome amplification (SGA), we cloned a large number of viral envelopes from plasma of individuals who underwent ATI. Furthermore, we have assessed whether macrophage-tropic viruses contributed to viral rebound in individuals who exhibited prolonged remission after receiving reduced intensity bone marrow transplant When these envelopes were used to construct recombinant molecular clones, they conferred the ability to fuse with, and replicate within primary macrophages. We believe that these results provide strong evidence for the existence of a myeloid cell reservoir in infected individuals on suppressive ART and furthermore, that this reservoir contributes to viral rebound when ART is interrupted. The longevity and the anatomic source of the reservoir from which these macrophage-tropic viruses originate, is under investigation.

Identification of Unequally Represented Founder Viruses Among Tissues in Very Early SIV Rectal Transmission.

Characterizing the transmitted/founder (T/F) viruses of multi-variant SIV infection may shed new light on the understanding of mucosal transmission. We intrarectally inoculated six Chinese rhesus macaques with a single high dose of SIVmac251 (3.1 × 104 TCID50) and obtained 985 full-length env sequences from multiple tissues at 6 and 10 days post-infection by single genome amplification (SGA). All 6 monkeys were infected with a range of 2 to 8 T/F viruses and the dominant variants from the inoculum were still dominant in different tissues from each monkey. Interestingly, our data showed that a cluster of rare T/F viruses was unequally represented in different tissues. This cluster of rare T/F viruses phylogenetically related to the non-dominant SIV variants in the inoculum and was not detected in any rectum tissues, but could be identified in the descending colon, jejunum, spleen, or plasma. In 2 out of 6 macaques, identical SIVmac251 variants belonging to this cluster were detected simultaneously in descending colon/jejunum and the inoculum. We also demonstrated that the average CG dinucleotide frequency of these rare T/F viruses found in tissues, as well as non-dominant variants in the inoculum, was significantly higher than the dominant T/F viruses in tissues and the inoculum. Collectively, these findings suggest that descending colon/jejunum might be more susceptible than rectum to SIV in the very early phase of infection. And host CG suppression, which was previously shown to inhibit HIV replication in vitro, may also contribute to the bottleneck selection during in vivo transmission.

Single cell on-chip whole genome amplification via micropillar arrays for reduced amplification bias.

Single cell whole genome amplification is susceptible to amplification biases that impact the accuracy of single cell sequencing data. To address this, we have developed a microfluidic device for the isolation and purification of genomic DNA from individual cells. The device uses a micropillar array to physically capture single cells and its chromosomal DNA upon extraction. The extracted DNA is immobilized within the micropillar array in a way that allows isothermal amplification. In this system, whole genome amplification of the single cell is carried out under a continual fluid flow within the microfluidic channel. We have demonstrated the process for amplification of individual human cancer cell genomes from the HeLa cell line. By sampling select gene loci along the human genome and performing whole exome sequencing, we demonstrate improved genome coverage and reduced amplification bias compared to amplification of single cells deposited in wells by fluorescence activated cell sorting.

Comprehensive chromosomal and mitochondrial copy number profiling in human IVF embryos

Single cell whole genome sequencing helps to decipher the genome heterogeneity within a cell population and facilitates the analysis of trace amounts of genetic material, such as is found in human embryos. The mitochondrial genome, although an important part of the genetic composition of eukaryotic cells, is often neglected in single cell genome analysis. A recently developed single cell whole genome amplification method was used, known as multiple annealing and looping based amplification cycles (MALBAC-NGS), for simultaneous analysis of chromosomal and mitochondrial genomes at the single cell level. The platform was validated by a series of technical and biological replicates and used for chromosomal and mitochondrial copy number analysis in 399 in-vitro fertilized embryos from 81 couples. A positive correlation of maternal age with increased mitochondria quantity (β = 0.176, P = 0.001) was observed after adjusting for the impact of cell type. Lower numbers of mitochondria were detected in successfully implanted embryos, although the difference was not significant. It is proposed that MALBAC-NGS could potentially be used for an advanced pre-implantation genetic screening procedure with both chromosomal constitution and mitochondrial copy number being evaluated.

Probing the compartmentalization of HIV-1 in the central nervous system through its neutralization properties.

Compartmentalization of HIV-1 has been observed in the cerebrospinal fluid (CSF) of patients at different clinical stages. Considering the low permeability of the blood-brain barrier, we wondered if a reduced selective pressure by neutralizing antibodies (NAb) in the central nervous system (CNS) could favor the evolution of NAb-sensitive viruses in this compartment. Single genome amplification (SGA) was used to sequence full-length HIV-1 envelope variants (453 sequences) from paired CSF and blood plasma samples in 9 subjects infected by HIV variants of various clades and suffering from diverse neurologic disorders. Dynamics of viral evolution were evaluated with a bayesian coalescent approach for individuals with longitudinal samples. Pseudotyped viruses expressing envelope glycoproteins variants representative of the quasi-species present in each compartment were generated, and their sensitivity to autologous neutralization, broadly neutralizing antibodies (bNAbs) and entry inhibitors was assessed. Significant compartmentalization of HIV populations between blood and CSF were detected in 5 out of 9 subjects. Some of the previously described genetic determinants for compartmentalization in the CNS were observed regardless of the HIV-1 clade. There was no difference of sensitivity to autologous neutralization between blood- and CSF-variants, even for subjects with compartmentalization, suggesting that selective pressure by autologous NAb is not the main driver of HIV evolution in the CNS. However, we observed major differences of sensitivity to sCD4 or to at least one bNAb targeting either the N160-V1V2 site, the N332-V3 site or the CD4bs, between blood- and CSF-variants in all cases. In particular, HIV-1 variants present in the CSF were more resistant to bNAbs than their blood counterpart in some cases. Considering the possible migration from CSF to blood, the CNS could be a reservoir of bNAb resistant viruses, an observation that should be considered for immunotherapeutic approaches.

The comparison of the performance of four whole genome amplification kits on ion proton platform in copy number variation detection.

With the development and clinical application of genomics, more and more concern is focused on single-cell sequencing. In the process of single-cell sequencing, whole genome amplification is a key step to enrich sample DNA. Previous studies have compared the performance of different whole genome amplification (WGA) strategies on Illumina sequencing platforms, but there is no related research aimed at Ion Proton platform, which is also a popular next-generation sequencing platform. Here by amplifying cells from six cell lines with different karyotypes, we estimated the data features of four common commercial WGA kits (PicoPLEX WGA Kit, GenomePlex Single Cell Whole Genome Amplification Kit, MALBAC Single Cell Whole Genome Amplification Kit, and REPLI-g Single Cell Kit), including median absolute pairwise difference, uniformity, reproducibility, and fidelity, and examined their performance of copy number variation detection. The results showed that both MALBAC and PicoPLEX could yield high-quality data and had high reproducibility and fidelity; and as for uniformity, PicoPLEX was slightly superior to MALBAC.