Blood-based tumor mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab.

Although programmed death-ligand 1-programmed death 1 (PD-L1-PD-1) inhibitors are broadly efficacious, improved outcomes have been observed in patients with high PD-L1 expression or high tumor mutational burden (TMB). PD-L1 testing is required for checkpoint inhibitor monotherapy in front-line non-small-cell lung cancer (NSCLC). However, obtaining adequate tumor tissue for molecular testing in patients with advanced disease can be challenging. Thus, an unmet medical need exists for diagnostic approaches that do not require tissue to identify patients who may benefit from immunotherapy. Here, we describe a novel, technically robust, blood-based assay to measure TMB in plasma (bTMB) that is distinct from tissue-based approaches. Using a retrospective analysis of two large randomized trials as test and validation studies, we show that bTMB reproducibly identifies patients who derive clinically significant improvements in progression-free survival from atezolizumab (an anti-PD-L1) in second-line and higher NSCLC. Collectively, our data show that high bTMB is a clinically actionable biomarker for atezolizumab in NSCLC.

Virus genomes reveal factors that spread and sustained the Ebola epidemic.

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics.

Fluid Spatial Dynamics of West Nile Virus in the United States: Rapid Spread in a Permissive Host Environment.

UNLABELLED: The introduction of West Nile virus (WNV) into North America in 1999 is a classic example of viral emergence in a new environment, with its subsequent dispersion across the continent having a major impact on local bird populations. Despite the importance of this epizootic, the pattern, dynamics, and determinants of WNV spread in its natural hosts remain uncertain. In particular, it is unclear whether the virus encountered major barriers to transmission, or spread in an unconstrained manner, and if specific viral lineages were favored over others indicative of intrinsic differences in fitness. To address these key questions in WNV evolution and ecology, we sequenced the complete genomes of approximately 300 avian isolates sampled across the United States between 2001 and 2012. Phylogenetic analysis revealed a relatively star-like tree structure, indicative of explosive viral spread in the United States, although with some replacement of viral genotypes through time. These data are striking in that viral sequences exhibit relatively limited clustering according to geographic region, particularly for those viruses sampled from birds, and no strong phylogenetic association with well-sampled avian species. The genome sequence data analyzed here also contain relatively little evidence for adaptive evolution, particularly of structural proteins, suggesting that most viral lineages are of similar fitness and that WNV is well adapted to the ecology of mosquito vectors and diverse avian hosts in the United States. In sum, the molecular evolution of WNV in North America depicts a largely unfettered expansion within a permissive host and geographic population with little evidence of major adaptive barriers. IMPORTANCE: How viruses spread in new host and geographic environments is central to understanding the emergence and evolution of novel infectious diseases and for predicting their likely impact. The emergence of the vector-borne West Nile virus (WNV) in North America in 1999 represents a classic example of this process. Using approximately 300 new viral genomes sampled from wild birds, we show that WNV experienced an explosive spread with little geographical or host constraints within birds and relatively low levels of adaptive evolution. From its introduction into the state of New York, WNV spread across the United States, reaching California and Florida within 4 years, a migration that is clearly reflected in our genomic sequence data, and with a general absence of distinct geographical clusters of bird viruses. However, some geographically distinct viral lineages were found to circulate in mosquitoes, likely reflecting their limited long-distance movement compared to avian species.

Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples.

We have developed a robust RNA sequencing method for generating complete de novo assemblies with intra-host variant calls of Lassa and Ebola virus genomes in clinical and biological samples. Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA. This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries. We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries. These protocols have enabled rapid deep sequencing of both Lassa and Ebola virus and are broadly applicable to other viral genomics studies.

Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak.

In its largest outbreak, Ebola virus disease is spreading through Guinea, Liberia, Sierra Leone, and Nigeria. We sequenced 99 Ebola virus genomes from 78 patients in Sierra Leone to ~2000× coverage. We observed a rapid accumulation of interhost and intrahost genetic variation, allowing us to characterize patterns of viral transmission over the initial weeks of the epidemic. This West African variant likely diverged from central African lineages around 2004, crossed from Guinea to Sierra Leone in May 2014, and has exhibited sustained human-to-human transmission subsequently, with no evidence of additional zoonotic sources. Because many of the mutations alter protein sequences and other biologically meaningful targets, they should be monitored for impact on diagnostics, vaccines, and therapies critical to outbreak response.

Within-host whole-genome deep sequencing and diversity analysis of human respiratory syncytial virus infection reveals dynamics of genomic diversity in the absence and presence of immune pressure.

UNLABELLED: Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in infants and young children and an important respiratory pathogen in the elderly and immunocompromised. While population-wide molecular epidemiology studies have shown multiple cocirculating RSV genotypes and revealed antigenic and genetic change over successive seasons, little is known about the extent of viral diversity over the course of an individual infection, the origins of novel variants, or the effect of immune pressure on viral diversity and potential immune-escape mutations. To investigate viral population diversity in the presence and absence of selective immune pressures, we studied whole-genome deep sequencing of RSV in upper airway samples from an infant with severe combined immune deficiency syndrome and persistent RSV infection. The infection continued over several months before and after bone marrow transplant (BMT) from his RSV-immune father. RSV diversity was characterized in 26 samples obtained over 78 days. Diversity increased after engraftment, as defined by T-cell presence, and populations reflected variation mostly within the G protein, the major surface antigen. Minority populations with known palivizumab resistance mutations emerged after its administration. The viral population appeared to diversify in response to selective pressures, showing a statistically significant growth in diversity in the presence of pressure from immunity. Defining escape mutations and their dynamics will be useful in the design and application of novel therapeutics and vaccines. These data can contribute to future studies of the relationship between within-host and population-wide RSV phylodynamics. IMPORTANCE: Human RSV is an important cause of respiratory disease in infants, the elderly, and the immunocompromised. RSV circulating in a community appears to change season by season, but the amount of diversity generated during an individual infection and the impact of immunity on this viral diversity has been unclear. To address this question, we described within-host RSV diversity by whole-genome deep sequencing in a unique clinical case of an RSV-infected infant with severe combined immunodeficiency and effectively no adaptive immunity who then gained adaptive immunity after undergoing bone marrow transplantation. We found that viral diversity increased in the presence of adaptive immunity and was primarily within the G protein, the major surface antigen. These data will be useful in designing RSV treatments and vaccines and to help understand the relationship between the dynamics of viral diversification within individual hosts and the viral populations circulating in a community.

Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells.

Recent molecular studies have shown that, even when derived from a seemingly homogenous population, individual cells can exhibit substantial differences in gene expression, protein levels and phenotypic output, with important functional consequences. Existing studies of cellular heterogeneity, however, have typically measured only a few pre-selected RNAs or proteins simultaneously, because genomic profiling methods could not be applied to single cells until very recently. Here we use single-cell RNA sequencing to investigate heterogeneity in the response of mouse bone-marrow-derived dendritic cells (BMDCs) to lipopolysaccharide. We find extensive, and previously unobserved, bimodal variation in messenger RNA abundance and splicing patterns, which we validate by RNA-fluorescence in situ hybridization for select transcripts. In particular, hundreds of key immune genes are bimodally expressed across cells, surprisingly even for genes that are very highly expressed at the population average. Moreover, splicing patterns demonstrate previously unobserved levels of heterogeneity between cells. Some of the observed bimodality can be attributed to closely related, yet distinct, known maturity states of BMDCs; other portions reflect differences in the usage of key regulatory circuits. For example, we identify a module of 137 highly variable, yet co-regulated, antiviral response genes. Using cells from knockout mice, we show that variability in this module may be propagated through an interferon feedback circuit, involving the transcriptional regulators Stat2 and Irf7. Our study demonstrates the power and promise of single-cell genomics in uncovering functional diversity between cells and in deciphering cell states and circuits.

Complete viral RNA genome sequencing of ultra-low copy samples by sequence-independent amplification.

RNA viruses are the causative agents for AIDS, influenza, SARS, and other serious health threats. Development of rapid and broadly applicable methods for complete viral genome sequencing is highly desirable to fully understand all aspects of these infectious agents as well as for surveillance of viral pandemic threats and emerging pathogens. However, traditional viral detection methods rely on prior sequence or antigen knowledge. In this study, we describe sequence-independent amplification for samples containing ultra-low amounts of viral RNA coupled with Illumina sequencing and de novo assembly optimized for viral genomes. With 5 million reads, we capture 96 to 100% of the viral protein coding region of HIV, respiratory syncytial and West Nile viral samples from as little as 100 copies of viral RNA. The methods presented here are scalable to large numbers of samples and capable of generating full or near full length viral genomes from clone and clinical samples with low amounts of viral RNA, without prior sequence information and in the presence of substantial host contamination.

Genome-wide patterns of intrahuman dengue virus diversity reveal associations with viral phylogenetic clade and interhost diversity.

Analogous to observations in RNA viruses such as human immunodeficiency virus, genetic variation associated with intrahost dengue virus (DENV) populations has been postulated to influence viral fitness and disease pathogenesis. Previous attempts to investigate intrahost genetic variation in DENV characterized only a few viral genes or a limited number of full-length genomes. We developed a whole-genome amplification approach coupled with deep sequencing to capture intrahost diversity across the entire coding region of DENV-2. Using this approach, we sequenced DENV-2 genomes from the serum of 22 Nicaraguan individuals with secondary DENV infection and captured ∼75% of the DENV genome in each sample (range, 40 to 98%). We identified and quantified variants using a highly sensitive and specific method and determined that the extent of diversity was considerably lower than previous estimates. Significant differences in intrahost diversity were detected between genes and also between antigenically distinct domains of the Envelope gene. Interestingly, a strong association was discerned between the extent of intrahost diversity in a few genes and viral clade identity. Additionally, the abundance of viral variants within a host, as well as the impact of viral mutations on amino acid encoding and predicted protein function, determined whether intrahost variants were observed at the interhost level in circulating Nicaraguan DENV-2 populations, strongly suggestive of purifying selection across transmission events. Our data illustrate the value of high-coverage genome-wide analysis of intrahost diversity for high-resolution mapping of the relationship between intrahost diversity and clinical, epidemiological, and virological parameters of viral infection.

Highly sensitive and specific detection of rare variants in mixed viral populations from massively parallel sequence data.

Viruses diversify over time within hosts, often undercutting the effectiveness of host defenses and therapeutic interventions. To design successful vaccines and therapeutics, it is critical to better understand viral diversification, including comprehensively characterizing the genetic variants in viral intra-host populations and modeling changes from transmission through the course of infection. Massively parallel sequencing technologies can overcome the cost constraints of older sequencing methods and obtain the high sequence coverage needed to detect rare genetic variants (< 1%) within an infected host, and to assay variants without prior knowledge. Critical to interpreting deep sequence data sets is the ability to distinguish biological variants from process errors with high sensitivity and specificity. To address this challenge, we describe V-Phaser, an algorithm able to recognize rare biological variants in mixed populations. V-Phaser uses covariation (i.e. phasing) between observed variants to increase sensitivity and an expectation maximization algorithm that iteratively recalibrates base quality scores to increase specificity. Overall, V-Phaser achieved > 97% sensitivity and > 97% specificity on control read sets. On data derived from a patient after four years of HIV-1 infection, V-Phaser detected 2,015 variants across the -10 kb genome, including 603 rare variants (< 1% frequency) detected only using phase information. V-Phaser identified variants at frequencies down to 0.2%, comparable to the detection threshold of allele-specific PCR, a method that requires prior knowledge of the variants. The high sensitivity and specificity of V-Phaser enables identifying and tracking changes in low frequency variants in mixed populations such as RNA viruses.

Whole genome deep sequencing of HIV-1 reveals the impact of early minor variants upon immune recognition during acute infection.

Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia.

Human papillomavirus-like particles mediate functional delivery of plasmid DNA to antigen presenting cells in vivo.

Because recombinant empty viral capsids are potentially attractive vectors for gene therapy, here we examined the ability of human papillomavirus (HPV) virus-like particles (VLPs) to mediate delivery and expression of DNA plasmids in vitro and in vivo. VLP-mediated delivery and expression of a GFP reporter construct in vitro was found to be highly dependent upon the presence of full-length L2 protein within the VLPs. Similarly, expression of GFP and luciferase reporter plasmids in vivo was strongly enhanced by co-administration of L1/L2 VLPs. Interestingly, in these experiments we routinely observed GFP expression in migrating antigen presenting cells (APC) recovered from mice inoculated with GFP plasmid in combination with VLPs, but not in APC recovered from mice inoculated with the plasmid alone. Additional evidence to support this concept was generated in experiments in which co-administration of VLPs with a plasmid designed to express HPV16 E6 oncoprotein was associated with significant enhancement of plasmid-encoded E6-specific cellular immune responses. These findings have implications for the design of vaccines for combined prophylaxis and therapy of HPV-associated diseases, and for other vaccines that rely on the administration of DNA-based immunogens, adjuvants, and/or other factors.

Cyclooxygenase-2 inhibition attenuates antibody responses against human papillomavirus-like particles.

Vaccination to generate protective humoral immunity against infectious disease is becoming increasingly important due to emerging strains of virus, poorly immunogenic vaccines, and the threat of bioterrorism. We demonstrate that cyclooxygenase-2 (Cox-2) is crucial for optimal Ab responses to a model vaccine, human papillomavirus type 16 virus-like particles (HPV 16 VLPs). Cox-2-deficient mice produce 70% less IgG, 50% fewer Ab-secreting cells, and 10-fold less neutralizing Ab to HPV 16 VLP vaccination compared with wild-type mice. The reduction in Ab production by Cox-2(-/-) mice was partially due to a decrease in class switching. SC-58125, a structural analog of the Cox-2-selective inhibitor Celebrex reduced by approximately 70% human memory B cell differentiation to HPV 16 VLP IgG-secreting cells. The widespread use of nonsteroidal anti-inflammatory drugs and Cox-2-selective inhibitory drugs may therefore reduce vaccine efficacy, especially when vaccines are poorly immunogenic or the target population is poorly responsive to immunization.