Predicting Cancer Immunotherapy Response From Gut Microbiomes Using Machine Learning Models

Cancer immunotherapy has significantly improved patient survival. Yet, half of patients do not respond to immunotherapy. Gut microbiomes have been linked to clinical responsiveness of melanoma patients on immunotherapies; however, different taxa have been associated with response status with implicated taxa inconsistent between studies. We used a tumor-agnostic approach to find common gut microbiome features of response among immunotherapy patients with different advanced stage cancers. A combined meta-analysis of 16S rRNA gene sequencing data from our mixed tumor cohort and three published immunotherapy gut microbiome datasets from different melanoma patient cohorts found certain gut bacterial taxa correlated with immunotherapy response status regardless of tumor type. Using multivariate selbal analysis, we identified two separate groups of bacterial genera associated with responders versus non-responders. Statistical models of gut microbiome community features showed robust prediction accuracy of immunotherapy response in amplicon sequencing datasets and in cross-sequencing platform validation with shotgun metagenomic datasets. Results suggest baseline gut microbiome features may be predictive of clinical outcomes in oncology patients on immunotherapies, and some of these features may be generalizable across different tumor types, patient cohorts, and sequencing platforms. Findings demonstrate how machine learning models can reveal microbiome-immunotherapy interactions that may ultimately improve cancer patient outcomes

Compartmented neuronal cultures reveal two distinct mechanisms for alpha herpesvirus escape from genome silencing.

Alpha herpesvirus genomes encode the capacity to establish quiescent infections (i.e. latency) in the peripheral nervous system for the life of their hosts. Multiple times during latency, viral genomes can reactivate to start a productive infection, enabling spread of progeny virions to other hosts. Replication of alpha herpesviruses is well studied in cultured cells and many aspects of productive replication have been identified. However, many questions remain concerning how a productive or a quiescent infection is established. While infections in vivo often result in latency, infections of dissociated neuronal cultures in vitro result in a productive infection unless lytic viral replication is suppressed by DNA polymerase inhibitors or interferon. Using primary peripheral nervous system neurons cultured in modified Campenot tri-chambers, we previously reported that reactivateable, quiescent infections by pseudorabies virus (PRV) can be established in the absence of any inhibitor. Such infections were established in cell bodies only when physically isolated axons were infected at a very low multiplicity of infection (MOI). In this report, we developed a complementation assay in compartmented neuronal cultures to investigate host and viral factors in cell bodies that prevent establishment of quiescent infection and promote productive replication of axonally delivered genomes (i.e. escape from silencing). Stimulating protein kinase A (PKA) signaling pathways in isolated cell bodies, or superinfecting cell bodies with either UV-inactivated PRV or viral light particles (LP) promoted escape from genome silencing and prevented establishment of quiescent infection but with different molecular mechanisms. Activation of PKA in cell bodies triggers a slow escape from silencing in a cJun N-terminal kinase (JNK) dependent manner. However, escape from silencing is induced rapidly by infection with UVPRV or LP in a PKA- and JNK-independent manner. We suggest that viral tegument proteins delivered to cell bodies engage multiple signaling pathways that block silencing of viral genomes delivered by low MOI axonal infection

Retrograde axonal transport of rabies virus is unaffected by interferon treatment but blocked by emetine locally in axons.

Neuroinvasive viruses, such as alpha herpesviruses (αHV) and rabies virus (RABV), initially infect peripheral tissues, followed by invasion of the innervating axon termini. Virus particles must undergo long distance retrograde axonal transport to reach the neuron cell bodies in the peripheral or central nervous system (PNS/CNS). How virus particles hijack the axonal transport machinery and how PNS axons respond to and regulate infection are questions of significant interest. To track individual virus particles, we constructed a recombinant RABV expressing a P-mCherry fusion protein, derived from the virulent CVS-N2c strain. We studied retrograde RABV transport in the presence or absence of interferons (IFN) or protein synthesis inhibitors, both of which were reported previously to restrict axonal transport of αHV particles. Using neurons from rodent superior cervical ganglia grown in tri-chambers, we showed that axonal exposure to type I or type II IFN did not alter retrograde axonal transport of RABV. However, exposure of axons to emetine, a translation elongation inhibitor, blocked axonal RABV transport by a mechanism that was not dependent on protein synthesis inhibition. The minority of RABV particles that still moved retrograde in axons in the presence of emetine, moved with slower velocities and traveled shorter distances. Emetine's effect was specific to RABV, as transport of cellular vesicles was unchanged. These findings extend our understanding of how neuroinvasion is regulated in axons and point toward a role for emetine as an inhibitory modulator of RABV axonal transport

Low MOI axonal PRV infection in compartmented neuronal cultures results in a quiescent infection in a small number of neurons.

<p><b>(A)</b> Tri-chamber compartments, S: soma (cell bodies), M: methocel (middle), N: axonal. PRV 233 infection was made in the N compartment at an MOI of 0.01. DiI (red) was added to the N compartment media 6 hpi to label red cell bodies that project axons to the N compartment. Circles highlight primarily infected neurons. Insets show single infected neurons (green) among surrounding non-infected neurons. <b>(B)</b> GFP positive, DiI labeled or non-labeled cell bodies were counted at 3 dpi (separate or merged channels are shown, arrowhead points to one GFP positive cell body, ph: phase contrast). Ratios of either red cell bodies to all S compartment neurons (connectivity), or green cell bodies to dual color (green and red) cell bodies (infectivity) were calculated and shown in the graph. <b>(C)</b> RNA was isolated after 7 days of either S compartment or N compartment infection with PRV180 at an MOI of 0.01. LAT and EP0 transcripts were quantitated. S compartment (cell body) infection resulted in a productive infection (red capsid accumulation in all of the cells), whereas N compartment (axonal) infection was silent (no detectable red fluorescent signal anywhere in the S compartment). The graph shows the ratio of each transcript in axonal to cell body infection after normalization to 28S rRNA. <b>(D)</b> Illustration of the complementation assay: N compartments are infected with PRV180 at an MOI of 0.01 while S compartments are treated with drugs, inhibitors, viruses or virus-like particles.</p

Simultaneous infection of cell bodies with UVPRV enables axonally infecting PRV180 to escape from silencing.

<p><b>(A)</b> Control and UVPRV959 (MOI 10) complemented S compartments are shown (3 dpi). Red channel is shown after background filtration. Raw image is used for the green channel (no signal). <b>(B)</b> Quantitation of imaging data was graphed including control dishes (7 dpi), UV959 (3 dpi), UVgBnull or UVgDnull PRV complementation (7 dpi), and UV959 plus H89 (UVH89) complementation (3 dpi). Each data point represents one dish (ns is not significant and **** is p<0.0001). <b>(C)</b> Western blot analysis of PKA targets after 3 hours of PRV180 and UVPRV infection in S compartments at an MOI of 10 with (+) or without (-) H89 treatment are shown in comparison to forskolin and dbcAMP treatments. <b>(D)</b> Control dishes with axonal PRV180 infection (MOI 0.01) were harvested either 3 or 10 dpi. S compartments infected only with UVPRV (MOI 10), and complementation assay samples were harvested at 3 dpi. The presence of viral capsid (VP5) and tegument (UL47 and EP0) proteins was determined by WB using half of the lysates (each lane represents contents of one S compartment). Beta-actin was used as a loading control. The other half of the lysates was used to quantitate the amount of viral DNA. <b>(E)</b> PRV DNA was quantitated using UL54 primers. DNA amounts calculated based on threshold cycle values are shown in the graph. Red line shows the detection limit.</p

LP promote escape from PRV180 silencing independent of PKA activity.

<p><b>(A)</b> 20 μl of PRV 180, complemented PRV 495 (produced after infecting UL25 expressing PK15 cells) stocks, and light particles (LP) prepared from PRV 495 infected PK15 supernatant (SN) were fractionated by SDS-PAGE. The presence and quantity of envelope (gD and Us9), tegument (UL36, UL47, EP0, and Us3), and capsid (VP5 and VP26) proteins were determined using specific antibodies (first two lanes were overexposed to be able to detect the proteins in the LP lane). <b>(B)</b> 50 μl LP were added to axons in the N compartment, and gM-pHluorin positive particles were visualized (no red particles were detected). The same amount of LP was also put on a glass coverslip and screened for green, red or dual color particles. <b>(C)</b> Complementation assays were performed using different amounts of LP with or without H89 (3 dpi). Each data point represents one dish (ns is not significant and ** is p = 0.0035). <b>(D)</b> Western blot analysis of PKA targets was done after 3 hrs of UVPRV or LP infection with (+) or without (-) H89 treatment. Beta-actin was used as a loading control.</p

PRV tegument proteins, US3 and EP0 are not required to promote PRV180 escape from silencing.

<p><b>(A)</b> 20 μl PRV EP0null (PRVEP06) and Us3null (PRV 823) virus stocks were run on SDS-PAGE in comparison with PRV959 stocks. <b>(B)</b> Complementation assays were performed using UV inactivated PRV EP06 (UVEP06) and PRV 823 (UV823) with or without H89 (3 dpi). Each data point represents one dish (ns is not significant, **** is p<0.0001, *** is p = 0.0004 and ** is p = 0.0021). Replication incompetent DNA viruses expressing GFP (baculovirus and adenovirus) were also included. Inset shows the transduction efficiencies of baculovirus and adenovirus in S compartments. <b>(C)</b> Western blot analysis of PKA targets during 3 hrs of UVPRV (PRV 959), UV823 or UVEP06 infection with (+) or without (-) H89 treatment. The samples are compared to control and forskolin treatment. Beta-actin was used as a loading control.</p

Forskolin and dbcAMP promote escape from silencing in a PKA and JNK dependent manner.

<p><b>(A)</b> S compartments were treated either with forskolin or dbcAMP for 3 hours or untreated as a control. The PKA inhibitor H89 was added either at 30 min. post forskolin or dbcAMP treatment (+) or not (-). 3 S compartments were harvested per condition and samples were run on SDS-PAGE. PKA activity was tested using a polyclonal antibody that detected PKA targets. Beta-actin was used as loading control. <b>(B)</b> The complementation assay was performed with forskolin treatment and entire S compartment scans for red fluorescence are shown for 1 control and 1 forskolin treated chamber 6 dpi. <b>(C)</b> The fluorescence intensity was quantitated for each S compartment scan after background was removed (AU: arbitrary units). Each data point represents one dish (ns is not significant, **** is p<0.0001 and *** is p = 0.0004). <b>(D)</b> The efficiency of inhibitors was tested in dissociated neurons in 12 wells (3 wells were pooled per condition). Inhibitors were added 1 h post forskolin treatment. Cells were harvested at 3 hpi (H: H89, J: JNKII). JNK activity was monitored by phospho-c-Jun (P-cJun) and total c-Jun (T-cJun) antibodies. Beta actin was used as loading control. <b>(E)</b> Untreated neurons (control) or neurons treated with forskolin plus H89 (F+H) or forskolin plus JNKII (F+J) were imaged at 6 days post treatment (dpt), and then infected with PRV 180 at an MOI of 5. Fluorescent images showing red capsid accumulation in cell bodies were taken 24 hpi.</p

Emetine restricts axonal transport of RABV particles but does not limit transport of Rab5- or Rab7-positive vesicles.

<p><b>(A)</b> 100 μM Emetine was added to N, 1 h prior to RABV P-mCherry infection in N. <b>(B)</b> Quantification of the number of RABV particles moving retrograde per FOV (+/-) emetine pretreatment in N. Open circles represent individual FOVs along the M compartment barrier. Vertical lines and error bars represent the mean ± SD for each condition with ****p < 0.0001 using unpaired t-test. Total FOVs counted were 224 (no treatment) and 166 (emetine) across 3 independent replicate chambers per condition. <b>(C)</b> S compartment cell bodies were transduced with adenoviruses expressing either Venus-Rab5 or Venus-Rab7 for 4 days. <b>(D)</b> Quantification of % moving Rab5 or Rab7 particles per axon (+/-) 100 μM emetine. Each dot represents one axon in the N compartment. A minimum of 22 axons were imaged per condition. Horizontal lines and error bars represent the mean ± SD for each condition (ns = not significant using two-way ANOVA).</p

Construction of a CVS-N2c RABV recombinant for live-cell imaging in PNS neurons.

<p><b>(A)</b> Genome schematics of the parental strain and P-mCherry-expressing, spread-deficient RABV recombinant. <b>(B)</b> Protein composition of sucrose-purified RABV P-mCherry particles (lanes 1 and 3) using SDS-PAGE and western blotting with anti-RABV P, G, N, or M. N2c mCherry particles (G-complemented N2cΔG expressing diffusible mCherry) are included for comparison (lanes 2 and 4). Asterisk indicates P-mCherry fusion protein. <b>(C)</b> Pie chart shows the proportion of enveloped virions (dual colored; 888) to non-enveloped nucleocapsids (red-only; 426) in the RABV P-mCherry stock (n = 1314 particles total). P-mCherry-positive particles (red) from purified supernatants were stained with anti-RABV G and Alexa Fluor 488-conjugated 2° antibody (green) (scale bars = 10 μm) (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007188#ppat.1007188.s001" target="_blank">S1 Fig</a>). <b>(D)</b> Dissociated SCG were infected with RABV P-mCherry (10⁵ ffu) (red) and stained at 48 hpi with FITC-conjugated anti-RABV targeting the N protein (green). DAPI stains nuclei (blue). White arrow indicates a cytoplasmic inclusion body (scale bar = 25μm).</p