Herpes simplex virus type 1 (HSV-1) not only causes painful recurrent oral-labial infections, it can also cause permanent brain damage and blindness. There is currently no HSV-1 vaccine. An effective vaccine must stimulate coordinated T cell responses, but the large size of the genome and the low frequency of HSV-1-specific T cells have hampered the search for the most effective T cell antigens for inclusion in a candidate vaccine. We have now developed what we believe to be novel methods to efficiently generate a genome-wide map of the responsiveness of HSV-1-specific T cells, and demonstrate the applicability of these methods to a second complex microbe, vaccinia virus. We used cross-presentation and CD137 activation-based FACS to enrich for polyclonal CD8 + T effector T cells. The HSV-1 proteome was prepared in a flexible format for analyzing both CD8 + and CD4 + T cells from study participants. Scans with participant-specific panels of artificial APCs identified an oligospecific response in each individual. Parallel CD137-based CD4 + T cell research showed discrete oligospecific recognition of HSV-1 antigens. Unexpectedly, the two HSV-1 proteins not previously considered as vaccine candidates elicited both CD8 + and CD4 + T cell responses in most HSV-1-infected individuals. In this era of microbial genomics, our methods -also demonstrated in principle for vaccinia virus for both CD8 + and CD4 + T cells -should be broadly applicable to the selection of T cell antigens for inclusion in candidate vaccines for many pathogens. IntroductionHerpes simplex virus type 1 (HSV-1) infects 60% of the US population and has a significant cumulative health care burden in addition to causing painful recurrent oral-labial infections. For example, brain and eye infections can cause permanent damage or blindness (1). HSV-1 also causes approximately 50% of clinical first-episode genital herpes in the United States. Vaccines for HSV that have been tested thus far have failed in clinical trials, including a recent phase III trial of an adjuvanted glycoprotein D (gD2) product (2). This vaccine elicits antibody and CD4 + T cell responses but fails to induce CD8 responses. Newer platforms can elicit CD8 + and CD4 + cells, but they require rationally selected T cell antigens. We therefore developed methods to permit measurement of both CD8 and CD4 responses to the complete HSV-1 proteome to begin rational prioritization of next-generation vaccine candidates.Several recent observations support the concept that an effective HSV vaccine will need to induce coordinated CD8 + and CD4 + T cell responses. HSV-1-specific CD8 + T cells localize to the site of HSV-1 infection in human and murine trigeminal ganglia (TG) (3-5), and both HSV-specific CD8 + and CD4 + T cells localize to acute and healed sites of skin infection in mice and humans, sug-

Neurons communicate through Ca 2+ -dependent neurotransmitter release at presynaptic active zones (AZs). Neurotransmitter release properties play a key role in defining information flow in circuits and are tuned during multiple forms of plasticity. Despite their central role in determining neurotransmitter release properties, little is known about how Ca 2+ channel levels are modulated to calibrate synaptic function. We used CRISPR to tag the Drosophila CaV2 Ca 2+ channel Cacophony (Cac) and investigated the regulation of endogenous Ca 2+ channels during homeostatic plasticity in males in which all endogenous Cac channels are tagged. We found that heterogeneously distributed Cac is highly predictive of neurotransmitter release probability at individual AZs and differentially regulated during opposing forms of presynaptic homeostatic plasticity. Specifically, Cac levels at AZ are increased during chronic and acute presynaptic homeostatic potentiation (PHP), and live imaging during acute expression of PHP reveals proportional Ca 2+ channel accumulation across heterogeneous AZs. In contrast, endogenous Cac levels do not change during presynaptic homeostatic depression (PHD), implying that the reported reduction in Ca 2+ influx during PHD is achieved through functional adaptions to pre-existing Ca 2+ channels. Thus, distinct mechanisms bi-directionally modulate presynaptic Ca 2+ levels to maintain stable synaptic strength in response to diverse challenges, with Ca 2+ channel abundance providing a rapidly tunable substrate for potentiating neurotransmitter release over both acute and chronic timescales.

Neurons communicate through Ca2+-dependent neurotransmitter release at presynaptic active zones (AZs). Neurotransmitter release properties play a key role in defining information flow in circuits and are tuned during multiple forms of plasticity. Despite their central role in determining neurotransmitter release properties, little is known about how Ca2+ channel levels are modulated to calibrate synaptic function. We used CRISPR to tag the Drosophila CaV2 Ca2+ channel Cacophony (Cac) and, in males in which all Cac channels are tagged, investigated the regulation of endogenous Ca2+ channels during homeostatic plasticity. We found that heterogeneously distributed Cac is highly predictive of neurotransmitter release probability at individual AZs and differentially regulated during opposing forms of presynaptic homeostatic plasticity. Specifically, AZ Cac levels are increased during chronic and acute presynaptic homeostatic potentiation (PHP), and live imaging during acute expression of PHP reveals proportional Ca2+ channel accumulation across heterogeneous AZs. In contrast, endogenous Cac levels do not change during presynaptic homeostatic depression (PHD), implying that the reported reduction in Ca2+ influx during PHD is achieved through functional adaptions to pre-existing Ca2+ channels. Thus, distinct mechanisms bidirectionally modulate presynaptic Ca2+ levels to maintain stable synaptic strength in response to diverse challenges, with Ca2+ channel abundance providing a rapidly tunable substrate for potentiating neurotransmitter release over both acute and chronic timescales.SIGNIFICANCE STATEMENT Presynaptic Ca2+ dynamics play an important role in establishing neurotransmitter release properties. Presynaptic Ca2+ influx is modulated during multiple forms of homeostatic plasticity at Drosophila neuromuscular junctions to stabilize synaptic communication. However, it remains unclear how this dynamic regulation is achieved. We used CRISPR gene editing to endogenously tag the sole Drosophila Ca2+ channel responsible for synchronized neurotransmitter release, and found that channel abundance is regulated during homeostatic potentiation, but not homeostatic depression. Through live imaging experiments during the adaptation to acute homeostatic challenge, we visualize the accumulation of endogenous Ca2+ channels at individual active zones within 10 min. We propose that differential regulation of Ca2+ channels confers broad capacity for tuning neurotransmitter release properties to maintain neural communication.

Fife is a Piccolo-RIM–related protein that regulates neurotransmission and motor behavior through an unknown mechanism. Here, Bruckner et al. show that Fife organizes synaptic vesicle docking and coupling to calcium channels to establish and modulate synaptic strength.

Neuronal communication depends on the precisely orchestrated release of neurotransmitter at specialized sites called active zones (AZs). A small number of scaffolding and cytoskeletal proteins comprising the cytomatrix of the active zone (CAZ) are thought to organize the architecture and functional properties of AZs. The majority of CAZ proteins are evolutionarily conserved, underscoring the fundamental similarities in neurotransmission at all synapses. However, core CAZ proteins Piccolo and Bassoon have long been believed exclusive to vertebrates, raising intriguing questions about the conservation of the molecular mechanisms that regulate presynaptic properties. Here, we present the identification of a piccolo-rim-related gene in invertebrates, together with molecular phylogenetic analyses that indicate the encoded proteins may represent Piccolo orthologs. In accordance, we find that the Drosophila homolog, Fife, is neuronal and localizes to presynaptic AZs. To investigate the in vivo function of Fife, we generated a deletion of the fife locus. We find that evoked neurotransmitter release is substantially decreased in fife mutants and loss of fife results in motor deficits. Through morphological analysis of fife synapses, we identify underlying AZ abnormalities including pervasive presynaptic membrane detachments and reduced synaptic vesicle clustering. Our data demonstrate the conservation of a Piccolo-related protein in invertebrates and identify critical roles for Fife in regulating AZ structure and function. These findings suggest the CAZ is more conserved than previously thought, and open the door to a more complete understanding of how CAZ proteins regulate presynaptic structure and function through genetic studies in simpler model systems.

Little is known concerning immunodominance within the CD4 T-cell response to viral infections and its persistence into longterm memory. We tested CD4 T-cell reactivity against each viral protein in persons immunized with vaccinia virus (VV), Immunodominance refers to the proportion of T cells specific for a defined epitope in relation to the entire set of T cells reacting to a complex antigen (1). For infectious pathogens encoding many polypeptides, the immunodominance of an open reading frame (ORF) is the proportion of the total pathogen-specific response accounted for by T cells reacting with this ORF. Immunoprevalence is a related concept, referring to the proportion of a population responding to an immunogen (2). The CD8 T-cell response to complex microbes can show remarkably strong immunodominance in humans and inbred animals.As antigen processing differs between T-cell subsets, it is not clear that immunodominance also applies to CD4 T-cell responses. For VV, memory CD4 T-cell responses in inbred mice are quite polyclonal and do not exhibit dominance. The top 14 epitopes account for only 20% of the total VV-specific CD4 T-cell response (3). Data for the human CD4 T-cell response to cytomegalovirus, in contrast, were somewhat consistent with immunodominance. Subjects recognized a median of 12 ORFs per person (of 213 ORFs studied), with the top 6 ORFs accounting for about 40% of the overall response (4). In humans, HLA variation is expected to influence the identity of immunodominant and immunoprevalent antigens in specific individuals. Model systems have identified additional factors controlling epitope choice for CD4 T cells, including naïve T-cell repertoire (5), antigen abundance (6), antigen folding (7), protease processing and epitopeflanking regions (8), and antigenic competition (9).Vaccinia virus is an orthopoxvirus that causes an infection that resolves completely in several weeks in immunocompetent hosts. CD4 T-cell memory persists for decades despite the absence of antigen reexposure, but little is known about the detailed architecture of long-term memory. The monotonic decline of specific antibody levels supports lack of intermittent boosting (10). VV has over 200 ORFs, so each human has a myriad of potential CD4 reactive T-cell specificities. Herpes simplex virus 1 (HSV-1) also has a complex proteome, but in contrast to self-limited VV infections, HSV-1 infections are chronic with intermittent reactivations. Following initial epithelial replication, the virus establishes persistence in the innervating sensory ganglia. Intermittent reactivation of latent HSV-1 in essentially all HSV-1-infected persons (11) results in periodic viral antigen exposure to HSV-1-specific memory T cells (12).To determine the breadth and persistence of CD4 T-cell immunodominance in acute and chronic human viral infections, we compared patterns of CD4 T-cell immunodominance between recent and remote VV recipients. The immune responses of persons chronically infected with HSV-1 were also investigated as an example of a c...

Nervous wreck (Nwk) is a conserved F-BAR protein that attenuates synaptic growth and promotes synaptic function in Drosophila. In an effort to understand how Nwk carries out its dual roles, we isolated interacting proteins using mass spectrometry. We report a conserved interaction between Nwk proteins and BAR-SH3 sorting nexins, a family of membrane-binding proteins implicated in diverse intracellular trafficking processes. In mammalian cells, BAR-SH3 sorting nexins induce plasma membrane tubules that localize NWK2, consistent with a possible functional interaction during the early stages of endocytic trafficking. To study the role of BAR-SH3 sorting nexins in vivo, we took advantage of the lack of genetic redundancy in Drosophila and employed CRISPR-based genome engineering to generate null and endogenously tagged alleles of SH3PX1. SH3PX1 localizes to neuromuscular junctions where it regulates synaptic ultrastructure, but not synapse number. Consistently, neurotransmitter release was significantly diminished in SH3PX1 mutants. Double-mutant and tissue-specific-rescue experiments indicate that SH3PX1 promotes neurotransmitter release presynaptically, at least in part through functional interactions with Nwk, and might act to distinguish the roles of Nwk in regulating synaptic growth and function.

We combined cryopreservation of intact Drosophila larvae and electron tomography with comprehensive segmentation of key features to reconstruct the complete ultrastructure of a model glutamatergic synapse in vivo. Presynaptically, we detail a complex network of filaments that connects and organizes synaptic vesicles. We link the complexity of this synaptic vesicle network to proximity to the active zone cytomatrix, consistent with the model that these protein structures function together to regulate synaptic vesicle pools. We identify a net-shaped network of electron-dense filaments spanning the synaptic cleft that suggests conserved organization of transsynaptic adhesion complexes at excitatory synapses. Postsynaptically, we characterize a regular pattern of macromolecules that yields structural insights into the scaffolding of neurotransmitter receptors. Together, these analyses extend our understanding of the ultrastructural correlates of the molecular machines that regulate synaptic communication and reveal an unexpected level of conservation in the nanoscale organization of diverse glutamatergic synapses.