Pre-existing T Cell Memory against Zika Virus.

The mosquito-borne Zika virus (ZIKV) has spread rapidly into regions where dengue virus (DENV) is endemic, and flavivirus cross-reactive T cell responses have been observed repeatedly in animal models and in humans. Preexisting cellular immunity to DENV is thought to contribute to protection in subsequent ZIKV infection, but the epitope targets of cross-reactive T cell responses have not been comprehensively identified. Using human blood samples from the regions of Nicaragua and Sri Lanka where DENV is endemic that were collected before the global spread of ZIKV in 2016, we employed an in vitro expansion strategy to map ZIKV T cell epitopes in ZIKV-unexposed, DENV-seropositive donors. We identified 93 epitopes across the ZIKV proteome, and we observed patterns of immunodominance that were dependent on antigen size and sequence identity to DENV. We confirmed the immunogenicity of these epitopes through a computational HLA binding analysis, and we showed that cross-reactive T cells specifically recognize ZIKV peptides homologous to DENV sequences. We also found that these CD4 responses were derived from the memory T cell compartment. These data have implications for understanding the dynamics of flavivirus-specific T cell immunity in areas of endemicity.IMPORTANCE Multiple flaviviruses, including Zika virus (ZIKV) and the four serotypes of dengue virus (DENV), are prevalent in the same large tropical and equatorial areas, which are inhabited by hundreds of millions of people. The interplay of DENV and ZIKV infection is especially relevant, as these two viruses are endemic in largely overlapping regions, have significant sequence similarity, and share the same arthropod vector. Here, we define the targets of preexisting immunity to ZIKV in unexposed subjects in areas where dengue is endemic. We demonstrate that preexisting immunity to DENV could shape ZIKV-specific responses, and DENV-ZIKV cross-reactive T cell populations can be expanded by stimulation with ZIKV peptides. The issue of potential ZIKV and DENV cross-reactivity is of relevance for understanding patterns of natural immunity, as well as for the development of diagnostic tests and vaccines.

Transcriptional signatures of Zika virus infection in astrocytes.

Astrocytes are an early and important target of Zika virus (ZIKV) infection in the developing brain, but the impacts of infection on astrocyte function remain controversial. Given that nonhuman primate (NHP) models of ZIKV infection replicate aspects of neurologic disease seen in human infections, we cultured primary astrocytes from the brain tissue of infant rhesus macaques and then infected the cells with Asian or African lineage ZIKV to identify transcriptional patterns associated with infection in these cells. The African lineage virus appeared to have greater infectivity and promote stronger antiviral signaling, but infection by either strain ultimately produced typical virus response patterns. Both viruses induced hypoxic stress, but the Asian lineage strain additionally had an effect on metabolic and lipid biosynthesis pathways. Together, these findings describe an NHP astrocyte model that may be used to assess transcriptional signatures following ZIKV infection.

Maintenance of AP-2-Dependent Functional Activities of Nef Restricts Pathways of Immune Escape from CD8 T Lymphocyte Responses.

Nef-specific CD8+ T lymphocytes (CD8TL) are linked to extraordinary control of primate lentiviral replication, but the mechanisms underlying their efficacy remain largely unknown. The immunodominant, Mamu-B*017:01+-restricted Nef195-203MW9 epitope in SIVmac239 partially overlaps a sorting motif important for interactions with host AP-2 proteins and, hence, downmodulation of several host proteins, including Tetherin (CD317/BST-2), CD28, CD4, SERINC3, and SERINC5. We reasoned that CD8TL-driven evolution in this epitope might compromise Nef's ability to modulate these important molecules. Here, we used deep sequencing of SIV from nine B*017:01+ macaques throughout infection with SIVmac239 to characterize the patterns of viral escape in this epitope and then assayed the impacts of these variants on Nef-mediated modulation of multiple host molecules. Acute variation in multiple Nef195-203MW9 residues significantly compromised Nef's ability to downregulate surface Tetherin, CD4, and CD28 and reduced its ability to prevent SERINC5-mediated reduction in viral infectivity but did not impact downregulation of CD3 or major histocompatibility complex class I, suggesting the selective disruption of immunomodulatory pathways involving Nef AP-2 interactions. Together, our data illuminate a pattern of viral escape dictated by a selective balance to maintain AP-2-mediated downregulation while evading epitope-specific CD8TL responses. These data could shed light on mechanisms of both CD8TL-driven viral control generally and on Mamu-B*017:01-mediated viral control specifically.IMPORTANCE A rare subset of humans infected with HIV-1 and macaques infected with SIV can control the virus without aid of antiviral medications. A common feature of these individuals is the ability to mount unusually effective CD8 T lymphocyte responses against the virus. One of the most formidable aspects of HIV is its ability to evolve to evade immune responses, particularly CD8 T lymphocytes. We show that macaques that target a specific peptide in the SIV Nef protein are capable of better control of the virus and that, as the virus evolves to escape this response, it does so at a cost to specific functions performed by the Nef protein. Our results help show how the virus can be controlled by an immune response, which could help in designing effective vaccines.

Diversity Plans and Postmarketing Studies: First Impressions of Anticipated Diversity Requirements in the United States.

INTRODUCTION: Recent Food and Drug Administration (FDA) draft guidelines are intended to improve representation and formalize the assessment of race and ethnicity in drug development, but how regulators and industry stakeholders plan to implement and enforce new requirements is still being determined. MATERIALS AND METHODS: Here, a 10-question survey was developed to assess the experiences of industry stakeholders in developing diversity plans. These survey results informed an analysis of postmarketing studies to understand how diversity requirements have been enforced to date. RESULTS AND DISCUSSION: Among 13 survey responders, experience submitting and receiving feedback on diversity plans was limited. A variety of challenges have been associated with developing these plans, including questions regarding regulatory guidance. Sponsors have utilized several data sources, including real-world datasets, to define enrollment goals. Diversity-related postmarketing studies most commonly related to oncologic diseases, and endpoints frequently related to efficacy. Most marketing applications associated with diversity-related postmarketing studies received Orphan drug designation (ODD) and Accelerated Approval. CONCLUSIONS: These results show that industry experience with diversity plans remains limited in the absence of finalized regulatory guidance. Sponsors are beginning to develop strategies for submitting diversity plans, which include identifying key functions and data sources to support enrollment goals, although definitive conclusions were difficult to draw from the small responder pool. In the postmarketing setting, studies are already underway to improve the understanding of racial and ethnic differences in responses to approved drugs. Development programs relating to oncology, which has historically suffered from a lack of diverse representation, have been a primary focus of such studies thus far.

In vivo delivery of engineered synthetic DNA-encoded SARS-CoV-2 monoclonal antibodies for pre-exposure prophylaxis in non-human primates.

COVID-19 remains a major public health concern. Monoclonal antibodies have received emergency use authorization (EUA) for pre-exposure prophylaxis against COVID-19 among high-risk groups for treatment of mild to moderate COVID-19. In addition to recombinant biologics, engineered synthetic DNA-encoded antibodies (DMAb) are an important strategy for direct in vivo delivery of protective mAb. A DMAb cocktail was synthetically engineered to encode the immunoglobulin heavy and light chains of two different two different Fc-engineered anti-SARS-CoV-2 antibodies. The DMAbs were designed to enhance in vivo expression and delivered intramuscularly to cynomolgus and rhesus macaques with a modified in vivo delivery regimen. Serum levels were detected in macaques, along with specific binding to SARS-CoV-2 spike receptor binding domain protein and neutralization of multiple SARS-CoV-2 variants of concern in pseudovirus and authentic live virus assays. Prophylactic administration was protective in rhesus macaques against signs of SARS-CoV-2 (USA-WA1/2020) associated disease in the lungs. Overall, the data support further study of DNA-encoded antibodies as an additional delivery mode for prevention of COVID-19 severe disease. These data have implications for human translation of gene-encoded mAbs for emerging infectious diseases and low dose mAb delivery against COVID-19.

Imaging of surface microdomains on individual extracellular vesicles in 3-D.

Extracellular vesicles (EVs) are secreted from all cell types and are intimately involved in tissue homeostasis. They are being explored as vaccine and gene therapy platforms, as well as potential biomarkers. As their size is below the diffraction limit of light microscopy, direct visualizations have been daunting and single-particle studies under physiological conditions have been hampered. Here, direct stochastic optical reconstruction microscopy (dSTORM) was employed to visualize EVs in three-dimensions and to localize molecule clusters such as the tetraspanins CD81 and CD9 on the surface of individual EVs. These studies demonstrate the existence of membrane microdomains on EVs. These were confirmed by Cryo-EM. Individual particle visualization provided insights into the heterogeneity, structure, and complexity of EVs not previously appreciated.

Zika virus persistence in the male macaque reproductive tract.

Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in that it is also vertically and sexually transmitted by humans. The male reproductive tract is thought to be a ZIKV reservoir; however, the reported magnitude and duration of viral persistence in male genital tissues vary widely in humans and non-human primate models. ZIKV tissue and cellular tropism and potential effects on male fertility also remain unclear. The objective of this study was to resolve these questions by analyzing archived genital tissues from 51 ZIKV-inoculated male macaques and correlating data on plasma viral kinetics, tissue tropism, and ZIKV-induced pathological changes in the reproductive tract. We hypothesized that ZIKV would persist in the male macaque genital tract for longer than there was detectable viremia, where it would localize to germ and epithelial cells and associate with lesions. We detected ZIKV RNA and infectious virus in testis, epididymis, seminal vesicle, and prostate gland. In contrast to prepubertal males, sexually mature macaques were significantly more likely to harbor persistent ZIKV RNA or infectious virus somewhere in the genital tract, with detection as late as 60 days post-inoculation. ZIKV RNA localized primarily to testicular stem cells/sperm precursors and epithelial cells, including Sertoli cells, epididymal duct epithelium, and glandular epithelia of the seminal vesicle and prostate gland. ZIKV infection was associated with microscopic evidence of inflammation in the epididymis and prostate gland of sexually mature males, pathologies that were absent in uninfected controls, which could have significant effects on male fertility. The findings from this study increase our understanding of persistent ZIKV infection which can inform risk of sexual transmission during assisted reproductive therapies as well as potential impacts on male fertility.

Prime-boost vaccination regimens with INO-4800 and INO-4802 augment and broaden immune responses against SARS-CoV-2 in nonhuman primates.

The enhanced transmissibility and immune evasion associated with emerging SARS-CoV-2 variants demands the development of next-generation vaccines capable of inducing superior protection amid a shifting pandemic landscape. Since a portion of the global population harbors some level of immunity from vaccines based on the original Wuhan-Hu-1 SARS-CoV-2 sequence or natural infection, an important question going forward is whether this immunity can be boosted by next-generation vaccines that target emerging variants while simultaneously maintaining long-term protection against existing strains. Here, we evaluated the immunogenicity of INO-4800, our synthetic DNA vaccine candidate for COVID-19 currently in clinical evaluation, and INO-4802, a next-generation DNA vaccine designed to broadly target emerging SARS-CoV-2 variants, as booster vaccines in nonhuman primates. Rhesus macaques primed over one year prior with the first-generation INO-4800 vaccine were boosted with either INO-4800 or INO-4802 in homologous or heterologous prime-boost regimens. Both boosting schedules led to an expansion of T cells and antibody responses which were characterized by improved neutralizing and ACE2 blocking activity across wild-type SARS-CoV-2 as well as multiple variants of concern. These data illustrate the durability of immunity following vaccination with INO-4800 and additionally support the use of either INO-4800 or INO-4802 in prime-boost regimens.

Zika virus infection during pregnancy protects against secondary infection in the absence of CD8+ cells.

While T cell immunity is an important component of the immune response to Zika virus (ZIKV) infection generally, the efficacy of these responses during pregnancy remains unknown. Here, we tested the capacity of CD8 lymphocytes to protect from secondary challenge in four macaques, two of which were depleted of CD8+ cells prior to rechallenge with a heterologous ZIKV isolate. The initial challenge during pregnancy produced transcriptional signatures suggesting complex patterns of immune modulation as well as neutralizing antibodies that persisted until rechallenge, which all animals efficiently controlled, demonstrating that the primary infection conferred adequate protection. The secondary challenge promoted activation of innate and adaptive immune cells, possibly suggesting a brief period of infection prior to clearance. These data confirm that ZIKV infection during pregnancy induces sufficient immunity to protect from a secondary challenge and suggest that this protection is not dependent on CD8 T cells.

Tetherin downmodulation by SIVmac Nef lost with the H196Q escape variant is restored by an upstream variant.

The H196 residue in SIVmac239 Nef is conserved across the majority of HIV and SIV isolates, lies immediately adjacent to the AP-2 (adaptor protein 2) binding di-leucine domain (ExxxLM195), and is critical for several described AP-2 dependent Nef functions, including the downregulation of tetherin (BST-2/CD317), CD4, and others. Surprisingly, many stocks of the closely related SIVmac251 swarm virus harbor a nef allele encoding a Q196. In SIVmac239, this variant is associated with loss of multiple AP-2 dependent functions. Publicly available sequences for SIVmac251 stocks were mined for variants linked to Q196 that might compensate for functional defects associated with this residue. Variants were engineered into the SIVmac239 backbone and in Nef expression plasmids and flow cytometry was used to examine surface tetherin expression in primary CD4 T cells and surface CD4 expression in SupT1 cells engineered to express rhesus CD4. We found that SIVmac251 stocks that encode a Q196 residue in Nef uniformly also encode an upstream R191 residue. We show that R191 restores the ability of Nef to downregulate tetherin in the presence of Q196 and has a similar but less pronounced impact on CD4 expression. However, a published report showed Q196 commonly evolves to H196 in vivo, suggesting a fitness cost. R191 may represent compensatory evolution to restore the ability to downregulate tetherin lost in viruses harboring Q196.

A Zika virus primary isolate induces neuroinflammation, compromises the blood-brain barrier and upregulates CXCL12 in adult macaques.

Zika virus (ZIKV) is a flavivirus that can cause neuropathogenesis in adults and fetal neurologic malformation following the infection of pregnant women. We used a nonhuman primate model, the Indian-origin Rhesus macaque (IRM), to gain insight into virus-associated hallmarks of ZIKV-induced adult neuropathology. We find that the virus causes prevalent acute and chronic neuroinflammation and chronic disruption of the blood-brain barrier (BBB) in adult animals. ZIKV infection resulted in specific short- and long-term augmented expression of the chemokine CXCL12 in the central nervous system (CNS)of adult IRMs. Moreover, CXCL12 expression persists long after the initial viral infection is apparently cleared. CXCL12 plays a key role both in regulating lymphocyte trafficking through the BBB to the CNS and in mediating repair of damaged neural tissue including remyelination. Understanding how CXCL12 expression is controlled will likely be of central importance in the definition of ZIKV-associated neuropathology in adults.

Immune outcomes of Zika virus infection in nonhuman primates.

Although the Zika virus (ZIKV) epidemic is subsiding, immune responses that are important for controlling acute infection have not been definitively characterized. Nonhuman primate (NHP) models were rapidly developed to understand the disease and to test vaccines, and these models have since provided an understanding of the immune responses that correlate with protection during natural infection and vaccination. Here, we infected a small group of male rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques with a minimally passaged Brazilian ZIKV isolate and used multicolor flow cytometry and transcriptional profiling to describe early immune patterns following infection. We found evidence of strong innate antiviral responses together with induction of neutralizing antibodies and T cell responses. We also assessed the relative importance of CD8 T cells in controlling infection by carrying out CD8 T cell depletion in an additional two animals of each species. CD8 depletion appeared to dysregulate early antiviral responses and possibly increase viral persistence, but the absence of CD8 T cells ultimately did not impair control of the virus. Together, these data describe immunological trends in two NHP species during acute ZIKV infection, providing an account of early responses that may be important in controlling infection.

A positive role of c-Myc in regulating androgen receptor and its splice variants in prostate cancer.

Increased expression of the full-length androgen receptor (AR-FL) and AR splice variants (AR-Vs) drives the progression of castration-resistant prostate cancer (CRPC). The levels of AR-FL and AR-V transcripts are often tightly correlated in individual CRPC samples, yet our understanding of how their expression is co-regulated is limited. Here, we report a role of c-Myc in accounting for coordinated AR-FL and AR-V expression. Analysis of gene-expression data from 159 metastatic CRPC samples and 2142 primary prostate tumors showed that the level of c-Myc is positively correlated with that of individual AR isoforms. A striking positive correlation also exists between the activity of the c-Myc pathway and the level of individual AR isoforms, between the level of c-Myc and the activity of the AR pathway, and between the activities of the two pathways. Moreover, the c-Myc signature is highly enriched in tumors expressing high levels of AR, as is the AR signature in c-Myc-high-expressing tumors. Using shRNA knockdown, we confirmed c-Myc regulation of expression and activity of AR-FL and AR-Vs in cell models and a patient-derived xenograft model. Mechanistically, c-Myc promotes the transcription of the AR gene and enhances the stability of the AR-FL and AR-V proteins without altering AR RNA splicing. Importantly, inhibiting c-Myc sensitizes enzalutamide-resistant cells to growth inhibition by enzalutamide. Overall, this study highlights a critical role of c-Myc in regulating the coordinated expression of AR-FL and AR-Vs that is commonly observed in CRPC and suggests the utility of targeting c-Myc as an adjuvant to AR-directed therapy.

Postnatal Zika virus infection of nonhuman primate infants born to mothers infected with homologous Brazilian Zika virus.

Recent data in a nonhuman primate model showed that infants postnatally infected with Zika virus (ZIKV) were acutely susceptible to high viremia and neurological damage, suggesting the window of vulnerability extends beyond gestation. In this pilot study, we addressed the susceptibility of two infant rhesus macaques born healthy to dams infected with Zika virus during pregnancy. Passively acquired neutralizing antibody titers dropped below detection limits between 2 and 3 months of age, while binding antibodies remained detectable until viral infection at 5 months. Acute serum viremia was comparatively lower than adults infected with the same Brazilian isolate of ZIKV (n = 11 pregnant females, 4 males, and 4 non-pregnant females). Virus was never detected in cerebrospinal fluid nor in neural tissues at necropsy two weeks after infection. However, viral RNA was detected in lymph nodes, confirming some tissue dissemination. Though protection was not absolute and our study lacks an important comparison with postnatally infected infants born to naïve dams, our data suggest infants born healthy to infected mothers may harbor a modest but important level of protection from postnatally acquired ZIKV for several months after birth, an encouraging result given the potentially severe infection outcomes of this population.

Nef Secretion into Extracellular Vesicles or Exosomes Is Conserved across Human and Simian Immunodeficiency Viruses.

Extracellular vesicles (EVs) or exosomes have been implicated in the pathophysiology of infections and cancer. The negative regulatory factor (Nef) encoded by simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) plays a critical role in the progression to AIDS and impairs endosomal trafficking. Whether HIV-1 Nef can be loaded into EVs has been the subject of controversy, and nothing is known about the connection between SIV Nef and EVs. We find that both SIV and HIV-1 Nef proteins are present in affinity-purified EVs derived from cultured cells, as well as in EVs from SIV-infected macaques. Nef-positive EVs were functional, i.e., capable of membrane fusion and depositing their content into recipient cells. The EVs were able to transfer Nef into recipient cells. This suggests that Nef readily enters the exosome biogenesis pathway, whereas HIV virions are assembled at the plasma membrane. It suggests a novel mechanism by which lentiviruses can influence uninfected and uninfectable, i.e., CD4-negative, cells.IMPORTANCE Extracellular vesicles (EVs) transfer biologically active materials from one cell to another, either within the adjacent microenvironment or further removed. EVs also package viral RNAs, microRNAs, and proteins, which contributes to the pathophysiology of infection. In this report, we show that both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) incorporate the virus-encoded Nef protein into EVs, including EVs circulating in the blood of SIV-infected macaques and that this presents a novel mechanism of Nef transfer to naive and even otherwise non-infectable cells. Nef is dispensable for viral replication but essential for AIDS progression in vivo Demonstrating that Nef incorporation into EVs is conserved across species implicates EVs as novel mediators of the pathophysiology of HIV. It could help explain the biological effects that HIV has on CD4-negative cells and EVs could become biomarkers of disease progression.

Effects of exosome on the activation of CD4+ T cells in rhesus macaques: a potential application for HIV latency reactivation.

Exosomes are small extracellular vesicles (EVs), released by a wide variety of cell types, carry donor origin-proteins, cytokines, and nucleic acids, transport these cargos to adjacent or distant specific recipient cells, and thereby regulate gene expression and activation of target cells. In this study, we isolated and identified exosomes in rhesus macaques, and investigated their effects on cell tropism and activation, especially their potential to reactivate HIV latency. The results indicated that plasma-derived exosomes preferentially fuse to TCR-activated T cells and autologous parent cells. Importantly, the uptake of exosomes, derived from IL-2 stimulated CD4+ T cells, effectively promoted reactivation of resting CD4+ T-cell, as indicated by an increased viral transcription rate in these cells. These findings provide premise for the potential application of exosome in the reactivation of HIV latency, in combination its use as functional delivery vehicles with antiretroviral therapy (ART).