Single dose of chimeric dengue-2/Zika vaccine candidate protects mice and non-human primates against Zika virus.

The development of a safe and effective Zika virus (ZIKV) vaccine has become a global health priority since the widespread epidemic in 2015-2016. Based on previous experience in using the well-characterized and clinically proven dengue virus serotype-2 (DENV-2) PDK-53 vaccine backbone for live-attenuated chimeric flavivirus vaccine development, we developed chimeric DENV-2/ZIKV vaccine candidates optimized for growth and genetic stability in Vero cells. These vaccine candidates retain all previously characterized attenuation phenotypes of the PDK-53 vaccine virus, including attenuation of neurovirulence for 1-day-old CD-1 mice, absence of virulence in interferon receptor-deficient mice, and lack of transmissibility in the main mosquito vectors. A single DENV-2/ZIKV dose provides protection against ZIKV challenge in mice and rhesus macaques. Overall, these data indicate that the ZIKV live-attenuated vaccine candidates are safe, immunogenic and effective at preventing ZIKV infection in multiple animal models, warranting continued development.

Purified Inactivated Zika Vaccine Candidates Afford Protection against Lethal Challenge in Mice.

In response to the 2016 global public health emergency of international concern announced by the World Health Organization surrounding Zika virus (ZIKV) outbreaks, we developed a purified inactivated Zika virus vaccine (PIZV) candidate from ZIKV strain PRVABC59, isolated during the outbreak in 2015. The virus isolate was plaque purified, creating six sub-isolated virus stocks, two of which were selected to generate PIZV candidates for preclinical immunogenicity and efficacy evaluation in mice. The alum-adjuvanted PIZV candidates were highly immunogenic in both CD-1 and AG129 mice after a 2-dose immunization. Further, AG129 mice receiving 2 doses of PIZV formulated with alum were fully protected against lethal ZIKV challenge and mouse immune sera elicited by the PIZV candidates were capable of neutralizing ZIKVs of both African and Asian genetic lineages in vitro. Additionally, passive immunization of naïve mice with ZIKV-immune serum showed strong positive correlation between neutralizing ZIKV antibody (NAb) titers and protection against lethal challenge. This study supported advancement of the PIZV candidate toward clinical development.

Histone Chaperone Nap1 Is a Major Regulator of Histone H2A-H2B Dynamics at the Inducible GAL Locus.

Histone chaperones, like nucleosome assembly protein 1 (Nap1), play a critical role in the maintenance of chromatin architecture. Here, we use the GAL locus in Saccharomyces cerevisiae to investigate the influence of Nap1 on chromatin structure and histone dynamics during distinct transcriptional states. When the GAL locus is not expressed, cells lacking Nap1 show an accumulation of histone H2A-H2B but not histone H3-H4 at this locus. Excess H2A-H2B interacts with the linker DNA between nucleosomes, and the interaction is independent of the inherent DNA-binding affinity of H2A-H2B for these particular sequences as measured in vitro When the GAL locus is transcribed, excess H2A-H2B is reversed, and levels of all chromatin-bound histones are depleted in cells lacking Nap1. We developed an in vivo system to measure histone exchange at the GAL locus and observed considerable variability in the rate of exchange across the locus in wild-type cells. We recapitulate this variability with in vitro nucleosome reconstitutions, which suggests a contribution of DNA sequence to histone dynamics. We also find that Nap1 is required for transcription-dependent H2A-H2B exchange. Altogether, these results indicate that Nap1 is essential for maintaining proper chromatin composition and modulating the exchange of H2A-H2B in vivo.

Eviction of linker histone H1 by NAP-family histone chaperones enhances activated transcription.

BACKGROUND: In the Metazoan nucleus, core histones assemble the genomic DNA to form nucleosome arrays, which are further compacted into dense chromatin structures by the linker histone H1. The extraordinary density of chromatin creates an obstacle for accessing the genetic information. Regulation of chromatin dynamics is therefore critical to cellular homeostasis, and histone chaperones serve as prominent players in these processes. In the current study, we examined the role of specific histone chaperones in negotiating the inherently repressive chromatin structure during transcriptional activation. RESULTS: Using a model promoter, we demonstrate that the human nucleosome assembly protein family members hNap1 and SET/Taf1ß stimulate transcription in vitro during pre-initiation complex formation, prior to elongation. This stimulatory effect is dependent upon the presence of activators, p300, and Acetyl-CoA. We show that transcription from our chromatin template is strongly repressed by H1, and that both histone chaperones enhance RNA synthesis by overcoming H1-induced repression. Importantly, both hNap1 and SET/Taf1ß directly bind H1, and function to enhance transcription by evicting the linker histone from chromatin reconstituted with H1. In vivo studies demonstrate that SET/Taf1ß, but not hNap1, strongly stimulates activated transcription from the chromosomally-integrated model promoter, consistent with the observation that SET/Taf1ß is nuclear, whereas hNap1 is primarily cytoplasmic. Together, these observations indicate that SET/Taf1ß may serve as a critical regulator of H1 dynamics and gene activation in vivo. CONCLUSIONS: These studies uncover a novel function for SET that mechanistically couples transcriptional derepression with H1 dynamics. Furthermore, they underscore the significance of chaperone-dependent H1 displacement as an essential early step in the transition of a promoter from a dense chromatin state into one that is permissive to transcription factor binding and robust activation.

Molecular characterization of the Tax-containing HTLV-1 enhancer complex reveals a prominent role for CREB phosphorylation in Tax transactivation.

Transcriptional activation of human T-cell leukemia virus type 1 (HTLV-1) is mediated by the viral oncoprotein Tax, which utilizes cellular transcriptional machinery to perform this function. The viral promoter carries three cyclic AMP-response elements (CREs), which are recognized by the cellular transcription factor cAMP-response element-binding protein (CREB). Tax binds to GC-rich sequences that immediately flank the CREs. The coactivator CREB-binding protein (CBP)/p300 binds to this promoter-bound ternary complex, which promotes the initiation of HTLV-1 transcription. Protein kinase A phosphorylation of CREB at serine 133 facilitates transcription from cellular CREs by recruiting CBP/p300 via its KIX domain. However, it remains controversial whether CREB phosphorylation plays a role in Tax transactivation. In this study, we biochemically characterized the quaternary complex formed by Tax, CREB, KIX, and the viral CRE by examining the individual molecular interactions that contribute to Tax stabilization in the complex. Our data show KIX, Ser(133)-phosphorylated CREB, and vCRE DNA are all required for stable Tax incorporation into the complex in vitro. Consonant with a fundamental role for CREB phosphorylation in Tax recruitment to the complex, we found that CREB is highly phosphorylated in a panel of HTLV-1-infected human T-cell lines. Significantly, we show that Tax is directly responsible for promoting elevated levels of CREB phosphorylation. Together, these data support a model in which Tax promotes CREB phosphorylation in vivo to ensure availability for Tax transactivation. Because pCREB has been implicated in leukemogenesis, enhancement of CREB phosphorylation by the virus may play a role in the etiology of adult T-cell leukemia.

Tax recruitment of CBP/p300, via the KIX domain, reveals a potent requirement for acetyltransferase activity that is chromatin dependent and histone tail independent.

Robust transcription of human T-cell leukemia virus type 1 (HTLV-1) genome requires the viral transactivator Tax. Although Tax has been previously shown to interact with the KIX domain of CBP/p300 in vitro, the precise functional relevance of this interaction remains unclear. Using two distinct approaches to interrupt the physical interaction between Tax and KIX, we find that Tax transactivation from chromatin templates is strongly dependent on CBP/p300 recruitment via the KIX domain. Additionally, we find that the primary functional contribution of CBP/p300 to Tax transactivation resides in the intrinsic acetyltransferase activity of the coactivators. These studies unexpectedly uncover a specific requirement for CBP/p300 acetyltransferase activity on chromatin templates assembled with nucleosomes lacking their amino-terminal tails. Together, these data indicate that the KIX domain of CBP/p300 is essential for targeting the acetyltransferase activity of the coactivator to the Tax-CREB (Tax/CREB) complex. Significantly, these observations reveal the presence of one or more CBP/p300 acetyltransferase targets that function specifically on chromatin templates, are independent of the histone tails, and are critical to Tax transactivation.