Route of self-amplifying mRNA vaccination modulates the establishment of pulmonary resident memory CD8 and CD4 T cells.

Respiratory tract resident memory T cells (TRM), typically generated by local vaccination or infection, can accelerate control of pulmonary infections that evade neutralizing antibody. It is unknown whether mRNA vaccination establishes respiratory TRM. We generated a self-amplifying mRNA vaccine encoding the influenza A virus nucleoprotein that is encapsulated in modified dendron-based nanoparticles. Here, we report how routes of immunization in mice, including contralateral versus ipsilateral intramuscular boosts, or intravenous and intranasal routes, influenced influenza-specific cell-mediated and humoral immunity. Parabiotic surgeries revealed that intramuscular immunization was sufficient to establish CD8 TRM in the lung and draining lymph nodes. Contralateral, compared with ipsilateral, intramuscular boosting broadened the distribution of lymph node TRM and T follicular helper cells but slightly diminished resulting levels of serum antibody. Intranasal mRNA delivery established modest circulating CD8 and CD4 T cell memory but augmented distribution to the respiratory mucosa. Combining intramuscular immunizations with an intranasal mRNA boost achieved high levels of both circulating T cell memory and lung TRM. Thus, routes of mRNA vaccination influence humoral and cell-mediated immunity, and intramuscular prime-boosting establishes lung TRM that can be further expanded by an additional intranasal immunization.

Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation.

Therapeutic messenger RNA vaccines enable delivery of whole antigens, which can be advantageous over peptide vaccines. However, optimal efficacy requires both intracellular delivery, to allow antigen translation, and appropriate immune activation. Here, we developed a combinatorial library of ionizable lipid-like materials to identify mRNA delivery vehicles that facilitate mRNA delivery in vivo and provide potent and specific immune activation. Using a three-dimensional multi-component reaction system, we synthesized and evaluated the vaccine potential of over 1,000 lipid formulations. The top candidate formulations induced a robust immune response, and were able to inhibit tumor growth and prolong survival in melanoma and human papillomavirus E7 in vivo tumor models. The top-performing lipids share a common structure: an unsaturated lipid tail, a dihydroimidazole linker and cyclic amine head groups. These formulations induce antigen-presenting cell maturation via the intracellular stimulator of interferon genes (STING) pathway, rather than through Toll-like receptors, and result in limited systemic cytokine expression and enhanced anti-tumor efficacy.

Targeted antigen delivery by an anti-class II MHC VHH elicits focused αMUC1(Tn) immunity.

Unusual patterns of glycosylation on the surface of transformed cells contribute to immune modulation and metastasis of malignant tumors. Active immunization against them requires effective antigen presentation, which is complicated by a lack of access to tumor-specific posttranslational modifications through standard genetic approaches and by the low efficiency of passive antigen sampling. We found that antigen targeted to antigen presenting cells via class II MHC products can elicit a robust immune response against MUC1(Tn) bearing a defined tumor-associated glycoform, Tn. The two-component vaccine construct was prepared by sortase-mediated protein ligation of a synthetic MUC1(Tn) fragment to a class II MHC-binding single-domain antibody fragment (VHH7) as targeting moiety. We show that VHH7 targets antigen presenting cells in vivo, and when conjugated to MUC1(Tn) can elicit a strong αMUC1(Tn) immune response in mice. The resulting sera preferentially recognized the MUC1 epitope with the tumor-associated carbohydrate antigen Tn and were capable of killing cancer cells in a complement-mediated cytotoxicity assay. Immunoglobulin isotype analysis and cytokine release assays suggested a favorable Th1 response. A single boost 12 months after primary immunization triggered a recall response of the same quality, suggesting that long-term αMUC1(Tn) memory had been achieved.

An RNA nanoparticle vaccine against Zika virus elicits antibody and CD8+ T cell responses in a mouse model.

The Zika virus (ZIKV) outbreak in the Americas and South Pacific poses a significant burden on human health because of ZIKV's neurotropic effects in the course of fetal development. Vaccine candidates against ZIKV are coming online, but immunological tools to study anti-ZIKV responses in preclinical models, particularly T cell responses, remain sparse. We deployed RNA nanoparticle technology to create a vaccine candidate that elicited ZIKV E protein-specific IgG responses in C57BL/6 mice as assayed by ELISA. Using this tool, we identified a unique H-2Db-restricted epitope to which there was a CD8+ T cell response in mice immunized with our modified dendrimer-based RNA nanoparticle vaccine. These results demonstrate that this approach can be used to evaluate new candidate antigens and identify immune correlates without the use of live virus.

Dendrimer-RNA nanoparticles generate protective immunity against lethal Ebola, H1N1 influenza, and Toxoplasma gondii challenges with a single dose.

Vaccines have had broad medical impact, but existing vaccine technologies and production methods are limited in their ability to respond rapidly to evolving and emerging pathogens, or sudden outbreaks. Here, we develop a rapid-response, fully synthetic, single-dose, adjuvant-free dendrimer nanoparticle vaccine platform wherein antigens are encoded by encapsulated mRNA replicons. To our knowledge, this system is the first capable of generating protective immunity against a broad spectrum of lethal pathogen challenges, including H1N1 influenza, Toxoplasma gondii, and Ebola virus. The vaccine can be formed with multiple antigen-expressing replicons, and is capable of eliciting both CD8(+) T-cell and antibody responses. The ability to generate viable, contaminant-free vaccines within days, to single or multiple antigens, may have broad utility for a range of diseases.

Extensive post-translational modification of active and inactivated forms of endogenous p53.

The p53 tumor suppressor protein accumulates to very high concentrations in normal human fibroblasts infected by adenovirus type 5 mutants that cannot direct assembly of the viral E1B 55-kDa protein-containing E3 ubiquitin ligase that targets p53 for degradation. Despite high concentrations of nuclear p53, the p53 transcriptional program is not induced in these infected cells. We exploited this system to examine select post-translational modifications (PTMs) present on a transcriptionally inert population of endogenous human p53, as well as on p53 activated in response to etoposide treatment of normal human fibroblasts. These forms of p53 were purified from whole cell lysates by means of immunoaffinity chromatography and SDS-PAGE, and peptides derived from them were subjected to nano-ultra-high-performance LC-MS and MS/MS analyses on a high-resolution accurate-mass MS platform (data available via ProteomeXchange, PXD000464). We identified an unexpectedly large number of PTMs, comprising phosphorylation of Ser and Thr residues, methylation of Arg residues, and acetylation, ubiquitinylation, and methylation of Lys residues-for example, some 150 previously undescribed modifications of p53 isolated from infected cells. These modifications were distributed across all functional domains of both forms of the endogenous human p53 protein, as well as those of an orthologous population of p53 isolated from COS-1 cells. Despite the differences in activity, including greater in vitro sequence-specific DNA binding activity exhibited by p53 isolated from etoposide-treated cells, few differences were observed in the location, nature, or relative frequencies of PTMs on the two populations of human p53. Indeed, the wealth of PTMs that we have identified is consistent with a far greater degree of complex, combinatorial regulation of p53 by PTM than previously anticipated.

The p53 protein does not facilitate adenovirus type 5 replication in normal human cells.

Although several adenovirus type 5 (Ad5) proteins prevent deleterious consequences of activation of p53, it has been reported that viral replication proceeds more efficiently when human tumor cells produce wild-type compared to mutant p53. We have now exploited RNA interference and lentiviral vectors to achieve essentially complete knockdown of p53 in normal human cells: no effects on the kinetics or efficiency of viral gene expression or production of infectious particles were observed.

The repression domain of the E1B 55-kilodalton protein participates in countering interferon-induced inhibition of adenovirus replication.

To begin to investigate the mechanism by which the human adenovirus type 5 E1B 55-kDa protein protects against the antiviral effects of type 1 interferon (IFN) (J. S. Chahal, J. Qi, and S. J. Flint, PLoS Pathog. 8:e1002853, 2012 [doi:10.1371/journal.ppat.1002853]), we examined the effects of precise amino acid substitution in this protein on resistance of viral replication to the cytokine. Only substitution of residues 443 to 448 of E1B for alanine (E1B Sub19) specifically impaired production of progeny virus and resulted in a large defect in viral DNA synthesis in IFN-treated normal human fibroblasts. Untreated or IFN-treated cells infected by this mutant virus (AdEasyE1Sub19) contained much higher steady-state concentrations of IFN-inducible GBP1 and IFIT2 mRNAs than did wild-type-infected cells and of the corresponding newly transcribed pre-mRNAs, isolated exploiting 5'-ethynyluridine labeling and click chemistry. These results indicated that the mutations created by substitution of residues 443 to 448 for alanine (Sub19) impair repression of transcription of IFN-inducible genes, by the E1B, 55-kDa protein, consistent with their location in a segment required for repression of p53-dependent transcription. However, when synthesized alone, the E1B 55-kDa protein inhibited expression of the p53-regulated genes BAX and MDM2 but had no impact whatsoever on induction of IFIT2 and GBP1 expression by IFN. These observations correlate repression of transcription of IFN-inducible genes by the E1B 55-kDa protein with protection against inhibition of viral genome replication and indicate that the E1B 55-kDa protein is not sufficient to establish such transcriptional repression.

Reduced infectivity of adenovirus type 5 particles and degradation of entering viral genomes associated with incomplete processing of the preterminal protein.

To investigate further the contribution of the adenovirus type 5 (Ad5) E1B 55-kDa protein to genome replication, viral DNA accumulation was examined in primary human fibroblasts and epithelial cells infected with Ad5 or the E1B 55-kDa-null mutant Hr6. Unexpectedly, all cell types were observed to contain a significantly higher concentration of entering Hr6 than of Ad5 DNA, as did an infectious unit of Hr6. However, the great majority of the Hr6 genomes were degraded soon after entry. As this unusual phenotype cannot be ascribed to the Hr6 E1B frameshift mutation (J. S. Chahal and S. J. Flint, J. Virol. 86:3064-3072, 2012), the sequences of the Ad5 and Hr6 genomes were compared by using high-throughput sequencing. Seven previously unrecognized mutations were identified in the Hr6 genome, two of which result in substitutions in virion proteins, G315V in the preterminal protein (preTP) and A406V in fiber protein IV. Previous observations and the visualization by immunofluorescence of greater numbers of viral genomes entering the cytosol of Hr6-infected cells than of Ad5-infected cells indicated that the fiber mutation could not be responsible for the low-infectivity phenotype of Hr6. However, comparison of the forms of terminal protein present in purified virus particles indicated that the production of mature terminal protein from a processing intermediate is impaired in Hr6 particles. We therefore propose that complete processing of preTP within virus particles is necessary for the ability of viral genomes to become localized at appropriate sites and persist in infected cells.

The human adenovirus type 5 E1B 55 kDa protein obstructs inhibition of viral replication by type I interferon in normal human cells.

Vectors derived from human adenovirus type 5, which typically lack the E1A and E1B genes, induce robust innate immune responses that limit their therapeutic efficacy. We reported previously that the E1B 55 kDa protein inhibits expression of a set of cellular genes that is highly enriched for those associated with anti-viral defense and immune responses, and includes many interferon-sensitive genes. The sensitivity of replication of E1B 55 kDa null-mutants to exogenous interferon (IFN) was therefore examined in normal human fibroblasts and respiratory epithelial cells. Yields of the mutants were reduced at least 500-fold, compared to only 5-fold, for wild-type (WT) virus replication. To investigate the mechanistic basis of such inhibition, the accumulation of viral early proteins and genomes was compared by immunoblotting and qPCR, respectively, in WT- and mutant-infected cells in the absence or presence of exogenous IFN. Both the concentration of viral genomes detected during the late phase and the numbers of viral replication centers formed were strongly reduced in IFN-treated cells in the absence of the E1B protein, despite production of similar quantities of viral replication proteins. These defects could not be attributed to degradation of entering viral genomes, induction of apoptosis, or failure to reorganize components of PML nuclear bodies. Nor was assembly of the E1B- and E4 Orf6 protein- E3 ubiquitin ligase required to prevent inhibition of viral replication by IFN. However, by using RT-PCR, the E1B 55 kDa protein was demonstrated to be a potent repressor of expression of IFN-inducible genes in IFN-treated cells. We propose that a primary function of the previously described transcriptional repression activity of the E1B 55 kDa protein is to block expression of IFN- inducible genes, and hence to facilitate formation of viral replication centers and genome replication.

Timely synthesis of the adenovirus type 5 E1B 55-kilodalton protein is required for efficient genome replication in normal human cells.

Previous studies have indicated that the adenovirus type 5 E1B 55-kDa protein facilitates viral DNA synthesis in normal human foreskin fibroblasts (HFFs) but not in primary epithelial cells. To investigate this apparent difference further, viral DNA accumulation was examined in primary human fibroblasts and epithelial cells infected by the mutant AdEasyE1Δ2347, which carries the Hr6 frameshift mutation that prevents production of the E1B 55-kDa protein, in an E1-containing derivative of AdEasy. Impaired viral DNA synthesis was observed in normal HFFs but not in normal human bronchial epithelial cells infected by this mutant. However, acceleration of progression through the early phase, which is significantly slower in HFFs than in epithelial cells, eliminated the dependence of efficient viral DNA synthesis in HFFs on the E1B 55-kDa protein. These observations suggest that timely synthesis of the E1B 55-kDa protein protects normal cells against a host defense that inhibits adenoviral genome replication. One such defense is mediated by the Mre11-Rad50-Nbs1 complex. Nevertheless, examination of the localization of Mre11 and viral proteins by immunofluorescence suggested that this complex is inactivated similarly in AdEasyE1Δ2347 mutant-infected and AdEasyE1-infected HFFs.

Specific DNA-binding by apicomplexan AP2 transcription factors.

Malaria remains one of the most prevalent infectious diseases worldwide, affecting more than half a billion people annually. Despite many years of research, the mechanisms underlying transcriptional regulation in the malaria-causing Plasmodium spp., and in Apicomplexan parasites generally, remain poorly understood. In Plasmodium, few regulatory elements sufficient to drive gene expression have been characterized, and their cognate DNA-binding proteins remain unknown. This study characterizes the DNA-binding specificities of two members of the recently identified Apicomplexan AP2 (ApiAP2) family of putative transcriptional regulators from Plasmodium falciparum. The ApiAP2 proteins contain AP2 domains homologous to the well characterized plant AP2 family of transcriptional regulators, which play key roles in development and environmental stress response pathways. We assayed ApiAP2 protein-DNA interactions using protein-binding microarrays and combined these results with computational predictions of coexpressed target genes to couple these putative trans factors to corresponding cis-regulatory motifs in Plasmodium. Furthermore, we show that protein-DNA sequence specificity is conserved in orthologous proteins between phylogenetically distant Apicomplexan species. The identification of the DNA-binding specificities for ApiAP2 proteins lays the foundation for the exploration of their role as transcriptional regulators during all stages of parasite development. Because of their origin in the plant lineage, ApiAP2 proteins have no homologues in the human host and may prove to be ideal antimalarial targets.