High-density Microarrays Research Articles

8213 A Splicing Modulator Inhibits Proliferation and Steroid Production of H295R Adrenocortical Cancer Cells

Abstract Disclosure: T. Tanaka: None. K. Ohe: None. T. Yanase: None. S. Kodama: None. Adrenocortical carcinoma (ACC) is a rare cancer, and treatment options are limited. The anti-tumor drug CX-4945 (Silmitasertib) is an inhibitor of Casein kinase 2 (CK2). It also inhibits cdc2-like kinase 2 (CLK2). CLK2 phosphorylates Serine/arginine rich splicing factors (SRSFs)/SR proteins and enhances exonic splicing enhancer (ESE) recognition during the pre-RNA splicing process. Anti-tumor agents targeting RNA splicing have been developed recently, and CLK inhibitors have shown anti-tumor effects against blood cancers. In this study, we analyzed the impact of CX-4945 on NCI-H295R cells. CX-4945 inhibited cell proliferation of H295R (IC50 = 6.4 µM, 72hr), and cortisol and aldosterone secretion in vitro. siRNA-induced CK2 inhibition did not alter H295R proliferation and steroid secretion. CX-4945 (150 mg/kg/day oral, 28 days) significantly reduced tumor weight in mice inoculated with H295R subcutaneously on the back. High-density microarray analysis showed reduced Nuclear receptor 5A1 (NR5A1) expression levels, indicating a possible splicing variant. RT-PCR, using a primer set that amplified the coding region, detected short variants of NR5A that were multiple exon-skipped and were not induced by suppression of CK2. The results of western blotting using an antibody that recognizes phospho-epitope of SRSFs/SR proteins showed that the phosphorylation of SRSF4 was reduced in H295R treated with CX-4945. These results suggest that CX-4945 inhibits CLK2 and decreases phosphorylated SRSF4 and ESE recognition, resulting in NR5A1 multiple exon-skipping. The prognosis of ACC patients with high NR5A1 expression levels is poor, independent of stage. CX-4945 may induce NR5A1 multiple exon-skipping, reduce full-length functional NR5A1, and suppress ACC growth and steroidogenesis, as a possible adjuvant in tackling this devastating cancer. Presentation: 6/1/2024

8213 A Splicing Modulator Inhibits Proliferation And Steroid Production Of H295R Adrenocortical Cancer Cells

Abstract Disclosure: T. Tanaka: None. K. Ohe: None. T. Yanase: None. S. Kodama: None. Adrenocortical carcinoma (ACC) is a rare cancer, and treatment options are limited. The anti-tumor drug CX-4945 (Silmitasertib) is an inhibitor of Casein kinase 2 (CK2). It also inhibits cdc2-like kinase 2 (CLK2). CLK2 phosphorylates Serine/arginine rich splicing factors (SRSFs)/SR proteins and enhances exonic splicing enhancer (ESE) recognition during the pre-RNA splicing process. Anti-tumor agents targeting RNA splicing have been developed recently, and CLK inhibitors have shown anti-tumor effects against blood cancers. In this study, we analyzed the impact of CX-4945 on NCI-H295R cells. CX-4945 inhibited cell proliferation of H295R (IC50 = 6.4 µM, 72hr), and cortisol and aldosterone secretion in vitro. siRNA-induced CK2 inhibition did not alter H295R proliferation and steroid secretion. CX-4945 (150 mg/kg/day oral, 28 days) significantly reduced tumor weight in mice inoculated with H295R subcutaneously on the back. High-density microarray analysis showed reduced Nuclear receptor 5A1 (NR5A1) expression levels, indicating a possible splicing variant. RT-PCR, using a primer set that amplified the coding region, detected short variants of NR5A that were multiple exon-skipped and were not induced by suppression of CK2. The results of western blotting using an antibody that recognizes phospho-epitope of SRSFs/SR proteins showed that the phosphorylation of SRSF4 was reduced in H295R treated with CX-4945. These results suggest that CX-4945 inhibits CLK2 and decreases phosphorylated SRSF4 and ESE recognition, resulting in NR5A1 multiple exon-skipping. The prognosis of ACC patients with high NR5A1 expression levels is poor, independent of stage. CX-4945 may induce NR5A1 multiple exon-skipping, reduce full-length functional NR5A1, and suppress ACC growth and steroidogenesis, as a possible adjuvant in tackling this devastating cancer. Presentation: 6/1/2024

Enhancing testing efficacy of high-density SNP microarrays to distinguish pedigrees belonging to the same kinship class

Kinship testing, which involves genotyping genetic markers and comparing their profiles between individuals, holds significant applications in forensic science. However, the prevalent use of independent markers often lacks the discriminatory power to distinguish pedigrees belong to the same kinship class. While numerous studies have attempted to address this challenge through diverse approaches, the testing efficacy of high-density SNP microarrays in combination with the likelihood approach remains unclear.In this study, we further explored the utilization of linked autosomal SNPs derived from microarrays with the likelihood approach. Several SNP panels with differing numbers of loci were developed and putative pedigrees were constructed to evaluated to test their efficacy in distinguishing second-degree relationships, including grandparent-grandchild, half-siblings, and avuncular. Our findings indicate that the use of high-density SNP microarrays is theoretically feasible for discriminating second-degree relationships, with balanced classification rates ranging from 0.444 to 0.853.Moreover, to optimize the practical effectiveness of discriminating pedigrees belonging to the same kinship class, several other aspects such as adding additional SNPs or an additional relative and examining the effects of genotype errors and population selection were discussed. Our results revealed that the employment of denser marker sets with more accurate genotyping methods may be beneficial. Additionally, the inclusion of additional relatives and the selection of an appropriate reference population also appear to be crucial factors for enhancing the accuracy of kinship testing.In conclusion, our study provides insights into the potential of high-density SNPs in kinship testing and highlights the need for further optimization and examination into various factors that may contribute to enhancing testing efficacy.

Exploring the biological responses involved in the genetic resistance to Rhipicephalus microplus in Argentine Creole cattle.

The common cattle tick Rhipicephalus microplus causes severe limitations to livestock production. Bovine genetics could be a decisive component for the success or failure of control programs for ticks and diseases transmitted. The objective of this work was to detect chromosomal regions associated with host resistance to R. microplus through an associative mapping study using medium and high density microarrays in a population of Argentine Creole cattle. The phenotypic record of the number of ticks that completed their development on the host, after artificial infestations, was obtained during 2015 to 2020. Genomic DNA was extracted for genotyping from 192 animals using Affymetrix high (Axiom™ Bos 1) and medium density (ArBos1) microarrays. In an exploratory study, chromosomal regions containing putative quantitative trait loci (QTLs) were recognized on chromosomes 27, 11, 10, 9, 16, 13, 3, 19, 8 and 18, associated with the variation of R. microplus load. Gene ontology based on genes located on these regions revealed an enrichment of terms and pathways for the immune system, blood coagulation, tissue regeneration, endopeptidase activity and protein phosphorylation. The information obtained in this work constitutes a first report of QTLs for tick count in the Argentine Creole cattle, and contributes with the knowledge about the underlying process involved in tick resistance.

Role of the X Chromosome in Alzheimer Disease Genetics

The X chromosome has remained enigmatic in Alzheimer disease (AD), yet it makes up 5% of the genome and carries a high proportion of genes expressed in the brain, making it particularly appealing as a potential source of unexplored genetic variation in AD. To perform the first large-scale X chromosome-wide association study (XWAS) of AD. This was a meta-analysis of genetic association studies in case-control, family-based, population-based, and longitudinal AD-related cohorts from the US Alzheimer's Disease Genetics Consortium, the Alzheimer's Disease Sequencing Project, the UK Biobank, the Finnish health registry, and the US Million Veterans Program. Risk of AD was evaluated through case-control logistic regression analyses. Data were analyzed between January 2023 and March 2024. Genetic data available from high-density single-nucleotide variant microarrays and whole-genome sequencing and summary statistics for multitissue expression and protein quantitative trait loci available from published studies were included, enabling follow-up genetic colocalization analyses. A total of 1 629 863 eligible participants were selected from referred and volunteer samples, 477 596 of whom were excluded for analysis exclusion criteria. The number of participants who declined to participate in original studies was not available. Risk of AD, reported as odds ratios (ORs) with 95% CIs. Associations were considered at X chromosome-wide (P < 1 × 10-5) and genome-wide (P < 5 × 10-8) significance. Primary analyses are nonstratified, while secondary analyses evaluate sex-stratified effects. Analyses included 1 152 284 participants of non-Hispanic White, European ancestry (664 403 [57.7%] female and 487 881 [42.3%] male), including 138 558 individuals with AD. Six independent genetic loci passed X chromosome-wide significance, with 4 showing support for links between the genetic signal for AD and expression of nearby genes in brain and nonbrain tissues. One of these 4 loci passed conservative genome-wide significance, with its lead variant centered on an intron of SLC9A7 (OR, 1.03; 95% CI, 1.02-1.04) and colocalization analyses prioritizing both the SLC9A7 and nearby CHST7 genes. Of these 6 loci, 4 displayed evidence for escape from X chromosome inactivation with regard to AD risk. This large-scale XWAS of AD identified the novel SLC9A7 locus. SLC9A7 regulates pH homeostasis in Golgi secretory compartments and is anticipated to have downstream effects on amyloid β accumulation. Overall, this study advances our knowledge of AD genetics and may provide novel biological drug targets. The results further provide initial insights into elucidating the role of the X chromosome in sex-based differences in AD.

Inheritance and QTL mapping identified multi-effects loci for fatty acid related traits in peanut (Arachis hypogaea L.)

Peanut (Arachis hypogaea L.) is an important oil and edible protein crop. The fatty acid composition not only influences the quality of peanut oil but also impacts flavor, shelf life, and consumer health. Peanut oil comprises approximately 80% oleic acid (C18:1) and linoleic acid (C18:2), 10% palmitic acid (C16:0), and the remaining 10% includes stearic acid (C18:0), arachidic acid (C20:0), gadoleic acid (C20:1), behenic acid (C22:0), and lignoceric acid (C24:0). To unravel the genetic foundation of fatty acid content and delve into QTL localization, we utilized high-density SNP microarrays for genotyping the RIL population of ‘SunOleic 97R’ × ‘NC94022’. A genetic linkage map was constructed with 3141 SNP markers, covering a total genetic distance of 3051.81 cM. We identified 60 quantitative trait loci (QTLs) associated with fatty acids which distributed in 11 linkage group, with phenotypic variance explained (PVE) ranging from 1.37% to 44.92%. Notably, the QTL qFAT_A05.1 and qFAT_A08.1 are multiple-effect loci contributing to various fatty acid compositions. Moreover, we identified 15 haplotypes for the QTL qFAT_A05.1 and qFAT_A08.1 through genotyping 178 peanut germplasms. Haplotypes analysis in natural population confirmed the closely relationship of the QTLs with the content of oil, oleic acid, lignoceric acid, palmitic acid and behenic acid. This study serves as a valuable reference for selecting improved peanut genotypes with superior oil quality and desirable fatty acid composition.

Genetic hallmarks and clinical implications of chromothripsis in childhood T-cell acute lymphoblastic leukemia

Chromothripsis (cth) is a form of genomic instability leading to massive de novo structural chromosome rearrangements in a one-time catastrophic event. It can cause cancer-promoting alterations, such as loss of sequences for tumor-suppressor genes, formation of oncogenic fusions, and oncogene amplifications. We investigated the genetic background and clinical significance of cth in childhood T-cell acute lymphoblastic leukemia (T-ALL) patients. For this purpose, whole-genome copy number alterations were analyzed in 173 children with newly diagnosed T-ALL using high-density microarrays. Cth was identified in 10 T-ALL samples (5.78%). In six of them, cth occurred in a constitutional background of Nijmegen breakage syndrome (n = 5) or Li-Fraumeni syndrome (n = 1). Cth generated alterations, including deletions of CDKN2A/B (n = 4) and EZH2 (n = 4), amplifications of CDK6 (n = 2), and NUP214::ABL1 and TFG::GPR128 fusions. Cth-positive leukemias exhibited deletions involving the tumor-suppressor genes RB1 (n = 3), TP53 (n = 1) and MED12 (n = 2). Cth-positive T-ALL patients had a lower probability of 5-year overall survival (OS) [0.56 vs. 0.81; hazard ratio (HR) = 4.14 (1.42–12.02) p = 0.017] as did 5-year event-free survival [0.45 vs. 0.74; HR = 3.91 (1.52–10.08); p = 0.012]. Chromothripsis is an infrequent genomic phenomenon in pediatric T-ALL but is significantly associated with cancer-predisposing syndromes and may associate with inferior prognosis.

Accelerated, high-quality photolithographic synthesis of RNA microarrays in situ.

Nucleic acid photolithography is the only microarray fabrication process that has demonstrated chemical versatility accommodating any type of nucleic acid. The current approach to RNA microarray synthesis requires long coupling and photolysis times and suffers from unavoidable degradation postsynthesis. In this study, we developed a series of RNA phosphoramidites with improved chemical and photochemical protection of the 2'- and 5'-OH functions. In so doing, we reduced the coupling time by more than half and the photolysis time by a factor of 4. Sequence libraries that would otherwise take over 6 hours to synthesize can now be prepared in half the time. Degradation is substantially lowered, and concomitantly, hybridization signals can reach over seven times those of the previous state of the art. Under those conditions, high-density RNA microarrays and RNA libraries can now be synthesized at greatly accelerated rates. We also synthesized fluorogenic RNA Mango aptamers on microarrays and investigated the effect of sequence mutations on their fluorogenic properties.

Herpes simplex virus spreads rapidly in human foreskin, partly driven by chemokine-induced redistribution of Nectin-1 on keratinocytes.

HSV infects keratinocytes in the epidermis of skin via nectin-1. We established a human foreskin explant infection model to investigate HSV entry and spread. HSV1 entry could only be achieved by the topical application of virus via high density microarray projections (HD-MAPs) to the epidermis, which penetrated beyond one third of its thickness, simulating in vivo microtrauma. Rapid lateral spread of HSV1 to a mean of 13 keratinocytes wide occurred after 24 hours and free virus particles were observed between keratinocytes, consistent with an intercellular route of spread. Nectin-1 staining was markedly decreased in foci of infection in the epidermis and in the human keratinocyte HaCaT cell line. Nectin-1 was redistributed, at the protein level, in adjacent uninfected cells surrounding infection, inducible by CCL3, IL-8 (or CXCL8), and possibly CXCL10 and IL-6, thus facilitating spread. These findings provide the first insights into HSV1 entry and spread in human inner foreskin in situ.

High-Throughput Antigen Microarray Identifies Longitudinal Prognostic Autoantibody for Chemoimmunotherapy in Advanced Non-Small Cell Lung Cancer

Chemoimmunotherapy has evolved as a standard treatment for advanced non-small cell lung cancer (aNSCLC). However, inevitable drug resistance has limited its efficacy, highlighting the urgent need for biomarkers of chemoimmunotherapy. A three-phase strategy to discover, verify, and validate longitudinal predictive autoantibodies (AAbs) for aNSCLC before and after chemoimmunotherapy was employed. A total of 528 plasma samples from 267 aNSCLC patients before and after anti-PD1 immunotherapy were collected, plus 30 independent formalin-fixed paraffin-embedded samples. Candidate AAbs were firstly selected using a HuProt high-density microarray containing 21,000 proteins in the discovery phase, followed by validation using an aNSCLC-focused microarray. Longitudinal predictive AAbs were chosen for ELISA based on responders versus non-responders comparison and progression-free survival (PFS) survival analysis. Prognostic markers were also validated using immunohistochemistry and publicly available immunotherapy datasets. We identified and validated a panel of two AAbs (MAX and DHX29) as pre-treatment biomarkers and another panel of two AAbs (MAX and TAPBP) as on-treatment predictive markers in aNSCLC patients undergoing chemoimmunotherapy. All three AAbs exhibited a positive correlation with early responses and PFS (p < 0.05). The kinetics of MAX AAb showed an increasing trend in responders (p < 0.05) and a tendency to initially increase and then decrease in non-responders (p < 0.05). Importantly, MAX protein and mRNA levels effectively discriminated PFS (p < 0.05) in aNSCLC patients treated with immunotherapy. Our results present a longitudinal analysis of changes in prognostic AAbs in aNSCLC patients undergoing chemoimmunotherapy.

Identification of highly conserved Trypanosoma cruzi antigens for the development of a universal serological diagnostic assay

ABSTRACT Chagas Disease is an important neglected tropical disease caused by Trypanosoma cruzi. There is no gold standard for diagnosis and commercial serological tests perform poorly in certain locations. By aligning T. cruzi genomes covering parasite genetic and geographic diversity, we identified highly conserved proteins that could serve as universal antigens for improved diagnosis. Their antigenicity was tested in high-density peptide microarrays using well-characterized plasma samples, including samples presenting true infections but discordant serology. Individual and combination of epitopes were also evaluated in peptide-ELISAs. We identified >1400 highly conserved T. cruzi proteins evaluated in microarrays. Remarkably, T. cruzi positive controls had a different epitope recognition profile compared to serologically discordant samples. In particular, multiple T. cruzi antigens used in current tests and their strain-variants, and novel epitopes thought to be broadly antigenic failed to be recognized by discordant samples. Nonetheless, >2000 epitopes specifically recognized by IgGs from both positive controls and discordant samples were identified. Evaluation of selected peptides in ELISA further illustrated the extensive variation in antibody profiles among subjects and a peptide combination could outperform a commercial ELISA, increasing assay sensitivity from 52.3% to 72.7%. Individual variation in antibody profiles rather than T. cruzi diversity appears to be the main factor driving differences in serological diagnostic performance according to geography, which will be important to further elucidate. ELISA with a combination of peptides recognized by a greater number of individuals could better capture infections, and further development may lead to an optimal antigen mixture for a universal diagnostic assay.

Enhancement of cellular immunity following needle-free vaccination of mice with SARS-CoV-2 spike protein.

The COVID-19 pandemic has highlighted the need for vaccines capable of providing rapid and robust protection. One way to improve vaccine efficacy is delivery via microarray patches, such as the Vaxxas high-density microarray patch (HD-MAP). We have previously demonstrated that delivery of a SARS-CoV-2 protein vaccine candidate, HexaPro, via the HD-MAP induces potent humoral immune responses. Here, we investigate the cellular responses induced by HexaPro HD-MAP vaccination. We found that delivery via the HD-MAP induces a type one biassed cellular response of much greater magnitude as compared to standard intramuscular immunization.

Multiplex detection and identification of viral, bacterial, and protozoan pathogens in human blood and plasma using an expanded high-density resequencing microarray platform.

Introduction: Nucleic acid tests for blood donor screening have improved the safety of the blood supply; however, increasing numbers of emerging pathogen tests are burdensome. Multiplex testing platforms are a potential solution. Methods: The Blood Borne Pathogen Resequencing Microarray Expanded (BBP-RMAv.2) can perform multiplex detection and identification of 80 viruses, bacteria and parasites. This study evaluated pathogen detection in human blood or plasma. Samples spiked with selected pathogens, each with one of 6 viruses, 2 bacteria and 5 protozoans were tested on this platform. The nucleic acids were extracted, amplified using multiplexed sets of primers, and hybridized to a microarray. The reported sequences were aligned to a database to identify the pathogen. To directly compare the microarray to an emerging molecular approach, the amplified nucleic acids were also submitted to nanopore next generation sequencing (NGS). Results: The BBP-RMAv.2 detected viral pathogens at a concentration as low as 100 copies/ml and a range of concentrations from 1,000 to 100,000 copies/ml for all the spiked pathogens. Coded specimens were identified correctly demonstrating the effectiveness of the platform. The nanopore sequencing correctly identified most samples and the results of the two platforms were compared. Discussion: These results indicated that the BBP-RMAv.2 could be employed for multiplex detection with potential for use in blood safety or disease diagnosis. The NGS was nearly as effective at identifying pathogens in blood and performed better than BBP-RMAv.2 at identifying pathogen-negative samples.

Identification of novel prognostic autoantibodies in diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone via a high-throughput antigen microarray.

R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) is a standard first-line treatment for diffuse large B-cell lymphoma (DLBCL). However, 20%-40% of patients survive less than 5years. Novel prognostic biomarkers remain in demand. Baseline plasma autoantibodies (AAbs) were assessed in 336 DLBCLs. In the discovery phase (n=20), a high-density antigen microarray (∼21,000 proteins) was used to expound AAb profiles. In the verification phase (n=181), with a DLBCL-focused microarray, comparative results based on event-free survival at 24months (EFS24) and lasso Cox regression models of progression-free survival (PFS) and overall survival (OS) were integrated to identify potential biomarkers. They were further validated by enzyme-linked immunosorbent assay in validation phase 1 (n=135) and a dynamic cohort (n=12). In validation phase 2, a two-AAb-based risk score was established. They were further validated in an immunohistochemistry cohort (n=55) and four independent Gene Expression Omnibus datasets (n=1598). Four AAbs (CREB1, N4BP1, UBAP2, and DEAF1) were identified that showed associations with EFS24 status (p<.05) and superior PFS and OS (p<.05). A novel risk score model based on CREB1 and N4BP1 AAbs was developed to predict PFS with areas under the curve of 0.72, 0.71, 0.76, and 0.82 at 1, 3, 5, and 7years, respectively, in DLBCL treated with R-CHOP independent of the International Prognostic Index (IPI) and provided significant additional recurrence risk discrimination (p<.05) for the IPI. CREB1 and N4BP1 proteins and messenger RNAs were also associated with better PFS and OS (p<.05). This study identified a novel prognostic panel of CREB1, N4BP1, DEAF1, and UBAP2 AAbs that is independent of the IPI in DLBCL.

Transcriptomic analysis of neural stem and progenitor cells in comparison with glioblastoma stem cells

Introduction. There is currently no effective therapy for the treatment of glioblastoma. This is partly explained by the high degree of intra- and intertumor heterogeneity of GB, the source of which is believed to be glioblastoma stem cells (GSC). The question of the origin of GSC, which is important for improving clinical outcomes, still remains open. It is believed that GSCs can be formed as a result of oncogenic transformation of neural stem and progenitor cells (NSPcs), which have morphological and functional properties similar to them. Despite significant progress in elucidating the nature of GSCs, little is yet known about the specifically expressed genes and transcripts in these cells in comparison with NSPcs. In this regard, it becomes relevant to study the molecular mechanisms of gliomagenesis using model cell systems based on various clones of GSC.Aim. To conduct a comparative transcriptomic analysis of CD133+-NSPCs and CD133+-GSCs to study the molecular genetic differences between the phenotypes of these cells and identify potential targets for therapeutic effects on GSCs.Materials and methods. Used: highly sensitive transcriptomic analysis on high-density microarrays, cellular technologies, modern bioinformatics analysis.Results. Transcriptomic analysis of CD133+-GSCs and CD133+-NSPCs identified 1825 differentially expressed genes. The biological processes and signaling cascades activated in CD133+-GSCs have been established. It was shown that significant transcriptomic aberrations in CD133+-GSC compared to CD133+-NSPC are primarily due to a group of transcripts regulated by the Shh (Sonic hedgehog), mTOR (mammalian target of rapamycin), ALK (anaplastic lymphoma kinase) signaling cascades, transcription factors E2F1, PRC2, HOXA9, MYC, as well as oncogenes ERBB2 and KRAS. Six transcripts (AQP9, TOX15, HOXB2, STEAP3, TREM1, RFC2) highly expressed in CD133+-GSC and closely associated with the survival of patients with glioblastoma, which may be potential targets for therapeutic effects on CD133+-GSC associated with gliomagenesis, which may be potential targets for therapeutic effects on CD133+-GSC, have been identified and annotated.Conclusion. The data obtained indicate a number of significant molecular genetic differences between the two cell phenotypes, which can be used in the development of new therapeutic approaches for the treatment of glioblastoma.

Safety, Tolerability, and Immunogenicity of Measles and Rubella Vaccine Delivered with a High-Density Microarray Patch: Results from a Randomized, Partially Double-Blinded, Placebo-Controlled Phase I Clinical Trial

Microarray patches (MAPs) have the potential to be a safer, more acceptable, easier-to-use, and more cost-effective means for the administration of vaccines than injection by needle and syringe. Here, we report findings from a randomized, partially double-blinded, placebo-controlled Phase I trial using the Vaxxas high-density MAP (HD-MAP) to deliver a measles rubella (MR) vaccine. Healthy adults (N = 63, age 18–50 years) were randomly assigned 1:1:1:1 to four groups: uncoated (placebo) HD-MAPs, low-dose MR HD-MAPs (~3100 median cell-culture infectious dose [CCID50] measles, ~4300 CCID50 rubella); high-dose MR-HD-MAPs (~9300 CCID50 measles, ~12,900 CCID50 rubella); or a sub-cutaneous (SC) injection of an approved MR vaccine, MR-Vac (≥1000 CCID50 per virus). The MR vaccines were stable and remained viable on HD-MAPs when stored at 2–8 °C for at least 24 months. When MR HD-MAPs stored at 2–8 °C for 24 months were transferred to 40 °C for 3 days in a controlled temperature excursion, loss of potency was minimal, and MR HD-MAPs still met World Health Organisation (WHO) specifications. MR HD-MAP vaccination was safe and well-tolerated; any systemic or local adverse events (AEs) were mild or moderate. Similar levels of binding and neutralizing antibodies to measles and rubella were induced by low-dose and high-dose MR HD-MAPs and MR-Vac. The neutralizing antibody seroconversion rates on day 28 after vaccination for the low-dose HD-MAP, high-dose HD-MAP and MR-Vac groups were 37.5%, 18.8% and 35.7%, respectively, for measles, and 37.5%, 25.0% and 35.7%, respectively, for rubella. Most participants were seropositive for measles and rubella antibodies at baseline, which appeared to negatively impact the number of participants that seroconverted to vaccines delivered by either route. The data reported here suggest HD-MAPs could be a valuable means for delivering MR-vaccine to hard-to-reach populations and support further development. Clinical trial registry number: ACTRN12621000820808.

Genome-wide epitope mapping across multiple host species reveals significant diversity in antibody responses to Coxiella burnetii vaccination and infection.

Coxiella burnetii is an important zoonotic bacterial pathogen of global importance, causing the disease Q fever in a wide range of animal hosts. Ruminant livestock, in particular sheep and goats, are considered the main reservoir of human infection. Vaccination is a key control measure, and two commercial vaccines based on formalin-inactivated C. burnetii bacterins are currently available for use in livestock and humans. However, their deployment is limited due to significant reactogenicity in individuals previously sensitized to C. burnetii antigens. Furthermore, these vaccines interfere with available serodiagnostic tests which are also based on C. burnetii bacterin antigens. Defined subunit antigen vaccines offer significant advantages, as they can be engineered to reduce reactogenicity and co-designed with serodiagnostic tests to allow discrimination between vaccinated and infected individuals. This study aimed to investigate the diversity of antibody responses to C. burnetii vaccination and/or infection in cattle, goats, humans, and sheep through genome-wide linear epitope mapping to identify candidate vaccine and diagnostic antigens within the predicted bacterial proteome. Using high-density peptide microarrays, we analyzed the seroreactivity in 156 serum samples from vaccinated and infected individuals to peptides derived from 2,092 open-reading frames in the C. burnetii genome. We found significant diversity in the antibody responses within and between species and across different types of C. burnetii exposure. Through the implementation of three different vaccine candidate selection methods, we identified 493 candidate protein antigens for protein subunit vaccine design or serodiagnostic evaluation, of which 65 have been previously described. This is the first study to investigate multi-species seroreactivity against the entire C. burnetii proteome presented as overlapping linear peptides and provides the basis for the selection of antigen targets for next-generation Q fever vaccines and diagnostic tests.

Superior efficacy of a skin-applied microprojection device for delivering a novel Zika DNA vaccine

Zika virus (ZIKV) infections are spreading silently with limited global surveillance in at least 89 countries and territories. There is a pressing need to develop an effective vaccine suitable for equitable distribution globally. Consequently, we previously developed a proprietary DNA vaccine encoding secreted non-structural protein 1 of ZIKV (pVAX-tpaNS1) to elicit rapid protectionin a Tcell-dependent manner in mice. In the current study, we evaluated the stability, efficacy, and immunogenicity of delivering this DNA vaccine into the skin using a clinically effective and proprietary high-density microarray patch (HD-MAP). Dry-coating of pVAX-tpaNS1 on the HD-MAP deviceresulted in no loss of vaccine stability at 40°C storage over the course of 28days. Vaccination of mice (BALB/c) with the HD-MAP-coated pVAX-tpaNS1 elicited a robust anti-NS1 IgG response in both the cervicovaginal mucosa and systemically and afforded protection against live ZIKV challenge. Furthermore, the vaccination elicited a significantly higher magnitude and broader NS1-specific T helper and cytotoxic Tcell response invivo compared with traditional needle and syringe intradermal vaccination. Overall, the study highlights distinctive immunological advantages coupled with an excellent thermostability profile of using the HD-MAP device to deliver a novel ZIKV DNA vaccine.

In situ enzymatic template replication on DNA microarrays.

DNA microarrays are very useful tools to study the realm of nucleic acids interactions at high throughput. The conventional approach to microarray synthesis employs phosphoramidite chemistry and yields unmodified DNA generally attached to a surface at the 3' terminus. Having a freely accessible 3'-OH instead of 5'-OH is desirable too, and being able to introduce nucleoside analogs in a combinatorial manner is highly relevant in the context of nucleic acid therapeutics and in aptamer research. Here, we describe an enzymatic approach to the synthesis of high-density DNA microarrays that can also contain chemical modifications. The method uses a standard DNA microarray, to which a DNA primer is covalently bound through photocrosslinking. The extension of the primer with a DNA polymerase yields double-stranded DNA but is also amenable to the incorporation of modified dNTPs. Further processing with T7 exonuclease, which catalyzes the degradation of DNA in a specific (5'→3') direction, results in template strand removal. Overall, the method produces surface-bound natural and non-natural DNA oligonucleotides, is applicable to commercial microarrays and paves the way for the preparation of combinatorial, chemically modified aptamer libraries.

Constructing high-density tissue microarrays with a novel method and a self-made tissue-arraying instrument.

Tissue microarrays (TMAs), also called tissue chips, contain hundreds to thousands of tissue cores obtained from different tissue donor blocks. By using TMA technology, a molecular marker, such as protein, RNA or DNA, can be simultaneously examined in hundreds of different specimens under the same experimental conditions. A growing number of previous studies have introduced different methods for constructing TMAs. Many authors tried to use various methods to implant more tissue cores in a single recipient block, and most of these methods involved reducing the diameter of the tissue cores and/or the spacing between adjacent tissue cores. However, when creating TMAs, it is difficult to reduce the distance between tissue cores to zero except with extremely expensive automatic TMA arrayers. Here, we introduce a novel method to construct a high-density TMA that does not have spacing between the tissue cores. We also introduce a method for preparing a self-made tissue-arraying instrument. With this method and the tissue-arraying instrument, we successfully created a TMA containing 126 tissue cores that were 2mm in diameter. H&E staining and immunohistochemical staining were performed on the sections cut from the TMA without any tissue spot loss. This method is easy to operate, and the materials for creating the tissue-arraying instrument are inexpensive and can be purchased anywhere. Therefore, this method can be applied in all laboratories.