Genetic relationships of three species of black flies (Diptera: Simuliidae) in Taiwan.

As an island about 150 km from the mainland, Taiwan would be expected to have endemic species. About 64 % of its 36 species of black flies are considered endemic, more than twice the level of endemicity that has been recorded for all insects on the island. To begin assessing the validity of the high level of endemism for the Simuliidae, we used giant chromosome banding patterns and cytochrome c oxidase I (COI) sequences, against a well-defined morphological backdrop, to evaluate three of Taiwan's black flies, Simulium chungi Takaoka & Huang, S. pingtungense Huang & Takaoka, and S. sakishimaense Takaoka. Molecular data revealed high similarity of populations of S. sakishimaense in Taiwan and at the type locality on Ishigaki Island, Japan, about 180 km to the east. Thus, populations referred to as S. sakishimaense in Taiwan are conspecific with typical S. sakishimaense in Japan, confirming their non-endemicity in Taiwan. Simulium sakishimaense might have reached Ishigaki by island hopping via Taiwan from the Chinese mainland. Chromosomes and the COI gene agree with morphology that S. sakishimaense is a member of the S. multistriatum species group although the chromosomal banding patterns do not indicate that it is distinct from S. fenestratum Edwards on the mainland. Although molecular sequences indicate S. sakishimaense is monophyletic, this taxon falls within the same Operational Taxonomic Unit as nine other members of the S. multistriatum group, including S. fenestratum. Simulium pingtungense, in agreement with morphology, is molecularly distinct from the 10 other analyzed members of the S. striatum species group, tentatively suggesting that it is endemic to Taiwan, pending analysis of samples from mainland China. Simulium chungi in Taiwan is chromosomally and molecularly unique, with larvae resembling those of S. saskishimaense. It is not, however, a member of either the S. multistriatum or S. striatum species groups. For now, the S. chungi species group remains a legitimate taxon consisting of two species. Strengthening the case for endemic taxa in Taiwan awaits analysis of key samples from the Chinese mainland.

Development of a recombinant non-replicating Newcastle disease virus.

RESEARCH HIGHLIGHTS: Development of nr-NDV.Reverse transfection was applied for the recovery of nr-NDV.Propagation of nr-NDV was done by sub-passaging transfected BSR T7/5 cells.Safety profile was done to prove that the nr-NDV is non-replicating.

Genomic testing for differences of sex development: Practices and perceptions of clinicians.

OBJECTIVES: To investigate the approach taken by clinicians involved in the diagnosis and management of individuals with Differences of Sex Development (DSD), particularly with regard to genomic testing, and identify perceived gaps/strengths/barriers in current practice. DESIGN AND METHODS: An anonymous online survey was developed, with questions exploring demographics, perceptions of genomic testing, availability of genetics services and opinions on the role and utility of genomic testing in DSD. All responses were anonymous. Clinicians involved in the diagnosis and management of individuals with DSD were recruited from relevant societies and departments across Australia and New Zealand. RESULTS: 79 eligible clinicians commenced the survey, with 63 completing it and 16 providing a partial response. The perceived benefit of having a genetic diagnosis for DSD was almost unanimous (97%). Almost half (48%) of respondents reported barriers in genomic testing. 81% of respondents reported they order genomic tests currently. Approaches to genomic testing when faced with four different clinical scenarios varied across respondents. Clinicians perceived genomic testing to be underutilised (median 36 on sliding scale from 0 to 100). CONCLUSIONS: Despite 97% of respondents reporting benefit of a genetic diagnosis for individuals with DSD, this was not reflected throughout the survey with regard to clinical implementation. When faced with clinical scenarios, the recommendations for genomic testing from respondents was much lower, indicating the discrepancy between perception and clinical practice. Genomic testing in the context of DSD is seen as both beneficial and desired, yet there are multiple barriers impacting its integration into standard clinical care.

Genome Sequencing for Diagnosing Rare Diseases.

BACKGROUND: Genetic variants that cause rare disorders may remain elusive even after expansive testing, such as exome sequencing. The diagnostic yield of genome sequencing, particularly after a negative evaluation, remains poorly defined. METHODS: We sequenced and analyzed the genomes of families with diverse phenotypes who were suspected to have a rare monogenic disease and for whom genetic testing had not revealed a diagnosis, as well as the genomes of a replication cohort at an independent clinical center. RESULTS: We sequenced the genomes of 822 families (744 in the initial cohort and 78 in the replication cohort) and made a molecular diagnosis in 218 of 744 families (29.3%). Of the 218 families, 61 (28.0%) - 8.2% of families in the initial cohort - had variants that required genome sequencing for identification, including coding variants, intronic variants, small structural variants, copy-neutral inversions, complex rearrangements, and tandem repeat expansions. Most families in which a molecular diagnosis was made after previous nondiagnostic exome sequencing (63.5%) had variants that could be detected by reanalysis of the exome-sequence data (53.4%) or by additional analytic methods, such as copy-number variant calling, to exome-sequence data (10.8%). We obtained similar results in the replication cohort: in 33% of the families in which a molecular diagnosis was made, or 8% of the cohort, genome sequencing was required, which showed the applicability of these findings to both research and clinical environments. CONCLUSIONS: The diagnostic yield of genome sequencing in a large, diverse research cohort and in a small clinical cohort of persons who had previously undergone genetic testing was approximately 8% and included several types of pathogenic variation that had not previously been detected by means of exome sequencing or other techniques. (Funded by the National Human Genome Research Institute and others.).

The presence of broadly neutralizing anti-SARS-CoV-2 RBD antibodies elicited by primary series and booster dose of COVID-19 vaccine.

Antibody-mediated immunity plays a key role in protection against SARS-CoV-2. We characterized B-cell-derived anti-SARS-CoV-2 RBD antibody repertoires from vaccinated and infected individuals and elucidate the mechanism of action of broadly neutralizing antibodies and dissect antibodies at the epitope level. The breadth and clonality of anti-RBD B cell response varies among individuals. The majority of neutralizing antibody clones lose or exhibit reduced activities against Beta, Delta, and Omicron variants. Nevertheless, a portion of anti-RBD antibody clones that develops after a primary series or booster dose of COVID-19 vaccination exhibit broad neutralization against emerging Omicron BA.2, BA.4, BA.5, BQ.1.1, XBB.1.5 and XBB.1.16 variants. These broadly neutralizing antibodies share genetic features including a conserved usage of the IGHV3-53 and 3-9 genes and recognize three clustered epitopes of the RBD, including epitopes that partially overlap the classically defined set identified early in the pandemic. The Fab-RBD crystal and Fab-Spike complex structures corroborate the epitope grouping of antibodies and reveal the detailed binding mode of broadly neutralizing antibodies. Structure-guided mutagenesis improves binding and neutralization potency of antibody with Omicron variants via a single amino-substitution. Together, these results provide an immunological basis for partial protection against severe COVID-19 by the ancestral strain-based vaccine and indicate guidance for next generation monoclonal antibody development and vaccine design.

Proactive vaccination using multiviral Quartet Nanocages to elicit broad anti-coronavirus responses.

Defending against future pandemics requires vaccine platforms that protect across a range of related pathogens. Nanoscale patterning can be used to address this issue. Here, we produce quartets of linked receptor-binding domains (RBDs) from a panel of SARS-like betacoronaviruses, coupled to a computationally designed nanocage through SpyTag/SpyCatcher links. These Quartet Nanocages, possessing a branched morphology, induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented in the vaccine. Equivalent antibody responses are raised to RBDs close to the nanocage or at the tips of the nanoparticle's branches. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increase the strength and breadth of an otherwise narrow immune response. A Quartet Nanocage including the Omicron XBB.1.5 'Kraken' RBD induced antibodies with binding to a broad range of sarbecoviruses, as well as neutralizing activity against this variant of concern. Quartet nanocages are a nanomedicine approach with potential to confer heterotypic protection against emergent zoonotic pathogens and facilitate proactive pandemic protection.

Exome copy number variant detection, analysis, and classification in a large cohort of families with undiagnosed rare genetic disease.

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and, with new innovative methods, can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the Genomics Research to Elucidate the Genetics of Rare Diseases consortium and analyzed using the seqr platform. The addition of CNV detection to exome analysis identified causal CNVs for 171 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb. The causal CNVs consisted of 140 deletions, 15 duplications, 3 suspected complex structural variants (SVs), 3 insertions, and 10 complex SVs, the latter two groups being identified by orthogonal confirmation methods. To classify CNV variant pathogenicity, we used the 2020 American College of Medical Genetics and Genomics/ClinGen CNV interpretation standards and developed additional criteria to evaluate allelic and functional data as well as variants on the X chromosome to further advance the framework. We interpreted 151 CNVs as likely pathogenic/pathogenic and 20 CNVs as high-interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher-resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.

A metabolic signature for NADSYN1-dependent congenital NAD deficiency disorder.

Nicotinamide adenine dinucleotide (NAD) is essential for embryonic development. To date, biallelic loss-of-function variants in 3 genes encoding nonredundant enzymes of the NAD de novo synthesis pathway - KYNU, HAAO, and NADSYN1 - have been identified in humans with congenital malformations defined as congenital NAD deficiency disorder (CNDD). Here, we identified 13 further individuals with biallelic NADSYN1 variants predicted to be damaging, and phenotypes ranging from multiple severe malformations to the complete absence of malformation. Enzymatic assessment of variant deleteriousness in vitro revealed protein domain-specific perturbation, complemented by protein structure modeling in silico. We reproduced NADSYN1-dependent CNDD in mice and assessed various maternal NAD precursor supplementation strategies to prevent adverse pregnancy outcomes. While for Nadsyn1+/- mothers, any B3 vitamer was suitable to raise NAD, preventing embryo loss and malformation, Nadsyn1-/- mothers required supplementation with amidated NAD precursors (nicotinamide or nicotinamide mononucleotide) bypassing their metabolic block. The circulatory NAD metabolome in mice and humans before and after NAD precursor supplementation revealed a consistent metabolic signature with utility for patient identification. Our data collectively improve clinical diagnostics of NADSYN1-dependent CNDD, provide guidance for the therapeutic prevention of CNDD, and suggest an ongoing need to maintain NAD levels via amidated NAD precursor supplementation after birth.

Narrowing the diagnostic gap: Genomes, episignatures, long-read sequencing, and health economic analyses in an exome-negative intellectual disability cohort.

PURPOSE: Genome sequencing (GS)-specific diagnostic rates in prospective tightly ascertained exome sequencing (ES)-negative intellectual disability (ID) cohorts have not been reported extensively. METHODS: ES, GS, epigenetic signatures, and long-read sequencing diagnoses were assessed in 74 trios with at least moderate ID. RESULTS: The ES diagnostic yield was 42 of 74 (57%). GS diagnoses were made in 9 of 32 (28%) ES-unresolved families. Repeated ES with a contemporary pipeline on the GS-diagnosed families identified 8 of 9 single-nucleotide variations/copy-number variations undetected in older ES, confirming a GS-unique diagnostic rate of 1 in 32 (3%). Episignatures contributed diagnostic information in 9% with GS corroboration in 1 of 32 (3%) and diagnostic clues in 2 of 32 (6%). A genetic etiology for ID was detected in 51 of 74 (69%) families. Twelve candidate disease genes were identified. Contemporary ES followed by GS cost US$4976 (95% CI: $3704; $6969) per diagnosis and first-line GS at a cost of $7062 (95% CI: $6210; $8475) per diagnosis. CONCLUSION: Performing GS only in ID trios would be cost equivalent to ES if GS were available at $2435, about a 60% reduction from current prices. This study demonstrates that first-line GS achieves higher diagnostic rate than contemporary ES but at a higher cost.

Co-occurrence of dual lineages within Simulium (Gomphostilbia) atratum De Meijere in the Indonesian Archipelago along Wallace's Line.

Mitochondrial cytochrome c oxidase subunit I (COI) sequences were utilized to infer the population genetic structure of Simulium (Gomphostilbia) atratum De Meijere, an endemic simulid species to Indonesia. Both median-joining haplotype network and maximum-likelihood tree revealed two genetic lineages (A and B) within the species, with an overlap distribution in Lombok, which is situated along Wallace's line. Genetic differentiation and gene flow with varying frequencies (FST = 0.02-0.967; Nm = 0.01-10.58) were observed between populations of S. (G.) atratum, of which population pairs of different lineages showed high genetic differentiation. Notably, the high genetic distance of up to 5.92 % observed within S. (G.) atratum in Lombok was attributed to the existence of two genetically distinct lineages. The co-occurrence of distinct lineages in Lombok indicated that Wallace's line did not act as faunistic border for S. (G.) atratum in the present study. Moreover, both lineages also exhibited unimodal distributions and negative values of neutrality tests, suggesting a pattern of population expansion. The expansion and divergence time estimation suggested that the two lineages of S. (G.) atratum diverged and expanded during the Pleistocene era in Indonesia.

Intellectual Functioning of Children With Isolated PRS, PRS-Plus, and Syndromic PRS.

OBJECTIVE: Describe the intelligence quotient (IQ) of children with Pierre Robin sequence (PRS). DESIGN: Prospective cohort study. SETTING: Neurodevelopmental follow-up clinic within a hospital. PATIENTS: Children with PRS (n = 45) who had been in the Neonatal Intensive Care Unit (NICU) were classified by a geneticist into 3 subgroups of isolated PRS (n = 20), PRS-plus additional medical features (n = 8), and syndromic PRS (n = 17) based on medical record review and genetic testing. MAIN OUTCOME MEASURE: Children with PRS completed IQ testing at 5 or 8 years of age with the Wechsler Preschool and Primary Scale of Intelligence, Third Edition (WPPSI-III) or Fourth Edition (WPPSI-IV) or the Wechsler Intelligence Scale for Children, Fourth Edition (WISC-IV) or Fifth Edition (WISC-V). RESULTS: IQ scores were more than 1 to 2 standard deviations below the mean for 36% of the overall sample, which was significantly greater compared to test norms (binomial test P = .001). There was a significant association between PRS subtype and IQ (Fisher's exact P = .026). While only 20% of children with isolated PRS were within 1 standard deviation below average and 35% of children with syndromic PRS were below 1 to 2 standard deviations, 75% of PRS-plus children scored lower than 1 to 2 standard deviations below the mean. CONCLUSION: PRS subgroups can help identify children at risk for cognitive delay. The majority of children with PRS-plus had low intellectual functioning, in contrast to the third of children with syndromic PRS who had low IQ and the majority of children with isolated PRS who had average or higher IQ.

Exome copy number variant detection, analysis and classification in a large cohort of families with undiagnosed rare genetic disease.

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and with new innovative methods can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the GREGoR consortium. Each family's CNV data was analyzed using the seqr platform and candidate CNVs classified using the 2020 ACMG/ClinGen CNV interpretation standards. We developed additional evidence criteria to address situations not covered by the current standards. The addition of CNV calling to exome analysis identified causal CNVs for 173 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb with estimates that 44% would not have been detected by standard chromosomal microarrays. The causal CNVs consisted of 141 deletions, 15 duplications, 4 suspected complex structural variants (SVs), 3 insertions and 10 complex SVs, the latter two groups being identified by orthogonal validation methods. We interpreted 153 CNVs as likely pathogenic/pathogenic and 20 CNVs as high interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.

Single-cycle influenza virus vaccine generates lung CD8+ Trm that cross-react against viral variants and subvert virus escape mutants.

Influenza virus-specific tissue-resident memory (Trm) CD8+ T cells located along the respiratory tract provide cross-strain protection against a breadth of influenza viruses. We show that immunization with a single-cycle influenza virus vaccine candidate (S-FLU) results in the deposition of influenza virus nucleoprotein (NP)-specific CD8+ Trm along the respiratory tract that were more cross-reactive against viral variants and less likely to drive the development of cytotoxic T lymphocyte (CTL) escape mutants, as compared to the lung memory NP-specific CD8+ T cell pool established following influenza infection. This immune profile was linked to the limited inflammatory response evoked by S-FLU vaccination, which increased TCR repertoire diversity within the memory CD8+ T cell compartment. Cumulatively, this work shows that S-FLU vaccination evokes a clonally diverse, cross-reactive memory CD8+ T cell pool, which protects against severe disease without driving the virus to rapidly evolve and escape, and thus represents an attractive vaccine for use against rapidly mutating influenza viruses.

Clinical exome sequencing efficacy and phenotypic expansions involving anomalous pulmonary venous return.

Anomalous pulmonary venous return (APVR) frequently occurs with other congenital heart defects (CHDs) or extra-cardiac anomalies. While some genetic causes have been identified, the optimal approach to genetic testing in individuals with APVR remains uncertain, and the etiology of most cases of APVR is unclear. Here, we analyzed molecular data from 49 individuals to determine the diagnostic yield of clinical exome sequencing (ES) for non-isolated APVR. A definitive or probable diagnosis was made for 8 of those individuals yielding a diagnostic efficacy rate of 16.3%. We then analyzed molecular data from 62 individuals with APVR accrued from three databases to identify novel APVR genes. Based on data from this analysis, published case reports, mouse models, and/or similarity to known APVR genes as revealed by a machine learning algorithm, we identified 3 genes-EFTUD2, NAA15, and NKX2-1-for which there is sufficient evidence to support phenotypic expansion to include APVR. We also provide evidence that 3 recurrent copy number variants contribute to the development of APVR: proximal 1q21.1 microdeletions involving RBM8A and PDZK1, recurrent BP1-BP2 15q11.2 deletions, and central 22q11.2 deletions involving CRKL. Our results suggest that ES and chromosomal microarray analysis (or genome sequencing) should be considered for individuals with non-isolated APVR for whom a genetic etiology has not been identified, and that genetic testing to identify an independent genetic etiology of APVR is not warranted in individuals with EFTUD2-, NAA15-, and NKX2-1-related disorders.

Protein Profiling of Aedes aegypti Treated with Streptomyces sp. KSF103 Ethyl Acetate Extract Reveals Potential Insecticidal Targets and Metabolic Pathways.

The insecticidal activity of Streptomyces sp. KSF103 ethyl acetate (EA) extract against mosquitoes is known; however, the underlying mechanism behind this activity remains elusive. In this study, liquid chromatography with tandem mass spectrometry (LC-MS/MS) was employed to investigate changes in the protein profile of Aedes aegypti larvae and adults treated with lethal concentrations of 50 (LC50) EA extract. By comparing the treated and untreated mosquitoes, this study aimed to identify proteins or pathways that exhibit alterations, potentially serving as targets for future insecticide development. Treatment with a lethal concentration of EA extract upregulated 15 proteins in larvae, while in adults, 16 proteins were upregulated, and two proteins were downregulated. These proteins were associated with metabolism, protein regulation/degradation, energy production, cellular organization and structure, enzyme activity, and catalysis, as well as calcium ion transport and homeostasis. Notably, ATP synthase, fructose-bisphosphate aldolase (FBA), and ATP citrate synthase were significantly expressed in both groups. Gene ontology analysis indicated a focus on energy metabolic processes. Molecular docking revealed a strong interaction between dodemorph, selagine (compounds from the EA extract), and FBA, suggesting FBA as a potential protein target for insecticide development. Further studies such as Western blot and transcriptomic analyses are warranted to validate the findings.

Integrated multi-omics for rapid rare disease diagnosis on a national scale.

Critically ill infants and children with rare diseases need equitable access to rapid and accurate diagnosis to direct clinical management. Over 2 years, the Acute Care Genomics program provided whole-genome sequencing to 290 families whose critically ill infants and children were admitted to hospitals throughout Australia with suspected genetic conditions. The average time to result was 2.9 d and diagnostic yield was 47%. We performed additional bioinformatic analyses and transcriptome sequencing in all patients who remained undiagnosed. Long-read sequencing and functional assays, ranging from clinically accredited enzyme analysis to bespoke quantitative proteomics, were deployed in selected cases. This resulted in an additional 19 diagnoses and an overall diagnostic yield of 54%. Diagnostic variants ranged from structural chromosomal abnormalities through to an intronic retrotransposon, disrupting splicing. Critical care management changed in 120 diagnosed patients (77%). This included major impacts, such as informing precision treatments, surgical and transplant decisions and palliation, in 94 patients (60%). Our results provide preliminary evidence of the clinical utility of integrating multi-omic approaches into mainstream diagnostic practice to fully realize the potential of rare disease genomic testing in a timely manner.

DNA methylation episignatures are sensitive and specific biomarkers for detection of patients with KAT6A/KAT6B variants.

Accurate diagnosis for patients living with neurodevelopmental disorders is often met with numerous challenges, related to the ambiguity of findings and lack of specificity in genetic variants leading to pathology. Genome-wide DNA methylation analysis has been used to develop highly sensitive and specific 'episignatures' as biomarkers capable of differentiating and classifying complex neurodevelopmental disorders. In this study we describe distinct episignatures for KAT6A syndrome, caused by pathogenic variants in the lysine acetyltransferase A gene (KAT6A), and for the two neurodevelopmental disorders associated with lysine acetyl transferase B (KAT6B). We demonstrate the ability of our models to differentiate between highly overlapping episignatures, increasing the ability to effectively identify and diagnose these conditions.

Pathogenic variants in the paired-related homeobox 1 gene (PRRX1) cause craniosynostosis with incomplete penetrance.

PURPOSE: Studies have previously implicated PRRX1 in craniofacial development, including demonstration of murine Prrx1 expression in the preosteogenic cells of the cranial sutures. We investigated the role of heterozygous missense and loss-of-function (LoF) variants in PRRX1 associated with craniosynostosis. METHODS: Trio-based genome, exome, or targeted sequencing were used to screen PRRX1 in patients with craniosynostosis; immunofluorescence analyses were used to assess nuclear localization of wild-type and mutant proteins. RESULTS: Genome sequencing identified 2 of 9 sporadically affected individuals with syndromic/multisuture craniosynostosis, who were heterozygous for rare/undescribed variants in PRRX1. Exome or targeted sequencing of PRRX1 revealed a further 9 of 1449 patients with craniosynostosis harboring deletions or rare heterozygous variants within the homeodomain. By collaboration, 7 additional individuals (4 families) were identified with putatively pathogenic PRRX1 variants. Immunofluorescence analyses showed that missense variants within the PRRX1 homeodomain cause abnormal nuclear localization. Of patients with variants considered likely pathogenic, bicoronal or other multisuture synostosis was present in 11 of 17 cases (65%). Pathogenic variants were inherited from unaffected relatives in many instances, yielding a 12.5% penetrance estimate for craniosynostosis. CONCLUSION: This work supports a key role for PRRX1 in cranial suture development and shows that haploinsufficiency of PRRX1 is a relatively frequent cause of craniosynostosis.

Multiviral Quartet Nanocages Elicit Broad Anti-Coronavirus Responses for Proactive Vaccinology.

Defending against future pandemics may require vaccine platforms that protect across a range of related pathogens. The presentation of multiple receptor-binding domains (RBDs) from evolutionarily-related viruses on a nanoparticle scaffold elicits a strong antibody response to conserved regions. Here we produce quartets of tandemly-linked RBDs from SARS-like betacoronaviruses coupled to the mi3 nanocage through a SpyTag/SpyCatcher spontaneous reaction. These Quartet Nanocages induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented on the vaccine. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increased the strength and breadth of an otherwise narrow immune response. Quartet Nanocages are a strategy with potential to confer heterotypic protection against emergent zoonotic coronavirus pathogens and facilitate proactive pandemic protection.