Biophysical studies do not reveal direct interactions between human PF4 and Ad26.COV2.S vaccine.

BACKGROUND: COVID-19 vaccines have been widely used to control the SARS-CoV-2 pandemic. In individuals receiving replication-incompetent, adenovirus vector-based COVID-19 vaccines (eg, ChAdOx1 nCoV-19 [AstraZeneca] or Ad26.COV2.S [Johnson & Johnson/Janssen] vaccines), a very rare but serious adverse reaction has been reported and described as vaccine-induced immune thrombotic thrombocytopenia (VITT). The exact mechanism of VITT following Ad26.COV2.S vaccination is under investigation. Antibodies directed against human platelet factor 4 (PF4) are considered critical in the pathogenesis of VITT, suggesting similarities with heparin-induced thrombocytopenia. It has been postulated that components of these vaccines mimic the role of heparin by binding to PF4, triggering production of these anti-PF4 antibodies. OBJECTIVES: This study aimed to investigate the potential interaction between human PF4 and Ad26.COV2.S vaccine using several biophysical techniques. METHODS: Direct interaction of PF4 with Ad26.COV2.S vaccine was investigated using dynamic light scattering, biolayer interferometry, and surface plasmon resonance. For both biosensing methods, the Ad26.COV2.S vaccine was immobilized to the sensor surface and PF4 was used as analyte. RESULTS: No direct interactions between PF4 and Ad26.COV2.S vaccine could be detected using dynamic light scattering and biolayer interferometry. Surface plasmon resonance technology was shown to be unsuitable to investigate these types of interactions. CONCLUSION: Our findings make it very unlikely that direct binding of PF4 to Ad26.COV2.S vaccine or components thereof is driving the onset of VITT, although the occurrence of such interactions after immunization (potentially facilitated by unknown plasma or cellular factors) cannot be excluded. Further research is warranted to improve the understanding of the full mechanism of this adverse reaction.

Booster vaccination with Ad26.COV2.S or an Omicron-adapted vaccine in pre-immune hamsters protects against Omicron BA.2.

Since the original outbreak of the SARS-CoV-2 virus, several rapidly spreading SARS-CoV-2 variants of concern (VOC) have emerged. Here, we show that a single dose of Ad26.COV2.S (based on the Wuhan-Hu-1 spike variant) protects against the Gamma and Delta variants in naive hamsters, supporting the observed maintained vaccine efficacy in humans against these VOC. Adapted spike-based booster vaccines targeting Omicron variants have now been authorized in the absence of human efficacy data. We evaluated the immunogenicity and efficacy of Ad26.COV2.S.529 (encoding a stabilized Omicron BA.1 spike) in naive mice and in hamsters with pre-existing immunity to the Wuhan-Hu-1 spike. In naive mice, Ad26.COV2.S.529 elicited higher neutralizing antibody titers against SARS-CoV-2 Omicron BA.1 and BA.2, compared with Ad26.COV2.S. However, neutralizing titers against the SARS-CoV-2 B.1 (D614G) and Delta variants were lower after primary vaccination with Ad26.COV2.S.529 compared with Ad26.COV2.S. In contrast, we found comparable Omicron BA.1 and BA.2 neutralizing titers in hamsters with pre-existing Wuhan-Hu-1 spike immunity after vaccination with Ad26.COV2.S, Ad26.COV2.S.529 or a combination of the two vaccines. Moreover, all three vaccine modalities induced equivalent protection against Omicron BA.2 challenge in these animals. Overall, our data suggest that an Omicron BA.1-based booster in rodents does not improve immunogenicity and efficacy against Omicron BA.2 over an Ad26.COV2.S booster in a setting of pre-existing immunity to SARS-CoV-2.

Immunogenicity and efficacy of one and two doses of Ad26.COV2.S COVID vaccine in adult and aged NHP.

Safe and effective coronavirus disease-19 (COVID-19) vaccines are urgently needed to control the ongoing pandemic. While single-dose vaccine regimens would provide multiple advantages, two doses may improve the magnitude and durability of immunity and protective efficacy. We assessed one- and two-dose regimens of the Ad26.COV2.S vaccine candidate in adult and aged nonhuman primates (NHPs). A two-dose Ad26.COV2.S regimen induced higher peak binding and neutralizing antibody responses compared with a single dose. In one-dose regimens, neutralizing antibody responses were stable for at least 14 wk, providing an early indication of durability. Ad26.COV2.S induced humoral immunity and T helper cell (Th cell) 1-skewed cellular responses in aged NHPs that were comparable to those in adult animals. Aged Ad26.COV2.S-vaccinated animals challenged 3 mo after dose 1 with a SARS-CoV-2 spike G614 variant showed near complete lower and substantial upper respiratory tract protection for both regimens. Neutralization of variants of concern by NHP sera was reduced for B.1.351 lineages while maintained for the B.1.1.7 lineage independent of Ad26.COV2.S vaccine regimen.

Detailed genetic and functional analysis of the hDMDdel52/mdx mouse model.

Duchenne muscular dystrophy (DMD) is a severe, progressive neuromuscular disorder caused by reading frame disrupting mutations in the DMD gene leading to absence of functional dystrophin. Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach aimed at restoring the reading frame at the pre-mRNA level, allowing the production of internally truncated partly functional dystrophin proteins. AONs work in a sequence specific manner, which warrants generating humanized mouse models for preclinical tests. To address this, we previously generated the hDMDdel52/mdx mouse model using transcription activator like effector nuclease (TALEN) technology. This model contains mutated murine and human DMD genes, and therefore lacks mouse and human dystrophin resulting in a dystrophic phenotype. It allows preclinical evaluation of AONs inducing the skipping of human DMD exons 51 and 53 and resulting in restoration of dystrophin synthesis. Here, we have further characterized this model genetically and functionally. We discovered that the hDMD and hDMDdel52 transgene is present twice per locus, in a tail-to-tail-orientation. Long-read sequencing revealed a partial deletion of exon 52 (first 25 bp), and a 2.3 kb inversion in intron 51 in both copies. These new findings on the genomic make-up of the hDMD and hDMDdel52 transgene do not affect exon 51 and/or 53 skipping, but do underline the need for extensive genetic analysis of mice generated with genome editing techniques to elucidate additional genetic changes that might have occurred. The hDMDdel52/mdx mice were also evaluated functionally using kinematic gait analysis. This revealed a clear and highly significant difference in overall gait between hDMDdel52/mdx mice and C57BL6/J controls. The motor deficit detected in the model confirms its suitability for preclinical testing of exon skipping AONs for human DMD at both the functional and molecular level.

Suppression of Mutant Protein Expression in SCA3 and SCA1 Mice Using a CAG Repeat-Targeting Antisense Oligonucleotide.

Spinocerebellar ataxia type 3 (SCA3) and type 1 (SCA1) are dominantly inherited neurodegenerative disorders that are currently incurable. Both diseases are caused by a CAG-repeat expansion in exon 10 of the Ataxin-3 and exon 8 of the Ataxin-1 gene, respectively, encoding an elongated polyglutamine tract that confers toxic properties to the resulting proteins. We have previously shown lowering of the pathogenic polyglutamine protein in Huntington's disease mouse models using (CUG)7, a CAG repeat-targeting antisense oligonucleotide. Here we evaluated the therapeutic capacity of (CUG)7 for SCA3 and SCA1, in vitro in patient-derived cell lines and in vivo in representative mouse models. Repeated intracerebroventricular (CUG)7 administration resulted in a significant reduction of mutant Ataxin-3 and Ataxin-1 proteins throughout the brain of SCA3 and SCA1 mouse models, respectively. Furthermore, in both a SCA3 patient cell line and the MJD84.2 mouse model, (CUG)7 induced formation of a truncated Ataxin-3 protein species lacking the polyglutamine stretch, likely arising from (CUG)7-mediated exon 10 skipping. In contrast, skipping of exon 8 of Ataxin-1 did not significantly contribute to the Ataxin-1 protein reduction observed in (CUG)7-treated SCA1154Q/2Q mice. These findings support the therapeutic potential of a single CAG repeat-targeting AON for the treatment of multiple polyglutamine disorders.

The expanded CAG repeat in the huntingtin gene as target for therapeutic RNA modulation throughout the HD mouse brain.

The aim of these studies was to demonstrate the therapeutic capacity of an antisense oligonucleotide with the sequence (CUG)7 targeting the expanded CAG repeat in huntingtin (HTT) mRNA in vivo in the R6/2 N-terminal fragment and Q175 knock-in Huntington's disease (HD) mouse models. In a first study, R6/2 mice received six weekly intracerebroventricular infusions with a low and high dose of (CUG)7 and were sacrificed 2 weeks later. A 15-60% reduction of both soluble and aggregated mutant HTT protein was observed in striatum, hippocampus and cortex of (CUG)7-treated mice. This correction at the molecular level resulted in an improvement of performance in multiple motor tasks, increased whole brain and cortical volume, reduced levels of the gliosis marker myo-inositol, increased levels of the neuronal integrity marker N-aceyl aspartate and increased mRNA levels of the striatal marker Darpp-32. These neuroanatomical and neurochemical changes, together with the improved motor performance, suggest that treatment with (CUG)7 ameliorates basal ganglia dysfunction. The HTT-lowering was confirmed by an independent study in Q175 mice using a similar (CUG)7 AON dosing regimen, further demonstrating a lasting reduction of mutant HTT protein in striatum, hippocampus and cortex for up to 18 weeks post last infusion along with an increase in motor activity. Based on these encouraging results, (CUG)7 may thus offer an interesting alternative HTT-lowering strategy for HD.