Agrobiogen (Germany)

Genetic diseases in goats are less well characterized than in other livestock species. This study reports a previously unrecognized congenital neurological disorder in Bunte Deutsche Edelziege goats and identifies its likely genetic cause. Between 2019 and 2023, a dairy goat farm observed 21 kids with congenital vestibular disease. The affected kids displayed pronounced, sometimes exuberant, wide bilateral excursions of the head and neck, delayed standing, wide-based stance, and poor postural control, while remaining alert and physically strong. Motor coordination improved with age. One retained female displayed mild persistent deficits and suspected hearing impairment in adulthood. Postmortem examinations were unremarkable, and infectious or nutritional causes were excluded. The repeated occurrence and pedigree structure suggested an inherited autosomal recessive disorder. Whole-genome sequencing of two affected kids, compared with large control cohorts, revealed a single rare nonsense variant in exon 5 of the PCDH15 gene encoding protocadherin-15. This variant truncates approximately 93% of the protein and is homologous to a pathogenic variant that causes Usher syndrome type 1F in humans. Sanger sequencing confirmed homozygosity in affected animals and heterozygosity in obligate carriers. The variant was absent from 90 unrelated goats of the same breed and over 1600 global controls. These findings indicate a naturally occurring Usher syndrome-like disorder in goats, representing the first such report, and highlight the value of genomic diagnostics for disease prevention and comparative research. The study provides a basis for the molecular diagnosis of similarly affected animals, as well as for genetic testing to prevent further cases.

A multivalent vaccine targeting high-consequence infectious diseases in Sub-Saharan Africa (SSA), which are linked to high mortality, morbidity and overlapping clinical manifestations, would significantly improve health security and economic stability in this region. Trivalent vector vaccines were devised to deliver digitally optimized versions of Orthoebolavirus, Orthomarburgvirus glycoproteins (GPs) and a Lassa mammarenavirus (LASV) nucleoprotein (NP) by a single Modified Vaccinia Ankara (MVA) known to protect against mpox virus (MPXV) along with a matched DNA vaccine. Three immunizations in mice and Hartley guinea pigs with MVA only or a DNA prime followed by two MVA administrations induced comparable levels of binding antibodies and LASV-specific T-cells, respectively. While DNA priming mitigated MVA-specific antibody responses, GP- and NP-specific antibodies developed already after a single MVA vaccination. Although a post-outbreak Ebola virus vaccine is available, outbreaks by other filoviruses, annual LASV epidemics and increased incidence of MPXV infections support the rationale for an MVA-based trivalent haemorrhagic fever vaccine for endemic and high-risk human populations in SSA.

Updates of SARS-CoV-2 vaccines are required to generate immunity in the population against constantly evolving SARS-CoV-2 variants of concerns (VOCs). Here we describe three novel in-silico designed spike-based antigens capable of inducing neutralising antibodies across a spectrum of SARS-CoV-2 VOCs. Three sets of antigens utilising pre-Delta (T2_32), and post-Gamma sequence data (T2_35 and T2_36) were designed. T2_32 elicited superior neutralising responses against VOCs compared to the Wuhan-1 spike antigen in DNA prime-boost immunisation regime in guinea pigs. Heterologous boosting with the attenuated poxvirus - Modified vaccinia Ankara expressing T2_32 induced broader neutralising immune responses in all primed animals. T2_32, T2_35 and T2_36 elicited broader neutralising capacity compared to the Omicron BA.1 spike antigen administered by mRNA immunisation in mice. These findings demonstrate the utility of structure-informed computationally derived modifications of spike-based antigens for inducing broad immune responses covering more than 2 years of evolved SARS-CoV-2 variants.

The delivery of vaccines plays a pivotal role in influencing the strength and longevity of the immune response and controlling reactogenicity. Mucosal immunization, as compared to parenteral vaccination, could offer greater protection against respiratory infections while being less invasive. While oral vaccination has been presumed less effective and believed to target mainly the gastrointestinal tract, trans-buccal delivery using mucoadhesive films (MAF) may allow targeted delivery to the mucosa. Here we present an effective strategy for mucosal delivery of several vaccine platforms incorporated in MAF, including DNA plasmids, viral vectors, and lipid nanoparticles incorporating mRNA (mRNA/LNP). The mRNA/LNP vaccine formulation targeting SARS-CoV-2 as a proof of concept remained stable within MAF consisting of slowly releasing water-soluble polymers and an impermeable backing layer, facilitating enhanced penetration into the oral mucosa. This formulation elicited antibody and cellular responses comparable to the intramuscular injection, but also induced the production of mucosal IgAs, highlighting its efficacy, particularly for use as a booster vaccine and the potential advantage for protection against respiratory infections. The MAF vaccine preparation demonstrates significant advantages, such as efficient delivery, stability, and simple noninvasive administration with the potential to alleviate vaccine hesitancy.

Modified vaccinia virus Ankara is a versatile vaccine vector, well suited for transgene delivery, with an excellent safety profile. However, certain transgenes render recombinant MVA (rMVA) genetically unstable, leading to the accumulation of mutated rMVA with impaired transgene expression. This represents a major challenge for upscaling and manufacturing of rMVA vaccines. To prevent transgene-mediated negative selection, the continuous avian cell line AGE1.CR pIX (CR pIX) was modified to suppress transgene expression during rMVA generation and amplification. This was achieved by constitutively expressing a tetracycline repressor (TetR) together with a rat-derived shRNA in engineered CR pIX PRO suppressor cells targeting an operator element (tetO) and 3’ untranslated sequence motif on a chimeric poxviral promoter and the transgene mRNA, respectively. This cell line was instrumental in generating two rMVA (isolate CR19) expressing a Macaca fascicularis papillomavirus type 3 (MfPV3) E1E2E6E7 artificially-fused polyprotein following recombination-mediated integration of the coding sequences into the DelIII (CR19 M-DelIII) or TK locus (CR19 M-TK), respectively. Characterization of rMVA on parental CR pIX or engineered CR pIX PRO suppressor cells revealed enhanced replication kinetics, higher virus titers and a focus morphology equaling wild-type MVA, when transgene expression was suppressed. Serially passaging both rMVA ten times on parental CR pIX cells and tracking E1E2E6E7 expression by flow cytometry revealed a rapid loss of transgene product after only few passages. PCR analysis and next-generation sequencing demonstrated that rMVA accumulated mutations within the E1E2E6E7 open reading frame (CR19 M-TK) or deletions of the whole transgene cassette (CR19 M-DelIII). In contrast, CR pIX PRO suppressor cells preserved robust transgene expression for up to 10 passages, however, rMVAs were more stable when E1E2E6E7 was integrated into the TK as compared to the DelIII locus. In conclusion, sustained knock-down of transgene expression in CR pIX PRO suppressor cells facilitates the generation, propagation and large-scale manufacturing of rMVA with transgenes hampering viral replication.

Vectored vaccines based on highly attenuated modified vaccinia Ankara (MVA) are reported to be immunogenic, tolerant to pre-existing immunity, and able to accommodate and stably maintain very large transgenes. MVA is usually produced on primary chicken embryo fibroblasts, but production processes based on continuous cell lines emerge as increasingly robust and cost-effective alternatives. An isolate of a hitherto undescribed genotype was recovered by passage of a non-plaque-purified preparation of MVA in a continuous anatine suspension cell line (CR.pIX) in chemically defined medium. The novel isolate (MVA-CR19) replicated to higher infectious titers in the extracellular volume of suspension cultures and induced fewer syncytia in adherent cultures. We now extend previous studies with the investigation of the point mutations in structural genes of MVA-CR19 and describe an additional point mutation in a regulatory gene. We furthermore map and discuss an extensive rearrangement of the left telomer of MVA-CR19 that appears to have occurred by duplication of the right telomer. This event caused deletions and duplications of genes that may modulate immunologic properties of MVA-CR19 as a vaccine vector. Our characterizations also highlight the exceptional genetic stability of plaque-purified MVA: although the phenotype of MVA-CR19 appears to be advantageous for replication, we found that all genetic markers that differentiate wildtype and MVA-CR19 are stably maintained in passages of recombinant viruses based on either wildtype or MVA-CR.

One important aim in cell culture-based viral vaccine and vector production is the implementation of continuous processes. Such a development has the potential to reduce costs of vaccine manufacturing as volumetric productivity is increased and the manufacturing footprint is reduced. In this work, continuous production of Modified Vaccinia Ankara (MVA) virus was investigated. First, a semi-continuous two-stage cultivation system consisting of two shaker flasks in series was established as a small-scale approach. Cultures of the avian AGE1.CR.pIX cell line were expanded in the first shaker, and MVA virus was propagated and harvested in the second shaker over a period of 8–15 days. A total of nine small-scale cultivations were performed to investigate the impact of process parameters on virus yields. Harvest volumes of 0.7–1 L with maximum TCID50 titers of up to 1.0×109 virions/mL were obtained. Genetic analysis of control experiments using a recombinant MVA virus containing green-fluorescent-protein suggested that the virus was stable over at least 16 d of cultivation. In addition, a decrease or fluctuation of infectious units that may indicate an excessive accumulation of defective interfering particles was not observed. The process was automated in a two-stage continuous system comprising two connected 1 L stirred tank bioreactors. Stable MVA virus titers, and a total production volume of 7.1 L with an average TCID50 titer of 9×107 virions/mL was achieved. Because titers were at the lower range of the shake flask cultivations potential for further process optimization at large scale will be discussed. Overall, MVA virus was efficiently produced in continuous and semi-continuous cultivations making two-stage stirred tank bioreactor systems a promising platform for industrial production of MVA-derived recombinant vaccines and viral vectors.

Subunit vaccines often require adjuvants to elicit sustained immune activity. Here, a method is described to evaluate the efficacy of single vaccine candidates in the preclinical stage based on cytokine and gene expression analysis. As a model, the recombinant human respiratory syncytial virus (RSV) fusion protein (RSV-F) was produced in CHO cells. For comparison, wild-type and glycoengineered, afucosylated RSV-F were established. Both glycoprotein vaccines were tested in a commercial Human Artificial Lymph Node in vitro model (HuALN®). The analysis of six key cytokines in cell culture supernatants showed well-balanced immune responses for the afucosylated RSV-F, while immune response of wild-type RSV-F was more Th1 accentuated. In particular, stronger and specific secretion of interleukin-4 after each round of re-stimulation underlined higher potency and efficacy of the afucosylated vaccine candidate. Comprehensive gene expression analysis by nCounter gene expression assay confirmed the stronger onset of the immunologic reaction in stimulation experiments with the afucosylated vaccine in comparison to wild-type RSV-F and particularly revealed prominent activation of Th17 related genes, innate immunity, and comprehensive activation of humoral immunity. We, therefore, show that our method is suited to distinguish the potency of two vaccine candidates with minor structural differences.