Levels of Fibrinogen Variants Are Altered in Severe COVID-19.

Background Fibrinogen variants as a result of alternative messenger RNA splicing or protein degradation can affect fibrin(ogen) functions. The levels of these variants might be altered during coronavirus disease 2019 (COVID-19), potentially affecting disease severity or the thrombosis risk. Aim To investigate the levels of fibrinogen variants in plasma of patients with COVID-19. Methods In this case-control study, we measured levels of functional fibrinogen using the Clauss assay. Enzyme-linked immunosorbent assays were used to measure antigen levels of total, intact (nondegraded Aα chain), extended Aα chain (α E ), and γ' fibrinogen in healthy controls, patients with pneumococcal infection in the intensive care unit (ICU), ward patients with COVID-19, and ICU patients with COVID-19 (with and without thrombosis, two time points). Results Healthy controls and ward patients with COVID-19 ( n = 10) showed similar fibrinogen (variant) levels. ICU patients with COVID-19 who later did ( n = 19) or did not develop thrombosis ( n = 18) and ICU patients with pneumococcal infection ( n = 6) had higher absolute levels of functional, total, intact, and α E fibrinogen than healthy controls ( n = 7). The relative α E fibrinogen levels were higher in ICU patients with COVID-19 than in healthy controls, while relative γ' fibrinogen levels were lower. After diagnosis of thrombosis, only the functional fibrinogen levels were higher in ICU patients with COVID-19 and thrombosis than in those without, while no differences were observed in the other fibrinogen variants. Conclusion Our results show that severe COVID-19 is associated with increased levels of α E fibrinogen and decreased relative levels of γ' fibrinogen, which may be a cause or consequence of severe disease, but this is not associated with the development of thrombosis.

Fibrinogen γ' promotes host survival during Staphylococcus aureus septicemia in mice.

BACKGROUND: Staphylococcus aureus is a common gram-positive bacterium that is the causative agent for several human diseases, including sepsis. A key virulence mechanism is pathogen binding to host fibrinogen through the C-terminal region of the γ-chain. Previous work demonstrated that FggΔ5 mice expressing mutant fibrinogen γΔ5 lacking a S. aureus binding motif had significantly improved survival following S. aureus septicemia. Fibrinogen γ' is a human splice variant that represents about 10% to 15% of the total fibrinogen in plasma and circulates as a fibrinogen γ'-γ heterodimer (phFibγ'-γ). The fibrinogen γ'-chain is also expected to lack S. aureus binding function. OBJECTIVE: Determine if human fibrinogen γ'-γ confers host protection during S. aureus septicemia. METHODS: Analyses of survival and the host response following S. aureus septicemia challenge in FggΔ5 mice and mice reconstituted with purified phFibγ'-γ or phFibγ-γ. RESULTS: Reconstitution of fibrinogen-deficient or wildtype mice with purified phFibγ'-γ prior to infection provided a significant prolongation in host survival relative to mice reconstituted with purified phFibγ-γ, which was superior to that observed with heterozygous FggΔ5 mice. Improved survival could not be accounted for by quantitative differences in fibrinogen-dependent adhesion or clumping, but phFibγ'-γ-containing mixtures generated notably smaller bacterial aggregates. Importantly, administration of phFibγ'-γ after infection also provided a therapeutic benefit by prolonging host survival relative to administration of phFibγ-γ. CONCLUSION: These findings provide the proof-of-concept that changing the ratio of naturally occurring fibrinogen variants in blood could offer significant therapeutic potential against bacterial infection and potentially other diseases.

Quantification of residual host cell DNA in adenoviral vectors produced on PER.C6 cells.

Recombinant adenoviral vectors for gene therapy and vaccination are routinely prepared on cultures of immortalized cells, allowing the production of vector batches of high titer and consistent quality. Quantification of residual DNA from the producing cell line is part of the purity tests for clinical lots. Stringent guidelines stipulate the maximum acceptable level of DNA per dose of vector, and this quantification is therefore a crucial piece of information for researchers and manufacturers alike. In this paper we describe an optimized assay based on real-time polymerase chain reaction (PCR) for the quantification of residual PER.C6 DNA in recombinant adenoviral vectors. In order to reduce the risk of introducing contaminations and to increase the throughput, the assay was designed to require minimum sample handling. Furthermore, DNA extraction from the samples is not necessary, thereby eliminating the need to account for possible sample losses. We also report the results of the assay qualification, demonstrating that the assay is accurate, precise, and sensitive. Finally, we applied the assay successfully to determine the level of host cell DNA in an adenovirus vector produced on PER.C6 cells throughout a standard purification process. Because of its specifications, we anticipate that the assay can have broad applicability to biologics other than adenoviral vectors produced on PER.C6 cells.