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Kareinen, L.

Publications and source records attributed to Kareinen, L..

7 recordsLinked to original sources
bioRxiv2026

A rational design strategy and validation for protease-resistant fusion-inhibitor antiviral peptides

Peptide-based fusion inhibitors are promising pharmaceuticals in the fight against enveloped viruses relying on membrane fusion for host infection. However, peptide therapeutic applications have long been hindered by their poor stability in vivo. Here, we discovered that peptide inhibitors with the wildtype sequence of the heptad repeat 2 (HR2) domain of the SARS-CoV-2 spike protein are efficiently cleaved by Transmembrane Protease, Serine 2 (TMPRSS2), a key protease involved in the SARS-CoV-2 virus-cell fusion pathway. We then identified the corresponding cleavage sites and designed three protease-resistant peptides using ranking based on deep mutational scanning and natural occurrence. The three candidates all exhibit inhibitory activity in a cell-cell fusion assay. A high-resolution cryo-EM structure of the top candidate, HR2-NHN, bound to its HR1 target reveals the molecular basis for its potent activity. The top candidate of the cell-based screening assay significantly improved efficacy relative to the wildtype peptide when administered 12 h before infection in both an authentic virus-cell infection assay and a mouse assay. More broadly, our results suggest that the design strategies for protease-resistant peptides could be applied to a broad spectrum of other enveloped viruses and pave the way for the development of safe, prophylactic antivirals that can be administered before exposure.

microbiology↗
bioRxiv2025

American mink as an animal model to study SARS-CoV-2 and vaccine response

Selecting a suitable animal model is crucial in understanding infectious diseases and developing vaccines. Here, we developed a receptor-binding domain -based SARS-CoV-2 vaccine with mouse Fc an immunopotentiator in a mink model. Four different variations of the vaccine were tested in groups of 30-31 American mink and followed for IgG and neutralizing antibodies (nAb) up to 27 weeks. Subcutaneous version induced a strong IgG and nAb response within two weeks and was still detectable at 27 weeks. Intranasal version also caused a detectable, although weaker, immune response. A simultaneously given subcutaneous vaccine against virus enteritis, botulism and hemorrhagic pneumonia potentially caused a lower SARS-CoV-2 antibody response, highlighting the need for further studies on co-effects of vaccines. In virus challenge with Alpha variant (B.1.1.7), vaccinated mink had a stronger antibody response than unvaccinated mink. Despite not preventing the infection, vaccinated mink had milder clinical signs and less virus in saliva. Another challenge of unvaccinated mink with Omicron variant showed similar results to alpha (BA.1) variant. Virus RNA was detected in the brain of unvaccinated mink but not vaccinated mink by in situ hybridization, indicating a suitability of mink to study neurological effects of SARS-CoV-2 and potentially long COVID as well. Author summaryFinding a good animal model is very important in studying infectious diseases and their treatment. The recent SARS-CoV2 pandemic highlighted this dilemma. We have developed an animal model based on mink due to their close match to humans in the symptomology of this disease. Such model will enable the study of relative susceptibility, transmission, tissue tropism, complex pathogenesis and long COVID, as well as prophylaxis and vaccines. This model will also help reduce the use of primates in this research. We have further developed a new vaccine for SARS-CoV2 based on the receptor binding domain of the S-protein with an inbuilt immune-enhancer. This vaccine underwent extensive testing in mink to determine response, long term protection, and safety. The vaccine was found to provide excellent titers of neutralizing antibodies with wide range in target variants. It also reduced the severity and duration of visible symptoms in the animals significantly. We propose this vaccine candidate for further study and future commercialization.

microbiology↗
bioRxiv2023

Complete Protection from SARS-CoV-2 Lung Infection in Mice Through Combined Intranasal Delivery of PIKfyve Kinase and TMPRSS2 Protease Inhibitors

Emerging variants of concern of SARS-CoV-2 can significantly reduce the prophylactic and therapeutic efficacy of vaccines and neutralizing antibodies due to mutations in the viral genome. Targeting cell host factors required for infection provides a complementary strategy to overcome this problem since the host genome is less susceptible to variation during the life span of infection. The enzymatic activities of the endosomal PIKfyve phosphoinositide kinase and the serine protease TMPRSS2 are essential to meditate infection in two complementary viral entry pathways. Simultaneous inhibition in cultured cells of their enzymatic activities with the small molecule inhibitors apilimod dimesylate and nafamostat mesylate synergistically prevent viral entry and infection of native SARS-CoV-2 and vesicular stomatitis virus (VSV)-SARS-CoV-2 chimeras expressing the SARS-CoV-2 surface spike (S) protein and of variants of concern. We now report prophylactic prevention of lung infection in mice intranasally infected with SARS-CoV-2 beta by combined intranasal delivery of very low doses of apilimod dimesylate and nafamostat mesylate, in a formulation that is stable for over 3 months at room temperature. Administration of these drugs up to 6 hours post infection did not inhibit infection of the lungs but substantially reduced death of infected airway epithelial cells. The efficiency and simplicity of formulation of the drug combination suggests its suitability as prophylactic or therapeutic treatment against SARS-CoV-2 infection in households, point of care facilities, and under conditions where refrigeration would not be readily available.

microbiology↗
bioRxiv2023

The infectivity of SARS-CoV-2 progeny virions requires the activity of host cell N-myristoyltransferases and it is severely compromised by their inhibition

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused the coronavirus disease 2019 (COVID-19) pandemic, remains a global health concern despite vaccines, neutralizing antibodies, and antiviral drugs. Emerging mutations can reduce the effectiveness of these treatments, suggesting that targeting host cell factors may be a valuable alternative. N-myristoyltransferases (NMT) are essential enzymes for protein N-myristoylation, affecting stability, interaction, localization, and function of numerous proteins. We demonstrate that selective inhibition of host cell NMT decreases SARS-CoV-2 infection by 90% in human lung and primary nasal epithelial cells, and choroid plexus-cortical neuron organoids. NMT inhibition does not affect viral entry, replication or release, but impairs the maturation and incorporation of viral envelope proteins into newly assembled virions, leading to compromised infectivity of released virions. The inhibition of host NMT triggers a Golgi-bypassing pathway for SARS-CoV-2 progeny virion egress, which occurs through endoplasmic reticulum and lysosomal intermediates.

microbiology↗
bioRxiv2022

Nanobody engineering for SARS-CoV-2 neutralization and detection

In response to the ongoing SARS-CoV-2 pandemic, the quest for coronavirus inhibitors has inspired research on a variety of small proteins beyond conventional antibodies, including robust single-domain antibody fragments, nanobodies. Here, we explore the potential of nanobody engineering in the development of antivirals and diagnostic tools. Through fusion of nanobody domains that target distinct binding sites, we engineered multimodular nanobody constructs that neutralize wild-type SARS-CoV-2 and the Alpha and Delta variants with high potency, with IC50 values up to 50 pM. However, we observed a limitation in the efficacy of multimodular nanobodies against the Beta (B.1.351) and Omicron variants (B.1.1.529), underlining the importance of accounting for viral evolution in the design of biologics. To further explore the applications of nanobody engineering in outbreak management, we present a novel detection assay, based on fusions of nanobodies with fragments of NanoLuc luciferase that can detect sub-nanomolar quantities of the SARS-CoV-2 spike protein in a single step. Our work showcases the potential of nanobody engineering to combat emerging infectious disease.

microbiology↗
bioRxiv2021

Intranasal inhibitor blocks Omicron and other variants of SARS-CoV-2

The recent emergence of novel SARS-CoV-2 variants capable of efficiently escaping neutralizing antibodies emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Nasal epithelium is rich in the ACE2 receptor and important for SARS-CoV-2 transmission by supporting early viral replication before seeding to the lung1. Intranasal administration of SARS-CoV-2 neutralizing antibodies or antibody fragments has shown encouraging potential as a protective measure in animal models2-5. However, there remains a dire need for SARS-CoV-2 blocking agents that are less vulnerable to mutational variation in the neutralization epitopes of the viral spike glycoprotein and more economical to produce in large scale. Here we describe TriSb92, a highly manufacturable and extremely stable trimeric human SH3 domain-derived antibody mimetic targeted against a conserved region in the receptor-binding domain of spike. TriSb92 potently neutralizes SARS-CoV-2 and its variants of concern, including Omicron BA.5 as well as the latest and most immunoevasive variants like BF.7, XBB, and BQ.1.1. Intranasal administration of a modest dose of TriSb92 (5 or 50 micrograms) as early as eight hours before a challenge with SARS-CoV-2 efficiently protected mice from infection, and was still effective even when given 4 h after the viral challenge. The target epitope of TriSb92 was defined by cryo-EM, which revealed triggering of a conformational shift in the spike trimer rather than competition for ACE2 binding as the molecular basis of its strong inhibitory action. The high potency and robust biochemical properties of TriSb92 together with the remarkable resistance of its inhibitory action against viral sequence evolution suggest that TriSb92 could be useful as a nasal spray for protecting susceptible individuals from SARS-CoV-2 infection.

microbiology↗