Biology subjects

Saksela, K.

Publications and source records attributed to Saksela, K..

6 recordsLinked to original sources

bioRxiv2024

Altered spike dynamics drives immune evasion of SARS-CoV-2

Escape from protective immunity has been a defining feature of the COVID-19 pandemic and is widely attributed to mutations in neutralizing antibody epitopes within the receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. We investigated whether this paradigm accounts for the escape of Omicron lineage viruses from broadly neutralizing antibodies (bNAbs) targeting conserved RBD epitopes. We found that mutational erosion of bNAb binding epitopes was not a major driver of Omicron immune escape. Viruses carrying RBDs from highly resistant contemporary Omicron variants were potently neutralized by bNAbs when those RBDs were placed into an ancestral Wuhan-like spike backbone. Similarly, sera collected during the pre-Omicron era from individuals vaccinated with Wuhan-based vaccines retained robust RBD-specific neutralizing activity against these chimeric viruses. Mechanistically, bNAb treatment triggered exposure of the S2 cleavage site in neutralization-sensitive but not resistant spike proteins, indicating that bNAb susceptibility reflects differences in spike propensity for antibody-induced premature conformational transition. These findings show that, rather than escaping through mutation of bNAb epitopes, SARS-CoV-2 has evolved spike variants with altered conformational dynamics that resist bNAb-induced destabilization of the prefusion spike trimer.

immunology↗

bioRxiv2023

Structural insight into rabies virus neutralization revealed by an engineered antibody scaffold

Host-cell entry of the highly pathogenic rabies virus (RABV) is mediated by trimeric glycoprotein (G) spikes, which also represent the primary target for the humoral immune response. RABV-G displays several antigenic sites targeted by neutralizing antibodies, including monoclonal antibodies (mAbs) which have been proposed as quality-controlled alternatives to traditional polyclonal rabies immunoglobulin treatment. In this study, we determine the epitope of a potently neutralizing human anti-rabies mAb, CR57, which we engineered into a diabody to facilitate crystallization. We report the crystal structure of the CR57 diabody alone at 2.38 [A] resolution, and in complex with RABV-G domain III at 3.15 [A] resolution. CR57 is demonstrated to bind RABV through a predominantly hydrophobic interface, with essential interactions targeting a conserved six-residue peptide sequence KLCGVL on the RABV-G. Further, our structural analysis suggests that CR57 sterically hinders receptor recognition and the fusogenic transitions of the spike protein that are required for host-cell entry. Altogether, this investigation provides a structural perspective on rabies inhibition by a potent antibody and delineates a functionally significant region in the spike. This understanding could pave the way for the development of prophylactic antibodies and other therapeutic strategies. Author summaryRabies virus (RABV) and many other lyssaviruses possess the ability to invade the central nervous system, leading to fatal encephalitis in mammals. Initiation of the infectious cycle depends on host cell recognition and entry, which is mediated by viral surface glycoprotein (G) spikes and can be inhibited by spike-targeting neutralizing antibodies. In our study, we elucidated the crystal structure of an antigenic domain from RABV-G in complex with a diabody derived from the potently neutralizing antibody CR57. This investigation revealed the molecular interactions by which CR57 binds to RABV-G and outlined a site of vulnerability comprising a conserved peptide in RABV-G domain III, where antibody binding is likely to inhibit RABV by obstructing host cell entry. Insights into the binding modalities of antibodies like CR57 deepen our understanding of how RABV and other lyssaviruses are neutralized, aiding the development of potential therapeutics. Furthermore, our study showcases the utility of engineering antibodies into diabodies to obtain crystal structures of antibody-antigen complexes.

molecular biology↗

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↗

bioRxiv2021

Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV 1/SIVcpz lentiviral lineage

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy - termed the "R-clamp" - that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution. AUTHOR SUMMARYViral replication depends on interactions with a plethora of host cell proteins. Cellular protein interactions are typically mediated by specialized binding modules, such as the SH3 domain. To gain access to host cell regulation viruses have evolved to contain SH3 domain binding sites in their proteins, a notable example of which is the HIV-1 Nef protein. Here we show that during the primate lentivirus evolution the structural strategy that underlies the avid binding of Nef to cellular SH3 domains, which we have dubbed the R-clamp, has been generated via alternative but functionally interchangeable molecular designs. These patterns of SH3 recognition depend on the amino acid combinations at the positions corresponding to residues 83 and 120 in the consensus HIV-1 Nef sequence, and are distinctly different in Nef proteins from SIVs of Eastern and Central chimpanzees, gorillas, and the four groups of HIV-1 that have independently originated from the latter two. These results highlight the evolutionary plasticity of viral proteins, and have implications on therapeutic development aiming to interfere with SH3 binding of Nef.

microbiology↗

bioRxiv2021

Antidepressant and antipsychotic drugs reduce viral infection by SARS-CoV-2 and fluoxetine show antiviral activity against the novel variants in vitro

Background and PurposeRepurposing of currently available drugs is a valuable strategy to tackle the consequences of COVID-19. Recently, several studies have investigated the effect of psychoactive drugs on SARS-CoV-2 in cell culture models as well as in clinical practice. Our aim was to expand these studies and test some of these compounds against newly emerged variants. Experimental ApproachSeveral antidepressant drugs and antipsychotic drugs with different primary mechanisms of action were tested in ACE2/TMPRSS2-expressing human embryonic kidney cells against the infection by SARS-CoV-2 spike protein-dependent pseudoviruses. Some of these compounds were also tested in human lung epithelial cell line, Calu-1, against the first wave (B.1) lineage of SARS-CoV-2 and the variants of concern, B.1.1.7 and B.1.351. Key ResultsSeveral clinically used antidepressants, including fluoxetine, citalopram, reboxetine, imipramine, as well as antipsychotic compounds chlorpromazine, flupenthixol, and pimozide inhibited the infection by pseudotyped viruses with minimal effects on cell viability. The antiviral action of several of these drugs was verified in Calu-1 cells against the (B.1) lineage of SARS-CoV-2. By contrast, the anticonvulsant carbamazepine, and novel antidepressants ketamine and its derivatives as well as MAO and phosphodiesterase inhibitors phenelzine and rolipram, respectively, showed no activity in the pseudovirus model. Furthermore, fluoxetine remained effective against pseudo viruses with N501Y, K417N, and E484K spike mutations, and the VoC-1 (B.1.1.7) and VoC-2 (B.1.351) variants of SARS-CoV-2. Conclusion and ImplicationsOur study confirms previous data and extends information on the repurposing of these drugs to counteract SARS-CoV-2 infection including different variants of concern.

pharmacology and toxicology↗

bioRxiv2021

T cell circuits that sense antigen density with an ultrasensitive threshold

Overexpressed tumor associated antigens (e.g. HER2 and EGFR) are attractive targets for therapeutic T cells, but toxic cross-reaction with normal tissues expressing low antigen levels has been observed with Chimeric Antigen Receptor (CAR) T cells targeting such antigens. Inspired by natural ultrasensitive response circuits, we engineer a two-step positive feedback circuit that allows T cells to discriminate targets based on a sigmoidal antigen density threshold. In this circuit, a low affinity SynNotch receptor for HER2 controls the expression of a high affinity CAR for HER2. Increasing HER2 density thus has cooperative effects on T cells it both increases CAR expression and activation leading to a sigmoidal response. T Cells with this circuit show sharp discrimination between target cells expressing normal and disease levels of HER2, both in vitro and in vivo. One Sentence SummaryA two-step positive feedback circuit generates engineered T cells capable of killing target cells with an ultrasensitive antigen density threshold.

synthetic biology↗