Phenotypic lentivirus screens to identify functional single domain antibodies

Manipulation of proteins is key in assessing their in vivo function. Although genetic ablation is straightforward, reversible and specific perturbation of protein function remains a challenge. Single domain antibody fragments, such as camelid-derived VHHs, can serve as inhibitors or activators of intracellular protein function, but functional testing of identified VHHs is laborious. To address this challenge, we have developed a lentiviral screening approach to identify VHHs that elicit a phenotype when expressed intracellularly. We identified 19 antiviral VHHs that protect human A549 cells from lethal infection with influenza A virus (IAV) or vesicular stomatitis virus (VSV), respectively. Both negative-sense RNA viruses are vulnerable to VHHs uniquely specific for their respective nucleoproteins. Antiviral VHHs prevented nuclear import of viral ribonucleoproteins or mRNA transcription, respectively, and may provide clues for novel antiviral reagents. In principle, the screening approach described here should be applicable to identify inhibitors of any pathogen or biological pathway. To identify the proteins essential to a biological pathway, small-molecule inhibitors or activators may be used to manipulate protein function transiently. Alternatively, screens involving mutagenesis, a reduction in levels or complete elimination of gene products are common. As applied to mammalian cells, these methods usually seek to achieve the removal of a protein from its normal biological context. Many proteins are multifunctional, or are components of multi-subunit complexes. Depletion of any single component may cause unexpected phenotypes due to the collapse of entire protein complexes. Small-molecule inhibitors often lack specificity and at best can target a fraction of all the proteins of interest. The screening of chemically diverse libraries must be paired with sophisticated methods to identify the molecular targets of any hit identified. Antibodies have been used as intracellular perturbants of protein function after microinjection or cytosolic expression of single-chain variable antibody fragments, but technical challenges have limited their application to few selected cases. In addition to conventional antibodies, the immune system of camelids generates heavy-chain-only antibodies. Their antigen binding site only consists of the variable domain of the heavy chain. This domain can be expressed on its own and is referred to as a VHH or nanobody, an entity that can retain its function in the reducing environment of the cytosol, independent of glycosylation. Many VHHs bind to their targets with affinities comparable to conventional antibodies. VHHs expressed in the cytosol can therefore act as molecular perturbants by occluding the interfaces involved in protein–protein interactions, by binding in the active sites of enzymes, or through the recognition or stabilization of distinct conformations of their targets. Both phage and yeast display, as well as mass spectrometry in combination with high-throughput sequencing, allow the identification of VHHs based on their binding properties. However, the identification of inhibitory VHHs remains a time-consuming process. VHHs obtained through biochemical screening methods must be expressed individually in the relevant cell type to test for the functional consequences of VHH expression. To address this challenge, we developed a phenotypic VHH screening method in living cells.National Institutes of Health (U.S.) (Health Pioneer Award

Immunoglobulin germline gene polymorphisms influence the function of SARS-CoV-2 neutralizing antibodies

The human immunoglobulin heavy-chain (IGH) locus is exceptionally polymorphic, with high levels of allelic and structural variation. Thus, germline IGH genotypes are personal, which may influence responses to infection and vaccination. For an improved understanding of inter-individual differences in antibody responses, we isolated SARS-CoV-2 spike-specific monoclonal antibodies from convalescent health care workers, focusing on the IGHV1-69 gene, which has the highest level of allelic variation of all IGHV genes. The IGHV1-69∗20-using CAB-I47 antibody and two similar antibodies isolated from an independent donor were critically dependent on allele usage. Neutralization was retained when reverting the V region to the germline IGHV1-69∗20 allele but lost when reverting to other IGHV1-69 alleles. Structural data confirmed that two germline-encoded polymorphisms, R50 and F55, in the IGHV1-69 gene were required for high-affinity receptor-binding domain interaction. These results demonstrate that polymorphisms in IGH genes can influence the function of SARS-CoV-2 neutralizing antibodies

A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo

Experiments with replication-competent SARS-CoV-2 were performed in the Biomedicum BSL3 core facility, Karolinska Institutet. We thank Jonas Klingström for providing Calu-3 cells and sharing the Swedish SARS-CoV-2 isolate, and Alex Sigal from the Africa Health Research Institute for providing the beta variant (B.1.351/501Y.V2) isolate. We thank Penny Moore and the NICD (South Africa) for providing the B.1.351/beta variant spike plasmid, which was generated using funding from the South African Medical Research Council. We gratefully acknowledge the G2P-UK National Virology consortium funded by MRC/UKRI (grant ref: MR/W005611/1.) and the Barclay Lab at Imperial College for providing the B.1.617.2 spike plasmid. All cryo-EM data were collected in the Karolinska Institutet’s 3D-EM facility. We thank Agustin Ure for assistance with figure generation and Tomas Nyman (Protein Science Facility at KI) for providing access to SPR instruments. L.H. was supported by the David och Astrid Hageléns stiftelse, the Clas Groschinskys Minnesfond and a Jonas Söderquist’s scholarship. This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 101003653 (CoroNAb), to B.M. and G.M.M. B.M.H. is supported by the Knut and Alice Wallenberg Foundation (KAW 2017.0080 and KAW 2018.0080). The work was supported by project grants from the Swedish Research Council to E.S. (2020-02682), B.M.H. (2017-6702 and 2018-3808), B.M. (2018-02381) and to G.M.M. (2018-03914 and 2018-03843). E.S. is supported by Karolinska Institutet Foundation Grants, National Molecular Medicine Program Grants, and the grants from the SciLifeLab National COVID-19 Research Program, financed by the Knut and Alice Wallenberg Foundation. We thank National Microscopy Infrastructure, NMI (VR-RFI 2016-00968).N

Theory of exciton-exciton correlation in nonlinear optical response

We present a systematic theory of Coulomb interaction effects in the nonlinear optical processes in semiconductors using a perturbation series in the exciting laser field. The third-order dynamical response consists of phase-space filling correction, mean-field exciton-exciton interaction, and two-exciton correlation effects expressed as a force-force correlation function. The theory provides a unified description of effects of bound and unbound biexcitons, including memory-effects beyond the Markovian approximation. Approximations for the correlation function are presented.Comment: RevTex, 35 pages, 10 PostScript figs, shorter version submitted to Physical Review

A Method for Efficient Calculation of Diffusion and Reactions of Lipophilic Compounds in Complex Cell Geometry

A general description of effects of toxic compounds in mammalian cells is facing several problems. Firstly, most toxic compounds are hydrophobic and partition phenomena strongly influence their behaviour. Secondly, cells display considerable heterogeneity regarding the presence, activity and distribution of enzymes participating in the metabolism of foreign compounds i.e. bioactivation/biotransformation. Thirdly, cellular architecture varies greatly. Taken together, complexity at several levels has to be addressed to arrive at efficient in silico modelling based on physicochemical properties, metabolic preferences and cell characteristics. In order to understand the cellular behaviour of toxic foreign compounds we have developed a mathematical model that addresses these issues. In order to make the system numerically treatable, methods motivated by homogenization techniques have been applied. These tools reduce the complexity of mathematical models of cell dynamics considerably thus allowing to solve efficiently the partial differential equations in the model numerically on a personal computer. Compared to a compartment model with well-stirred compartments, our model affords a more realistic representation. Numerical results concerning metabolism and chemical solvolysis of a polycyclic aromatic hydrocarbon carcinogen show good agreement with results from measurements in V79 cell culture. The model can easily be extended and refined to include more reactants, and/or more complex reaction chains, enzyme distribution etc, and is therefore suitable for modelling cellular metabolism involving membrane partitioning also at higher levels of complexity

A Novel Vaccine Strategy Employing Serologically Different Chimpanzee Adenoviral Vectors for the Prevention of HIV-1 and HCV Coinfection

Background: Nearly 3 million people worldwide are coinfected with HIV and HCV. Affordable strategies for prevention are needed. We developed a novel vaccination regimen involving replication-defective and serologically distinct chimpanzee adenovirus (ChAd3, ChAd63) vector priming followed by modified vaccinia Ankara (MVA) boosts, for simultaneous delivery of HCV non-structural (NSmut) and HIV-1 conserved (HIVconsv) region immunogens.Methods: We conducted a phase I trial in which 33 healthy volunteers were sequentially enrolled and vaccinated via the intramuscular route as follows: 9 received ChAd3-NSmut [2.5 × 1010 vp] and MVA-NSmut [2 × 108 pfu] at weeks 0 and 8, respectively; 8 received ChAdV63.HIVconsv [5 × 1010 vp] and MVA.HIVconsv [2 × 108 pfu] at the same interval; 16 were co-primed with ChAd3-NSmut [2.5 × 1010 vp] and ChAdV63.HIVconsv [5 × 1010 vp] followed at week 8 by MVA-NSmut and MVA.HIVconsv [both 1 × 108 pfu]. Immunogenicity was assessed using peptide pools in ex vivo ELISpot and intracellular cytokine assays. Vaccine-induced whole blood transcriptome changes were assessed by microarray analysis.Results: All vaccines were well tolerated and no vaccine-related serious adverse events occurred. Co-administration of the prime-boost vaccine regimens induced high magnitude and broad T cell responses that were similar to those observed following immunization with either regimen alone. Median (interquartile range, IQR) peak responses to NSmut were 3,480 (2,728–4,464) and 3,405 (2,307–7,804) spot-forming cells (SFC)/106 PBMC for single and combined HCV vaccinations, respectively (p = 0.8). Median (IQR) peak responses to HIVconsv were 1,305 (1,095–4,967) and 1,005 (169–2,482) SFC/106 PBMC for single and combined HIV-1 vaccinations, respectively (p = 0.5). Responses were maintained above baseline to 34 weeks post-vaccination. Intracellular cytokine analysis indicated that the responding populations comprised polyfunctional CD4+ and CD8+ T cells. Canonical pathway analysis showed that in the single and combined vaccination groups, pathways associated with antiviral and innate immune responses were enriched for upregulated interferon-stimulated genes 24 h after priming and boosting vaccinations.Conclusions: Serologically distinct adenoviral vectors encoding HCV and HIV-1 immunogens can be safely co-administered without reducing the immunogenicity of either vaccine. This provides a novel strategy for targeting these viruses simultaneously and for other pathogens that affect the same populations.Clinical trial registration:https://clinicaltrials.gov, identifier: NCT0236221

Probabilistic classification of anti-SARS-CoV-2 antibody responses improves seroprevalence estimates.

OBJECTIVES: Population-level measures of seropositivity are critical for understanding the epidemiology of an emerging pathogen, yet most antibody tests apply a strict cutoff for seropositivity that is not learnt in a data-driven manner, leading to uncertainty when classifying low-titer responses. To improve upon this, we evaluated cutoff-independent methods for their ability to assign likelihood of SARS-CoV-2 seropositivity to individual samples. METHODS: Using robust ELISAs based on SARS-CoV-2 spike (S) and the receptor-binding domain (RBD), we profiled antibody responses in a group of SARS-CoV-2 PCR+ individuals (n = 138). Using these data, we trained probabilistic learners to assign likelihood of seropositivity to test samples of unknown serostatus (n = 5100), identifying a support vector machines-linear discriminant analysis learner (SVM-LDA) suited for this purpose. RESULTS: In the training data from confirmed ancestral SARS-CoV-2 infections, 99% of participants had detectable anti-S and -RBD IgG in the circulation, with titers differing > 1000-fold between persons. In data of otherwise healthy individuals, 7.2% (n = 367) of samples were of uncertain serostatus, with values in the range of 3-6SD from the mean of pre-pandemic negative controls (n = 595). In contrast, SVM-LDA classified 6.4% (n = 328) of test samples as having a high likelihood (> 99% chance) of past infection, 4.5% (n = 230) to have a 50-99% likelihood, and 4.0% (n = 203) to have a 10-49% likelihood. As different probabilistic approaches were more consistent with each other than conventional SD-based methods, such tools allow for more statistically-sound seropositivity estimates in large cohorts. CONCLUSION: Probabilistic antibody testing frameworks can improve seropositivity estimates in populations with large titer variability

SARS-CoV-2 spike HexaPro formulated in aluminium hydroxide and administered in an accelerated vaccination schedule partially protects Syrian Hamsters against viral challenge despite low neutralizing antibody responses

SARS-CoV-2 continues to pose a threat to human health as new variants emerge and thus a diverse vaccine pipeline is needed. We evaluated SARS-CoV-2 HexaPro spike protein formulated in Alhydrogel® (aluminium oxyhydroxide) in Syrian hamsters, using an accelerated two dose regimen (given 10 days apart) and a standard regimen (two doses given 21 days apart). Both regimens elicited spike- and RBD-specific IgG antibody responses of similar magnitude, but in vitro virus neutralization was low or undetectable. Despite this, the accelerated two dose regimen offered reduction in viral load and protected against lung pathology upon challenge with homologous SARS-CoV-2 virus (Wuhan-Hu-1). This highlights that vaccine-induced protection against SARS-CoV-2 disease can be obtained despite low neutralizing antibody levels and suggests that accelerated vaccine schedules may be used to confer rapid protection against SARS-CoV-2 disease

Migraine in women: the role of hormones and their impact on vascular diseases

Migraine is a predominantly female disorder. Menarche, menstruation, pregnancy, and menopause, and also the use of hormonal contraceptives and hormone replacement treatment may influence migraine occurrence. Migraine usually starts after menarche, occurs more frequently in the days just before or during menstruation, and ameliorates during pregnancy and menopause. Those variations are mediated by fluctuation of estrogen levels through their influence on cellular excitability or cerebral vasculature. Moreover, administration of exogenous hormones may cause worsening of migraine as may expose migrainous women to an increased risk of vascular disease. In fact, migraine with aura represents a risk factor for stroke, cardiac disease, and vascular mortality. Studies have shown that administration of combined oral contraceptives to migraineurs may further increase the risk for ischemic stroke. Consequently, in women suffering from migraine with aura caution should be deserved when prescribing combined oral contraceptives

Measurement of the tau lepton lifetime

The mean lifetime of the tau lepton is measured in a sample of 25700 tau pairs collected in 1992 with the ALEPH detector at LEP. A new analysis of the 1-1 topology events is introduced. In this analysis, the dependence of the impact parameter sum distribution on the daughter track momenta is taken into account, yielding improved precision compared to other impact parameter sum methods. Three other analyses of the one- and three-prong tau decays are updated with increased statistics. The measured lifetime is 293.5+/-3.1+/-1.7 fs. Including previous (1989-1991) ALEPH measurements, the combined tau lifetime is 293.7+/-2.7+/-1.6 fs