Nanobodies to multiple spike variants and inhalation of nanobody-containing aerosols neutralize SARS-CoV-2 in cell culture and hamsters.

The ongoing threat of COVID-19 has highlighted the need for effective prophylaxis and convenient therapies, especially for outpatient settings. We have previously developed highly potent single-domain (VHH) antibodies, also known as nanobodies, that target the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein and neutralize the Wuhan strain of the virus. In this study, we present a new generation of anti-RBD nanobodies with superior properties. The primary representative of this group, Re32D03, neutralizes Alpha to Delta as well as Omicron BA.2.75; other members neutralize, in addition, Omicron BA.1, BA.2, BA.4/5, and XBB.1. Crystal structures of RBD-nanobody complexes reveal how ACE2-binding is blocked and also explain the nanobodies' tolerance to immune escape mutations. Through the cryo-EM structure of the Ma16B06-BA.1 Spike complex, we demonstrated how a single nanobody molecule can neutralize a trimeric spike. We also describe a method for large-scale production of these nanobodies in Pichia pastoris, and for formulating them into aerosols. Exposing hamsters to these aerosols, before or even 24 h after infection with SARS-CoV-2, significantly reduced virus load, weight loss and pathogenicity. These results show the potential of aerosolized nanobodies for prophylaxis and therapy of coronavirus infections.

A nanobody toolbox to investigate localisation and dynamics of Drosophila titins and other key sarcomeric proteins.

Measuring the positions and dynamics of proteins in intact tissues or whole animals is key to understanding protein function. However, to date, this is challenging, as the accessibility of large antibodies to dense tissues is often limited, and fluorescent proteins inserted close to a domain of interest may affect protein function. These complications apply in particular to muscle sarcomeres, arguably one of the most protein-dense assemblies in nature, which complicates studying sarcomere morphogenesis at molecular resolution. Here, we introduce a toolbox of nanobodies recognising various domains of the two Drosophila titin homologs, Sallimus and Projectin, as well as the key sarcomeric proteins Obscurin, α-Actinin, and Zasp52. We verified the superior labelling qualities of our nanobodies in muscle tissue as compared to antibodies. By applying our toolbox to larval muscles, we found a gigantic Sallimus isoform stretching more than 2 µm to bridge the sarcomeric I-band, while Projectin covers almost the entire myosin filaments in a polar orientation. Transgenic expression of tagged nanobodies confirmed their high affinity-binding without affecting target protein function. Finally, adding a degradation signal to anti-Sallimus nanobodies suggested that it is difficult to fully degrade Sallimus in mature sarcomeres; however, expression of these nanobodies caused developmental lethality. These results may inspire the generation of similar toolboxes for other large protein complexes in Drosophila or mammals.

Our muscles are not just for lifting weights. They also keep us alive. For example, our heartbeat is powered by the muscles in the heart wall. Just like other organs in the body, muscles are made up of cells called muscle fibres. Each muscle fibre is divided into many smaller units, or 'sarcomeres', which contain specialised proteins that pull on each other to produce muscle contractions. Although the structure of mature muscles is rather well understood, we know much less about how muscles develop or how they are maintained throughout adult life. Understanding this is especially important in the case of the heart, because its muscle cells are not replaced throughout our lives. Instead, the heart muscle cells we are born with are maintained as we age while working continuously. This means that the proteins within the heart muscle sarcomeres are continuously under mechanical stress and may need to be repaired. How this repair might happen is not well understood. Nanobodies are very small versions of antibodies that recognise and bind to specific protein targets. In biological research, they are used as a tool to observe proteins of interest within cells. This is done by labelling nanobodies, for example, with chemical fluorophores or fluorescent proteins; once labelled, the nanobody binds to its target protein, and scientists can monitor its location and behaviour within the cell. Cells, and even flies, can also be genetically manipulated to produce labelled nanobodies themselves, which has the advantage of visualising the dynamic behaviour of the target protein in the living cell or organism. To better study the proteins in muscle cells, scientists from two different research groups developed a nanobody 'toolbox' that specifically targets sarcomere proteins. First, Loreau et al. made a 'library' of labelled nanobodies targeting different sarcomere proteins in Drosophila melanogaster fruit flies. Second, they used this library of nanobodies to locate several sarcomere proteins in the mature sarcomeres of different fly muscles. Third, using flies that had been genetically altered to produce the labelled nanobodies in their muscle cells, Loreau et al. were able to observe the behaviour of the target proteins in the living muscle. Together, these experiments showed that one protein in Drosophila that is similar to the human sarcomere protein titin has a similar size to the human version, whereas a second Drosophila titin-like protein is shorter and located at a different place in the sarcomere. Both of these proteins work together to stabilise muscle fibres, which is also the role of human titin. The nanobodies generated here are a significant contribution to the tools available to study muscle development and maintenance. Loreau et al. hope that they will help reveal how sarcomere proteins like titin are maintained, especially in the heart, and ultimately how the heart muscle manages to continue working throughout our lives.

Neutralization of SARS-CoV-2 by highly potent, hyperthermostable, and mutation-tolerant nanobodies.

Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein, we isolated 45 infection-blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS-CoV-2 at 17-50 pM concentration (0.2-0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X-ray and cryo-EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune-escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low-picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such "fold-promoting" nanobodies may allow for simplified production of vaccines and their adaptation to viral escape-mutations.

Genotoxic stress increases cytoplasmic mitochondrial DNA editing by human APOBEC3 mutator enzymes at a single cell level.

Human cells are stressed by numerous mechanisms that can lead to leakage of mitochondrial DNA (mtDNA) to the cytoplasm and ultimately apoptosis. This agonist DNA constitutes a danger to the cell and is counteracted by cytoplasmic DNases and APOBEC3 cytidine deamination of DNA. To investigate APOBEC3 editing of leaked mtDNA to the cytoplasm, we performed a PCR analysis of APOBEC3 edited cytoplasmic mtDNA (cymtDNA) at the single cell level for primary CD4+ T cells and the established P2 EBV blast cell line. Up to 17% of primary CD4+ T cells showed signs of APOBEC3 edited cymtDNA with ~50% of all mtDNA sequences showing signs of APOBEC3 editing - between 1500-5000 molecules. Although the P2 cell line showed a much lower frequency of stressed cells, the number of edited mtDNA molecules in such cells was of the same order. Addition of the genotoxic molecules, etoposide or actinomycin D increased the number of cells showing APOBEC3 edited cymtDNA to around 40%. These findings reveal a very dynamic image of the mitochondrial network, which changes considerably under stress. APOBEC3 deaminases are involved in the catabolism of mitochondrial DNA to circumvent chronic immune stimulation triggered by released mitochondrial DNA from damaged cells.

Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage.

Foreign and self-cytoplasmic DNA are recognized by numerous DNA sensor molecules leading to the production of type I interferons. Such DNA agonists should be degraded otherwise cells would be chronically stressed. Most human APOBEC3 cytidine deaminases can initiate catabolism of cytoplasmic mitochondrial DNA. Using the human myeloid cell line THP-1 with an interferon inducible APOBEC3A gene, we show that cytoplasmic DNA triggers interferon α and ß production through the RNA polymerase III transcription/RIG-I pathway leading to massive upregulation of APOBEC3A. By catalyzing C→U editing in single stranded DNA fragments, the enzyme prevents them from re-annealing so attenuating the danger signal. The price to pay is chromosomal DNA damage in the form of CG→TA mutations and double stranded DNA breaks which, in the context of chronic inflammation, could drive cells down the path toward cancer.

Increased BST2 expression during simian immunodeficiency virus infection is not a determinant of disease progression in rhesus monkeys.

BACKGROUND: Bone marrow stromal cell antigen 2 (BST2), also known as tetherin, HM1.24 or CD317 represents a type 2 integral membrane protein, which has been described to restrict the production of some enveloped viruses by inhibiting the virus release from the cell surface. This innate antiviral mechanism is counteracted by the HIV-1 viral factor Vpu, targeting BST2 for cellular degradation. Since antiviral BST2 activity has been mainly confirmed by in vitro data, we investigated its role in vivo on the disease progression using the SIV/macaque model for AIDS. We determined BST2 expression in PBMC and leukocyte subsets of uninfected and SIV-infected rhesus macaques by real-time PCR and flow cytometry and correlated it with disease progression and viral load. RESULTS: Compared to pre-infection levels, we found increased BST2 expression in PBMC, purified CD4(+) lymphocytes and CD14(+) monocytes of SIV-infected animals, which correlated with viral load. Highest BST2 levels were found in progressors and lowest levels comparable to uninfected macaques were observed in long-term non-progressors (LTNPs). During acute viremia, BST2 mRNA increased in parallel with MX1, a prototype interferon-stimulated gene. This association was maintained during the whole disease course. CONCLUSION: The detected relationship between BST2 expression and viral load as well as with MX1 indicate a common regulation by the interferon response and suggest rather limited influence of BST2 in vivo on the disease outcome.

Human APOBEC3A isoforms translocate to the nucleus and induce DNA double strand breaks leading to cell stress and death.

Human APOBEC3 enzymes deaminate single stranded DNA. At least five can deaminate mitochondrial DNA in the cytoplasm, while three can deaminate viral DNA in the nucleus. However, only one, APOBEC3A, can hypermutate genomic DNA. We analysed the distribution and function of the two APOBEC3A isoforms p1 and p2 in transfected cell lines. Both can translocate to the nucleus and hypermutate CMYC DNA and induce DNA double strand breaks as visualized by the detection of ©H2AX or Chk2. APOBEC3A induced G1 phase cell cycle arrest and triggered several members of the intrinsic apoptosis pathway. Activation of purified human CD4+ T lymphocytes with PHA, IL2 and interferon α resulted in C->T hypermutation of genomic DNA and double stranded breaks suggesting a role for APOBEC3A in pro-inflammatory conditions. As chronic inflammation underlies many diseases including numerous cancers, it is possible that APOBEC3A induction may generate many of the lesions typical of a cancer genome.

Human Tribbles 3 protects nuclear DNA from cytidine deamination by APOBEC3A.

The human polydeoxynucleotide cytidine deaminases APOBEC3A, APOBEC3C, and APOBEC3H are capable of mutating viral DNA in the nucleus, whereas APOBEC3A alone efficiently edits nuclear DNA. Deamination is rapidly followed by excision of uracil residues and can lead to double-stranded breaks. It is not known to which protein networks these DNA mutators belong. Using a yeast two-hybrid screen, we identified the human homolog of Drosophila Tribbles 3, TRIB3, as an interactor for APOBEC3A and APOBEC3C. The interaction was confirmed by co-affinity purification. Co-transfection of APOBEC3A with a TRIB3 expression vector reduced nuclear DNA editing whereas siRNA knockdown of TRIB3 increased the levels of nuclear DNA editing, indicating that TRIB3 functioned as a repressor of A3A. It also repressed A3A-associated γH2AX positive double-stranded breaks. The interaction results in degradation of A3A in a proteasome-independent manner. TRIB3 has been linked to cancer and via its own interactors and links the A3A DNA mutators to the Rb-BRCA1-ATM network. TRIB3 emerges as an important guardian of genome integrity.

Increased APOBEC3G and APOBEC3F expression is associated with low viral load and prolonged survival in simian immunodeficiency virus infected rhesus monkeys.

BACKGROUND: The cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) are innate cellular factors that inhibit replication of a number of viruses, including HIV-1. Since antiviral activity of APOBEC3 has been mainly confirmed by in vitro data, we examined their role for disease progression in the SIV/macaque model for AIDS. RESULTS: We quantified A3G and A3F mRNA in PBMC and leukocyte subsets of uninfected and SIVmac-infected rhesus macaques. Compared with uninfected animals, we found increased A3G and A3F mRNA levels in PBMC, purified CD4+ T-cells and CD14+ monocytes as well as lymph node cells from asymptomatic SIV-infected macaques. APOBEC3 mRNA levels correlated negatively with plasma viral load, and highest amounts of APOBEC3 mRNA were detected in long term non-progressors (LTNPs). During acute viremia, A3G mRNA increased in parallel with MxA, a prototype interferon-stimulated gene indicating a common regulation by the initial interferon response. This association disappeared during the asymptomatic stage. CONCLUSION: Our findings suggest a protective effect of APOBEC3 for HIV and SIV in vivo and indicate regulation of APOBEC3 by interferon during early infection and by contribution of other, hitherto undefined factors at later disease stages. Elucidating the regulatory mechanisms leading to increased APOBEC3 mRNA levels in LTNPs could help to develop new therapies against HIV.

The possible role of the Jak/STAT pathway in lymphocytes at the fetomaternal interface.

Pregnancy is accompanied by a Th2-prone immune modulation, which is a major puzzle piece among maternofetal tolerance-promoting factors. A large number of cytokines is physiologically or pathologically present in the decidua and is potentially able to act on lymphocytes and NK cells, which express a variety of respective receptors. Intracellular signals from these receptors are to a major part transduced via the Janus kinases (JAK) and signal transducers and activators of a transcription (STAT) system, which consists of at least 4 different kinases and 7 STATs plus several subtypes and splicing variants. A network of suppressors of cytokine signaling (SOCS) controls their balance. The interactions of all these intracellular factors and cross-linking with further signaling systems seem to be crucial for the maintenance of a maternal cytokine profile which promotes the tolerance of the fetus.