A high-throughput cell- and virus-free assay shows reduced neutralization of SARS-CoV-2 variants by COVID-19 convalescent plasma

The detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies in the serum of an individual indicates previous infection or vaccination. However, it provides limited insight into the protective nature of this immune response. Neutralizing antibodies recognizing the viral spike protein are more revealing, yet their measurement traditionally requires virus- and cell-based systems that are costly, time-consuming, inflexible, and potentially biohazardous. Here, we present a cell-free quantitative neutralization assay based on the competitive inhibition of trimeric SARS-CoV-2 spike protein binding to the angiotensin-converting enzyme 2 (ACE2) receptor. This high-throughput method matches the performance of the gold standard live virus infection assay, as verified with a panel of 206 seropositive donors with varying degrees of infection severity and virus-specific immunoglobulin G titers, achieving 96.7% sensitivity and 100% specificity. Furthermore, it allows for the parallel assessment of neutralizing activities against multiple SARS-CoV-2 spike protein variants of concern. We used our assay to profile serum samples from 59 patients hospitalized with coronavirus disease 2019 (COVID-19). We found that although most sera had high activity against the 2019-nCoV parental spike protein and, to a lesser extent, the alpha (B.1.1.7) variant, only 58% of serum samples could efficiently neutralize a spike protein derivative containing mutations present in the beta (B.1.351) variant. Thus, we have developed an assay that can evaluate effective neutralizing antibody responses to SARS-CoV-2 spike protein variants of concern after natural infection and that can be applied to characterize vaccine-induced antibody responses or to assess the potency of monoclonal antibodies.

Antiviral Defense in Mice Lacking Both Alpha/Beta and Gamma-Interferon Receptors

Michel Aguet

Alpha/beta interferon (IFN) and gamma IFN exert widely overlapping biological effects. Still, mice with individually inactivated alpha/beta or gamma receptors exhibit variably severely reduced resistance to infection and altered immune responses. To investigate to what extent the two IFN systems are functionally redundant, we generated mice with a combined receptor defect (AG129 mice), Like mice with individual mutations, AG129 mice had no apparent anomalies, confirming that in the mouse the IFN system is not essential for normal development. These mice showed an additive phenotype with respect to antiviral defense and exhibited an increased susceptibility to lymphocytic choriomeningitis virus (LCMV) and notably vaccinia virus infection, Because of unlimited replication and subsequent rapid exhaustion of cytotoxic T lymphocyte (CTL) precursors, these mice were unable to mount a CTL response to LCMV. CDSC-mediated immunopathology was absent in LCMV-infected mice, and virus persisted, Vaccinia virus replicated much faster in AG129 mice, and a 10(4)-fold lower dose of vaccinia virus was sufficient to prime these mice, With the normal priming dose of 10(6) PFU, cytopathic effects and overwhelming infection possibly causing partial exhaustion of CTL interfered with the anti-vaccinia virus response. Even though global antiviral immunoglobulin G (IgG) titers were within normal ranges, the IgG subclass distribution was heavily biased toward IgG1.

1995

Role and regulation of the melanization reaction in Drosophila immune response

Olivier Binggeli

In arthropods, the melanization reaction consists in the rapid synthesis of melanin at the site of infection and injury. A key enzyme in the melanization process is phenoloxidase (PO), which catalyzes the oxidation of phenols to quinones that subsequently polymerize into melanin. PO is synthesized under an inactive form called pro-PO (PPO) that is activated upon cleavage by a terminal protease of a serine protease cascade. The main objective of my PhD thesis is to understand the role and regulation of the melanization reaction in the Drosophila immune response. The Drosophila genome encodes three PPOs, two of which, PPO1 and PPO2, are produced by crystal cells, a larval-specific type of hemocyte. I generated flies carrying null mutations in PPO1 and PPO2 as well as PPO1, PPO2 double mutant flies. My results show that both PPO1 and PPO2 contribute to the PO activity in the hemolymph induced by septic injury. While PPO1 is involved in the early delivery of PO activity, PPO2 is stored in crystalline form in the crystal cells and can be deployed at a later phase of the immune response. My genetic analysis also reveals an important role of PPO1 and PPO2 in the resistance of flies to infection with Gram-positive bacteria and fungi. This study definitively establishes the importance of melanization in insect host defense. Serine proteases regulate many immune reactions, including blood clotting, the complement system in mammals, and melanization and Toll pathway activation in insects. A second objective of my thesis is to identify new serine proteases or serine protease inhibitors (serpins) participating in the Drosophila immune response. I performed an RNAi screen to identify genes encoding serine protease or serpins whose inactivation caused an immune phenotype. In parallel, I identified or generated mutations affecting some serine proteases and serpins and described their immune phenotype. This led to the identification of SPH9, a gene strongly induced by microbial infection, which encodes a serine protease homologue that negatively regulates the melanization reaction. Together, my results provide new insights on the melanization reaction in Drosophila.

EPFL2013

Anti-angiogenic immunotherapy for lung cancer

Ece Kadioglu

Lung cancer is the leading cause of cancer-associated deaths worldwide. Platinum-based chemotherapy is the most common therapeutic approach in advanced non-small cell lung cancer (NSCLC), particularly for tumors that carry non-druggable activating mutations. However, these tumors are generally not eradicated when diagnosed at an advanced stage and are frequently resistant to chemotherapy. Therefore, there is an urgent medical need for new treatments for this frequent cancer type. Immunotherapies are treatments based on increasing the strength of immune responses against cancer. In particular, monoclonal antibodies that block inhibitory "immune-checkpoints" expressed on T cells (e.g., programmed cell death protein 1, or PD1) show clinical efficacy in increasing tumor types. However, these therapies improve survival in only a minority of NSCLC patients, and ongoing efforts are being made for increasing their efficacy through combinatorial treatments. In this regard, growing evidence indicates that tumor angiogenesis is closely associated with immunosuppression. Pro-angiogenic factors, such as vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (ANG2), cause abnormalities and dysfunctions in tumor blood vessels that impair T cell extravasation and anti-tumoral activity. Recent findings in preclinical cancer models show that reprogramming the features of tumor-associated blood vessels by anti-angiogenic drugs may help to increase intratumoral T cell abundance and sensitize refractory tumors to immunotherapy. Nevertheless, the potential of anti-angiogenics in the context of lung cancer immunotherapy is poorly understood. Therefore, the main objective of my project was to audit combinations of anti-angiogenic therapy and immune checkpoint blockade in a genetically engineered mouse model of NSCLC. I first studied the anti-tumoral activity of dual VEGFA and ANG2 inhibition using a bispecific antibody (A2V) in the KrasLSL-G12D/+;p53fl/fl (KP) NSCLC model. I found that A2V, but not single VEGFA or ANG2 blockade, had robust tumor-inhibitory activity in KP mice, which was at least comparable to standard-of-care chemotherapy. However, A2V only moderately increased cytotoxic T cells in the tumors. To potentially increase the immunogenicity of KP tumors, I also generated two KP mouse variants by either co-expressing a surrogate neoantigen or genetically deleting a DNA mismatch repair enzyme in the cancer cells. However, the therapeutic efficacy of A2V was not further improved in spite of a slightly more activated immune response. I next investigated combination therapies involving A2V and PD1 blockade. In contrast to previous findings in other cancer types, the addition of PD1 antibodies deteriorated the efficacy of A2V, likely through enhancing immunosuppressive mechanisms sustained, at least in part, by regulatory T cells and T cell-antagonizing cytokines. In summary, my work supports the potential clinical efficacy of combined VEGFA and ANG2 blockade for lung cancer therapy but provided little evidence for its synergy in association with anti-PD1 therapy.