Boosting of SARS-CoV-2 immunity in nonhuman primates using an oral rhabdoviral vaccine.

An orally active vaccine capable of boosting SARS-CoV-2 immune responses in previously infected or vaccinated individuals would help efforts to achieve and sustain herd immunity. Unlike mRNA-loaded lipid nanoparticles and recombinant replication-defective adenoviruses, replicating vesicular stomatitis viruses with SARS-CoV-2 spike glycoproteins (VSV-SARS2) were poorly immunogenic after intramuscular administration in clinical trials. Here, by G protein trans-complementation, we generated VSV-SARS2(+G) virions with expanded target cell tropism. Compared to parental VSV-SARS2, G-supplemented viruses were orally active in virus-naive and vaccine-primed cynomolgus macaques, powerfully boosting SARS-CoV-2 neutralizing antibody titers. Clinical testing of this oral VSV-SARS2(+G) vaccine is planned.

IMMUNO-COV v2.0: Development and Validation of a High-Throughput Clinical Assay for Measuring SARS-CoV-2-Neutralizing Antibody Titers.

Neutralizing antibodies are key determinants of protection from future infection, yet well-validated high-throughput assays for measuring titers of SARS-CoV-2-neutralizing antibodies are not generally available. Here, we describe the development and validation of IMMUNO-COV v2.0, a scalable surrogate virus assay, which titrates antibodies that block infection of Vero-ACE2 cells by a luciferase-encoding vesicular stomatitis virus displaying SARS-CoV-2 spike glycoproteins (VSV-SARS2-Fluc). Antibody titers, calculated using a standard curve consisting of stepped concentrations of SARS-CoV-2 spike monoclonal antibody, correlated closely (P < 0.0001) with titers obtained from a gold standard 50% plaque-reduction neutralization test (PRNT50%) performed using a clinical isolate of SARS-CoV-2. IMMUNO-COV v2.0 was comprehensively validated using data acquired from 242 assay runs performed over 7 days by five analysts, utilizing two separate virus lots, and 176 blood samples. Assay performance was acceptable for clinical use in human serum and plasma based on parameters including linearity, dynamic range, limit of blank and limit of detection, dilutional linearity and parallelism, precision, clinical agreement, matrix equivalence, clinical specificity and sensitivity, and robustness. Sufficient VSV-SARS2-Fluc virus reagent has been banked to test 5 million clinical samples. Notably, a significant drop in IMMUNO-COV v2.0 neutralizing antibody titers was observed over a 6-month period in people recovered from SARS-CoV-2 infection. Together, our results demonstrate the feasibility and utility of IMMUNO-COV v2.0 for measuring SARS-CoV-2-neutralizing antibodies in vaccinated individuals and those recovering from natural infections. Such monitoring can be used to better understand what levels of neutralizing antibodies are required for protection from SARS-CoV-2 and what booster dosing schedules are needed to sustain vaccine-induced immunity. IMPORTANCE Since its emergence at the end of 2019, SARS-CoV-2, the causative agent of COVID-19, has caused over 100 million infections and 2.4 million deaths worldwide. Recently, countries have begun administering approved COVID-19 vaccines, which elicit strong immune responses and prevent disease in most vaccinated individuals. A key component of the protective immune response is the production of neutralizing antibodies capable of preventing future SARS-CoV-2 infection. Yet, fundamental questions remain regarding the longevity of neutralizing antibody responses following infection or vaccination and the level of neutralizing antibodies required to confer protection. Our work is significant as it describes the development and validation of a scalable clinical assay that measures SARS-CoV-2-neutraling antibody titers. We have critical virus reagent to test over 5 million samples, making our assay well suited for widespread monitoring of SARS-CoV-2-neutralizing antibodies, which can in turn be used to inform vaccine dosing schedules and answer fundamental questions regarding SARS-CoV-2 immunity.

Development and validation of IMMUNO-COV™: a high-throughput clinical assay for detecting antibodies that neutralize SARS-CoV-2.

We here describe the development and validation of IMMUNO-COV™, a high-throughput clinical test to quantitatively measure SARS-CoV-2-neutralizing antibodies, the specific subset of anti-SARS-CoV-2 antibodies that block viral infection. The test measures the capacity of serum or purified antibodies to neutralize a recombinant Vesicular Stomatitis Virus (VSV) encoding the SARS-CoV-2 spike glycoprotein. This recombinant virus (VSV-SARS-CoV-2-S-Δ19CT) induces fusion in Vero cell monolayers, which is detected as luciferase signal using a dual split protein (DSP) reporter system. VSV-SARS-CoV-2-S-Δ19CT infection was blocked by monoclonal α-SARS-CoV-2-spike antibodies and by plasma or serum from SARS-CoV-2 convalescing individuals. The assay exhibited 100% specificity in validation tests, and across all tests zero false positives were detected. In blinded analyses of 230 serum samples, only two unexpected results were observed based on available clinical data. We observed a perfect correlation between results from our assay and 80 samples that were also assayed using a commercially available ELISA. To quantify the magnitude of the anti-viral response, we generated a calibration curve by adding stepped concentrations of α-SARS-CoV-2-spike monoclonal antibody to pooled SARS-CoV-2 seronegative serum. Using the calibration curve and a single optimal 1:100 serum test dilution, we reliably measured neutralizing antibody levels in each test sample. Virus neutralization units (VNUs) calculated from the assay correlated closely (p < 0.0001) with PRNT EC50 values determined by plaque reduction neutralization test against a clinical isolate of SARS-CoV-2. Taken together, these results demonstrate that the IMMUNO-COV™ assay accurately quantitates SARS-CoV-2 neutralizing antibodies in human sera and therefore is a potentially valuable addition to the currently available serological tests. The assay can provide vital information for comparing immune responses to the various SARS-CoV-2 vaccines that are currently in development, or for evaluating donor eligibility in convalescent plasma therapy studies.

Mapping of Ion and Substrate Binding Sites in Human Sodium Iodide Symporter (hNIS).

The human sodium iodide symporter (hNIS) is a theranostic reporter gene which concentrates several clinically approved SPECT and PET radiotracers and plays an essential role for the synthesis of thyroid hormones as an iodide transporter in the thyroid gland. Development of hNIS mutants which could enhance translocation of the desired imaging ions is currently underway. Unfortunately, it is hindered by lack of understanding of the 3D organization of hNIS and its relation to anion transport. There are no known crystal structures of hNIS in any of its conformational states. Homology modeling can be very effective in such situations; however, the low sequence identity between hNIS and relevant secondary transporters with available experimental structures makes the choice of a template and the generation of 3D models nontrivial. Here, we report a combined application of homology modeling and molecular dynamics refining of the hNIS structure in its semioccluded state. The modeling was based on templates from the LeuT-fold protein family and was done with emphasis on the refinement of the substrate-ion binding pocket. The consensus model developed in this work is compared to available biophysical and biochemical experimental data for a number of different LeuT-fold proteins. Some functionally important residues contributing to the formation of putative binding sites and permeation pathways for the cotransported Na+ ions and I- substrate were identified. The model predictions were experimentally tested by generation of mutant versions of hNIS and measurement of relative (to WT hNIS) 125I- uptake of 35 hNIS variants.

Antibody neutralization of retargeted measles viruses.

The measles virus (MV) vaccine lineage is a promising oncolytic but prior exposure to the measles vaccine or wild-type MV strains limits treatment utility due to the presence of anti-measles antibodies. MV entry can be redirected by displaying a polypeptide ligand on the Hemagglutinin (H) C-terminus. We hypothesized that retargeted MV would escape neutralization by monoclonal antibodies (mAbs) recognizing the H receptor-binding surface and be less susceptible to neutralization by human antisera. Using chimeric H proteins, with and without mutations that ablate MV receptor binding, we show that retargeted MVs escape mAbs that target the H receptor-binding surface by virtue of mutations that ablate infection via SLAM and CD46. However, C-terminally displayed domains do not mediate virus entry in the presence of human antibodies that bind to the underlying H domain. In conclusion, utility of retargeted oncolytic measles viruses does not extend to evasion of human serum neutralization.

Epitope dampening monotypic measles virus hemagglutinin glycoprotein results in resistance to cocktail of monoclonal antibodies.

The measles virus (MV) is serologically monotypic. Life-long immunity is conferred by a single attack of measles or following vaccination with the MV vaccine. This is contrary to viruses such as influenza, which readily develop resistance to the immune system and recur. A better understanding of factors that restrain MV to one serotype may allow us to predict if MV will remain monotypic in the future and influence the design of novel MV vaccines and therapeutics. MV hemagglutinin (H) glycoprotein, binds to cellular receptors and subsequently triggers the fusion (F) glycoprotein to fuse the virus into the cell. H is also the major target for neutralizing antibodies. To explore if MV remains monotypic due to a lack of plasticity of the H glycoprotein, we used the technology of Immune Dampening to generate viruses with rationally designed N-linked glycosylation sites and mutations in different epitopes and screened for viruses that escaped monoclonal antibodies (mAbs). We then combined rationally designed mutations with naturally selected mutations to generate a virus resistant to a cocktail of neutralizing mAbs targeting four different epitopes simultaneously. Two epitopes were protected by engineered N-linked glycosylations and two epitopes acquired escape mutations via two consecutive rounds of artificial selection in the presence of mAbs. Three of these epitopes were targeted by mAbs known to interfere with receptor binding. Results demonstrate that, within the epitopes analyzed, H can tolerate mutations in different residues and additional N-linked glycosylations to escape mAbs. Understanding the degree of change that H can tolerate is important as we follow its evolution in a host whose immunity is vaccine induced by genotype A strains instead of multiple genetically distinct wild-type MVs.

Use of attenuated paramyxoviruses for cancer therapy.

Paramyxoviruses, measles virus (MV), mumps virus (MuV) and Newcastle disease virus (NDV), are well known for causing measles and mumps in humans and Newcastle disease in birds. These viruses have been tamed (attenuated) and successfully used as vaccines to immunize their hosts. Remarkably, pathogenic MuV and vaccine strains of MuV, MV and NDV efficiently infect and kill cancer cells and are consequently being investigated as novel cancer therapies (oncolytic virotherapy). Phase I/II clinical trials have shown promise but treatment efficacy needs to be enhanced. Technologies being developed to increase treatment efficacy include: virotherapy in combination with immunosuppressive drugs (cyclophosphamide); retargeting of viruses to specific tumor types or tumor vasculature; using infected cell carriers to protect and deliver the virus to tumors; and genetic manipulation of the virus to increase viral spread and/or express transgenes during viral replication. Transgenes have enabled noninvasive imaging or tracking of viral gene expression and enhancement of tumor destruction.

Retrovirus vectors.

In this chapter, we present an outline of retroviruses and retroviral vectors - the concepts and applications. In particular, we discuss lentiviral vectors and the suitability of these vectors for the treatment of renal pathologies. We review vector design and the data on the use of lentiviral vectors for gene transfer to the kidney. Finally, we discuss potential pathologies and avenues for the optimization of the technology for gene transfer to a complex organ such as the kidney.

Isolation and characterization of human cells resistant to retrovirus infection.

BACKGROUND: Identification of host cell proteins required for HIV-1 infection will add to our knowledge of the life cycle of HIV-1 and in the development of therapeutics to combat viral infection. We and other investigators have mutagenized rodent cells and isolated mutant cell lines resistant to retrovirus infection. Since there are differences in the efficiency of single round infection with VSVG pseudotyped HIV-1 on cells of different species, we conducted a genetic screen to isolate human cells resistant to HIV-1 infection. We chemically mutagenized human HeLa cells and validated our ability to isolate mutants at test diploid loci. We then executed a screen to isolate HeLa cell mutants resistant to infection by an HIV-1 vector coding for a toxic gene product. RESULTS: We isolated two mutant cell lines that exhibit up to 10-fold resistance to infection by HIV-1 vectors. We have verified that the cells are resistant to infection and not defective in gene expression. We have confirmed that the resistance phenotype is not due to an entry defect. Fusion experiments between mutant and wild-type cells have established that the mutations conferring resistance in the two clones are recessive. We have also determined the nature of the block in the two mutants. One clone exhibits a block at or before reverse transcription of viral RNA and the second clone has a retarded kinetic of viral DNA synthesis and a block at nuclear import of the preintegration complex. CONCLUSION: Human cell mutants can be isolated that are resistant to infection by HIV-1. The mutants are genetically recessive and identify two points where host cell factors can be targeted to block HIV-1 infection.

Isolation, characterization, and genetic complementation of a cellular mutant resistant to retroviral infection.

By using a genetic screen, we have isolated a mammalian cell line that is resistant to infection by retroviruses that are derived from the murine leukemia virus, human immunodeficiency virus type 1, and feline immunodeficiency virus. We demonstrate that the cell line is genetically recessive for the resistance, and hence it is lacking a factor enabling infection by retroviruses. The block to infection is early in the life cycle, at the poorly understood uncoating stage. We implicate the proteasome at uncoating by completely rescuing the resistant phenotype with the proteasomal inhibitor MG-132. We further report on the complementation cloning of a gene (MRI, modulator of retrovirus infection) that can also act to reverse the inhibition of infection in the mutant cell line. These data implicate a role for the proteasome during uncoating, and they suggest that MRI is a regulator of this activity. Finally, we reconcile our findings and other published data to suggest a model for the involvement of the proteasome in the early phase of the retroviral life cycle.

Construction and use of retroviral vectors encoding the toxic gene barnase.

Suicide genes for negative selection of cells have been powerful tools in somatic cell genetic studies and in gene therapy. Here we report on the construction, characterization, and utilization of retroviral vectors encoding barnase, a ribonuclease from Bacillus amyloliquefaciens, expression of which results in apoptosis of transduced mammalian cells. High-titer viral vector production was enabled by expression of an inhibitor of barnase (barstar) in transfected cells generating murine leukemia virus (MLV)- and HIV-1-based vectors. To identify cellular genes required for infection we used barnase-encoding vectors in a genetic screen to isolate mutant mammalian cells that are resistant to infection by MLV and HIV-1. We describe one such mutant clone that is inhibited in the infection process after reverse transcription. These results suggest that barnase-encoding vectors should be useful for negative selection strategies examining retroviral infection from entry to integration. Furthermore these vectors could have utility in approaches for gene therapy that require specific cell ablation.

DNA-repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a.

The Rad23 family of proteins, including the human homologs hHR23a and hHR23b, stimulates nucleotide excision repair and has been shown to provide a novel link between proteasome-mediated protein degradation and DNA repair. In this work, we illustrate how the proteasomal subunit S5a regulates hHR23a protein structure. By using NMR spectroscopy, we have elucidated the structure and dynamic properties of the 40-kDa hHR23a protein and show it to contain four structured domains connected by flexible linker regions. In addition, we reveal that these domains interact in an intramolecular fashion, and by using residual dipolar coupling data in combination with chemical shift perturbation analysis, we present the hHR23a structure. By itself, hHR23a adopts a closed conformation defined by the interaction of an N-terminal ubiquitin-like domain with two ubiquitin-associated domains. Interestingly, binding of the proteasomal subunit S5a disrupts the hHR23a interdomain interactions and thereby causes it to adopt an opened conformation.